_____________________________________________________________________________________________________ *corresponding author: e-mail: mariajnad66@gmail.com; asian journal of immunology 3(1): 305-310, 2020; article no.aji.61363 the relationship between the levels of anti cyclic citrullinated protein antibodies type2 (acpa2) and the severity of erosive rheumatoid arthritis (ra) m. jnad1*, k. aldou1 and h. yazaji2 1 division of rheumatology, department of internal medicine, tishreen university hospital, lattakia, syria. 2 department of laboratory, tishreen university hospital, lattakia, syria. authors’ contributions this work was carried out in collaboration among all authors. author mj designed the study, performed the statistical analysis, wrote the protocol and wrote the first draft of the manuscript, managed the analyses of the study and managed the literature searches. author ka was supervisor professor. author hy was assistant supervisor professor. all authors read and approved the final manuscript. article information editor(s): (1) dr. wagner loyola, brazilian agricultural research corporation, brazil. reviewers: (1) ibrahim mohamad saeed shnawa, university of qasim, iraq. (2) k. ramya, tamil nadu veterinary and animal sciences university, india. complete peer review history: http://www.sdiarticle4.com/review-history/61363 received 15 july 2020 accepted 20 september 2020 published 25 september 2020 abstract background: rheumatoid arthritis (ra) is a chronic systemic disease, characterized by autoimmunity. one of the most specific and important diagnostic and prognostic markers of ra are antibodies, anti cyclic citrullinated protein antibodies (acpa). objective: the purpose of this study was to investigate the association between acpa (anti-ccp) levels and radiological damage. methods: an observational cross-sectional study included 54 patients fulfilling the acr-eular 2010 criteria for ra, with minimum disease duration of 1 year. radiographs were scored using a modified sharp score (van der heijde modification of sharp's score 1989). acpa levels were determined using immunofluorescence assay. results: acpa levels were strongly associated with radiographic severity and there was a significant relationship between acpa levels and total sharp score (p=0.0001). original research article jnad et al.; aji, 3(1): 305-310, 2020; article no.aji.61363 306 conclusion: this study concluded that acpa is an independent severity factor for ra, and our data supports the association between acpa and radiographic severity. keywords: anti cyclic citrullinated protein; type2 (acpa2); rheumatoid arthritis; chronic systemic disease. abbreviations ra : rheumatoid arthritis acpa : anti citrullinated peptides antibody anti-ccp : anti cyclic citrullinated protein antibodies acr : american college of rheumatology eular : european league against rheumatism rf : rheumatoid factors dmard : disease modifying anti rheumatic drugs 1. introduction rheumatoid arthritis (ra) is a chronic systemic disease of unknown origin, characterized by autoimmunity, with the presence of autoantibodies in the blood and joint fluid. one of them is acpa, which state high disease specificity and may contribute to the pathological process of the disease such as erosiveness and synovitis [1]. if untreated, it usually leads to progressive joint damage and reduction of functional capacity and quality of life [2,3]. early diagnosis and effective intervention may prevent joint damage, increase the possibility of achieving remission and improve the outcome [4]. radiography is the most common method for evaluating the existence of structural damage, which results from cumulative disease activity, and determining the effectiveness of treatment by estimating bone erosions and joint space narrowing on conventional radiography [5]. acpa positive patients are at particularly high risk for developing severe forms of ra and severe radiographic damage, as they are independent severity factor for rheumatoid arthritis (ra), and may be very useful biomarkers to predict disease damage [6,7]. there is no research on association between acpa and structural damage in ra patients in syria. the objective of the present work was to map the possible association betweenacpa2 and the radiographic severity of the ra patient joints. 2. methods and patients an observational cross-sectional study included patients (n=54; 45 female, 9 male) with ra (duration more than 1 year) fulfilling the acreular 2010 criteria for ra, at the department of rheumatology, tishreen university hospital, lattakia, syria. over 1year period from april 2019 to march 2020, patients having an overlap of ra with other rheumatic disease like gout were excluded from the study. radiographs of hand and feet were scored using a modified sharp score (van der heijde modification of sharp's score). 2.1 antibody measurement (acpa/anti ccp) anti ccp were measured, in a blind fashion, at the immunological laboratory in tishreen hospital using an (i-chromα anti-ccp plus) kit based on the immunofluorescence assay method. a value of > 5.0 u/ml was considered positive as per the manufacturers' recommendations. rf was measured using the standard latex agglutination technique. 2.2 measure of disease outcome (structural damage) the radiographs were evaluated by two senior rheumatologists in a blind fashion regarding serology and clinical status, with high concordance between them. posterior anterior radiographs of the hands, wrists, and feet were obtained and were examined blind by a two rheumatologists, and graded according to modified sharp score (van der heijde modification of sharp's score) in comparison with standard reference films. jnad et al.; aji, 3(1): 305-310, 2020; article no.aji.61363 307 the scoring system included erosion score: 16 locations in each hand and wrist, and 12 location in each foot were scored. each location in the hand/wrist and foot was scored individually using a 6-point scale from 0 to 5 based on the number and size of discrete erosions in each location. joint space narrowing (jsn) score: 15 locations in each hand and wrist, and 6 locations in each foot were scored using a 5-point scale (from 0 to 4). the maximum erosion score is 160 for hands and wrists, and 120 for feet. the maximum jns score is 120 for hands and wrists, and 48 for feet. the total score ranges from 0 to 448 [8]. 2.3 statistics all data were analyzed using the statistical package for social sciences (spss, version 20). data were presented in simple measures of frequency, percentage, mean, and standard deviation. non-parametric test mann whitney used for nonnormally distributed data, while correlation between quantitative data were tested using person correlation. results were considered statistically significant with a p-value<5%. fig. 1. distribution of patients according to sex 3. results the cross-sectional study includes 54 patients (45 female, 9 male) with ra, the median (iqr, interquartile range) age was 50.5 (27-80) yr. the median (iqr, interquartile range) disease duration was 2.7 year (1-20) yr, the proportion of females in the study was 83.3%. 94.4% of the patients were treated with dmards. about 68.5 per cent (n=38) patients were seropositive (positive for rf), and 81.5 per cent (n=44) patients had erosive disease. on bivariate analysis, patients with erosive disease had a significantly higher disease duration, dmard naïve period, rf positivity and acpa titers than patients with non-erosive disease (table 1). 3.1 association among radiographic outcome and anti-ccp2 (acpa) status in our study acpa titers were strongly associated with radiographic severity as measured using sharp van der heijde score (pvalue=0.0001). 4. discussion  we founded that disease duration, dmard naïve period, rf titers, and acpa titers were significantly higher in patients with erosive disease compared with nonerosive disease (table 1). the most significant and important factor of them all was acpa titers (p=0.001). this result compatible with the study that was done by shankar et al [9].  the long duration without compatible treatment with dmards can be attributed in syria to the lack of medications in the light of the economic situation.  the most significant difference was in acpa, rf titers between patients with erosive and non-erosive disease, what confirms that the combination of acpa and rf are associated with more aggressive articular disease and that the association of both rf and acpa is independent factor for more severe destructive disease [10,1,6].  the high positive correlation between acpa titers and sharp scores (r=0.7, p=0.0001) (fig. 2) indicates that acpa has a significant role in predicting the radiographic and structural damage in rheumatoid patients, and that corresponds with a previous study by meyer et al. that included 191 patients with recent oncet ra followed for 5 years. their serum samples were examined for acpa, and erosions female 83% male 17% distribution of patients according to sex sex ratio(f:m)=4.9:1 female male jnad et al.; aji, 3(1): 305-310, 2020; article no.aji.61363 308 were scored using sharp scores modified as described by van der heijde. their results suggested that odds of a complete sharp score increase after 5 years was significantly higher in patients with acpa [11].  it confirms that patients with erosive disease had significantly higher acpa titers than those with non-erosive disease; consequently, high titers of acpa antibodies are associated with more aggressive disease, as measured by significantly higher sharp scores[6].  finally, this study showed the effect of dmard naïve period and the disease duration on erosion formation, as the median of dmard naïve period and the median disease duration were significantly higher in patients with erosive disease compared to patients with non-erosive disease [12]. table 1. characteristics of patients with erosive and non-erosive disease characteristics erosive disease(n=44) non erosive disease(n=10) test used pvalue the statistical significance disease duration (yr) median (iqr) 3.5 (1-20) 2 (1-3) mann whitney 0.03 s dmard naïve period (month) median (iqr) 12 (0-60) 10.5 (0-24) mann whitney 0.04 s rf positivity (%) 31 (70.45) 6 (60) chi-square 0.07 n.s rf positivity (u/ml) median (iqr) 47 (1-192) 30 (10-210) mann whitney 0.002 s median acpa titer (u/ml) median (iqr) 16.2 (5.1-300) 7.5 (5.4-38) mann whitney 0.001 s dmard: disease modifying anti rheumatic drugs acpa: anti citrullinated peptides antibody s: statistically significant n.s: statistically not significant fig. 2. showing high positive correlation between acpa titers and sharp scores. r=0.7, p=0.0001 0 50 100 150 200 250 300 350 400 0 50 100 150 200 250 300 350 sh a r p acpa u/ml sharp jnad et al.; aji, 3(1): 305-310, 2020; article no.aji.61363 309 5. conclusion  acpa titers are reliable predictors of erosive disease, in addition to the fact of their ability to predict the cumulative structural and radiological damage is superior to rf [6,13].  acpa is very important serological marker in identifying patients with worse prognosis who may develop more severe structural damage, and need early aggressive treatment and consequently improves their clinical outcome and reduce joint damage and disability[14].  determination of acpa are extremely valuable marker which is especially useful in rf negative patients with early arthritis who did not yet fulfill the diagnostic criteria of ra [1,14]. consent and ethical approval this study was reviewed and approved by the moral committee of the tishreen university hospital. as per international standard or university standard guideline participant consent has been collected and preserved by the authors. acknowledgements we wish to thank doctors and technicians in laboratory and radiology departments of tishreen university hospital who worked with us. we would like to thank miss elissar hejazia, dr. leen doya, and dr. rawa saad for technical help. we would like to thank the assistance provided by prof. dr. ahmad chreitah. competing interests authors have declared that no competing interests exist. references 1. stainer g. autoantibodies: diagnostic helpers and pathogenetic players. in: hochberg m, silman a, smolen j. (eds) rheumatoid arthritis. philadelphia: mosbey. 2008;180-190. 2. weisman m. definition and classification. rheumatoid arthritis. new york: oxford university press. 2011;5-10. 3. živkovi n, stankovi a, krsti i, kosti t, ili n. influence of joint damage on functional capacities in patients suffering from rheumatoid arthritis. acta med media. 2009;48(2):18-21. 4. heidari b. rheumatoid arthritis: early diagnosis and treatment outcomes. caspian journal of internal medicine. 2011;2(1):161. 5. weisman m. laboratory and imaging assessments. rheumatoid arthritis. new york: oxford university press. 2011;55-60 6. song yw, kang eh. autoantibodies in rheumatoid arthritis: rheumatoid factors and anticitrullinated protein antibodies. qjm: an international journal of medicine. 2010;103(3):139-46. 7. rocha sd, baldo dc, andrade le. clinical and pathophysiologic relevance of autoantibodies in rheumatoid arthritis. advances in rheumatology. 2019;59. 8. peterfy cg, wu c, lu y, genovese m. comparison of the genant-modified sharp and van der heijde-modified sharp scoring methods for radiographic assessment in rheumatoid arthritis. international journal of clinical rheumatology. 2011;6(1):15. 9. shankar s, grover r, handa r. role of anti cyclic citrullinated peptide antibodies in erosive disease in patients with rheumatoid arthritis. indian journal of medical research. 2006;124(6):689-96. 10. andrade f, darrah e, rosen a. autoantibodies in rheumatoid arthritis. in: gary s. firestein, sherine e. gabriel, iain b. mcinnes, james r. o’dell. (eds) kelley and firestein’s textbook of rheumatology, tenth edition. philadelphia: elsevier. 2017;831-845. 11. vossenaar er, van venrooij wj. anti-ccp antibodies, a highly specific marker for (early) rheumatoid arthritis. clinical and applied immunology reviews. 2004;4(4):239-62. 12. bahadur m, singh p, satyawali v, joshi a. role of dmard naïve period in the development of severe rheumatoid arthritis. indian journal of orthopaedics. 2020;1-5. 13. jilani aa, mackworth-young cg. the role of citrullinated protein antibodies in predicting erosive disease in rheumatoid arthritis: a systematic literature review and meta-analysis. international journal of rheumatology; 2015. jnad et al.; aji, 3(1): 305-310, 2020; article no.aji.61363 310 14. niewold tb, harrison mj, paget sa. anti-ccp antibody testing as a diagnostic and prognostic tool in rheumatoid arthritis. journal of the association of physicians. 2007;100(4): 193-201. _________________________________________________________________________________ © 2020 jnad et al.; this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: http://www.sdiarticle4.com/review-history/61363 http://creativecommons.org/licenses/by/4.0 _____________________________________________________________________________________________________ *corresponding author: e-mail: ezenelson24@gmail.com; asian journal of immunology 1(1): 14-19, 2018; article no.aji.44952 missed immunisation and immunisation drop-outs among infants in rural health facilities in abakaliki, nigeria n. c. eze1* 1 department of community medicine, federal teaching hospital abakaliki, nigeria. author’s contribution author nce designed, analysed, interpreted and prepared the manuscript. article information doi: 10.9734/aji/2018/44952 editor(s): (1) dr. wagner loyola, department of immunology, brazilian agricultural research corporation (embrapa) concordia, canada. reviewers: (1) abram wagner, university of michigan, usa. (2) a. shaikh amir, indira college of pharmacy, savitribai phule pune university, india. (3) jose manuel jaramillo ortiz, national university jauretche, argentina. complete peer review history: http://www.sciencedomain.org/review-history/27207 received 15 th august 2018 accepted 5 th november 2018 published 14 th november 2018 abstract background: missed immunisation, immunisation drop-out and coverage rates at primary health care (phc) level indicate the level at which communities utilise the preventive services and thus serve as a measure of the strength of the public health system. they also measure the effectiveness of the immunisation programme. the extent of missed immunisation and immunisation drop-out is not well known in the study area. this study, therefore, determined the extent of missed immunisation and immunisation drop-outs in abakaliki. materials and methods: descriptive cross-sectional analytical study design was used for the survey. total number of infants in the health facilities was used as a sample size in this study (406 infants at mile-four hospital and 281 infants at st. vincent hospital). data were extracted from the existing immunisation registers in the two health facilities studied. statistical package for social sciences (spss) version 22 was used for data analysis. ethical approval for this study was obtained from the research and ethics committee (rec) of the federal teaching hospital abakaliki (fetha). original research article eze; aji, 1(1): 14-19, 2018; article no.aji.44952 15 results: showed significant differences in the proportion of missed immunisations (39.7% in milefour and 48.4% in st. vincent respectively, p=0.02) and immunisation drop-outs (35.7% in milefour and 47.7% in st.vincent respectively, p=0.02). the drop-out rate is 64.3% in mile-four and 52.3% in st. vincent. it also showed that 36.6% of male infants when compared to 34.8% of female infants dropped-out of the 3 rd dose of pentavalent vaccines in mile-four while 44.5% of male infants and 51.1% of female infants dropped-out 3 rd dose of pentavalent vaccines in st.vincent hospital. there was no significant difference in the immunisation drop-out rates between male and female infants in the study groups (p>0.05). conclusion: a higher proportion of infants missed immunisation and dropped out of vaccination at st.vincent than mile-four. this calls for an aggressive public campaign on the need to ensure immunisation timeliness for effective immunisation in such rural areas. keywords: missed immunisation; immunization drop-out; infants; rural health facilities; abakaliki. 1. introduction immunisation drop-outs refer to infants who have used or missed immunisation services and do not return for subsequent vaccinations. when drop-outs exceed 10 percent, it indicates a problem of utilisation of services. in most settings where full immunisation coverage is low, most infants receive at least one dose of pentavalent vaccines, but the proportion that receives the needed second and third doses drops significantly. drop-out rates are calculated as the percentage point difference between successive doses of a vaccine, expressed as a percentage of the first dose [1]. immunisation coverage which is the indicator of access to the preventive services at phc level is measured by the percentage of infants who have received the appropriate immunisations. immunisation coverage is calculated as the total number of infants that have received all their immunisations up to the measles vaccine, divided by the total population of children under one year old in a given area [1]. however, differences in the indicators of access and utilisation of immunisation services exist. indicators of access to health services among other factors include a level of education, sex, patriarchal social arrangement, rural residence, poverty, religious and cultural beliefs about certain diseases and location of health facilities etc. indicators of utilisation of immunisation services include quality of staff skills, protocols of treatment, availability of supplies and environment of health facilities, physical and financial accessibility of services, knowledge of which services exist, education about how to best utilise self and practitioner-provided services and cultural norms of treatment [2,3]. vaccination is typically offered free in primary health care centres and are usually not compulsory for school enrolment. childhood vaccines are given at interval of 0, 6 th , 10 th , 14 th week and 9 th month of birth. immunisation drop-out usually are due to far distance to health facility. the extent of missed immunisation and immunisation drop-out is not well known in the study area. this study, therefore, determined the extent of missed immunisation and immunisation drop-outs in abakaliki. 2. materials and methods this study was carried out at mile four and st. vincent hospitals (in ebonyi and izzi local government areas respectively) in ebonyi state. both private facilities are in the rural area. descriptive crosssectional analytical study design was used for the survey. data were extracted from the existing immunisation registers in the two health facilities studied. a total number of infants in the health facilities was used as sample size for the study (406 infants at mile-four hospital and 281 infants at st. vincent hospital, both in abakaliki, ebonyi state). statistical package for social sciences (spss) version 22.0 was used for data analysis. chisquared test was to determine association or differences between proportion of the variables and the level of statistical significance was set at p< 0.05 and confidence level at 95%. ethical approval for this study was obtained from the research and ethics committee (rec), federal teaching hospital abakaliki (fetha), ebonyi state, nigeria. 3. results a total of 687 infants were studied, 406 (59%) in mile-four and 281 (41%) in st. vincent. these figures were the number of infants recorded as at eze; aji, 1(1): 14-19, 2018; article no.aji.44952 16 the time of data extraction. review of immunisation register showed significant differences in the proportion of missed immunisations (39.7% in mile four and 48.4% in st. vincent respectively, p=0.02) and immunisation drop-outs (35.7% in mile four and 47.7% in st. vincent respectively, p=0.02). table 1 showed that the proportion of infants who missed immunisations was 39.7% in milefour and 48.4% in st. vincent. the difference in proportion of infants who missed immunisation was statistically significant (p=0.02). table 2 showed that the proportion of male and female infants who missed penta-valent vaccines. the difference in their proportion was not significant. table 3 showed that 35.7% of infants dropped out of pentavalent vaccines 3 in mile-four when compared with 47.7% who dropped out of pentavalent vaccines 3 in st. vincent. the difference in their proportions was statistically significant (p=0.02). the drop-out rate is 64.3% in mile-four and 52.3% in st.vincent. table 4 showed that 36.6% of male infants and 34.8% of female infants dropped-out of 3 rd dose of pentavalent vaccines in st. vincent while 44.5% of male infants and 51.1% of female infants dropped-out 3 rd dose of pentavalent vaccines in mile-four. there was no significant difference in the immunisation drop-out rates between male and female infants in the study groups (p>0.05). 4. discussion in this study, a significantly lower proportion of infants (39.7%) in mile four than st.vincent (48.4%) missed immunisation. more infants missed immunisations in increasing interval (most commonly missed are 14 th week vaccines compared to 10 th week vaccines and least missed are 6 th week vaccines). this may be due to the fact that as immunisation time intervals increase, caregivers tend to forget the exact date of immunisation of their infants thereby making immunisation timeliness unrealisable and putting such infants temporally at risk of infection by vaccine preventable diseases [4]. this finding is at variance with that found in nnewi in a study of mother-neonate pairs where most commonly missed immunisations were bcg, opv0 and opv1, hbv1and dpt1 compared to the 10 th week and 14 th week vaccines. in that study, mother’s age, education and knowledge of immunisation were not significantly associated with missed immunisation. there was comparable proportion of missed immunisations (pentavalent vaccines 1 and 3) between the male and female infants who were vaccinated three (3) months before the intervention. this may explain the importance attached to both male and female infants now unlike olden days when preference was given to male infants in the african society. a higher proportion of infants missed opv3, pentavalent 3 vaccines and pcv3 than the 6 th and 10 th week vaccines. the prevalence of missed immunisation in mile-four (39.7%) is higher compared to those reported in nnewi (17%) [5], benin city (27.6%) [6] among mothers of infants 6 months to 1 year and consistent with that in calabar (39.1%) [7] but lower than the 57.1% reported in india [8]. the missed vaccines would invariably reduce herd immunity of such population and consequently make such infants prone to vaccine preventable diseases [4]. the commonest vaccines missed were opv1, hbv1 and dpt1 (40.38%), followed by bcg and opv0 (38.46%), and opv2, hbv2, and dpt2 (11.54%). the finding of higher rate of missed immunisation is in keeping with other study elsewhere [9] suggesting that the reasons for this high level of missed immunisation have not been adequately addressed. these should be addressed through adequate communication between mothers and health workers, training of health workers and policy flexibility [5]. lower proportion of infants in mile-four (35.7%) than st.vincent (47.7%) dropped-out of vaccination. there was a statistically significant difference in the proportion of infants who dropped-out of vaccination in the groups. this significant dropout rate among the infants would invariably compromise the herd immunity of such population and consequently make such infants prone to vaccine preventable diseases with attendant morbidity and mortality [4]. there was no significant difference in the immunisation drop-out rates between male (36.6%) and female (34.8%) infants in mile-four and st.vincent (male; 44.5% and female; 51.1%). the cumulative drop-out rates were 64.3% in mile-four and 52.3% in st.vincent. this may be due to the fact that most infants were delivered in the facility and as their mother’s postnatal care services ended, they continued the immunisation in a nearby health facility [5]. eze; aji, 1(1): 14-19, 2018; article no.aji.44952 17 table 1. proportion of infants who missed immunisations in both facilities variables mile-four (n=406) freq. (%) st. vincent (n=281) freq. (%) χ 2 (p-value) number who missed immunisation yes 161 (39.7) 136 (48.4) 5.17 (0.02)٭ no 245 (60.3) 145 (51.6) number who missed each vaccine bcg 0 (0.0) 0 (0.0) opv0 0 (0.0) 0 (0.0) hb0 2 (0.5) 0 (0.0) opv1 60 (14.8) 22 (7.8) pentavalent1 11 (2.7) 5 (1.8) pcv1 26 (6.4) 7 (2.5) opv2 89 (21.9) 57 (20.1) pentavalent2 80 (19.7) 58 (20.6) pcv2 99 (24.4) 57 (20.3) opv3 152 (37.4) 136 (48.4) pentavalent3 146 (36.0) 135 (48.0) pcv3 161 (39.7) 136 (48.4) statistically significant٭ table 2. within group comparison between sex of infants and missed immunisation in both facilities variables mile four (n=406) st.vincent (n=281) missed immunisation missed immunisation yes freq. (%) no freq. (%) total χ 2 (p-value) yes freq. (%) no freq. (%) total χ 2 (p-value) sex of infants pentavalent vaccines1 pentavalent vaccines1 male 4 (2.0) 201 (98.0) 205 (100) 0.90 (0.34) 4 (2.7) 142 (97.3) 146 (100) ft (0.37) female 7 (3.5) 194 (96.5) 201 (100) 1 (0.7) 134 (99.3) 135 (100) pentavalent vaccines 3 pentavalent vaccines 3 male 76 (37.1) 129 (62.9) 205 (100) 0.22 (0.63) 65 (44.5) 81 (55.5) 146 (100) 1.51 (0.21) female 70 (34.8) 131 (65.2) 201 (100) 70 (51.9) 65 (48.1) 135 (100) ft=fisher’s exact test eze; aji, 1(1): 14-19, 2018; article no.aji.44952 18 table 3. proportion of clients who droppedout of vaccines in both facilities variables mile-four (n=406) freq. (%) st.vincent (n=281) freq. (%) χ 2 (p-value) number who dropped-out vaccines yes 145 (35.7) 134 (47.7) 6.10 (0.02)٭ no 251 (64.3) 147 (52.3) drop-out rate 64.3% 52.3% number who dropped-out each vaccine opv3 147 (36.2) 134 (47.7) 9.05 (<0.01)* pentavalent3 145 (35.7) 134 (47.7) 9.87 (<0.01)* pcv3 152 (37.4) 136 (48.4) 7.11 (0.01)* statistically significant٭ table 4. within group comparison between sex of infants and immunisation drop-outs in both facilities (3 rd dose of pentavalent vaccines) variables mile four (n = 406) st.vincent (n=281) immunisation drop-out immunisation drop-out yes freq. (%) no freq. (%) total χ 2 (p-value) yes freq. (%) no freq. (%) total χ 2 (p-value) sex of infants pentavalent 3 pentavalent 3 male 75 (36.6) 130 (63.4) 205 (100) 0.13 (0.71) 65 (44.5) 81 (55.5) 146 (100) 1.22 (0.26) female 70 (34.8) 131 (65.2) 201 (100) 69 (51.1) 66 (48.9) 135 (100) eze; aji, 1(1): 14-19, 2018; article no.aji.44952 19 5. conclusion higher proportion of infants missed immunisation and dropped out of vaccination in st.vincent than mile-four hospital. this finding calls for an aggressive public campaign on need to ensure immunisation timeliness for effective immunisation in such rural areas. consent as per international standard or university standard, patient’s written consent has been collected and preserved by the author(s). ethical approval ethical approval for this study was obtained from the research and ethics committee (rec), federal teaching hospital abakaliki (fetha), ebonyi state, nigeria. competing interests author has declared that no competing interests exist. references 1. immunisation drop-out. health management information system news. sept 22 2011;1. 2. nnonyelu an, nwankwo iu. social determinants of differential access to health services across five states southeast nigeria. europ. scientific j. 2014;3: 1857-7881. 3. ebonyi state ministry of health, health management information system unit; ebonyi state health systems development project. 2015;27. 4. balogun mr, sekoni ao, okafor ip, odukoya oo, ezeiru ss, ogunnowo be, campbell pc. access to information technology and willingness to receive text message reminders for childhood immunisation among mothers attending a tertiary facility in lagos, nigeria. afr jch. 2012;6(3):76-80. 5. ubajaka fc1, ukegbu au, okafor nj1, ejiofor o. the prevalence of missed opportunities for immunisation among children utilising immunisation services in nnamdi azikiwe university teaching hospital, nnewi, journal of biology, agriculture and healthcare. 2012;2(6):113114. 6. onyiriuka an. vaccination default among children attending a static immunisation clinic in benin city, nigeria. jmbr. 2005; 4(1):71-77. 7. anah mu, etuk is, udo jj. opportunistic immunisation with in-patient programme: eliminating a missed opportunity in calabar, nigeria. annals of afr. medicine. 2006;5(4):188-191. 8. coreil j, augustin a, holt e, halsey na. use of ethnographic research for instrument development in a case control study of immunisation in haiti. int. j. epidemiol. 1989;18:33-37. 9. ngini ae. missed opportunity to ep1 scheduled immunization in enugua health facility survey (dissertation). west african postgraduate medical college; 1999. _________________________________________________________________________________ © 2018 eze; this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: http://www.sciencedomain.org/review-history/27207 http://creativecommons.org/licenses/by/4.0 _____________________________________________________________________________________________________ *corresponding author: e-mail: albertopk2000@yahoo.co.uk; asian journal of immunology 3(1): 278-282, 2020; article no.aji.58254 discussion on the concept of immunity and how human body system could build immunity against covid 19 and it relevance to public health education albert opoku1*, joana owusu danso1, olivia nyarko mensah1, nicholas amoah owusu2, prince twene2, jefferson asare dankwa3 and andrews owusu4 1 nursing and midwifery training college, kumasi, ghana. 2 food and drug authority, ghana. 3 ghana health service, ghana. 4 konongo odumasi hospital, ghana health service, ghana. authors’ contributions this work was carried out in collaboration among all authors. author ao designed the study and did the literature review with authors jod and onm. authors nao and pt wrote the protocol and wrote the first draft of the manuscript. authors jad and ao edited the manuscript. all authors read and approved the final manuscript. article information editor(s): (1) dr. wagner loyola, brazilian agricultural research corporation, brazil. reviewers: (1) asmaa abdallah darwish, desert research center, egypt. (2) hasta handayani idrus, universitas muslim indonesia, indonesia. complete peer review history: http://www.sdiarticle4.com/review-history/58254 received 03 june 2020 accepted 21 june 2020 published 01 july 2020 abstract the aim of this article is to explain different types of immunity and discuss how the human body could build immunity against covid 19. this would also serve as a guide for public health education on immunity in this era of covid 19 pandemic. keywords: immunity; public health; covid 19. mini-review article ………… article opoku et al.;aji, 3(1): 278-282, 2020; article no.aji.58254 279 1. introduction every second of day, an enemy of aggressive pathogens (disease causing organisms) such as bacteria, fungi, and viruses crowd on our skin and we stay remarkable healthy most of the time. the body seems to have evolved a single minded approach to such rivalsif you are not with us, you are against us. to implement that stance, it relies heavily on the two intrinsic defense systems and that act both independently and cooperatively to provide resistance or immunity against disease causing pathogens [1]. the above introduction therefore suggest that one’s immune system is very vital to fight the new covid 19 pandemic. the discussion in this article seek to explain types of immunity and their application to fight covid 19. 2. immunity immunity is defined as the body’s own ability to destroy pathogens or other foreign material and to prevent further cases of certain infectious diseases. this ability is of vital importance because the body is exposed to pathogens from the moment of birth. immunitytherefore means being protected from something and being unaffected or not bothered by it. let's say you have immunity to heat this means heat can't bother you whatsoever walking up to hot molten lava (about 2,000 degrees f!) would be no different than walking up to a river. our immune system protects us and helps fight off disease causing microorganisms such as virus, bacteria, fungi and others [2]. immunity has two main components: innate immunity and adaptive immunity [3]. 2.1 innate immunity innate immunity may be called nonspecific, does not create memory, and its responses are always the same irrespective of the target. the innate immune system consist of structures that defend body against infections and could be activated immediately pathogens invade the body [4]. the innate immune system is essentially made up of barriers that aim to keep viruses, bacteria, parasites, and other foreign particles out of your body or limit their ability to spread and travel throughout the body. the innate immune system consist of: 2.1.1 physical barriers physical barriers that prevent entry of pathogens such as intact skin, intact the gastrointestinal tract, the intact respiratory tract including nasopharynx and cilia, eyelashes and other body hair [5]. 2.1.2 defense mechanisms defense mechanismssuch as various secretions, mucous, bile, gastric acid, saliva, tears, and sweat. 2.1.3 general immune general immune responses such as inflammation, complement, and non-specific cellular responses. the inflammatory response vigorously brings immune cells to the location of an infection by increasing blood flow to the area [6]. 2.1.4 complement complement system is an immune response that marks pathogens for them to be destroyed and makes holes in the cell membrane of the pathogen. the innate immune system is always general, or nonspecific, meaning anything that is identified as foreign or non-self is a target for the innate immune response. the innate immune system is stimulated by the existence of antigens and their chemical properties [3]. the innate immunity is very effective against all kinds of infections including viruses such as covid 19. thus people with very strong and efficient innate immune system could therefore withstand the covid 19 infection and this explain why the young are not normally dying from the covid 19 infection as compare to the aged. this is because the innate immune system deteriorate with ageing especially among those with comorbidity. the reason there is high morbidity and mortality with covid 19 among the aged with comorbidity [6]. in addition to the above, the preventive measure such as face masks combined with other preventive measures, such as frequent hand-washing and social distancing, help slow the spread of the virus because they actually enforce the physical barrier provided by innate immunity [7]. again the preventive measures help to stop the spread of the infection through people coming into contact opoku et al.;aji, 3(1): 278-282, 2020; article no.aji.58254 280 with defense secretion like mucus, saliva, tears, and sweat that may be used as vehicle to spread the covid 19 from person to person. 2.2 adaptive immunity adaptive immunity is very specific as to its target, may involve antibodies, does create memory, and may become more efficient. unlike the innate immune system, the adaptive immune system depend on fewer types of cells to carry out its responsibilities: b cells and t cells. both b cells and t cells are lymphocytes that are produced from specific types of stem cells, called hematopoietic stem cells or tissues, in the red bone marrow. after they are production in the bone marrow, they need to mature and become stimulated [3]. when activated during an immune response, some b cells will divide many times and become plasma cells that produce antibodies to a specific foreign antigen. the mechanisms of immunity that involve t cells and b cells are specific, meaning that one foreign antigen is the target each time a mechanism is activated. have you ever wondered how your recovery time for the common cold, the flu, or small infections seems to get shorter after you’ve been exposed and successfully recovered the first time? the adaptive immune system, also called acquired immunity, uses specific antigens to strategically mount an immune response. unlike the innate immune system, which attacks only based on the identification of general threats, the adaptive immunity is activated by exposure to pathogens, and uses an immunological memory to learn about the threat and enhance the immune response accordingly. the adaptive immune response is much slower to respond to threats and infections than the innate immune response, which is primed and ready to fight at all times [8]. the adaptive immune system is exactly that it's adaptive, meaning it can adapt to a specific threat, or antigen. for example, if the opposing team brings in a substitute, the entire team can adapt to that new substitute's unique threat. however, the adaptive immune system takes time to develop; there's a lag of sorts, meaning the trainer of the team needs to coach his players on how to adapt to an antigen, or the recognizable and visible aspect of a threat, like a bacterium's surface receptors. the good thing is once the players are trained well enough, they develop a good memory with respect to that threat. this means that whenever they encounter that substitute, or specific antigen or threat, again, they will know exactly what to do with it and will not have to waste time training for it. basically, the delay is only really significant the first time around, not as much thereafter. all of this is in contrast to the innate immune system, which suffers no delay, has no memory and isn't trained to respond to a specific threat; it goes after everything that moves [9]. this means that for one to get immunity against covid 19, the individual should be exposed to the virus first before the body is stimulated to produce antibodies against it. this may require the person being infected with the virus and after recovery the person produce antibodies for subsequent infections of covid 19. on the other hand, one could also produce antibodies through covid 19 vaccination after which could build resistance against the virus. 3. types of immunity based source of immunity if we consider the source of immunity, that is, where it comes from, we can begin with two major categories: genetic immunity and acquired immunity (adaptive immunity) [10]. 3.1 genetic immunity genetic immunity does not involve antibodies or the immune system but conferred by our dna; it is the result of our genetic makeup. what this means is that some pathogens cause disease in certain host species but not in others [11]. dogs and cats, for example, have genetic immunity to the measles virus, which is a pathogen only for human. on the other hand, mouse leukemia viruses affect only mice and not human; thus human have genetic immunity to these viruses [12]. this is not because human have antibodies against these mouse viruses, but rather we have genes that are the codes for proteins that make it impossible for such pathogens to reproduce in the human cells and tissues. monkeys have similar protective genes and proteins for the human aids virus and therefore hiv does not cause disease in these monkeys. because this is a genetic characteristic programmed in dna, genetic immunity always lasts a lifetime [10]. the above explanation means that some group of people may have this type of immunity which could make them build some resistance to the opoku et al.;aji, 3(1): 278-282, 2020; article no.aji.58254 281 covid 19. the reasons black people in africa and for that matter ghana are perhaps recording low mortality from covid 19. 3.2 acquired immunity acquired immunity does involve antibodies and consist of passive immunity and active immunity. acquired immunity is developed or acquired by natural or artificial means. 3.3 passive immunity there are two types passive immunity which arenaturally acquired passive immunity and artificially acquired passive immunity. 3.3.1 naturally acquired passive immunity it means that the antibodies are from another source but naturally transmitted. one type of naturally acquired passive immunity is the placental transmission of antibodies (igg) from maternal blood to fetal circulation [10]. this means a pregnant woman could transmit antibodies of covid 19 to fetus before the baby is born. such a child would be bornimmunity against covid 19. the baby will then be born temporarily immune to the diseases (covid 19) that the mother is immune to. such passive immunity may be prolonged by breast-feeding, because breast milk also contains maternal antibodies (iga). 3.3.2 artificially acquired passive immunity it immunization induced by the transfer of antibodies is obtained by the injection of immune globulins (gamma globulins or preformed antibodies) after presumed exposure to a particular pathogen. such immune globulins are available for german measles, hepatitis a and b, tetanus and botulism (anti-toxins), and rabies. these are not vaccines; they do not stimulate immune mechanisms, but rather provide immediate antibody protection. passive immunity is always temporary, lasting a few weeks to a few months, because antibodies from another source eventually break down [13]. with this type of immunity means that scientist would have develop immunoglobins to fight covid 19 which may be quiet expensive. 3.4 active immunity acquired immunity is the production of one’s own antibodies and may be stimulated by natural or artificial means. active immunity means that the individual produces his own antibodies [14]. 3.4.1 naturally acquired active immunity it means that a person has recovered from a disease and now has antibodies and memory cells specific for that pathogen. naturally acquired active immunity occurs when a person is exposed to a live pathogen and develops a primary immune response, which leads to immunological memory [14]. this type of immunity is "natural" because deliberate exposure does not induce it. the covid 19 patients who have been recovered from the virus could acquire this type of immunity and therefore may build resistance against subsequent infections of covid 19. 3.4.2 artificially acquired active immunity it is the result of a vaccine that has stimulated production of antibodies and memory cells [15]. artificially acquired active immunity can be induced by a vaccine, a substance that contains antigen. a vaccine stimulates a primary response against the antigen without causing symptoms of the disease [16]. for this immunity be acquired against covid 19, there is the need to develop vaccine that could be administered after people have tested negative to the covid 19. it should be noted that, no general statement can be made about the duration of active immunity. recovering from plague, for example, confers lifelong immunity, but the plague vaccine does not. duration of active immunity, therefore, varies with the particular disease or vaccine [17]. 4. conclusion from the above discussions it is clear that there are different dimensions to immunity and therefore various means of acquiring each of the immunity explained. this could be used by health professionals to educate the public on the prevention of the spread of covid 19. consent it is not applicable. ethical approval it is not applicable. opoku et al.;aji, 3(1): 278-282, 2020; article no.aji.58254 282 competing interests authors have declared that no competing interests exist. references 1. marieb en. human anatomy and physiology (10 th ed) san francisco, pearson benjamin cummings; 2014. 2. reed d. what is immunity? – definition & types study.com retrieved 18 june; 2020. 3. delves pj. overview of the immune system, last full review/revision may 2019| content last modified may msd manual; 2019. 4. massimo mangino, mario roederer, margaret h. beddall, frank o. nestle, tim d. spector. innate and adaptive immune traits are differentially affected by genetic and environmental factors. nature communications. 2017;8:13850. 5. guyton ac, hall je. text book for medical physiology, (11 ed) philadelphia, usa elsevier inc; 2006. 6. opoku a, antwi js, yiadom akb, danso jo, twene p. biological and comorbidity as risk factors for covid 19 high morbidity and mortality among the aged population and its implications for public health education and research in ghana. journal of advances in medicine and medical research. 2020;32(6):88-95. 7. mayo clinic staff, covid-19: how much protection do face masks offer? mayo clinic, may 28; 2020. 8. scanlon vc, sanders t. essentials of anatomy and physiology (12 th ed) usa, f. a. davis company; 2018. 9. cheprasov a. what is adoptive immunity? definition and types. study.com retrieved 20 june; 2020. 10. dezube r. defense mechanisms of the respiratory system johns hopkins university last full review/revision jun 2019 content last modified jun; 2019. 11. lazzaro bp, schneider ds. the genetics of immunity, genetics. 2014;197(2):467– 470. 12. felipe a. vieira braga, sarah a. teichmann, xi chen. genetics and immunity in the era of single-cell genomics human molecular genetics, volume 25, issue r2, 1 october; 2016. 13. seeley rr, stephen td, tate p. anatomy and physiology 6 th (ed) usa, new york, mcgraw – hill companies, inc; 2003. 14. van de graft km, fox si. concept of human anatomy and physiology (5 th ed) usa, mcgraw hill companies inc; 1999. 15. scepanovic p, alanio c, hammer c, et al. human genetic variants and age are the strongest predictors of humoral immune responses to common pathogens and vaccines. genome med. 2018;10 (59). 16. saladin ks. anatomy and physiology, the unity of form and function, (8 th ) boston, mcgraw hill companies inc; 2017. 17. lambert nd, haralambieva ih, kennedy rb, ovsyannikova ig, pankratz vs, poland ga. polymorphisms in hla-dpb1 are associated with differences in rubella virus-specific humoral immunity after vaccination. j infect dis. 2015;211. _________________________________________________________________________________ © 2020 opoku et al.; this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: http://www.sdiarticle4.com/review-history/58254 https://www.ncbi.nlm.nih.gov/pubmed/?term=schneider%20ds%5bauthor%5d&cauthor=true&cauthor_uid=24939992 javascript:; javascript:; javascript:; javascript:; http://creativecommons.org/licenses/by/4.0 _____________________________________________________________________________________________________ *corresponding author: e-mail: ezenelson24@gmail.com; asian journal of immunology 3(1): 253-258, 2020; article no.aji.57925 knowledge, attitude and uptake of hepatitis b vaccine among clinical medical students of a tertiary institution in southeast nigeria n. c. eze1*, e. c. egba2, j. e. ogbanna2, s. n. nwamini2, p. u. nweke2 and j. s. amasianya2 1 department of community medicine, federal teaching hospital, abakaliki, ebonyi state, nigeria. 2 department of community medicine, ebonyi state university, abakaliki, ebonyi state, nigeria. authors’ contributions this work was carried out in collaboration among all authors. all authors read and approved the final manuscript. article information editor(s): (1) dr. wagner loyola, canada. reviewers: (1) sandi alfa wiga arsa, indonesia. (2) nathalia costa gonzaga saraiva, federal university of paraiba, brazil. complete peer review history: http://www.sdiarticle4.com/review-history/57925 received 06 april 2020 accepted 13 june 2020 published 24 june 2020 abstract background: hepatitis b virus infection is a global public health problem especially in developing countries like nigeria. good knowledge of hepatitis b virus, its infection and positive attitude by the public are key to the prevention of hepatitis b infection the infection can be prevented with hepatitis b virus vaccine. this study aimed at assessing the knowledge, attitude and uptake of hepatitis b virus vaccine among clinical medical students of ebonyi state university, abakaliki. materials and methods: the study is descriptive cross-sectional in design and comprised of 187 clinical medical students selected by consecutive sampling technique. data was collected using pretested, semi-structured, self-administered questionnaires and analysed using statistical package for social sciences (spss) for microsoft window version 22. results: the mean age of the respondents was 25.6 ± 3.2 years. about 80.7% of the respondents (clinical medical students of ebsu) had good knowledge of hbv infection. also, all the respondents (100%) had good knowledge of hbv vaccine. nearly, 74.3% of our respondents were concerned about being infected with hbv. however only 55.6% of them had tested for hbv surface original research article eze et al.; aji, 3(1): 253-258, 2020; article no.aji.57925 254 antigen. also 84.2% of the respondents said they would like to be vaccinated. although the participants in this study had positive attitude towards hbv vaccination, only 29.4% of the respondents had received hbv vaccine. most of those vaccinated received incomplete doses of the vaccine while only very few received complete doses of the vaccine. conclusions: there should be sustained health education to clinical medical students on the need to be vaccinated against hbv and also hbv vaccine should be made free to the general public so as to remove the challenge created by cost. keywords: knowledge; attitude; uptake; hb vaccine; clinical medical students; abakaliki. 1. introduction hepatitis b virus (hbv) infection is a global public health problem, especially in developing countries. globally, over 2 billion people have been infected with hbv, and there are over 350 million carriers [1]. good knowledge of hepatitis b virus, its infection and positive attitude by the public are key to the prevention of hepatitis b infection. hbv infection poses a health risk to health care workers who are in close proximity to infected individuals and their bodily fluids [2]. medical students in tertiary health institutions are particularly high-risk group in the current literature [2]. hepatitis b is a serious blood born infection caused by hepatitis b virus (hbv). it is the most common cause of chronic hepatitis, liver cirrhosis and hepato-cellular carcinoma [3-5]. hbv is spread through body fluids such as blood, vaginal secretions, semen. its routes of infection include sexual intercourse, unintentional needle sticks or using of infected needles, organ transplantation and blood transfusions [6]. infected pregnant women can also transmit the infection to their newborns during delivery [7]. who recommends that everybody belonging to an already identified high-risk group should be vaccinated. some of these high-risk groups include: people who frequently require blood and/or blood products, dialysis patients, people interned in prisons, intravenous drug users, people with multiple sexual partners, healthcare workers, household and sexual contacts of people with chronic hbv infection [7]. the infection can be prevented with hepatitis b vaccine uptake [7]. good knowledge of hepatitis b virus, its infection and positive attitude by the public are key to its prevention. this study therefore determined knowledge, attitude and uptake of hbv vaccine among clinical medical students of ebsu. 2. materials and methods a descriptive cross-sectional design was used for the study. the study population comprised clinical medical students of ebsu. a minimum sample size of 168 respondents was calculated using the formula: n= z 2 pq/d 2 . however, to allow for 10% non-response rate, a sample size of 187 was used. consecutive sampling technique was used for subject selection. ethical approval was obtained from the research and ethics committee (rec) of ebsu. consent was obtained from the respondents. data was collected using pretested, semi-structured, selfadministered questionnaires and analyzed using statistical package for social sciences (spss) for microsoft window version 20. data collection lasted for four weeks (july 2019). 3. results table 1 showed that the mean age of the respondents was 25.6 ± 3.2 years. fifty seven point two percent (57.2%) of the respondents were within age range of 18-27 years with a significant proportion (89.8%) being single. table 2 below showed that a significant proportion of respondents (88.2%) have heard of hb vaccine. about 80.7% and 69% of the respondents knew that hb infection is caused by virus and the best way of prevention is by vaccination respectively. in table 3 below, majority (74.3%) of the respondents were worried about being infected with hbv. however, only 55.6% have been tested for hbv surface antigen. also, 89.8% of the respondents expressed willingness to be vaccinated. in table 4 below, only 29.4% of the respondents had received hb vaccine. majority (64.2%) have not received hbv vaccine. about 55.8% gave ‘not readily accessible’ as reason for not been vaccinated. eze et al.; aji, 3(1): 253-258, 2020; article no.aji.57925 255 table 1. socio-demographic characteristics of respondents (n=187) age group (years) frequency percentage 18-27 107 57.2 ≥ 27 80 42.8 mean age (25.6 ± 3.2 years) sex male 104 55.6 female 83 44.4 marital status single 168 89.8 married 19 10.2 class level 400 85 45.5 500 81 43.3 600 21 11.2 table 2. awareness and knowledge of hepatitis b virus vaccine (n=187) have you heard of hbv vaccination frequency percentage yes 165 88.2 no 16 8.6 don’t know 6 3.2 hbv is a viral infection yes 151 80.7 no 36 19.3 source of information hospital 65 34.8 newspaper/television/radio 15 8.0 workplace 5 2.7 school 102 54.5 best way of preventing hbv is by vaccination no 58 31.0 yes 129 69.0 table 3. attitude towards hbv infection and its vaccination (n=187) worried about being infected by hbv frequency percentage yes 139 74.3 no 36 19.3 don’t know 12 6.4 tested for hbv yes 104 55.6 no 77 41.2 don’t know 6 3.2 would like to be vaccinated against hbv yes 168 89.8 no 6 3.2 don’t know 13 7.0 4. discussion awareness and knowledge assessment in this study showed that a significantly high proportion of the respondents were aware of hb vaccine and a large proportion also had good knowledge of hepatitis b vaccine. this proportion of respondents with good knowledge may be due to their daily participation in educational programs on hepatitis b virus infection and prevention within and outside the academic setting as quite above half of the respondents got their information from the school. this finding is in agreement with the findings in another study at eze et al.; aji, 3(1): 253-258, 2020; article no.aji.57925 256 table 4. uptake of hbv vaccine have you been vaccinated against hbv frequency percentage yes 55 29.4 no 120 64.2 don’t know 12 6.4 if not vaccinated, reasons for not being vaccinated (n=120) expensive 17 14.2 not readily accessible 67 55.8 not interested 22 18.3 others 14 11.7 reasons for being vaccinated (n=55) it was free 22 40.0 fear of being infected 17 30.9 advice from friend doctor 16 29.1 no of doses received (n=55) 1 dose 26 47.3 2 doses 24 43.6 3 doses 5 9.1 irua, nigeria [8] and also comparable with the results of two other separate studies in osun [9] and lagos [2] both in western nigeria. it is however at variance with the findings in similar studies done at university of jos [10] in cameroon among medical students [11]. poor knowledge was also reported among medical and health science students in a study done at haramaya university, ethiopia [4]. the findings in this study are however in contrast with two other separate studies done in india [12]. a good knowledge of hb virus, its modes of infection as well as adequate vaccination may reduce infection rate. studies carried out among health care workers in sudan and morocco revealed that most of them knew blood as a medium of infection but lacked adequate vaccine coverage. inadequate knowledge of hbv among health workers may reflect their behavioural pattern to vaccination and safety measure [5,9]. in india, a study revealed that majority of the third year students knew about the vaccine type, vaccination schedule, type of syringes, route of administration, safe disposal of syringe and needles and other preventive strategies. on the contrary, very few second year students had correct knowledge regarding the same [12]. among medical students at northern border university, arar kingdom of saudi arabia, a study showed that a significantly large proportion knew that vaccine could prevent hbv infection [13], but at variance with those in north west ethiopia where a large proportion of the respondents were aware that they are at risk of contracting hbv and believed that hbv vaccine is effective and safe [14,15]. vaccine uptake and immune response to hbv infection study done in southwest region of cameroon revealed that health care workers though few were the most vaccinated. upon assessing the reasons for not being vaccinated, lack of awareness was topmost. some of the participants had never heard about hepatitis b infection while others were not aware there was a vaccine against hbv infection. this automatically implies that there is low sensitization coverage on hbv infection in cameroon despite the high hbv prevalence in the country. although health care workers topped the number of vaccinated cases among the study groups, majority of them were still not vaccinated despite their awareness [16]. significantly high proportion of the respondents in this study affirmed they would like to be vaccinated. however, a large proportion were concerned about being infected by hb virus. this positive attitude could be adduced by their good knowledge of hbv infection and vaccine. these findings were comparable with the result of a study done in iran [17] but however in discordance with the findings in other studies carried out in port-harcourt [18] and jos [10]. although, a significantly large proportion of respondents in this study had positive attitudes towards hbv vaccination, only few had received hbv vaccine. this low level of uptake may due to unavailability and expensive nature of the vaccine. these eze et al.; aji, 3(1): 253-258, 2020; article no.aji.57925 257 findings (reasons) were at variance with the results of studies done in greece [19] and iran [17] but comparable with the findings from other similar studies done in port-harcourt [18], irua [14] and jos [20] on the uptake of hbv vaccine. these variations in greece and iran could be due to better availability and accessibility of hbv vaccine among respondents. 5. conclusions this study found good knowledge among the respondents. there was positive attitude towards hbv vaccination among the respondents. however, uptake of hbv vaccine was poor. more intensive health education programs for the clinical medical students and indeed the general public on the need to be vaccinated against hbv is recommended. the hbv vaccine should be subsidized and/or made free to ameliorate the barrier created by cost. hbv infection is of public health importance in nigeria. health workers and clinical medical students who are at high risk of the infection should be tested and vaccinated against hbv infection accordingly. consent and ethical approval ethical approval was obtained from the research and ethics committee (rec) of ebsu. consent was obtained from the respondents. data was collected using pretested, semi-structured, selfadministered questionnaires and analyzed using statistical package for social sciences (spss) for microsoft window version 20. data collection lasted for four weeks (july 2019). competing interests authors have declared that no competing interests exist. references 1. alshammari ms, alshamari ng, alshammari as, kareem m. electronic physicians. 2017;(9):5388–94. 2. algadheeb as, al-alsheikh as, al-hamoudi wk, alswat ka. medical students’ awareness of and compliance with the hepatitis b vaccine in a tertiary care academic hospital : an epidemiological study. j infect public health 2016;9:60-65. 3. bello fm, health p, anne cp. health workers’ knowledge, attitude and practice towards hepatitis b infection in northern nigeria. 2017;9(3):939–54. 4. mesfin ym, kibret kt. assessment of knowledge and practice towards hepatitis b among medical and health science students in haramaya university, ethiopia. scientific research publishing. 2013; 8(11):1–6. 5. reddy rs, swapna la, ramesh t, pradeep k. knowledge, attitude and practice on hepatitis b prevention among dental professionals in india. pan african medical journal. 2011;10(4). 6. mohammed a, aldeen z, alelyani am, alshammari ks, abdulaziz m, alsaif b, et al. assessment of knowledge, attitudes and practices toward prevention of hepatitis b virus infection among medical students in hail region, saudi arabia sky jounral of medicine and medical sciences. 2017;3(4):31–5. 7. taher ha, mansuri s. hepatitis b infection control knowledge and practice of undergraduate students at college of dentistry, madinah, saudi arabia. bmc. 2015;3(4):47–51. 8. kesieme eb, uwakwe k, irekpita e, dongo a, bwala kj, alegbeleye bj. knowledge of hepatitis b vaccine among operating room personnel in nigeria and their vaccination status.hepat res treat. 2011;2011:7–12. 9. adekanle o, ndububa da, olowookere sa, ijarotimi o, ijadunola kt. knowledge of hepatitis b virus infection, immunization with hepatitis b vaccine, risk perception, and challenges to control hepatitis among hospital workers in a nigerian tertiary hospital. hepat res treat. 2015;2015:1–6. 10. daboer jc, chingle mp, banwat me. knowledge, risk perception and vaccination against hepatitis b infection by primary healthcare workers in jos, north central nigeria. pan african medical journal. 2017;6–10. 11. noubiap jjn, nansseu jrn, kengne kk, ndoula st, agyingi la. occupational exposure to blood, hepatitis b vaccine knowledge and uptake among medical students in cameroon.open access; 2013. 12. singh a, jain s. prevention of hepatitis bknowledge and practices among medical students. bmc. 2012;(2):52–6. 13. reang t, chakraborty t, sarker m, tripura a. a study of knowledge and practice regarding hepatitis b among nursing students attending tertiary care hospitals in agartala city. international jurnal of research in medical sciences 2015;3(7): 1641–9. eze et al.; aji, 3(1): 253-258, 2020; article no.aji.57925 258 14. abdela a, woldu b, haile k, mathewos b, deressa t. assessment of knowledge, attitudes and practices toward prevention of hepatitis b virus infection among students of medicine and health sciences in northwest ethiopia. bmc res notes. 2016;1–7. 15. demsiss w, seid a, fiseha t. hepatitis b and c: seroprevalence, knowledge, practice and associated factors among medicine and health science students in northeast ethiopia. plos one. 2018;1– 12. 16. meriki hd, tufon ka, anong dn, tony j, kwenti te, bolimo af, et al. vaccine uptake and immune responses to hbv infection amongst vaccinated and non vaccinated healthcare workers, household and sexual contacts to chronically infected hbv individuals in the south west region of bmc. 2018;185:1–18. 17. alavian sm, mahboobi n, savadrudbari mm, azar ps, daneshvar s. iranian dental students’ knowledge of hepatitis b virus infection and its control practices. world journal of vaccines. 2011;12:1627– 34. 18. paul n, peterside o. hepatitis b vaccination rate among medical students at the university of port harcourt teaching hospital (upth). world journal of vaccines. 2015;(2):1–7. 19. papagiannis d, tsimtsiou z, chatzichristodoulou i, adamopoulou m. hepatitis b virus vaccination coverage in medical, nursing and paramedical students: bmc. 2015;8(5):1–9. 20. fufore mb, cook pa, kirfi am. health workers’ knowledge, attitude and practice towards hepatitis b infection in northern nigeria. international journal of caring sciences. 2016;9(3):939-54. ________________________________________________________________________________ © 2020 eze et al.; this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: http://www.sdiarticle4.com/review-history/57925 http://creativecommons.org/licenses/by/4.0 _____________________________________________________________________________________________________ *corresponding author: e-mail: augustineairaodion@yahoo.com; asian journal of immunology 3(1): 84-90, 2020; article no.aji.55862 common household insecticides used in nigeria induced oxidative stress in wistar rats augustine i. airaodion1*, kenneth o. ngwogu2, ada c. ngwogu3, anthony u. megwas4, john a. ekenjoku5 and olaide o. awosanya6 1 department of biochemistry, federal university of technology, owerri, imo state, nigeria. 2 department of pathology, abia state university, uturu, nigeria. 3 department of microbiology, abia state university, uturu, nigeria. 4 department of optometry, federal university of technology, owerri, imo state, nigeria. 5 department of pharmacology and therapeutics, abia state university, uturu, nigeria. 6 department of biochemistry, university of ibadan, oyo state, nigeria. authors’ contributions this work was carried out in collaboration among all authors. author aia conceptualized, designed the study and also wrote the manuscript. authors acn and ooa managed the analyses of the study. author kon managed the literature searches. author jae wrote the protocol while author aum performed the statistical analysis. all authors read and approved the final manuscript article information editor(s): (1) dr. jaffu othniel chilongola, kilimanjaro christian medical university college, tanzania. reviewers: (1) eze ejike daniel, kabale university, uganda. (2) julia pérez ramos, universidad autónoma metropolitana, mexico. complete peer review history: http://www.sdiarticle4.com/review-history/55862 received 24 january 2020 accepted 28 march 2020 published 08 april 2020 abstract background: the use of household insecticides for the eradication of insects especially mosquitoes in nigeria is increasing. these insecticides are used without consideration of their adverse effect on human health. aim: this study is therefore sought to investigate the effect of common household insecticides used in nigeria on oxidative stress biomarkers. methodology: thirty (30) male wistar rats were divided into five groups of six (6) each. rats in group 1 were exposed to rambo, those in group 2 were exposed to mortein, those in group 3 were exposed to raid, those in group 4 were exposed to baygon while those in group 5 were not exposed to any insecticide and served as the control group. the exposure was done twice daily via inhalation route. throughout the experiment, animals were fed ad libitum with standard feed and original research article airaodion et al.; aji, 3(1): 84-90, 2020; article no.aji.55862 85 drinking water. after twenty-one (21) days of exposure, they were sacrificed after an overnight fast under diethyl ether as anesthesia. blood samples were collected via cardiac puncture. oxidative stress biomarkers (lipid peroxidation, glutathione, catalase, superoxide dismutase and glutathione peroxidase) were determined using standard methods. results: the results obtained showed that exposure of the animals to all types of insecticides (rambo, mortein, raid and baygon) significantly (p <0.05) increased lipid peroxidation an index of oxidative biomarkers in rats when compared to the control group. conversely, the concentration of glutathione was significantly (p <0.05) reduced compared to the corresponding control group.while the activities of antioxidant enzymes (catalase, superoxide dismutase and glutathione peroxidase) were observed to significantly (p < 0.05) increase as a result of insecticides exposure to animals when compared with the control group animals. conclusion: from the findings of the present study, it can be concluded that common household insecticides used in nigeria induced oxidative stress in wistar rats and might compromise the immune system. suggesting that such oxidative stress inducing effects of these insecticides as observed in animals used in this study may be extrapolated in humans who use these insecticides to protect against various insects. therefore, staying in a room fumigated with these insecticides when the effect has not completely subsided may be hazardous to humans and hence, this should be discouraged. keywords: antioxidants; free radicals; household insecticides; oxidative stress. 1. introduction oxidative stress (os) is the results of production of reactive oxygen species (ros). sometimes they are also referred to as reactive oxygen intermediates (roi). they occur as a result of metabolism of tissues [1]. ros act as signaling mediators as most of the time may be beneficial. most of the exogenous substances may activate the production of these ros. cigarette smoke, uv radiation in the atmosphere, alcohol, drugs and cancer chemotherapeutic agents and radiotherapy treatments induce oxidative stress. petroleum combustion products, heavy metals as well as pesticide particles and their metabolic end products also initiate the oxidative stress. infections, tissue injury, and ischaemia also contribute in the elevated levels of ros [2]. cigarette smoke is one of the exogenous agents that induce oxidative damage in cell line. cigarette smoke induces oxidation of structural and functional components and also able to decrease the endothelial growth. in a recent study, airaodion et al. [3] reported that hydrocarbon induced os when animals were fed with crude oil treated-diet. several studies have also reported that alcohol has the propensity to induce oxidative stress [4,5,6]. insecticides are substances used to kill, repel or mitigate one or more species of insects. they also include ovicides and larvicides used against insect eggs and larvae, respectively [7]. insecticides are used in agriculture, healthcare, industry and by individuals. insecticides have been reported to be a major factor behind the increase in the 20 th century's agricultural productivity [8]. nearly all insecticides have the potential to significantly alter ecosystems; many are toxic to humans and/or animals; some are accumulate in the body and bio-magnify as they pass along the food chain [8]. insecticides can be classified into two major groups: systemic insecticides, which have residual or long term activity; and contact insecticides, which have no residual activity. the mode of action describes how the pesticide kills or inactivates a pest. it provides another way of classifying insecticides [7]. mode of action can be important in understanding whether an insecticide will be toxic to unrelated species, such as fish, birds and mammals. insecticides may be repellent or non-repellent. social insects such as ants cannot detect non-repellents and readily crawl through them. as they return to the nest they take insecticide with them and transfer it to their nestmates. over time, this eliminates all of the ants including the queen. this is slower than some other methods, but usually completely eradicates the ant colony [9]. insecticides are distinct from non-insecticidal repellents, which repel but do not kill. the common household insecticides used in nigeria are systemic insecticides. they include rambo produced by gongoin and co, mortein produced by reckitt benckiser, raid and baygon both produced by s.c. johnson and co. people use these insecticides without consideration of their adverse effect on health. airaodion et al. [10] has reported that these insecticides had adverse effect on male reproductive hormones. this airaodion et al.; aji, 3(1): 84-90, 2020; article no.aji.55862 86 present study therefore sought to evaluate the effect of these insecticides on oxidative stress biomarkers in wistar rats. 2. materials and methods 2.1 collection of insecticides baygon, raid and mortein insecticides were purchased from ‘pinnacle’ supermarketwhile rambo was purchased from ‘medhelp’ pharmacy both in ibadan, nigeria. they were kept at room temperature before and during the experiment. 2.2 experimental design and animal treatment thirty (30) male wistar rats weighing between 220 and 245 g were used for this study. they were acclimatized for seven (7) days to laboratory conditions before the commencement of the experiment. during this period, they were fed ad libitum with standard feed and drinking water and were housed in clean cages placed in well-ventilated housing conditions (under humid tropical conditions) throughout the experiment. all the animals received humane care according to the criteria outlined in the ‘guide for the care and use of laboratory animals’ prepared by the national academy of science and published by the national institute of health. they were randomly divided into five groups of six rats each and kept in different rooms. rats in group 1 were exposed to rambo insecticide, those in group 2 were exposed to mortein insecticide, those in group 3 were exposed to raid insecticide, those in group 4 were exposed to baygon insecticide while those in group 5 were not exposed to any insecticide and served as the control group. the exposure was done twice daily via inhalation route. throughout the experiment, they were fed ad libitum with standard feed and drinking water.after twenty-one days of exposure, the rats were sacrificed after an overnight fast under diethyl ether as anesthesia. blood samples were collected via cardiac puncture. 2.3 determination of oxidative stress biomarkers determinationof lipid peroxidation (lpo), reduced glutathione (gsh), catalase (cat), superoxide dismutase (sod) and glutathione peroxidase (gpx) were carried out on the serum of animals according to the methods previously described by airaodion et al. [11]. 2.4 statistical analysis data were subjected to analysis using analysis of variance (anova) with the aid of graph pad prism. data from each parameter was expressed as mean value ± standard error of the mean (sem). data were considered to be significantly different at 95% confidence level (p ≤ 0.05). 3. results the results of the effect of common household insecticides used in nigeria lpo, gsh, cat, sod and gpx are presented in figs. 1-5 respectively. fig. 1. effect of household insecticides on the lipid peroxidation in animals after 21 days of exposure results are presented as mean ± sem with n = 6. bars with different letters are significantly different at p < 0.05 airaodion et al.; aji, 3(1): 84-90, 2020; article no.aji.55862 87 fig. 2. effect of household insecticides on the concentration of reduced glutathione (gsh) in animals after 21 days ofexposure results are presented as mean ± sem with n = 6. bars with different letters are significantly different at p<0.05 fig. 3. effect of household insecticides on the activity of catalase (cat) in animals after 21 days of exposure results are presented as mean ± sem with n = 6. bars with different letters are significantly different at p<0.05 airaodion et al.; aji, 3(1): 84-90, 2020; article no.aji.55862 88 fig. 4. effect of household insecticides on the activity of superoxide dismutase (sod) in animals after 21 days of exposure results are presented as mean ± sem with n = 6. bars with different letters are significantly different at p<0.05 fig. 5. effect of household insecticides on the activity of glutathione peroxidase (gpx) in animals after 21 days of exposure results are presented as mean ± sem with n = 6. bars with different letters are significantly different at p<0.05 4. discussion in this study, it was observed that all the insecticides used in this study significantly elevated the malondialdehyde (mda) levels in animals indicating enhanced peroxidation and breakdown of the antioxidant defense mechanisms. decomposition products of lipid hydroperoxide such as mda and 4hydroxynonenal can cause chaotic cross-linkage airaodion et al.; aji, 3(1): 84-90, 2020; article no.aji.55862 89 with proteins and nucleic acids, which plays an important role in the process of carcinogenesis. in this investigation, serum lipid peroxidation (lpo) activities showed significant increase due to insecticide exposure [12]. furthermore, extensive damage to tissues in a free radical mediated lpo results in membrane damage and subsequently decreases the membrane fluid content [13]. glutathione (gsh) is a tripeptide (l-αglutamylcysteinol glycine) which is highly abundant in all cell compartments and it is the major soluble antioxidant. glutathione directly quenches ros such as lipid peroxides, and also plays a major role in xenobiotic metabolism [11,14]. glutathione detoxifies hydrogen peroxide and lipid peroxide by donating electron to hydrogen peroxide to reduce it to water and oxygen protecting macromolecules such as lipids from oxidation. in this study, the decrease in the reduced serum glutathione level in animals exposed to insecticides for 21 days might be connected to insecticide-induced oxidative stress and direct conjugation of gsh with reactive intermediates of insecticide oxidation. in this present study, all the insecticides were observed to significantly (p < 0.05) increase the activities of antioxidant enzymes (sod, cat gpx) investigated when compared to those in their respective control group (figs. 3-5). sod plays an important role in reducing the effect of free radicals’ attack. it is the only enzymatic system quenching o2to oxygen and h2o2 and plays a significant role against oxidative stress [14]. these radicals have been reported to be deleterious to polyunsaturated fatty acids and proteins [3,11]. cat and gpx are other enzymatic antioxidants that act as a defense mechanism against oxidative stress [14]. the significant increase in the activities of antioxidant enzymes in animals exposed to insecticides might be an indication that these insecticides increased the generation of free radicals which these enzymes tend to combat, thereby increasing their activities. these insecticides have been reported to induce nephronand hepato-toxicity in wistar rats due to the generation of free radicals [15]. the mechanism in which insecticides increased the activities of these enzymes is unclear but airaodion et al. [3] reported that hydrocarbon exposure significantly increased these enzymes. thus, the mechanism of action of insecticides in this present study might be similar to that of hydrocarbon. 5. conclusion from the result of this present study, common household insecticides used in nigeria induced oxidative stress and might compromise the immune system. thus, staying in a room fumigated with these insecticides when the effect has not completely subsided may be hazardous, and thus should be discouraged. disclaimer the products used for this research are commonly and predominantly use products in our area of research and country. there is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. also, the research was not funded by the producing company rather it was funded by personal efforts of the authors. consent it is not applicable. ethical approval as per international standard written ethical permission has been collected and preserved by the author(s). competing interests authors have declared that no competing interests exist. references 1. repine je, bast al. oxidative stress in chronic obstructive pulmonary disease. oxidative stress study group. american journal of respircrit care med. 1997;156: 341–357. 2. kasahara y, tuder rm, cool cd, lynch da, flores sc, voelkel nf. endothelial cell death and decreased expression of vascular endothelial growth factor and vascular endothelial growth factor receptor 2 in emphysema. am j respircrit care med. 2001;163:737–744. 3. airaodion ai, ogbuagu u, ekenjoku ja, ogbuagu eo, airaodion eo, okoroukwu vn. hepato-protective efficiency of airaodion et al.; aji, 3(1): 84-90, 2020; article no.aji.55862 90 ethanol leaf extract of moringa oleifera against hydrocarbon exposure. international journal of advances in herbal and alternative medicine. 2019; 03(01):32-41. 4. airaodion ai, ogbuagu eo, ewa o, ogbuagu u, awosanya oo, adekale oa. ameliorative efficacy of methanolic extract of corchorusolitorius leaves against acute ethanol-induced oxidative stress in wistar rats. asian journal of biochemistry, genetics and molecular biology. 2019; 7(6):1-9. 5. ogbuagu eo, airaodion ai, ogbuagu u, airaodion eo. prophylactic propensity of methanolic extract of vernonia amygdalina leaves against acute ethanol-induced oxidative stress in wistar rats. international journal of bio-science and biotechnology. 2019;11(7):37-46. 6. airaodion ai, akinmolayan jd, ogbuagu eo, esonu ce, ogbuagu u. preventive and therapeutic activities of methanolic extract of talinum triangulare leaves against ethanol-induced oxidative stress in wistar rats.international journal of bioscience and bio-technology. 2019;11(7): 85-96 7. iupac. glossary of terms relating to pesticides. international union of pure and applied chemistry. 2006;2123. 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[isbn 978-0-412-72800-6] 9. murray b. isman botanical insecticides, deterrents, and repellents in modern agriculture and an increasingly regulated world. annual review of entomology. 2013;51:45-66. 10. airaodion ai, ngwogu ac, megwas au, ekenjoku ja, ngwogu ko. effect of common household insecticides used in nigeria on rat male reproductive hormones. international journal of research and reports in gynaecology. 2020;2(1):1-8. 11. airaodion ai, ogbuagu eo, ogbuagu u, adeniji ar, agunbiade ap, airaodion eo. hepatoprotective effect of parkia biglobosaon acute ethanol-induced oxidative stress in wistar rats. international research journal of gastroenterology and hepatology. 2019;2(1):1-11. 12. airaodion ai, ngwogu ac, ekenjoku ja, ngwogu ko. hepatoprotective potency of ethanolic extract of garciniakola (heckel) seed against acute ethanol-induced oxidative stress in wistar rats. international research journal of gastroenterology and hepatology. 2020;3(2):1-10. 13. oyenihi or, afolabi ba, oyenihi ab, ogunmokun oj, oguntibeju oo. hepato and neuro-protective effects of watermelon juice on acute ethanol-induced oxidative stress in rats. toxicology reports. 2016;3: 288–294. 14. airaodion ai, ogbuagu eo, ekenjoku ja, ogbuagu u, airaodion eo. therapeutic effect of methanolic extract of telfairia occidentalis leaves against acute ethanolinduced oxidative stress in wistar rats. international journal of bio-science and bio-technology. 2019;11(7):179-189. 15. airaodion ai, ekenjoku ja, megwas au,ngwogu ko, ngwogu ac. nephroand hepato-toxicity of common household insecticides used in nigeria. international research journal of gastroenterology and hepatology. 2020;3(3):1-8. _________________________________________________________________________________ © 2020 airaodion et al.; this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: http://www.sdiarticle4.com/review-history/55862 http://creativecommons.org/licenses/by/4.0 _____________________________________________________________________________________________________ *corresponding author: e-mail: rajini.kurup@uog.edu.gy; asian journal of immunology 3(1): 39-45, 2020; article no.aji.55501 a pilot study on pre-diabetes and associated central obesity among students of university of guyana, guyana dakari jordan1, dacia simpson1, rajini kurup1*, audrey anderson1 and cecil boston1 1 faculty of health science, university of guyana, turkeyen campus, georgetown, guyana. authors’ contributions this work was carried out in collaboration among all authors. authors aa, ds and dj oversaw study design, data collection and initial data analysis. authors rk and cb participated in study design and final data analysis and interpretation. in addition, authors rk and cb revised the manuscript and approved final version. article information editor(s): (1) dr. darko nozic, university of belgrade, serbia. reviewers: (1) mra aye, melaka manipal medical college, malaysia. (2) ng hak yung, tseung kwan o hospital, china. complete peer review history: http://www.sdiarticle4.com/review-history/55501 received 09 january 2020 accepted 15 march 2020 published 25 march 2020 abstract objective: the purpose of this study was to examine the incidence of pre-diabetes and associated central obesity among sampled university of guyana students. methods: the study was a cross sectional study where a questionnaire was distributed to each participant to assess predisposing risk factors. anthropometric measurements like bmi and hba1c were used to ascertain if the participant was overweight or obese. nycocard® hba1c test kit was used for collecting hba1c. data was analyzed using spss 20.0 to calculate descriptive statistics and analysis of variance were used to investigate the research questions. a p value of <0.05 was considered to be significant for all analysis. results: a total of seventy-four students were randomly selected for the study. the findings of this study revealed pre-diabetes incidence of 40.5% and a weak positive correlation between central obesity and pre-diabetes, with r-values of 0.25 (males), 0.27 (females) & 0.26 (overall). original research article jordan et al.; aji, 3(1): 39-45, 2020; article no.aji.55501 40 conclusion: there was a weak association between central obesity and pre-diabetes incidence. therefore, a large follow up study would be important to understand association between pre-diabetes and central obesity and to create nationwide awareness or development of an operational policy/strategy/action plan to reduce overweight. keywords: incidence; pre-diabetes; central obesity; type 2 diabetes. 1. introduction the epidemic of type 2 diabetes continues to pose a challenge to both developed and developing countries, accounting for 95% of all diabetes cases [1]. the morbidity, mortality and the cost of care associated with type 2 diabetes makes it an important global public health concern. globally, the number of people with diabetes is expected to almost double in the next two decades, increasing from 415 million in 2015 to 642 million in 2040 [2]. moreover, given the burden of type 2 diabetes and its complications, much attention has been given to prevention, beginning with identifying at risk individuals. this has led to the designation of the term “prediabetes.” pre-diabetes characterizes an individual having blood glucose levels higher than normal but not high enough to be classified as diabetes. without weight loss measures, healthy eating and moderate physical activity many people living with pre-diabetes will go on to develop diabetes [3]. approximately 5-10% of people per year with pre-diabetes progress to diabetes; with similar proportion converting back to normoglycemia [4]. observational evidence suggests an association between pre-diabetes and several adverse health outcomes, which includes: early forms of nephropathy, small fiber neuropathy, diabetic retinopathy, chronic kidney disease and increased risk of macrovascular disease [4]. pre-diabetes is also associated with being overweight or obese and carries an excess risk for cardiovascular disease (cvd) and death. this is of tremendous public health concern as the global burden of obesity has significantly increased. the most recent analysis by the world health organization (who) reports that the number of adults globally who are overweight or obese is 1.9 billion and 650 million, respectively [5]. the pan american health organization has also estimate that more than half the population in latin america and the caribbean is overweight, with 58% or 360 million people being overweight and 23% or 140 million people are estimated to be obese. obesity in women is 10% higher than in men [6]. due to this increase in the epidemic of obesity; understanding body fat distribution and its clinical implications is critical to a timely intervention. adipose tissue is a special type of loose connective tissue in which adipose cells (adipocytes) predominates. primitively, it was considered simply as storage organ for triacylglycerol, however, over the last decade there has been considerable experimental data about the biology and biochemistry of adipose tissue that it is no longer considered to be an inert tissue that just stores fat. it is a metabolically dynamic organ that is not only the primary site of storage for excess energy, but also serves as an endocrine organ capable of synthesizing a number of biologically active compounds that regulate metabolic homeostasis [7]. diabetes continues to be a major health issue in guyana; since it’s a relatively small population and continues to be a burden on the healthcare system. to date, no study was conducted to evaluate and assess the incidence of prediabetes among the guyanese population and therefore combat the rate of progression to diabetes. 2. materials and methods the study was a cross sectional study where university of guyana (ug) students ≥18 years old, who were overweight or obese or had waist circumferences exceeding 80 cm (females) and 90 cm (males) were randomly selected for the study. all participants were told about the study and an informed consent form was obtained before the study. the study was conducted during march 2018. a total of 92 participants were enrolled in the study of which 74 participants completed the study. others were excluded due to various reasons like incomplete information, reluctance to perform tests. inclusion criteria: students enrolled at ug with an identification card, no known disease condition, willingness to enroll in the study. jordan et al.; aji, 3(1): 39-45, 2020; article no.aji.55501 41 instrument for weight assessment: portable electronic weighing scale. instrument for height and waist assessment: constant tension tape, for waist circumference measurement was taken 2.5 cm above the umbilicus. criteria for prediabetes: patients with prediabetes are defined by the presence of impaired fasting glucose (ifg) and/or impaired glucose tolerance (igt) and/or a1c 5.7–6.4% (39–47 mmol/mol). measurement of blood pressure: digital blood pressure monitor was used. normal blood pressure was considered measured if systolic under 140 mmhg and diastolic under 90 mmhg. a questionnaire was distributed to each participant to assess predisposing risk factors. bmi was used to ascertain if the participant was overweight or obese by measuring the height in cm and weight in kg. nycocard® hba1c test kit was used to assess the hba1c levels of each participant. data was analyzed using spss version 20.0 software for frequencies, descriptive statistics, and analysis of variance. a p value of ≤0.05 was considered to be significant for all analysis. ministry of public health, institutional review board (irb) gave approval for conducting the study, medical arts laboratory and the university of guyana (ug) to carry out this study. 3. results of the 92 students participated in the study, 74 met the inclusion criteria, with 80% participation rate. a higher percentage of participants (67.6%) were females and 32.4% were males (table 1). table 1 shows the baseline characteristics and anthropometric indices of the study population. mean ± sd height and weight were 167.7±1.1 cm and 88.9±3.9 kg respectively, with the mean bmi being 31.4±1.1 kg/m2. the incidence of overweight and obesity was estimated to be 40.5% and 44.6% respectively. the mean waist circumference for men and women were 101.4± 2.3 cm and 96.4±1.6 cm, respectively. out of a total of 74 participants, 30 (40.5%) were considered pre-diabetic, 6 (8.1%) were considered diabetic and 38 (51.3%) were considered non-diabetic/pre-diabetic (table 2). a weak positive correlation was recorded between central obesity and pre-diabetes (r=0.25 for males, r=0.27 for females and r=0.26 for total participants). among female participants, 21 (42.0%) were pre-diabetic and 4 (8.0%) were diabetic. however, for males 9 (37.7%) were pre-diabetic and 2 (8.3%) were diabetic. females accounted for the highest number of pre-diabetes person in the study. the mean hba1c was 5.8±0.1%. correlation of hba1c among male students were r=0.25 (p≥0.05), for females r=0.27 (≥0.05) and for total participants r=0.26 (≤0.005). only 16 (21.6%) participants indicated that they engage in high level of physical activity, 24 (32.4%) in a low level of physical activity, while 34 (45.9%) reported a moderate level of physical activity. only three participants indicated that they are smokers. interestingly, a significant proportion (66.3%) of persons reported that they have a family history of diabetes. overall, 30 (40.5%) participants were classified as having pre-diabetes and 6 (8.1%) as being diabetic. amerindian ethnic group had higher risk of being pre-diabetic compared to african ethnic group (odds ratio=2.9). in addition, the odd of someone being pre-diabetic is 2.86 for the amerindian ethnicity compared to the african ethnicity. overall there was a strong positive correlation (r=0.8) between family history and pre-diabetes and weak positive correlation (r=0.4) between physical activity and the development of pre-diabetes. 4. discussion this is the first cross-sectional study that has examined the incidence of pre-diabetes among a sampled guyanese population and its association with central/abdominal obesity. several important findings were observed in this study. firstly, incidence of pre-diabetes was estimated at 30 (40.5%) within the sample. similarly, a study from iran, also a country with a high prevalence of diabetes (11.4%) [8], showed that pre-diabetes had an incidence of 40.6 per 1000-person years of those aged 20 and above [9]. within this study females (28.3%) had the highest incidence of pre-diabetes as compared to males (12.1%). this was mainly due to the larger portion of our study population constituted by females, (i.e., 67.6%). moreover, this may be a good representation of the large number of women at risk of developing future diabetes in guyana, a country with a high prevalence of diabetes among women (10.9%) [5]. the incidence of central obesity was higher than jordan et al.; aji, 3(1): 39-45, 2020; article no.aji.55501 42 overall obesity, which indicates a significant portion of the population, may not be classified as obese by bmi level. overall the incidence of central obesity was found higher among the study participants. these finding are consistent with studies conducted in china and the united states of america [10,11]. the association of central obesity and the incidence of pre-diabetes were weakly correlated and overall were statistically significant. table 1. shows the baseline characteristics and anthropometric indices variable % (n) sex: female 67.6 (50) male 32.4 (24) faculty: fhs 16.2 (12) fns 16.2 (12) fss 10.8 (8) fees 13.5 (10) foa 13.5(10) fot 16.2 (12) feh 13.5 (10) ethnicity: african 51.4 (38) east indian 18.9 (14) amerindian 4.1 (3) mixed 25.7 (19) bmi group: underweight ( <18.5) 0.0 (0) normal (18.5 24.9) 16.2 (12) overweight (25 29.9) 40.5 (29) obese (≥30) 44.6 (33) mean waist circumference: female 96.4 cm male 101.4 cm mean weight 88.9 kg mean height 167.7 cm mean hba1c values: female 5.7 % male 5.2 % pre-diabetic: yes 40.5 (30) no 59.5 (44) family history: yes 68.9 (51) no 31.1 (23) physically active: low 32.4 (24) moderate 45.9 (34) high 21.6 (16) smoking status: yes 4.0 (3) no 95.9 (71) hypertension: yes 2.7 (2) no 97.3 (72) jordan et al.; aji, 3(1): 39-45, 2020; article no.aji.55501 43 variable % (n) symptoms of increase thirst yes 31.1 (23) no 68.9 (51) dry mouth yes 20.3 (15) no 79.7 (59) itchy skin yes 27.0 (20) no 73.0 (54) blurred vision yes 32.4 (24) no 17.6 (50) loss of appetite yes 23.0 (17) no 77.0 (57) frequent urination yes 33.8 (25) no 66.2 (49) fhs-faculty of health sciences, fns-faculty of natural sciences, fss-faculty of social sciences, foa-faculty of agriculture, fot-faculty of technology, feh-faculty of education & humanities table 2. bmi and diabetes status among participants bmi status n (%) 95% ci p-value normal 12 (16.2) 8.7-26.6 overweight 29 (39.2) 28.0-51.2 obese 33 (44.6) 33.2-56.6 ≤0.05 diabetes status normal 38 (51.4) 39.4-63.1 pre diabetes 30 (40.5) 29.3-52.6 diabetes 6 (8.1) 3.0-16.8 ≤0.001 previous studies have assessed the relationship between obesity and pre-diabetes incidence [11]. in most of these studies reported to date, obesity was measured using body mass index (bmi) as it has shown many advantages as a surrogate of body fat, such as simplicity and reproducibility. however, a significant limitation of using bmi is its inability to differentiate between lean mass and fat mass, especially in patients with a bmi <30 kg/m2 across age, sex and race [12]. secondly, fat distribution could not be distinguished by bmi, whilst it has been generally accepted that visceral adiposity plays a more important role in developing insulin resistance and diabetes rather than overall adiposity. when comparing asians and europeans with similar bmis, subjects with bmis less than 25 kg/m2 need to be considered as high-risk for type 2 diabetes or heart diseases [13]. waist circumference (wc) is considered a good anthropometric indicator for abdominal obesity because it is an aggregate measurement of the actual amount of total and abdominal fat accumulation. it has long been assumed that diabetes is associated with abdominal obesity and anthropometric measurements such as waist circumference that correlate with regional body fat distribution more so than bmi. correlations of intra-abdominal visceral fat with waist circumference is thought to be the biological explanation for the superiority of waist circumference as a predictor of diabetes compared to bmi. coelho et al. [14] reported that accumulation of visceral fat stores affects insulin metabolism by releasing a number of bioactive substances such as free fatty acids which when elevated, induce hepatic insulin resistance, particularly by enhancing gluconeogenesis. this was further supported by the fact that jordan et al.; aji, 3(1): 39-45, 2020; article no.aji.55501 44 gluconeogenesis increases in proportion to visceral fat [15]. other studies have also suggested that the waist-to-height ratio is the best measure of obesity, compared to other anthropometric measures [16]. females accounted for the highest number of pre-diabetes person in the study, which can be tied to the fact that the larger portion of the study population constituted females. moreover, this may be a good representation of the large number of women at risk of developing future diabetes in guyana, a country with an increasing prevalence of diabetes among women [5]. this study showed a high incidence of pre-diabetes (40.5%), which is a worrisome especially due to the fact that participants are young adults from the university. international diabetes federation (idf) estimates 587 million individuals with pre diabetes by 2045 [9]. in addition, the odd of someone being prediabetic is 2.86 for the amerindian ethnicity compared to the african ethnicity. this implies that a pre-diabetic person is 2.86 times more likely to be from the amerindian descendant compared to the african descendant from the study population. this study has several limitations that should be considered. the major limitation was the crosssectional design, which describes the exposure and outcomes at a time. therefore, our data had shown only the associations with present risk factors but did not predict the future risk of prediabetes. longitudinal follow-up studies are needed to further examine the relationship found in our study. secondly the small sample size and the few numbers of cases with prediabetes that decrease the statistical power of the analysis. furthermore, our study population was not a representative of the general population. lastly, caution should be taken in interpreting our results as even though the use of waist circumference (wc) provided a simple measurement for visceral fat (vf), it does not represent only vf, as subcutaneous fat (scf) also contributes to it. computerized tomography (ct) is the gold standard for the measurement of visceral fat volume, however, it is expensive and involves radiation which does not justify its use as a screening tool. nevertheless, this study has several strengths including sizable response rate (80%), followed by simple random procedure for sample collection and using anthropometric variables, not self-reported values, in a sample of university students with very reliable data. furthermore, confounding effects of various confounders and mediators were considered in data analyses. 5. conclusion although the magnitude of pre-diabetes can differ across nations and by personal characteristics, there is an international consensus that the health complications associated with pre-diabetes in young adults are alarming and therefore strategies aimed at preventing abdominal obesity are urgently needed to reduce the increasing burden of diabetes, cvd, and metabolic diseases. we the researchers recommend the following:  conduct follow-up studies with a larger sample size.  prioritization of pre-diabetes care and prevention such as hosting nationwide awareness sessions.  development of an operational policy/ strategy/action plan to reduce overweight and obesity.  extend health promotion to reduce prediabetes and its complications. consent all authors declare that ‘written informed consent was obtained from the patient. ethical approval approval was granted from irb, ministry of public health, guyana. acknowledgement authors are thankful to staffs and management of medical art’s laboratory. heartfelt thanks are also extended to the law firm of hughes, fields and stoby. competing interests authors have declared that no competing interests exist. references 1. american diabetes association. classification and diagnosis of diabetes: standards of medical care in diabetes— jordan et al.; aji, 3(1): 39-45, 2020; article no.aji.55501 45 2020. diabetes care 43. supplement 1. 2020;s14-s31. 2. ogurtsova k, da rocha fernandes j, huang y, linnenkamp u, guariguata l, cho n, et al. idf diabetes atlas: global estimates for the prevalence of diabetes for 2015 and 2040. diabetes res clin pract. 2017;128:40-50. 3. tuso p. prediabetes and lifestyle modification: time to prevent a preventable disease. perm j. 2014;18(3):88–93. 4. tabak a, herder c, rathmann w, brunner e, kivimaki m. prediabetes: a high-risk state for developing diabetes. lancet. 2012; 379(9833):2279-2290. 5. world health organization. who: obesity and overweight; 2016. 6. fao and paho. panorama of food and nutritional security. healthy food systems to end hunger and malnutrition; 2017. 7. ottaviani e, malagoli d, franceschi c. the evolution of the adipose tissue: a neglected enigma. general and comparative endocrinology. 2011;174(1):1-4. 8. esteghamati a, larijani b, aghajani mh, ghaemi f, kermanchi j, shahrami a, ismail-beigi f. diabetes in iran: prospective analysis from first nationwide diabetes report of national program for prevention and control of diabetes (nppcd-2016). scientific reports. 2017; 7(1). 9. hostalek u. global epidemiology of prediabetes present and future perspectives. clinical diabetes and endocrinology. 2019;5(5). 10. zhang p, wang r, gao c, jiang l, lv x, song y, li b. prevalence of central obesity among adults with normal bmi and its association with metabolic diseases in northeast china. plos one. 2016;11(7). 11. mainous ag, tanner rj, jo a, anton sd. prevalence of prediabetes and abdominal obesity among healthy-weight adults: 18year trend. annals of family medicine. 2016;14(4):304–310. 12. peltz g, aguirre mt, sanderson m, fadden mk. the role of fat mass index in determining obesity. am j hum biol. 2010; 22(5):639-647. 13. yoon kh, lee jh, kim jw, cho jh, choi yh, ko sh, et al. epidemic obesity and type 2 diabetes in asia. lancet. 2006;368(9548): 1681–1688. 14. coelho m, oliveira t, fernandes r. biochemistry of adipose tissue: an endocrine organ. arch med sci. 2013;9: 191–200. 15. nielsen s, guo zk, johnson cm, hensrud dd, jensen md. splanchnic lipolysis in human obesity. journal of clinical investigation. 2004;113(11):1582–1588. 16. garnett sp, baur la, cowell ct. waist-toheight ratio: a simple option for determining excess central adiposity in young people. international journal of obesity. 2008;32(6): 1028–1030. _________________________________________________________________________________ © 2020 jordan et al.; this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: http://www.sdiarticle4.com/review-history/55501 _____________________________________________________________________________________________________ *corresponding author: e-mail: salamakasang2@gmail.com; asian journal of immunology 3(1): 291-297, 2020; article no.aji.60171 immunological response to recombinant hepatitis b virus (hbv) vaccine among vaccinated adults in kaduna south, nigeria salama k. ibrahim1*, yetunde faidero isa2, hosea s. hamafyelto3, lohya nimzing4 and zakka sheyin5 1 hope for the village child foundation, km 2 along kachia road, p.m.b. 500, kaduna state, nigeria. 2 apin/pepfar laboratory, juth, p.m.b. 2076, jos plateau state, jos, nigeria. 3 department of medical microbiology, university of jos, plateau state, nigeria. 4 department of microbiology, university of maiduguri, p.m.b. 1069, maiduguri, borno state, nigeria. 5 department of medical laboratory science, university of jos, plateau state, jos, nigeria. authors’ contributions this work was carried out in collaboration among all authors. author ski designed the study, wrote the protocol and the first draft of the manuscript. author hsh performed the statistical analysis. author yfi managed the laboratory analyses of the study. authors ln and zs supervised and monitored the entire work. all authors read and approved the final manuscript. article information editor(s): (1) dr. darko nozic, university of belgrade, serbia. reviewers: (1) manoj sharma, csk himachal pradesh agriculture university palampur, india. (2) ibrahim m. s. shnawa, qassim university, iraq. complete peer review history: http://www.sdiarticle4.com/review-history/60171 received 10 june 2020 accepted 17 august 2020 published 27 august 2020 abstract aim: the aim of this study was to determine the rate of immunological response to recombinant hepatitis b virus (hbv) vaccine among vaccinated residents of kaduna south senatorial district. materials and methods: this was a cross-sectional study of 180 consented residents of kaduna south senatorial district, kaduna state who have been vaccinated with hbv vaccines. systematic sampling technique and written informed consent were used in recruiting subjects for this study. five (5mls) of venous blood was collected from each subject after filling a structured questionnaire. sera obtained from 180 subject were qualitatively assayed for hbv markers using skytech profile and quantitatively tested for anti-hbs using elisa kit (enzyme linked immunoassay for qualitative and quantitative determination of antibodies to hepatitis b surface antigen by dia.pro original research article ibrahim et al.; aji, 3(1): 291-297, 2020; article no.aji.60171 292 in italy). the results from the laboratory analysis of the specimens were computed using spss version 21. results: the results represent 51.7% seropositive of hbsab among subjects with male having 18.9% and female had 32.8% which statistically showed no significant difference between the groups (χ 2 =3.612,p = .43) see table 1 .in respect to age, 26 – 30years age grouped had the highest sero-conversion rate of 10.0%. this however, was not statistically significant (table 2) (χ 2 =5.604, p = .70). for the number of vaccine shots (table 3) taken, 40.6% of those who completed their vaccination were sero-converted followed by those who took two shots with 4.4% while those who had one shot had 6.7% hbsab (χ 2 =30.665, p< .001). sero-conversion in relation to the quantity of hbv vaccine with titre values of ≥100iu/ml had 34.6%while ≤ 100iu/ml had 16.1% respectively. the result therefore showed statistically significant difference to the quantity of the vaccine administered at χ2 = 6.98, p = .08. in table 4. conclusion: the findings in the research show that none of the subjects tested positive for hepatitis b envelope antigen or hepatitis b envelop antibody. this could due to prior resolved hbv infection before the onset of vaccination or a resolved hbv infection mid-way into the vaccination process. low sero-conversion rate was observed which could be due to the inclusion of subjects who failed to complete their vaccination. keywords: hepatitis b virus; sero-conversion; sero-protection; vaccination; adults. 1. introduction hepatitis is a general term meaning inflammation of the liver. this can be caused by viruses, drugs, and poisons e.tc. among the many known causes of hepatitis, viruses are the most important, with hepatitis b virus being the most critical in terms of complications, morbidity and mortality. it is 50 to 100 times more infectious than hiv and can survive outside the body for at least 7 days [1]. hepatitis b virus is a member of the hepadnavirus family [2]. the virus particle called dane particle / virion [3] consist of an outer lipid envelope and an icosahedral nucleocapsid core composed of protein. the nucleocapsid encloses the viral dna (a dna polymerase that has reverse transcriptase activity similar to retroviruses). the outer envelope contains embedded proteins which are involved in viral binding of, and entry into susceptible cells [4]. the hepatitis b virus is known as a blood-borne virus because it is transmitted from one person to another via blood or fluids contaminated with blood. another important route of transmission is from an infected mother to a newborn child, which occurs during or shortly after birth [5]. currently, an estimated 2 billion people worldwide have had contact with the virus. 360 million of such infected persons progress to chronicity with about25% of chronic cases developing cirrhosis and hepatocellular carcinoma [6]. hbv infection is of great concern in most developing countries. sub-saharan countries are reported to be hyper endemic (i.e.>8% of the population are infected) [7]. among these sub-saharan countries, nigeria has a pooled prevalence rate of 13.6% [8]. though drugs are now available for treatment of hbv infection, present treatments are very costly and not readily available. however, vaccination is now the most effective and economical means for the prevention of this disease [9]. who in 1991 recommended the inclusion of hepatitis b vaccines in the national vaccination program in all countries with the aim of reducing the global impact of hbv infections. hepatitis b vaccine is a genetically engineered (man-made in the laboratory) piece of the virus. it does not contain live virus, thus cannot cause hepatitis due to reversion as in some live attenuated vaccines. this vaccine works by helping the body produce immunity (through antibody production) that prevents infection from hepatitis b virus. it is pertinent to note hepatitis b vaccine does not protect from other viral infections 1 . there are two types of products available for immunization against hepatitis b: a vaccine that confers active immunity and a specific immunoglobulin that provides passive and temporary immunity while awaiting response to vaccine. hepatitis b vaccines are routinely given intramuscularly in the upper arm or anterolateral thigh. the buttock must not be used because vaccine efficacy may be reduced [10]. protection by hbv vaccine is based on the production of immunity or antibodies to the ibrahim et al.; aji, 3(1): 291-297, 2020; article no.aji.60171 293 surface protein or outer coat of the virus. this outer coat is called hepatitis b surface antigen or hbsag [11]. successfully vaccinated individuals will be positive for only antihbs while persons who got immunity as a result of hbv infection will be positive for both antihbc and antihbs [12]. however, around 10% to 15% of adults fail to respond to three shots of vaccine or respond poorly. poor responses are mostly associated with age (over 40 years), obesity and smoking [13]. lower sero-conversion rates have also been reported in alcoholics, particularly those with advanced liver disease [14]. thus the need for post vaccination testing for hbsag and antihbs following hbv vaccination. though this vaccine is commonly used in nigeria, there is no sufficient data on nigerian subjects regarding immunologic response or lack thereof in terms of its immunogenicity [15]. and many among those who start to receive the vaccine fail to complete the 3 recommended doses of the vaccine.thus the need to study the immunological response to hbv vaccine, among vaccinated persons, in kaduna south senatorial district. 2. materials and methods 2.1 study area blood random samples were collected from residents of kaduna south senatorial district in kaduna state. kaduna state is located at 10°31”n7° 26’25”e in north-western nigeria. the population of kaduna as at the 2006 nigerian census was 760,084 but this is believed to have grown to over 1.8 million as of 2013. collected samples will be appropriately. 2.1.1 study population persons with any history of hepatitis b vaccination who had no knowledge of being hbsag positive and consented to be part of the study were included and also excluded subjects who had knowledge of being hbsag-positive, above the age of 60, non hbv vaccinated, between july and november 20 [16] from kaduna south senatorial district. 2.1.2 study design the study was a hospital based descriptive, cross-sectional study that recruited 180 consenting adults in kaduna south senatorial district, kaduna state. 2.2 sample collection five (5) ml of venous blood was collected from the forearm of one hundred and eighty residents of kaduna south senatorial district, kaduna state after the signing of a consent form and the administration of questionnaire. collected samples were placed in sterile plain tubes and labeled appropriately. the blood samples were centrifuged at 2000 rpm for five minutes. sera were separated using automated pipette and stored in the freezer at -20°c prior to usage. all samples were qualitatively tested for five hepatitis b markers hbsag, hbsab, hbeag, hbeab and hbcab using hbv 5-panel test kit (skytech, usa). quantity of hbsab was also tested using elisa kit (enzyme linked immunoassay for qualitative and quantitative determination of antibodies to hepatitis b surface antigen in human serum and plasma by dia.pro in italy). 2.3 qualitative analysis using hbv profile test kit 2.3.1 principle of hbv detection with hbv profile test kit the product uses the colloidal gold and membrane chromatography technology, measures hbsag, hbeag in whole blood, serum and plasma with dual-antibody sandwich method, measures hbsab with dual – antigen sandwich method and measures hbeab and hbcab with neutralization competitive inhibition method. 2.3.2 assay procedure of hbv profile test the right side of the hbv profile test card were placed horizontally from the original package, from left to right, respectively corresponding to hbsag, hbsab, hbeag, hbeab, hbcab. serum was dropped into each of the five sample wells of the test board using micropipettes. a drop of buffer was added to each of the five wells on the test card. the results were observed and recorded within fifteen minutes. 2.3.3 results determination negative results for hbsag, hbsab, hbeag (sandwich method), were represented with only one purple bar at the control line in the control (c) zone. while positive results were by detecting two purple bars, one in the test (t) zone and the other in the control (c) zone. negative results for hbeab and hbcab (competition method) was done by detecting two ibrahim et al.; aji, 3(1): 291-297, 2020; article no.aji.60171 294 purple bars in t and c zone and positive results were by detecting only one purple bar (control line) in the control zone. 2.4 data analysis the data obtained from the study were analysed using statistical package for social sciences (spss) version 21 (ibm spss inc, usa).proportions were compared using chisquare with confidence limit (p-value) of < 0.05considered significant. 3. results of the 180 respondents, 61 were male while 119 were female. the overall sero-conversion rate of hbsab 51.7% out of which male had 18.9% representing 34 positive rate while female with 32.8% had 59 tested positive. the results there showed no statistically significant difference between male and female (χ 2 =3.612, p = .43) see table 1. the study revealed the highest hbsab sero-conversion rate of 10.0% among the age group 26 – 30 years with the lowest rate of 3.3% among 16 – 20 years age group. this however, was not statistically significant (table 2) (χ 2 =5.604, p = .70). for the number of vaccine shots, 40.6% of those who completed their vaccination were seroconverted followed by those who took two shots with 4.4% while those who had one shot had 6.7% hbsab at χ 2 =30.665, p< .001. this showed a significant difference between the vaccine shots (table 3). protective seroprotection in relation to the quantity of hbv vaccine with titre values of ≥100iu/ml had 34.6% while ≤ 100iu/ml had 16.1% respectively. the result therefore showed statistically significant difference to the quantity of the vaccine administered at χ2 = 6.98, p = .08. in table 4. this study did not recruit equal number of respondents in the one shot, two shots and three shots category and did not take into consideration other the cellular and genetic factors that influence sero-conversion. table 1. sero-conversion of adult subjects to hbv vaccine in relation to gender gender no. tested for hbsab no positive for hbsab % positive male 61 34 18.9 female 119 59 32.8 total 180 93 51.7 (χ 2 =3.612, p= .43) table 2. sero-conversion of adult subjects to hbv vaccine in relation to age age group no. tested for hbsab no positive for hbsab % positive 16-20 14 6 3.3 21-25 20 11 6.1 26-30 34 18 10 31-35 11 8 4.4 36-40 20 12 6.7 41-45 30 16 8.9 46-50 22 8 4.4 50-55 14 6 3.3 ≥55 15 8 4.4 total 180 103 57.2 chi-square (χ 2 ) =5.604, p=.70 table 3. sero-conversion of adult subjects to hbv vaccine in association with the number vaccine shots received no of shots received no. tested for hbsab no positive for hbsab % positive one 47 12 6.7 two 27 8 4.4 three 106 73 40.6 total 180 93 51.7 ibrahim et al.; aji, 3(1): 291-297, 2020; article no.aji.60171 295 (χ 2 =30.665, df = 2, p< .001) table 4. protective sero-conversion in association with quantity of hbsab produced after vaccination quantity of hbsab no. tested for hbsab no positive for hbsab % positive <100 94 29 16.1 >100 86 64 34.6 total 180 93 51.7 χ 2 =13.98, p = .08 4. discussion the rate of sero-conversion for all subjects observed by this study was 51.7% (93/180) regardless of the number of vaccine shots received. none of the subjects tested was positive for hepatitis b envelope antigen (hbeag) or hepatitis b envelop antibody. this showed that none of the subjects was at the infectivity phase or has recovered from the phase. this could be as a result of prior resolved hbv infection before the onset of vaccination or a resolved hbv infection mid-way into the vaccination process. in this study, the 18.9% rate of sero – conversion among male subjects lower than 32.8% sero – conversion rates recorded in female subjects. this was in agreement with findings of [16] who suggested that the high level of testosterone in men represses transcription factors implicated in immune activation and with findings by [17] that found higher rates of seroconversion in women than men. however, this study agrees with that by 15 who found that men were more likely to sero-convert to hbsab than women. the highest rate of sero –conversion of 10.0% in age group 26 – 30 years had no relationship with sero – conversion after hbv vaccination as was found by [18,19]. this finding may be as a result of active immunity as a result of physical exercise possessed by the older subjects in this study. the number of hbv vaccine shots received by subjects was analyzed in this study. result of the analysis showed that there is a relationship between the rate of sero conversion and the number of vaccine shots received [7]. this buttresses the importance of completing the course of vaccination as 40.6%of the subjects who completed their vaccination sero-converted and just 6.5% of those who received just one shot of the vaccine seroconverted. in this study, sero-conversion of 34.6% (64/180) with titre values of ≥100iu/ml of hbsab (level deemed protective by the european health sector) was lower than the 85-90% of full response of vaccinated individuals as reported [20] and the 82.9% [21]. this low sero-conversion rate reported by this study may be as a result of the inclusion of subjects who received one or two shots of the vaccine. 5. conclusion it was concluded based on the findings that none of the subjects tested were positive for hepatitis b envelope antigen (hbeag) or hepatitis b envelop antibody. this could be as a result of prior resolved hbv infection before the onset of vaccination or a resolved hbv infection mid-way into the vaccination process. low sero-conversion rate was observed which could be as a result of the inclusion of subjects who received one or two shots of the vaccine. consent all the authors reviewed and gave their consent for this article to be submitted for publication. ethical approval before the commencement of the project, relevant ethical approval was sought for and obtained from the kaduna state ministry of health. competing interests authors have declared that no competing interests exist. ibrahim et al.; 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(2) awofadeju stephen olajide, obafemi awolowo university teaching hospitals complex, nigeria. complete peer review history: http://www.sdiarticle4.com/review-history/54405 received 22 november 2019 accepted 27 january 2020 published 03 february 2020 abstract due to conflicting reports on the impact of exercise on haematological and immunological indices, this study investigated the effects of exercise on some haematological and cellular immune markers in male athletes. blood samples were collected from 86 apparently healthy male athletes before and after exercise training using standardized methods. similarly, blood samples were obtained from 100 male non-athletes and served as control. blood samples collected from athletes and non-athletes were subjected to experimental evaluation of some haematological and cellular immune biomarkers using standard techniques. results showed that, with the exception of neutrophils that significantly increased after exercise in athletes, lymphocytes, monocytes, eosinophils and clusters of differentiation counts significantly (p<0.05) decreased in athletes after exercise. also, with the exception of platelets, other haematological parameters assayed in this study significantly (p<0.05) decreased in athletes after exercise. however, there was no significant change in these parameters between athletes at rest and non-athletes. this study concludes that heavy training could lead to an open window of immunodepression leading to susceptibility to infection in athletes and non-athletes alike. original research article nwoke et al.; aji, 3(1): 23-29, 2020; article no.aji.54405 24 keywords: immuno-physiological; immunodepression; haematological; immune biomarkers. 1. introduction it is widely believed that physical activity enhances the cardiovascular system. however; new studies have pointed to what seemed like adverse effects of prolonged heavy exercise upon both resistance to and the course of various viral and bacterial diseases [1]. upper respiratory tract infection (urti) is a major ailment that have been reported in athletes following participation in marathon or ultra marathon events [2,3]. examinations show that the pathogens normally responsible for urti could not be identified in these athletes, suggesting that changes in the immune system and not pathogens could be responsible for these post exercise traumas [4]. to further explain this, some studies targeted at the specific immune response to exercise, have reported that light regular physical exercises have favourable effects on the physiological, psychological and immunological functions [5] and therefore could increase resistance of the body against infections [6]. conversely, vigorous exercise produces negative effects on the aforementioned functions. moderate exercise has been reported to boosts immune functions via chemotaxis and phagocytosis whereas extreme exercise on the other hand reduces these functions, with the exception of chemotaxis and degranulation [7]. increased lymphocyte concentration has been reported to be likely due to the recruitment of all lymphocyte subpopulations (cd41 t cells, cd81 t cells, cd191 b cells, cd161 natural killer (nk) cells, and cd561 nk cells) to the vascular compartment [8,9]. earlier studies have shown that nk cells with a high il-2 response capacity were recruited to the blood during bicycle exercise [10]. on immunoglobulin, lower concentrations of the salivary iga have been reported in cross-country skiers after a race [11]. this finding was confirmed by a 70% decrease in salivary iga that persisted for several hours after completion of intense, long-duration ergometer cycling [12]. decreased salivary iga was also found after intense swimming [13] and after incremental treadmill running to exhaustion [14]. submaximal exercise had no effect on salivary iga [14]. the percentage of b cells among blood mononuclear cells (bmnc) does not change in relation to exercise; suggesting that the suppression of immunoglobulin-secreting cells is not due to changes in numbers of b cells. many of these reported studies were on caucasian non-athletes in the temperate environment. research needs to be extended to performing athletes especially in the tropical environment. this is therefore partly covered in this study. 2. materials and methods 2.1 study population and sample size with ethical approval from the university of port harcourt research ethics committee, the targeted population for this study was the 112 registered nigerian athletes that partook in the 14th west african university games (waug) held at the university of port harcourt between october and november, 2018. the sampling adopted was total population purposive-sampling technique, in which case, there is the possibility of selection all the elements in the study population. the basis for purposive sampling is to concentrate on subjects with particular desired characteristics that would be able to assist with the generation of relevant information to achieve the research objectives. therefore, the justification for the adoption of total population purposive sampling technique is because it is a non-probability sampling technique that could include all members, within the population of interest. hence, all the elements in the population were given equal opportunity to participate in the selection. based on the sampling technique, 86 apparently healthy subjects between the ages of 22 and 30 years volunteered and were recruited for the study. the subjects were drawn from the following sporting events: fitness exercises, soccer, long-distance running, short-distance running and swimming. the athletes have been in the profession for not less than two years; and were at the peak of their training, in preparation for the west african university games (waug) held at the university of port harcourt between october and november, 2018. training sessions were not less than 3 days per week for a minimum of 60 minutes per session as recommended by the [15]. in addition, the subjects were encouraged to avoid smoking, use of tobacco products or anti-inflammatory drugs. subjects also completed a health history, drug usage, and physical activity questionnaires to determine eligibility. also, 100 volunteer non-athletes were recruited and served as the control. prior to participation in this study, each subject was informed of all procedures, potential risks, and benefits associated with the study and an informed consent form was signed. nwoke et al.; aji, 3(1): 23-29, 2020; article no.aji.54405 25 2.2 anthropometrics for the participants that met the selection criteria, weight was measured using an electronic scale (hanover, md) while the subjects were wearing shorts with bare feet. height was measured using a seca stadiometer with 1 cm spaced leaned to the wall. body mass index (bmi) for each participant was calculated using the formula: weight/height². all measurements were taken in the morning from 7 a.m. to 8 a.m. 2.3 collection of blood sample and analysis of biomarkers using standard venipuncture procedures [16], about 5 ml of blood was collected from the antecubital veins of subjects using sterile syringes before exercise; between the hours of 7 am-8 am and immediately after exercise between 10-11 am. the blood was dispensed in ethylenediaminetetraacetic acid (edta) and stored at -4c pending analysis. the cells that make up the cellular immune system; namely: lymphocytes, neutrophils, monocytes and eosinophils were assayed. also, total white blood cell (wbc) count, red blood cell (rbc) count, hemoglobin (hb), packed cell volume (pcv), platelets (plt) were also assayed. these parameters were assayed using the hematology autoanalyzer (celldyn 3700 (abbott, chicago, il, usa), which makes use of the coulter principle [17]. the cd4 count was determined manually by microbead separation of cd4 t lymphocytes from other blood cells, followed by standard manual cell counting techniques using a light microscope. 2.4 statistical analysis data collated on selected variables from the study were statistically analyzed using statistical package and service solution (spss, version 20.0). the mean of descriptive statistics was recorded as mean± standard deviation. comparison of parameters between athletes and non-athletes were done using paired t-test with the level of statistical significance accepted at p<0.05. multiple comparison between nonathletes, athletes before exercise and athletes after was done using post-hoc multiple comparison test (lsd). 3. results the result of the study is presented in tables and the figure below. table 1 showed the anthropometric measurement of the athletes and non-athletes. table 2 showed the effect of exercise on haematological parameters. table 3 showed the effect of exercise on cellular immuno-physiological marker while fig. 1 showed percentage changes in both haematological and some immuno-physiological parameters of male athletes after exercise. 4. discussion body mass index (bmi): the bmi is a calculation that compares weight relative to height. the index however, is not perfect because a body builder’s bmi may indicate overweight, but the extra weight is muscle rather than fat [18]. for everyone else, however, bmi is a reasonable measure of fitness level, demonstrating whether one is overweight, underweight or just right. the result of this study showed that there was no statistically significant p<0.05 difference between the bmi of male athletes compared with male non-athletes (table 1); suggesting that bmi alone cannot be used to determine fitness levels in athletes because hypertrophied muscle may give perception of overweight. this agrees with a study of which concluded that the intensity of physical activity does not matter in lowering or maintaining bmi [19]; therefore, one can appear to be fat and yet be fit. haematological parameters: investigators have suggested that the value of haematological indices in athletes varies as a result of different training regimes [20,21]; usually higher at the beginning of the competition, then declined in well-trained athletes [22]. therefore, haematological indices is key determinants of optimal exercise performance in athletes. result of this study showed that with the exception of platelets, haemoglobin, pcv, rbc and wbc diminished significantly in athletes after exercise; as shown in table 2. this result is consistent with a study which reported low values of haematological variables in athletes during intensive training periods compared with clinical norms [23]. however, the study disagrees with other investigations that reported normal concentrations of hematological indices throughout training programmes [20,24]. the significant decrease in hb, pcv and rbc immediately after heavy exercise might be due to training induced haemodilution as a result of plasma volume expansion associated with aerobic sports, endurance and ultra-endurance nwoke et al.; aji, 3(1): 23-29, 2020; article no.aji.54405 26 events. plasma volume expansion is a compensatory mechanism to increase cardiac output and reduces blood viscosity, thereby optimizing microcirculation and improving oxygen delivery to the working muscles. innate immunological markers: figure 1 showed that lymphocyte, monocyte, esinophil and cd4 counts significantly p<0.05 decreased after exercise. however, there was significant p<0.05 increase in neutrophils count. the significant decrease in lymphocyte and cd4 (cluster of differentiation) cells seen in this study is consistent with the studies which reported a rapid exercise-induced decrease in lymphocyte number within 30 minutes following a prolonged or high-intensity exercise [25,26]. however, there are contrary reports of lymphocytosis occurring during or immediately after exercise; depending on the intensity and the duration of the exercise [25,27]. the significant decrease in lymphocyte count, rather than being a real reduction, may be secondary to lymphocyte redeployment to peripheral tissues as a result of stress induced cortisol release. monocytes are mobilized during exercise; they are rapidly redeployed to skeletal muscle and differentiate into tissue-resident macrophages that facilitate repair and regeneration after an intensive bouts of exercise that cause significant skeletal muscle damage [28]. this redeployment of monocytes to muscles may account for the significant decrease in monocyte count immediately after exercise. eosinophils are a type of white blood cells that defend against parasites and infectious agents. in this study, we observed a significant decrease in eosinophil count which is consistent with a similar study in the elderly. however, it differs with the result of a similar study in asthmatic subjects [29]. the significant increase in neutrophil count seen in this study agrees with a report which showed that running at 75% of maximum heart rate (hrmax.) for 15 min increased neutrophil number [30]. neutrophils recruited into circulation during exercise may be responsible for controlling the elevated levels of oxidative stress in plasma after exercise [31]. this impact of exercise on neutrophil count may be mediated by the activation of catecholamine and cortisol which are known to have adverse effect on the immune system [32,33]. table 1. anthropometric measurement of athletes and non-athletes variables male athletes (n=86) male non-athletes (n=100) percentage change (%) weight (kg) 72.43±5.76 75.20±6.47 3.68 height (m) 1.69±0.05 1.7101±0.06 1.18 bmi (kg/m 2 ) 25.41±1.52 25.69±1.56 1.09 values are expressed as mean ± s.d. *=statistically significant at p < 0.05, n= sample size fig. 1. changes in haematological and some immuno-physiological parameters of male athletes after exercise * shows that there was a statistically significant change at p≤0.05 nwoke et al.; aji, 3(1): 23-29, 2020; article no.aji.54405 27 table 2. effect of exercise on haematological parameters variables non-athletes ( n=100) athletes before exercise (n=86) athletes after exercise (n=86) change in athletes after exercise (%) change between athletes after exercise and non-athletes(%) change between athletes before exercise and nonathletes(%) rbc (x10^12/l) 6.22±0.29 6.19±0.27 5.08±0.40* -21.85 -22.44 -0.48 pcv (%) 40.59±1.71 41.76±1.68 39.45±1.16 -5.89 -2.89 2.80 hb(g/dl) 13.91±0.41 13.98±0.46 12.37±0.55* -13.02 -12.45 0.50 platelets (x10^7/l) 210.52±11.71 210.06±12.24 253.20±6.20* 17.04 16.85 -0.22 wbc(x10^9/l) 6.40±0.21 6.39±0.19 4.07±0.38* -57.00 -57.25 -0.16 all values are expressed as mean ± s.d. *=statistically significant at p < 0.05, n= sample size table 3. effect of exercise on cellular immuno-physiological markers variables nonathletes (n=100) athletes before exercise(n=86) athletes after exercise(n=86) change in athletes before and after exercise (%) change between athletes after exercise and nonathletes(%) change between athletes before exercise and nonathletes(%) neut (%) 46.15±4.97 46.20±4.77 60.17±3.45* 23.23 23.30 0.11 lymph (%) 32.69±1.95 32.95±2.14 22.98±1.62* -43.39 -42.25 0.79 mono (%) 6.10±1.11 6.08±1.23 4.07±0.27* -49.39 -49.88 -0.33 eosino (%) 2.09±0.85 2.09±0.85 2.07±0.82 -0.97 -0.97 0 cd4(cells/�l) 704.21±31.96 688.05±54.14* 508.98±38.85* -35.18 -38.36 -2.35 results are presented as mean±standard deviation; n= sample size; * shows that there was a statistically significant change at p<0.05 when compared with the non-athletes nwoke et al.; aji, 3(1): 23-29, 2020; article no.aji.54405 28 5. conclusion the available evidence from this study showed that exercise decreases hematological indices in the vascular compartment and also has modulatory effects on immunocyte dynamics. these effects may cause a temporary decline in the cellular immune system and create an open window, during which the exerciser may be susceptible to infections. consent prior to participation in this study, each subject was informed of all procedures, potential risks, and benefits associated with the study through both verbal and written form and signed an informed consent form. ethical approval with ethical approval from the university of port harcourt research ethics committee. competing interests authors have declared that no competing interests exist. references 1. cox ag, gleeson m, pyne db, callister r, hopkings wg, fricker pa. clinical and laboratory evaluation of upper respiratory symptoms in elite athletes. clin j sports med. 2008;18:438-445. 2. peters em, bateman ed. ultramarathon running and upper respiratory tract infections. an epidemiological survey. s after med j. 1983;64:582–584. 3. nielsen hb, secher nh, kappel m, hanel b, pedersen bk. lymphocyte nk and lak cell responses to maximal exercise. int j sports med. 1996;17:60–65. 4. bermon s. airway inflammation and upper respiratory tract infection in athletes: is there a link? exercise immunology review. 2007;13:6-14. 5. mackinnon lt. chronic exercise training effects on immune function. med sci sports exerc. 2000;32(7):369-376. 6. shinkai s, shore s, shek pn, shephard rj. acute exercise and immune function. relationship between lymphocyte activity and changes in subset counts. int j sports med. 1992;13:452-461. 7. pedersen bk, steensberg a, fischer c, keller c, keller p, plomgaard p, febbraio m, saltin b. searching for the exercise factor: is il-6 a candidate? j mus res cell motil. 2003;24:113–9. 8. li tl, gleeson m. the effect of single and repeated bouts of prolonged cycling on leukocyte redistribution, neutrophil degranulation, il-6, and plasma stress hormone responses. int j sport nutr exerc metab. 2004;14:501–516. 9. fondell e, lagerros yt, sundberg cj, lekander m, bälter o, rothman kj, bälter k. physical activity, stress, and selfreported upper respiratory tract infection. med sci sports exerc. 2011;43(2):272-9. 10. nieman dc. marathon training and immune function. spo med. 2007;37:412– 5. 11. libicz s, mercier b, bigou n, le gallais d, castex f. salivary iga response of triathletes participating in the french iron tour. int j sports med. 2006;27:389-94. 12. michishita r, shono n, inoue t, tsuruta t, node k. effect of exercise therapy on monocyte and neutrophil counts in overweight women. the american journal of the medical sciences. 2010;339(2):152– 156. 13. bishop nc, gleeson m. acute and chronic effects of exercise on markers of mucosal immunity. front biosci. 2009;14:4444-56. 14. nakamura d, akimoto t, suzuki s, kono i. daily changes of salivary secretory immunoglobulin a and appearance of upper respiratory symptoms during physical training. j sports med phys fitness. 2006;46(1):152-7. 15. physical activity guidelines for americans. office of disease prevention & health promotion, us department of health and human services; 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6:333–63. 27. peake jm, neubauer o, walsh np, simpson rj. recovery of the immune system after exercise. j appl physiol. 2017;122:1077–87. 28. peake j, nosaka k, suzuki k. characterization of inflammatory responses to eccentric exercise in humans. exercimmunol rev. 2005;11:64– 85. 29. massoud a, rezaei n. immunology of aging. springer science & business media; 2013. 30. cannon jg, orencole sf, fielding ra, meydani m, meydani sn, fiatarone ma. acute phase response in exercise: interaction of age and vitamin e on neutrophils and muscle enzyme release. am j physiol. 1990;259(6pt 2):r1214–9. 31. quindry jc, stone wl, king j, broeder ce. the effects of acute exercise on neutrophils and plasma oxidative stress. med sci sports exerc. 2003;35:1139–45. 32. matson kd, horrocks npc, tieleman bi, haase e. intense flight and endotoxin injection elicit similar effects on leukocyte distributions but dissimilar effects on plasmabased immunological indices in pigeons. j exp biol. 2012;215:3734–41. 33. brown gp, shine r. immune response varies with rate of dispersal in invasive cane toads (rhinella marina). plos one. 2014;9:e99734. _________________________________________________________________________________ © 2020 nwoke et al.; this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: http://www.sdiarticle4.com/review-history/54405 _____________________________________________________________________________________________________ *corresponding author: e-mail: elizabeth.finbarrs-bello@esut.edu.ng; asian journal of immunology 3(1): 77-83, 2020; article no.aji.55832 evaluation of hematological and histopathological effects of dennettia tripetala fruit extract on isoniazid-induced seizure in wistar rats finbarrs-bello e.1*, odo c. e.1 and ojuolape s. g.1,2 1 department of anatomy, faculty of basic medical sciences, enugu state university of science and technology, parklane, enugu state, nigeria. 2 department of anatomy, kampala international university, western campus, ishaka-bushenyi, uganda. authors’ contributions this work was carried out in collaboration among all authors. author fbe gave the concept, designed the study and did the acquisition of data. author oce analyzed and interpreted the data. author osg drafted the manuscript. all authors critical revised the manuscript for important intellectual content. all authors read and approved the final manuscript. article information editor(s): (1) dr. jaffu othniel chilongola, tumaini university, tanzania. reviewers: (1) s. danish kadir, university of pharmacy, bangladesh. (2) atiq-ur-rehman, the university of lahore, pakistan. complete peer review history: http://www.sdiarticle4.com/review-history/55832 received 27 january 2020 accepted 02 april 2020 published 07 april 2020 abstract seizure is one of the neurological and episodic clinical challenges and, one underutilized option for medication-resistant in seizure is the use of medicinal plants. dennettia tripetala has been found to possess anti-seizure effects. this study estimates the hematological parameters and examines the histopathological effect of ethanolic extract of dennettia tripetala fruit on isonaizid-induced seizure on the temporal lobe. twenty-four (24) adult wistar rats were randomized into six groups (1-6) of four rats each (n=4). the positive and negative control groups (group 1 and 2) received isoniazid 300 mg/kg, i.p (inducing agent) and 1 ml, i.p normal saline (vehicle); while the rats in groups 3, 4, 5 and 6 were induced with 300 mg/kg of isoniazid and treated with 250 mg/kg, 500 mg/kg and 750 mg/kg of the extract and 50 mg/kg of sodium valproate, o.p respectively. in the hematological study, the blood parameters rbc, pcv and hb decreased, while wbc remained normal and decrease eosinophils count was noted. histopathological changes observed in the untreated seizure induced (positive control) group presented atrophied neurons, shrunken neurons, original research article finbarrs-bello et al.; aji, 3(1): 77-83, 2020; article no.aji.55832 78 coagulative necrosis and microcystic spaces. the extract treated groups exhibited dose dependent reduction in the above histopathological features as well as the sodium valproate standard drug group. this implies that, dennettia tripettala fruit extract at high dose exerts hematotoxic effect, while neuroprotective effect was observed at low dose. in conclusion, it is safe and effective at low dosage. keywords: dennettia tripettala; seizure; isoniazid; atrophy; hematotoxocity; neuroprotective. abbreviations gaba: gamma amino-butyric acid; pcv: packed cell volume; hb: hemoglobin concentration; wbc: white blood cell; d.p.x: dibutylphthalate polystyrene xylene. 1. introduction seizure is one of the most common neurological conditions in the world [1]. it has an estimated neuro-epidemiological index of at least 65 million people worldwide living with seizures with large proportion of sufferers’ resident within the developing countries [2]. its recognizable feature is an excessive neuronal activity in the cortical areas of the brain such as the frontal and temporal lobes [3]. the etiology/pathogenesis is traceable to several factors including: brain damage (traumatic brain injury, stroke, brain tumors), infections scarring (gliosis), hypoxia, severe hypoglycemia, very high fever and abnormal electrical activity in the brain [4]. sometimes, it could be initiated by drug related adverse reaction or overdose, in the case with isoniazid [5]. primarily, there is loss of gamma amino-butyric acid (gaba) mediated inhibitory interneurons and increased hyper excitability of the neurons leading to recurrent seizure episodes [5,6]. isoniazid is one of the most commonly prescribed tuberculosis chemotherapy regimens [7]. isoniazid overdose is known to causes recurrent seizures, profound metabolic acidosis, coma and even death [7]. the isoniazid-induced seizure represents a pathophysiological state of decrease availability of gamma aminobutyric acid (inhibitory neurotransmitter) and increase the level of glutamate (excitatory neurotransmitter) in the affected brain areas [8]. this dysfunctional milieu disrupts the glutamate and gamma aminobutyric acid balance which consequently leads to excessive excitatory signals in the brain and altered brain electroencephalography wave patterns [9]. conventional anticonvulsants are the typical pharmacotherapy used for seizure which target different membrane channels, receptors and neurotransmitters in the brain. sodium valproate as an anticonvulsant, acts via the voltage dependent sodium channels blockage and inhibition of gamma-aminobutyric acid transaminase with increases brain levels of gaba [10]. the increase in the turnover of gamma-aminobutyric acid (gaba) potentiates a gabaergic function which is able to be in control of seizure generation and propagation [11]. but, the refractory rate of sodium valproate is high, coupled to incidence of side effect and inadequate of curative potential. pepper fruit botanically known as dennettia tripetala of family annonaceae, is known with local names as: ako in edo, opipi in idoma, nkarika in ibibio, mmimi in igbo and ata igbere in yoruba language. d. tripetala is a well-known medicinal in the southern parts of nigeria and consumed due to the spicy taste of the fruit and leaf [5,12]. phytochemical screening of the ethanolic extract showed the presences of tannins, alkaloids, steroids, flavonoids, cardiac glycosides, saponins, and terpenoids. it also contains antioxidant vitamins c and e, as well as the b group of vitamins b1, b6, b9 and b12 [5,13], which provides a scientific basis for the use of d. tripetala in traditional medicine. previous studies also documented its hypnotic, anticonvulsant, and anxiolytic effects in mice and rats [5,14]. most synthetic anticonvulsants including sodium valproate had been found to have adverse effects on the on the targeted organs. furthermore, about 30% of patients with epilepsy have seizures that do not react adequately to conventional antiepileptic drugs [11]. these restrictions with conventional antiepileptic drugs showcases the need to seek new drugs that could be more efficacious as the action of conventional antiepileptic drugs, with less side effects so as to achieve a better treatment outcome for seizures. this therefore calls the need to seek for an effective herbal medicine for finbarrs-bello et al.; aji, 3(1): 77-83, 2020; article no.aji.55832 79 seizure that can possibly offer the required opportunities for the isolation of important compounds which can be used in the development of better, affordable and well tolerated antiepileptic drugs. thus, the ineffectiveness of these synthetic drugs, along with their serious side effects makes any safe and effective herbal medicine an attractive possibility. therefore, this work investigates the effect of the ethanolic extract of d. tripetala fruits on the isoniazid induced seizure on the hematological parameters and temporal lobe histology. it showcases the comparative therapeutic potentials of d. tripetala extract and sodium valproate. this knowledge can be used by pharmaceutical industries to produce cheaper and less toxic drug for the populace or serve as an alternative for seizure therapy. dennettia tripetala fruits 2. materials and methods 2.1 plant collection, identification and preparation fresh samples of d. tripetala fruit were harvested from farmlands, in igbo-eze-south local government area of enugu state nigeria. the fruits were identification and authentication by plant curator in the department of plant science and biotechnology of the university nigeria nsukka. the fruits were selected of impurities, washed and air dried for 7 days. the dried fruits were pulverized into powdered form; 863.54 g of the powder was soaked in 2.2 litre of 90% ethanol and allowed to stand for 48 hours with intermittent stirring. thereafter, the mixture was filtered with a meshwork and what-man paper no 1. the filtrate was left in an open space for ethanol to evaporate and form paste with percentage yield of 4.26%. it was stored in a refrigerator at 4°c and constituted to desired volume when required. 2.2 drugs and chemicals isoniazid (isonamede india, pubchem id: 3767) was procured from a registered pharmaceutical store and the stock solution of inh was prepared by dissolving 300mg of the tablets in 10 ml of distilled water at room temperature. 2.3 experimental animal and ethical approval twenty four (24) wistar rats were obtained from the animal house of the zoology department of university of nigeria, nsukka. the rat were housed at the animal facility of esut college of medicine where the study was conducted, acclimatized for two weeks with free access to food and water, under light and dark phase of 12 hours within 24 hours at a room temperature during the period of the experiment [15]. thereafter, the rats were randomized into six groups of four (4) rats’ per-cage. 2.4 experimental design group 1 (positive control): 300 mg/kg of isoniazid (single dose, i.p). group 2 (negative control): 0.1 ml normal saline (vehicle, i.p). group 3 (low dose treatment): 300 mg/kg of isoniazid (single dose, i.p) and 250 mg/kg of extract, p.o. group 4 (medium dose treatment): 300 mg/kg of isoniazid (single dose, i.p) and 500 mg/kg of extract, p.o. group 5 (high dose treatment): 300 mg/kg of isoniazid (single dose, i.p) and 750 mg/kg of extract, p.o. group 6 (standard treatment): 300 mg/kg of isoniazid (single dose, i.p) and 50 mg/kg, p.o of sodium valproate. 2.5 haematological study the rats were anesthetised by xylazine and ketamine (v/v); 2 ml blood samples was collected via the retro-orbital vessels from each rat into heparinized tubes and immediately used for the determination of rbc, pvc, hb, wbc the leucocytes differential counts. total red blood cell (rbc) and white blood cell (wbc) counts were estimated using the visual method. the percentage packed cell volume (pcv) and blood finbarrs-bello et al.; aji, 3(1): 77-83, 2020; article no.aji.55832 80 hemoglobin concentrations (hb) and luecocytes differential counts of all samples were estimated as specified the physiology practical manuel for esut college of medicine. the film were evenlycovered with leishmans’ stain and after 2 minutes by means of a clean pipette an equal quantity of water added, mixedand allowed to stand for 10 minutes. the mixture was poured off and the film was washed in a gentle stream of water, dried with filter paper andexamined under a high power microscope; the different types of white cells (leucocytes) were systematically counted and categorized until a total of 100 white cells have been counted. 2.6 histological studies the brain tissues were harvested at the end of blood sample collection and fixed in 10% neutral formal saline for 48 hrs, afterwards the temporal lobes were dissected and processed manually for paraffin wax embedding, sections of 5 um thickness were obtained using the rotary microtomy, deparaffinized sections were stained using hematoxylin and eosin (hand e). the stained sections were then dried and mounted in dibutylphthalate polystyrene xylene (d.p.x), micrographed and interpreted. 2.7 statistical analysis data was analyzed by one way analysis of variance (anova) using the statistical package for social sciences (spss version 21) for windows (spss inc., chicago, usa). the results were presented as mean ± standard deviation (sd). statistical significance was considered at p=.05. 3. results hematological findings: table 1. effect of ethanolic extract of dennettia tripetala on rbc, pcv and hb on seizure model test group rbc count pcv % hb g/dl isoniazid (300 mg/kg) 5.80±0.75 44.50±2.65 14.83±0.88 normal saline 5.00±0.82 39.75±5.56 13.25±1.86 250 mg/kg extract &300 mg/kg isoniazid 4.64±0.56 * 38.25±5.06 12.53±1.82 500 mg/kg extract& 300 mg/kg isoniazid 4.39±0.47 36.75±4.11 * 11.95±1.45 * 750 mg/kg extract& 300 mg/kg isoniazid 6.00±0.00 50.00±0.00 16.70±0.00 50 mg/kg sodium valproate & 300 mg/kg isoniazid 2.00±0.14 47.25±2.06 16.07±0.70 key: *p=.05, when compared with group 1. a p=.05rbc counts showed statistically significant decrease in group 3(p=.05), non-significant decrease were also observed in groups 3 and 4. pcv and hb also showed a statistical significant decrease (p=.05) in group 4 when compared with group 1, while only group 6 showed a significant increase in hb (p=.05) when compared with group 2 table 2. effect of ethanolic extract of dennettia tripetala on total wbc and differential counts on test groups test groups twbc count neutrophils % lymphocytes % monocytes % eosinophils % basophils % isoniazid (300 mg/kg) 6.28±3.96 51.75±7.68 41.75±8.66 3.50±0.58 2.25±0.50 0.75±0.50 normal saline 3.23±1.45 58.50±7.51 34.75±8.96 3.75±0.96 2.50±0.58 0.50±0.58 250 mg/kg extract & 300 mg/kg isoniazid 3.33±0.48 48.25±13.72 46.75±12.37 3.75±1.50 1.25±0.50 * 0.00±0.00 500 mg/kg extract & 300 mg/kg isoniazid 3.68±0.76 55.25±14.36 41.25±13.94 2.75±0.96 0.75±0.96 * 0.00±0.00 750 mg/kg extract & 300 mg/kg isoniazid 4.60±0.00 37.00±0.00 60.00±0.00 3.00±0.00 0.00±0.00 0.00±0.00 50 mg/kg valproate & 300 mg/kg isoniazid 8.30±0.50 66.00±3.65 30.50±5.74 0.25±0.50 0.50±0.58 * 0.00±0.00 key:*(p=.05) no significant effect on the wbc across the groups , for differential leucocytes counts only eosinophil count was significantly( p=.05) decreased in groups 3, 4 and 6 which represents the low , medium dose of the extract and standard drug the sodium valproate respectively finbarrs-bello et al.; aji, 3(1): 77-83, 2020; article no.aji.55832 81 histological observations: fig. 1. photomicrograph of temporal lobe (a) positive control (isoniazid 300 mg/kg) coagulative necrosis (white arrow), microcystic space (black arrow), neurons atrophy (green arrow) and shrinking neurons (sn). (b) negative control (1 ml normal saline) normal neuronal cell (black arrow). (c) 250 mg/kg of d. tripetala and 300 mg/kg of isoniazid, few atrophic neurons (white arrow) and intracytoplasmic inclusion (black arrow). (d) 500 mg of d. tripetala and isoniazid 300 mg/kg, few atrophic neurons (green arrow), intracytoplasmic inclusion (black arrow) and shrinking neurons (white arrow).(e) 750 mg/kg and isoniazid reduced microcystic space (black arrow), atrophic neurons (green arrow) and mild shrinking neurons (white arrow). (f) sodium valproate treated large nuclei and basophilic inclusions (black arrow) with few atrophic neurons (white arrow). h&e. x400 4. discussion hematological assessment is vital in the evaluation of causes, effect of treatments and recovery in most illnesses. thus, the total blood parameter testing is carried out on seizure patients to access the overall health and identify their likely conditions such as; infections and anemia that might trigger seizure, and to monitor the possible occurrence of medication-induced side effects [16]. in this study, we evaluated the hematologic parameters and found that the rbc counts was significantly decreased in the low extract group (p<0.05), following the seizure induction by isoniazid, while the diseased observed in the rest groups was insignificant. in a similar study, daniel and clement [17] earlier reported decrease in rbc counts with the d. tripetala fruit in the absence of seizure in rats. the role of the extract on the rbc is primarily due to the presence of certain phytochemical constituent like alkaloids. although, secondary effect of direct bone marrow suppression might also be attributed to the effect on rbc count in the case of sodium valproate, for example, the aplastic anemia and pheripheral cytopenia could explain the characterised hematologic toxicity associated with sodium valproate [18,19]. conversely, pcv and blood hb counts showed insignificant decreases in the treated model (table 1). a direct modulatory relationship exists between rbc, pcv and hb concentration physiologically, therefore, an alteration in one parameter alternately impact another. hence, the importance of evaluating blood parameters while on certain medications such as herbal and anticonvulsants. hematotoxic effect has also a b d e finbarrs-bello et al.; aji, 3(1): 77-83, 2020; article no.aji.55832 82 been reported on isoniazid overdose which might have interplayed to cause further disease in the treatment groups. the lack of statistical evidence on the effect of the total white blood count possesses a question of whether this suggests that the extract exhibited a protective effect or the harmful effect of isoniazid. this is not likely the case with sodium valproate which has been shown to suppress leucocytosis, induce thrombocytopenia and agranulocytosis [18]. sodium valproate group showed a significant increase compared with negative control (table 2), this may be a representation of a normal physiological response of the body which triggers the migration of wbc to defend the rbc against hemolysis, eosinophil counts decreased in the in sodium valproate, the low and medium dose of the extract. this effect might be attributed to the phytochemical components of the extracts and the valproate could be related to its effect on the hematopoietic stem cells. consistently, loss of neurons has been a histopathological feature associated with focal temporal seizure particularly in humans [20]. the positive control or non treated seizure group (fig. 1a) revealed characteristic features of the neuronal degenerative changes: atrophied and shrunken neurons, coagulative necrosis and microcystic spaces. these can be attributed to the acute neurotoxic effect of the high dose of isoniazid (inh). the atrophied neurons observed in the non treated seizure group were reduced following extract treatments with varying doses. thus, the extract exhibited dose dependant effect, its low to medium dosages displayed potent property while the high dose could be toxic. in this regards, only the low and medium dose of the extract could be comparable to sodium valproate treatment. this implies the extract exerts similar activity as the standard drug sodium valproate by providing some measures of neuroprotective effects on the temporal lobe histology. furthermore, isoniazid toxicity is refractory to anticonvulsant and responsive to treatment with high dose of pyridoxine (vitamin b6) as demonstrated in our previous studies on the anticonvulsant effect of this extract [5]. however, the study is a pointer to the fact that d. tripetala fruit extract has promising potential in the search for an entity with anti-seizure activity but this might require further studies on the fractionated components or the isolated compound. 5. conclusion the d. tripetala extract has dose dependant effects on the seizure model (both hematologically and histologically) of the temporal lobe compare to sodium valproate. consent it is not applicable. ethical approval the study protocol was reviewed and approved by the esucom animal research committee. animals were cared for in accordance with the institutional and international guidelines. acknowledgement the authors would like to thank the staff of the physiology and histology laboratories for the technical assistance. competing interests authors have declared that no competing interests exist. references 1. who. epilepsy care in the world. geneva; world health organization. 2005;8-16. 2. nwani po, nwosu mc, asomugha la, enwereji ko, arinzechi eo, ogunniyi ao. epidemiology of active epilepsy in a suburban community in southeast nigeria: a door-to-door survey. nigeria journal of clinical practice. 2015;18(4):527-533. 3. fisher r, van-emde bw, blume w, elger c, genton p, lee p, engel j. epileptic seizures and epilepsy: definitions proposed by the international league against epilepsy (ilae) and the international bureau for epilepsy (ibe). epilepsia. 2005;46(4):470-472. 4. athanassios v, john eg, dimitrios n. risk factors associated with epilepsy: case study-control study. health science journal. 2012;6(3):509-517. 5. elizabeth fb, ignatius io, samsudeen go, abonyi eo. antioxidant and anticonvulsant effect of dennettia tripetala on rat model of isoniazid–induced seizure. european finbarrs-bello et al.; aji, 3(1): 77-83, 2020; article no.aji.55832 83 journal of medicinal plants. 2019;30(2):110. 6. bertram gk, serzan bm, anthony jt. sedative-hypnotic drugs: basic and clinical pharmacology. 11 th international ed, mcgraw hill companies inc. singapore. 2009;371-509. 7. puri mm, lokender k, vishwakarma pd, behera d. seizures with single therapeutic dose of isoniazid. indian journal of tuberculosis. 2012;59:100-102. 8. tuba şafak, ali ek, şeref kc, yunsur c. what is the cause of seizure: isoniazid poisoning or epidural hematoma? a case report. jemcr. 2015;1:3-5. 9. tuba şafak, ali ek, şeref kc, yunsur c. what is the cause of seizure: isoniazid poisoning or epidural hematoma? a case report. jemcr. 2015;1:3-5. 10. treiman dm. gabaergic mechanism in epilepsy. epilepsia. 2001;42(3):8-12. 11. vinay k, sandeep ks, nagarajan k, praveen kd. effects of lycopene and sodium valproate on pentylenetetrazolinduced kindling in mice. iranian journal of medical sciences. 2016;41(5):430-36. 12. egharevba ho, idah ea. major compounds from the essential oil of the fruit and comparative phytochemical studies of the fruits and leaves of dennettia tripetala barker f. found in north central nigeria. international journal of pharmacognosy and phytochemical research. 2015;7(6):1262-1266. 13. sylvia oi. a review of the uses and medicinal properties of dennettia tripetala (pepper fruit). medical sciences. 2015;3(4):104-111. 14. oyemitan ia, iwalewa eo, akanmu ma, olugbade ta. antinociceptive and antiinflammatory effects of essential oil of dennettia tripetala g. baker (annonaceae) in rodents. afric. j. tradit. complement. altern. med. 2008;5:355-362. 15. esut college of medicine. manual for practical physiology. teamwork publications int’l ltd. enugu. 2016;65-67. 16. nass rd, sassen r, elger ce, surges r. the role of postictal laboratory blood analyses in the diagnosis and prognosis of seizures. seizure. 2017;47:51-65. 17. daniel ei, clement on. effect of ethanolic extract of dennettia tripetala fruit on haematological parameters in albino wistar rat. nigerian journal of physiological sciences. 2008;23(1-2):13-17. 18. acharya s, bussel jb. heamtologic toxicity of sodium valproate. journal of pediatric hematology and oncology. 2000;22(1):6265. 19. kaipainen p, westermarck t, atroshi f, kaski m, iivanainen m. clinical and hematological profiles during valproate treatment of epileptic patients with intellectual disability-case study and mini review. provisional chapter; intech open science. 2014;1-10. 20. ingmar b, roberto s, gernil h, rolan c, katja k, christian c, et al. histopathological findings in brain tissue obtained during epilepsy surgery. n. engl. j. med. 2017;377:1648-1655. _________________________________________________________________________________ © 2020 finbarrs-bello et al.; this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: http://www.sdiarticle4.com/review-history/55832 https://www.researchgate.net/journal/0253-0716_iranian_journal_of_medical_sciences https://www.researchgate.net/journal/0253-0716_iranian_journal_of_medical_sciences https://www.ncbi.nlm.nih.gov/pubmed/?term=nass%20rd%5bauthor%5d&cauthor=true&cauthor_uid=28288363 https://www.ncbi.nlm.nih.gov/pubmed/?term=sassen%20r%5bauthor%5d&cauthor=true&cauthor_uid=28288363 https://www.ncbi.nlm.nih.gov/pubmed/?term=elger%20ce%5bauthor%5d&cauthor=true&cauthor_uid=28288363 https://www.ncbi.nlm.nih.gov/pubmed/?term=surges%20r%5bauthor%5d&cauthor=true&cauthor_uid=28288363 http://creativecommons.org/licenses/by/4.0 _____________________________________________________________________________________________________ *corresponding author: e-mail: gideonbenjamin.y@gmail.com; asian journal of immunology 2(1): 64-71, 2019; article no.aji.53159 interleukin-12 (p70) concentrations in malaria patients attending some hospitals in zaria, kaduna state, nigeria gideon yakusak benjamin1*, b. e. moses2, e. d. jatau1 and c. m. z. whong1 1 department of microbiology, ahmadu bello university, zaria, nigeria. 2 department of general studies, school of agricultural technology, nuhu bamalli polytechnic, zaria, nigeria. authors’ contributions this work was carried out in collaboration among all authors. author gyb designed the study, wrote the protocol and performed the laboratory experiments and statistical analysis. author bem read and contributed to literature searches. authors edj and cmzw co-supervised the research. all authors read and approved the final manuscript. article information editor(s): (1) dr. tania mara pinto dabes guimaraes, associate professor, faculty of pharmacy, federal university of minas gerais, belo horizonte, minas gerais, brazil. (2) dr. cynthia aracely alvizo baez, professor, laboratory immunology and virology, faculty of biological sciences, autonomous university of nuevo leon, mexico. (3) dr. darko nozic, professor, higher medical school in belgrade, university of belgrade, serbia. reviewers: (1) muhammad ghafoor ali, pakistan. (2) fadia m. attia, suez canal university, egypt. complete peer review history: http://www.sdiarticle4.com/review-history/53159 received 15 october 2019 accepted 19 december 2019 published 25 december 2019 abstract background and aim: malaria is the most important parasitic disease of man, and it remains one of the major threats to public health and economic development in africa. interleukin-12 is a heterodimeric cytokine which has potent effects on innate and adaptive immunity. this study was aimed at determining interleukin-12 (p70) concentrations among malaria patients attending some hospitals in zaria, kaduna state. methods: a cross sectional hospital based study was conducted on consenting participants in zaria. four hundred blood samples were collected, from which giemsa-stained thick and thin blood films were prepared and examined for the presence of plasmodium species by microscopy. enzyme linked immunosorbent assay (elisa) was used to determine concentrations of interleukin-12 (p70) in malaria positive samples and control samples. original research article benjamin et al.; aji, 2(1): 64-71, 2019; article no.aji.53159 65 results: males had higher malaria prevalence (37.2%) than females (24.7%). the difference was statistically significant (p = 0.01). pregnant women had a prevalence of 17.8% which was lower than the 27.9% obtained in non-pregnant women (p = 0.07), and the highest malaria prevalence (20.0%) was found in pregnant women in their third trimester (p = 0.65). interleukin-12 (p70) was present at a significantly (p = 0.00) higher level in the plasma of participants in the malaria positive group than in the control group (those who tested negative for malaria). conclusion: gender was significantly associated with malaria in this study. the prevalence of malaria was higher in males than females; males are therefore encouraged to take more precautions to prevent malaria. despite the fact that the exact role of cytokines in malaria pathogenesis is unclear, we can infer from the findings of this study that more interleukin-12 (p70) is produced during malaria infection. keywords: interleukin-12(p70); plasmodium; prevalence; malaria; cytokine. 1. introduction malaria is a life-threatening disease caused by parasites that are transmitted to people through the bites of infected female anopheles mosquitoes. it is preventable and curable [1]. malaria is the most important parasitic disease of man. approximately 5% of the world’s population is infected. it remains one of the major threats to public health and economic development in africa. it is estimated that three million deaths result from malaria throughout the world, with africa having more than 90% of this burden [2]. about half of the world’s population is at risk of malaria [3]. in 2016, an estimated 445 000 malaria deaths occurred worldwide from an estimated 216 million reported cases of malaria; 90% of these cases were in the world health organization (who) african region. of the 91 countries reporting indigenous malaria cases in 2016, 15 countries – all in sub-saharan africa, except india – carried 80% of the global malaria burden [1]. in 2017, plasmodium falciparum accounted for 99.7% of estimated malaria cases in the who african region, as well as in the majority of cases in the who regions of southeast asia (62.8%), the eastern mediterranean (69%) and the western pacific (71.9%) [1]. according to the level of malaria transmission and immunity acquisition, vulnerable populations differ in endemic areas. in highly endemic settings, children under five years and pregnant women are the most affected, constituting the main target population of new malaria control strategies as recommended by the world health organization [4]. there are now a large number of regular prevalence surveys of childhood parasitemia [5], as most malaria deaths occur in children. however, the prevalence of parasitemia in adults remains of scientific interest, not only because clinical attacks in adults remain an important cause of death in adults [6,7], as well as of morbidity and health service use [8], but also because adults form a community reservoir of infection for children. with the current sustained implementation of malaria control and prevention strategies across most african countries and the consideration of elimination in some settings [9,10], the impact of this adult reservoir in these control strategies needs to be assessed. in pregnancy, there is a transient depression of cell-mediated immunity that allows fetal allograft retention which in the other hand interferes with resistance to various infectious diseases such as malaria [11]. on the top of host, pregnant women, immunossupression; studies showed that immunological interactions between protozoan and helminths infection can intensify the impact of parasitic infection when they co-exist [12]. in addition, as the epidemiology of malaria changes across africa there are likely to be changes in the disease pattern with adults becoming susceptible to severe disease and this trend should be monitored. while there are many studies that associate patterns of cytokines to disease, results may be different depending on the cohort population. thus, there is an association between elevations in certain cytokines and disease outcomes, but it is hard to generalize these associations to different patient populations. il-12 acts on antigen stimulated cd4+ t cells, promoting the differentiation of t cells into the th1 subset [13], which acts on macrophages not only to stimulate their microbicidal functions, but also to increase their production of il-12. the elevated levels of il-12 also modulate the macrophage activity, which is associated with the increased erythrocyte destruction, bone marrow dyserythropoiesis [14] and thrombocytopenia [15]. during the intraerythrocytic life cycle of benjamin et al.; aji, 2(1): 64-71, 2019; article no.aji.53159 66 plasmodium falciparum, macrophages avidly phagocytize parasite specific products, leading to the impairment of macrophage functions [16] and cytokine production [17]. 2. materials and methods 2.1 study area the study was carried out in four selected hospitals in zaria nigeria; major ibrahim b. abdullahi memorial hospital zaria, hajiya gambo sawaba hospital kofan gaya zaria, salama hospital and st. luke’s hospital wusasa zaria. zaria is a city found in kaduna state, nigeria. it is located at 11.11 latitude and 7.72 longitude and it is situated at elevation 644 meters above sea level. zaria has a population of 975,153 making it the second largest city in kaduna [18]. 2.2 sample size the sample size was determined using a prevalence of 23.45% [19] and the following formula as described by naing, et al. [20]: n = n= number of samples p=prevalence rate of previous study = 23.45% = 0.2345 z=standard normal distribution at 95% confidence limit = 1.96 d=absolute desired precision of 5% = 0.05 z=1.96 n=1.96 2 *0.2345(1-0.2345) 0.05 2 n=275 samples four hundred (400) blood samples were however collected for this study. 2.3 administration of structured questionnaire a structured questionnaire was used to collect data from consenting participants. 2.4 sample collection a total of 400 blood samples were collected from major ibrahim b. abdullahi memorial hospital zaria (former limi hospital), hajiya gambo suwaba hospital kofan gaya zaria, salama hospital and st. luke’s hospital wusasa zaria (100 samples from each hospital). venipuncture technique was used for blood sample collection. a soft tubing tourniquet was fastened to the upper arm of the patients to enable the index finger to feel a suitable vein. the puncture site was then cleansed with methylated spirit (methanol) and venipuncture was made with the aid of a 21 g needle attached to a 5 ml syringe. when sufficient blood (3 ml) was collected, the tourniquet was then released and the needle removed immediately while the blood was transferred into an edta bottle [21]. 2.5 determination of malaria parasitemia the malaria parasitemia was determined using a grading scheme of + =1-10 parasites, ++ = 11-20 parasites, +++ = more than 20 parasites per microscopic field was used to establish the levels of parasitemia [19]. 2.6 determination of interleukin-12 (p70) concentrations the concentrations of interleukin-12 (p70) were determined in eighty eight (88) serum samples, using boster’s interleukin-12 elisa kit purchased from boster biological technology co. ltd. (fremont, ca usa). the samples were divided into two groups; the malaria positive group (59) and the control group; without parasitemia (29). the interleukin-12 concentrations were determined according to manufacturer’s instruction. 2.7 statistical analysis the data obtained were analyzed using statistical package for social sciences (spss inc., chicago, il,usa). chi square and odds ratio were used to check for association. p≤0.05 was considered significant. 3. results table 1 shows malaria prevalence in relation to gender. out of the 113 males examined; 42 (37.2%) were positive, while out of the 287 females screened; 71 (24.7%) were positive. therefore males had a higher prevalence (37.2%) than females (24.7%). the p value and odds ratio showed significant statistical association (p = 0.01, odds ratio=1.800, confidence interval= 1.129-2.869, chi square = 6.180). benjamin et al.; aji, 2(1): 64-71, 2019; article no.aji.53159 67 the age related prevalence of malaria among the study population is shown in table 2. the prevalence rate in the table reveals that the highest prevalence of 50.0% was found in the age group 66 years and above, followed by the age group 56-60 years with 44.4% prevalence, 51-55 years with 40.0% prevalence, 11-15 years with 37.5% prevalence, 41-45 years with 33.3% prevalence and 16-20 years with 32.9% prevalence. the lowest prevalence was in the age group 46-50 years (0.00%). the age group 31-35 years also had a low prevalence of 13.3%. statistically, there was no significant difference among the age groups (χ 2 =11.620, p value=0.56). table 3 shows the prevalence of malaria in relation to pregnancy status. pregnant women had a prevalence of 17.8% which was lower than the 27.9% obtained in non-pregnant women. the difference was not statistically significant (p>0.05). table 4 shows the distribution of malaria according to pregnancy trimester. pregnant women in their third trimester had the highest prevalence (20.0%), followed by those in their second trimester (19.6%) and those in their first trimester (10.5%). the difference was not statistically significant (p>0.05). table 5 shows the mean concentrations of il-12 (p70) among malaria positive individuals and control group. the malaria positive group had a mean concentration of 28.31 pg/ml which was high compared to the 19.23 pg/ml mean concentration of the control group (those without malaria infection). the statistical difference was not significant (p=0.06). fig. 1 shows the mean concentrations of il-12 (p70) in relation to malaria parasitemia. the ++ parasitemia group had the highest mean concentration of 39.39 pg/ml. the + parasitemia group had 22.59 pg/ml mean concentration of il12 (p70) which was lower than the 27.67 pg/ml mean il-12 (p70) concentration found in the +++ parasitemia group. the mean il-12 (p70) concentration in each of the three parasitemia groups (+,++,+++) was more than the mean il12 (p70) concentration in the control group (19.23 pg/ml). the difference was statistically significant (df=3, p value=0.00). table 1. malaria prevalence in relation to gender gender no. examined no. positive % prevalence χ 2 p value or 95%ci male 113 42 37.2 6.180 0.01* 1.800 1.129-2.869 female 287 71 24.7 total 400 113 28.3 key: no=number, or=odds ratio, ci=confidence interval, *=significant table 2. age-related prevalence of malaria among the study population age(years) no. examined no. positive % prevalence χ 2 p value 0-5 43 11 25.6 11.620 0.56 6-10 28 9 32.1 11-15 24 9 37.5 16-20 70 23 32.9 21-25 74 20 27.0 26-30 61 17 27.9 31-35 30 4 13.3 36-40 26 7 26.9 41-45 18 6 33.3 46-50 8 0 0.00 51-55 5 2 40.0 56-60 9 4 44.4 61-65 2 0 00.0 66> 2 1 50.0 total 400 113 28.3 key: no=number, χ 2 =chi square benjamin et al.; aji, 2(1): 64-71, 2019; article no.aji.53159 68 table 3. malaria prevalence in relation to pregnancy pregnant no. examined no. positive % prevalence χ 2 p value yes 90 16 17.8 3.412 0.07 no 197 55 27.9 total 287 71 24.7 key: no=number χ 2 = chi square table 4. distribution of malaria according to pregnancy trimester trimester no. examined no. positive % prevalence χ2 p value first 19 2 10.5 0.867 0.65 second 56 11 19.6 third 15 3 20.0 total 90 16 17.8 key: no=number χ 2 = chi square table 5. mean concentrations of il-12 (p70) among malaria positive individuals and control group malaria no. mean(pg/ml) se p value positive 59 28.31 2.003 0.06 cg 29 19.23 2.125 total 88 25.32 1.576 key: cg= control group, se=standard error, no.= number of samples fig. 1. mean concentrations of il-12 (p70) in relation to malaria parasitemia (p value<0.05) key: cg= control group, a= +, b=++, c=+++, (+=1-10, ++=11-20, +++=20> parasites per microscopic field) 4. discussion in the current study, we found a high prevalence of malaria in males than females. the difference observed was statistically significant (p<0.05). this agrees with the findings of muntaka and opoku-okrah [22] who reported the percentage of males with malaria to be higher than females; 19.4% and 10.7% for males and females respectively. reza and taghi [23] also reported a similar finding. our finding is in contrast to that of otajevwo [24] who reported a higher infection rate in females than males. the high prevalence in males may be due to the fact that men are less likely to sleep under the insecticide treated bed nets than females [4]. according to olapeju, et al. [25], itn use tends to be higher among females than males especially in households without sufficient itns. in some societies, men have a greater occupational risk of contracting malaria than women if they work in mines, fields or forests at peak biting times. leisure activities and 0 10 20 30 40 cg a b c 19.23 22.59 39.39 27.67 m ea n c o n ce n tr a ti o n (p g /m l) parasitemia benjamin et al.; aji, 2(1): 64-71, 2019; article no.aji.53159 69 sleeping arrangements may also be contributing factors, as men are more likely to sleep outdoors or be found outdoors during the active biting hours of anopheles mosquito [26,27]. this can increase the human-vector contact, and consequently lead to plasmodium infection [26]. interleukin-12 (p70) was present at a significantly (p=0.000) higher level in the plasma of those in the malaria positive group than in the control group (those who tested negative for malaria). this is in agreement with the findings of lyke, et al. [28]. however, adrian, et al. [29] reported that concentration of il-12 (p70) was significantly higher in the plasma of those with mild malaria than in the plasma of those with severe malaria. on comparing the different parasitemia groups and their mean interleukin-12 (p70) concentrations, the control group still had the lowest mean concentration of il-12 (p70). the + and +++ parasitemic group had mean il-12 (p70) concentrations which were less than the il-12 (p70) concentrations in the ++ parasitemia group. this is possible because low interleukin-12 (p70) activity has also been associated with severe plasmodium falciparum malaria [30]. our finding is however in contrast to that of adrian, et al. [29] who reported that the acute-phase, pretreatment plasma il-12 and alpha interferon (ifn-a) levels, as well as the acute-phase mitogen-stimulated whole-blood production capacity of il-12, were significantly lower in children with severe rather than mild malaria. it has been reported that early events in the cell-mediated immune response required for protection against malaria are initiated by the release of interleukin-12 (il-12) from monocytes/macrophages, b cells, and perhaps other cell types [31,32]. in plasmodium falciparum infection, il-12 has immunoregulatory functions with effects on the immune response to the blood stage of disease, but also induces protection and reduces malarial anemia [33,34]. il-12 has been shown to be involved in protective immunity against malaria by regulating gamma interferon. pro-inflammatory cytokines like il-12 are thought to be critical for controlling the erythrocytic and hepatic stages of plasmodium infection [34] this may be one of the reasons why we had higher concentrations in the parasitemia group than the control group in this study. 5. conclusion this study found a significant association between the gender of participants and malaria. the prevalence of malaria was higher in males than females; males are therefore encouraged to take more precautions to prevent malaria. although the concentrations of interleukin-12 (il12 (p70) in this study differed with parasitemia, its concentrations reflect the role it plays in malaria infection. and despite the fact that the exact role of cytokines in malaria pathogenesis is unclear, the findings of this study suggest that more il-12 (p70) cytokine is produced during malaria infection. consent as per international standard, patient’s written consent has been collected and preserved by the author(s). ethical approval the research protocol was read and approved by the ethics and research committee (erc) of kaduna state ministry of health, nigeria. competing interests authors have declared that no competing interests exist. references 1. world health organization. world malaria report; 2017. 2. breman jg, alilio ms, mills a. conquering the intolerable burdine of malaria; what is new, what is needed. american journal of tropical medicine and hygiene. 2004; 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asian journal of immunology 1(1): 36-43, 2018; article no.aji.46581 antifungal and immunomodulatory activity of bryophyllum pinnatum leaf extracts jacinta edesiri okpoho1, lucky evbuomwan1* and fortune itojie ebiala1 1 department of microbiology, faculty of life sciences, university of benin, benin city, p.m.b. 1154, nigeria. authors’ contributions this work was carried out in collaboration between all authors. author jeo designed the study, performed the statistical analysis, wrote the protocol, and wrote the first draft of the manuscript. authors le and fie managed the analyses and literature searches of the study. all authors read and approved the final manuscript. article information doi: 10.9734/aji/2018/v1i130092 editor(s): (1) dr. jaffu othniel chilongola, department of biochemistry and molecular biology, kilimanjaro christian medical university college, tumaini university, tanzania. (2) dr. darko nozic, professor, higher medical school in belgrade, university of belgrade, serbia. reviewers: (1) muhammad shahzad aslam, xiamen university, china. (2) mahendran sekar, universiti kuala lumpur, malaysia. (3) vivek kumar singh, public health and infectious disease research center (phidrec), nepal. complete peer review history: http://www.sdiarticle3.com/review-history/46581 received 26 th october 2018 accepted 30 th january 2019 published 26 th february 2019 abstract this study was carried out to investigate the antifungal and immunomodulatory activities of bryophylum pinnatum. both aqueous and ethanol solvents were used for extraction. five fungal species including aspergillus niger, aspergillus flavus, cladosporium herbarium, candida albicans and penicillium italicum were obtained from the university of benin teaching hospital and they were preliminarily identified using standard microbiological methods. wistar rat models for the study were purchased and acclimatized for a period of two weeks. the rats were divided into five groups and orally administered with the ethanol extract of the plant while one group served as control. antimicrobial and hematological parameters including packed cell volume (pcv), hemoglobin, white blood cell counts, platelets and cd4 count were assayed using standard methods. the only fungus sensitive to aqueous extract was aspergillus niger, with zone of inhibition ranging from 6.00±0.58-11.33±0.33 mm at concentration range of 50-100 mg/ml. mean zones of inhibition of ethanolic extract ranged from 11.67±0.67-20.33±0.33 mm against original research article okpoho et al.; aji, 1(1): 36-43, 2018; article no.aji.46581 37 cladosporium herbarium at concentration range of 6.25-100 mg/ml. minimum inhibitory concentrations (mic) of ethanol extract ranged from 6.2550 mg/ml against fungal isolates. while mic of aqueous extract was 50 mg/ml against susceptible fungal isolate. minimum fungicidal concentrations of ethanol and aqueous were 25100 mg/ml and 100 mg/ml respectively. significant difference was observed between the treatment and control groups in platelet counts (range: 95.40±1.86-126.20±5.40% and control: 108.60±4.19%), pcv (range: 39.00±0.71-44.20±0.58%; control: 39.00±0.71%) and hemoglobin (range:12.94±0.21-14.62±0.24 g/dl; control: 12.94±0.21 g/dl). there was no significant difference between the treatment and control groups in cd4 counts (75.40±19.32-99.00±6.33cells/ml and control 75.4±19.32 cells/ml). bryophylum pinnatum has been shown in this work to possess both antimicrobial and hematological properties. keywords: fungicidal; immunomodulatory; phytochemical; extract, inhibition. 1. introduction bryophyllum pinnatum (kalanchoe pinnata; lamarch crassulaceae) is an erect, succulent, perennial shrub that grows about 1.5m tall and reproduces through seeds and also vegetatively from leaf bubils. it has a tall hollow stem, freshly dark green leaves that are distinctively scalloped and trimmed in red and dark bell-like pendulous flowers [1]. bryophyllum pinnatum can easily be propagated through stems or leaf cutting. it is an introduced ornamental plant that is now growing as a weed around plantation crop and widely used in traditional medicines [2]. nigeria is richly endowed with indigenous plants which are used in herbal medicine to cure diseases and heal injuries. some of the plants are used as food or medicine. these plants exhibit a wide range of biological and pharmacological activities such as anti-cancer, anti-inflammatory, diuretic, oxytocic, laxative, antispasmodic, antihypertensive, antidiabetic, and anti-microbial and immunomodulatory functions [3]. the secondary metabolites of plants provides humans with numerous biological active products which have been used extensively as drugs, foods, additives, flavours, insecticides, colorants, fragrances and chemicals. these secondary metabolites include several classes such as saponins, terpenoids, phenolic compounds, steroids, alkaloids and flavonoids [4]. in recent years, researches into new sources of chemotherapeutic agents have intensified due to failure or non-effectiveness of conventional drugs to which many pathogens have developed resistance. many compounds with potential biological activities have been isolated from many plants. in this regard, bryophyllum pinnatum is used in ethnomedicine for the treatment of earache, burns, abscesses, ulcers, insect bites, whitlow, diarrhoea and cithiasis [5]. in south eastern nigeria, this herb is used to facilitate the dropping of the placenta of new born baby [6]. the lightly roasted leaves are used externally for skin fungus and inflammations. the leaf infusions are an internal remedy for fever [4]. different naturally occurring flavonoids have been described and subcategorized into flavones, falvans, flavanones, isoflavonoids, chalcones, aurones and anthocyanidines. these flavonoids exhibit remarkable biological activities including inhibitory effects on enzymes, modulatory effects on some cell types, protection against allergies, antiviral, anti-malarial, antioxidant, antiinflammatory and anti-carcinogenic properties [7]. phytochemical screenings of bryophyllum pinnatum have yielded alkaloids, triterpenes, glycosides, flavonoids, steroids, butadienolides, lipids, and organic acids, phenol and tannis, free amino acid and terpenoids. arachidic acid, astragalin, behenic acid, beta amyrin, benzenoids, bersaldegenin, betasitosterol, bryophollenone, bryophollone, bryophyllin, caffeic acid, ferulic acid, quercetin, steroids, taraxerol have also been found from extracts of brophyllum pinnatum [8]. two novel flavanoids; 5 methyl 4,5,7 trihydroxyl flavones and 4,3,5,7 tetrahydroxy 5 methyl 5 propenamine anthocyanidines from this plant have shown potential antimicrobial activities against pseudomonas aeruginosa, klebsiella pneumoniae, e. coli, staphylococcus aureus, candida albicans and aspergillus niger. when 60% methanolic extract of bryophyllum pinnatum leaf was used to inhibit the growth of bacteria, at a concentration of 25 mg/ml it showed good antibacterial effects. further the plant is effective in the treatment of typhoid fever and other bacterial infections, particularly those caused by s. aureus, e. coli, b. subtilis, p. aeruginosa, k. aerogenes, k. pneumonia and s. typhi due to the presence of phenolic compounds [9]. the antiinflammatory potential of bryophyllum pinnatum okpoho et al.; aji, 1(1): 36-43, 2018; article no.aji.46581 38 was investigated by [10]. however, the antifungal and immunomodulating activity of the plant has not been well studied. therefore, this work was carried out to determine the immunomodulatory properties and antifungal activities of bryophyllum pinnatum leaf extract. 2. materials and methods 2.1 plant material bryophylum pinnatum leaves were obtained from adolor street in benin city and identified at the herbarium, department of plant biology and biotechnology, university of benin, benin city, edo state. the leaves were air-dried, ground using sterilized laboratory blender. the powdered leaf was kept in a sterile bottle container until required. 2.2 preparation of crude extracts fifty grams (50 g) of the grinded bryophylum pinnatum leaves was soaked in 250 ml each of distilled water and ethanol for 24 hrs. the extract was filtered through a sieve with pore size of 250µm to remove debris. the filtrate was then filtered through membrane filter paper. the final filtrate was evaporated in a water bath at 40°c to get the crude extract. the crude aqueous and ethanol extracts were stored at 4°c until required [11]. 2.3 preparation of concentration of plant extract one gram (1g) each of both ethanol and aqueous extract was added to 10ml of ethanol and distilled water respectively to give a concentration of 100mg/ml. other concentrations of 50, 25 and 12.5 and 6.25mg/ml were prepared by double dilution method. in this procedure, 1ml of content in the stock concentration (containing 100mg/ml) was added to 1ml each of distilled water and ethanol in test tubes to give 50mg/ml concentration. 1ml of this 50mg/ml is added to another test tube of 1ml distilled water and ethanol to give 25mg/ml and so on [12]. 2.4 test microorganisms five fungal isolates, aspergillus niger, aspergillus flavus, cladosporium herbarium, candida albicans and penicillium italicum were used in this study. the fungi were obtained from the microbiology laboratory stocks in university of benin teaching hospital. they were then identified based on their cultural and microscopic characteristic before further work on them were carried out. 2.5 fungal inoculum preparation the fungi inocula were prepared by inoculating the test organisms on potato dextrose broth and kept at room temperature for 24hr. 0.2 ml of the different fungal broth culture was used for the antifungal study [12]. 2.6 agar well diffusion technique the ability of the various extracts to inhibit the growth of the clinical test organisms was determined using the agar well technique. the inoculated potato dextrose agar plates were allowed to dry. after which, wells were bored on the surface of inoculated agar plates using 4mm cork borer. zero point one millilitres (0.1ml) of the different concentration of each extracts was transferred into the well using pasteur pipette. the wells were sufficiently spaced to prevent the resulting zones of inhibition from overlapping. the plates were kept at room temperature for 24hr. the experiment was performed in triplicate and the resulting zones of inhibition were recorded as mean ± standard error [13]. 2.7 determination of minimum inhibitory concentration (mic) and minimum fungicidal concentration (mfc) the minimum inhibitory concentration (mic) of the extracts was determined for each of the test organisms at varying concentrations of 100, 50, 25, 12.5 and 6.25mg/ml. 1 ml of potato dextrose broth and 1 ml each of the extracts were added to test tubes and a loopful of fungi isolates was introduced. a tube containing potato dextrose broth only was seeded with the fungi isolates to serve as control and all tubes were kept at room temperature for 24hr to check for growth. the minimum fungicidal concentration of the plant extract was carried out according to [14]. briefly, 1 ml fungal culture was pipetted from the mixture obtained in the determination of mic tubes which did not show any growth and subcultured on to potato dextrose agar. the agar plates were left at room temperature. after incubation the concentration at which there was no single growth of fungi was taken as mfc. okpoho et al.; aji, 1(1): 36-43, 2018; article no.aji.46581 39 2.8 experimental animals eight weeks old wistar rats weighing between 200-270 g were used in this experiment. the rats were housed at the department of microbiology animal house and were fed with pelleted rat food and water daily. after acclimatization for two weeks, rats were divided into six groups, with five rats in each group. temperature ranged from 30°c to 37°c during the period of experiment. the groups were labelled a to f. group f served as the control group and did not receive administration of extract. groups a to e received different dosage of the ethanolic plant extract. group a received 300mg/kg, group b-250mg/kg, group c – 200mg/kg, group d – 150mg/kg and group e -100mg/kg body weight. on day 15, 2.5ml of blood sample was collected from each rat into edta anticoagulant container and was used to estimate the counts of cd4, neutrophils, lymphocytes, eosinophils and total leukocytes of each group [15]. 2.9 full blood count (fbc) this was carried out using symex21n japan model auto-analyzer. automated cell counters was used to quantify and classify the different cell populations according to [16]. 2.10 enumeration of cd4count this was carried out using partec cyflow counter (cy-s-3022 model). briefly, 20µl whole blood was added to a partec test tube and 20µl of cd4 mab pe was added. this was gently mixed and incubated for 15min at room temperature in the dark. after incubation, 800µl of no lyse buffer was added and shaken gently. the sample was then analyzed on the partec device [16]. 2.11statistical analysis data were analysed using statistical package for social sciences (spss) version 23.0. descriptive statistic was used to present values in means and standard errors. one way anova was used to find significant difference among different parameters [17]. 3. results the zone of inhibition of aqueous extract of brophyllum pinnatum against funga isolates is shown in table 1. many fungi species were resistant to all concentrations of aqueous extract of b. pinnatum except the fungus aspergillus niger with zones of inhibition ranging from 6.0±0.58 – 11.33±0.33 mm at concentration range of 50-100mg/ml. a comparatively higher antifungal activity for ethanolic extract was observed with cladosporium herbarium, candida albicans and aspergillus niger as shown in table 2. the order of antifungal activity was aspergillus niger (50100mg/ml) ˂ candida albicans (25-100mg/ml) ˂ cladosporium herbarium (6.25-100mg/ml). minimum antifungal activity of aqueous extract was also observed against fungal isolates when compared with ethanolic extract and higher zones of inhibition were observed at higher concentrations in susceptible fungi species. the minimum inhibitory concentration of ethanolic extract against fungi species as shown in table 3 ranged from 6.25-50mg/ml while that of aqueous extract was 50mg/ml. the minimum fungicidal concentration of ethanolic extract ranged from 25-50mg/ml while that of aqueous extract was 100mg/ml against susceptible fungal isolates. effects of ethanolic extract of b. pinnatum on haematological parameters of wistar rat models is shown in table 4. the pcv values ranged from 39.00±0.7144.20±0.58% with the control being 39.00±0.71%. haemoglobin values ranged from 12.94±0.21-14.62±0.24g/dl, while the control was 12.94±0.21g/dl. effect of ethanolic extract of b. pinnatum on cd4 count as shown in table 5 has values ranging from 81.60±2.8499.00±6.33 cells/ml while the control was 75.40±19.32 cells/ml. there was no significant difference between treated and control groups. 4. discussion plants have been reported to be vast repertoire of bioactive phytochemical compound. these compounds which include flavonoids, alkaloids, tannins etc., are usually responsible for the various biologic properties of the plant, including antimicrobial and other medicinal properties. it has been reported that organic solvent such as ethanol, will usually extract more of the bioactive phytochemical component of the plant compared to aqueous solvent, hence the reason for higher antibacterial activity in the ethanolic fraction of the leaf extract [18]. okpoho et al.; aji, 1(1): 36-43, 2018; article no.aji.46581 40 table 1. zone of inhibition of aqueous extract of bryophyllum pinnatum (mm) against fungal isolates test organism concentrations (mg/ml) 100 50 25 12.5 6.25 penicillium italicum 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 cladosporium herbarium 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 candida albicans 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 aspergillus flavus 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 aspergillus niger 11.33±0.33 6.0±0.58 0.0±0.0 0.0±0.0 0.0±0.0 table 2. zone of inhibition of ethanolic extract of bryophyllum pinnatum (mm) against fungal isolates test organisms concentration (mg/ml) 100 50 25 12.5 6.25 penicillium italicum 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 cladosporium herbarium 20.33±0.33 15.67±0.67 12.33±0.33 12.0±0.58 11.67±0.67 candida albicans 19.0±0.58 18.0±0.58 14.67±0.33 0.0±0.0 0.0±0.0 aspergillus flavus 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 aspergillus niger 14.0±0.58 9.33±0.33 0.0±0.0 0.0±0.0 0.0±0.0 table 3. minimum inhibitory concentration and minimum fungicidal concentrations of ethanolic and aqueous extracts of b. pinnatum test organisms mic(mg/ml) mfc (mg/ml) ethanol aqueous ethanol aqueous penicillium italicum nd nd nd nd cladosporium herbarium 6.25 nd 25 nd candida albicans 25 nd 50 nd aspergillus flavus nd nd nd nd aspergillus niger 50 50 100 100 ndnot determined table 4. effect of ethanol extract of b. pinnatum on haematological parameters of rat groups parameters pcv(%) hb(g/dl) a b 42.6 ± 0.93 b 14.16±0.31 b b 42.2±1.16 b 14.0±0.39 c b 43.0±1.70 b 14.3±0.53 d b 44.2±0.58 b 14.62±0.24 e b 42.8± 0.86 b 142.24± 0.29 f a 39.0± 0.71 a 12.94±0.21 letters a-e stands for the groups of organism administered 300, 250, 200, 150 and 100 mg/kg of the ethanolic extract respectively while group f is the control. groups with similar superscript as control has no significance while groups with different superscript describes the significance at p <0.05 table 5. immuno-modulatory effects of ethanol extract of b. pinnatum on white blood cells proliferation parameters groups a b c d e f wbc(×10 3 ) 6.04±0.12 b 5.16±0.25 a 5.52±0.24 a 4.90±0.25 a 4.88±0.21 a 4.96±0.10 a neut(%) 44.8±3.69 a 36.2±1.53 a 36.4±9.18 a 43.4±1.86 a 33.3±1.48 a 39.2±4.40 a lymp(%) 49.2±3.15 a 59.2±1.24 a 57.4±10.78 a 52.2±1.88 a 62.8±2.67 a 53.2±3.02 a platelet(×10 3 ) 105.8±4.10 a 105.8±4.60 a 126.2±5.40 c 103.0±3.38 a 95.4±1.86 a 108.6±4.19 b cd4(cells/ml) 81.6±2.84 a 95.8±2.08 a 99.0±6.33 a 86.2±4.59 a 91.6±4.07 a 75.4±19.32 a letters a-e stands for the groups of organism administered 300, 250, 200, 150 and 100 mg/kg of the ethanolic extract respectively while group f is the control. groups with similar superscript as control has no significance while groups with different superscript describes the significance at p <0.05 okpoho et al.; aji, 1(1): 36-43, 2018; article no.aji.46581 41 many of the fungi species were resistant to the all concentrations of the aqueous plant extract. the only sensitive fungus was aspergillus niger. this resistance may have resulted from the inability of the plant extract to penetrate the cell wall of the fungal isolates. comparatively higher antifungal activities were observed in the ethanol extract. only penicillium italicum and aspergillus flavus were completely resistant to all concentrations of the plant extract. minimum inhibitory concentrations of ethanol extract was lower than that of the aqueous extract indicating a higher efficacy of the ethanol extract. similarly, minimum fungicidal concentrations of ethanol extract was also lower than that of aqueous extract. zakharchenko et al. [19] isolated the antimicrobial peptide cecropin p1 from bryophyllum pinnatum and reported its fungicidal activity against dermatomycoses and even in treatment of wound infected with fungi. in their work, the antifungal activity observed against candida albicans agrees with findings from this work as the ethanol extract of the plant was active against candida albicans in this work. however, there was no in vivo study in this work compared to their work. there were changes in haematology parameters in experimental animals administered with ethanol extract of bryophyllum pinnatum, compared to the control group. observable changes between experimental and control animals are indication that ethanol extract of the plant has modulatory propensity on haematological parameters. ethanolic extract of brophyllum pinnatum has been shown in this work to increase the pcv and haemoglobin levels. no significant variation was observed between white blood cells proliferation of experimental animals and control group. increase in haemoglobin enhances the oxygen transporting properties of the red blood cells as a result of increased number of red cell. the observed increase in the pcv recorded in this study on administration of extract of bryophyllum pinnatum may have been due to effect on bone marrow stem cell by improving its proliferative activity [20]. there was a significant difference in platelet numbers between treated and control groups. the reduction in platelet counts suggests that continuous intake of this extract needs to be checked. there was no significant difference in white blood cell (wbc) parameters between treated and control group except for group a that received highest concentration of the extract (300mg/kg body weight). this means that the ethanolic extract of b. pinnatum may increase the wbc at higher concentrations. no significant difference was observed in the cd4 counts of treated and control groups. this could also be due to the concentration of the extract used as higher concentration may register significant increase. results showed that ethanolic extract of leaf caused increased haematological parameters that help in primary and secondary clearance of invading pathogens. this is in line with the work of [18] who reported that crude methanolic leaf extract of b. pinnatum has properties that increase the haemoglobin, packed cell volume and total white blood cells while decreasing the platelets. however, in contrast to their findings, the amount of platelet was found to increase at extract concentration 200mg/kg of experimental animals. in a research findings by [21], aqueous extract of bryophyllum pinnatum elevated white blood cell count, reduced neutrophil count without affecting lymphocyte count and packed cell volume, when compared to control, thus agreeing with the findings from this study that the extract possesses immunomodulating potentials. the phytochemical constituents of the leaf extract of b. pinnatum may have stimulatory effect on the bone marrow for leucocyte proliferation and haemoglobin production. this may be as a result of tannin, ascorbic acid [6] and phenolic content. other phytochemicals that may have affected the haematological parameters in this study include flavonoids, zinc, riboflavin and niacin [22]. 5. conclusion this work has shown that bryophyllum pinnatum ethanol and aqueous extracts have moderate antifungal activity. however, the ethanol extract was more active compared to the aqueous extract. furthermore, moderate immunemodulatory activity was observed in wistar rat models with an increase in platelet count, haemoglobin and pcv. b. pinnatum has been shown to possess hematological properties in this study. ethical approval ethical approval for the experimental protocol was obtained from the university of benin ethics committee and care of animals was taken as per guidelines of committee for the purpose of control and supervision on experiments on animals (cpcsea) (with reference number: fls/17/101). http://www.sciencedomain.org/journal/86/authors-instruction#sdi-ethical-approval okpoho et al.; 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(family malvaceae) in a mouse model. phytother res. 2008;22:664–668. https://www.ncbi.nlm.nih.gov/pubmed/?term=yadav%20m%5bauthor%5d&cauthor=true&cauthor_uid=28446830 https://www.ncbi.nlm.nih.gov/pubmed/?term=gulkari%20vd%5bauthor%5d&cauthor=true&cauthor_uid=28446830 https://www.ncbi.nlm.nih.gov/pubmed/?term=wanjari%20mm%5bauthor%5d&cauthor=true&cauthor_uid=28446830 https://www.ncbi.nlm.nih.gov/pmc/articles/pmc5382824/ okpoho et al.; aji, 1(1): 36-43, 2018; article no.aji.46581 43 21. aprioku js, igbe i. effects of aqueous bryophyllum pinnatum leaf extract on haematological, renal and sperm indices in wistar rats. indian journal pharmaceutical science. 2017;79(4):521526 22. salahdeen hm, yemitan ok. neuropharmacological effects of aqueous leafextract of bryophyllum pinnatum in mice. african journal of biomedical research. 2006;9:101-107. _________________________________________________________________________________ © 2018 okpoho et al.; this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: http://www.sdiarticle3.com/review-history/46581 http://creativecommons.org/licenses/by/4.0 _____________________________________________________________________________________________________ *corresponding author: e-mail: pkorisadiran@pgschool.lautech.edu.ng; asian journal of immunology 2(1): 1-10, 2019; article no.aji.52086 assessment of post exposure of benzene on some hematology parameters and dna lesions on adult wistar rats orisadiran patrick kunle1*, adedeji adebayo lawrence1, adedeji abiodun oluwabusola2 and abdulrahman abdulfatai3 1 department of biochemistry, ladoke akintola university of technology, ogbomoso, nigeria. 2department of anatomy, olabisi onabano university, ago iwoye, nigeria. 3 department of anatomy, ladoke akintola university of technology, ogbomoso, nigeria. authors’ contributions this work was carried out in collaboration among all authors. author opk designed the study and wrote the first draft of the manuscript. author aao performed the statistical analysis. author aal wrote the protocol, author araf managed the analyses of the study. all authors read and approved the final manuscript. article information editor(s): (1) dr. jaffu othniel chilongola, department of biochemistry and molecular biology, kilimanjaro christian medical university college, tumaini university, tanzania. reviewers: (1) raquel salazar-lugo, universidad de oriente, venezuela. (2) byron baron, university of malta, malta. complete peer review history: http://www.sdiarticle4.com/review-history/52086 received 08 august 2019 accepted 16 october 2019 published 22 october 2019 abstract aims: the study sought to investigate the effect of post-exposure of benzene on some haematology parameters and dna lesions on adult wistar rats. methods: a total of twenty-eight rats were grouped into 4 groups, with group 1 serving as a control. the remaining 3 groups were interperitoneally administered 0.2 ml of benzene 48 hourly for 4 weeks and were left of 0, 3 and 6 weeks respectively before sacrifice. results: the result obtained showed that white blood cell (wbc), red blood cell (rbc) and platelet counts were significantly reduced in benzene post exposed groups (p<0.05). 8hydroxydeoxyguanosine generation in liver and bone marrow were significantly higher in benzene post exposed groups. the photomicrograph of blood film of benzene exposed group showed promyelocyte and myeloblast. original research article orisadiran et al.; aji, 2(1): 1-10, 2019; article no.aji.52086 2 conclusion: it is concluded that benzene and its metabolite are highly toxic and are potentially damaging agents to the hematopoietic system. evidence from our study suggested that elevated levels of 8-ohdg in liver and bone marrow compare to control would be a sign of increased oxidative stress, impaired antioxidant defence or inadequate repair of oxidative damaged dna. keywords: interperitoneally; hematology; 8-hydroxydeoxyguanosine; promyelocyte; myeloblast; benzene. 1. introduction benzene is an aromatic hydrocarbon and a component of crude oil and gasoline. it is a widely used chemical formed from both natural processes and human activities and a ubiquitous contaminant in the environment [1]. occupational exposure to benzene occurs through solvent exposure in the chemical industry, in petroleum refineries, oil pipelines, on ships and tankers, bus garages and auto repair shops [2]. human exposure to benzene occurs in diverse ways such as through dermal absorption, inhalation, ingestion of contaminated food and water, and active and passive tobacco smoking [3]. benzene exposure causes many adverse effects, such as skin irritation, inflammation of the nasal airways and throat, central nervous system depression, immunotoxicity and hematoxicity [4]. the cells of the hematopoietic system and bone marrow are the most sensitive target organs of benzene toxicity. repeated occupational and non-occupational exposure to benzene over long periods may affect several hematopoietic parameters such as red blood cell, white blood cell and platelet counts [5] and eventually induce toxicity of the blood and blood forming organs. benzene exposure is associated with increased risk of acute myeloid leukemia [6], multiple myeloma and non-hodgkin’s lymphoma. studies indicate that exposure to benzene produce toxicity and an increased risk of malignancies of the blood and blood-forming organs [7]. benzene toxicity is linked to bone marrow depression, aplastic anemia, acute myeloblastic leukaemia and acute non-lymphocytic leukemia [8] and even at low doses can affect white blood cell and platelet counts [9]. this research was designed to study the immunotoxicity and hematotoxicity of post exposure to benzene in adult rats of wistar strain. 2. materials and methods 2.1 chemicals and reagents citric acid, sodium citrate, sodium chloride and tris-hydrochloric acid were products of british drug house (pool, england). benzene was obtained from sigma, st. louis mo while 8hydroxydeoxyguanosine assay kit was obtained from cloud-clone corp, usa. the total protein kit was obtained from fortress diagnostics limited (muckamore, united kingdom). all the chemicals and reagents were of analytical grade and were used and stored according to the manufacture’s instructions. 2.2 preparation of reagents 2.2.1 citrate buffer 21.01 g of citric acid and 29.41 g of sodium citrate were dissolved in 1 liter of distilled water each. 9.5 ml of citric acid solution was added to 41.5 ml of sodium citrate solution and was made up to 100 ml and the ph was adjusted to 6.0 and stored at 4°c. 2.2.2 homogenizing buffers 7.86 g of 50 mm tris-hcl and 11.2 g of potassium chloride was dissolved in 900mls of distilled water and the ph was adjusted to 7.4. the solution was made up to 1 l and stored at 4°c. 2.3 experimental design a total of 28 albino rats of wistar strain, with an average weight of 190 g were obtained from the animal house of physiology department, ladoke akintola university of technology, nigeria. the animals were divided into 4 groups and housed in separate cages in the same environment. the animals were allowed to acclimatize in the laboratory for two weeks before the commencement of the experiments. benzene was administered to wistar rats by interperitoneally injection of 0.2 ml of benzene solution, given every 48 hours for 4 weeks. the animals were randomly selected into 4 groups, each containing 7 rats. group i: rats in this group served as the negative control. group ii: rats in this group orisadiran et al.; aji, 2(1): 1-10, 2019; article no.aji.52086 3 were administered benzene for 4 weeks and were sacrificed immediately (0 week) after administration. group iii: rats in this group were administered benzene for 4 weeks and were left for 3 weeks before sacrifice. group iv: rats in this group were administered benzene for 4 weeks and were left for 6 weeks before sacrifice. 2.4 collection of blood and organs the rats were fasted overnight and sacrificed according to the experimental protocol. blood was collected by cardiac puncture. part of the blood was collected inside plain sample bottles (without anticoagulant), allowed to coagulate and then centrifuged at 3000 rpm for 15 minutes to obtain serum. the serum was kept under refrigeration at 4°c for biochemical parameters. the other part of the blood was collected into vacuum edta bottles, to be used for haematological parameters. 2.5 tissue preparation the livers of the rats were excised, washed with normal saline solution and weighed. 1 g of the washed liver was homogenized in 5 ml homogenizing buffer (7.86 g of 50 mm tris-hcl and 11.2 g of potassium chloride dissolve in 900ml of distilled water, ph 7.4) using mortar and pestle. the homogenates were centrifuged at 3000 rpm for 15 minto obtain the clear supernatant. the resulting homogenates were kept at 4ºc for biochemical parameters. 2.6 preparation of bone marrow aspirate the femur and humorous bone of the sacrificed experimental mice were dissected and both the proximal and distal ends were removed by using the modified method of saha, et al. [10]. briefly, citrate buffer solution of ph 6.0 was injected gently into one end of the shaft. this process was followed to flush out the bone marrow through the opposite end into eppendoff tube to prepare and centrifuge at 3000 rpm for 15 minutes to obtain the clear supernatant and pellet and were kept at 4°c for biochemical analysis. 2.7 determination of haematological parameters haematological parameters were assessed by flow cytometry (direct current method) using suitable cell packs according to the manufacturer’s specification for the desired cell population on the symex kx-21n autoanalyzer. 2.8 biochemical examination the concentration of 8-hydroxyldeoxylguanosine (8-ohdg) in liver homogenate, bone marrow cell and bone marrow supernatant were measured using immunoenzymatic colorimetric method of diametra, italy (2008) and total protein concentration was determined in the serum and tissue homogenates by the biuret (colorimetric) method [11] using fortress total protein kit. 2.9 statistical analysis results are presented as mean ± sd. paired student’s t-test was used to compare variations amongst groups. the minimum level of significance was considered at p˂0.05. statistical analysis was carried out using a software program (graphpad prism ver. 5; graphpad software, san diego, ca). 3. results 3.1 effects of post benzene exposure on haematological parameters there was a significant decrease (p<0.05) in red blood cell counts of groups post exposed to benzene at week 0 (8.19 x 103 mm3), week 3 (7.38 x 10 3 mm 3 ) and week 6 (5.02 x 10 3 mm 3 ; figure 1) compared to the control (11.3 x 103 mm3). similarly, there was a significant (p<0.05) decrease in white blood cell counts of groups post exposed to benzene at week 0 (9.85 x 10 9 /l), week 3 (4.46 x 10 9 /l) and week 6 (4.42 x 103 mm3; fig. 2) compared to the control (12.0 x 10 9 /l). platelet counts were significant (p<0.05) decrease in groups post exposed to benzene at week 0 (636 x 109/l), week 3 (563 x 109/l) and week 6 (510 x 10 9 /l) (fig. 3) compared to the control (981 x 109/l) 3.2 effect of post benzene exposure on 8-hydroxydeoxyguanosin (8-ohdg) concentration there was a significant increase (p<0.05) in liver 8-hydroxydeoxyguanosin concentration of group post exposed to benzene for 0 (0.61 ng/mg protein), 3 (0.59 ng/mg protein) and 6 (0.35 ng/mg protein) weeks respectively (fig. 4) compared to the control. production of liver 8ohdg reduced during the post-exposure period; this it decreased between 4% and 43% from initial week to third week and third week to sixth week, respectively. there was also significant (p<0.05) increase in generation of 8-ohdg by orisadiran et al.; aji, 2(1): 1-10, 2019; article no.aji.52086 4 bone marrow cell in groups post exposed to benzene for 0 (0.29 ng/mg protein), 3 (0.46 ng/mg protein) and 6 (0.71 ng/mg protein) weeks (fig. 5) compared to the control (0.1651 ng/mg protein). the generation of 8-ohdg by bone marrow cell in groups exposed benzene increase during post-exposure period, (between 37% and 58% at week 3 and week 6, respectively). similarly there was a significant (p<0.05) increase in 8-ohdg concentration in bone marrow supernatant groups post exposed to benzene for 0 (3.75 ng/mg protein), 3 (3.90 ng/mg protein) and 6 (4.50 ng/mg protein) weeks respectively, compared to the control (0.242 ng/mg protein; fig. 6). 3.3 effect of benzene exposure on blood film the histological study on blood film and bone marrow smear revealed different morphological changes in blood cells structures. the photomicrograph of blood film of benzene exposed group showed promyelocyte and myeloblast compared to normal control, promyelocyte (10-20 m) is slightly larger than a blast. its nucleus, although similar to a myeloblast shows slight chromatin condensation and less prominent nucleoli. the cytoplasm contains striking azurophilic granules or primary granules. these granules contain fig. 1. effects of post treatment on red blood cell counts each value is mean ± sd. bars with different alphabets ( a, b, c, d ) are significantly different from each other at p<0.05 fig. 2. effects of post treatment duration of benzene on white blood cell counts each value is mean ± sd. bars with different alphabets (a, b, c, d) are significantly different from each other at p<0.05 orisadiran et al.; aji, 2(1): 1-10, 2019; article no.aji.52086 5 fig. 3. effects of post treatment duration of benzene on platelet counts each value is mean ± sd. bars with different alphabets ( a, b, c, d ) are significantly different from each other at p<0.05 fig. 4. effects of post treatment duration of benzene on liver 8-ohdg concentration each value is mean ± sd. bars with different alphabets (a, b, c, d) are significantly different from each other at p<0.05 myeloperoxidase, acid phosphatase, and esterase enzymes. normally no promyelocytes are seen in the peripheral blood. myelocytes (1018m) are slightly smaller than promyelocytes and have eccentric round-oval nuclei, often flattened along one side. the chromatin is fine but shows evidence of condensation. nucleoli may be seen in early stages but not in the late myelocyte. the photomicrograph of blood films of the exposed group also shows hypersegmented neutrophil compared to normal control. neutrophils are generally the most abundant white cell. they are round, are 10–14 µm wide, and contain a lobulated nucleus with two to four lobes connected by a thin chromatin thread. 4. discussion a study has revealed that blood is the most important tissue in the body by which metabolic changes are reproduced [12], any changes in blood parameters and indices have always been considered to be the most reliable indicator of toxic effects of any substance, such as drugs and heavy metals. changes in some haematological orisadiran et al.; aji, 2(1): 1-10, 2019; article no.aji.52086 6 parameters and indices were recorded in rats post expose to benzene. the principal function of wbcs as phagocytes is to defend the body against invading microorganisms or is xenobiotic by ingesting and destroying them and, hence, contributing to cellular mechanism [13]. the decrease in total wbc suggests a decrease in immune system in fighting foreign substances [14]. these findings were consistent with studies in which leucopoenia was shown to be the most sensitive effect on blood cells associated with benzene exposure [15,16]. a similar conclusion was reported by tsai, et al. [17] in a large study on 1,200 employees exposed to benzene. the mechanism underlying reduction in wbc is direct cytotoxicity of benzene metabolites on the production of wbc in the bone marrow. benzene is known as immunotoxic and blood cell carcinogen, inducing anaemia, and blood formula modification [18]. fig. 5. effects of post treatment duration of benzene on bone marrow cell 8-ohdg concentration each value is mean ± sd. bars with different alphabets ( a, b, c, d ) are significantly different from each other at p<0.05 fig. 6. effects of post-treatment duration on bone marrow supernatant 8-ohdg concentration each value is mean ± sd. bars with different alphabets ( a, b, c, d ) are significantly different from each other at p<0.05 orisadiran et al.; aji, 2(1): 1-10, 2019; article no.aji.52086 7 fig. 7. photomicrograph of blood film of rat showing normal neutrophil (nn), hypersegmented neutrophil (hn), promyelocyte (pm) and myeloblast (mb) (leishman stain x1000) key: nn (normal neutrophil), hn (hypersegmented neutrophil), pm (promyelocyte), mb (myeloblast) the rbc values showed a significant reduction in benzene post expose groups. the rbc reduction was duration dependent. this was consistent with the study of sun, et al. [19] who reported a decrease in hematopoietic stem cells, reduced burst-forming unit erythroid (bfu-e), committed progenitors granulocytes-erythroid-monocyte-megakaryocyte (cfu-gemm) and granulocytemacrophages (cfu-gm) progenitors in mice with subchronic benzene exposure. this result indicated that significant hematotoxicity was successfully induced by benzene exposure rat within 0, 3 or 6 weeks of post-exposure. the observed reduction in rbc may be attributed to the cytotoxic effects of benzene. oxidative stress may be induced by benzene with its effect on red cell membrane, this could possibly have accounted for the susceptibility of the red cell membrane to oxidative attack giving way to hemolysis. in studies carried out by shakirov and farkhutdinov [20], exposure or contact with chemicals in the oil-refining industry have been established to caused alterations in the red cell adenyl and blood monooxygenase system. they suggested that such an effect could alter the integrity of the red cell membrane to cause cellular haemolysis. therefore, the result of this study agreed with their claim. this studied showed decreased in platelet counts in benzene post exposed rats. megakaryocytes are large bone marrow cells which are responsible for platelet production [21]. suppression of megakaryocytes or bone marrow suppression in benzene exposed rats decreased the production of platelets leading to thrombocytopenia. the severity of suppression is directly proportional to post-exposure duration in benzene exposed rats in the present study. low platelet count observed in benzene exposed rats suggested that the process of clot-formation will orisadiran et al.; aji, 2(1): 1-10, 2019; article no.aji.52086 8 be prolonged resulting in excessive loss of blood in the case of injury as reported by gresele et al. [21]. reduction in the values of rbc, wbc and platelet content as recorded in this study is suggestive of anaemic conditions which agree with the report of eyong, et al. [22] on the hematotoxicity of petroleum product. the haemopoietic system, in response to this likely anaemic condition, may have flooded the system with reticulocytes which can carry oxygen to meet the body's demand as earlier stated. benzene is among other toxicants, suppresses the immune system, and causes disruption or suspension of haematopoiesis [23]; which corroborate the results in this study. it has also been established that the toxic constituents of benzene are activated in the bone marrow, where these substances exert cytotoxic effects that could be mediated through disturbance in dna function [24]. the resultant bone marrow depression is characterized by inadequate production of red cell and other formed elements. this is in line with the findings in this study, as post exposure to benzene showed a significant reduction of rbc from the control value. when dna is oxidized, a number of damage products such as base damage, sugar damage, and protein-dna crosslinks are formed [25]. one of these products, 8-ohdg, has been proposed to be an indicator of oxidative damage in dna both in vitro and in vivo. our results showed a significant increase in liver and bone marrow 8ohdg levels compared to the control. this may be largely attributed to a high number of mitochondria and higher metabolism of liver cells [26]. also, higher production of ros in the liver cells is associated with a high density of mitochondria [27]. the quantity of 8-ohdg generated from dna was dependent on the 8ohdg levels in the dna, almost all 8-ohdg in the dna was released as 8-ohdg by rat liver homogenates. therefore, generation of 8-ohdg correlated with the degradation of dna. the majority of benzene metabolism occurs in liver where cytochrome p-450 oxidizes benzene to phenol, catechol, hydroquinone and benzoquinone [27]. these phenolic metabolites and muconic acid are then transported from the liver to bone marrow. high accumulation of benzene and its metabolites over a long period in the bone marrow resulted in increased dna lesion in benzene post exposed group. furthermore, the chronic and progressive nature of benzene hematotoxicity suggests genomic reprogramming that would result in aberrant gene expression inducing extensive apoptosis in bone marrow resulting to increase generation of 8-ohdg [28] as observed during the posttreatment duration of benzene in the study 5. conclusion the present study has provided insight into the hematotoxicity of benzene and the degree of alteration of the integrity of haematological responses. evidence from our study suggested that elevated levels of 8-ohdg in liver and bone marrow compare to control would be a sign of increased oxidative stress, impaired antioxidant defence or inadequate repair of oxidative damaged dna. consent it is not applicable. ethical approval the protocol of the study was approved by the local ethical committee for animal experimentation of the faculty of basic medical sciences, ladoke akintola university of technology, nigeria. acknowledgement we appreciate sharphor enterprises and bolab laboratories for their support throughout the period of this research. competing interests authors have declared that no competing interests exist. references 1. schettgen t, ochsmann e, alt a, kraus t. a biomarker approach to estimate the daily intake of benzene in non-smoking and smoking individuals in germany. j expo sci environ epidemiol. 2009;20:427433. 2. weisel cp. benzene exposure: an overview of monitoring methods and their findings. chem biol interact. 2010;184:5866. 3. goldstein bd, shalat sl. the causal relation between benzene exposure and multiple myeloma. blood. 2000;95(4): 1512–4. orisadiran et al.; 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this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: http://www.sdiarticle4.com/review-history/52086 _____________________________________________________________________________________________________ *corresponding author: e-mail: oluohauche@yahoo.com; asian journal of immunology 1(1): 20-29, 2018; article no.aji.44878 a cross-sectional study on awareness, attitude and utilisation of immunisation services by mothers of under-five in elele, rivers state, nigeria kenechi a. uwakwe1, uche r. oluoha1*, anthony c. iwu1, chukwuma b. duru1, ernest nwaigbo2 and ijeoma n. alex-okedo3 1 department of community medicine, college of medicine, imo state university, owerri, nigeria. 2 department of community medicine, imo state university teaching hospital, orlu, nigeria. 3 department of community medicine, federal teaching hospital, abakaliki, ebonyi state, nigeria. authors’ contributions this work was carried out in collaboration between all authors. authors kau and uro developed the research question, questionnaire design, write up and general coordination. author aci managed the data analysis. author cbd did the literature review. authors en and inao collected the data. all authors read and approved the final manuscript. article information doi: 10.9734/aji/2018/44878 editor(s): (1) dr. wagner loyola, department of immunology, brazilian agricultural research corporation (embrapa) concordia, canada. (2) dr. jaffu othniel chilongola, department of biochemistry and molecular biology, kilimanjaro christian medical university college, tumaini university, tanzania. reviewers: (1) vinodkumar mugada, vignan institute of pharmaceutical technology, india. (2) alexandrina cardoso, escola superior de enfermagem do porto, portugal. complete peer review history: http://www.sciencedomain.org/review-history/27671 received 19 th september 2018 accepted 24 th november 2018 published 8 th december 2018 abstract introduction: approximately 6.2 million under-five children die globally on annual basis and immunisation having been recognised as the most successful and cost-effective public health intervention of the 20 th century regarding the number of deaths averted can help to prevent approximately 2 million of these deaths if coverage is optimal. objectives: to assess the awareness and attitude of mothers towards utilisation of immunisation services in elele, rivers state. methodology: it was a descriptive cross-sectional study conducted between april and july 2015. the study population comprised of mothers with at least one under-five-year-old child. stratified sampling technique was used, and data collection was with a pre-tested, semi-structured, original research article uwakwe et al.; aji, 1(1): 20-29, 2018; article no.aji.44878 21 interviewer-administered questionnaire. data analysis was done using ibm spss version 20. level of statistical significance was set at p-value ≤ 0.05. results: the average age of the respondents was 28.6 ± 5.3 years. most (89.3%) had at least a secondary level of education. respondents level of awareness of childhood immunisation was high (95.0%) and the major sources of information on immunisation were antenatal clinic (61.0%) and health workers (20.0%). respondents' knowledge of vaccine-preventable diseases (vpd) was highest with poliomyelitis (76.0%) while diphtheria at 34.4% was the least known. approximately seven percent (7.3%) of the respondents' children were unimmunized while 18.1% were partially immunised. tetanus toxoid utilisation was significantly associated with respondents' level of education (χ 2 = 9.44, p-value = 0.0240) while the odds of completing their children immunisation was higher if done in a hospital setting compared to home service {or (95% ci): 4.03 (1.09 – 14.95)}. conclusion: health education on some of the vpds by healthcare personnel is advocated for the community. greater efforts should be placed on strengthening routine immunisation as against supplemental immunisation since the former has a better immunisation completion rate. keywords: childhood; immunisation; mothers; utilisation; nigeria. 1. introduction approximately 6.2 million children under the age of five died globally in 2013 with sub-saharan africa contributing almost half (3 million) of these deaths [1]. the world health organisation had in 2009 estimated that if global vaccine coverage increased to 90% by 2015, then approximately two million deaths of children under the age of five would be prevented [2]. immunisation has been recognised as the most successful and cost-effective public health intervention of the 20 th century regarding the number of deaths prevented per year [3]. zangene et al. have also reported that childhood immunisation indirectly prevents infectious diseases in adults through herd immunity [4]. they found that the use of pneumococcal protein conjugate vaccine among children reduced the total number of invasive pneumococcal disease (ipd) cases and resulted in a 38% decrease in the rate of ipd among nonvaccinated elderly adults through herd immunity [4]. immunisation campaigns became more popular since 1988 when world health organisation (who) in conjunction with united nations children fund (unicef), rotary international, bill and melinda gates foundation and the united states centre for disease control and prevention (cdc) launched the polio eradication programme. immunisation campaigns against polio and measles have yielded tremendous results globally and in nigeria. global polio cases have been reduced from 350,000 in 1988 to 74 reported cases in 2015 (> 99% reduction) [5] likewise, and global measles deaths have decreased by 79% from an estimated 651,600 in the year 2000 to 134,200 in 2015 [6]. despite the success of expanded programme on immunisation (epi), such as eradication of smallpox and global lowering of the incidence of polio and measles; many vaccine-preventable diseases remain prevalent especially in developing countries [7]. child immunisation in nigeria is provided through routine immunisation and catch-up supplemental immunisation campaigns (also known as national immunisation days) organised across the country or sub-nationally in selected areas [8,9]. a fully immunized child in nigeria is expected to have received one dose of bacillus calmette-guerin (bcg) at birth or soon after, 3 doses each of diphtheria, pertussis and tetanus (dpt) and oral polio vaccines at 6, 10 and 14 weeks and one dose of measles vaccine at 9 months of age or thereabout [10,11]. yellow fever vaccination is also given at 9 months. vaccines introduced more recently and administered during the first year of life include hepatitis b, pneumococcus and rotavirus vaccines [9]. also, vitamin a is administered at 9 and 15 months [11]. as part of the polio eradication and endgame strategic plan, inactivated polio vaccine was introduced in the routine immunisation schedule in 2015 and nigeria participated in the april 2016 switch from trivalent to bivalent polio vaccine [12]. furthermore, given the introduction of the second dose of measles vaccine and other booster doses by countries, improved coverage of routine immunisation is expected in the second year of life and beyond as this provides opportunities to uwakwe et al.; aji, 1(1): 20-29, 2018; article no.aji.44878 22 catch up on any missed immunisation from the first year [11]. vaccine-preventable diseases account for about one in five child deaths in nigeria, amounting to over 200,000 deaths per year [13]. despite recent improvement, immunisation coverage in nigeria is still abysmally low. according to nigerian demographic and health survey of 2013, only 25% of children aged 12 – 23 months were fully vaccinated with bcg, measles and three doses each of dpt and polio vaccines [10]. specifically, 51% received bcg vaccination, 38% received the recommended three doses of dpt, 54% were fully vaccinated against polio, and only 42% received measles vaccine. twenty-one percent (21%) of nigerian children aged 12 – 23 months received no vaccination at all according to this survey [10]. given that immunisation is not 100% effective [14], this high level of under-immunised and unimmunized children will impact negatively on the herd immunity thereby significantly increasing the risk of infection for vaccinated children. asides operational factors relating to policies, vaccine funding, vaccine availability and health workers related factors, some researchers [7,15] have identified awareness, attitude and perception of parents/caregivers as major obstacles to high immunisation coverage. in spite of efforts directed at solving operational problems, immunisation coverage in nigeria has persistently remained unacceptably low [16,17], examining maternal factors that could impede utilisation of immunisation cannot be overemphasised. the aim of this study is therefore to assess the awareness and attitude of mothers towards immunisation services and their utilisation of the services in elele, rivers state. 2. materials and methods this was a cross-sectional descriptive study that was conducted in elele community in ikwerre local government area of rivers state between april and july 2015. the community has a total population of 20,620 according to the 2006 national population census and a projected population of 27,712 in 2016 using 3% annual growth rate. mothers with at least one child less than five years of age and who consented to participate in the study were included. mothers who refused to give consent despite adequate explanations were excluded from this study. sample size was determined using the cochrane sample size formula for single proportion in population greater than 10,000 people; n = zα 2 p(1p)/d 2 , where n is the minimum sample size, zα is the standard normal deviate at 95% confidence level (1.96), p is the proportion of fully vaccinated children from previous study (0.25) and d is the level of precision required, set at 0.05. the calculated sample size was 288. considering a potential non-response rate of 10%, the minimum sample size required is 317, however, 400 participants were enrolled for this study. stratified sampling technique was utilized for this study. respondents were stratified by those interviewed in their homes, hospitals, primary and secondary schools, university (madonna university, elele) and markets. a total of eighty eligible mothers were interviewed by trained interviewers in each of the five stratums on the first seen basis. data was collected using a questionnaire design adapted from 2013 nigerian demographic and health survey [10] and modified to suit the study environment following a pre-test. a semi-structured interviewer-administered questionnaire was used for this study. data collected from mothers included mother’s age, mother’s highest educational level, marital status and occupation. others were on awareness, knowledge, attitude and utilisation of immunisation services. ethical approval for this research was obtained from madonna university ethical review committee with reference number adm/e15/a vol. 111/816 and informed consents were given by eligible mothers. data entry and analysis were done using spss version 20. data were analysed as proportions of responses and results presented as tables and charts. association between variables was tested using the chisquare test and level of significance was set at p < 0.05. 3. results 3.1 sociodemographic profile of respondents the average age of the respondents was 28.6 ± 5.3 years and christianity (89.8%) was the dominant religion. the majority (96.3%) of the uwakwe et al.; aji, 1(1): 20-29, 2018; article no.aji.44878 23 participants were married and of igbo (39.8%) and ikwerre (38.8%) ethnic nationalities. fairly equal proportions were unskilled (32.5%), and skilled (31.0%) workers and majority (89.3%) had post-primary education (table 1). table 1. sociodemographic profile of respondents variable frequency n = 400 (%) age (years) 20 – 30 270 (67.5) 31 – 40 124 (31.0) 41 – 50 6 (1.5) mean age ± sd 28.6 ± 5.3 religion christianity 358 (89.7) islam 36 (9.0) others 6 (1.3) marital status single 10 (2.5) married 385 (96.3) divorced 1 (0.3) widowed 4 (1.0) ethnicity igbo 159 (39.8) ikwerre 155 (38.8) yoruba 23 (5.8) hausa 15 (3.8) others 48 (12.0) occupation housewife 92 (23.0) unskilled 130 (32.5) semi-skilled 54 (13.5) skilled 124 (31.0) educational status none 14 (3.5) primary 29 (7.3) secondary 238 (59.5) tertiary 119 (29.8) 3.2 respondents level of awareness of immunisation most of the respondents (95.0%) were aware of immunisation, but the majority (61.0%) do not know that there could be vaccination failure (table 2). the major sources of information on immunisation were antenatal clinic (61.0%) and health workers (20.0%), (fig. 1). 3.3 knowledge and attitude of respondents towards immunisation services most of the respondents believe that vaccination can prevent diseases in individuals and that immunisation is important (97.0% and 98.8% respectively). concerning knowledge of vaccinepreventable diseases (vpd), poliomyelitis was the one mothers had the most knowledge of (76.0%) while the least was diphtheria (34.4%). most of the respondents (98.3%) believed that immunising their children will help avert vpd and will be encouraging other mothers to immunise their children (99.2%) just as 75.2% do not think that reactions from vaccination are lethal and 98.5% believed that pregnant women should be vaccinated when necessary. table 2. respondents level of awareness of immunisation variable frequency n = 400 (%) aware of immunisation yes 380 (95.0) no 20 (5.0) awareness of vaccination failure yes 156 (39%) no 244 (61%) the most common reason for not fully immunising a child was ignorance (50.0%). others were fever/illness (27.0%) and fear of injection abscess (19.0%), (fig. 2). 3.4 utilization of immunisation services by respondents most of the respondents received tetanus toxoid during pregnancy (90.2%), had their under-fiveyear-olds immunised (92.7%) and had immunisation cards (92.0%). however, only 81.9% of respondents completed the immunisation schedule for their underfive-yearolds children. bcg (90.0%) and opv (77.3%) were the most common vaccines received by these children while the least common were dpt (22.3%) and yellow fever vaccine (46.5%). 3.5 effects of mothers’ educational status on selected parameters the uptake of tetanus toxoid vaccination during pregnancy is significantly associated with educational status of mothers (χ 2 = 9.44, p = 0.0240) just as mothers with some level of education have greater odds of ensuring completion of scheduled vaccination for their under 5 children compared to mothers without formal education through this failed to reach statistical significance (χ 2 = 2.02, p = 0.5690), table 5. uwakwe et al.; aji, 1(1): 20-29, 2018; article no.aji.44878 24 fig. 1. source of information on immunisation table 3. knowledge and attitude of respondents towards immunisation services variable frequency n = 400 (%) can vaccine prevent diseases in individuals? yes 388 (97.0) no 12 (3.0) is immunisation important? yes 395 (98.8) no 5 (1.2) knowledge of vaccine-preventable diseases (vpd) ** tuberculosis 256 (64.0) poliomyelitis 304 (76.0) whooping coughs 168 (42.0) diphtheria 137 (34.3) tetanus 239 (59.8) measles 275 (68.8) yellow fever 224 (56.0) will immunising your child help in averting vpd yes 393 (98.3) no 7 (1.7) will you be advising other mothers to immunise their children yes 397 (99.2) no 3 (0.8) can reactions from the vaccine kill yes 99 (24.8) no 301 (75.2) should pregnant women receive the vaccine yes 394 (98.5) no 6 (1.5) 3.6 effects of place of immunisation on completion of vaccination place of immunisation of under 5 children by their mothers has a statistically significant influence on the completion of vaccination (χ 2 = 9.69, p = 0.0080). mothers whose children were vaccinated in a hospital setting were 4 times more likely to complete the vaccination compared to those vaccinated at home (or = 4.03, p = 0.0369), table 6. 0% 6% 7% 6% 61% 20% source of information on immunisation (n = 400) mass media parents friends antenatal clinic health worker uwakwe et al.; aji, 1(1): 20-29, 2018; article no.aji.44878 25 fig. 2. reasons were given by some respondents for not fully vaccinating their children table 4. utilisation of immunisation services by respondents variable frequency n = 400 (%) received tetanus toxoid during pregnancy yes 361 (90.2) no 39 (9.8) children under 5 years of age immunised yes 371 (92.7) no 29 (7.3) have immunisation cards yes 368 (92.0) no 32 (8.0) place of immunisation at home 13 (3.5) hospital 131 (35.3) health centre 227 (61.1) completion of immunisation (n = 371) yes 304 (81.9) no 67 (18.1) vaccines received by under 5 children bcg 360 (90.0) opv 309 (77.3) dpt 89 (22.3) hbv 188 (47.0) pentavalent vaccine 216 (54.0) yellow fever vaccine 186 (46.5) measles vaccine 253 (63.3) 4. discussion this study describes the knowledge, attitude and utilisation of immunisation services by mothers of under-five children in elele, a suburban community in rivers state. it has been known that successful immunisation of children depends substantially on mothers’ existing knowledge and positive disposition [18]. 0% 27% 19% 50% 1% 3% reasons for not fully vaccinating their children (n = 67) fever / illness fear of injection abscess ignorance cost religious / cultural beliefs uwakwe et al.; aji, 1(1): 20-29, 2018; article no.aji.44878 26 table 5. effects of mothers’ educational status on selected parameters variable yes n =351(%) no n = 49 (%) χ 2 or (95% ci) p-value tetanus toxoid utilization none primary secondary tertiary 10(2.8) 29(8.3) 212(60.4) 100(28.5) 4(8.2) 0(0.0) 26(53.1) 19(38.8) 9.44 1.00 25.25(1.25 510.67) 3.26(0.95 – 11.15) 2.11(0.60 – 7.41) 0.02 0.04 0.06 0.25 completion of immunisation by children n = 329 (%) n = 71(%) none primary secondary tertiary 10(3.0) 23(7.0) 200(60.8) 96(29.2) 4(5.6) 6(8.5) 38(53.5) 23(32.4) 2.02 1.00 1.53 (0.35 – 6.65) 2.11 (0.63 – 7.06) 1.67 (0.48 – 5.80) 0.57 0.57 0.23 0.42 table 6. effects of the place of immunisation on completion of vaccination variable yes n = 304 (%) no n = 67 (%) χ 2 or (95% ci) p value place of immunisation home hospital health centre 9 (3.0) 118 (38.8) 177 (58.2) 4 (6.0) 13 (19.4) 50 (74.6) 9.69 1.00 4.03 (1.09 – 14.95) 1.57 (0.47 – 5.32) 0.01 0.04 0.47 almost ninety percent (89.3%) of mothers in the current study had at least a secondary level of education. this probably explained the high level of awareness (95%) of immunisation services by respondents in this study. the above average literate level of participants in this study could also be responsible for their high level of knowledge and attitude towards immunisation services. most mothers in this study believe that immunisation is important (98.8%) and can prevent vaccine-preventable diseases (98.3%). also, their knowledge of the different vaccinepreventable diseases (vpd) is above average except for pertussis (whooping cough) and diphtheria. the rarity with which these two vpd are seen in the study area presently could be the reason for the low awareness. respondents’ educational status is significantly associated with tetanus toxoid utilisation during pregnancy, and educated mothers have higher odds of completing their children immunisation schedule compared to mothers with no formal education. the significant impact of maternal education on the utilisation of immunisation services have also been noted by other authors. tagbo et al. in their study on mothers' knowledge, perception and practice of childhood immunisation in enugu, south eastern nigeria observed that educated mothers are more likely to immunise their children at an appropriate age as well as utilise supplemental immunisation campaigns [19]. kabir et al. had also noted that mothers with formal education were more likely to be aware of the need for childhood immunisation compared to those who had no formal education [20]. it could then be inferred that the more educated a population is, the higher the immunisation coverage. however, a study by manjunath and pareek in india found that literacy status did not significantly influence immunisation coverage rates [7]. most of the respondents in this study immunised their children (92.7%) and have immunisation cards (92.0%). however, only 81.9% completed the immunisation schedule for their children. thus, 7.3% of the respondent's children received no immunisation, and almost one-fifth (18.1%) were partially immunised. the percentage of fully vaccinated children is appreciably higher than the 55% reported for rivers state in the 2013 nigeria demographic and health survey (ndhs) [10]. the ndhs 2013 also reported that nearly 21% of nigerian children were unimmunized [10]. the differences could be due to immense socioeconomic, ethnic and cultural diversity of the country. tagbo et al. reported routine immunisation rejection rate of 4% in enugu [19] compliance to routine immunisation is generally high in the south eastern part of nigeria [10]. ignorance (50%), child’s febrile illness (27%) and fear of injection abscess (19%) were given as the uwakwe et al.; aji, 1(1): 20-29, 2018; article no.aji.44878 27 major reasons for not fully immunising a child in this study. vonasek et al. identified being fearful of side effects (46%), ignorance, disinterest or laziness (42%) and travel or financial constraint (18%) as the major reasons parents do not fully immunise their children in their study on childhood immunisation in rural uganda [21]. other workers in ethiopia and kenya had reported busy schedules of parents as the major reason for not completing their children immunisation schedule [22,23]. these discrepancies may reflect true differences in barriers to immunising children in the different study communities. it may also be a reflection of the different study designs used. the major sources of information on immunisation were from antenatal clinic (61%) and health workers (20%). adeyinka et al. in their study in igbo-ora in oyo state, south western nigeria also reported antenatal care (65.7%) and health educators (19.2%) as the major sources of knowledge about immunisation [24]. this underlines the need for continuous training and re-training of health workers concerning immunisation services as this has been shown to have a direct impact on knowledge, awareness and utilisation of immunisation services by mothers [25]. place of immunisation also has a statistically significant impact on the completion of immunisation in this study. we observed that mothers generally preferred to have their children vaccinated in a hospital setting rather than at home. some researchers have reported that mothers preference for immunising their children in hospitals is based on their belief that the child will be properly assessed before the vaccination [19]. another reason given by mothers for preference of hospital vaccination was that they believed that hospital staff were more competent compared to campaign vaccinators [19]. it has been reported that the rejection rate is higher for supplemental immunisation compared to routine immunisation [19]. these findings are important in policy formulation and suggest that greater attention is focused on the more acceptable routine immunisation. 5. conclusion given that antenatal clinics and health workers were the major sources of information on immunisation, there is a need to continually update the knowledge of these categories of persons on immunisation. the importance of regular public enlightenment on immunisation cannot be overemphasised given that the major reasons some mothers deny their children immunisations were ignorance and unfounded fears. lastly, since immunisation completion rate is better among children immunised in hospital settings in comparison to those immunised at home, greater attention should be given to routine immunisation as against supplemental immunisation. ethical approval and consent ethical approval for this research was obtained from madonna university ethical review committee with reference number adm/e15/a vol. 111/816 and informed consents were given by eligible mothers. competing interests authors have declared that no competing interests exist. references 1. unicef. level and trends in child mortality. the un inter-agency group for child mortality estimation; 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asian journal of immunology 3(1): 182-190, 2020; article no.aji.56867 stem cell therapy in treatment of noncommunicable diseases: possibilities and challenges nahian rahman1*, mussamat mahbuba sultana1, marium sultana1, susmita ghosh1, urme mostary1, priya saha1 and md. ruhul kabir1 1 department of food technology and nutrition science, noakhali science and technology university, sonapur-3814, bangladesh. authors’ contributions this work was carried out in collaboration among all authors. authors mrk and nr conceptualized the idea, analyzed updated evidence, compared it, conducted the study and prepared the manuscript and drafting. authors mms and ms helped in drafting process and comparison. all authors read and approved the final manuscript. article information editor(s): (1) dr. cynthia aracely alvizo báez, autonomous university of nuevo leon, mexico. reviewers: (1) nina drizr, national research center for hematology, russia. (2) adam husein, universiti sains malaysia, malaysia. complete peer review history: http://www.sdiarticle4.com/review-history/56867 received 05 march 2020 accepted 09 may 2020 published 18 may 2020 abstract over recent years stem cells have stood out as a promising tool for regenerative medicine, providing alternative therapeutic solutions for many non-communicable diseases. many clinical trials using stem cells or induced pluripotent stem cells are focused on the refit and regeneration of various tissues and organs in degenerative diseases, whose current treatment only succeeds in delay down the progression of the disease. this review summarizes several current clinical and nonclinical information on the use of embryonic stem cells (escs), induced pluripotent stem cells (ipscs) and mesenchymal stem cells (mscs) in various diseases. the aim of this review was to expand on the background and therapeutic potential of escs, mscs and ipscs whilst linking this to their use within disease therapy with a specific focus on diabetes, kidney disease and cardiovascular disease with future possibilities and challenges. also aimed to explain the benefits of transplantation with side effects shortly after transplant and later and interruptions, possibilities and challenges of transplantation. review article rahman et al.; aji, 3(1): 182-190, 2020; article no.aji.56867 183 keywords: regenerative medicine; stem cells; stem cell therapy; pluripotent stem cells; embryonic stem cells; mesenchymal stem cells. abbreviations escs : embryonic stem cells ipscs : induced pluripotent stem cells mscs1 : mesenchymal stem cells dpscs : dental pulp stem cells avn : avascular necrosis adscs : adipose stem cells hscs : hematopoietic stem cells afscs : amniotic fluid stem cells rps : renal progenitors cvd : cardiovascular diseases chd : coronary heart disease gvhd : graft-versus-host disease 1. introduction stem cells are undifferentiated cells of a multicellular organism, that can turn into specific cells, as the body needs them. stem cells have the potential to revolutionize tissue regeneration and engineering [1]. cells in the body have specific purposes, but stem cells are cells that do not yet have a specific role and can become almost any cell that is required. there are three main types of stem cells depending on source: embryonic stem cells (escs), induced pluripotent stem cells (ipscs) and mesenchymal stem cells (mscs) [2]. for the first time in 1981, researchers could isolate stem cells from mouse embryos. more accurate studies on the biology of mouse stem cells led to discovery of methods for separation of stem cells from the human embryo in 1998. another name of stem cell therapy is regenerative medicine, it’s promotes the repair response of diseased, dysfunctional or injured tissue using stem cells or their derivatives [3]. the origin and function of these stem cells varies but they all hold diagnostic and therapeutic potential. a potential route of treatment is using stem cells that have the capability of differentiating into healthy tissue, replacing any lost through disease manifestation and thus justifying the avoidance of lifelong expensive treatments [4]. stem cells provide new cells for the body as it grows and replace specialized cells that damaged or lost. they have two unique properties that, they can divide repeatedly to produce new cell, and they can change into the other types of cell that make up the body [5]. 2. classification based on potency stem cells can be classified based on potency, by the extent to which they can differentiate into different cell types. the four main classifications are totipotent, pluripotent, multipotent, or unipotent. totipotent: the ability to differentiate into all possible cell types. examples are the zygote and the first few cells that result from the division of the zygote [5]. pluripotent: the ability to differentiate into almost all cell types. examples are embryonic stem cells and cells that are isolated from the mesoderm, endoderm and ectoderm germ layers that are formed in the beginning stages of embryonic stem cell differentiation [6]. multipotent: the ability to differentiate into a closely related family of cells. examples include hematopoietic (adult) stem cells that can become red and white blood cells or platelets. unipotent: the ability to only produce cells of their own type. examples include (adult) muscle stem cells [7]. 3. classification based on their sources embryonic stem cells: embryonic stem cells are self-replicating cells that are potentially immortal [7]. they are derived from embryos at a developmental stage before the time of implantation occur in the uterus. they can change into any cell in the body, so these stem cells are said to be pluripotent [8]. adult stem cells: adult stem cells are undifferentiated totipotent or multipotent cells that are found in the body after embryonic development and multiply by dividing cells. the main role of adult stem cells in vivo is to preserve and improve the tissue in which they are found. they can replace blood stem cells (or “blood formation”) only with blood cells and various types of skin cells (or “epithelium”) [8]. there are a number of adult stem cell types have been isolated from dental tissues, known as dental pulp stem cells (dpscs) [9] and these cells exhibited differentiation potential into rahman et al.; aji, 3(1): 182-190, 2020; article no.aji.56867 184 odontoblastic, adipogenic and neural citotype; the same group isolated a similar citotype in deciduous teeth that have been called sheds (stem cells from human exfoliated deciduous) [10]. induced pluripotent stem cells: recently, a third type of stem cell has emerged that has properties like embryonic stem cells. scientists have created these induced pluripotent stem cells (ips cells) by controlling the expression of specific genes and reprogramming somatic cells into pluripotency [11]. they are also pluripotent and can develop into cells of any type [12]. 4. applications of stem cell therapy the goal of stem cell therapy is to treat damaged tissue that can’t heal itself. recent stem cell research often encourages patients who have not been treated for diseases in order to alleviate the symptoms of chronic diseases. stem cell therapy involves more than just implanting cells into the body and growing new healthy tissue. it may also be possible to gratify stem cells already in the body to work overtime and produce new tissue [13]. the growth of stem cells after implantation into host tissues or organs is influenced with several natural polymers employed as biologic scaffolds carry stem cells for tissue repair, including alginate, collagen, fibrin, albumin, hyaluronan, platelet-rich plasma, and gelatin [14]. diseases and conditions where stem cell treatment is being investigated include: nonunion/delayed union fracture, osteonecrosis or avascular necrosis (avn),knee cartilage defect, rheumatoid arthritis (ra), spinal cord injury, spinal fusion treatment, cerebral palsy, autism, motor neuron disease, multiple sclerosis, parkinson’s disease, alzheimer/ dementia disease, cerebellar atrophy, cerebellar ataxia, spinal muscular atrophy, down syndrome, optic nerve damage, retinitis pigmentosa, macular degeneration, dystrophy glaucoma disease, diabetes (type 1 & 2), acute/chronic liver disease, muscular dystrophy, acute/chronic kidney disease, peripheral arterial disease, myocardial infarction, lung disease, erectile dysfunction, antiaging treatment, scleroderma disease, skin replacement, male infertility, female infertility, breast cancer, scar, colon cancer, etc [15-17]. 5. possibilities in some most occurring non-communicable diseases 5.1 stem cell therapy for kidney disease kidney diseases are caused by damage to nephrons, which can be sudden and short lived called acute kidney disease or slow and progressive called chronic kidney disease. chronic kidney disease can lead to renal failure and is fatal if not treated. scientists are studying how the kidneys can be restored and the types of kidney cells involved in this process [18]. after variety of insults the kidney has the capacity for regeneration. the factors that caused kidney damage over the past few decades have been carefully studied. there is currently no fda approved stem cell therapy for kidney disease [19]. however, clinical trials are being conducted to determine whether stem cellbased kidney therapy is safe and effective in humans. scientists have already successfully created an artificial rat kidney that produces urine once transplanted into the animal, making artificial organ transplantation a highly possible reality for human kidney [17,20]. 6. types of stem cells investigated to regenerate damaged tissue significant advances have been made in promoting stem cell products as a possible treatment of kidney disease, restoring various types of stem cells from renal function in preclinical models of acute and chronic renal failure [21]. both preclinical reports and clinical trials of stem cells used for treatment kidney disease are increasing rapidly. different types of stem cells, ranging from mesenchymal stem cells (mscs), adipose stem cells (adscs), hematopoietic stem cells (hscs), amniotic fluid stem cells (afscs), renal progenitors (rps) and so forth, can stimulate renal repair in vivo in models of acute and chronic kidney failure [19]. the promise of stem cell therapies in pre-clinical models of kidney diseases is yet to be translated into more persuasive proof of clinical efficacy. several clinical trials have confirmed the safety and tolerability of stem cells, and of msc-based therapies, in patients with renal diseases and kidney transplants. however, long-term monitoring is recommended to rule out the rahman et al.; aji, 3(1): 182-190, 2020; article no.aji.56867 185 potential risk of cancer and of developing antihla antibodies [22]. 7. stem cell technology for the treatment of diabetes diabetes is a disease with high blood sugar. most of the glucose comes from the food we eat. the hormone used to transport glucose to the cell as energy is insulin. when our body cannot produce insulin, it is called type 1 diabetes. in type 2, glucose is not available due to insulin resistance [23]. in the past, numerous diabetes treatment technologies have been used, including increased insulin delivery and glucose monitoring systems, new methods for the complete transplantation of the pancreas and graft, and the formation of b cells from the pancreatic ducts or stem cells. now-a-days these two conditions of diabetes are treated by stem cell technology [24]. 8. types of stem cells investigated to regenerate damaged tissue stem-cell therapy means the replacement of diseased cell or missing cells from progeny of pluripotent or multipotent cells. many groups of cells are used to turn into beta cells of the pancreas, which produce insulin during differentiation. both embryonic stem cells (derived from the inner cell mass of a blastocyst) and adult stem cells (found in the postnatal organism) have been used to produce β-cells or otherwise restore the functioning of β-cell [25]. embryonic stem cells (esc): stem cells follow appropriate developmental pathway in order become insulin producing cells by using embryonic stem cells transfusing with insulin promoter, resulting insulin producing cells in mouse esc. which permitted them to make insulin producing cells [24]. induced pluripotent cells (ipsc): ips has high reproducibility and pluripotency. these cells can be divided into cells that produce insulin. so, we can also make the pancreatic beta cells form these cells that can be used for the treatment of diabetes [25]. mesenchymal stem cell (msc) therapy: stem cell therapy is an alternative to small islet cell transplantation in patients with type 2 diabetes. mesenchymal stem cell (msc) can be obtained from patients with autologous transplantation. however, autologous mscs from diabetic patients are still remarkably different from escs, because of prolonged exposure to hyperglycemia [26]. by using these different kinds of stem cell technologies, we can make the insulin producing cells that will be helpful in the cure of diabetes that is worldwide disease. type 1 diabetes can successfully treated by using β cell from stem cells where type 2 diabetes is treated by using β cell in combination with drug therapy [27]. 9. stem cells and cardiac repair cardiovascular diseases (cvd), hypertension, coronary heart disease (chd), stroke, and cardiac arrest (chf) were the leading causes of death. finally, stem cells can satisfy a large unmet clinical need and improve the quality of life of millions of people with cardiovascular diseases [28]. reliable evidence suggests that stem cells promise to be a treatment for damaged myocardial regeneration. ischemic heart failure occurs when there is a lack of oxygen in the tissues of the heart [29]. 10. types of stem cells investigated to regenerate damaged myocardial tissue adult and embryonic stem cells have been investigated to regenerate damaged myocardial tissue in animal models and in a limited number of clinical studies. specific considerations for the application of various cell types will be discussed in the following sections [30]. embryonic stem (es) cells: pluripotent es cells can produce various types of cells that repair damaged myocardial tissue, including myocardial cells, endothelial cells and smooth muscle cells. for this purpose, it has been shown that mouse and human es cells spontaneously differentiate in vivo to form endothelial muscle and smooth muscle cells in the body. human es cells differentiate into myocytes with the structural and functional properties of myocardial cells. moreover, es cells that were transplanted into is chemically injured myocardium in rats differentiated into normal myocardial cells that remained viable for up to four months suggesting that these cells may be candidates for regenerative therapy in humans [31]. 11. other diseases stroke: stroke triggers the loss of neurons and glial cells in large numbers. in the treatment of this disease, cell therapy opens fresh horizons by rahman et al.; aji, 3(1): 182-190, 2020; article no.aji.56867 186 facilitating the process of neuronal regeneration. animal studies and several preclinical studies verify the effectiveness of cell therapy in poststroke functional enhancement [3]. spinal cord injury: spinal cord injury is one of the serious neurological damage caused by neuronal tissue loss and consequently sensory and motor function loss. there is no therapy for this damage to be regenerated. this harm can be remedied by replacing stem or progenitor cells [32]. inflammatory bowel disease: crohn and ulcerative colitis were referred to as inflammatory diseases of the intestine. the precise cause of these illnesses is still unknown, but one of their causes is immune system dysfunction. because stem cells are immunoregulatory cells, and also because of their capacity to transdifferentiate and fuse cells, they appear to have a beneficial impact on improving these illnesses [33]. liver diseases: stem cell transplantation has now been proposed in the therapy of cirrhosis as a novel technique. for this purpose, various types of stem cells such as embryonic stem cells, mesenchymal stem cells, annex stem cells and endothelial progenitor cells were used in laboratory studies. also, laboratory studies have shown that primary hepatocytes can be replaced in liver, spleen, peritoneal cavity and other sites outside the liver [34]. 12. benefits of stem cell therapy with so many treatment options out there, you may be surprised what benefits stem cell therapy provides. some of the benefits include minimal risk, minimal recovery time and minimal worry. here are 5 more specific benefits to be aware of no need of surgery and avoid its risks and complications because it is an invasive, nonsurgical procedure, required less post-procedural recovery time, does not require the use of general anesthesia, there is no risk of rejection because this therapy uses the biologics extracts from the patient, no risk of communicable disease transmission [35]. the potential growth of stem cells relies on effective recruitment of host stem or progenitor cells into the implanted biomaterial scaffolds and induction of the infiltrating cells into tissuespecific cell lineages for functional tissue regeneration [36]. growth factors released from the scaffolds could remarkably prompt stem cell growth and differentiation, but most of these proteins cannot bind with scaffolds and so require a bridge to covalently crosslink scaffolds on one end and bind growth factors on another end. heparin, one such element, has high levels of sulfated anionic glycosaminoglycans that contain a growth factor binding domain [37] which allows heparin to bind growth factors with high affinity while retaining its biological activity. using heparin with growth factors controls their release keeps stem cells viable after transplantation into host tissues or organs [38]. 13. side effects of stem cell transplant after the transplantation many of the side effects are happens to the patient shortly after the transplant and later. these side effects are briefly described below: shortly after the transplants: many of the problems can happens shortly after the transplants this includesmouth and throat pain, nausea and vomiting, infection, bleeding and transfusions, interstitial pneumonitis, other lung problems, graft failure and graft-versus-host disease. when the body does not accept the new stem cells the graft fails. graft failure is more common when the patient and donor are not well matched and when patients get stem cells that have had the t-cells removed. graft failure can lead to serious bleeding and/or infection and graft-versus-host disease (gvhd) can happen in allogeneic transplants when the immune cells from the donor see the recipient’s body as foreign. doctors think of gvhd as acute or chronic. acute gvhd starts immediately after transplant and lasts a short time. chronic gvhd starts later and lasts a long time [39]. acute gvhd: the average time is about 25 days, but acute gvhd may occur between 10 and 90 days after transplantation. skin rash, redness of the skin, redness of the palm and initial signs. it can spread throughout your body. other symptoms include nausea, vomiting, abdominal cramps, diarrhea (moisture, sometimes bleeding), loss of appetite, yellowing of the skin and eyes (jaundice), abdominal pain (abdominal pain) and weight loss [40]. chronic gvhd: 90 to 600 days after the stem cell transplant, chronic gvhd can start anywhere. rash on the palms of the hands or the soles of the feet are the first signs. the rash can spread and is usually itchy and dry. in severe cases, the skin may blister and peel, like a bad rahman et al.; aji, 3(1): 182-190, 2020; article no.aji.56867 187 sunburn. other symptoms of chronic gvhd can include: enlarged liver, bloated abdomen (belly), pain in the upper right part of the abdomen (belly), increased levels of liver enzymes in the blood (seen on blood tests), the skin feels tight, dry burning eyes, dryness or painful sores in the mouth, burning sensations when eating acidic foods, bacterial infections, and blockages in the smaller airways of the lungs, etc [41]. problems that may show up later: organ damage, relapse (the cancer comes back), secondary (new) cancers, abnormal growth of lymph tissues, infertility (the inability to produce children), hormone changes, such as changes in the thyroid or pituitary gland, cataracts (clouding of the lens of the eye, which causes vision loss) [42]. 14. dietary concerns during stem cell transplant patients with stem cell transplantation are very sensitive to microorganisms that can be transmitted through food and beverages. to provide a more protected environment, transplant patients are maintained on a diet upon admission which is secure for the patient. the stem cell transplant diet restrictions continue until the patient’s anc (anc: the absolute neutrophil count, the number of white blood cells (wbcs) that are neutrophils) is greater than 500 for three consecutive days and if graft vs. host disease is not present [43]. patients who underwent autologous or allogeneic hsct(hematopoietic stem cell therapy) had more pronounced changes in diet acceptance and longer hospital stays, as well as a greater delay from transplantation until engraftment because they are at increased nutritional risk due to the underlying disease, high metabolic demand and complications related to the conditioning regimen, which mostly affect the gastrointestinal tract, and are thus able to cause symptoms, such as nausea, vomiting, mucositis, odynophagia, diarrhea, abdominal pain and constipation, that make the ingestion of food and absorption of nutrients worse [44,45]. when the appropriate transplantation occurs, the special diet is terminated. the special diet will be stopped when adequate engraftment occurs; however, it is a good idea to continue to use safe food handling and preparation even after the special diet restrictions have ended. diet guidelines should be maintained before and after therapy [46]. 15. interruptions, challenges and prospects though stem cells offer breathtaking commitment for coming therapies, significant technical hurdles remain. one of the major interruptions is that mass proliferation of stem cells to generate enough quantities of tissue is required for useful transplant purposes. secondly differentiated stem cells should be able to integrate into the surrounding tissue after transplantation and their functionality for the total duration of the patient's life is another area of concern along with the fears of immune rejection. uses of autologous adult stem cells and lifelong immunosuppressive medicine can reduce the harmful side effects. another major concern is the development of cancer after sct due to selfrenewal and uncontrolled proliferation capabilities of stem cells, in this way making them able to malignant transformation as cscs. another major complexity after allogeneic hematopoietic stem cell transplantation is graftversus-host disease is due to donor t-cell recognition of recipient alloantigen’s. autologous hematopoietic stem cell transplantation also exposes the development of a syndrome like allogeneic graft-versus-host disease causing serious disease in the gi tract [47]. though this field is still in its primary stage, scientists are hopeful to use ipscs in transplantation therapy. use of retroviral vectors to initiate transcription factors for reprogramming objective can be harmful itself with a raise risk of cancer and viral disease development therefore researchers are currently investigating non-viral allotment techniques. although the nature of the reprogramming method is still not surely clear and the developmental potential of ipscs derived from various tissues using various methods is unknown. another main challenge is to recognize adult or cancer stem cells in a tissue population [48]. many preclinical studies regarding sct have moved to clinical trials and translational stage. such as to promote recovery of the damaged heart after myocardial infarction (mi), stem cells have been delivered by intracoronary infusion. where, recent reviews of these clinical trials report that less than half of the trials found only small improvements in cardiac function. significant tasks yet remain to enhance the usefulness of stem cell therapy for cvd including improved identification, recruitment, and in vitro growth of autologous stem cells, besides rahman et al.; aji, 3(1): 182-190, 2020; article no.aji.56867 188 identification of mobilizing and homing agents, and development of novel culture conditions to improve stem cell survival and engraftment. other clinical trials are planned to address treatment options for diabetes and kidney diseases [49]. to date (08/04/2019), over 880 msc-based clinical trials, complete or ongoing, have been registered in the us national institutes of health database, including hematological diseases, graft versus host disease, diabetes, organ transplantation, inflammatory diseases, and diseases in the lung, liver, bone as well as cardiovascular, neurological and autoimmune diseases. most of the above clinical trials are carried out in early phases (phase i–ii), suggesting that the efficiency of sc treatment remains to be further investigated in the longterm [50]. 16. conclusion there is a lot of research on investigating how stem cells can be used to treat different types of diseases. it is expected that; stem cell therapy might be used to develop for many types of diseases in future for which there are no effective treatments now. the obtained pluripotent stem cells and adult stem cells are important candidates for regenerative therapy because of the nature of self-healing and the wide range of properties of pluripotency. stem cells have been tested for use in various diseases, such as diabetes, kidney disease, spinal cord injuries, heart disease, stroke and parkinson's disease, as well as various forms of blood diseases. where kidney disease has been studied, there is no proven treatment for this disease with stem cells because of its complex structure. on the other hand, type 1 diabetes can successfully treated by using β cell from stem cells where type 2 diabetes is treated by using β cell in combination with drug therapy. and for the patient with different heart disease stem cell therapeutics are very promising. as stem cell therapy has many benefits for treating disease it also has some side effects too. stem cells have a bright future for the therapeutic world according to different literature. we hope to see new horizon of therapeutics in the form of bone marrow transplant, skin replacement, organ development, and replacement of lost tissue such as hairs, tooth, retina and cochlear cells which will add new dimension to the treatment and healing process of ailing patients. consent it is not applicable. ethical approval it is not applicable. acknowledgements the authors acknowledge the support of department of food technology & nutrition science, nstu for the opportunity to conduct this research work. competing interests authors have declared that no competing interests exist. references 1. menendez ja, joven j. energy metabolism and metabolic sensors in stem cells: the metabostem crossroads of aging and cancer, in oxidative stress and inflammation in non-communicable diseases-molecular mechanisms and perspectives in therapeutics. springer. 2014;117-140. 2. marks pw, witten cm, califf rm. clarifying stem-cell therapy’s benefits and risks. n engl j med. 2017;376(11):10071009. 3. larijani b, et al. stem cell therapy in treatment of different diseases; 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asian journal of immunology 3(1): 259-268, 2020; article no.aji.57314 role of inflammatory cytokines in the pathogenesis of rheumatoid arthritis and novel therapeutic targets shaayau shehu1*, abdulaziz umar kurya2, usama aliyu1 and dinesh c. sharma2 1 department of biochemistry, usmanu danfodiyo university, sokoto, nigeria. 2 school of life and allied health sciences, glocal university, saharanpur, uttar pradesh, india. authors’ contributions this work was carried out in collaboration among all authors. author ss designed and supervised the study. author auk wrote the first draft of the manuscript, outline the protocol and performed the statistical analysis while authors ua and dcs managed the literature search and analyses of the study. all authors read and approved the final manuscript. article information editor(s): (1) dr. jaffu othniel chilongola, tumaini university, tanzania. reviewers: (1) s. tripathy, sri vasavi institute of pharmaceutical sciences, india. (2) ingrid patricia, dávalos rodríguez, méxico. complete peer review history: http://www.sdiarticle4.com/review-history/57314 received 06 april 2020 accepted 12 june 2020 published 29 june 2020 abstract cytokines are low molecular weight secreted proteins that mediate and regulate immune responses, inflammation and hematopoiesis; they act by autocrine, paracrine, endocrine and antagonistic mode of actions. cytokines have strong correlation with autoimmune disease, the most prominent among others is rheumatoid arthritis (ra) which targets synovial joints, and often accompanied by an array of extra-articular manifestations which are ultimately major predictors of increased morbidity and mortality, ra affect about 1% of the world population and about 0.6% of the american population with annual incidence estimated to about 40 per 100,000 individuals and mostly women. persistent inflammation endorsed by major pro-inflammatory cytokines such as interleukins (il-1, il-6) and tumor necrosis factors (tnf-α) and imbalance between the proinflammatory and anti-inflammatory cytokines is the main pathogenesis of ra. evidence suggests that interaction between antigen-presenting cells and cd4+ t helper cells is involved in the induction of inflammation in ra. continuous recruitment and activation of macrophages and review article shehu et al.; aji, 3(1): 259-268, 2020; article no.aji.57314 260 monocytes occur with the recruitment of pro-inflammatory cytokines, specifically tnf-α, il-1 and il-6 into the synovial cavity resulting to loss of cartilage and bone erosion. these activated immune cells stimulate angiogenesis, which explains increased vascularity found in the synovium of patients with ra. novel therapeutic targets are developed to minimize the morbidity rate, the use of tnf-α blockade drugs such as adalimumab, etanercept and infliximab is widely approved globally with more precise therapeutic effect on inflammation. as the il-1 plays a critical role in joint damage by facilitating the degradation of cartilage which leads to ra, the use of interleukin-1 receptor antagonist drugs such as anakinra suppresses ra by inhibiting the release of il-1, likewise, the use of anti-il-6 receptor agent such as tocilizumab has become a major resource for the treatment of ra. keywords: interleukins; rheumatoid arthritis; tumor necrosis factor; therapeutics. 1. introduction the concept of cytokines was described in 1974 after the discovery of interferon for the first time. cytokines are small secreted proteins with a molecular weight less than 30 kda (˂200 amino acids) that mediate and regulate immune responses, inflammation, and hematopoiesis they are released by varieties of cells such as leukocytes, produced de novo(starting from the beginning) in response to an immune stimulus and act by binding to the surface membranes of target cells to evoke a cascade of responsive reactions [1-2]. they bind to specific receptors on target cells, triggering signal-transduction pathways that ultimately alter gene expression in these cells to produce cytokines that are involved in innate, adaptive immunity, the gene products (proteins) participate in the cell proliferation, differentiation, migration, and apoptosis activities [3-4]. to be more precise, cytokines regulate the intensity and duration of the immune response by stimulating or inhibiting proliferation, differentiation, trafficking or emigration of lymphocytes all the while acting as a messenger for both arms of the immune system [5]. cytokines either stimulate or suppress the responses of various cells involved in host immune mechanisms, they assist such cells to interact or convey the essential messages for the proper functioning of defense systems against numerous pathogens or disease conditions. cytokines and their receptors exhibit very high affinity for each other as a result they can exert their biological effects even at very low concentrations [6]. according to the secretion of cytokines, they are classified in table 1. 2. materials and methods this literature review titled ‘role of inflammatory cytokines in the pathogenesis of rheumatoid arthritis and novel therapeutic targets’’ was conducted using online database of scientific collections, key articles used were retrieved precisely from ncbi (pmc, pubmed), google scholar, using the terms ‘rheumatoid arthritis, cytokines, and therapeutics of rheumatoid arthritis as keywords for our search. we included scientific publications from december 2004 to november 2018. articles focusing on clinical characteristics, epidemiology, pathogenesis, and treatments for rheumatoid arthritis were eligible for inclusion. 2.1 mode of cytokines action a particular cytokine might bind to receptors on the membrane of the same cell that secreted it and exert its biological effect, a condition which is referred as autocrine action [8].it can also bind to receptors on a target cell nearby and exert its biological effect which is known as paracrine table 1. shows different classes of cytokine and their function chemokine function ref. lymphokines cytokines that are secreted by t cells and regulate the immune response [7] proinflammatory cytokines that amplify and perpetuate the inflammatory process [7] anti-inflammatory cytokines that negatively modulate the inflammatory response [7] growth factors cytokines that promote cell survival and result in structural changes in the airways [7] chemokine cytokines which are chemotactic for inflammatory cells [7] shehu et al.; aji, 3(1): 259-268, 2020; article no.aji.57314 261 action [8]. besides, those cytokines which bind to target cells in distant locations and exert their biological effect are known to exert endocrine mode of action [8]. however the synergistic effect is seen when the combined effect of two or more cytokines is greater than the additive effect of individual cytokine [9]. another mode of action exhibit by cytokine is their ability to activate multiple signaling pathways thereby affecting the activity of multiple cells which is known as pleiotropic mode of action [9]. besides, redundant mode of action is also seen when multiple cytokines have the ability to exert similar function [9]. under some circumstance two or more cytokines may act on one cell and the effect of one cytokine inhibit or offset the effects of another cytokine [10]. 2.2 pro-inflammatory cytokines pro-inflammatory cytokines or simply inflammatory cytokines are special types of signaling molecules that are excreted from immune cells like t helper cells and macrophages and certain other cells that promote inflammation [11]. the most prominent among them are interleukins (il-1, il-6) and tumor necrosis factor (tnf-α and tnf-β) which play an important role in mediating immune response, excess production of inflammatory cytokines contributes to inflammatory diseases [11]. inflammatory cytokines are proved to be beneficial for their bactericidal capacity of phagocytes, recruit additional innate cell populations to sites of infection, induce dendritic cell maturation and direct the subsequent specific immune response to the invading microbes [12]. three pro-inflammatory cytokines, interleukin-1, interleukin-6, and tumor necrosis factor-α, appear to have a central role in tissue destruction and are secreted by a variety of cell types comprising monocytes, macrophages, dendritic cells, epithelial cells, keratinocytes and fibroblasts [12]. interleukin-1 (il-1) is one of the primary inflammatory cytokines that mediate many local and systemic features of inflammation, they are made mainly by monocytes and macrophages in response to a range of stimuli including various microbial products, viruses, immune complexes, activated t cells and combine the action of other cytokines such as interleukin-2 and interferongamma [13]. il-1 exist in two isoformil-1 α and β both of which are prototypic pro-inflammatory cytokines that exert pleiotropic effects on a variety of cells and play key roles in acute and chronic inflammatory and autoimmune disorders [13]. il-1α is a regulator of intracellular events and a local inflammatory mediator, while il-1β is primarily an extracellular protein released from cells [13]. il-1 also stimulates the proliferation of keratinocytes, fibroblasts and endothelial cells of the tissues, therefore il-1 is a critical component in the homeostasis of tissues and its unrestricted production may lead to tissue damage [14]. il-6 is produced locally in the inflamed tissues following cellular activation by bacterial lipopolysaccharide or other cytokines such as il1β or tnfα [14]. there are two il-1 receptors, il-1ri and il-1rii. il-1α and il-1β signal through il-1ri, binding to il-1rii does not lead to cell signaling and is therefore considered as a decoy receptor, upon binding of il-1 to il-1ri, a second receptor termed il-1 receptor accessory protein (il1racp) gets recruited at the cell membrane to form a high-affinity binding receptor complex leading to intracellular signalling [15]. a third il-1 family member, il-1 receptor antagonist (il1ra), binds to il-1 receptors and prevents the interaction of il-1 with its receptors, acting as a natural il-1 inhibitor.il-1β has important homeostatic functions in the normal organism, such as regulation of feeding, sleep, and temperature, however, overproduction of il-1β is implicated in the pathophysiological changes that occur during different disease states such as rheumatoid arthritis, neuropathic pain, inflammatory bowel disease, osteoarthritis, vascular diseases, multiple sclerosis, and alzheimer's disease [15]. tumor necrosis factorα (tnfα): is a proinflammatory cytokine that possesses a wide range of immune regulatory functions and has the potential to stimulate the production of secondary mediators, including chemokine or cyclooxygenase products, which amplifies the degree of inflammation [16]. tnf-α was cloned over 2 decades ago and its identification in part led to the discovery of a superfamily of tumor necrosis factor (tnfs) and their receptors [17]. macrophages are major producers of tnfα and interestingly are also highly responsive to tnfα, it signals through two transmembrane receptors, tnfr1 and tnfr2 and regulate several critical cell functions including cell proliferation, survival, differentiation and apoptosis [17]. aberrant tnfα production and tnfα receptor signaling have been associated with the pathogenesis of several diseases, including rheumatoid arthritis, crohn's disease, shehu et al.; aji, 3(1): 259-268, 2020; article no.aji.57314 262 atherosclerosis, psoriasis, sepsis, diabetes and obesity [18]. tnfα has been shown to play a pivotal role in orchestrating the cytokine cascade in many inflammatory diseases and because of this role as a "master regulator" of inflammatory cytokine production, it has been proposed as a therapeutic target for several diseases [19]. indeed, anti-tnfα drugs are now licensed for treating certain inflammatory diseases including rheumatoid arthritis and inflammatory bowel disease [20]. interleukin 6 (il-6): is a pleiotropic inflammatory cytokine synthesized by a variety of cells such as t lymphocytes, macrophages, monocytes, and synovial fibroblasts [21]. il-6 was initially discovered and referred as b-cell differentiation factor identified in tissue culture supernatants which induce b cells to produce immune globulins and involved in diverse biological functions, such as t lymphocytes activation, induction of acute-phase response, growth stimulation, differentiation of hematopoietic precursor cells and proliferation of synovial fibroblasts [22]. il-6 is among the major cytokines which contribute to the progression of many chronic inflammatory diseases and a wellestablished target for pharmacological intervention [23-24]. mammalian il-6 exerts its biological effects by binding to two cell surface receptors, il-6rα and glycoprotein 130 receptors [25-27] the assembly of the il-6·il-6rα·gp130 on the cell surface forming a complex activates the jak family of tyrosine kinases and the downstream stat3 transcription factor [28-30]. in cells that express il-6rα, il-6-dependent activation of the signaltransducing gp130 receptor is mediated by membrane-bound il-6rα. alternatively; il-6 in a complex with soluble il-6rα (sil-6rα) can activate gp130 in cells [31]. 2.3 rheumatoid arthritis rheumatoid arthritis (ra) is an autoimmune disease that causes chronic inflammation of the joint. autoimmune diseases are illnesses that occur when the immune system mistakenly attacks the body [32]. the immune system contains a complex organization of cells and antibodies design to protect the body against infections, one of the most prominent among autoimmune diseases is rheumatoid arthritis; also refer to as systemic illness or rheumatoid disease [33]. the other type of rheumatoid arthritis is juvenile idiopathic arthritis which manifests in individuals less than 16 years of age [33]. when tissue inflammation occurs, the disease is active (relapsed) and the symptoms include fatigue, loss of appetite, muscle and joint pain, joint redness, joint swelling joint tenderness rheumatoid nodules and finally if the symptoms persist it lead to loss of joint function [33]. when tissue inflammation subsides, the disease is inactive (remission), remission may occur spontaneously with treatment and can last for weeks, month and years [33]. ra targets synovial joints and is often accompanied by an array of extra-articular manifestations which are ultimately major predictors of increased morbidity and mortality [34]. it was discovered that joint damage occurs very early in ra, and after 2 years, approximately 50% of patients will exhibit bone erosions [35]. early management of rheumatoid arthritis with disease-modifying antirheumatic drugs (dmards) is well recognized in regulating the disease severity, until recently, treatment options were limited to single or combination therapy with a relatively restricted therapeutic armament [36]. these comprise of immunosuppressive and dmards such as azathioprine, prednisolone sulfasalazine, and methotrexate [37]. many of these medications reduce disease activity and block the radiological progression of bone erosion, judging by the clinical experience they are regarded to be suitable in many patients [38]. 2.4 prevalence of rheumatoid arthritis globally and in the us ra is a common disease that affects about 1% of the world population and about 0.6% of the american population, many adults are impacted by the condition with prevalence of 0·8–1·0% and the disease continues to cause significant morbidity and premature mortality [39]. the annual incidence of ra has been estimated to about 40 per 100,000 individuals, this shows that in each 100,000 individuals 40 new cases of ra are been diagnosed [40]. ra often initially impacts many children and individuals between 30-50 years of age, surprisingly, about threequarters of all the individuals with ra are women [41]. 2.5 pathogenesis of rheumatoid arthritis in the synovial joint, there is an imbalance between pro-inflammatory cytokines and anti shehu et al.; aji, 3(1): 259-268, 2020; article no.aji.57314 263 inflammatory cytokines [42]. t lymphocytes play an essential role by the production of various cytokines with different properties which facilitate the process of inflammation at the disease state [42]. interestingly, the t-helper cells which have two main phenotypes are characterized by two distinct cytokine profiles namely th1 and th2 along with the third subset called th0 which is found to be the precursor of the two subsets [42]. th0 cytokine profile is intermediate, hence th1 cells significantly intercede the cell-mediated immune response (cmi) while the th2 cells intercede the humoral immune response, and these crucial steps determine whether the acquired immune response is dominated by activation of macrophage, cell mediated immune response, pro-inflammatory activity of humoral immune response and anti-inflammatory activity [42]. t cell-induced in the presence of tnf α, ifn-γ, and il-12 develop into th-1 type of phenotype whereas those induced in the presence of il-4, il-6 and il-10develops into th-2 phenotype [43]. evidence suggests that interaction between an unknown exogenous or endogenous antigen via antigen-presenting cells and cd4+ t helper cells are involved in the induction of inflammation in ra [43]. continuous recruitment and activation of macrophages and monocytes occur with the recruitment of pro-inflammatory cytokines, specifically tnf-α and il-1 and il-6 into the synovial cavity, the release of these cytokines mediates tissue destruction by activation of chondrocytes and fibroblasts which release collagenases and metalloproteinase with resultant cartilage loss and bone erosion [44]. these activated macrophages, monocytes, lymphocytes and fibroblasts, as well as their product, also stimulate angiogenesis, which explains increased vascularity found in the synovium of patients with ra [44]. 3. therapeutic target tnf-α blockade: tnf-αis a potent cytokine that promotes inflammation by stimulating fibroblasts to express intercellular adhesion molecules that play a substantial role in inflammation, immune response and intracellular signalling event [46]. interaction of these adhesion molecules with their respective ligands on the surface of leukocytes results in increased transport of leukocytes to inflammatory sites, including the joints in patients with rheumatoid arthritis [45]. tnf-αindirectly down-regulates inflammation by stimulating the release of the corticotrophinreleasing hormone, this hormone stimulates the adrenal cortex to release cortisol, which inhibits inflammation [46]. as an inflammatory cytokine, tnf-αhas an important role in rheumatoid synovitis as studied in the culture of synovial cells from patients with rheumatoid arthritis, by blocking the tnf-αwith antibodies significantly fig. 1. ra is characterized by proliferative synovium (pannus) and an excessive immune response of t-cells. pannus comprises t-cells, synovial fibroblasts, and macrophages that produce inflammatory cytokines, such as tumor necrosis factor (tnf)-α, interleukin-1 (il-1), il6, and il-17. these inflammatory cytokines activate osteoclasts, leading to bone destruction [45] shehu et al.; aji, 3(1): 259-268, 2020; article no.aji.57314 264 reduced the production of interleukin-1, interleukin-6, interleukin-8, and granulocyte– monocyte colony-stimulating factor [47]. thus, the blockade of tnf-αhas a more precise effect on inflammation than the blockade of other cytokines present in high concentrations in the synovial membrane, such as interleukin-1 [48]. the result of studies in the animal model shows the importance of tnf-αin rheumatoid arthritis, in transgenic mice that expressed a deregulated human tnf-αgene, there is the spontaneous development of polyarthritis which is another form of arthritis that affects many joints, and however first aid treatment of these animals with a monoclonal antibody against tnf-αprevents the development of ra [49]. interleukin-1 receptor antagonist: the il-1 receptor antagonist (il-1ra) is an endogenous ligand that binds the interleukin-1 receptor (il1r) without recruiting the interleukin-1 receptor accessory protein (il1-racp) to hinder the activation of the receptor, il-1ra has a higher affinity for the il-1r than il-1α or il-1β and serves to regulate the signaling of il-1 by preventing the binding of other active cytokines [51]. deficiency of il-1ra results in a reduction of regulatory function and can lead to severe inflammation and auto inflammatory disorders such as arthritis, vasculitis, and skin lesions in humans [52]. il-1 binds to two types of cellsurface receptors, type i receptors are found on many cells in trace amount, they have a cytoplasmic tail and are capable of intracellular signaling, while type ii receptors are expressed primarily on neutrophils, monocytes and b cells, they are decoy receptors which bind circulating il-1 without delivering any intracellular signal [53]. soluble forms of both types of il-1r compete with cell surface receptors thereby decreasing il1 mediated activation of cells [54]. il-1ra, binds the type i receptor with high affinity without triggering a signal, therefore providing another mechanism for inhibiting the il-1 activity [55]. il1 plays a critical role in joint damage by facilitating the degradation of cartilage which leads to rheumatoid arthritis, whereas the injection of antibodies against il-1 suppresses collagen-induced arthritis in mice and decreases the damage to the cartilage [56]. anti-interleukin 6 receptor: interleukin-6 (il-6) is a major pro-inflammatory cytokine with pleiotropic functions, il-6 plays a key role in immune activation and inflammation in ra, thus, inhibition of il-6 is a good strategy in controlling ra [57]. il-6 has diverse and protein effects on many cell types, involved in both homeostatic and inflammatory pathways, however anti-il-6 receptor agent tocilizumab (tcz) has become a major resource for the treatment of ra [58]. in patients with ra, tcz has a dramatic effect on b lineage cells, with decreases in levels of circulating switched and unswitched memory b cells and with overall decreases in measured levels of antibody gene hypermutation in blood b cells [59]. a humanized il-6r antibody, tocilizumab (tcz), was developed by a research team from chugai pharmaceutical company and osaka university in japan [59]. after several randomized clinical trials (rcts), an intravenous formulation of tcz (tcz-iv) was approved for ra treatment in japan in 2008, in europe in 2009, and in the usa in 2010 [58]. recently, a subcutaneous formulation of tcz (tcz-sc) was developed in consideration of patients’ preferences after a non-inferiority trial of tczsc and tcz-iv, tcz-sc was approved in japan and the usa in 2013 and europe in 2014 [60]. tcz displays a good efficacy in patients with ra, including disease-modifying antirheumatic drug (dmard) naïve patients and patients with an inadequate response to conventional synthetic dmards (csdmards), methotrexate (mtx), or tnf inhibitors (tnfi) [60]. table 2. showing tnf-α blockers as the first therapeutic agents approved for the treatment of rheumatoid arthritis; tnf-α blockade has become a central strategy of targeted antiinflammatory therapy in the disease agent class target structure ref. adalimumab cytokine inhibitor tnf-α human monoclonal antibody [50] certolizumab cytokine inhibitor tnf-α egylated humani ed fab fragment of an anti–tnf-α monoclonal antibody [50] etanercept cytokine inhibitor tnf-α tnf-α receptor–fc fusion [50] golimumab cytokine inhibitor tnf-α human monoclonal antibody [50] infliximab cytokine inhibitor tnf-α chimeric monoclonal antibody [50] shehu et al.; aji, 3(1): 259-268, 2020; article no.aji.57314 265 table 3. this therapeutic agent is considered one of the cytokine blockades in rheumatoid arthritis; it has profound effects on systemic features, acute phase response and synovitis agent class target structure ref anakinra cytokine inhibitor interleukin-1 interleukin-1 receptor antagonist [57] table 4. this therapeutic agent is considered to be one of the cytokine blockades in rheumatoid arthritis; it has profound effects on systemic features, acute phase response and synovitis . agent class target structure ref tocilizumab cytokine inhibitor interleukin-6 receptor human monoclonal antibodies [63] the overall efficacy including clinical responses and the radiographic damage progression of tcz was comparable to that of other biological dmards such as tnfi [61]. il-6 was found to affects psychosomatic functioning, sleep-related symptoms, and fatigue, while on the other hand, tcz improves sleep quality and fatigue [62]. 4. conclusion this review has summarized the critical role played by the cytokines in the pathogenesis of ra. persistent recruitment and activation of macrophages and monocytes occur with the recruitment of pro-inflammatory cytokines, specifically tnf-α and il-1 and il-6 into the synovial cavity which leads to tissue destruction by activation of chondrocytes and fibroblasts thereby releasing collagenases and metalloproteinase with resultant cartilage loss and bone erosion. however, due to the increase morbidity and premature mortality of ra in the world population, many therapeutic strategies are adopted to overcome the disease. the novel therapeutic approaches to ra that are aimed at restoring this cytokines imbalance include the use of monoclonal antibodies to tnf-α as mentioned in (table 2) which mainly inhibit the proinflammatory cytokine activity, il-1rantagonist such as anakinra which suppresses collagen-induced arthritis thereby decreasing the damage to cartilage and the use of tocilizumab which displays a good efficacy in patients with ra, including 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(2) vijaya krishnan, mgm college of physiotherapy, india. (3) m. zamri saad, universiti putra malaysia, malaysia. complete peer review history: http://www.sdiarticle4.com/review-history/55881 received 20 march 2020 accepted 30 march 2020 published 01 april 2020 abstract the pandemic of novel corona virus disease (covid 19), is presently sweeping the world over. the causative virus is named, sars-cov-2. the rapidity with which the pandemic is advancing is catastrophic. that there is no known treatment for this killer disease is throwing public at large into a state of fear psychosis. the urgent need for preventive vaccine is felt now, more than ever before though promising research is going on, the goal to achieve a breakthrough vaccine is still a long way to go. since sars-cov-2 is an infection restricted to respiratory system of the affected, vaccines aimed at inducing local (mucosal) immunity may offer better chance of preventing the acquisition and spread of the disease to others, than induction of systemic immunity. with this background theme, this article briefly reviewed the epidemiology, the vaccine trials of the covid 19 as well as the immune responses to viral infection in general and with a special focus on the concept of local immunity advanced by prof besredka (1924). the historical perspective of the local immunity concept is recapitulated and the diseases, both viral and bacterial, to which vaccines are developed in the past basing on this concept are reviewed. a case is made out for applying this concept for the prevention of covid 19. it is suggested that an approach to boost local immunity, than systemic immunity, might be a better strategy of prevention of covid 19. opinion article prasad; aji, 3(1): 61-70, 2020; article no.aji.55881 62 keywords: covid 19; local immunity; viral immunity; sars-cov-2(severe acute respiratory syndrome corona virus 2); mers-cov(middle east respiratory syndrome coronavirus). 1. introduction 1.1 epidemiological considerations of covid 19  the massive awareness programme against the covid 19 world over, made any introduction to this disease superfluous. similarly the rapid changes from day to day of statistical data made any cited data meaningless, in no time. nevertheless, convention demands brief introduction to the disease. a pneumonia of unknown cause detected in wuhan, china was first reported to the who country office in china on 31 december 2019.the outbreak was declared a public health emergency of international concern on 30 january 2020. on 11 february 2020, who announced a name for the new coronavirus disease. the strain involved is considered novel as it is different from corona virus strains that are known before and hence.  the name covid 19. sars-cov-2 belongs to the beta coronavirus genus, which also includes sars cov (2003) and mers cov (2012). sars-cov first emerged in 2002-2003 in guangdong, china as an unusual pneumonia, that caused lifethreatening respiratory failure in certain cases. it quickly became pandemic. mers-cov epidemic appeared in saudi arabia in 2012, with people experiencing similar symptoms to sars-cov but dying at a much higher rate of 34 per cent. unlike sars-cov, which spread quickly and widely, mers-cov has been mainly limited to the middle east. on 11 february, the novel coronavirus that had provisionally been known as 2019-ncov, was given a new name, sars-cov-2. the acronym sars stands for severe acute respiratory syndrome. cov2 stands for corona virus 2. the name denotes its close relationship to the sars virus. according to who report as on 18 th march 2020, the total globally confirmed cases are 1,90,415 and total confirmed deaths are 7, 800. as on march 23,2020 the total number of cases and total deaths globally are respectively, 292142 and 12784 who, data as on 24 march 2020 is total number of cases-372757, deaths 16,231 and countries infected are 195. as on 30 march 2020, 715 660 cases have been reported.  including 33 579 deaths. the speed with which the disease is spreading can be inferred from the above figures. the mortality rate is between, 1 t0 3% the economic loss due to covid 19 as estimated in the beginning of march 2020 is about 1 to 3 trillion dollar. trade and tourism, world over are worst effected. it is considered as a zoonotic infection, reservoirs being the wild bats and certain snakes. the infection starts like flu with cold and dry cough and fever. soon shortness of breath occurs with developing pneumonia and the patient may require ventilator support. while cold and flu viruses take 2-3 days to develop once you contract the virus, coronavirus symptoms take anywhere from 2-14 days after being exposed to the virus, as per the centres of disease control and prevention (cdc). according to a study (march 10, 2020). the transmission from man to man is through direct spread by droplet infection and indirectly by coming into contact with objects on which the virus containing infective material is deposited. the survival of the virus outside the human host is variable, depending on the material concerned ranges from 1 to 5 days. the estimated median of ip is 5.1 days. comorbid conditions in general and respiratory diseases like asthma/ copd as well as old age appear to be the risk factors at the individual level. women and children are mostly spared, but not completely. presently thereisno specific drug against the virus is available, so, the present emphasis is on prevention of the spread of the infection. home isolation of the suspected cases, qwarentaining the positive cases, social isolation and self imposed curfew are some of the strategies adopted, which are showing promising results. some retrovial drugs and hydroxy chloroquin t-azithromycine combination are used emparically. there is intense research going on to find a vaccine against the disease. prasad; aji, 3(1): 61-70, 2020; article no.aji.55881 63 1.2 can covid 19 be transmitted by asymptomatic carriers? zhang et al. [1] have reported a familial cluster of covid-19 indicating virus can be transmitted by asymptomatic carriers. anbai’s study presumed that asymptomatic 2019 ncov carrier could be a transmitter. [2] a couple with covid-19 were transmitted infection by their asymptomatic daughter who had travelled from the epidemic center of wuhan. the sequence suggests that the coronavirus can be transmitted by asymptomatic carriers in their early and middle latent period. asymptomatic virus carriers should be monitored and isolated as early as possible to facilitate the control of the epidemic. a paper published on nejm about the first four people in germany infected with 2019 ncov found asymptomatic persons can still transmit the virus to others [3] but soon the german government’s public health agency announced that the information was wrong. the “asymptomatic” patient actually had some symptoms, it was found later [4]. 1.3 are there two strains of the novel corona virus? at the outset it is made clear that the who has not at accepted the 2 strains concept as on now. since some investigators found two types of mutations in the genome of the covid virus (see below) and few investigators tried to see difference in virulence patterns of the same virus at different geographical places, (for instance the infection is catastrophic in italy, but so far appears to be comparatively, mild in india) hazard a guess about the existence of more virulent and less virulent strains of the covid 19 virus. it is probable that the deficiency of timely interventional measures might be the cause, as felt in case of italy. xiaolu tang et al, at peking university in beijing and his colleagues studied the viral genome taken from 103 cases. they found common mutations at two locations on the genome. the team identified two types of the virus based on differences in the genome at these two regions. mutation at region 72 are considered to be the “l-type” and 29 were classed “s-type”. s type is believed to be the ancestral strain descended from the animal and s type its human variant that developed later. s is wide spread in distribution source:(https://www.newscientist.com/article/223 6544-coronavirus-are-there-two-strains-and-isone-more-deadly/#ixzz6hohp3wf7) 1.4 recovery of the novel, corona virus from the feces some researchers have reported the recovery of the virus from the feces, but its significance at present is not known. there is no evidence of feco-oral transmission. 1.5 reinfection the who has not yet confirmed any possibility of reinfection. 1.6 approaches to corona virus vaccine production 1.6.1 structure and antigens of corona virus the novel corona virus is an enveloped virus containing a single stranded m rna and a protein inside and spoke like projections of glycoprotein on the surface. the later earned the virus the name corona, which means a crown. genome of sars-cov-2 (2019-ncov) encodes the spike protein, the envelope protein, the membrane protein and the nucleocapsid protein. the spike protein (s-protein) mediates receptor binding and membrane fusion. spike protein contains two subunits, s1 and s2. s1 contains a receptor binding domain (rbd), which is responsible for recognizing and binding with the cell surface receptor. s2 subunit is the "stem" of the structure, which contains other basic elements needed for the membrane fusion. the spike protein is the common target for neutralizing antibodies and vaccines.euis been reported that sars-cov-2 (2019-ncov can infect the human respiratory epithelial cells through interaction with the human ace2 receptor. indeed, the recombinant spike protein can bind with recombinant ace2 protein. it has been reported that sars-cov-2 (2019-ncov) can infect the human respiratory epithelial cells through interaction with the human ace2 receptor. indeed, the recombinant spike protein can bind with recombinant ace2 protein. the spike protein (s) on the envelope of the virus and m-rna strand of the virus within the capsid are two novel approached in the vaccine industry (source: sino biological, inc with headquarters in beijing, china with branches in the united states and europe). 1.6.2 the vaccine trials according to the kaiser permanente report “the artificial mrna produced in the lab, prompts host prasad; aji, 3(1): 61-70, 2020; article no.aji.55881 64 cells to build a protein found on the surface of the virus, . a person’s immune system would react to this new protein by building up an arsenal of antibodies that target and latchonto this protein, tagging the virus for elimination. then, the mrna should break down and be eliminated by the body, leaving the vaccinated person better prepared to fight off sars-cov-2, if they are infected subsequently. a clinical trial is slated on april 2020 to conduct a preliminary human safety clinical trial of this vaccine a wall street journal reports. in february 2020, novavax started testing its recombinant nanoparticle vaccine candidate for coronavirus in animal models. the candidates produced antigens derived from the coronavirus spike (s) protein. novavax is planning to use its matrix-m adjuvant with its covid-19 vaccine candidates to boost immune responses. “curevac has successfully harnessed its biological insights of the lifeenabling molecule mrna to move medicine into the future. powered by our optimization technology.” curevac ag, a clinical stage biopharmaceutical company is pioneering mrna-based vaccine against the novel corona virus. recently the who approved hydroxy chloroquine vaccine for prophylaxis against covid 19. who has published a long list of counties working on corona virus vaccine and their different approaches, in this regard. interested can refer the same as it is out of the scope of this article. 2. discussion it is proposed to briefly review the broad principles of immune reaction s to viral infections in general as the specific immune reaction of corona virus 19 is to early to decipher. however, the intended focus is on the concept of local immunity and it’s possible role in the interventional strategy to contain the disease. principles of immune reactions against viral infections in general: there are two types of systemic immunity they are  innate immunity  adoptive immunity innate and adoptive immune mechanisms both operate against the virus or the infected cells of the host. cytotoxic cells: nk (natural killer) and cd8+cells cytotoxic t cell circulate and kills cells that are infected with viruses with toxic mediators. cytotoxic t cells have specialised proteins on their surface that help them to recognise virally-infected cells. these proteins are called t cell receptors (tcrs). each cytotoxic t cell has a tcr that can specifically recognise a particular antigenic peptide bound to an mhc 1 molecule. the virus have tcr receptors which express the viral proteins. the host cells have hla 1 antigens to which the vral proteins are bound bind. nk cytotoxic cells are innate defence mechanisms which have fewer than normal mhc 1 receptors on the surface of the cells. toxic mediators of t cells: preformed toxic mediators are stored called granules, in both cytotoxic t cells and nk cells, until contact with an infected cell triggers their release. one of these mediators is perforin, a protein that can make pores in cell membranes; these pores allow entry of other factors into a target cell to facilitate destruction of the cell. enzymes called granzymes are also stored in, and released from, the granules. granzymes enter target cells through the holes made by perforin. once inside the target cell, they initiate a process known as programmed cell death or apoptosis, causing the target cell to die. another released cytotoxic factor is granulysin, directly attacks the outer membrane of the table 1. showing the various viral immune mechanisms immune mechanisms in viral infections against the infected cells against the virus innate nk cells interferons (a, b and g) alternative compliment pathway adaptive adcc (antibody dependant cell mediated cytotoxicity classical complement pathway cd 8 cytotoxic cells phagocytosis prasad; aji, 3(1): 61-70, 2020; article no.aji.55881 65 target cell, destroying it by lysis. cytotoxic cells synthesise and release, cytokines, after making contact with infected cells. cytokines include interferon-g and tumour necrosis factor-a. interferon: it is one of the most effective innate defence mechanism produced by the infected. host cell. there are 3 types of interferon, a, b and g the first two are mainly produced by monocytes-macrophages and to a lesser extent by fibroblasts. but interferon-g is produced by cd 4 and cd 8 lymphocytes and nk cells. mechanisms of interferon action: "transitory resistance of cells; induction of different molecules with anti-viral activity; activation of genes expressing anti-viral proteins, and increasing the expression of sla i and sla ii“. role of antibodies (ig g, igm,): 1. neutralisation of the virus (both igg and igm) 2. agglutination (igm) effectiveness of humoral vs cell mediated immunity (cmi): some investigators have suggested that serum antibody is responsible for protection in the human, others have observed that influenza infection is not prevented by high levels of serum antibody still investigators believe that serum antibody correlates with, but does not cause, protection. cell-mediated immune responses are not only effective during acute phase of viral infection.when infected cells with virus are targeted by the cd 8+ subset of t cell population but also establish long-lived immunological memory by virtue of cd 4 + t cells.. these memory t cells become rapidly activated if they re-encounter virally infected cells and therefore function to protect against secondary exposures to the original viral pathogens. it can be host damaging if hypersensitive reaction develops as a part of immune reaction. it has also been proposed that cell-mediated immunity might be important in protection against viral infections as exemplified by the patients with hypogammaglobulinemia are not prone to more frequent or severe viral infections. 2.1 the concept of local immunity historical perspective of the concept of local immunity [5]: prof besredka is the founder of this type of immunity. working in pasture institute in 1924, he developed his concept and tested on different organisms. he showed that b anthracite inoculated intraperitonially in guinea pig neither produced antibodies in the blood nor protected the animal from subsequent challenge with the live virus. age also proved that the organism when applied to the skin, though it did not also develop any antibodies, protected the animal from subsequent challenge with the microorganism. this study has driven home two truths. one, that immunity against the infection can be achieved even in the absence of raising antibodies against it. second, vaccinating the site which is the portal of entry of the micro organism conferred immunity, independent of humoral immunity. this type of immunity, he called as "local immunity". 2.2 review of some of the diseases to which the concept of local immunity is applied and tested 2.2.1 local immunity in polio virus infection [6] the salk polio vaccine, which consists of killed virus administered systemically, elicits serum igg as the major antibody and induces little or no secretory response. as a result, the immunized individual resists systemic infection, but may become a temporary carrier, with virus persisting at the intestinal portal of entry because of the lack of secretory antibody. the orally administered, live sabin polio vaccine, on the other hand, induces secretory antibody in the intestine and is effective in preventing replication and subsequent mucosal penetration by the virus. however us stopped opv in favour of ipv (inactivated polio vaccine) to prevent resurgence of type 2 polio virus. 2.2.2 local immunity in the influenza virus infection influenza infection of the ferret resembles the disease in the human, [7] and has been studied extensively by henry barber and parker a. small, jr, who showed that influenza infection in the ferret is a local phenomenon, whereas recovery from active infection is influenced by systemic immune mechanism" [8]. several workers [9,10,11] have shown that natural infection with respiratory viruses stimulates the production of immunoglobulin a antibodies in secretions and leads to resistance to reinfection, it has been postulated that a vaccine’s ability to stimulate nasal secretary antibody is the deciding factor in protection of humans against influenza infection, and some prasad; aji, 3(1): 61-70, 2020; article no.aji.55881 66 field trials of local immunization gave promising results. [12,13,14,15]. on the contrary, trial of local immunization [16] as well as attempts to correlate secretary immunoglobulin a antibody with protection [17] was unsuccessful. the study suggested that srum antibody was irrelevant to prevention of influenza and recovery from influenza in the ferret [18]. antibodies directed to the 2 major viral surface membrane proteins, hemagglutinin (ha) and neuraminidase (na), mediate protection against reinfection following natural infection or vaccination. some investigators have suggested that serum antibody is responsible for protection in the human, [19] others have observed that influenza infection is not prevented by high levels of serum antibody [20]. several investigators believe that serum antibody correlates with, but does not cause, protection [21]. the lack of virus dissemination is consistent with the inability to detect viremia during influenza infection of ferrets and humans [22]. mucosal immunity in shigella et al. [23] explored the mechanisms of protection mediated by shigella lps-specific secretory iga (siga), the major mucosal ab (antibody) induced upon natural infection. they found that anti-shigella lps siga, mainly via immune exclusion, prevented shigella-induced inflammation responsible for the destruction of the intestinal barrier. in the form of immune complexes, siga guarantees both immune exclusion and neutralization of translocated bacteria, thus preserving the intestinal barrier integrity by preventing bacterial-induced inflammation. these findings add to the multiple facets of the non inflammatory properties of siga. 2.2.3 mucosal immunity in infection by v. cholerae [24] bloom pd, et al. reviewing the mucosal immunity against enteric bacterial pathogens states that "v cholerae, secretes cholera toxin (ct), a potent enterotoxin that induces a voluminous diarrhea via adenylate cyclase-dependent chloride secretion. protective immunity is based on secretory (s) immunoglobulin a directed against whole-cell components that prevent attachment to gut epithelial cells and is enhanced by ct, an immunogen with potent adjuvant activity". mycoplasma and local immunity: lisa m. hodge, jerry w. simecka et al. [25] in their study concluded that "local immunity along the respiratory tract plays a major role in resisting and controlling mycoplasma infection and should be considered in vaccine development against mycoplasma respiratory diseases. generation of immunity in the upper respiratory tract appears to be optimal in preventing the initial mycoplasma infection at this site, but adjuvants (like cholera toxin (ct) enhanced these protective responses. in the lower respiratory tract, pulmonary immune responses seem to be more effective than serum antibody responses and nasal immunization can confer some protection from pulmonary infection". role of the secretary iga in local immunity [26]: secretory iga (siga) is the principal immunoglobulin (ig) on mucosal surfaces of humans the respiratory contains equivalent amounts of iga and igg in addition to some igm (brandtzaeg et al., 1999). the main function of iga is the neutralization of pathogens and toxins without causing inflammation since it does not activate complement (cerutti, 2008; macpherson & slack, 2007). the mucosal microbiota, epithelial cells, and the mucosal immune system constitute a stable and interdependent “tripod” that maintains mucosal homeostasis by complex mechanisms (corthesy, 2007; hooper, 2004). 1. bacteria adhere to the surface receptors of the epithelial cells and express the basolateral membrane receptor (polymeric ig receptor; pigr) that transports locally produced polymeric (p)iga into the external secretions (kaetzel & mostov, 2005). 2. endogenous bacterial of the intestinal tract, and the respiratory and genital tracts, are coated in vivo with siga (bos, cebra, & kroese, 2000; van der waaij et al. 2004) that limits their epithelial adherence and penetration, thereby confining them to the mucosal surfaces. iga neutralizes inflammatory microbial products inside epithelial cells (fernandez et al., 2003). finally, if bacteria trespass the epithelial barrier, iga transports these bacteria back into the lumen via pigr or promotes their phagocytosis through fc (pasquier et al., 2005; phalipon corthésy, 2003). killed bacteria seem to be at least 100-fold less effective at inducing iga responses than live bacteria, presumably due to their inactivity and the digestion of dead bacteria during transit through the stomach and intestine (hapfelmeier et al. 2010; macpherson & uhr, 2004). prasad; aji, 3(1): 61-70, 2020; article no.aji.55881 67 2.2.4 concept on local immunity as applied to in covid 19 immunity 2.2.4.1 recapitulation of some considerations 1. belyakov et al. [27] opined that: a. "the route of vaccination is important in influencing immune responses at the initial site of pathogen invasion where protection is most effective. immune responses required for mucosal protection can differ vastly depending on the individual pathogen. for some mucosal pathogens, high-titer neutralizing antibodies (abs) that enter tissue parenchyma or transude into the mucosal lumen are sufficient for clearing cells, free of the virus." b. induction of the mucosal innate and adaptive immune systems, including cd4+ t helper cells, th17, high avidity cd8+ ctl and secretory iga and igg1 neutralizing abs, at the site of pathogen entry may be required for effective protection against highly invasive pathogens. 2. the main function of the innate mucosal immune system, to discriminate between dangerous and innocuous organisms, is determined by the recognition of specific pathogen-associated molecular patterns via activation of tlrs, nod-like receptors, retinoic acid (ra)-inducible gene i-like helicases, and c-type lectins [28]. 3. studies evaluating mucosal infection and immunization in humans and animals have demonstrated the existence of a common mucosal immune system (cmis) that consists of gastrointestinal, respiratory, and genital mucosa [29]. 4. the cmis implies the ability of ag-specific lymphocytes to home to mucosal effector sites in addition to the site where initial ag exposure occurred [30] compartmentalized mucosal immune responses (cmirs) that consist of innate responses, mucosal abs (secretory ig (sig) a and sigg) and cd8+ ctls localized to tissues proximal to the mucosal site of immunization are necessary for protection from mucosal pathogens (belyakov et al.). 5. protective immunity against mucosal pathogens will require novel vaccine strategies to induce mucosal immune responses tailored to the anatomic location and the threat of the invading pathogen 6. protective immunity against mucosal pathogens will require novel vaccine strategies to induce mucosal immune responses tailored to the anatomic location and the threat of the invading pathogen pathogens may require mucosal vaccine strategies that activate multiple arms of the innate and adaptive immune systems [31,32,33,34,35]. 7. optimum mucosal vaccination leading to compartmentalized mucosal immune responses might ensure that the appropriate cells are armed and ready to respond immediately to infection and to confer protection not achieved following natural infection. 8. protective mucosal immune responses are most effectively induced by mucosal immunization through oral, intranasal (i.n.), intrarectal, or intravaginal routes, and an optimized mucosal vaccination strategy may have a much greater potential for generating local protective mucosal immune responses. 9. mucosal immunity concept has worked well in case of viral infections like influenza, poliomyelitis and bacterial infections like salmonella and v. cholerae and mycoplasma infections, as outlined above. 10. the cdc has not made any comments regarding the relative merits of the inactivated and recombinant vaccine and nasal spray vaccine for influenza. the proposed hypothesis: covid 19 is different from other deadly viral infections, say for instance hiv. in the later, the virus is found in all body secretions including semen with variable infectivity of each body fluid. there is no suggestion of any tissue "tropism" in hiv. as against hiv, the corona virus (cov) enters the body through the nose, multiplies there, and excreted through nasal secretion. in fact, they are limited almost exclusively in the upper respiratory epithelium initially and later if not prevented, spreads locally to lower respiratory epithelium, suggesting some sort of "tissue tropism". additionally, the novel corona virus is shown not to be disseminated beyond the respiratory tract. the recent reports of involvement of cardiovascular system (myocarditis) and a couple of cases of involvement of the brain, have no proof of direct involvement of the cov, beyond reasonable doubt. it could’ve be due to fallout of the immune reactio mounted against the virus. consequently prasad; aji, 3(1): 61-70, 2020; article no.aji.55881 68 the cause of high mortality associated with covid 19, is restricted to a deadly type of pneumonia. if the infection could be arrested at portal of entry, i.e. the nasal epithelial level, the disastrous consequences of spread to lower respiratory tract; ĺmay be averted. this makes a strong case for creating local immunity at the level of the nasal epithelium itself. local mucosal infections of the respiratory or gastro-intestinal tract may elicit local cell-mediateld and humoral (iga) immune responses, but not necessarily systemic immunity. conversely, systemic immunity does not always lead to local mucosal immunity. localized infections of mucosal surfaces, and the protection derived , does not correlate with the presence of serum antibody, but it does correlate with the presence of local iga antibody, as has been shown in human studies of viruses restricted to the respiratory tract (e.g., respiratory syncytial virus and influenza virus) or to the gastrointestinal tract (e.g., enteroviruses). the route of administering the vaccine is the major determinant of the issue immunity. the past experience of intranasal vaccine as is approved for influenza may be taken as an example, in the case of covid 19. it is also shown that prior systemic vaccination may interfere with the recall memory exercised by the nasal mucosa, should the pathogen cause reinfection the same person. there is no permanent infection-induced immunity conferred by the covid 19. this factor also supports the suggestion of raising local immunity of nasal epithelium by administering the vaccine intranasally. it is logical that raising local immunity in nasal epithelium may give a multi pronged protection from the covid 19. against entry multiplication. as well excretion in nasal sections. the last one is very important from public health point of view , in preventing the spread of the infection to others. local immunity is found to be effective at least in two diseases. to elaborate this point, the polio virus, whose portal of entry of the infection is the gastrointestinal tract, oral polio vaccine is the time tested vaccine that conferred immunity to polio virus. it is shown to be effective by raising mucosa immunity both at pharyngeal and intestinal levelled –ipv (inactivated polio vaccine) was less effective than opv in preventing and limiting intestinal infection, even though it induced higher post vaccination serum antibody levels. while opv (oral polio vaccine) checks the excretion of polio virus through faeces (there by preventing the spread of infection to others, [36] the continued excretion of the polio virus in case of immunisation by the inactivated vaccine administered by parenteral route, poses risk of continued deco-oral transmission, though it offered immunity at the personal level. secondly, the influenza vaccine administered intranasally as a spray type vaccine has been accepted efficacy-wise by the cdc. 3. conclusion the covid 19, as seen from the tropism to airway (nasal) epithelium through which it gains entry, multiplies and even shed in the nasal secretions, indicates that, efforts to raise the local immunity of the respiratory epithelium, especially at the very portal of its entry, may be fruitful in ‘nipping the infection in the bud’. accordingly in the case of covid 19, vaccine developed might be effective, if administered intranasally, rather than other routes of vaccine administration, it is felt. consent and ethical approval it is not applicable. competing interests author has declared that no competing interests exist. references 1. zhang s, na j, zhou w, liu b, yang w, zhou p, et al. a familial cluster of covid19 indicating virus can be transmitted by asymptomatic carriers. 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this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: http://www.sdiarticle4.com/review-history/55881 http://creativecommons.org/licenses/by/4.0 _____________________________________________________________________________________________________ *corresponding author: e-mail: pkorisadiran@pgschool.lautech.edu.ng; asian journal of immunology 3(1): 11-22, 2020; article no.aji.53370 effects of d-ribose-l-cysteine on lipid profile, atherogenic index and infertility in streptozotocininduced male diabetic wistar rats abiodun oluwabusola adedeji1,2 and patrick kunle orisadiran3* 1 department of anatomy, olabisi onabanjo university, ago iwoye, nigeria. 2department of anatomy, ladoke akintola university of technology, ogbomoso, nigeria. 3department of biochemistry, ladoke akintola university of technology, ogbomoso, nigeria. authors’ contributions this work was carried out in collaboration between both authors. author aoa participated in study design, data collection and interpretation, contributed to drafting/ revising of manuscript. author pko participated in study design, data analysis and interpretation, did literature search and drafted/edited/ revised manuscript. both authors read and approved the final manuscript. article information editor(s): (1) dr. jaffu othniel chilongola, department of biochemistry and molecular biology, kilimanjaro christian medical university college, tumaini university, tanzania. reviewers: (1) rupali sengupta, india. (2) ann nkeiruka, national root crops research institute, nigeria. complete peer review history: http://www.sdiarticle4.com/review-history/53370 received 02 november 2019 accepted 07 january 2020 published 20 january 2020 abstract aims: the present study aimed at determining the effects of d-ribose-l-cysteine on lipid profile, atherogenic index, and infertility in streptozotocin-induced diabetic male wistar rats. methods: a total of twenty-eight adult male wistar rats were divided into four groups of seven rats each. 1: normal control group, 2: diabetic control group, 3: normal rats treated with 30 mg/kg body weight of d-ribose-l-cysteine, 4: diabetic rats treated with 30 mg/kg body weight of d-ribose-lcysteine. group 2 and 4 were injected intraperitoneally with a single dose of streptozotocin (stz) (65 mg/kg in 0.1 m cold citrate buffer, ph 7.5) prior to d-ribose-l-cysteine treatment. group 4 were subsequently administered d-ribose-l-cysteine orally 72 hours post administration of streptozotocin, twice daily for 28 days. parameters tested include: fasting blood and serum glucose, malondialdehyde (mda) concentration, sperm motility, morphology and count. testosterone (tt), follicle stimulating hormone (fsh) and luteinizing hormone (lh) were also examined. original research article adedeji and orisadiran; aji, 3(1): 11-22, 2020; article no.aji.53370 12 results: the results showed that diabetic rats showed weights loss, increased mda, increase blood and serum glucose levels, elevated lipid profile, altered tt, fsh and lh as well as reduced sperm count, motility and morphology. these effects were ameliorated in diabetic rats treated with d-ribose-l-cysteine. conclusion: current study revealed that d-ribose-l-cysteine attenuates the oxidative stress in streptozotocin-induced diabetic rats on blood glucose, lipid profile, reproductive hormone and sperm parameters. keywords: d-ribose and l-cysteine; wistar rats; diabetic rat; sperm; hormone; streptozotocin. 1. introduction diabetes mellitus is a group of metabolic disorders resulting from a deficiency in insulin secretions and/or actions [1]. this deficiency, in turn, leads to accumulation of glucose in the bloodstream with obstruction of carbohydrate, fat and protein metabolism. disease progression results in tissue damage leading to severe diabetic complications such as impairment or loss of vision, impairment of kidney function, cardiovascular and microbial complications [2]. the world prevalence of diabetes among adults between 20 and 79 years was estimated to be 6.4%, affecting 285 million adults, in 2010, and will increase to 7.7% and 439 million adults by 2030 [3]. out of the two types of diabetes (insulin dependent and non-insulin dependent), the incidence of non-insulin dependent diabetes mellitus is much higher than the insulindependent diabetes mellitus [4]. glucose is the main oxidizable substrate in various cell types. homeostasis is maintained in a diabetic state by the activation of catabolic pathways such as gluconeogenesis and glycogenolysis [5]. as a result, lipolysis is also increased, which trigger dyslipidemia, a risk factor for the development of atherosclerosis that affects 97 percent of diabetic patients [6,7]. dyslipidemia is characterized by elevated levels of triacylglycerol plasma and total cholesterol, increasing the concentration of very low-density lipoproteins (vldl-cholesterol) and low-density lipoproteins (ldl-cholesterol) and decreasing the concentration of high-density lipoproteins (hdlcholesterol) [7,8]. in different studies by valko et al. [9] and schilling [10], they showed that an increase in ldl-cholesterol that occurs due to the release of oxidizing agents during the metabolic pathways activated by hyperglycemia is directly related to the development of atherosclerosis, the inflammatory process triggered by the oxidation of ldl-cholesterol. oxidized ldls are phagocytosed by macrophages in the subendothelial layer, thereby transforming into foam cells that contribute to the formation of atherosclerotic plaque and, consequently, atherosclerosis [6,11]. reproductive disorders in diabetic males have been widely studied. experimental studies by o’neill et al. [12] and ricci et al. [13] revealed different structural and physiological reproductive dysfunctions in cases of diabetes in males. diabetes mellitus affects male reproductive functions at multiple levels as well as its negative effects on endocrine control of spermatogenesis and/or by impairing erection and ejaculation [14]. ricci et al. [13] established that insulindependent diabetes is accompanied by reduced semen volume and decreased sperm motility and vitality. this corroborates with the report of agbaje et al. [15] which stated that a high level of blood sugar in the bloodstream may affect sperm quality and consequently decreases male fertility potentials. also, joao and co. [16] confirmed high rates of infertility and poor reproductive outcomes in diabetic men compared with healthy men. therefore, effective control of the blood glucose level is crucial in preventing, controlling or reversing diabetic complications associated with diabetics, thus improving the quality of life in diabetic patients [17]. a study has shown that antioxidant substances can reduce chronic degenerative diseases such as diabetes mellitus and its complications [18]. antioxidants are stable substances that act as reducing agents capable of reducing oxidized substances and making them stable, thus repairing the structure of the cellular macromolecules [19]. d-ribose is an antioxidant and a prodrug form of l-cysteine known to aid the elevation of intracellular levels of glutathione (gsh) [20]. gsh is a coenzyme that mediates the protection against free radicals generated during the adedeji and orisadiran; aji, 3(1): 11-22, 2020; article no.aji.53370 13 oxidative metabolism of acetaminophen by the hepatic cytochrome p-450 system [21]. whole glutathione consumption cannot be effective because it would be destroyed in the digestion process before reaching the cell. the ribose component of the d-ribose-l-cysteine solves these challenges allowing the cells to produce glutathione when needed by effectively protecting and delivering the fragile cysteine molecule [22]. in view of the discussed above, this study aimed to determine the beneficial effects of d-ribose-lcysteine on lipid profile, atherogenic index, and infertility in streptozotocin-induced diabetic male wistar rats. 2. materials and methods 2.1 chemicals streptozotocin (stz) was a product of sigma chemical company st. louis u.s.a. all assay kits were from randox laboratories limited uk. chemicals and reagents used were of analytical grade. d-ribose-l-cysteine supplement was obtained from max international, salt lake city, utah, usa. 2.2 experimental design a total of 28 albino rats of wistar strain, with an average weight of 160 g were obtained from the animal house of physiology department, ladoke akintola university of technology, nigeria. the animals were divided into 4 groups of 7 rats each and housed in separate cages in the same environment. the animals were allowed to acclimatize in the laboratory for two weeks before the commencement of the experiments. 2.3 diabetes induction streptozotocin (65 mg/kg in 0.1 m cold citrate buffer, ph 7.5) was administered to wistar rats fasted overnight by interperitoneally injection of freshly prepared solution. the animals were considered as diabetic if the blood glucose values of the overnight fasted rats, were > 250 mg/dl on the third day. group 1: control rats given citrate buffer only (0.01 m, ph 7.5), group 2: diabetic control (untreated rats) group 3: rats treated with 30 mg/kg body weight of d-ribose-l-cysteine. group 4: diabetic rats treated with 30 mg/kg body weight of d-ribose-l-cysteine the diabetic rats were then kept for 24 hours on 5% glucose solution bottles in their cages to prevent hypoglycemia. blood was collected every 3 days through the rat’s tail vein for glucose estimation using one touch glucometer; after 28 days of treatment, the body weight and fasting blood glucose, serum glucose of the animals were again determined. 2.4 collection of blood the rats were fasted overnight after 28 day, anaesthetized with chloroform and sacrificed. blood was collected by cardiac puncture into plain sample bottles, allowed to coagulate and then centrifuged at 3000 rpm for 15 minutes to obtain serum. the serum was kept under refrigeration at 4ºc for biochemical examination. 2.5 biochemical examination serum total cholesterol was determined using the method of zak [23], plasma triglyceride estimated by the method of mendez et al. [24], hdl-c by lopezvitrella et al. [25], ldl-c and vldl triglyceride values were calculated by the modified method of friedewald formula [26]. serum mda was measured by a thiobarbituric acid assay procedure [27]. 2.6 semen analysis the caudal epididymis of the rats was incised and a drop of epididymal fluid was smeared onto a glass slide, covered by a 22 · 22 mm. the slide was examined under the light microscope at 100 magnification to evaluate different fields [28]. after assessing different microscopic fields, the relative percentage of motile sperm was estimated and reported to the nearest 5% using the subjective determination of motility [29]. the sperm count was determined using the neubauer improved hemocytometer. the epididymal fluid ratio of 1:20 was prepared by adding 0.1 ml of fluid to 1.9 ml of water. the dilution was mixed thoroughly and both sides of the counting chamber were scored and the average was taken. spermatozoa within five of the red blood cell squares including those which lie across the outermost lines at the top and right sides were counted, while those at the bottom and left sides were left out. the number of spermatozoa counted was expressed in millions/ml [30]. adedeji and orisadiran; aji, 3(1): 11-22, 2020; article no.aji.53370 14 2.7 reproductive hormones the serum levels of testosterone, luteinizing hormone (lh) and follicle-stimulating hormone (fsh) were measured using enzyme-linked immunoassay kit (abcam) according to the manufacturer’s instructions. 2.8 statistical analysis results were presented as mean ± sd. paired student’s t-test was used to compare variations amongst groups. the minimum level of significance was considered at p˂0.05. statistical analysis was carried out using a software program (graphpad prism ver. 5; graphpad software, san diego, ca). 3. results 3.1 effect of d-ribose-l-cysteine on bodyweight and serum glucose on stz induced diabetic rats there was a significant increased (p<0.05) in the bodyweight of normal control and d-ribose-lcysteine treated group whereas the body weight of diabetic rats significantly reduced during the period of the experiment (fig. 1). the blood glucose levels increased significantly (p < 0.05) after the induction of diabetes but after the administration of d-ribose-l-cysteine the blood glucose levels also decreased significantly (p < 0.05) compared to the diabetic control (fig. 2a). the serum glucose level in the diabetic control group (15.5±1.3 mmol/l) was significantly (p<0.05) higher compared to normal control group (6.4±1.3 mmol/l), while treated diabetic rats showed a slight significant decreased (p<0.05) in the serum glucose (9.7±1.2 mmol/l) compared to the diabetic control rats (fig. 2b). 3.2 effect of d-ribose-l-cysteine on lipid peroxidation in stz induced diabetic rats figure 3 shows the changes in serum mda concentration. the mean value of serum mda levels in the diabetic rats (7.77±0.78 nmol/ml) significantly increased (p<0.05) when compared with the normal control (5.7±0.90 nmol/ml) but significantly decreased (p<0.05) in diabetic rats treated with d-ribose-l-cysteine (5.93±0.89 nmol/ml). 3.3 effect of d-ribose-l-cysteine on lipid profile in stz induced diabetic rats serum levels of normal and diabetic rats treated with d-ribose-l-cysteine are shown in figure 4. diabetic control rats exhibited higher serum total cholesterol, ldl-cholesterol, triglycerides, and low hdl-cholesterol levels compared to those of normal rats. the total cholesterol, ldlcholesterol and triglycerides levels were significantly higher (p<0.05) in diabetic control rats compared to the d-ribose-l-cysteine treated rats (97.50±10.50, 45.23±12.34 and 8 5.49±13.80 mg/dl), d-ribose-l-cysteine treated diabetic rats (105.20±7.30, 33.67±12.54 and 92.62±14.30 mg/dl), and normal control rats (110.50±7.80, 43.98±15.23 and 89.407.80, mg/dl) respectively. whereas ldl-cholesterol levels of rats treated with d-ribose-l-cysteine are not significantly different when compared with the normal control rats. 3.4 effect of d-ribose-l-cysteine on sperm parameters in stz induced diabetic rats the mean percentage of sperm motility after 28 days of treatment is shown in figure 5. sperm motility in the diabetic control group (35.3±7.5%) was significantly lower (p<0.05) when compared to the normal control group (87.7±9.1%). treatment of diabetic group with d-ribose-lcysteine produced a more pronounced effect with an increased in sperm motility (69.5 ±9.6%). the movement of sperm in d-ribose-l-cysteine treated group were similar to the normal control group (fig. 5). as shown in figure 5, streptozotocin-induced diabetic control group showed a significant decreased (p<0.05) in sperm morphology (37.6±6.3%) as compared with the normal control group (84.2±6.9). there was significantly increased (p<0.05) in mean sperm morphology in d-ribose-l-cysteine treated group and dribose-l-cysteine treated diabetic group (85.90 ± 7.00 and 70.0±10.1) in comparison with the diabetic control group (fig. 5). there was a significant decreased in sperm count value in the diabetic control group (33.5± 7.50, p<0.05) when compared with normal control group ((86.8± 7.70), d-ribose-l-cysteine treated rats (93.7±7.00) and d-ribose-l adedeji and orisadiran; aji, 3(1): 11-22, 2020; article no.aji.53370 15 cysteine treated diabetic rats (75.5± 7.11.8) (fig. 5). 3.5 effect of d-ribose-l-cysteine on reproductive hormones in stz induced diabetic rats testosterone levels decreased significantly (p < 0.05) in diabetic group compared to non-diabetic normal control. there was no significant difference in testosterone levels of animals that received d-ribose-l-cysteine compared with normal control, but it increased significantly (p < 0.05) compared to diabetic control group (fig. 6). there was no significant difference in fsh of normal control and d-ribose-l-cysteine groups; however, there was a significant increase of fsh in animals that received d-ribose-l-cysteine group and d-ribose-l-cysteine diabetic group compared to diabetic group (fig. 6). luteinizing hormone levels increased significantly (p < 0.05) in the d-ribose-lcysteine group compared to diabetic control groups. however, diabetic animals administered d-ribose-l-cysteine had significantly decreased (p < 0.05) levels of lh compared to normal control. fsh decreased significantly (p < 0.05) in the diabetic control group and compared with non-diabetic normal control (fig. 6). nc dc drc drcd 0 50 100 150 200 250 initial final * * ** parameters figure 1: effect of d-ribose-l-cysteine on body weight in stz induced diabetic rats. values are expressed as mean ± sd, * significantly different from normal control group (p<0.05). ** significantly different from normal and diabetic controls (p<0.05). nc: normal control, dc: diabetic control, drc: d-ribose-l-cysteine treated rats, drcd: d-ribose-l-cysteine treated diabetic rats w ei gh t (g ) nc dc drc drcd 0 100 200 300 400 500 initial final * ** figure 2a: effect of d-ribose-l-cysteine on blood glucose in stz induced diabetic rats. values are expressed as mean ± sd, * significantly different from normal control group (p<0.05). ** significantly different from normal and diabetic controls (p<0.05).  significantly different from initial fasting blood glucose (p<0.05) nc: normal control, dc: diabetic control, drc: d-ribose-l-cysteine treated rats, drcd: d-ribose-l-cysteine treated diabetic rats f as ti ng b lo o d g lu co se ( m gd l-1 ) adedeji and orisadiran; aji, 3(1): 11-22, 2020; article no.aji.53370 16 nc dc drc drcd 0 5 10 15 20 ** ** * figure 2b: effect of d-ribose-l-cyste ine on serum glucose in stz induced diabetic rats. values are expressed as mean ± sd, * significantly different from normal control group (p<0.05). ** significantly different from normal and diabetic controls (p<0.05). nc: normal control, dc: diabetic control, drc: d-ribose-l-cysteine treated rats, drcd: d-ribose-l-cysteine treated diabetic rats s er um c on ce nt ra ti on (m m ol l -1 ) 0 2 4 6 8 10 nc dc drc drcd * ** ** mda figure 3: effect of d-ribose-l-cysteine on lipid peroxidation in stz induced diabetic rats. values are expressed as mean ± sd, * significantly different from normal control group (p<0.05). ** significantly different from normal and diabetic controls (p<0.05). nc: normal control, dc: diabetic control, drc: d-ribose-l-cysteine treated rats , drcd: d-ribose-l-cysteine treated diabetic rats c on ce nt ra ti on ( nm ol m l-1 ) tc hdl-c ldl-c tg 0 50 100 150 nc dc drc drcd * ** * * ** ** * * * * * figure 4: effect of d-ribose-l-cysteine on lipid profile in stz induced diabetic rats. values are expressed as mean ± sd, * significantly different from normal control group (p<0.05). ** significantly different from normal and diabetic controls (p<0.05). nc: normal control, dc: diabetic control, drc: d-ribose-l-cysteine treated rats, drcd: d-ribose-l-cysteine treated diabetic rats. tc: total cholesterol, hdl-c: high density lipoprotein cholesterol, ldl-c: low density lipoprotein cholesterol tg: triglycerides c o n ce nt ra ti on ( m gd l -1 ) adedeji and orisadiran; aji, 3(1): 11-22, 2020; article no.aji.53370 17 tt (ngdl-1) fsh (miul-1) lh (miul-1) 0 2 4 6 8 10 nc dc drc drcd * * * ** ** ** ** ** figure 6: effect of d-ribose-l-cysteine on reproductive hormone in stz induced diabetic rats. values are expressed as mean ± sd, * signif icantly different from normal control group (p<0.05). ** signif icantly dif ferent from normal and diabetic controls (p<0.05). nc: normal control, dc: diabetic control, drc: d-ribose-l-cysteine treated rats, drcd: d-ribose-l-cysteine treated diabetic rats. tt: testosterone, fsh: follicle stimulating hormone, lh: lutenizing hormone c o n c e n tr a ti o n 4. discussion diabetes mellitus is a chronic metabolic disease with debilitating complications. two types are mostly described in the literature; type 1(insulindependent diabetes mellitus) and type-2 (insulinresistant diabetes mellitus) [1]. these two interestingly, though have distinct pathogenesis, share similar life-threatening complications such as long-term hyperglycemia, which is associated with many other complications, including male reproductive dysfunctions and infertility [29]. over 90% of diabetic patients are known to suffer from severe insulin resistance, which leads to severe metabolic and reproductive complications [30,31]. hwang et al. [32] and chen et al. [33] in a different study reported the toxic effects of stz on pancreatic beta cells. these effects lead to a decrease in insulin production as well as less cellular absorption of glucose resulting in glucose toxicity, increased ros and beta cells apoptosis. hence, the usage of chemical substances that will aid the cellular sensitivity to insulin consequently ameliorate and prevent glucose toxicity [34]. the present study aims to investigate the ameliorative effect of d-ribose-lcysteine on the blood glucose, lipid peroxidation, lipid profile and reproductive system in adult male wistar rats. kalaiarasi and pugalendi [35] reported a severe loss in body weight in streptozotocin-induced diabetes. the decrease in the bodyweight of diabetic rats in this study was due to the cytotoxic effects of streptozotocin on cells leading to loss or degradation of structural motility (%) morphology (%) sperm count (x106/ml) 0 50 100 150 nc dc drc drcd * ** * ** * ** figure 5:effect of d-ribose-l-cysteine on sperm parameters in stz induced diabetic rats. values are expressed as mean ± sd, * significantly different from normal control group (p<0.05). ** significantly different from normal and diabetic controls (p<0.05). nc: normal control, dc: diabetic control, drc: d-ribose-l-cysteine treated rats, drcd: d-ribose-l-cysteine treated diabetic rats adedeji and orisadiran; aji, 3(1): 11-22, 2020; article no.aji.53370 18 proteins to provide amino acids for gluconeogenesis during insulin deficiency resulting to muscle wasting and weight loss. the protein content is decreased in muscular tissue by proteolysis due to insulin deficiency [36]. in the present study, diabetic control rats showed a marked reduction in their body weights when compared to normal control rats. the weight loss was reversed by the administration of d-ribosel-cysteine to the diabetic rats. in addition, glucose reacts with proteins in a non-enzymatic manner leading to the development of amadori products followed by the formation of advanced glycation end-products ages; ros is generated at multiple steps during this process [37]. elevated lipid peroxidation in tissues will result in a concomitant decrease in body weights [38] as seen in this study. d-ribose-l-cysteine administered maintained the body weights of the animals protecting them from the cytotoxic effects of streptozotocin. these results suggest a possibility of d-ribose-l-cysteine to either improve pancreatic beta cells function or prevent lipid peroxidation by impairing the formation of ros or increasing the production of antioxidants to neutralize ros generated. increased oxidative stress and changes in the antioxidant capacity as observed in this study have been implicated in the etiology of chronic diabetes complications as reported by van et al. [39] and omotayo et al. [38]. this study demonstrated that streptozotocin increased lipid peroxidation. the elevated levels of oxidative stress markers have been associated with hyperglycemia which is due to the shortfall in insulin as a result of beta cell dysfunction [40,41]. this anomaly has been associated with complications including cardiovascular diseases in diabetes mellitus as reported by omotayo et al. [38] and george et al. [42]. these negative effects of streptozotocin were ameliorated by d-ribose-l-cysteine. since streptozotocin is known to destroy pancreatic beta cells leading to a concomitant increase in glucose availabilityhyperglycemia [43], therefore, the possible mechanism of action of dribose-l-cysteine is mediated through influencing glucose uptake or utilization by tissues and probably regeneration of beta cells. hyperglycemia may result in glucose toxicity and increase in ros activity as well as increased lipid peroxidation especially in the pancreas with low antioxidants level [44]. this is in tandem with the increased serum mda levels of the diabetic controls rats in this study. furthermore, the decreased mda levels as seen in animals that received d-ribose-l-cysteine showed an abrogation of cellular redox. in this study, there was an increased in total cholesterol, ldl-cholesterol, triacylglycerides, and low hdl-cholesterol in diabetic rats, confirming the development of dyslipidemia which is supported by previous studies [45,46]. insulin deficiency in diabetes mellitus results in increased lipolysis and subsequent �-oxidation of acetyl-coa, a key enzyme in cholesterol biosynthesis used in lipogenesis together with hmg-coa reductase [46], thereby promoting the hepatic formation of vldl-cholesterol and consequently increasing serum levels of cholesterol and ldl-cholesterol. fatty acids that are not �-oxidated are esterified into triacylglycerols which are incorporated into vldl-cholesterol in the liver and exported to the bloodstream [47]. these metabolic events increased lipoproteins to a value above normal in the present study, identifying dyslipidemia as a risk factor for the development of atherosclerosis [46]. improved dyslipidemia in diabetic rats treated with d-ribose-l-cysteine can be explained by the greater insulin secretion in the presence of this antioxidant, d-ribose-lcysteine, since insulin decreases blood sugar levels and lipolysis in adipose tissue. studies have shown that d-ribose-l-cysteine increases insulin secretion. on the other hand, ayyasamy and leelavinothan [46] revealed that insulin increases the activity of lipoprotein lipase, which catalyzes the breakdown of triacylglycerol ester bonds thereby increasing the clearance of vldlcholesterol. also, the increased insulin level as seen in diabetic control rats elevates the activity of lecithin cholesterol acyltransferase and the enzyme responsible for extracellular cholesterol esterification, thus increasing the efficiency of reverse cholesterol transport, indicating an inverse correlation with cardiovascular accidents [48,46]. studies have identified a relationship between antioxidants and reduced cholesterol levels, due to inhibition of hmg-coa reductase activity and cholesterol biosynthesis [49,46]. thus, the d-ribose-l-cysteine administered in diabetic rats was able to normalize the atherogenic index because it was possible to control the lipid profile, a finding that is corroborated by yang et al. [50], who reported that an increased atherogenic index is related to low antioxidant activity. thus, d-ribose-lcysteine was able to reduce the formation of atherosclerotic plaque by lowering blood glucose adedeji and orisadiran; aji, 3(1): 11-22, 2020; article no.aji.53370 19 levels, the glycation of ldl-cholesterol, and its consequent oxidation in the present study. the increased antioxidant levels by d-ribose-lcysteine in diabetic rats enhanced the production of fsh and lh by the anterior pituitary gland [51] which promotes follicle development and testosterone synthesis. administration of d-ribose-l-cysteine in the study was found to improve the percentage sperm motility and sperm count. the antioxidant activity of this substance may be attributed to the reduction or amelioration of oxidative-stress induced diabetic complications such as lipid peroxidation, by elevation of antioxidant enzyme activities as observed from this study. in addition, previous studies involving treatments with antioxidant compounds demonstrate their importance in regulating βpancreatic cell functions and growth, thereby reducing the complications due to diabetes [52,53]. the antioxidant activities of d-ribose-l-cysteine may also be responsible for the restoration of βcells’ integrity and metabolic functions, while at the same time ensuring maximum synthesis of insulin by these cells necessary for glucose tolerance [52]. therefore, helps to reverse the reproductive dysfunctions associated with diabetes as seen in the present study [54,55]. this study further strengthens previous findings by ballester et al. [56] and suthagar et al. [57] that intraperitoneal administration of high doses of alloxan in male rats induces type-1 diabetogenic conditions, which leads to reproductive complications such as reduced testicular and epididymal weights, decreased testosterone production, reduced sperm motility and sperm counts, and also decrease in the gonadal function of both leydig (testosterone producing) cells and sertoli (supporting) cells. 5. conclusion in conclusion, d-ribose-l-cysteine therefore, revealed in the current study to attenuate the oxidative stress in streptozotocin-induced diabetic rats on blood glucose, lipid profile, spermatogenesis, and steroidogenesis. dribose-l-cysteine could be used as adjuvant therapy for the reduction of atherosclerosis and infertility in diabetic subject because of its potent antioxidant property. consent it is not applicable. ethical approval the protocol of the study was approved by the local ethical committee for animal experimentation of the faculty of basic medical sciences of olabisi onabanjo university, nigeria. acknowledgement the authors especially thank mr. akinrinola and sharphor enterprises for their support during the course of this study. competing interests authors have declared that no competing interests exist. references 1. american diabetes association, diagnosis and classification of diabetes mellitus, diabetes care. 2014;37:s81–s90. 2. bastaki s. review: diabetes mellitus and its treatment. intl j diabetes metab. 2005; 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this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: http://www.sdiarticle4.com/review-history/53370 _____________________________________________________________________________________________________ *corresponding author: e-mail: akibul433@gmail.com; asian journal of immunology 3(1): 269-277, 2020; article no.aji.58128 role and effects of micronutrients supplementation in immune system and sars-cov-2(covid-19) akibul islam chowdhury1* 1 department of food technology and nutrition science, noakhali science and technology university, sonapur-3814, bangladesh. author’s contribution the sole author designed, analyzed and interpreted and prepared the manuscript. article information editor(s): (1) dr. tania mara pinto dabés guimarães, federal university of minas gerais, brazil. reviewers: (1) ghada mostafa lofty el ashy, egypt. (2) ritu ghosh, rabindranath tagore university, india. (3) hanchu dai, university of illinois, usa. complete peer review history: http://www.sdiarticle4.com/review-history/58128 received 31 may 2020 accepted 21 june 2020 published 30 june 2020 abstract covid-19 is a serious disease caused by the virus sars-cov-2 which is easily transmitted to humans. there are no vaccines or drugs discovered yet to control its transmission and prevent the disease. in that case, it is important to find the methods of preventing and controlling it. to fight against the virus a well-functioning immune system is necessary. the immune system is always active but if an individual becomes infected then the activity of immune system is enhanced to fight against the virus and recover the body from infection. adequate intake of micronutrients through diet (vitamins a, b-complex, c, d and zinc, selenium, copper, iron) can help to boost up the immune system as each of the nutrients named above has roles in supporting antiviral and antibacterial defense. although the supplementation of micronutrients can be given, it is necessary to provide the right amount of supplements after assessing the nutritional status of each individual as supplementation of micronutrient have some adverse effects. therefore, it is recommended to follow a healthy diet to prevent covid-19 and also assess the nutritional status of covid-19 patients before prescribing treatments. keywords: micronutrients; immune system; micronutrient supplementation; respiratory tract infection; sars-cov-2; covid-19. review article chowdhury; aji, 3(1): 269-277, 2020; article no.aji.58128 270 abbreviations il : interleukin cd : cluster of differentiation covid-19 : corona virus disease 2019 sars cov-2 :severe acute respiratory syndrome who : world health organization pmnl : polymorphonuclear leukocytes dth : delayed-type hypersensitivity 1. introduction covid-19, a severe acute respiratory syndrome is caused by the virus sars-cov-2 and who declared this as a global pandemic [1]. previous epidemics related to cov included that severe acute respiratory syndrome (sars)-cov was started in china in 2002 [2] and the middle east respiratory syndrome (mers)-cov in the middle east, first reported in 2012 [3]. not all types of people are suffering from this virus. people with weak immune system are much exposed to sascov-2 [4]. there are many recommendations to prevent the spread of covid-19 from the who [5], the uk [6], usa [7] governments and the european commission [8] as well as public health agencies including the key direction of self-isolation [9]. some potential treatment options (anti-viral, anti-malarial, herbals) are also introduced to treat covid-19 patients [10]. nutrition especially micronutrients play a vital role to alleviate both mortality and morbidity in covid-19 as micronutrients boost up our immunity system. nutritional status is an important factor influencing the outcome of patients with covid-19 [11]. in a study, a list of nutrients were identified with possible anticoronavirus effects based on in vitro and clinical studies [11]. several micronutrients have antioxidants feature such as vitamin c, vitamin e, and beta-carotene. antioxidants increase response to influenza virus vaccine compared with placebo and also increase the number of tcell subsets, enhance lymphocyte response to mitogen [12]. the activity of the immune system in our body is accompanied by an increasing rate of metabolism, requiring energy sources, substrates for biosynthesis, and regulatory molecules that are derived from the diet. adequate supply of essential nutrients is required in this pandemic situation [13,14]. this review is based on published data and information and major searches were done using google scholar and pubmed. this review aims to describe the role of specific micronutrients in supporting the immune system and also describes the effect of micronutrients supplementation in this pandemic situation. 2. role of immunity in viral infection when an individual exposed to viral infection, the immunity system becomes vital. first of all, the innate immunity system of the body such as macrophages, monocytes and dendritic cells recognize the foreign particles or pathogens. the presence of pattern recognition receptors (prrs) and microbe-associated molecular patterns (mamps) help in the identification of the pathogens. tlr7 and tlr8 of innate immune system are more important for recognition of corona virus. after viral infection some cells release ifn-α and ifn-β. natural killer cells are directly activated after viral infection to kill the infected cells. the maturation of dendritic cells is induced by prrs signaling which functions to precess and present viral antigen. the presentation of viral antigens is resulted by upregulation of mhc i on virally infected cells which kill the virally affected cells releasing the pore forming protein like perforin. the production of il-2 by cd4+ helper t lymphocytes and t helper 1 phenotype promotes cytotoxic t lymphocytes activity which produce antival antibodies by the differentiation of b lymphocytes to plasma cells. then these antibodies can bind to the viruses and destroy them [15-18]. 3. role of vitamin a in immunity the roles of vitamin a in the immunity system and host susceptibility to infection are identified in many studies [19-23]. the main functions of vitamin a are helping in visions, providing immunity, contributing in gene expression, etc. vitamin a is required for immune cell maturation and functioning as boosting the immune system is the main focus to prevent the spread of covid-19. deficiency of vitamin a may impair barrier functions and immune response. vitamin a and its metabolites help to modulate innate immunity along with barrier function and also control neutrophil maturation. the functions of neutrophil are to ingest and kill bacteria. vitamin a also supports in phagocytic activity to promote bacteria killing. vitamin a helps to increase the activity of natural killer cells which have antiviral defenses. other functions of vitamin a are to control dendritic cell, cd4+ t lymphocyte maturation and maintaining the balance between t helper 1 and t helper 2 lymphocytes. there are various chemical forms of vitamin a and each chowdhury; aji, 3(1): 269-277, 2020; article no.aji.58128 271 of the form has different activity in immune system. movements of t lymphocyte to the gutassociated lymphoid tissue is promoted by retinoic acid and it is also required for cd8+ t lymphocyte survival and proliferation [24]. many systemic reviews supported that vitamin a could reduce the rate of mortality and mobility from measles and improved symptoms in acute pneumonia. a systemic review study is conducted by imdad et al. [25] found that vitamin a decreased all causes of mortality and morbidity from diarrhea and measles and decreased the incidence of diarrhea and measles. another study found that vitamin a decreased morbidity and mortality with pneumonia and also increased the clinical response and shortened length of hospital stay [26]. common sources of vitamin a are liver, eggs, oily fish, fortified margarine, dairy products, carrots, orange fruits, green and yellow vegetables and tomato juice and the recommendation of vitamin a intake for adults is 3000-5000iu [27]. the adverse effects of vitamin a supplementation are identified in table 2. 4. role of vitamin d in immunity vitamin d is an important micronutrient for health because of its function in bone, immune etc. several studies included the role of vitamin d in immune system [28-30]. the active form of vitamin d is 1, 25-dihydroxyvitamin d3 which has important immune-regulatory properties. however, the functions of vitamin d on the cellular components of immunity are rather complex. vitamin d improves antimicrobial peptide synthesis in epithelial tissue [31] and also inhibits the proliferation of t cell, and production of cytokines by t helper 1 lymphocyte. it also has little impact on cd8+ t lymphocyte. low level of vitamin d in the body has been associated with upper respiratory tract infections [32,33] including influenza [34], chronic obstructive pulmonary disease [35] and allergic asthma [36]. many studies showed that supplementation of vitamin d can reduce the risk of viral respiratory tract infection [37] and many meta-analysis studies have been concluded this subjects. according to bergman et al. and martineau et al., vitamin d decreases the risk of respiratory tract infections [38,39]. a systematic review and meta-analysis conducted by zhou et al. [40] concluded that risk of pneumonia had been increased with deficiency of vitamin d. so, vitamin d can be important micronutrients to treat covid-19 patients. the common sources of vitamin d are sunlight, liver, eggs, oily fish, fortified margarine and dairy products and recommendation of vitamin d intake for adults is 400-1000iu [27]. the adverse effects of vitamin d supplementation are identified in table 2. table 1. effects of vitamin and mineral supplementation on immunity immune system vitamins and minerals models effects macrophages c supplementation [41] human stimulation of chemotaxis and phagocytosis e dietary supplementation [42] rat inhibited glomerular sclerosis zinc supplementation [43] human increased il-1, il-6, tnf-α, inf-γ natural killer cell c dietary supplementation [44] human enhance activity e dietary supplementation [45] human enhance activity zinc [46] human enhance activity selenium [47] mouse enhance activity and il-2 protein iron [48] mouse decreased activity pmnl b12 supplementation [49] human increased bacterial killing c supplementation [50] human enhanced phagocytosis, decreased superoxide production c,e supplementation [51] human suppressed production of oxygen free radical copper, zinc and iron supplementation [52-54] human increased neutrophil function dth c supplementation [55] human increased e supplementation [56] human increased t cell c, e and a supplementation [57] human increased zinc supplementation [58] human increased t lymphocyte abbreviations: pmnl: polymorphonuclear leukocytes, dth: delayed-type hypersensitivity chowdhury; aji, 3(1): 269-277, 2020; article no.aji.58128 272 table 2. adverse effects of vitamin and mineral supplementation [27,59-61] vitamin and minerals adverse effects associated with supplements vitamin a hepatotoxic effects, visual change, hair and skin change, risk of lung cancer among smokers, diarrhea vitamin b6 sensory neuropathy, ataxia vitamin b12 no upper limit unknown vitamin d hypocalcaemia, soft tissue calcifation vitamin c diarrhea, gastric vitamin e nausea, vomiting, diarrhea, headache, fatigue, blurred vision zinc nausea and vomiting, immune-suppression and impaired copper uptake selenium brittle hair and nails, peripheral neuropathies and gastrointestinal upset iron nausea, vomiting, reduced zinc uptake and constipation 5. role of vitamin c in immunity vitamin c is a water soluble vitamin which has antioxidant properties. it plays a major role in the immune system of the human body. it is required for collagen biosynthesis and also helps in the migration of leucocytes to the sites of infection [62]. other roles of vitamin c in immunity are phagocytosis, bacteria killing, and natural killer cells activity [62,63]. vitamin c supplementation enhances the activity of natural killer cells [44]. variety of studies stated that the severity of upper respiratory tract infections can be lowered by vitamin c supplementation. a meta-analysis stated that vitamin c decreased the incidence of pneumonia as well as reduced severity and morbidity from pneumonia [64]. for its functional properties, many doctors and researchers suggested to increase the intake of vitamin c to boost up the immunity and to fight against the corona virus. the common sources of vitamin c are citrus fruits, broccoli, kiwi, yams, strawberries, melons and recommendation of vitamin c intake for adults is 60-90mg [27]. the adverse effects of vitamin c supplementation are identified in table 2. 6. role of vitamin b-complex in immunity b vitamins are mainly involved in intestinal immune regulation and help in the gut-barrier functions. folic acid increases the number of circulating t lymphocyte, but the activity of neutrophils appears unchanged. vitamin b12 increases the phagocytic and bacteria killing capacity of neutrophils while vitamin b6 helps in the lymphocyte proliferation and increases the number of t lymphocyte in the blood. folic acid, vitamin b6 and b12 enhanced the activity of natural killer cells which would be important in antiviral defense [65,66]. a study found that deficiency of vitamin b6 decreased the t lymphocyte and b lymphocyte proliferation and il-2 production [67]. the common sources of vitamin b complex are poultry, fish, meat, nuts, legumes, whole grains, potatoes, meat, egg, seaweed etc and the recommendation of vitamin b6 and b12 for adults are 1.3-2 mg and 2.4-6 μg respectively [27]. the adverse effects of vitamin b complex supplementation are identified in table 2. 7. role of vitamin e in immunity vitamin e acts as a scavenger of free radical by blocking the per-oxidation of polyunsaturated fatty acids (pufa) and also acts as antioxidants. it is suggested that vitamin e is an important nutrient in the immune system [68]. due to vitamin e deficiency, t-cell mitogenesis, il-2 production, pmn phagocytosis, and pmn chemotaxis are decreased [56]. it is found that there is a positive association between vitamin e and cell-mediated immune responses and vitamin e supplementation appears to be beneficial for adults people [69]. vitamin e supplementation also enhances the activity of helper t lymphocyte and improves vaccine responses [70]. several studies identified that vitamin e supplementation reduced the risk of respiratory tract infections and decreased the duration of respiratory tract infections among adults [71]. the common sources of vitamin e are plant oils (soya, corn, olive), nuts, seeds, wheat germ and the recommendation of vitamin e for adults people is 15-20 mg [27]. supplementation of vitamin e also has some adverse effects which are identified in table 2. 8. role of zinc in immunity in this covid-19 situation, zinc is considered as a supportive treatment therapy as it has direct antiviral effects [72]. it is found that zinc supplementation may have positive effects in the chowdhury; aji, 3(1): 269-277, 2020; article no.aji.58128 273 treatment of covid-19 patient [73]. the role of zinc in immunity is explained in many studies [74-76]. zinc deficiency can cause loss of t helper cells and also responsible for atrophy of thymus and spleen. zinc increases the igm plaque response and igg response. cell mediated immunity is also increased with zinc supplementation. t lymphocyte proliferation is also maintained by proper dietary intake of zinc [77]. some meta-analysis found that zinc decreased the prevalence and incidences of pneumonia as well as duration of common cold [78,79]. the common sources of zinc are meat, cheese, cereals and grains, shellfish etc. 9. role of selenium in immunity selenium has important effect on both innate and acquired immunity. selenium enhances the function of t lymphocyte and b lymphocyte and also increases the activity of natural killer cell [80,81]. a study found that selenium supplementation improved immune function in the human body [82]. the common sources of selenium are fish, meat, egg and nuts. supplementation of selenium also has some adverse effects on the body. 10. role of copper in immunity some studies described the importance of copper in the immune system. copper has antimicrobial properties, and also increases the activity of natural killer cell and neutrophils, monocyte function [83,84]. some studies showed that lymphocyte proliferation and il-2 production are promoted by copper [85]. a systematic review found that respiratory tract infection can be reduced by copper supplementation [86]. the common sources of copper are nuts, shellfish and some vegetables. 11. conclusion nutrition is very important for every steps of life and proper intake of both micronutrients and macronutrients can help to prevent and treat several infectious diseases. all nutrients named above have special role in immune system of the body, and consumption of adequate amount nutrients is necessary to boost up our immune function. immune system can also be boosted up by supplementation of these nutrients. however, there are some adverse effects of micronutrients supplementation in case of overdosing. many studies showed both positive and negative effects of micronutrients supplementation on immune response and human health. however, there are no published studies which described the function of micronutrients for the prevention and treatment of corona-virus but some studies showed that intake right amount of micronutrients can reduce the duration of the disease severity. consent it is not applicable. ethical approval it is not applicable. competing interests author has declared that no competing interests exist. references 1. sohrabi c, alsafi z, o’neill n, khan m, kerwan a, al-jabir a, et al. world health organization declares global emergency: a review of the 2019 novel coronavirus (covid-19). international journal of surgery; 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asian journal of immunology 2(1): 72-77, 2019; article no.aji.53137 malaria-related anaemia among children and adolescents attending general hospitals in obi and oju local government areas of benue state v. u. obisike1*, o. j. agbo2, m. o. ogbu3, j. i. chikwendu1 and e. u. amuta1 1 department of animal and environmental biology, abia state university, uturu, abia, nigeria. 2 department of biological sciences, federal university wukari, nigeria. 3 department of biological sciences, benue state university, makurdi, nigeria. authors’ contributions this work was carried out in collaboration among all authors. authors vuo, moo and eua designed the study, performed the statistical analysis, wrote the protocol and wrote the first draft of the manuscript. authors oja and jic managed the analyses of the study. authors vuo, moo and eua managed the literature searches. all authors read and approved the final manuscript. article information editor(s): (1) dr. wagner loyola, department of immunology, brazilian agricultural research corporation (embrapa), concordia, canada. reviewers: (1) nwagu marcellinus uchechukwu, edo university, nigeria. (2) esraa ashraf ahmed elhawary, ain shams university, egypt. (3) florencia del puerto, universidad nacional de asuncion, paraguay. complete peer review history: http://www.sdiarticle4.com/review-history/53137 received 08 october 2019 accepted 13 december 2019 published 25 december 2019 abstract anaemia is one of the leading complications due to malaria infections. it is defined in terms of packed cell volume (pcv) as a pcv <32%. this study was focused on the prevalence of malariarelated anaemia among children and adolescents attending general hospital in obi and oju local government areas of benue state. whole blood samples for malaria test were collected by fingertip pricking and vein punctured from a total of 738 children and adolescent patients that visited these hospitals between the months of october, 2015 to february, 2016. rapid diagnostic test kit (rdt) and haematocrit reader were used to determine malaria infections and pcv respectively. those tested positive for malaria were 207 (106 and 101 for obi and general hospitals respectively). positive participants were further tested for anaemia and 73 (35.2%) were found anaemic. distribution in the general hospitals were 34 (46.6%) and 39 (53.4%) for obi and oju original research article obisike et al.; aji, 2(1): 72-77, 2019; article no.aji.53137 73 respectively. october, 2015 had the highest prevalence. amongst those anaemic females had a higher prevalence of 43 (58.9%) than males 30 (41.1%), (r=0.95). also age group 0-5 years were most anaemic, 31 (53.4%), (p>0.05). conclusively, the prevalence of malaria-related anaemia among children and adolescents attending these hospitals of the study areas was high. reducing the prevalence of malaria should be prioritized by individuals and government. keywords: anaemia; rapid diagnosis; children; malaria. 1. introduction the tropics and developing countries have been faced with severe public health problems which anaemia is one of the health challenges facing the area. anaemia occurs at all stages of the life cycle, but is more prevalent in young children and pregnant women [1] about 3.5 billion persons are affected by anaemia in developing countries [2] loss of blood, decrease in the red blood cells production, and increase in red blood cells breakdown [3] could lead to anaemia. plasmodium falciparum infection is considered the major cause of anaemia, especially in children. it retards the normal development in children, and constitutes a major public health problem in young children in the developing world with wide social and economic implications. therefore decreased physical exercise tolerance and intellectual performance have been associated with mild anaemia, which may lead to a slowdown of growth in children [4]. a lot of work has been done on malaria such as in the areas of prevention and control, clinical and laboratory diagnosis, treatment, pathology and pathogenesis, epidemiology and genetic sequencing, yet the disease is still endemic in the tropics and developing nations, causing so much pain and sorrow to several families on the african continent and beyond [5]. malaria is caused by parasites that are transmitted to people through the bites of infected female mosquitoes; p. faciparum is the most deadly malaria parasite and the most prevalence [1]. about 70% of the disease burden worldwide and her pregnant women, children are found in africa [6]. the death accounts for 3-5 million each year and majority are children below five years, infected mainly by p. falciparum. malaria associated anaemia is a major health problem in setting of intense malaria transmission [7]. the current study was aimed to investigate regarding the malariarelated anaemia in children and adolescents attending general hospitals in obi and oju local government areas of benue state. 2. materials and methods 2.1 study areas the study areas for this project work were obi and oju local government areas of benue state of north-central, nigeria. they are both located in the savannah zone. obi local government area is found in the coordinates 8º22’n and 8º46’e [3]; her local government headquarter been obarike-ito, with 2006 population census of 9,707 people. oju on the other hand is located in the coordinates 6º51’n and 8º25’e [3]; with oju as her local government headquarter, 2006 population census of 168,491. the people of the two local government areas speak igede as their major dialect; and their major occupation is farming. 2.2 study population and sample size the study was conducted among the patients (children and adolescents) attending general hospital obarike-ito, in obi local government area and general hospital oju, in oju local government area. a total of 763 whole blood samples were collected from different patients that attended the hospitals from october 2015 to february 2016. amongst these samples collected, 426 and 337 were obtained in obi and oju respectively. malaria test was carried out, and followed by packed cell volume (pcv) test on the samples that were tested positive to malaria. 2.3 experimental design the experimental design used was variance, particularly completely randomized design; from which anova table was done for analysis. also chi-square used as well as correlation. the methods employed in this study involved two haematological tests – chromatographic test (rapid diagnostic test), for detecting evidence of malaria parasites (antigens) in human blood; and packed cell volume (pcv) tests for anemia. obisike et al.; aji, 2(1): 72-77, 2019; article no.aji.53137 74 fig. 1. map of obi and oju l.g.as showing the study locations [8] packed cell volume (pcv) also called hematocrit – the proportion of blood volume that is occupied by erythrocytes (the ratio of red blood cells to the whole blood volume, usually expressed in percentage) was determined with the use of hematocrit centrifuge and microhematocrit reader. anaemia was defined as pcv of <30%. mild anaemia pcv: 25-29% moderate anaemia pcv: 19-24% and severe anaemia pcv: ≤18%. whole blood sample was used for the two tests, (malaria and pcv) involved. the blood was either gotten through pricking of fingertip or by venous puncture. fingertip pricking: the finger of a patient’s hand (mostly third finger of the left hand) was firmly held and cleansed using alcohol pad. a sterile lancet was then used to prick the fingertip, and blood drop was collected in a micro pipette; ready to be used [3]. venous blood: the patient’s arm was tightened with a tunicate and palpated for vein to become prominent. the skin surface of over the prominent vein was cleansed using alcohol pad or swab, then allowed to dry. the vein was punctured with a sterile needle, attached to a syringe. whole blood was then collected in the syringe and transferred into an edta tube; ready to be used [3]. 2.4 test procedures malaria rapid diagnostic test: the whole blood, collected in a micropipette by fingertip pricking (as described above), was transferred onto the sample pad on malaria immunoassay test kit. immediately, two drops reagent was applied onto the reagent pad, directly above sample pad; and allowed to run for 15 minutes. appearance of two pink strips or bands was taken to be an indication that the patient is infected with either plasmodium falciparum or one of the other three species responsible for human malaria [9]. for hematocrit (packed cell volumme-pcv) test: the whole blood collected in edta tube (as described above) was fed into a capillary tube (2/3 half-filled), from one end. the other end of the capillary tube was sealed with a sealant and mounted onto the micro-haematocrit centrifuge and centrifuged for 3 minutes at 10,000 rpm (revolution per minute). the capillary, having the packed erythrocytes after centrifugation was then dismounted from the centrifuged and placed on the haematocrit reader, and the reader, and the reading was taken and recorded. 3. results from the months of october, 2015 to february, 2016, a total of 738 blood samples were obisike et al.; aji, 2(1): 72-77, 2019; article no.aji.53137 75 collected from children and adolescents for malaria test; out of which 207 were tested positive for malaria tables 1 and 2. in general hospital, obi, a total number 106 was obtained; while general hospital, oju had a total number of 101; with the prevalence of 24.9% and 30.0% respectively. among the 207 samples tested positive to malaria, 73 were tested anaemic, with percentage prevalence of 35.2%. also, obi had a number of 34 amongst those tested anaemic; while oju had a number of 39, with prevalence of 32.1% and 38.6% respectively. from the fig. 2, october, 2015 had the highest prevalence of 50.7%; while december, 2016 had the lowest prevalence of 9%; with p>0.05. fig. 3 shows females prevalence to be 43 (58.9%), as against 30 (41.1%) for males, with chi-square value showing p>0.05. table 3 showed that age range 0 – 5 had the highest prevalence of 40 (54.8%); while age range 12 – 17 has the lowest prevalence of 14 (17.8%). but variance analysis showed that p>0.05; which revealed a nonsignificant difference in the prevalence between the age ranges. table 1. monthly distribution of malaria-related anaemia in obi month no examined positive for malaria anaemia october 98 39 (36.8%) 12 (35.8%) november 65 18 (17.0%) 5 (14.7) december 75 22 (20.8%) 7 (20.6%) january 96 17 (16.0%) 7 (20.6%) february 92 10 (9.4%) 3 (8.8%) total 426 106 (24.9%) 34 (32.1%) table 2. monthly distribution of malaria-related anaemia in oju month no examined positive for malaria anaemia october 162 64 (63.4%) 25 (64.1%) november 55 18 (17.8%) 7 (17.9) december 20 3 (3.0%) 0 january 47 7 (6.9%) 3 (7.7%) february 53 9 (8.9%) 4 (10.3%) total 337 101 (30.0%) 39 (38.6%) fig. 2. prevalence for malaria-related anaemia of each month for obi and oju l.g.as r = 0.96; x 2 cal = 1.05. p>0.05; d.f = 4 obisike et al.; aji, 2(1): 72-77, 2019; article no.aji.53137 76 table 3. the prevalence of malaria-related anaemia with respect to age (years) month 0 – 5 6 – 11 12 17 october 17 (42.5%) 10 (50%) 4 (30.8%) november 8 (20.0%) 2 (10%) 2 (15.4%) december 4 (10.6%) 4 (20.0%) 2 (15.4%) january 5 (12.5%) 3 (15.0%) 4 (30.8%) february 6 (15%) 1 (5.0%) 1 (7.6%) total 40(54.8%) 20 (27.4%) 13 (17.8%) grand total: 73 f – calculated = 1.98; p>0.05 fig. 3. prevalence for malaria-related anaemia with respect to sex r = 0.95; x 2 cal = 6.33; p>0.05; d.f = 4 4. discussion this work revealed the prevalence of malariarelated anaemia among the patients attending general hospitals in obi and oju local government areas of benue state. the result is higher than that reported in port harcourt [10], nigeria; but lower compared to previous report in kano, nigeria [11] and benin republic and mali [12]. the result however, agrees with earlier report in the brazil [13]. the reason could be the environmental conditions of the area, such factors like bushes around the residential areas, the sanitary conditions of the locality and belief. october, 2015 had the highest prevalence. this could be due to the fact that october was partly a rainy season; and rainy season favours breeding of malaria vector (mosquito). age distribution revealed that age range 0 – 5 years had the highest prevalence, but statistical analysis revealed a non-significant difference in the prevalence between the age groups. this is lower than the report of imam, [11] in kano, nigeria, but higher than that of earlier study in vietnam, [14]. the high prevalence recorded in this age group could be attributed to the active nature of the group which makes them often play outside leaving their bodies uncovered, thereby exposing themselves to mosquito bite. with respect to sex, female population had the higher prevalence for anaemia, however, statistical analysis revealed a non-significant difference in the prevalence between the two sexes; as well as a very close correlation, unlike the previous finding [7], in kenya. this could also be attributed to the fact that most women in the developing nations do a whole lot of outdoor activities ranging from farm work to domestic works, making them equally exposed to mosquitoes. obisike et al.; aji, 2(1): 72-77, 2019; article no.aji.53137 77 5. conclusion this study revealed a high prevalence of malariarelated anaemia among children and adolescents attending the hospitals in obi and oju l.g. areas, benue state. it is an indication that anaemia as a complication due to malaria infection is a threat to the studied population of these areas. the prevalence of anaemia was highest among the females, and among age group 0 to 5 years. though the prevalence of was high in both local government areas, oju had a higher prevalence than obi. consent as per international standard, patient’s written consent has been collected and preserved by the author(s). ethical approval ethical permission was granted by the hospital management. competing interests authors have declared that no competing interests exist. references 1. who. malaria fact sheet; 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2013. available: www.maps oju and obi local gov benue state 9. cdc; 2014. available: www.cdc.gov/malaria/diagnosis treatment/rdt.html 10. george io, otaigbe be. anaemia in critically ill children. a case study from nigeria. international journal of tropical disease and health. 2012;2(1):79-84. 11. imam ts. anaemia and malaria in children attending two selected paediatric clinics in kano metropolis, northern nigeria. international journal of biomedical and health sciences. 2009;5(3):18-22. 12. ngnie-teta i, receveur o, kuate-defo b. risk factors for moderate to severe amaemia among children in blenin and mali: insights from a multilevel analysis. food and nutrition bulletin. 2007;28(1): 12-18. 13. leal pl, filhol mb, cabral de liral pi, figueiroa jn, osόrio mm. prevalence of anemia and associated factors in children aged 6-59 months in pernambuco, northeastern brazil. rev saúde pública. 2011;45(3):22-27. 14. nguyen ph, nguyen kc, le mai b, nguyen tv, ha kh, bern c. risk factors for anemia in vietnam. southeast asian journal of tropical medicine and public health. 2006;37(6):329-348. _________________________________________________________________________________ © 2019 obisike et al.; this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: http://www.sdiarticle4.com/review-history/53137 http://www.who.int/mediacentre/factshets/fs094/en/on http://www.who.int/mediacentre/factshets/fs094/en/on http://www.researchgate.net/publication/221928490 http://www.researchgate.net/publication/221928490 http://www.maps/ http://www.cdc.gov/malaria/diagnosis%20treatment/rdt.html http://www.cdc.gov/malaria/diagnosis%20treatment/rdt.html http://creativecommons.org/licenses/by/4.0 _____________________________________________________________________________________________________ *corresponding author: e-mail: xjg71@163.com; asian journal of immunology 3(1): 160-165, 2020; article no.aji.56639 polyphenol and flavonoids content and antioxidant activity of different solvent extracts from artemisia argyi dong-qin wang1,2, jing yang2 and jianguo xu1,2* 1 modern college of humanities and sciences, shanxi normal university, linfen 041004, china. 2 school of food sciences, shanxi normal university, linfen 041004, china. authors’ contributions this work was carried out in collaboration among all authors. author dqw designed the study, performed the statistical analysis, wrote the protocol and wrote the first draft of the manuscript. authors jy and jx managed the analyses of the study. author jx managed the literature searches. all authors read and approved the final manuscript. article information editor(s): (1) dr. wagner loyola, brazilian agricultural research corporation (embrapa), brazil. reviewers: (1) douati togba etienne, université felix houphouet boigny, côte d’ivoire. (2) yves latune, university of abidjan, côte d’ivoire. complete peer review history: http://www.sdiarticle4.com/review-history/56639 received 27 february 2020 accepted 04 may 2020 published 11 may 2020 abstract the effects of extraction solvents (methanol, ethanol, acetone, petroleum ether and n-hexane) on the content of total polyphenols and flavonoids, as well as antioxidant activities of artemisia argyi were investigated. the results showed that, the ethanol extract had the highest total polyphenols and flavonoids content, and it exhibited stronger antioxidant activities, followed by methanol and acetone extracts. correlation analysis revealed that the content of phytochemicals was well correlated with antioxidant activities of extracts from artemisia argyi, which indicates that different solvents had a great influence on the level of total polyphenols, flavonoids and antioxidant activities of extracts. therefore, selective extraction from artemisia argyi, by an appropriate solvent, is important for obtaining fractions with high antioxidant activity, which will be useful for the developing and application of artemisia argyi. keywords: artemisia argyi; extraction solvent; polyphenols; flavonoid; antioxidant activity. short communication wang et al.; aji, 3(1): 160-165, 2020; article no.aji.56639 161 1. introduction artemisia argyi (a. argyi) is known in chinese as “aicao” and in japanese as “gaiyou” and belongs to the arteraceae family that comprises over 500 species [1], which is mainly distributed in asia, europe and north america [2]. as a traditional medicinal and edible plant, ancient chinese commonly pick the buds and leafs of a. argyi consumed as a food supplement or a flavoring and colorant for the chinese dish qingtuan [3]. a. argyi also contains many active compounds such as volatile oil [1], flavonoids [4], phenols, organic acids [4], polysaccharides [5], terpenoids [6] and glycosides [4,7-9]. several studies have reported that a. argyi extracts possess various biological activ ities, such as antioxidant [8], anti-inflammatory [10], antibacterial, anti-tumor [11], hemostatic, antihypertensive effects, immunoregulatory, antiasthmatic, oncogene inhibitory and antiosteoporotic activities [7,8,12,13]. extract solvents is one of the most important factors affecting the chemical composition and biological activity of plant extracts [14,15]. therefore, the objective of this work was to investigate the content of total polyphenols, flavonoids and antioxidant activity of different solvent extracts (methanol, ethanol, acetone, petroleum ether, nhexane) from artemisia argyi. 2. materials and methods 2.1 preparation of extracts fresh leaves of artemisiae argyi were collected around the fifth day of the fifth lunar month in 2019 from linfen (shanxi province) and were dried at 40°c. the dried leaves (100 g) were grounded and were extracted with 250 ml solvents (methanol, ethanol, acetone, petroleum ether, n-hexane) and kept in a shaker at 25°c for 1 h. and then the mixture was centrifuged at 4000 rpm/min for 15 min at 4°c. the precipitation was extracted with 250 ml solvent once again and mixed supernatants. the extracts were dried under vacuum and stored at 4°c until analysis. 2.2 determination of polyphenolic content polyphenolic content of extracts was determined according to method described by bettaieb et al. with some modifications [14]. briefly, an aliquot (0.1 ml) extract was mixed with 2.8 ml of distilled water and 0.1 ml of folin-ciocalteu reagent (1.0 mol/l), and then the solution was mixed and incubated at room temperature (25°c) for eight minutes. following that, 2 ml of 7.5% sodium carbonate (na2co3) solution was added and shaken thoroughly. the mixture was incubated for 2 h in the dark at room temperature (25°c) and the absorbance was determined at 765 nm. gallic acid was used for calibration of the standard curve and total phenolic content was expressed as milligram gallic acid equivalent per gram dried weight. 2.3 determination of flavonoid content an aliquot (1 ml) extract was mixed with 0.3 ml of 3% nano3 solution and incubated for 6 min. after that, 0.3 ml of 10% al(no3)3 was added and the solution was kept at room temperature (25°c) for 6 min. finally, mix the solution with 4 ml of 4% naoh solution and add water to 10 ml. after 20 minute of incubation, the absorbance of the mixture at 510 nm was measured. rutin was used for calibration of the standard curve, and flavonoid content was expressed as milligram rutin equivalents per gram dried weight. 2.4 dpph radical scavenging assay dpph radical scavenging assay was measured the method described by xu et al. with some modifications [16]. extract was serially diluted to different concentrations and 0.5 ml of diluted extract mixed with 2.5 ml of 60 μmol/l dpph solution dissolving in methanol. the mixture was shaken thoroughly and incubated in the dark at room temperature (25°c) for 30 min, and the absorbance was measured at 517 nm. trolox was used for calibration of the standard curve and the dpph radical scavenging activity of sample was expressed as milligram trolox equivalent per gram dried weight (mg trolox/g). 2.5 abts radical scavenging assay abts radical scavenging assay was determined according to the method described by xu et al [16]. briefly, a certain quality of abts and potassium persulfate was dissolved in water to keep the final concentrations of the two substances to be 7 mmol/l and 2.45 mmol/l respectively. the mixture was kept in the dark for 16~24 h to make the abts radical working solution and its absorbance at 734 nm was adjusted to 0.700±0.050. the abts radical scavenging ability was measured by adding 50 μl of diluted extract to 1.9 ml of abts radical working solution and the absorbance at 734 nm after 6 min was recorded. trolox was used for app:ds:rutin app:ds:rutin app:ds:potassium app:ds:persulfate wang et al.; aji, 3(1): 160-165, 2020; article no.aji.56639 162 calibration of the standard curve and the abts radical scavenging activity of sample was expressed as milligram trolox equivalent per gram dried weight (mg trolox/g). 2.6 ferric reducing antioxidant power (frap) assay the reducing ability was determined as described by xu et al [16]. briefly, the frap reagent was freshly prepared from 300 mm sodium acetate buffer (ph 3.6), 10 mm 2,4,6tri(2-pyridyl)-s-triazine (tptz) solution in 40 mm hcl and 20 mm fecl3 solution in proportions of 10:1:1 (v/v), respectively. the frap reagent was prepared fresh daily and was warmed to 37 o c in a water bath prior to use. then 0.1 ml sample solution was mixed with 1.8 ml frap reagent and 3.1 ml ultra-pure water. the absorption of the reaction mixture was measured at 593 nm after incubation for 30 min at 37 o c. the standard curve was constructed using feso4 solution (100-1000 μm), and frap value was expressed as micromoles fe(ii) per gram dw. 3. results and discussion 3.1 contents of total polyphenols and flavonoids the contents of phytochemicals of different solvent extracts were showed in fig. 1. depending on the solvent used, the total polyphenol content ranged from 6.58 to 25.6 mg gae/g. ethanol extract had the highest polyphenol content, followed by methanol, acetone, the lowest for petroleum ether and nhexane. however, no significant difference in total phenolic content was found between petroleum ether and n-hexane extracts. the range of flavonoid content in different solvent extracts was 12.6-40.2 mg re/g and their order corresponded to the same order in the polyphenol content. generally speaking, differences in the content of total polyphenols and flavonoids from different solvent extracts may be come from differences in the polarity of solvents. however, there was not a one-to-one relationship between the polarity of solvent and the content of phytochemicals. these differences in the content of total phenolics and flavonoids from different extracts may be come from differences in the polarity, chemical structure, dielectric constant of solvents [17], which can influence the extraction efficiency and compositions of extracts. 3.2 dpph and abts radicals scavenging abilities of different extracts due to the differences in the compositions and contents of the phytochemicals, the extracts showed the different antioxidant abilities. table 2 showed that there were significant differences of dpph radical scavenging abilities of different solvent extracts. consistent with the results in phytochemical test, ethanol extract owned the highest dpph radical scavenging ability, followed by methanol and acetone extracts. petroleum ether and n-hexane extracts had lowest dpph radical scavenging ability, of which were 0.63 and 0.86 mg trolox/g respectively. but there was no significant difference between petroleum ether and n-hexane extracts. the results of abts radical scavenging abilities were similar to that of dpph assay. the difference was that there was no significant differences between methanol and ethanol extracts. 3.3 ferric reducing antioxidant power (frap) table 1 also showed that different extracts from artemisia argyi exhibited different reducing power. there was a significant difference (p < 0.05) in the reducing power among different extracts, and ranged from 16.7 to 55.6 μmol fe(ii)/g dw for n-hexane, petroleum ether, acetone, ethanol and methanol extracts, respectively. as observed in the dpph and abts, the reducing power of methanol extracts was the highest, followed by ethanol, acetone, and petroleum ether extracts, the lowest for hexane extracts. the results suggested that extracts from artemisia argyi had a potency to donate electron to reactive free radicals, converting them into more stable non-reactive species and terminating the free radical chain reaction [16]. 3.4 correlation among total phenolics, flavonoids and antioxidant activities to further investigate their interrelationship, the correlation between the active compounds and biological activities were established, and correlation coefficients (r) are shown in table 2. in this study, the dpph and abts were highly and positively correlated to the content of both total phenolics and flavonoids (r ≥ 0.893), indicating total phenolics and flavonoids are the main constituents contributing to the antioxidant wang et al.; aji, 3(1): 160-165, 2020; article no.aji.56639 163 fig. 1. the contents of total phenolics and flavonoids of extracts from artemisia argyi table 1. the antioxidant activities of different extracts from artemisia argyi dpph (mg trolox/g) abts (mg trolox /g) frap (μmol fe(ii)/g) petroleum ether 0.63±0.06d 2.88±0.63c 18.6±1.3d n-hexane 0.86±0.16d 2.55±0.52c 16.7±1.4 d acetone 2.76±0.42c 6.84±0.73 b 31.5±1.9 c ethanol 6.93±0.44a 14.25±1.42a 48.5±2.2 b methanol 4.35±0.52b 12.72±1.57 a 55.6±3.1 a table 2. correlation analysis of polyphenols, flavonoids and antioxidant activities phenolics flavonoids antioxidant activities dpph abts frap phenolics 1 flavonoids 0.964 * 1 dpph 0.992 ** 0.988 * 1 abts 0.930 0.893 0.935 1 frap 0.557 0.501 0.568 0.821 1 values are correlation coefficient r. a significantly different: ** p < 0.01, * p < 0.05 and antibacterial activities of extracts, which was supported by previous reports studied on some cereals or plants [18,19]. however, frap was moderately correlated to the content of phenolics and flavonoids. 4. conclusions plant polyphenols and flavonoids have been considered as one of the most widely distributed secondary metabolites which contains various complicated compounds. based on the principle of the dissolution in the similar material structure, different solvent extracts differ in its active constituents as well as their contents due to the different polarity and solubility of extracting solvents. our findings indicated that ethanol extract had the highest content of polyphenol and flavonoid among all of five solvent extracts. consistent with results described above, ethanol extract showed the highest antioxidant abilities in dpph, abts radicals scavenging and frap assays. these results indicated that selective extraction from artemisia argyi, by an appropriate solvent, is important for the development and utilization of artemisia argyi. consent it is not applicable. d d c b a b d c d a 0 10 20 30 40 50 0 10 20 30 40 50 petroleum ether hexane acetone ethanol methanol f la v o n o id s (m g r e /g d w ) p o ly p h en o ls ( m g g a e / g d w ) extracts polyphenols flavonoids wang et al.; aji, 3(1): 160-165, 2020; article no.aji.56639 164 ethical approval it is not applicable. acknowledgements this work was financially supported by a project of the natural science foundation of modern college of humanities and sciences of shanxi normal university, china (project no. 2019jcyj18). competing interests authors have declared that no competing interests exist. references 1. abad mj, bedoya lm, apaza l, bermejo p. the artemisia l. genus: a review of bioactive essential oils. molecules. 2012;17(3):2542–2566. 2. bora ks, sharma a. the genus artemisia: a comprehensive review. pharm biol. 2011;49(1):101–109. 3. he x. study on argy wormwood food production and processing. j anhui agr sci. 2004;32(5):995–996. 4. han bs, xin zq, ma ss, liu wb, zhang by, ran l, yi lz, ren db. comprehensive characterization and identification of antioxidants in folium artemisiae argyi using high-resolution tandem mass spectrometry. j chromatogr b. 2017;1063: 84–92. 5. zhang p, shi b, li t, xu y, jin x, guo x, yan s. immunomodulatory effect of artemisia argyi polysaccharide on peripheral blood leucocyte of broiler chickens. j anim physiol anim nutr. 2018;102(4): 939–946. 6. yoshikawa m, shimada h, matsuda h, yamahara j, murakami n. bioactive constituents of chinese natural medicines. i. new sesquiterpene ketones with vasorelaxant effect from chinese moxa, the processed leaves of artemisia argyi levl. et vant.: moxartenone and moxartenolide. chem pharm bull. 1996;44(9):1656–1662. 7. song xw, wen x, he jw, zhao h, li sm, wang my. phytochemical components and biological activities of artemisia argyi. j funct foods. 2019;52:648–662. 8. xiao jq, liu wy, sun hp, li w, koike k, kikuchi t, yamada t, li d, feng f, zhang j. bioactivity-based analysis and chemical characterization of hypoglycemic and antioxidant components from artemisia argyi. bioorg chem. 2019;92:103268. doi: 10.1016/j.bioorg.2019.103268 9. zhang lb, lv jl, chen hl, yan xq, duan ja. chemical constituents from artemisia argyi and their chemotaxonomic significance. biochem syst ecol. 2013; 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asian journal of immunology 3(1): 166-172, 2020; article no.aji.56699 ameliorative effects of turmeric extract against ccl4 induced liver and kidney injury in adult wistar rat e. u. eric1* and e. adolphus2 1 department of medical laboratory sciences, niger delta university, wilberforce island, bayelsa state, nigeria. 2 department of histopathology, federal medical center, yenagoa, bayelsa state, nigeria. authors’ contributions this work was carried out in collaboration between both authors. author eue designed the study, performed the statistical analysis, wrote the protocol, and wrote the first draft of the manuscript. authors eue and ea managed the analyses of the study. author ea managed the literature searches. both authors read and approved the final manuscript. article information editor(s): (1) dr. cynthia aracely alvizo báez, autonomous university of nuevo leon, mexico. reviewers: (1) gayatri c. gawade, bharati vidyapeeth deemed university, india. (2) dharma lindarto, universitas sumatra utara, india. complete peer review history: http://www.sdiarticle4.com/review-history/56699 received 25 february 2020 accepted 02 may 2020 published 13 may 2020 abstract objectives: to examine and ascertain the toxic effects of ccl4 on the histology of the liver and kidney and to investigate the ameliorative of turmeric against his injury. materials: thirty (30) male adult wistar rats weighing 150 ± 2.6 g 317 ± 3.5 g were procured from the animal house of the department of pharmacology, college of health sciences niger delta university, bayelsa state. nigeria, assigned into five (5) major groups with five (5) animals in each group after the period of acclimatization: a control group "a" and three test groups (c, d and e) except group b with ten (10) rats. animals in group a (control): received pelleted growers mash (feed) and water .group b (positive control): received ccl4 only (0.5 ml/100 g). group c: received ccl4 (0.5 ml/100 g) and turmeric extract 200 mg/kg. group d: received 200 mg/kg of turmeric extract and then treated with ccl4 (0.5 ml/100 g ) group e: received 200 mg/kg of turmeric extract only, at the end of the treatment, the liver and the kidney of each sacrificed rat were processed for paraffin sectioning and stained with harris hematoxylin and eosin. original research article eric and adolphus; aji, 3(1): 166-172, 2020; article no.aji.56699 167 result: photomicrograph of groups b, c and d show moderate inflammatory cells and fat infiltration which are features of hepatic injury but the result further shows that there were no noticeable histopathological changes observed in the histology of the kidney of all animal groups as compared with the control group. conclusion: the study demonstrates that turmeric extract has no ameliorative effect against ccl4induced liver damage in rats. keywords: liver; kidney; turmeric; carbon tetrachloride; oral; ameliorative. 1. introduction carbon tetrachloride (ccl4) is one of the most potent toxins, which is widely used in scientific research to produce experimental model that mimic the oxidative stress in many pathophysiological situation [1]. ccl4 is a possible carcinogen; inhalation or ingestion can cause damage to the brain, liver, kidneys, and can even cause death. ccl4 also contributes to ozone layer depletion [2]. carbon tetrachloride (ccl4) is a toxin that was used extensively to induce liver, renal and cardiac toxicity. ccl4 administered to rats induces histologically proven severe hepatopathology, nephropathy, cardiopathy and an increase of serum concentrations of urea, creatinine and liver enzymes such as aspartate aminotransferase (ast), alanine aminotransferase (alt), that are indicators of renal and liver tissue damage [3]. the biochemical mechanisms to ccl4 exposure is induction of an increase in lipid peroxide and free peroxide radical concentrations that are highly reactive and cause injury or necrosis [4]. ccl4 is metabolized by p450 cytochrome to ccl3 radical that induces the peroxidation of membrane lipids and disturbs ca 2+ homeostasis thus inducing liver tissue damage [5]. it has been reported that ccl4 administration increases the silver-stained nucleolar organizer region, alters its size, morphology or spreading in the nucleus, which may be utilized as an indicator of genotoxicity, neoplasia and hyperplasia to complement other histological procedures [6]. turmeric (curcuma longa) is the rhizome or underground stem of ginger like plant. turmeric has also been used for centuries in ayurvedic medicine, which integrates the medicinal properties of herbs with food. this extraordinary herb has found its way into the spotlight in the west and rest of globe, because of its wide range of medicinal benefits. it is extensively used in ayurveda, unani and siddha medicine as home remedy for various diseases [7]. turmeric is a potent blood purifier and helps to create new blood. turmeric also protects liver from toxins and pathogens. it is known to destroy major hepato toxins, like aflatoxin and to rebuild the liver. turmeric has a protective effect against kidney and liver toxicity caused by certain medications. in addition, it is used as herbal remedy due to the prevalent belief that the plant has medical properties. in folk medicine, the rhizome juice from c. longa is used in the treatment of many diseases such as anthelmintic, asthma, gonorrhea, and its essential oil is used in the treatment of carminative, stomachic and tonic [8]. in traditional medicine, several plants and herbs have been used experimentally to treat liver and kidney disorders, including liver cirrhosis and chronic kidney disease. turmeric possesses antioxidant, anti-tumor, antimicrobial, antiinflammatory; wound healing, and gastroprotective activities. several studies have shown that the aqueous extract of turmeric has hepatoprotective and nephroprotective activity against carbon tetrachloride toxicity (suzy et al. 2013). the main aim of this research is to investigate the curative, hepatoprotective and nephroprotective effect of turmeric extract on albino wistar rats. 2. materials and methods 2.1 location of study this study was carried out in the department of medical laboratory science, faculty of basic medical sciences, college of health science, niger delta university, wilberforce island amassoma, bayelsa state of nigeria. 2.2 procurement of turmeric extract commercially prepared turmeric extract (5 g) was purchased from loba chem pvt ltd (india) was used for this studies. https://www.ncbi.nlm.nih.gov/pubmed/?term=salama%20sm%5bauthor%5d&cauthor=true&cauthor_uid=23496995 eric and adolphus; aji, 3(1): 166-172, 2020; article no.aji.56699 168 2.3 animal housing thirty (30) male albino wistar rats weighing between 150 ± 2.6 g-317 ± 3.5 g were procured from the animal house of the department of pharmacology, college of health sciences niger delta university, amassoma bayelsa state and moved to the animal house of the department of medical laboratory science niger delta university amassoma, bayelsa state, nigeria, where they were housed under standard condition of temperature (27 ± 2°c) with twelve hours light/dark periodicity in plastic cages. the rats were allowed to acclimatize for two weeks and were fed ad libitum during this period, with water and grower mesh feed. animals where handled throughout study according to institutions guidelines for an experiment involving the use of laboratory animals. 2.4 experimental design the animals were weighed and assigned into five (5) major groups with five (5)animals in each group after the period of acclimatization: a control group "a" and three test groups (c, d and e) except group b with ten (10) rats. 2.5 substance administration group a (control): rats were administered orally with pelleted growers mash (feed) and water throughout the experiment (21 days). group b (positive control): rats were intraperitoneally injected with of ccl4 only (0.5 ml/100 g body weight)and given pelleted growers mashes (feed) and water for 2 days, five (5) animals sacrificed on the 14 th day and the remaining five were given pelleted growers (feed) for 21 days. group c: rats were injected intraperitoneally with ccl4 (0.5 ml/100 g body weight) for 2 days, and then given orally with turmeric extract 200 mg/kg body weight, pelleted growers mash (feed) and water throughout the experiment (21 days). group d: rats were administered orally with 200 mg/kg of turmeric extract for 21 days and injected intraperitoneally with ccl4 (0.5 ml/100 g body weight) for 2 days, and they were also given pelleted growers mash (feed) and water throughout the experiment (21 days). group e: rats were administered orally with 200 mg/kg of turmeric extract and given pelleted growers mash and water for twenty days (21 days). 2.6 collection of sample at the end of three weeks of administration, the rats were sacrificed by administering chloroform as anesthesia. the rats were then dissected to harvest the liver and the kidney which were then fixed immediately in 10% formalin. 2.7 histopathological processing the excised organs (liver and kidney) were cut in slabs of about 0.5 µm thick and fixed in 10% formal saline. routine tissue processing was carried out using automatic tissue processor histokinette (leica tp 1020).the tissues were embedded in paraffin wax in tissue embedder (leica eg 1160) and sectioned in a rotary microtome (heitz 150 rotary microtome) at 5 microns and stained in hematoxylin and eosin using the method for general tissue architecture [9]. the stained slides were examined using the compound light microscope at ×100 and x400 magnifications. 2.8 microscopy and photomicrography the sections were examined using olympus binocular microscope with in-built lighting system. the sections were then photomicrograph using a digital microscope camera (samsung model ss850) attached to an olympus trinocular microscope. table 1. treatment dose for studied sample group a (control) group b group c group d group e feed and water only ccl4 (0.5 ml/100 g bw) + feed and water. ccl4 (0.5 ml/100 g bw) first + turmeric extract (200 mg/kg) + feed and water. turmeric extract (200 mg/kg) first + ccl4 (0.5 ml/100 g bw) + feed and water. turmeric extract (200 mg/kg) first + feed and water. eric and adolphus; aji, 3(1): 166-172, 2020; article no.aji.56699 169 3. results photomicrograph of kidney sections stained with hematoxylin and eosin x400. sections shows normal bowman’s capsule (bc), the renal tubules (rt) all are consistent with normal kidney histology. photomicrograph of liver section stained with hematoxylin and eosin x 400. sections shows portal vein (pv), central vein (cv) and sinusoids (s) consistent of liver histology. plate b shows fat infiltration (f) indication fatty liver degeneration. plate c and d show occlusion (o) of sinusoidal spaces with enlargement of hepatocytes. fig. 1 shows the histology of the kidney stained with hematoxylin and eosin. slide a (control) shows normal kidney stroma with bowman’s capsule (bc) with glomerulus (renal corpuscle) and renal tubules (rt). slide b, c and d is similar to the control. fig. 2 shows the histology of the liver stained with hematoxylin and eosin. slide a (control) shows normal hepatic stroma with portal vein (pv), central vein (cv) and sinusoids (s) consistent of liver histology. slide b shows fat infiltration (f) indication fatty liver degeneration. slide c and d show occlusion (o) of sinusoidal spaces with enlargement of hepatocytes. slide f shows similar morphology with the control. 4. discussion ccl4 is a well-known hepatotoxin that is widely used to induce toxic liver injury in a range of laboratory animals. ccl4-induced hepatotoxicity is believed to involve two phases. the initial phase involves the metabolism of ccl4 by cytochrome p450 to the trichloromethyl radicals, which lead to membrane lipid peroxidation and finally to cell necrosis. the second phase of ccl4-induced hepatotoxicity involves the activation of kupffer cells, which is accompanied by the production of proinflammatory mediators [10]. turmeric therapeutic properties including antioxidant effects, anti-inflammatory effects, anti-cancer and anti-microbial effects, hepatoprotective effects, reno-protective effects, thrombo inhibitory effects, cardio-protective effects, an anti-inflammatory effects on the rheumatoid arthritis, has been confirmed by modern and advanced researches. fig. 1. shows the histology of the kidney stained with hematoxylin and eosin, x400 eric and adolphus; aji, 3(1): 166-172, 2020; article no.aji.56699 170 fig. 2. shows the histology of the liver stained with hematoxylin and eosin, x400 the livers of the rats from control group is consistent with normal liver histology (fig. 1a). histopathological changes such as fat infiltration (f) were consistently observed in liver of all the rats from ccl4-treated group (fig. 1b). also, predominant changes such as occlusion (o) of sinusoidal spaces with enlargement of hepatocytes was observed in livers of all rats treated with a combination of ccl4 and turmeric extract (fig. 1c & d). however, the histological appearance of the liver in the turmeric extract group had no morphological change (fig. 1e). administration of turmeric extract had no ameliorative effect on the changes induced by ccl4 in liver. in other studies, carried out by saad et al. (2017) on hepatoprotective effect of aqueous extracts of some medicinal plant mixtures on ccl4-induced liver toxicity, administering 1.5 ml/kg of ccl4 for 10 days. it reported that ccl4 induces severe histological changes such as extensive hepatocellular degeneration and necrosis, fatty changes, inflammatory cell infiltration, congestion, and sinusoidal dilatation in the hepatic tissues. he further observed that treatment with turmeric extract effectively prevented the necrosis and the other histopathological changes induced by ccl4 toxicity. the result further shows that there were no noticeable histopathological changes observed in the histology of the kidney of all animal groups as compared with the control group (fig. 1a-1d). this may be due to the duration of administration (twice a week for one week) of 0.5 ml/kg ccl4. other study conducted by naima et al. [11] reported that ccl4 induced nephrotoxicity in rats in the duration of administration of 0.5 ml/kg (twice a week for six weeks) and 5 ml/kg (once a week for six weeks) of ccl4 respectively. also curcumin prevented the decrease in the following enzymatic activities: aconitase, antioxidant enzymes and mitochondrial respiratory complexes i, ii, ii–iii and v [12]. rosita et al. [13] also reported that c. mangga extract was able to inhibit the increase of creatinine level and showed a significantly different from negative control (p <0.05). the result was supported by histopathology examination which did not show any cell damage, in a similar study carried out by https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/enzyme-assay https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/aconitase eric and adolphus; aji, 3(1): 166-172, 2020; article no.aji.56699 171 alireza and daryoush [14] on preventive effects of turmeric (curcuma longa linn) on renal ischemia-reperfusion injury in rats showed that renal structure is normal in the control group and there were not pathologic changes. in the group 2, degenerative changes of tubular cells, acute tubular necrosis, edema, hyperemia and sever hemorrhage were more prevalent. 5. conclusion although turmeric has also been used for centuries in ayurvedic medicine, which integrates the medicinal properties of herbs with food. this extraordinary herb has found its way into the spotlight in the west and rest of globe, because of its wide range of medicinal benefits. it is extensively used in ayurveda, unani and siddha medicine as home remedy for various diseases [15]. turmeric is a potent blood purifier and helps to create new blood. turmeric also protects liver from toxins and pathogens. it is known to destroy major hepatoxins, like aflatoxin and to rebuild the liver. turmeric has a protective effect against kidney and liver toxicity caused by certain medications. in addition, it is used as herbal remedy due to the prevalent belief that the plant has medical properties. in folk medicine, the rhizome juice from c. longa is used in the treatment of many diseases such as anthelmintic, asthma, gonorrhea, and its essential oil is used in the treatment of carminative, stomachic and tonic [16]. tumeric possesses antioxidant, anti-tumor, antimicrobial, anti-inflammatory; wound healing, and gastroprotective activities [17]. however this study demonstrates that turmeric extract had no had no ameliorative effect against ccl4-induced hepatic damage in rats at the dose and duration of administration. disclaimer the products used for this research are commonly and predominantly use products in our area of research and country. there is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. also, the research was not funded by the producing company rather it was funded by personal efforts of the authors. consent it is not applicable. ethical approval animal ethics committee approval has been taken and preserved by the author for this study. competing interests authors have declared that no competing interests exist. references 1. yoshioka h, usuda h, fukuishi n, nonogaki t, onosaka s. carbon tetrachloride-induced nephrotoxicity in mice is prevented by pretreatment with zinc sulfate. biological and pharmaceutical bulletin. 2016;39(6):1042–1046. 2. hanaa-wafay g, el-saeed s, eltoukhy e, nabila e, mervat a, shereen e. potential effect of garlic oil and silymarinon carbon tetrachloride induced liverinjury. australian journal of basic and appliedsciences. 2012;6(3):409-414 3. teocharis se, margeli ap, skaltsas sd, spiliopoulou ca, koutselinis as. induction of metallothionein in the liver of carbon tetrachloride intoxicated rats: an immuno histo-chemical study. toxicology. 2001;161(3):129–138. 4. miyazaki t, bouscarel b, ikegami t, honda a, matsuzaki y. the protective effect of taurine against hepatic damage in a model of liver disease and hepatic stellate cells. advance experimental medical biology. 2009;643:293–303. 5. södergren e, cederberg j, vessby b, basu s. vitamin e reduces lipid peroxidation in experimental hepatotoxicity in rats. european journal of nutrition. 2001;40(1):10–16. 6. khan mr, rizvi w, khan gn, khan ra, shaheen s. carbon tetrachloride induced nephrotoxicity in rat: protective role of digeramuricata. journal of ethnopharmacology. 2009;122:91–99. 7. abas f, lajis nh, shaari k, israf da, stanslas j, yusuf uk, and raof sm. (2005). a labdane diterpene glucoside from the rhizomes of curcuma longa. journal of nature production. 2005;68: 1090-1093. 8. hwa-young l, seung-wook k, geumhwa l, min-kyung c, jung y, jeong k, han-jung c. turmeric extract and its active compound, curcumin, protect against chronic ccl4-induced liver damage eric and adolphus; aji, 3(1): 166-172, 2020; article no.aji.56699 172 by enhancing antioxidation. bmc complementary and alternative medicine bmc series open, inclusive and trusted. 2016;16:316-317 9. ochei j, kolhatkar a.routine hematoxylin and eosin staining method in: medical laboratory science, theory and practice. tata mcgraw-hill publishing company limited. new delhi. 2000;449-450. 10. preeti r, waseem r, ramteke, suchit a. john. turmeric: the golden spice of life ijpsr. 2012;3(7):1987-1994. 11. naima z, howaida i, hanan fa, mantawi, ni. ccl4-induced hepato nephrotoxicity: protective effect nutraceuticals on inflammatory factors and antioxidant status in rats. journal of applied pharmaceutical science. 2014;4(2):87-90. 12. molina-jijón ee, tapia c, zazueta m, elhafidi zl ,zatarain-barrón r, hernández-pando on, medina-campos g, zarco-márquez i, torres j, pedrazachaverri t. (curcumin prevents cr(vi)induced renal oxidant damage by a mitochondrial pathway, free radical biology & medicine. 2011;51:1543–1557. 13. rithaporn t, monga m, rajasekharan m. curcumin: a potential vaginal contraceptive. contraception. 2003;68:219– 223. 14. alireza ms, daryoush m. preventive effects of turmeric (curcuma longa linn) on renal ischemia-reperfusion injury in rats. advances in bioresearch. 2013;4(4):40-46. 15. abas f, lajis nh, shaari k, israf da, stanslas j, yusuf uk, raof sm. a labdane diterpene glucoside from the rhizomes of curcuma longa. journal of nature production. 2005;68:1090-1093. 16. ishita c, kaushik b, uday b, ranajit kb. turmeric and curcumin: biological actions and medicinal applications. current science. 2004;87(1):10-11. ________________________________________________________________________________ © 2020 eric and adolphus; this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: http://www.sdiarticle4.com/review-history/56699 http://creativecommons.org/licenses/by/4.0 _____________________________________________________________________________________________________ *corresponding author: e-mail: adedayoogunware21@gmail.com; asian journal of immunology 3(1): 99-105, 2020; article no.aji.56071 seroprevalence of hepatitis c virus infection amongst febrile patients attending selected public and private hospitals in lagos state, nigeria adedayo emmanuel ogunware1*, adedayo olawale onakomaiya2 and adesewa adeyinka adekoya1 1 department of biochemistry, lagos state university, ojo, p.m.b. 0001, lagos, nigeria. 2 department of microbiology, lagos state university, ojo, p.m.b. 0001, lagos, nigeria. authors’ contributions this work was carried out in collaboration among all authors. all authors read and approved the final manuscript. article information editor(s): (1) dr. jaffu othniel chilongola, tumaini university, tanzania. reviewers: (1) victor b. oti, nasarawa state university, nigeria. (2) naresh kumar, mewar university, india. complete peer review history: http://www.sdiarticle4.com/review-history/56071 received 03 february 2020 accepted 08 april 2020 published 12 april 2020 abstract hepatitis c virus (hcv) is the leading reason for liver transplantation in the world; patients infected with hcv are at increased risk of cirrhosis and hepatocellular cancer. the study was aimed at evaluating the seroprevalence of hepatitis c virus infection amongst febrile patients attending selected public and private hospitals in lagos state, nigeria. the hospital based cross-sectional study took place between october and december 2019. a total of 89 blood samples were collected from febrile patients after informed consent and self-administered questionnaires were completed. the samples were centrifuged, and screened for anti-hcv antibodies using the enzyme-linked immunosorbent assay (elisa) technique. the results of this study were analysed statistically and out of the 89participants screened, only 5(5.61%) were positive for anti-hcv ab. there was no significant difference (p>0.05) between the number of male and female patients positive and also other demographic characterization (age and temperature). keywords: hepatitis c virus; febrile; elisa; blood donors; lagos. original research article ogunware et al.; aji, 3(1): 99-105, 2020; article no.aji.56071 100 1. introduction hepatitis c is a major public health problem that must be controlled and possibly eradicated. hepatitis c virus (hcv) was first documented as a cause of transfusion associated with acute and chronic hepatitis in 1989 [1] and plays a key role as a cause of chronic liver injury with potential for neoplastic deterioration. it is mostly transmitted by parenteral route; although with lower effectiveness, it may also be transmitted by sexual intercourse as well as from mother to child. hcv infection is often asymptomatic, making it very hard to detect at an early stage. this is the major reason why early treatment is difficult, therefore, hepatitis c is often referred to as a “silent disease”. in many infected people, the virus infection does not resolve naturally. the neutralizing antibodies appear to be manufactured during a natural infection; yet, the virus transforms to escape surveillance. as hepatitis c virus infection is transmitted with high efficacy via blood to blood contact, the prevalence of hcv within different countries, regions and populations is closely related to the incidence of blood borne (mainly intravenous drug use) disease. hcv infection is an important public health problem today because it is an emerging epidemic disease which infects nearly 3% of the population worldwide and has emerged as a major causative agent of liver disease, resulting in acute and chronic infections that can lead to fibrosis, cirrhosis and hepatocellular carcinoma. it is estimated that more than 170 million individuals are infected with hcv worldwide, most of them chronically [2]. hcv is accountable for about 350,000 deaths annually; among western countries, southern europe and particularly italy is among the most affected areas [3]. most people (80%) with acute hcv infection are asymptomatic. if symptoms occur, they may includeabdominal pain, loss of appetite, nausea, fatigue, dark urine and jaundice. of those who develop chronic hcv infection, the most common symptom is fatigu e [4]. severe liver disease develops in approximately 10%–20% of chronically infected people, but progression to end-stage liver disease is slow and typically does not occur until ≥20 years after infection. this development is often clinically silent until late in the course of disease, and in the absence of hcv testing, most people are unaware of their infection. africa has the highest who estimated regional prevalence (5.3%) with egypt having the highest prevalence (17.5%) of hcv in the world; also, many hiv-positive persons in sub-saharan africa are co-infected, a systematic review and metaanalysis, showed anti-hcv prevalence rates of 7% among hiv infected individuals. the epidemiology of hcv infection in nigeria is not well understood although the prevalence among high risk groups was given as 12.3% [5]. majority of research on prevalence of hepatitis c in nigeria have focused on serological characterization of selected population groups, e.g. patients with diabetes mellitus, prison inmates, hiv-infected persons, blood donors, patients with chronic renal failure and those with sickle cell anemia, for whom risk for hcv infection in urban areas of nigeria is variable. previous studies established a broad hcvseroprevalence rate, ranging from 1.9% among pregnant women in benin city to 14.5% among apparently healthy individuals with a family history of diabetes in plateau state [6] or among hiv-positive patients in lagos [6]. hcv infections have been shown to play a significant role in the aetiology of chronic liver disease and hepatocellular carcinoma (hcc) in nigeria. however, a study of adolescent and adult patients with sickle cell anemia (sca) in benin by [7] showed 20% prevalence rate. another study in ibadan conducted among doctors recorded a seroprevalence of 11% [8]. it was also noted that hcv infections were found more in lower socio-economic class than other social classes. in nigeria, 18.7% of liver cancer patients carry markers of hcv and it is said that the results of seroprevalence studies of hcv in nigeria vary depending on the study population and the geographical setting having higher rates along the eastern borders and some in northern regions [9]. the challenge arises that there is dearth of information on burden and circulation of hcv in nigeria (lagos state). the risk factors for hcv transmission in nigeria have not been properly characterized and the seroprevalence of hcv infection amongst unhealthy, immunecompromised patients and carriers in lagos is unknown. therefore, this study was carried out to evaluate the seroprevalence of hepatitis c virus infection amongst febrile patients attending selected public and private hospitals in lagos state. ogunware et al.; aji, 3(1): 99-105, 2020; article no.aji.56071 101 2. materials and methods 2.1 study area alimosho local government area is home to the general hospital alimosho, igando. it is the largest local government in lagos state with coordinates 6°36’38’n/3°17’45"e. it has a total population of about 1,362,077 and land area of 185 km 2 with average density of 713 persons per square kilometer approximately. is bounded in the north and west by river owo and ifakoijaiye, agege respectively, and the east by ikeja local government area while it is bounded in the south by oshodi/isolo, amuwo-odofin and ojo local government areas of lagos state [10]. lagos state university health centre is the primary health care clinic available to the staffs and students of lagos state university at ojo. ojo local government area has a total population of about 598,071 comprising 310,100 males and 287,971 females (npc. 2006) and land area of 375 sq. km. it occupies the south western part of the lagos metropolis. it is located between latitude 60°22′n and 60°32′n and on longitude 30°4′e and 30°20′e. the local government area is bounded by six other local government areas and is among the seven local government areas occupying the coastal plain of lagos metropolis. the predominant land uses in this area are residential and commercial land use. commercial land use includes financial institution and market [11]. agbara is located at the west of ologe lagoon and north of badagry creek, in lagos state southwest nigeria, on longitude of 2°421 and 3°231e and latitude of 6°231 and 6°281n. it is an industrial area with various kinds of industries including pharma-deko and unileverand ibijola medical center is also situated there [12] 2.2 study centre this was a hospital based cross-sectional study that covered three local government areas in lagos state. the hospital samples were collected from are general hospital alimosho, igando (agh), lagos state university health center (lsuhc), ibijola medical center agbara (imc). eighty-nine (89) blood samples were collected from patients in the phlebotomy section of the hospitals mentioned above (20 samples from agh, 35 samples from imc and 34 samples from lsuhc). a structured questionnaire was designed and administered, in order to obtain demographic information, characteristics, personal (such as age and sex). 2.3 study population the collection of samples was based on adult and children febrile patients/clients attending alimosho general hospital, lagos state university health center, ibijola medical center lagos state. 2.4 sample collection and transportation blood samples were collected from each febrile patientfrom the month of october 2019.all study subjects were between 18-50 years of age who must have had fever at the point of collection or symptoms related over the last six months i.e. patients whose temperature was above 37.5°c.a total of 89 blood samples were collected by venipuncture using sterile 5 ml syringe /10ml edta vacutainer and transported in cold box to the centre of human virology and genomics, nigerian institute of medical research (nimr), yaba, lagos state. the samples were centrifuged (eppendorf) at 3500 rpm for 10 minutes and the plasma (supernatant) aliquot was transferred into cryogenic vials and stored in the refrigerator (4°c) until ready for analysis. all necessary precautions were taken to guide against interference that could negatively affect the result. 2.5 serological technique 2.5.1 principle of the test the micro plates were coated with hcv-specific antigens derived from “core” and “ns’’ region encoding from conservative and immune dominant antigenic determinants (core peptide, recombinant ns3, ns4 and ns5 peptides). the solid phase is first treated with the diluted sample, and the 2nd incubation bound hcv antibodies, igg and igm as well, are detected by the addition of polyclonal specific anti igg&m antibodies, labeled with horse radish peroxidase (hrp). the enzyme captured on the solid phase, acting on the substrate/ chromogen mixture, generates an optical signal that is proportional to the amount of anti-hcv antibodies present in the sample. a cut-off value let optical densities be interpreted into hcv antibody negative and positive results. ogunware et al.; aji, 3(1): 99-105, 2020; article no.aji.56071 102 2.5.2 hcv detection using elisa kit the hcv antibodies were detected using qualitative recombilisa hcv elisa kit manufactured by dia.pro diagnostics bioprobes. all reagents and controls (positive and negative) were brought to room temperature and the concentrated washing buffer was diluted 30-fold with distilled water. the hcv-ab test kit used was an enzyme immunoassay kit used for the determination of anti-hepatitis c virus antibody in human serum and plasma and it is an in-vitro diagnostic test. the procedure used is an enzyme linked immunosorbent assay (elisa). 2.5.3 assay procedure the first well was left empty for the operation of blanking. next 200ul of negative control was dispensed in triplicates, 200ul calibrator was placed in duplicate and 200ul of positive control is dispensed in a single well. then 200ul of sample diluent (dilspe) was added to all the sample wells except the calibrator and controls which were already pre-diluted, 10ul of sample was dispensed in each properly identified well and mixed gently. 50ul of assay diluent (dilas) was also dispensed into all the controls/calibrator and sample wells. the micro plate was incubated for 45min at 37°c and the plate was washed with an automatic washer 6 times. 100ul of the enzyme conjugate was pipetted into each well, except the 1st blanking well and was then covered with a sealer. the micro plate was incubated for 45 min at 37°c and after incubation the micro wells were washed 6 times using an automatic washer. 100ul chromogen/substrate mixture was pipetted into each well and the blank included. then incubated the micro plate at room temperature (18-24°c) for 15mins. 100ul of sulphuric acid was pipetted into the wells using the same sequence as the substrate to stop the enzymatic reaction. addition of acid will turn the positive control and positive samples from blue to yellow/brown. finally, the optical density (od) was read at 450nm wavelengths within 15 minutes of stopping the reaction using gfm3000 micro plate reader and results were read according to the manufacturer‘s manual. 3. results a total of 89 plasma samples were tested for hepatitis c virus antibodies in addition with the reagents used (controls and calibrations) on the micro plate and an enzyme linked immunosorbent assay (elisa) reader was implored for the reading of the optical density. among the 89 blood samples collected from febrile patients in the three selected hospitals, 5 (5.62%) were positive for hcv antibody (table 1). the demographic characteristics of the patients showed age range of 18 26, the overall mean age of the 89 patient’s blood samples collected across the 3 selected public and private hospital was 25years (age range 18 – 26). the highest proportion of hcv antibody seropositive result was recorded most among males whose age are below 30. the gender distribution of the positive patients was shown in fig. 1. 4. discussion hcv is widespread in the world and in lagos state. it is among the important hepatotropic viruses because of liver tropism which leads to cihorrsis or hepatocellular carcinoma. this has become an issue of public health concern. the recombilisa hcv ab elisa is able to qualitatively detect antibodies (both igg and igm) to hepatitis c virus in human serum or plasma and has become an important tool to identify individuals with early infection so as to administer early antiviral therapy. the prevalence of hcv ab among the febrile patients in this study was 5.61%. though no work has been done pertaining to febrile patients in lagos or nigeria, the prevalence is greater than zero (0%) recorded by enitan et al.,in 2019 [13] among undergraduate students of babcock university ogun state. work done by muhibi et al. [14] and alquatani et al. [15], reported zero (0%) prevalence of anti-hcv antibody among undergraduate students of achievers university, owo in south-west nigeria, as well as among health students in the najran region of southwestern saudi arabia, respectively. the outcome of this study however is also greater than the work of hebo et al. [16] who reported a prevalence of 0.42% among health workers of university medical center, southwest ethiopia, as well as that of jemilohun et al. [17] who reported a prevalence rate of 0.40% among undergraduate student of ladoke akintola university of technology (lautech), ogbomosho, oyo state, south-west nigeria. the results of this study is lower than the work of udeze et al. [18] who reported a prevalence rate of 8.0% among first year students of university of ilorin, kwara state, nigeria. in a recent study by tula et al. [19] a much higher prevalence rate of 11.5% was recorded among students of federal polytechnic mubi, adamawa; majority of whom had history of blood transfusion, medical surgery and circumcision. ogunware et al.; aji, 3(1): 99-105, 2020; article no.aji.56071 103 table 1. prevalence rate of hepatitis c virus antibodies in each study area hospital frequency (n=89) no of positive result no of negative result percentage of positive results (%) ibijola medical center 35 3 32 3.37 alimosho general hospital 34 0 34 0 lagos state university health center 20 2 18 2.24 total 89 5 84 5.61 fig. 1. gender distribution of hcv antibody positive patients from ibijola medical center and lagos state university health center gender was not found to be associated with the viral prevalence although the infection was higher among males than females (p > 0.05), but this may be connected to the fact that some men involve in homo sexualism which is a high risk factor for the transmission of hcv as risky sexual behaviors such as fisting and unprotected intercourse can be mucosally traumatic and may be associated with bleeding [20]. whether bleeding is necessary for hcv transmission is still debatable though some studies have identified hcv in seminal and rectal fluids of hiv infected men and providing evidence that these fluids can mediate hcv transmission [21] or due to having numerous sexual partners due to promiscuous lifestyle. the age stratification in this study shows no statistical significance with age in hcv prevalence. hcv ab was detected more among patients that were between the age 18 – 26 years this might be suggestive that those in such age are more active and likely to engage in unprotected sex and are also not aware of immunization. temperature was not found to be associated with the prevalence despite the fact that fever is a symptom of acute stage hcv but this might be that some patients had other serious infection such as typhoid or malaria at the time of blood collection. 5. conclusion after all test and serological analyses carried out the research indicate 5 positive hcv ab among tested blood patients, which gave a seroprevalence rate of 5.61% in the study area and this indicated that hcv is endemic in our environment though with a low prevalence rate, it should be taken into account by doctors and nurses in hospitals when treating febrile patients as drug interactions could potentially increase morbidity in this population. the information provided by the study can be used to provide baseline data that can contribute to knowledge on the magnitude of the disease, stimulate further research on the disease and inform policy on risk assessment. our study has some limitations. first, in the number of people we males 60% females 40% gender distribution ogunware et al.; aji, 3(1): 99-105, 2020; article no.aji.56071 104 involved, as more test subjects would yield more reliable results. second, it would have been useful to confirm the presence of the hepatitis viruses with a nucleic acid-based technique such as polymerase chain reaction, nonetheless, our results are consistent with other studies, and are relevant for improving the care of hiv/aids patients. compulsory public awareness campaigns against hcv infection and prevention programs should be intensified to eradicate future outbreak cases of hcv in the country. consent all authors declare that ‘written informed consent was obtained from the patient (or other approved parties) for publication of this case report. ethical approval an ethical approval with reference number (lshsc/2222/vol. vb/66) was sought from the lagos state health service commission (lshsc). an approval was also given from the nigerian institute of medical research (nimr) an institutional review board to carry out the laboratory work in their human genomics and molecular laboratory. an approval was also sought from the respective chief medical director (cmd) of the hospitals. the following information was given to each participant to ensure that they make an informed choice; a complete description of the aims of the study, infectious agent that was being screened, details of sample collection procedures, potential benefits and risks of their participation in the study and assurance of confidentiality of any information given as well as of the test results, all these were explained to the subject in english language and/or, their native languages and consent were sought through the signing of informed consent form. dignity of the study participants was upheld throughout the study. competing interests authors have declared that no competing interests exist. references 1. choo ql, kuo g, weiner aj, overby lr, bradley dw, houghton m. isolation of a cdna clone derived from a blood-borne non-a, non-b viral hepatitis genome. science. 1989;244(4902):359362. 2. lakošeljac d, rukavina t. epidemiology of hepatitis c viral infection. medicina. 2007; 43(2):112-117. 3. zaltron s, spinetti a, biasi l, baiguera c, castelli f. chronic hcv infection: epidemiological and clinical relevance. bmc infectious diseases. 2012;12(2):s2. 4. chlibek r, smetana j, sosovickova r, gal p, dite p, stepanova v, pliskova l, plisek s. prevalence of hepatitis c virus in adult population in the czech republic–time for birth cohort screening. plos one. 2017; 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93(6):404-409. 21. foster gr, gane e, asatryan a, asselah t, ruane pj, pol s, poordad f, stedman ca, dore g, roberts s. endurance-3: safety and efficacy of glecaprevir/pibrentasvir compared to sofosbuvir plus daclatasvir in treatmentnaïve hcv genotype 3-infected patients without cirrhosis. j hepatol. 2017;66(1): s33. _________________________________________________________________________________ © 2020 ogunware et al.; this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: http://www.sdiarticle4.com/review-history/56071 http://creativecommons.org/licenses/by/4.0 _____________________________________________________________________________________________________ *corresponding author: e-mail: hassanshora56@gmail.com; asian journal of immunology 2(1): 26-33, 2019; article no.aji.53002 asthma severity and serum leptin level in obese egyptian children enas f. elngar1, osama a. zekry1, mamdouh m. ali1 and hassan a. shora1* 1 department of pediatrics and neonatology, faculty of medicine, suez canal university and port-said university, egypt. authors’ contributions this work was carried out in collaboration among all authors. author efe designed the study, performed the statistical analysis, wrote the protocol and wrote the first draft of the manuscript, authors oaz and mma managed the analyses of the study. author has managed the literature searches and contributed in writing the final manuscript. all authors read and approved the final manuscript. article information editor(s): (1) dr. darko nozic, professor, higher medical school in belgrade, university of belgrade, serbia. reviewers: (1) hanan mohamed hamed, national research centre, egypt. (2) gayatri c. gawade, bharati vidyapeeth dental college and hospital, india. complete peer review history: http://www.sdiarticle4.com/review-history/53002 received 22 september 2019 accepted 28 november 2019 published 04 december 2019 abstract background: the relation between leptin and asthma is still unclear especially in obese children. we hypothesized that high serum leptin concentrations would also be associated with asthma in obese egyptian children. we aimed to evaluate serum leptin concentrations in asthmatic obese and none obese children to investigate their association with asthma and degree of asthma severity. subjects and methods: the study was carried out on 56 children attended pediatric outpatient clinic of suez canal university hospital on jan, 2016 to dec 2016. they were divided into three groups (group 1) asthmatic obese children involved 16 child (group 2) asthmatic nonobese child; 20 (group 3) 20 non asthmatic child as control. diagnosis of asthma was done according to global initiative of asthma. serum leptin level was assessed in all study groups by elisa. results: serum leptin levels in obese asthmatic patients was 91.3±9.3 ng/ml ,while in non-obese asthmatics it was 87.8±5.6 ng/ml, finally in non-asthmatic control it was71.3±8.2 ng/ml (p value 0.175). there was positive significant correlation between serum leptin levels and asthma severity (p = <0.001). there was also significant negative association of leptin with peak expiratory flow rate results; there was also significant relation between serum leptin levels and history of allergy as original research article elngar et al.; aji, 2(1): 26-33, 2019; article no.aji.53002 27 well as family history of asthma (p = <0.001). there was significantly higher body mass index in asthmatics than non-asthmatics (p = 0.004). conclusion: leptin is playing an important role in assessment of asthma severity and is the most predictive factor for asthma severity when compared to body mass index, family history of asthma and history of allergy. keywords: asthma; leptin; asthma severity; body mass index. abbreviations bmi : body mass index; pefr : peak expiratory flow rate test; elisa : enzyme-linked immunosorbent assay; 1. introduction asthma afflicts 339 million people worldwide and 8.3% of us children and is considered the most common disease of children. the annual healthcare expenditures for asthma is estimated $ 81 billion in us and eur72 billion in europe since it is a major reason for pediatric emergency room visits, admissions in hospitals’ pediatric wards, abstenance from schools in addition to the economic loss due to decreased parental work [1-3]. bronchial asthma is a chronic inflammation of the bronchial tree characterized, by completely or partially reversible airway obstruction, which may improve spontaneously or by specific therapy. airway hyper-responsiveness is a narrowing of the airways due to a variety of stimuli, such as allergens and nonspecific triggers and infections [4]. the prevalence of asthma and allergies is increasing in both western and developing countries. despite a large volume of clinical and epidemiological research within affected populations, the etiology and risk factors of these conditions remains poorly understood [5]. several epidemiological studies have shown that the prevalence of bronchial asthma and obesity is increasing concomitantly worldwide among children and young adults [6]. obesity may be associated with respiratory symptoms via cardiorespiratory deconditioning, physiological restriction of the chest wall by excess adipose tissue, or comorbidities, including gastro esophageal reflux and sleep-disordered breathing [7]. obesity is characterized by chronic low-grade systemic inflammation. obese adipose tissue is infiltrated by macrophages that are a source of inflammatory cytokines [8], more than 50 different adipocytokines are secreted by adipocytes. adipocytokines are proteins that help regulate various body functions [9]. serum leptin is pro inflammatory adipocytokine that affects both innate and adaptive immune responses, and its serum levels are markedly increased in obesity [10]. leptin is one of the adipose tissuederived energy-regulating hormones and a product of the obesity gene. circulating leptin is positively correlated with body fat percentage and body fat mass. in addition to its primary effects on energy regulation, which it exerts by inhibiting food intake and increasing energy expenditure, leptin has been found to play a regulatory role within the immune system [11]. the regulatory capacity of leptin is associated not only with adaptive immunity but also with the innate system [12]. leptin promotes the production of nitric oxide and pro-inflammatory cytokines in macrophages and monocytes [10]. the aim of this study was to evaluate serum leptin concentrations in asthmatic obese and none obese children and its association with degree of asthma severity. what is known?  serum leptin is pro inflammatory cytokine that affects both innate and adaptive immune responses, and serum levels are markedly increased in obesity.  circulating leptin is positively correlated with body fat percentage and body fat mass. what is new?  leptin is playing an important role in assessment of asthma severity.  there is a significant relation between serum leptin levels and history of allergy as well as family history of asthma. 2. materials and methods this study is observational cross-sectional case control. this study was conducted among asthmatic children in pediatric outpatients clinic elngar et al.; aji, 2(1): 26-33, 2019; article no.aji.53002 28 in suez canal university hospital. control group was normal healthy children. fifty-six children were included; 20 non asthmatic children (control group) and 36 asthmatic children; 16 obese asthmatic and 20 non obese asthmatic, all aged above 3 years old. diagnosis of asthma was done according to global initiative of asthma (gina 2015). obese children were identified as children with body mass index above 95th percentile for their age according to egyptian growth charts. inclusion criteria: asthmatic children above 3 years old who did not receive steroid treatment are included in the study without injury of vital organs including heart, liver and kidney. exclusion criteria: children who receive treatment with steroid either by inhalation or systemic route or children with infection and severe allergic reactions are excluded from the study. the ethical committee of suez canal university hospital approved the study and signed informed consent of children’s guardians is obtained. detailed history and full clinical examination was done. 2.1 pulmonary function test it was done by peak expiratory flow rate test (pefr) for children above five years old. 2.2 leptin assessment five ml blood samples was collected by a skilled and qualified technician at the outpatient clinic around 8 a.m. in the morning following an overnight fast. after collection, the blood samples was centrifuged for 10 minutes and serum leptin stored at −80 c. the materials used for collection was disposable, adequately labeled, and of recognized quality. leptin levels measured using a commercially available enzyme-linked immunosorbent assay (elisa) kit according to the manufacturer's instructions and standard guidelines. statistical analysis was performed using spss for windows statistical package, version 20. (spss, inc., chicago, ill., usa). data were expressed as mean ± sd. student’s t test, mannwhitney u test and χ 2 test were used for comparing mean values. a p value <0.05 was considered statistically significant. 3. results 3.1 demographic data this study was carried out on fifty-six children divided as; 20 non asthmatic children (control group) and 36 asthmatic children; 16 obese asthmatic and 20 non obese asthmatic, all aged above 3 years old, the mean bmi in was 21.3±5.8 and 16.6±2.4 in asthmatic and nonasthmatic groups respectively. the mean age of asthmatic group was 7.3±3.3 while it was 8.5±3.6 in non-asthmatic group, all these data are summarized in table 1. serum leptin was significantly higher in asthmatics than non-asthmatics (p=<0.001), but there was no significant relation between serum leptin in obese asthmatic and non-obese asthmatic (p=0.175) as shown in table 2. relation of serum leptin and degree of asthma severity: table 3 shows significantly higher serum leptin in severe asthmatics 97.6±8.5, than moderate 89.3±4.6 and mild asthmatics 82.0±3.2 (p =<0.001) respectively. table 1. socio demographic parameters of study groups p value non-obese asthmatics (20) obese asthmatics (16) p value nonasthmatic (20) asthmatic (36) 0.201 8.5±3.6 7.3±3.3 age (years) 0.584 30.8±14.5 33.0±15.5 body weight 0.081 132.3±21.4 122.0±19.8 height 0.004* 16.6±2.4 21.3±5.8 bmi sex 0.577 11(55.0%) 17(47.2%) males 0.201 9(45.0%) 19(52.8%) females 0.175 87.8±5.6 91.3±9.3 <0.001* 71.3±8.2 89.3±7.6 serum leptin data was presented as means ± sd or no. and % * statistically significant p <0.05; ≠ or for sex (male/female) elngar et al.; aji, 2(1): 26-33, 2019; article no.aji.53002 29 table 2. relation of serum leptin and degree of asthma severity p value asthma severity severe (8) moderate (19) mild (9) <0.001* 97.6±8.5 89.3±4.6 82.0±3.2 serum leptin table 3. relation between serum leptin levels and history of allergy p value serum leptin (mean ± sd) negative positive 010.0* 79.5±12.2 87.7±9.0 history of allergy <0100. .* 75.7±9.7 91.2±7.4 family history of asthma table 4. comparison between serum leptin in study groups serum leptin group p value p value asthmatics (36) non-asthmatics (20) obese asthmatics (16) non-obese asthmatics (20) 89.3±7.6 71.3±8.2 <0.001* 91.3±9.3 87.8±5.6 0.175 * statistically significant at p<0.05 table 5. relation of clinical parameters of asthmatics and asthma severity asthma severity p value mild (9) moderate (19) severe (8) age (years) 7.7±3.6 7.5±3.6 6.5±2.8 0.776 body weight 34.0±11.6 34.0±18.5 29.6±12.5 0.745 height 126.1±21.1 122.2±20.3 117.1±18.8 0.666 bmi 21.3±6.0 21.5±6.2 20.9±5.5 0.995 sex males 6(66.7%) 7(36.8%) 4(50.0%) 0.338 females 3(33.3%) 12(63.2%) 4(50.0%) history of allergy 3(33.3%) 8(42.1%) 5(62.5%) 0.503 family history of asthma 2(22.2%) 14(73.7%) 8(100.0%) 0.002* serum leptin 82.0±3.2 89.3±4.6 97.6±8.5 <0.001* pefr 146.1±50.9 140.2±67.9 151.1±62.9 0.824 data was presented as means ± sd or no. and %; * statistically significant p <0.05 there was positive significant correlation between serum leptin levels and asthma severity (correlation coefficient was 0.862* and p value was <0.001) while it is demonstrated a significant negative association of leptin with pefr results r=-0.468 and p<0.001. relation between serum leptin levels and history of allergy: table 2 shows a significant relation between serum leptin levels and history of allergy (p =0.019) as well as family history of asthma (p = <0.001). relation between body mass index and asthma severity: table 3 shows a significant moderate positive correlation between body mass index and asthma severity (rho=0.334, p=0.012). table 4 show serum leptin was significantly higher in asthmatics than nonasthmatics (p=<0.001) there was significant negative association between peak expiratory flow rate results and asthma severity. r=-0.427and p=0.001. roc curve of serum leptin for diagnosis of severe asthma showed serum leptin was 91.5% accurate in diagnosis of severe asthma (auc=0.915, p<0.001). at cutoff value of 75.5, serum leptin was shown 100% sensitivity and 75% specificity for diagnosis of severe asthma. 4. discussion leptin is an obesity gene product derived from adipocytes and activates proinflammtory adipocytokines including tumor necrosis factor α, interleukine-6 and interferon-γ. this study showed no statistically significant difference between the two study groups in terms elngar et al.; aji, 2(1): 26-33, 2019; article no.aji.53002 30 of socio demographic characteristics (age, body weight, height and sex) (p >0.05). in addition, the current study reported a significant difference between asthmatic and control groups in terms of bmi (21.3±5.8, 16.6±2.4 respectively, p = 0.004). these findings are similar to those reported by black et al who also found a significant difference between asthmatics and control groups in terms of bmi [13]. furthermore, a recent meta-analytic study of 18 studies which included 73,252 children reported that overweight or obesity was a significant risk factor for asthma in obese children [14]. however our study revealed no statistically significant difference in bmi between obese and non-obese asthmatic children. the present study showed that serum leptin levels of asthmatic children (89.3±7.6 ng/ml) were significantly higher than those of healthy control (71.3±8.2) p-value was <0.001. this coincides with the study done by about yousif, et al. who found a statistically significant difference in serum leptin values between asthmatics and controls being higher in asthmatics regardless to the body weight (p=0.034) [15] and also coincides with another two studies stated that even after controlling of body mass index, leptin levels were increased in asthmatic children compared to non-asthmatic children [16,17] (table 5). our study showed that there was no significant difference between serum leptin in obese asthmatics and non-obese asthmatics p=0.175 and this is consistent with results reported by shore, et al. who also found no significant difference between the two groups (p > 0.05) [18]. it was also reported by another study which demonstrated that no differences in leptin or adiponectin levels in obese and non-obese children with asthma [19]. as regard to bmi, the present study showed a significant positive correlation of leptin and bmi (r= 0.434, p=0.001) (table 6). this result is similar to the results reported by several researchers [20,21]. yosif, et al. suggested that bmi is the main determinants for the variations of leptin [22]. as demonstrated by french epidemiological study on indirect effects of leptin on genetics and environment of asthma egea revealed that leptin partially mediated the association between obesity and persistent asthma over time [23]. a recent longitudinal study used new mediation analysis to detect the direct and indirect effects mediated by leptin in asthma development confirmed the odds ratio of direct effect were 1.59 (95% ci: 0.95-2.97), 2.06 (1.06-4.00) and 3.25 (1.01-9.41), respectively while the indirect effect mediated by leptin odds ratio were 1.68 (1.09-2.46), 1.55 (0.99-2.57) and 1.99 (0.94-4.83), respectively [24]. leptin mediated its effects by augmenting phagocytic function. table 6. socio demographic data of asthmatic obese children age in year weight(kg) height(cm) bmi sex asthma severity 10 47 130 27.8 female mild 5 34 14 26.2 male mild 11 36.5 120 25.3 female moderate 5 28 103 26.4 female moderate 4 31 107 27.1 male mild 13 66 152 28.6 female moderate 8 45 126 28.3 female moderate 7 37 117 27 male severe 6 37 112 29.5 female moderate 10 47 134 26.2 male moderate 9 50 133 28.3 female severe 4 25 97 26.6 female severe 9 43 122 28.9 female mild 3.5 25 98 26 male moderate 10.5 58 141 29.2 female moderate 15 80 162 29.7 female moderate elngar et al.; aji, 2(1): 26-33, 2019; article no.aji.53002 31 fig. 1. roc curve of serum leptin for diagnosis of asthma severity proliferation and alternation of macrophages and consequently led to increased secretion of proinflammatory cytokines. the latter include tumor necrosis factor α, reactive oxygen species and interleukin 6 [25]. our study results showed that there was significantly negative correlation between leptin and pefr results in total asthmatic patients (r=0.468 and p=0.001). this result agreed with the result of sin and man work who also reported a negative correlation between serum leptin levels and pefr in asthmatic patients (r=-0.521and p=0.001) [26]. our study also showed significant relation between serum leptin levels and history of allergy (p=0.019) as well as family history of asthma (p= <0.001). the positive correlation between history of allergy and serum leptin level is consistent with these results reported by samra, et al. as there was a positive correlation between leptin levels and history of allergy (p=0.0058) [27]. concerning the severity of asthma, this study showed that the mean ± sd of serum leptin in moderate asthmatics (89.3±4.6 ng/ml) was significantly higher than that of mild asthmatics (82.0±3.2 ng/ml) p< 0.001. these results were in accordance with those reported by the american college of allergy, asthma and immunology study [28], that linked serum concentration of leptin to disease activity and severity, patients with intermittent asthma had value of 8.9 ng/dl, those with mild persistent asthma had levels of 14.5 ng/dl and moderately persistent asthma patients had 18.9 ng/dl with p-value less than 0.05. shore et al. suggested that increase serum leptin can increase airway hyperresponsivness and exacerbate asthma [29]. our results showed that the sensitivity of serum leptin for diagnosis of severe asthma showed serum leptin was 91.5% accurate (p <0.001). at cutoff value of 75.5, serum leptin was shown 100% sensitivity and 75% specificity for diagnosis of severe asthma. these findings were similar to those reported by tanju et al who reported 89% accuracy of serum leptin levels in severe asthma (p <0.001). our results suggest that leptin is playing an important role in diagnosis and determining the severity of asthma. the study showed also that leptin is the most predictive factor for severity of asthma when compared to body mass index, family history of asthma and history of allergy which has low significant prediction for severity of asthma. 5. conclusion asthma in obese children is significantly correlated with serum leptin levels and bmi. so, high leptin levels and bmi may serve as markers of asthma severity in obese children as well as potential future therapeutic targets in the era of advanced bionanotechnology.this approach can be possible via targeting vital cell signaling pathways by novel ligands that act as agonists or antagonists for crucial adaptor molecules in asthma pathogenesis mediated by leptin such as elngar et al.; aji, 2(1): 26-33, 2019; article no.aji.53002 32 parathyroid-hormone-related protein (pthrp) and prostaglandin e2 (pthrp/pparγ) signaling pathway inhibitors. 6. future research directions due to heterogeneity of obesity related asthma in children and its multifactorial nature of the disease with genetic-environmental interactions, it is difficult to find out precise markers. however, emerging novel biomarkers for severe asthma using multi-omics technologies such as proteomics, epigenetics, transcriptomics, metagenomics, gut microbiome and untargeted metabolomics is a promising approach. novel innovative advances in quantum computational sciences, biostatistics and molecular bioinformatics of asthma are needed for appropriate phenotypic subclassification and searching for more precise future digital biomarkers and enabling progress of precision medicine for asthma. consent informed consent was obtained from all individual participants included in the study. ethical approval all procedures performed in this study were in accordance with the ethical standards of the institutional and national research committee and with the 1964 helsinki declaration and its later amendments. competing interests authors have declared that no competing interests exist. references 1. centers for disease control and prevention. vital signs: asthma prevalence, disease characteristics, and self-management education: united states. 2001-2009. mmwr morb mortal wkly rep. 2011;60:547–52. available:https://www.cdc.gov/mmwr/previ ew/mmwrhtml/mm6017a4.htm 2. akinbami lj, moorman je, garbe pl, sondik ej. status of childhood asthma in the united states, 1980–2007. pediatrics. 2009;123(suppl3):s131–45. doi: 10.1542/peds.2008-2233c 3. akinbami lj, simon ae, rossen lm. changing trends in asthma prevalence among children. pediatrics. 2016; 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12(11):32–41. 23. loffreda s, yang sq, lin hz, et al. leptin regulates pro-inflammatory immune responses. faseb j. 1998;12. 24. li z, leynaert b, dumas o, diaz gil o, et al. role of leptin in the association between body adiposity and persistent asthma: a longitudinal study. obesity (silver spring). 2019;27(6):894-898. doi: 10.1002/oby.22466 tilg h, moschen ar. adipocytokines: mediators linking adipose tissue, inflammation and immunity. nat rev immunol. 2006;6:772-783. 25. sin dd, man sfp. impaired lung function and serum leptin in men and women with normal body weight: a population based study. thorax. 2003;58(8):695–8 . 26. samra nm, ei-sharkawi sz, abd el-ghany hm, mohammed zt. relationship between concentration of serum leptin and childhood asthma in egypt. res j med med sci. 2009;4(2):249–56. 27. tanju a, cekmez f, aydinoz s, karademir f, suleymanoglu s, gocmen i. association between clinical severity of childhood asthma and serum leptin levels. indian j pediatr. 2011;78(3):291–5. 28. shore sa, fredberg jj. obesity, smooth muscle, and airway hyper responsiveness. j allergy clin immunol. 2005;115(5):925– 7. _________________________________________________________________________________ © 2019 elngar et al.; this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: http://www.sdiarticle4.com/review-history/53002 https://www.ncbi.nlm.nih.gov/pubmed/?term=li%20z%5bauthor%5d&cauthor=true&cauthor_uid=31004416 https://www.ncbi.nlm.nih.gov/pubmed/?term=leynaert%20b%5bauthor%5d&cauthor=true&cauthor_uid=31004416 https://www.ncbi.nlm.nih.gov/pubmed/?term=dumas%20o%5bauthor%5d&cauthor=true&cauthor_uid=31004416 https://www.ncbi.nlm.nih.gov/pubmed/?term=diaz%20gil%20o%5bauthor%5d&cauthor=true&cauthor_uid=31004416 https://www.ncbi.nlm.nih.gov/pubmed/31004416 https://www.ncbi.nlm.nih.gov/pubmed/31004416 http://creativecommons.org/licenses/by/4.0 _____________________________________________________________________________________________________ *corresponding author: e-mail: johnisa66@yahoo.com; asian journal of immunology 2(1): 18-25, 2019; article no.aji.52667 immunomodulatory effects of honey in wistar rats infected with salmonella typhimurium justinah f. john-isa1*, tinuola t. adebolu1 and victor o. oyetayo2 1 department of microbiology, federal university of technology akure (futa), p.o.box 704, akure, ondo state, nigeria. 2 department of microbiology, federal university of technology, akure, nigeria. authors’ contributions this work was carried out in collaboration among all authors. author jfji designed the study, performed the statistical analysis, wrote the protocol and wrote the first draft of the manuscript. authors tta and author voo managed the analyses of the study. author tta managed the literature searches. all authors read and approved the final manuscript. article information editor(s): (1) dr. jaffu othniel chilongola, department of biochemistry and molecular biology, kilimanjaro christian medical university college, tumaini university, tanzania. reviewers: (1) asma bouasla, university souk-ahras, algeria. (2) oyiborhoro onoriode, university of medical sciences, nigeria. (3) syed umer jan, university of balochistan, pakistan. complete peer review history: http://www.sdiarticle4.com/review-history/52667 received 10 september 2019 accepted 14 november 2019 published 19 november 2019 abstract aim: to evaluate the immunomodulatory effects of honey on wistar rats infected with salmonella typhimurium. place and duration of study: research laboratory of the federal university of technology akure (futa), ondo state, nigeria between july, 2019 and september, 2019. methodology: a total thirty – nine (39) apparently healthy wistar rats, three (3) rats per group were used in this study. twelve (12) out of the rats were used to determine the infectivity dose of s. typhimurium on the rats and twenty – seven (27) rats for infection and treatment assay. the rats were divided into nine (9) groups of 3 rats per group, the first 8 groups were infected with s. typhimurium and treated for seven (7) days with honey, augmentin and oral rehydration solution (ors) (different treatment for different groups) except group 1 that was infected and not treated and group 9, that was not infected, not treated. the blood samples of all the rats was collected after treatment to study the effect of honey on the haematological parameters of the rats. original research article john-isa et al.; aji, 2(1): 18-25, 2019; article no.aji.52667 19 results: honey administered at 2ml and 3ml twice daily to the s. typhimurium infected rats exerted good therapeutic potential in combating diarrhoea in the animals. also, in these group of rats, honey caused an increase in the pcv, rbc, hb and lymphocytes which displays honey to be a good immunostimulator and immunomodulator. conclusion: honey exerted therapeutic, haematinic and immunomodulatory potentials in rats infected with s. typhimurium. these findings therefore could be exploited in the treatment of diarrhoeal diseases caused by this bacterium. keywords: s. typhimurium; wistar rats; honey; augmentin; oral rehydration solution (ors); immnuomodulation. 1. introduction salmonella typhimurium is a gram-negative, flagellated, aerobic (oxygen-consuming) bacteriium, the major cause of human salmonellosis [1], a type of gastroenteritis, or inflammation of the intestine [2]. s. typhimurium is also a frequent cause of acute, self-limiting food borne diarrhoea. it is spread primarily by contaminated food and drink, but it can come in contact with human via direct contact with an infected animal or pet [1]. salmonella typhimurium induces a systemic infection in rats, so s. typhimurium-infected rats have been extensively used as models for the understanding of the immunological and antibacterial effect of honey. diarrhoeal diseases are among the leading causes of morbidity and mortality in young children in developing countries [3]. it is characterized by frequent, loose and watery stool which may result in dehydration and in severe cases, death. each year, an estimated 2.5 billion cases of diarrhoea occur among the children under five years of age, and estimates suggest that overall incidence has remained relatively stable over the past two decades. although, diarrhoea is self-limiting however when it is as a result of bacterial infection, antibiotic therapy might be required but because of the high resistance rate of bacteria to available antibiotics, administration of antibiotics may not result in recovery of patients. moreover, some of these antibiotics can also induce diarrhoea known as “antibiotic induced diarrhoea” [4]. therefore, it becomes imperative to search for alternatives to conventional antibiotics to treat this disease. in most ancient cultures, honey has been used for both nutritional and medical purposes. the belief that honey is a nutrient, a drug and an ointment has been carried into our days, and thus, an alternative medicine branch, called ‘‘apitherapy’’, has been developed in recent years, offering treatments based on honey and other bee products against many diseases including bacterial infections. honey has been reported to have immunomodulatory and antibacterial activity on bacteria found in wounds [5], responsible for food spoilage [6], common diarrhoeagenic bacteria such as s. typhimurium [7] and many other bacterial species. it becomes worthwhile therefore to investigate whether honey has therapeutic and immunostimulatory potentials in wistar rats infected with s. typhimurium in addition to nits antibacterial potential. 2. materials and methods 2.1 location and duration of the research the research was carried out in the graduate research laboratory of department of microbiology, federal university of technology, akure, ondo state, nigeria between july and september, 2019. 2.2 honey sample the honey sample used was collected from funaab, abeokuta ogun state. it’s of wild flora source. 2.3 test organism the test organism used was salmonella typhimurium. 2.4 isolation and identification of the test organism s. typhimurium was isolated from poultry droppings, the droppings was serially diluted in sterile distilled water using the method of boateng and diunase [8]. the dilutions were plated on salmonella shigella agar to isolate the bacterium and was identified based on morphological and biochemical characteristics according to the method of rozanska [9]. 2.5 experimental animals a total of 39 female wistar rats of weight range 60-90 g were used for the study. the animals john-isa et al.; aji, 2(1): 18-25, 2019; article no.aji.52667 20 were purchased at animal production and health dept. of federal university of technology akure, ondo state. they were brought to the animal house of microbiology department, futa and acclimatized for 7 days before the commencement of this work. the animals were fed with broiler starter and clean water twice daily. 2.6 determination of infectivity dose (id) of salmonella typhimurium a total of twelve (12) female apparently wistar rats was used to determine infectivity dose. the rats were divided into four groups of 3 rats per cage. this was done using standard method described by adebolu et al. [10]. a colony of s. typhimurium of 24 hrs old was inoculated into 100 ml of nutrient agar, incubated at 37ºc for 18 – 24 hrs. the cells were harvested by centrifuging at 3000 rpm for 15 minutes. the supernatant was decanted and 10ml of sterile normal saline was poured into the tube and was further centrifuged to wash the cells, this was done three times. serial dilution was carried on the harvested cells and 1ml was taken from each of the different concentrations already prepared to infect different groups of the experimental animals respectively. the dilution that produced the symptoms of illness in all of the animals was taken as the infectivity dose (id) of the organism. 2.7 experimental design a total of 27 female apparently healthy wistar rats were assigned into nine (9) treatment groups designated as 1 – 9. i.e. 3 rats per cage. rats in group 1 were infected with the id of s. typhimurium and not treated, rats in group 2 were infected and treated with 1 ml raw honey 12 hourly, group 3 infected and treated with 2 ml raw honey 12 hourly, group 4 infected and treated with 3ml raw honey 12 hourly, group 5 infected and treated with 0.5 ml augmentin (30 mg/kg/day) 12hourly, group 6 infected and administered honey – ors 12 hourly, group 7 infected and administered 1ml commercial ors 12 hourly, group 8 infected and administered 1ml homemade ors 12 hourly and group 9 not infected, not treated (control group). 2.8 infection of rats with the id of s. typhimurium the infection of the animals was done using the infectivity dose (id) of the organism by orogastrically dosing them according to the method of adebolu et al. [10]. the infectivity dose used in this study was calculated to be 1.5 x10 8 cfu/ml. 2.9 treatment of infected rats treatment begins 24 hours after which infection has set in, specific volume of honey, augmentin, honey – ors, ors both commercial and home made variants were administered to the infected rats for 7 days according to oladunmoye [11]. 2.10 isolation, identification and enumeration of s. typhimurium in the faeces of infected rats one gram (1 g) of faeces of the infected rats were aseptically collected, serial dilution was done on them and plated on salmonella shigella agar in order to isolate the salmonella typhimurium present in the rats and monitor their bacterial count throughout the experiment [12]. 2.11 weighing of animals the weight of the animals were taken throughout the pre and post ingestion period using the method of momoh et al. [13]. 2.12 haematological assay the blood of the infected and uninfected rats was collected weekly into edta bottles after which the packed cell volume (pcv), haemoglobin (hb), red blood cell (rbc), white blood cell (wbc) and differential leukocytes counts of the collected blood samples were evaluated according to the method described by baker et al. [14]. 2.13 statistical analysis all experiments were done in triplicates, mean, standard deviation were calculated for all data using descriptive statistics and difference between means was determined by duncan’s new multiple range test at p≤.05. 3. results and discussion all the rats infected with s. typhimurium and treated with honey recovered by the 3 rd day while those ones that was administered honey – ors john-isa et al.; aji, 2(1): 18-25, 2019; article no.aji.52667 21 and the ones with homemade ors recovered by day 4 and those treated with augmentin and those administered commercial ors recovered by the 5 th day, those that were infected but not treated started to show signs of recovery by the 6 th day. the recovery without treatment by the 6 th day confirms that acute diarrhoea is self limiting according chen et al. [15]. the mean recovery times of rats infected with salmonella typhimurium and treated with honey –ors was significantly reduced when compared with infected and not treated group (table 1). this is agreement with the work of beretta et al. [16]. there was no evidence of s. typhimurium in the faeces of rats treated with 1 ml, 2 ml, 3 ml of honey and administered 1 ml of honey –ors 12 hourly for 7 days (fig. 1). there was no evidence of salmonella typhimurium in the faeces of rats treated with 1 ml, 2 ml, 3 ml of honey and administered 1 ml of honey – ors 12 hourly for 7 days. (fig. 1). table 1. physical observations of the wistar rats during infection with salmonella typhimurium and treatment group of rats treatment interval (days) 1 2 3 4 5 6 7 1 infected and not treated with honey ra, el, uf,pm,ss ra, el, uf, pm, ss ra, el, uf, pm, ss ra, el, uf, pm, ss ra, el, uf, pm, ss a, ew, fs, nm, sf a, ew, fs, nm, sf 2 infected and treated with 1 ml honey (12 hourly) ra, el, uf, pm, ss ra, el, uf, pm, ss a, ew, fs, nm a, ew, fs, nm a, ew, fs, nm a, ew, fs, nm a, ew, fs, nm 3 infected and treated with 2 ml honey (12 hourly) ra, el, uf, pm, ss ra, el, uf, pm, ss a, el, uf, pm, ss a, el, fs, nm, sf a, el, fs, nm, sf a, ew, fs, nm, sf a, ew, fs, nm, sf 4 infected and treated with 3 ml honey (12 hourly) ra, el, uf, pm, ss ra, el, uf, pm, ss a, el, uf, pm, ss a, el, fs, nm, sf a, el, uf, nm, sf a, ew, fs, nm, sf a, ew, fs, nm, sf 5 infected and treated with 0.5 ml augmentin (12 hourly) ra, el, uf, pm, ss ra, el, uf, pm, ss ra, el, uf, pm, ss ra, el, uf, pm, ss a, ew, fs, nm, sf a, ew, fs, nm, sf a, ew, fs, nm, sf 6 infected and treated with 1 ml honey ors (12 hourly) ra, el, uf, pm, ss ra, el, uf, pm, ss ra, el, uf, pm, ss a, ew, fs, nm, sf a, ew, fs, nm a, ew, fs, nm, sf a, ew, fs, nm, sf 7 infected and treated with 1 ml ors (12 hourly) ra, el, uf, pm, ss ra, el, uf, pm, ss ra, el, uf, pm, ss ra, el, uf, *pm, ss a, ew, fs, nm, sf a, ew, fs, nm, sf a, ew, fs, nm, sf 8 infected and treated with 1 ml homemade (12 hourly) ra, el, uf, pm, ss ra, el, uf, pm, ss ra, el, uf, pm, ss a, ew, fs, *pm, ss a, ew, fs, nm, sf a, ew, fs, nm, sf a, ew, fs, nm, sf 9 not infected and not treated a, ew, fs, nm, sf a, ew, fs, nm, sf a, ew, fs, nm, sf a, ew, fs, nm, sf a, ew, fs, nm, sf a, ew, fs, nm, sf a, ew, fs, nm, sf key: a = activeness, ra = reduced activity, el = eating little, ew = eating well, uf = informed stool, fs = formed stool, sf = smooth fur, ss = scattered fur, pm = presence of mucous, *pm = high presence of mucous, nm = no mucous john-isa et al.; aji, 2(1): 18-25, 2019; article no.aji.52667 22 fig. 1. total counts of s. typhimurium in the faeces of wistar faeces after infection and treatment key: a = infected and not treated, b = infected and treated with 1 ml honey, c = infected and treated with 2 ml honey, d = infected and treated with 3 ml honey, e = infected and treated with 0.5 ml augmentin, f = infected and administered with 1 ml honey – ors, g = infected and administered with 1ml commercial ors, h = infected and administered with 1 ml homemade ors, tm1 = treatment day 1, tm2 = treatment day 2. tm3 = treatment day 3, tm4 = treatment day 4 and tm7 = treatment after day 7 the infected rats lost weight as a result of the infection with s. typhimurium however, administration of honey to the infected rats caused a significant increase (p<0.05) in their weight but the infected and not treated rats recorded weight loss for a longer duration than those that were administered different volumes of honey (fig. 2a-e). the observation that the infected and not treated rats did not gain back their body weight throughout the duration of the research is in agreement with the work of momoh et al. [13]. a b 0 1 2 3 4 5 6 7 24 h/tm1 48 h/tm2 72 h/tm3 96 h / tm4 168 h / tm7 lo g cf u /m l infection period (h)/treatment a b c d e f g h 0 20 40 60 80 100 120 0 1 2 3 4 5 6 7 w ei gh t (g ) period (day) infected and not treated with honey not infected and not treated 0 20 40 60 80 100 120 0 1 2 3 4 5 6 7 w ei gh t (g ) period (day) infected and treated with 1ml honey not infected and not treated john-isa et al.; aji, 2(1): 18-25, 2019; article no.aji.52667 23 c d e fig. 2 a–e. average weight of wistar rats before infection with s. typhimurium, at the onset of infection and after treatment with honey and augmentin infection of rats with s. typhimurium caused a decrease in their pcv, hb and rbc and increase in their neutrophil counts, showing a sign of infection but after treatment with honey (between 2ml and 3ml), there was no significant difference in the pcv, wbc of the group of rats treated with honey and the group not infected, not treated (control) (tables 2a and b). administration of honey to apparently healthy rats (control) caused a significant (p<.05) increase in the pcv and lymphocytes of the rats. this shows that the honey has both haematinic and immunomodulatory potentials. 0 20 40 60 80 100 120 0 1 2 3 4 5 6 7 w ei gh t (g ) period (day) infected and treated with 2ml honey not infected and not treated 0 20 40 60 80 100 120 0 1 2 3 4 5 6 7 w ei gh t (g ) period (day) infected and treated with 3ml honey not infected and not treated 0 20 40 60 80 100 120 0 1 2 3 4 5 6 7 w ei gh t (g ) period (day) infected and treated with 0.5ml augmentin not infected and not treated john-isa et al.; aji, 2(1): 18-25, 2019; article no.aji.52667 24 table 2a. effect of honey on the haematological parameters of wistar rats infected with s. typhimurium group pcv (%) hb (g/l) wbc (10 9 /l) rbc (10 12 g/l) 1 32.50 ± 4.95 b 10.85 ± 1.66 b 12.70 ± 0.28 a 3.00 ± 0.82 b 2 40.00 ± 2.83 ab 13.50 ± 0.71 ab 11.70 ± 0.28 ab 3.20 ± 0.25 ab 3 42.50 ± 2.12 a 14.14 ± 0.66 a 8.77 ± 0.49 c 4.04 ± 0.87 ab 4 43.00 ± 4.24 a 14.30 ± 1.36 a 8.94 ± 0.42 c 4.29 ± 0.07 a 5 40.33 ± 4.51 ab 13.50 ± 1.51 ab 10.86 ± 0.17 abc 4.06 ± 0.30 ab 6 35.50 ± 0.71 ab 11.90 ± 0.28 ab 10.88 ± 0.05 abc 3.66 ± 0.03 ab 7 39.67 ± 4.51 ab 13.19 ± 1.56 ab 9.56 ± 1.92 bc 4.07 ± 0.34 ab 8 35.00 ± 6.00 ab 11.64 ± 2.00 ab 9.83 ± 1.47 bc 3.66 ± 0.53 ab 9 42.33 ± 2.08 a 13.66 ± 0.35 ab 8.82 ± 1.06 c 4.07 ± 0.58 ab key: 1 = infected and not treated, 2 = infected and treated with 1ml honey, 3 = infected and treated with 2ml honey, 4 = infected and treated with 3ml honey, 5 = infected and treated with 0.5ml augmentin, 6 = infected and administered with 1ml honey – ors, 7 = infected and administered with 1ml commercial ors, 8 = infected and administered with 1ml homemade ors, 9 = not infected, not treated, pcv = packed cell volume, hb = haemoglobin concentration, wbc = white blood cell and rbc = red blood cell table 2b. effect of honey on the haematological parameters of wistar rats infected with s. typhimurium contd group lymphocytes (%) neutrophils (%) monocytes (%) eosinophils (%) basophil (%) 1 31.50 ± 0.71 f 64.50 ± 0.71 a 3.00 ± 0.00 a 1.50 ± 0.71 a 1.00 ± 0.00 a 2 37.50 ± 0.71 bc 61.00 ± 1.41 ab 2.50 ± 0.71 a 2.00 ±1.41 a 1.00 ± 0.00 a 3 40.00 ± 2.83 ab 56.50 ± 0.71 b 1.50 ± 0.71 a 1.00 ± 0.00 a 0.00 ± 0.00 4 40.50 ± 0.71 a 57.00 ± 1.41 b 1.50 ± 0.71 a 1.00 ± 0.00 a 0.00 ± 0.00 5 39.33 ± 150 abc 56.00 ± 4.00 b 1.67 ± 1.15 a 1.00 ± 0.00 a 0.00 ± 0.00 6 37.00 ± 1.41 cd 60.00 ± 2.83 ab 2.00 ± 1.41 a 1.50 ± 0.71 a 0.00 ± 0.00 7 32.67 ± 1.15 ef 60.33 ± 3.79 ab 2.00 ± 1.00 a 1.67 ± 0.58 a 0.00 ± 0.00 8 34.67 ± 0.58 de 60.67 ± 1.15 ab 2.33 ± 0.58 a 1.67 ± 0.58 a 1.00 ± 0.00 a 9 40.00 ± 1.00 ab 57.33 ± 1.15 b 1.33 ± 0.58 a 1.33 ± 0.58 a 0.00 ± 0.00 key: 1 = infected and not treated, 2 = infected and treated with 1ml honey, 3 = infected and treated with 2ml honey, 4 = infected and treated with 3ml honey, 5 = infected and treated with 0.5ml augmentin, 6 = infected and administered with 1ml honey – ors, 7 = infected and administered with 1ml commercial ors, 8 = infected and administered with 1ml homemade ors and 9 = not infected, not treated 4. conclusion this study has shown that honey sample from funaab (hf) caused enhanced immune response in the rats against s. typhimurium. this honey also has haematinic, immunomodulatory, and immunostimulatory potentials in rats infected with s. typhimurium. these findings therefore could be exploited in boosting the immune system and in the treatment of diarrhoeal diseases caused by this bacterium. consent it is not applicable ethical approval as per international standard written ethical approval has been collected and preserved by the author(s). acknowledgements the authors appreciate the effort made by mr jimoh, kabiru ayobami for cross checking the statistical analysis. competing interests authors have declared that no competing interests exist. references 1. torpdahl m, tsai-ling l, shiu-yun l, ishien l, sung-hsi w, and chienshun c. human isolates of salmonella enterica serovar typhimurium from taiwan displayed significantly higher levels of antimicrobial resistance than those from denmark. international journal of food microbiology. 2013;69-75. john-isa et al.; 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[isbn-13:978-1118285954] [isbn-10:1118285956] 3. chen c, campbell lt, blair se, carter dc. the effect of standard heat and filtration processing procedures on antimicrobial activity and hydrogen peroxide levels in honey. frontiers microbiology. 2012;3(265): 1-8. 4. cheesbrough m. medical laboratory manual for tropical countries. microbiology. university of cambridge press. great britain. 2006;ii:479. 5. basualdo c, sgroy v, finola ms, junm m. comparison of the antibacterial activities of honey from different provenance against bacterial usually isolated from skin wounds. veterinary microbiology. 2007; 127:375-381. 6. lusby pe, coombes al, wilkinson jm. bbactericidal activity of different honeys against pathogenic bacteria. archieve medical resources. 2005;36:464-467. 7. adebolu tt. effects of natural honey on local isolates of diarrhoea causing bacteria in southwestern, nigeria. african journal of biotechnology. 2005;4(10):1172-1174. 8. boateng j, diunase kn. comparing the antibacterial and functional properties of cameroonian and manuka honey for potential wound healing. molecules, mdpi journals. 2015;9(20):16068-16084. 9. rozanska hanna. microbiological quality of polish honey. national veterinary research institute. pulway. 2011;55:443–445. 10. adebolu tt, adeoye oo, oyetayo vo. effect of garlic (allium sativum) on salmonella typhi infection, gastrointestinal flora and haematological parameters of albino rats. african journal of biotechnology. 2011;10(35):6804-6808. 11. oladunmoye mk. the imunostimulatory effect of ethanolic extract of cassia alata on immune system of albino rats dosed with staphylococcus aureus (ncib 8588). journal of pharmacology and toxicology. 2007;2(2):200-204. 12. fawole mo, oso ba. characterization of bacteria: laboratory manual of microbiology. 4 th edn. spectrum book ltd, ibadan nigeria. 2004;24-33. 13. momoh ao, adebolu tt, ogundare ao. therapeutic effects of beniseed extracts and fermented liquor in treating diarrhoea in albino rats infected with bacillus cereus. global advanced research journal of medicinal plants. 2012;1(1):007-012. 14. baker fj, silverstone re. medical laboratory science. 8 th edition. chris publisher, washington d.c., usa. 2006; 447. 15. chen c, campbell lt, blair se, carter dc. the effect of standard heat and filtration processing procedures on antimicrobial activity and hydrogen peroxide levels in honey. frontier microbiology. 2012;3(265): 1-8. 16. beretta g, gelmini f, lodi v, piazzalunga a, facino rm. profile of nitric oxide (no) metabolites (nitrate, nitrite and n-nitroso groups) in honeys of different botanical origins: nitrate accumulation as index of origin, quality and of therapeutic opportunities. journal of pharmaceutical and biomedical analysis. 2010;53(3):343349. _________________________________________________________________________________ © 2019 john-isa et al.; this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: http://www.sdiarticle4.com/review-history/52667 http://creativecommons.org/licenses/by/4.0 _____________________________________________________________________________________________________ *corresponding author: e-mail: makwethomas2@gmail.com; asian journal of immunology 3(1): 139-144, 2020; article no.aji.56215 preliminary report on the prevalence of intestinal parasites and malaria among people living with hiv/aids within makurdi metropolis, benue state, nigeria o. t. makwe1*, v. u. obisike2, o. amali3 and g. n. imandeh3 1 bioresources development center (national biotechnology development agency), abagana, anambra state, nigeria. 2 department of animal and environmental biology, abia state university, uturu, nigeria. 3 department of zoology, federal university of agriculture, makurdi, benue state, nigeria. authors’ contributions this work was carried out in collaboration among all authors. authors otm, oa and gni designed the study. authors otm and vuo performed the statistical analysis, wrote the protocol and wrote the first draft of the manuscript. authors otm, vuo and oa managed the analyses of the study. authors otm and vuo managed the literature searches. all authors read and approved the final manuscript. article information editor(s): (1) dr. darko nozic, university of belgrade, serbia. reviewers: (1) florencia del puerto, universidad nacional de asunción, paraguay. (2) ana júlia pinto fonseca sieuve afonso, universidade nova de lisboa, portugal. (3) ifeyinwa chijioke-nwauche, university of port harcourt, nigeria. complete peer review history: http://www.sdiarticle4.com/review-history/56215 received 18 february 2020 accepted 24 april 2020 published 02 may 2020 abstract this research was conducted to determine the prevalence of intestinal parasites and malaria among people living with hiv/aids within makurdi metropolis, benue state, nigeria. four hundred (400) blood and stool samples of people living with hiv/aids were collected from 45 naf base hospital and bishop murray hospital, makurdi and examined for intestinal parasites and malaria infections between the months of september and december 2014. formal-ether concentration technique was used for the stool examination; the thick film was prepared for blood examinations. the most prevalent parasitic infections found was malaria (plasmodium falciparum) (46.0%).this was followed by taenasis (taenia solium) (13.0%), amoebasis (entamoeba histolytica) (8.0%) and original research article makwe et al.; aji, 3(1): 139-144, 2020; article no.aji.56215 140 hookworm disease (4.0%). the result shows that the age groups1– 10 and 11– 20 years had the highest prevalence of malaria and intestinal parasites (93.0%) each. while 51– 60 years had the least prevalence of malaria and intestinal parasites (50.0%). chi-square analysis showed that there was significant differences in the distribution of intestinal parasite and malaria by age (χ 2 cal= 120.835 df 6, p˂0.05).chi-square test also revealed that there was significant differences between the infections in male and female individuals (χ 2 cal= 36.288df 60, p˂0.05 and χ 2 cal= 67.924df 60, p˂0.05) respectively. the prevalence rate of intestinal parasite and malaria is high among people living with hiv/aids within makurdi metropolis. keywords: malaria; intestinal parasite; hiv/aids; makurdi. 1. introduction helminthinfection is recognized as one of the important public health problems in tropical africa. intestinal helminths are among the commonest and most widespread of human infection. man acquires intestinal helminths when in contact with the infective stage of the parasites. infection can also be acquired through contaminated, unwashed fingers (fecal-oral), eggs can be spread by flies and other animals, circulation of bank notes and by wind during dry condition [1]. intestinal parasites are parasites that populate the gastro-intestinal tract in humans and other animals, they can live throughout the body, but most preferred the intestinal wall [2]. the major groups of parasites include protozoans (organisms having only one cell) and parasitic worms (helminth), of these, protozoans, including cryptosporidium, microsporidia, and isospora, are most common in hiv-infected persons. each of these parasites can infect the digestive tract, and sometimes two or more can cause infection at the same time [3]. children are particularly susceptible if they are not thoroughly cleaned after coming into contact with infected soil that is present in environments that they may frequently visit such as sandboxes and school playgrounds also people in developing countries are also at risk of intestinal and malaria parasites due to drinking water from sources that may be contaminated with parasites that colonize the gastrointestinal tract and the sleeping site. 2. methodology 2.1 study population sample size: sample size was determined using this formula: s= x 2 np (1-p) ÷ d 2 (n-1) + x 2 p (1-p) where: s = sample size being sought x 2 = table value for chi-square at 1 degree of freedom at the desired alpha level (0.05 = 3.84: 01 =6.64) n = population size p = the population proportion (usually 0.05 as this provides the maximum sample size). d = degree of accuracy desired, expressed as a proportion (usually 0.05). (rosie, 2006). a total number of 400 samples were collected and analyzed. the samples were collected from bishop murray hospital makurdi and 45 naf base hospital makurdi, these two hospital run hiv clinical services weekly and also tend to have the highest number of hiv patients attending clinical service. a total number of 200 samples were collected from bishop murray hospital makurdi while 200 from 45 naf base hospital makurdi. approval was gotten from the hospital management board and informed consent was obtained from each of the participants and or their parents/ guardians in case of children prior to specimen collection. 2.2 method of sample selection table of random numbers was used to randomly select participant for this investigation at the selected hospitals in makurdi metropolis. 2.3 collection of fecal samples each enrolled hiv/aids patient was asked to provide a fresh fecal sample in cleaned and dried sterile specimen bottles that were provided. the selected individuals were adequately instructed on how to get a little portion of their stool into the bottles using cardboard paper and applicator stick. each participant was interviewed for sociodemographic variables. http://en.m.wikipedia.org/wiki/parasite http://en.m.wikipedia.org/wiki/gastro-intestinal_tract http://en.m.wikipedia.org/wiki/protozoan http://en.m.wikipedia.org/wiki/parasitic_worm http://en.m.wikipedia.org/wiki/parasitic_worm http://en.m.wikipedia.org/wiki/cryptosporidium http://en.m.wikipedia.org/wiki/microsporidia http://en.m.wikipedia.org/wiki/isospora http://en.m.wikipedia.org/wiki/hiv makwe et al.; aji, 3(1): 139-144, 2020; article no.aji.56215 141 2.4 laboratory procedure for intestinal parasite (chesbrough (2006) each stool specimen was assessed for consistency. then, it was examined by direct wet mount method using normal saline (0.85% nacl solution) in order to prevent the loss of motile stage of parasites. lugol's iodine was used to detect the cyst of intestinal parasites, also formal ether concentration techniques was used for the diagnosis. faeces were emulsified by stirring 1g of stool suspension in 10ml of normal saline solution. the suspension was filtered into a centrifuge tube to remove large faecal particle before adding ether. the filtrate was centrifuged at 1,500 revolutions per minute for 3 minutes and the supernatant was discarded before keeping the tube in an upright position to allow water from the side to drain to the bottom. one to two (1-2) drops of sediment was transferred to a clean grease-free slide and then covered with a coverslip and the preparation was examined microscopically using ×10 objective lens. the laboratory investigation was carried out at city hospital old g.r.a makurdi. diagnosis was based on the identification of helminth ova and protozoan cyst in the sample during microscopic examination. a person was considered to have a multiple infection if they were found to be positive for more than one species. 2.5 data analysis ch-square test was use to test the association between intestinal helminthes and malaria infection amongst hiv patients at p=0.05. 3. results table 1 shows the age distribution of intestinal and malaria parasites. the age group of 11-20 years has the highest infection of (93%), while 51-60 years has the least infection of (50%). chisquare test reveal that there is significant difference in distribution of intestinal and malaria parasites by age (χ 2 cal= 120.835df 6, p˂0.05). table 2 distribution of intestinal and malaria parasites in male living with hiv/aid within makurdi metropolis. the age group 11-20 years recorded the highest infection of (95%), while 60 years and above has the least infection of (40%). chi-square test reveal there is significant difference in distribution of intestinal and malaria parasites by sex and age (χ 2 cal= 36.288df 6, p˂0.05). table 3 distribution of intestinal and malaria parasites in female living with aids within makurdi metropolis. the age group <1-10 years has the highest infection of (91%) while 60 years and above has the least infection of (50%). chi-square test reveal there is significant difference in distribution of intestinal and malaria parasites by sex and age (χ 2 cal= 63.308df 6, p˂0.05 4. discussion the result of this research work shows that the age groups 1-10 and 11-20years has the highest prevalence rate of infection (93%) of intestinal parasites and malaria parasites; this could be attributed to the fact that soil children play with contaminated soil. this agrees with russel and howson [4], who reported that the high-risk groups include young children, pregnant women, immune suppressed aids individuals, refugees, displaced persons and labourers entering endemic areas. age groups 1-10 and 11-20 years had the highest prevalence of entaoeba histolytica at the rate of 19% while was the age group 11-20years had 18% prevalence of taenia solium .children are susceptible to intestinal parasites and can be attributed to dirty behavioral pertain among school age children. this study is in agreement with reports of montresor et al. [5] who reported that children may also be particularly susceptible to the adverse effects of helminth infections due to their incomplete physical development and their greater immunological vulnerability. the study further agrees with albonico et al. [6] who reported that children age 0-20 years habour heavy intestinal parasites and thus are a good study group; they are the group most responsible for contaminating the environment and transmitting these infections. this also agrees with nwosu (1981), school children carry the heaviest burden of the associated morbidity, due to their dirty habits of playing or handling of infested soils, eating with soiled hands, unhygienic toilet practices, drinking and eating of contaminated water and food. hookworm, the age group 11-20 years recorded (12%), the result of this study contrast with the work albonico et al. [6] who reported that in subsaharan africa alone, there are 41 million hookworm-infected school-age children. makwe et al.; aji, 3(1): 139-144, 2020; article no.aji.56215 142 table 1. age distribution of intestinal and malarial parasites among people living with hiv/aids within makurdi metropolis parasite ˂1 10 n=54 a(%) 11-20 n=75 a(%) 21-30 n=74 a(%) 31-40 n=77 a(%) 41-50 n=54 a(%) 51-60 n=50 a(%) 60˄ n=16 a(%) total n=400 a(%) χ 2 cal p. value malarial 11(20) 20(26) 53(72) 41(53) 32(59) 21(42) 7(44) 185(46) 61.892 0.00 hookworm 4(7) 7(12) 2(3) 2(3) 1(1) 0(0) 0(0) 16(4) 7.12 0.129 fasciola spp 3(6) 2(3) 0(0) 0(0) 0(0) 0(0) 0(0) 5(1) 0.200 0.655 taenia solium 4(7) 13(18) 4(5) 17(22) 8(11) 3(6) 1(6) 50(13) 28.96 0.00 taenia seginata 3(6) 5(7) 4(5) 1(1) 3(4) 0(0) 1(6) 17(4) 4.529 0.476 entamoeba histolytica 10(19) 11(15) 3(4) 3(4) 3(4) 0(0) 0(0) 30(8) 11.333 0.023 hymenolepsis nana 3(6) 5(7) 0(0) 2(3) 0(0) 0(0) 0(0) 10(3) 1.400 0.497 giardia lamblia 3(6) 2(3) 0(0) 0(0) 1(1) 0(0) 0(0) 6(2) 1.000 0.607 ascaris lumbricoides 1(2) 0(0) 0(0) 0(0) 0(0) 0(0) 0(0) 1(0.3) 0.001 0.00 schistosoma mansoni 4(7) 4(5) 0(0) 1(1) 0(0) 0(0) 0(0) 9(2) 2.000 0.368 strongyloide stercoralis 4(7) 1(1) 0(0) 0(0) 0(0) 0(0) 0(0) 5(1) 1.800 0.180 total % 50(93) 70(93) 67(91) 68(88) 48(89) 24(48) 9(56) 334(84) 120.835 a is the number infected χ 2 cal= 120.835 df 6, p˂0.05.there is significant difference in distribution of intestinal and malaria parasites by age table 2. the distribution of intestinal and malarial parasites among hiv/aids male individuals within makurdi metropolis parasite ˂1 10 n=32 a(%) 11-20 n=43 a(%) 21-30 n=40 a(%) 31-40 n=34 a(%) 41-50 n=41 a(%) 51-60 n=19 a(%) 60˄ n=5 a(%) total n=400 a(%) χ 2 cal p. value malarial 11(34) 15(35) 18(5) 17(50) 24(59) 10(53) 2(40) 97(24) 21.278 0.002 hookworm 4(13) 4(9) 1(3) 1(1) 1(2) 0(0) 0(0) 11(3) 4.909 0.297 fasciola spp 2(6) 1(2) 0(0) 0(0) 0(0) 0(0) 0(0) 3(1) 0.333 0.564 taenia solium 2(6) 7(16) 3(8) 9(27) 6(15) 3(16) 0(0) 30(8) 7.600 0.180 taenia seginata 3(9) 4(9) 2(5) 0(0) 2(5) 0(0) 0(0) 11(3) 1.000 0.801 entamoeba histolytica 1(3) 3(7) 2(5) 1(3) 2(5) 0(0) 0(0) 9(2) 1.556 0.817 hymenolepsis nana 1(3) 2(5) 0(0) 1(3) 0(0) 0(0) 0(0) 4(1) 0.500 0.779 giardia lamblia 1(3) 0(0) 0(0) 0(0) 0(0) 1(5) 0(0) 2(1) 0.000 1.000 ascaris lumbricoides 1(3) 0(0) 0(0) 0(0) 0(0) 0(0) 0(0) 1(1) 0.001 0.087 schistosoma mansoni 2(6) 4(10) 0(0) 0(0) 0(0) 0(0) 0(0) 6(2) 0.667 0.414 strongyloide stercoralis 1(3) 1(2) 0(0) 0(0) 0(0) 0(0) 0(0) 2(1) 0.000 1.000 total % 29(91) 41(95) 26(65) 29(85) 35(85) 15(79) 2(40) 176(44) 36.288 χ 2 cal= 36.288 df60, p˂0.05.there is significant difference in distribution of intestinal and malaria parasites by sex and age makwe et al.; aji, 3(1): 139-144, 2020; article no.aji.56215 143 table 3. the distribution of intestinal and malarial parasites among hiv/aids female individuals within makurdi metropolis parasite ˂1 10 n=32 a(%) 11-20 n=43 a(%) 21-30 n=40 a(%) 31-40 n=34 a(%) 41-50 n=41 a(%) 51-60 n=19 a(%) 60˄ n=5 a(%) total n=400 a(%) χ 2 cal p. value malarial 5(23) 10(31) 20(59) 26(61) 15(46) 5(31) 2(33) 88(22) 39.711 0.000 hookworm 1(5) 2(6) 1(3) 1(2) 0(0) 0(0) 0(0) 5(1) 0.600 0.896 fasciola spp 1(5) 1(3) 0(0) 0(0) 0(0) 0(0) 0(0) 2(1) 0.000 1.000 taenia solium 1(5) 3(9) 2(6) 8(19) 2(6) 3(19) 1(17) 20(5) 12.200 0.058 taenia seginata 1(5) 1(3) 2(6) 1(2) 1(3) 0(0) 0(0) 6(2) 0.667 0.955 entamoeba histolytica 6(27) 5(16) 1(3) 2(5) 1(3) 0(0) 0(0) 15(4) 7.333 0.119 hymenolepsis nana 2(9) 3(9) 0(0) 1(2) 0(0) 0(0) 0(0) 6(2) 1.000 0.607 giardia lamblia 1(5) 2(6) 0(0) 0(0) 1(3) 0(0) 0(0) 4(1) 0.500 0.779 ascaris lumbricoides 0(0) 0(0) 0(0) 0(0) 0(0) 0(0) 0(0) 0(0) 0.001 0.087 schistosoma mansoni 1(5) 1(3) 0(0) 1(2) 0(0) 0(0) 0(0) 3(1) 0.000 1.000 strongyloide stercoralis 1(5) 1(3) 0(0) 1(2) 0(0) 0(0) 0(0) 3(1) 0.000 1.000 total % 20(91) 29(90) 26(65) 41(95) 20(49) 8(50) 3(50) 147(37) 63.308 χ 2 cal= 63.308 df 60, p˂0.05.there is significant difference in distribution of haemoparasite and intestinal parasites by sex and age the result shows a progressive decrease in the prevalence of malaria parasites among the younger age groups with (20%) and (26%) within the age group <1-10 years and 11-20 years, while the older age group 21-30 years and 41-50 years continuously increase progressively with (59%) and (72%); the result of this study contrast with the work of russel and howson, [4] who reported that the high-risk groups include young children, pregnant women, non-immune travelers, refugees, displaced persons and labourers entering endemic areas. the overall result shows that the prevalence rate of intestinal parasite infection is generally high among the school age children with hookworm (7%) and entamoeba histolytica (19%), this result agrees with world health organization (1996), children in the age groups 5-9 and 1014 years are more exposed to helminth infection since they usually play with soil and eat food without washing hands, as well as vegetables and fruits. the male within the age group<1-10 years have the highest infection (13%) of hookworm and the least is recorded among the age group 31-40 years with (1%), the age group <1-10 years recorded (1%) infection of ascaris lumbricoides while the remaining age groups has the same infection rate of (0%). this result contradict the work of martin et al. (1984) which show incidence of ascaris lumbricoides (58%), trichuris trichura (whip worm) (52%) and hookworm 52%. malaria parasites, the highest infection is recorded among the age group 41-50 years with (59%) , while the least infection is recorded among the age group with <1-10 years (34%). this tremendous prevalence among the older age groups in malaria parasites distribution could be attributed with the fact that most male stay outdoor in the night, thereby exposing them to mosquito bite. this result contradict the works of krogstad, [7] who reported that malaria is a major public health problem with an estimated two million children worldwide dying of malaria parasites yearly, primarily because of malaria parasites and its complications. the schistosoma mansoni, recorded (6%) for age group <1-10years while the remaining age groups has the same infection rate of (0%), this result contradict the work of kaba et al. [8] who revealed the overall prevalence of schistosoma mansoni (81.6%) and (81.3%) among males and females in uganda. in summary sex is not factor in parasitic distribution among male hiv/aids individuals. the affirmed parasite, hookworm, fasciola spp, taenia seginata, hymenolepsis nana, giardia lamblia, ascaris lumbricoides, schistosoma mansoni and strongyloides stercoralis do not show a significant difference in makwe et al.; aji, 3(1): 139-144, 2020; article no.aji.56215 144 the distribution of parasitic infection among people living with hiv/aids. this work agrees with who [3], reported that several environmental and socio-economic factors have been identified to be responsible for the continued persistence of intestinal parasites in children and people living with hiv/aids; some of these include poor sanitary conditions, unhygienic practices, absence of portable water, poor housing and poverty. 5. conclusion conclusively, the prevalence of intestinal and malaria parasites infection among the hiv/aids individual was high and there by posing danger or threats now and in near feature. this study has revealed that hiv/aids individuals are prone to higher prevalence of intestinal and malaria parasite. people living with hiv/aids are threatened by several diseases caused by different kinds of pathogens. consent it is not applicable. ethical approval it is not applicable. competing interests authors have declared that no competing interests exist. references 1. ukoli f. prevalence of intestinal parasites among school children in nigeria: african journal. 1990;52. 2. loukopoulos p, komnenou a, papadopoulos e, psychas v. lethal. ozolaimusmegatyphlon infection in a green iguana (iguana iguanarhinolopa). journal of zoo and wildlife medicine. 2007;38:131-134. 3. who. action for the control of soil transmitted helminthiasis in nigeria. proceeding of an international workshop on strategies for the control of soil transmitted helminthiasis in nigeria. ile-ife, nigeria 7 th –9 th may; 1991. 4. russel pk, howson cp. vaccines against malaria: hope in a gathering storm. national academy press, washington dc. 1996;30. 5. montresor a. helminth control in schoolage children: a guide for managers of control programs. world health organization; 2002. 6. albonico m, ramsan m, wright v, jape k, haji hj, taylor m, savioli l. and bickle q. soil-transmitted nematode infections and mebendazole; 2002. 7. krogstad dj. malaria as a re-emerging disease. epidemiology reviews. 1996;18: 77-89. 8. kaba tereien nb, arithoc, christen no. schistosoma in pach, nebbi district uganda 40 years after nelson tropical medical parasitology; 1992. _________________________________________________________________________________ © 2020 makwe et al.; this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: http://www.sdiarticle4.com/review-history/56215 http://www.who.int/wormcontrol/documents/en/001to011.pdf http://www.who.int/wormcontrol/documents/en/001to011.pdf http://www.who.int/wormcontrol/documents/en/001to011.pdf http://creativecommons.org/licenses/by/4.0 _____________________________________________________________________________________________________ *corresponding author: e-mail: sadeeqsheshe@yahoo.com; asian journal of immunology 3(1): 91-98, 2020; article no.aji.55971 mechanism of antiviral immune response and covid-19 infection sadeeq sheshe1*, abdullahi nazifi1, a. m. labbo2, g. m. khalid3, abubakar yahya4, umar muhammad5 and aliyu musa haruna6 1 department of biochemistry, kano university of science and technology, wudil, nigeria. 2 department of biochemistry, sokoto state university, nigeria. 3 department of pharmaceutics and pharmaceutical technology, bayero university kano, nigeria. 4 department of pediatrics, muhammad abdullahi wase specialist hospital, kano, nigeria. 5 department of pediatrics, federal medical centre, gusau, nigeria. 6 department of medicine, aminu kano teaching hospital, bayero university kano, nigeria. authors’ contributions this work was carried out in collaboration among all authors. author ss designed the study. authors an and aml performed critical analysis of the literature. author gmk analyzed the recent data updates. authors ay and um searched for the literature. author amh wrote the first draft of the manuscript. all authors read and approved the final manuscript. article information editor(s): (1) dr. cynthia aracely alvizo báez, autonomous university of nuevo leon, mexico. reviewers: (1) victor b. oti, nasarawa state university, nigeria. (2) pichon maxime, university hospital of poitiers, france. complete peer review history: http://www.sdiarticle4.com/review-history/55971 received 23 march 2020 accepted 04 april 2020 published 09 april 2020 abstract immune systems are responsible for the body’s protection against invading foreign agents that could bring harm to normal cellular activities. viral infection is one of the significant foreign attacks on the body and the immune system has developed key mechanisms to contain such attack. the recent pandemic of the coronavirus disease 2019 (covid-19) has stimulated studies on antiviral response with much emphasis on the corona viruses. this disease caused by the novel severe acute respiratory syndrome-coronavirus-2 (sars-cov-2) has been shown to have evolved with mechanisms to evade the normal immune responses. as such, this review therefore critically apprised recent literature on mechanisms of antiviral responses emphasizing on the coronaviruses particularly the sars-cov-2.the paper also highlighted the current situation regarding studies to find cure and vaccine development against the covid-19. mini-review article sheshe et al.; aji, 3(1): 91-98, 2020; article no.aji.55971 92 keywords: immunity; corona virus; covid-19; immune; antiviral; innate; adaptive; lymphocytes. 1. introduction the immune system is the major protective system of the body against invading foreign bodies and infectious agents [1]. human immune system is one of the most critical and complex systems responsible for mediating response against foreign agents [2]. such system could be the inborn innate immunity involving group of cells much organized up to organ levels that serve to protect the body against any infection. such innate immune system involves one of the first line of immune response [3]. the innate immune system comprises of organs including the skin responsible for the major protection of the body [4]. continuously, it includes group of cells protecting the body against any foreign invading material without any further categorization [4]. these cells mostly ferocious in their actions kill or damage the invading foreign body, release chemicals that ultimately kills the foreign agent [5]. adaptive immunity on the other hand which is also referred to as the acquired immunity is the other type of immune response [6]. it is mostly concerned with detailed and more specific action against invading microbes, toxins and even mediating immune response between donor and recipient during organ or tissue transplants [7]. it is much controlled and specified form of immunity and involves key mechanisms for activation of different cells. this acquired immunity could mediate a cellular based response or humoral immunity [8]. cell mediated immune response involves the activation of cells that could lead to destruction and elimination of the foreign bodies [9]. the cell mediated immune response comprises the key players; lymphocytes [10]. these cells particularly the t lymphocytes are responsible for the cell based immune responses. the t lymphocytes (t-cells) mediate response through presentation of the antigens of the invading cells particularly in viral infections [11]. the presentation involves action of proteins that culminates in formation of histocompatibility complex with the antigenic material. accordingly, the presentation of histocompatibility complex class i (mhc i) leads to activation of cytotoxic t cells [12]. these cells are significant in cell based immune responses. in essence, upon viral infection, the viral proteins are presented on the mhc i which in turn activates the cytotoxic t cells there by mounting a response against the virus [13]. humoral immune response is associated with b lymphocytes activation and subsequent release of specially produced antibodies [14]. in terms of antigen presentation, the proteins of the invading agent are presented on mhc ii thus leading to activation and subsequent proliferation of b-cells [12]. the b cells continue to produce antibodies otherwise known as immunoglobulins and facilitation of antigen-antibody complex formation [15]. this complex formation is paramount to elimination of the invading agent as the antibodies are specific towards the consequent antigen [16]. at the center of all these activations in the adaptive immunity are the helper t-cells. they are referred to as architects of all immune responses [17]. the b-cells also develop into specialized memory b-cells having the tendency to store the memory of the antigen which initially caused the immune response and subsequent production of the specified antibodies [12]. with the current surge in the pandemic corona virus disease-2019 (covid19) which is ravaging the globe, this review critically discussed the current literature regarding antiviral immune response in humans with particular attention on the covid19. literature was extracted from scopus, web of science, pubmed, and embase using the following keywords; coronavirus, covid-19, immune, antiviral, innate, adaptive, lymphocytes. 2. mechanism of different immune cells leucocytes or white blood cells are the cells responsible for immunity [18]. these cells have different actions and mechanisms from the onset of their activation to the consequent removal or destruction of the foreign invading agent [19]. the cells could morphologically be classified as granules-containing cells (granulocytes) and non-granules contains cells (agranulocytes) [20]. both cells depending on the invading agent particularly viral infections, mediate immune responses in various ways. granule-containing cells including neutrophils, basophils and eosinophils release the content of their granules which are mostly hydrolytic sheshe et al.; aji, 3(1): 91-98, 2020; article no.aji.55971 93 enzymes that could easily destroy the invading agent [21]. the agranulocytes are do not contain granules. however, they are mostly capable of endocytosis, engulfing the invading agent and or its antigen there by causing its destruction [22]. similarly, these cells including macrophages, monocytes, dendritic cells, natural killer cells, mast cells are also capable of releasing cytokines, interleukins and interferons which also cause the death and destruction of the invading agents [23]. these chemicals also play critical roles in b cells activation proliferation leading to secretion of different immunoglobulins [24]. 3. antiviral immune responses infections of viruses usually differ with other invading agents particularly in its mechanism and responses [24]. retro viruses are able to enter into cells and utilize the cellular machinery to replicate its genome which at the final end will cause the destruction of the host cell [25]. although there are systems that facilitate cellular recognition upon entry of invading foreign agents, viruses lack these specialized signals such as pathogen associated molecular patterns (pamps) [26]. they could only be recognized with interaction of toll-like receptors (tlrs) with their nucleic acids [27]. these interactions . fig. 1. description of immune system and activation of antiviral immune response sheshe et al.; aji, 3(1): 91-98, 2020; article no.aji.55971 94 between toll-like receptors and the viral nucleic acids initiates a positive immune response via the release of interferons consequently mediating a b-cell mediated immunity with the concomitant production of antibodies [26]. viral infections have been associated with the type of the hosting system and nature of organs. principally the nature of infection and mode of immune response could influence the pathogenesis of the disease [28]. considering the respiratory system, it comprises the airways which are formed by a layer of epithelial cells which in essence is part of the innate immunity. mucosal secretions by the epithelial wall serve also as part of the innate immunity. however, with a lot of viral population being part of the host microbiome particularly in the respiratory system, it may affect a possible antiviral immune response [29]. similarly, various modifications during leucocytes differentiation might allow for lack of responses against the viral population in the microbiome. with that said however, no available studies have shown direct relation between onset of viral immune response and the microbiome in respiratory tract [30]. 4. corona viruses coronaviruses are members of a large family of viruses causing illnesses associated with respiratory tract. these viruses belonging collectively to the order nidovirales, are positively stranded rna viruses [31]. the coronavirinae are further subdivided into alpha, beta and gamma subfamilies each representing a particular nuclear configuration of the viral rna [32]. the viral rna contains a 5’ cap and 3’ polya tail allowing to act as a messenger rna for expression of its replication machinery proteins [31]. thus a major part of the genome encodes for the replication machinery proteins hence its rapid replication upon entry into the host cell. structural organization of the viral cell showed the virus as cylindrical with 4 specified proteins encoded by the minor sections of the genome [33]. these include the membrane (m) protein, the spike (s) protein, envelop (e) protein and nucleocapsid(n) protein. a major distinction is seen in the beta subfamily where another (5 th ) structural protein is observed, the hemagglutinin esterase protein [31]. the protein is thought to enhance spike protein activity and establish interaction with sialic acids on glycoproteins present on the surface of host cells there by facilitating viral entry [34]. upon viral entry, the expression of the replication machinery is achieved. the replicase protein expression is achieved in a series of complex mechanisms starting with expression of two polyproteins ppla and pplb [31]. studies showed distinct controlled mechanisms in expression of these proteins suggesting the complicated nature of the replication machinery expression which powers the virus with its rapid spread [35]. next is the assembly of the viral replication complex to replicate the viral rna. the replication machinery utilizes viral rna to synthesize the viral genome again. the viral rna synthesis proceeds with formation of genomic rna and the sub genomic rna which is concerned with expression of structural and accessory proteins. their encoding genes are located along the 3’ terminal end representing nest of rna [36]. the process is completed with translation of the structural proteins of the virus which are then transferred to the endoplasmic reticulum, assembled and further released as mature varions. 5. covid-19 the corona virus disease-2019 was reported to be caused by a new strain of sars-cov2 in december 2019 [37]. patients have shown pneumonia like symptoms and others associated with severe respiratory syndrome [38]. according to who, the massive spread of covid-19 has reached more than 10 countries globally and could possibly infect 20 million people. as at march 2020, there have been 274,202 reported cases for covid-19 out of which 11,354 deaths have been reported. positively, about 90,000 individuals have completely recovered and for the first time no new indigenous case has been recovered in the chinese city of wuhan where it started. in nigeria, so far 135 cases have been confirmed with 2 deaths most of which have been from individuals with history of foreign travels [39]. measures have already been in place by various government agencies to contain and control the spread of the virus. sheshe et al.; aji, 3(1): 91-98, 2020; article no.aji.55971 95 fig. 2. summary of life cycle and pathogenesis of sars-cov2; the viral proteins together with yet to be elucidated mechanisms inhibit activation of transcriptional factors irf3 and nfkb. these factors initiate a signal cascade that results into blocking of viral replication and assembly (adopted from [5]) key molecular mechanisms of the sars-cov2 pathogenesis are still unknown [40]. however, its pathophysiology involves viral entry in respiratory droplets into the lungs via the airways [31]. major target of the virus is the alveolar cells which play critical roles in gaseous exchange during respiration [41]. interaction between the viral spike proteins and the ace receptor ensures viral entry into the alveolar cells with the concomitant of structural disintegration. the inflammatory response initiated was due to release of pro-inflammatory cytokines such as il6 and tnf-alpha [37]. these two cytokines interact with the brain’s hypothalamus resulting in physiological changes including increase in body temperature and fever. additionally, the response results in vasodilation and increase vascular permeability. as such, with corresponding fluid accumulation, causing increased breathing capabilities and by extension breathing difficulty [31]. as it is currently no specified treatment or vaccine for covid-19 [42]. however, various clinical trials have been conducted and have shown significant results. the united states’ sheshe et al.; aji, 3(1): 91-98, 2020; article no.aji.55971 96 food and drug administration has shown promising results regarding the use of the antimalarial drug chloroquine and its derivative as possible treatment for covid-19 [41]. however, the fda is yet to give a formal approval contrary to previous claims. possibly, target in development of treatment include study on molecular mechanism of the disease pathogenesis to target the viral key proteins [42]. however, as such processes could take longer period, the rapid spread of the disease may not allow for that. 6. how does sars-cov2 possibly evade antiviral immune response? the sars-cov2 is a novel corona viral strain responsible for the december 2019 pandemic. the key mechanisms through which these coronaviruses including the sars-cov2 tries to circumvent immune response are still under investigation [36]. however, as with most coronaviruses, the sars-cov2 utilizes its structural proteins to gain entry into the host cell cytosol as well as suppress signaling pathways particularly with the toll-like receptors (tlr) [31]. usually, the interaction between the viral nucleic acid is with tlr7 and tlr3 [27]. this interaction initiates a signal cascade involving the transcriptional factors irf3 and nfkb. these transcriptional factors are further translocated into the nuclei where they activate the machinery for expression of pro-inflammatory cytokines and interferons particularly ifn1 [33]. ifn in turn activates the jak-stat pathway via phosphorylation of stat-1 and -2. the combine activated forms of stat-1 and -2 further form complexes with irf9 with the immediate release of active interferon stimulating genes (isgs) resulting in a massive suppression of viral replication [33]. 7. conclusion the covid-19 pandemic has so far reached a very disturbing rate. measures have continued to be in place to prevent the continued spread of the virus, and so far there have been promising results. researchers have continued to investigate the mechanism of antiviral response to the sars-cov2 and there have been positive results. moreover, there is hope that in the near future, vaccine and definitive treatment to covid19 will come to fruition. consent it is not applicable. ethical approval it is not applicable. competing interests authors have declared that no competing interests exist. references 1. o’shea j, schwartz d, villarino a, gadina i, mcinnes b, laurence a. the jak-stat pathway: impact on human disease and therapeutic intervention. annu. rev. med. 2015;66:311–328. doi:10.1146/annurev-med-051113024537. 2. zheng c, chen j, chu f, zhu j, jin t. inflammatory role of tlr-myd88 signaling in multiple sclerosis. front. mol. neurosci. 2019;12:314. doi: 10.3389/fnmol.2019.00314 3. varga j, bui-marinos m, katzenback b. frog skin innate immune defences: sensing and surviving pathogens. front. immunol. 2018;9:3128. doi: 10.3389/fimmu.2018.03128. 4. kumar h, kawai t, and akira s. pathogen recognition by the innate immune system. int. rev. immunol. 2011;30(1):16–34. doi: 10.3109/08830185.2010.529976 5. quaresma j. organization of the skin immune system and compartmentalized immune responses in infectious diseases. clin. microbiol. rev. 2019;32(4). doi: 10.1128/cmr.00034-18 6. chaplin d. overview of the immune response. j. allergy clin. immunol. 2010;125(2):s3–s23. doi: 10.1016/j.jaci.2009.12.980 7. agrawal b. heterologous immunity: role in natural and vaccine-induced resistance to infections. frontiers in immunology. 2019;10:2631. 8. yu jc. innate immunity of neonates and infants. frontiers in immunology. 2018;9:1759. 9. ilatovskaya vd, halade g, deleonpennell ky. adaptive immunity-driven inflammation and cardiovascular disease. am. j. physiol. heart circ. physiol. 2019; 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2020. available:https://www.who.int/docs/defaultsource/coronaviruse/situationreports/20200330-sitrep-70-covid19.pdf?sfvrsn=7e0fe3f8_2 (access date: 21 st march, 2020) 42. centre for disease control. coronavirus disease-19. how to protect yourself; 2020. available:https://www.cdc.gov/coronavirus/ 2019-ncov/prevent-gettingsick/prevention.html?cdc_aa_refval=http s%3a%2f%2fwww.cdc.gov%2fcoronavir us%2f2019ncov%2fprepare%2fprevention.html (access date: 20 th march,2020) _________________________________________________________________________________ © 2020 sheshe et al.; this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: http://www.sdiarticle4.com/review-history/55971 http://creativecommons.org/licenses/by/4.0 _____________________________________________________________________________________________________ *corresponding author: e-mail: adediranmoras@gmail.com; asian journal of immunology 2(1): 45-55, 2019; article no.aji.53020 studies on the adherence to antiretroviral therapy (art) among hiv patients receiving treatment in a major hiv care unit in akure, ondo state, nigeria t. a. olusi1, m. b. adediran1* and m. o. oniya1 1 federal university of technology, akure, nigeria. authors’ contributions this work was carried out in collaboration among all authors. author tao designed the study, wrote the protocol and managed the literature searches. author mba performed the statistical analysis and wrote the first draft of the manuscript. author moo managed the analyses of the study. all authors read and approved the final manuscript. article information editor(s): (1) dr. darko nozic, professor, higher medical school in belgrade, university of belgrade, serbia. reviewers: (1) rajathi sakthivel, dr. m.g.r medical university, india. (2) alma m. corpuz, tarlac state university, philippines. complete peer review history: http://www.sdiarticle4.com/review-history/53020 received 01 october 2019 accepted 04 december 2019 published 11 december 2019 abstract there has been great improvement in the life expectancy for hiv patients since the introduction of antiretroviral therapy (art). however, good adherence to regimen is important to surmounting health problems associated with hiv/aids. while availability of drugs and accessibility to it are important, many other social, psychological and clinical factors needed to be considered. this crosssectional study seeks to determine adherence to art among 441 patients living with hiv and attending the state specialist hospital, akure for treatment. the participants included more females (79.1%) while the remaining 20.9% were males. majority of the participants were married (71.2%) and 73.0% of them earn less than ₦18000 (about us$52) monthly. only 29.7% of them enrolled for art less than six (6) months to the commencement of the study. adherence in this study was found to be 56% and was significantly related to age (χ 2 = 14.31, p = 0.03), marital status (χ 2 = 9.47, p = 0.01), medication burden (χ 2 = 9.07, p = 0.01) and “art type” (χ 2 = 19.09, p = 0.00). the adherence rate (56%) recorded in the study is however low and underscores the need for measures necessary in ensuring total adherence among people living with hiv in ondo state nigeria. original research article olusi et al.; aji, 2(1): 45-55, 2019; article no.aji.53020 46 keywords: adherence; antiretroviral therapy; hiv; ondo state; nigeria. 1. introduction the continuous increase in new hiv infections despite improved antiretroviral therapy (art) regimen calls for concern, especially in subsaharan africa, which bears the highest burden of the infection and particularly in nigeria, where the lowest record of reduction in new hiv infections was obtained since 2009 according to unaids 2016 report. issues bordering on factors including adherence, retention in care and issues on drug resistance were topics of hot debate few years ago while introducing art to sub-saharan africa [1]. the eventual advent of very highly active antiretroviral treatment has reduced morbidity and mortality, improved life expentancy among hiv infected persons, enhanced immunity, sustained viral load suppression and reduced the risk of transmission in countries where they have been made available and accessible. however, adherence to medication and retention in care are still factors needed to be addressed in order to achieve optimum benefits because instances of drug-resistant hiv strains and the continuous transmission of these strains in the population have long been attributed to sub-optimal adherence among hiv infected people [2]. adherence is the rate at which an individual’s behaviour, compliance to medication, sticking to a diet, changing lifestyle and compliance to schedule visits and clinicians assessment conforms to standard recommendations of health officials [3]. according to moges and kassa [4], adherence is classified as good if is greater than 95% and poor if it is less than 85%. poor compliance to medication not only put an individual’s health at risk but may also lead to increase in viral transmission and drug resistance which subsequently make treatment difficult [5]. the recommended percentage of adherence to maintain long term viral suppression is 95% and several studies conducted on hiv infected persons (children, adolescents and adults) were reported to fall below 95% [6,7,8,9,10,11,12]. several factors may be responsible for nonadherence among hiv infected persons. for example in nigeria, a structurally induced nonadherence was said to be created in the year 2004, when the national arv programme organized by the federal government of nigeria suffered a setback, after being affected by a shortage in the supply of drugs. this caused many patients to be out of drugs for few months [13]. there had been cases where only 15% of people in need of arv in nigeria had access to treatment [11]. all of these further lay credence to the fact that availability and accessibility of drugs are important in ensuring compliance. cost of medication, adverse side effects, alcohol abuse and social stigma are among other factors reportedly to have been responsible for nonadherence. castro [14] has however viewed barriers to adherence as dynamic interactions of biologic and social factors. in nigeria, reports of previous studies shows adherence to have ranged from as low as 44% to more than 95% [15,16]. several studies on adherence have been reported in cities such as ibadan, enugu, port-harcourt, benin, ijesha, keffi and kano [17]. reports on adherence of patients receiving art in ondo state are still limited and therefore necessitate enquiries to generate data for effective decision making on intervention measures that gives better hope for positive improvements. this study was therefore, designed to investigate adherence of hiv infected individuals to art in akure the capital city of ondo-state and factors influencing nonadherence. 2. materials and methods 2.1 study site the study was conducted at the hiv care unit of the ondo state specialist hospital in akure these facilities attend to patients living in akure and indeed most towns in the state on a daily basis but operate full clinic activities on tuesdays and thursdays. 2.2 study population the study population includes 441 individuals living with hiv who are on art whose ages ranged between six (6) and sixty-five (65) years. criteria for inclusion includes hiv positive status, art not less than three months to the time of study, willingness to participate and availability of parents of patients less than 18 years of age (most of these parents were found also to be living with hiv). appropriate sample size was calculated based on the number of patients enrolled in the facility. during the study period of four months, a total of 1,286 patients olusi et al.; aji, 2(1): 45-55, 2019; article no.aji.53020 47 visited the hiv clinics out of which 441 were recruited to take part in the study. the sample size was calculated based on the estimated population proportion of 50 percent, a confidence level of 90 percent and alpha value of 0.05 using z 2 1-α/2p (1 – p)/d [18]. 2.3 study design the study design was a facility based cross sectional study. consenting patients and parent of patients less than 15 years old were interviewed by the researcher using a pretested and structured questionnaire adapted from aactg (adults clinical trials group) that covered socio demographic information, psychosocial characteristics, art duration, medication burden, type of art (whether free treatment, paid treatment or both). 2.4 adherence measurement the golden standard for assessing adherence is the arv level in the blood. viral load could also be used as a measure for adherence but data emanating from self-reports are easily accessible (afe et al., 2018) and according to dolezal et al. [9] self reported data correlates with viral loads. therefore, for this study, adherence was rated through the self reported data obtained from the participants using a 4-day recall semi structured follow up questionnaire adapted from adults aids clinical trials group (aactg) [19]. the percentage of adherence was obtained by dividing the total number of drugs taken in the last four days by the total number of drugs recommended to be taken for the four days multiplied by 100 as summarized below. adherence was then categorized as greater or equal to 95% and non-adherence as less than 95%. ((total number of drugs taken / total number of drugs prescribed) x (100/1)) 2.5 statistical analysis data collected were entered into excel 2007 worksheet and imported into spss version 23.0 for windows. the chi-square test was used to measure adherence in relation to socio demographic variables, while the logistic regression was used to find association between risk factors that could have influenced nonadherence in hiv infected people. the strength of associations were measured using adjusted odd ratios (95% confidence interval). values less than 0.05 were considered statistically significant. 3. results 3.1 socio-economic and demographic characteristics of participants the 441 subjects recruited for this study comprises of 92(20.9%) males and 349(79.1%) females, an indication that the majority of them were females and all the subjects had formal education though not to tertiary level as majority 235(53.3%) had secondary education, while only 40(9.1%) had tertiary education and the remaining had primary education 166(37.6%). most of the participants 225 (50.5%) were engaged in business as means of income while a good number of them 103(23.4%) were not employed. a larger percentage of them were married 314(71.2%) with 72 (16.3%) of them separated from their spouses while 55 (12.5%) of them had never married. a larger percentage of the participants had less than ₦18,000 has their average monthly income 322(73.0%). 3.2 art type of participants majority of the participant 269 (61.0%) were on free medication while 63 (14.3%) preferred buying their medications and 24.7% of them engaged in both options i.e they were also on free medications but purchase when their antiretrovirals clinics were out of stock). almost all the participants of aged 61 and above (91.7%) received free medications. a larger percentage of the female participants 236 (67.6%) were on free medication. most patients that depended on free medication 113(68.1%) had only primary education while many participants who buys their medication (45.0%) were educated up to the tertiary education level. most of those who prefers free art (65.6%) were business men and women and they are married (64.6%) earning less than ₦18000 monthly income (68.3%). 3.3 hiv patients adherence to art as shown in table 3, the highest percentage of adherence was recorded among the female participants 210 (60.2%) while 51.1% of males adhered. adults aged between 41-50 adhered most while participant between ages 21-30 had the highest percentage for non-adherence olusi et al.; aji, 2(1): 45-55, 2019; article no.aji.53020 48 33(56.9%). participants who are educated up till tertiary level and those unemployed had the highest percentage of adherence 27(67.5%) and 58(63.7%) respectively. most of the married participants (62.7%) adhered to medication while participant who no longer live with their spouses had the least adherence (55.6%). participants who benefitted from free medication when accessible and pay for it when out of stock had the highest level of adherence (76.1%) while those who only prefers buying their medication at all times had the lowest adherence (49.2%). 3.4 adherence to art in relation to socio-demographic factors table 4 summarizes adherence among attendees of hiv care unit in akure, ondo state, based on their socio-demographic parameters. adherence was higher among female patients. adherence was higher among female patients 60.2% (210/349) with no significant difference in adherence on the basis of sex (χ 2 = 2.47, p=0.12). there is however a significant variation in the adherence of patients in relation to their ages (χ 2 = 14.31, p=0.03). adherence in relation to their level of education varies from 54.0% to 67.5% (χ 2 = 4.10, p=0.13). with regard to marital status and occupation of the patients, adherence varied between 44.4% and 62.7% (χ 2 = 9.47, p=0.01) and 38.8% and 63.7% (χ 2 = 9.55, p=0.05) respectively. married patients significantly adhered (62.7%, 197/314) compared to others, also patients who are not employed adhered significantly with 63.7% (58/91). 3.5 participant’s adherence in relation to psycho-socio factors patients with family support adhered significantly (66.8%) compared to those without family support (table 5). participant who reported no adverse effect of medication adhered more (61.1%) to treatment compared to 52.4% level recorded among patients who reported adverse effect of medication although the variation is statistically not significant (χ 2 =3.05, p = 0.08). adherence in relation to the overall clinic table 1. socio-economic and demographic characteristics of participants factors total examined (%) age group 0-10 39(8.8) 11-20 23(5.2) 21-30 58(13.2) 31-40 156(35.4) 41-50 116 (26.3) 51-60 37(8.4) 61 and above 12(2.7) sex male 92(20.9) female 349(79.1) educational level primary 166 (37.6) secondary 235(53.3) tertiary 40(9.1) employment student 61 (13.8) civil servant 37(8.4) business 223(50.5) farming 17(3.9) not employed 103(23.4) marital status married 314(71.2) separated 72(16.3) never married 55(12.5) average monthly income <18000 322(73.0) 18,000-40,000 73(16.6) >40,000 46(10.4) olusi et al.; aji, 2(1): 45-55, 2019; article no.aji.53020 49 table 2. distribution of patients living with hiv undergoing treatment based on their art type factors paid art treatment (%) free art treatment (%) free and paid treatment (%) total examined (%) age group 63(14.3) 269(61.0) 109(24.7) 441(100) 0-10 6(15.4) 28 (71.8) 5 (12.8) 39(8.8) 11-20 4 (17.4) 16 (69.6) 3 (13.0) 23(5.2) 21-30 12 (20.7) 29 (50.0) 17 (29.3) 58(13.2) 31-40 21 (13.5) 87 (55.8) 48 (30.8) 156(35.4) 41-50 11 (9.5) 76 (65.5) 29 (25.0) 116 (26.3) 51-60 8 (21.6) 22 (59.5) 7 (18.9) 37(8.4) 61 and above 1 (8.3) 11 (91.7) 0 (0.0) 12(2.7) sex male 24 (26.1) 33(35.9) 35 (38.0) 92(20.9) female 39 (11.2) 236 (67.6) 74 (21.2) 349(79.1) educational level primary 13 (7.8) 113 (68.1) 40 (24.1) 166 (37.6) secondary 38 (16.2) 138 (58.7) 59 (25.1) 235(53.3) tertiary 12 (30.0) 18 (45.0) 10 (25.0) 40(9.1) employment student 11 (18.0) 34 (55.7) 16 61 (13.8) civil servant 12 (32.4) 16 (43.2) 9 (24.3) 37(8.4) business 28 (12.6) 146 (65.6) 49 (22.0) 223(50.5) farming 3 (17.6) 7 (41.2) 7 (41.2) 17(3.9) not employed 9 (8.7) 66 (64.1) 28 (27.2) 103(23.4) marital status married 39 (12.4) 203 (64.6) 72 (22.9) 314(71.2) separated 16 (22.2) 34 (47.2) 22 (30.6) 72(16.3) never married 8 (14.5) 32 (58.2) 15 (27.3) 55(12.5) economic tatus <18000 24 (7.5) 220 (68.3) 78 (24.2) 322(73.0) 18,000-40,000 21 (28.8) 33 (45.2) 19 (26.0) 73(16.6) >40,000 18 (39.1) 16 (34.8) 12 (26.1) 46(10.4) time since starting art <6 20 (15.3) 96 (73.3) 15 (11.5) 131(29.7) 12-24 24 (15.4) 94 (60.3) 38 (24.4) 156(35.4) >24 19 (12.3) 79 (51.3) 56 (36.4) 154(34.9) satisfaction of the patients varied from excellent or very good (59.9%) to fair or poor (57.0%) with no significant difference (χ 2 =0.39, p=0.54). with regards to alcohol consumption, adherence rate of 59.6% was recorded among tattlers while 36.0% of those who reportedly take little quantities of alcohol adhered to art medication. 3.6 participant’s adherence in relation to medication patients taking just a single pill of medication adhered significantly (χ 2 =9.07,p= 0.01) compared to those burdened with between 23(60.9%) or more pills(44.3%). there is also a significant difference (χ 2 =19.09, p= 0.00) among patients preference of access to art. the highest percentage of adherence (76.1%) was obtained among those who sometimes pay for treatment whenever free supply is not accessible. with regard to knowledge of medication and number of hospitalizations due to hiv related illnesses, adherence varied between 58.1% and 58.9% (χ 2 =0.02; = 0.88) and between 67.0% and 55.9% (χ 2 = 3.76; = 0.15), respectively. 4. discussion adherence to medication regime is very important in the management of hiv infection because it helps to sustain viral suppression, olusi et al.; aji, 2(1): 45-55, 2019; article no.aji.53020 50 table 3. compliance to art among the various socioeconomic group of attendees (n=441) factors adherence (%) non-adherence(%) total examined (%) sex male 47 (51.1) 45 (48.9) 92(20.9) female 210 (60.2) 139 (39.8) 349(79.1) age group 0-10 21 (53.8) 18 (46.2) 39(8.8) 11-20 12 (52.2) 11 (47.8) 23(5.2) 21-30 25 (43.1) 33 (56.9) 58(13.2) 31-40 92 (59.0) 64 (41.0) 156(35.4) 41-50 81(69.8) 35 (30.2) 116(26.3) 51-60 18 (48.6) 19 (51.4) 37(8.4) 61 and above 8 (66.7) 4 (33.3) 12(2.7) educational level primary 103 (62.0) 63 (38.0) 166(37.6) secondary 127 (54.0) 108 (46.0) 235(53.3) tertiary 27 (67.5) 13 (32.5) 40(9.1) employment student 38 (62.3) 23 (37.7) 61(13.8) civil servant 19 (38.8) 30 (61.2) 49(11.1) business 133 (59.6) 90 (40.4) 223(50.6) farming 9 (52.9) 8 (47.1) 17(3.9) not employed 58 (63.7) 33 (36.3) 91(20.6) marital status married 197 (62.7) 117 (37.3) 314(71.2) separated 32 (44.4) 40 (55.6) 72(16.3) never married 28 (50.9) 27 (49.1) 55(12.5) type of art free art 142 (52.8) 127 (47.2) 269(61.0) pay out of pocket 32 (50.8) 31 (49.2) 63(14.3) both 83 (76.1) 26 (23.9) 109(24.7) family support yes 231 (66.8) 115 (33.2) 346(78.5) no 26 (27.4) 69 (72.6) 95(21.5) recover immunity, reduce morbidity due to infection and hinder the development of drug resistance strain of the virus [20]. the present study shows 56% (χ 2 =2.47, p=0.12) adherence among participants studied at the hiv care unit in the ondo state specialist hospital akure. some other studies across the country have reported a higher proportion of adherence between 70% to 86% [17,21,22,23], (uzochukwu et al., 2009). a recent study done in the southwestern states of nigeria reported 42% adherence among study participants [24]. some other studies have equally reported adherence of between 44%-65% [25, 26], (uzochukwu et al., 2009). adherence was found in this study to be significantly related to age (χ 2 = 14.31, p=0.03) where adults aged 41-50 years recorded the highest rate of adherence of 69.8%.this agrees with the findings of suleiman and momo [17] where adherence in participants studied in bayelsa state were found to be age related. most of the parents and guidance of children less than 15 years opined that forgetfulness is a major barrier to adherence on their part. this was also applicable to participants of between ages 20 and 40 where preoccupation with other domestic chores and fear of side effects were the major hindrance to optimum adherence. adherence on the basis of marital status of participants was also found to be statistically significant (χ 2 = 9.47, p=0.01), where participants who are married adhered to art more than others who were either single or separated from their spouses. this is different to the findings of afe et al., [24] where no relationship exist between marital status and adherence. family support have been identified as an important factor that determines adherence to art. this study observed that patient with good olusi et al.; aji, 2(1): 45-55, 2019; article no.aji.53020 51 family support adhered more in comparism with their counterparts who do not have support. interactions with the patients in the course of the study revealed that many participants without support are persons who conceal their status from their families. it was also observed that patients who were no longer living with their spouses admitted that their health status was the actual cause of separation and if given another chance, they would not disclose to their spouses. some other patients mostly from polygamous families had vowed never to disclose their status to anyone since they did not even know how they got infected with hiv. adherence based on medication burden was also found to be statistically significant as the rate of adherence decreased with increase in the number of pills. this is in support of the opinions of ogba [27], lucas and gilles [28] that increase in the complexity of regimen for many chronic diseases including hiv/aids often leads to decrease in adherence. based on “art type,” participants for the study were grouped into three. these include (i) those that solely depended on free anti-retroviral drugs (ii) those that prefer buying their medication (pay out of pocket) and (iii) those who get free drugs but buys when clinics were out of stock. adherence among the three groups was found to be statistically significant (χ 2 = 19.09, p=0.00) as 76.1% of the third category adhered to their medication. this group of patient can however be said to be selfmotivated to take art. this support the findings of achappa et al., [29] where patients, who were self-motivated recorded high level of adherence. in a study by sarna et al., [18], adherence was found to be low among patients receiving free art. according to ayenigbara [30], non-availability of arv drugs is table 4. univariate and multivariate analysis of participants’ adherence in relation to socio demographic factors (n=441) factors adherence (%) total examined χ 2 p aor (95% ci) p sex 2.47 0.12 0.69(0.44-1.09) 0.12 male 47 (51.1) 92 female 210 (60.2) 349 age group 14.31 0.03 0.88(0.77-1.01) 0.07 0-10 21 (53.8) 39 11-20 12 (52.2) 23 21-30 25 (43.1) 58 31-40 92 (59.0) 156 41-50 81(69.8) 116 51-60 18 (48.6) 37 61 and above 8 (66.7) 12 educational level 4.10 0.13 1.06(0.79-1.44) 0.69 primary 103 (62.0) 166 secondary 127 (54.0) 235 tertiary 27 (67.5) 40 marital status 9.47 0.01 1.40(1.07-1.84) 0.01 married 197 (62.7) 314 separated 32 (44.4) 72 never married 28 (50.9) 55 employment 9.55 0.05 0.92(0.79-1.07) 0.29 student 38 (62.3) 61 civil servant 19 (38.8) 49 business 133 (59.6) 223 farming 9 (52.9) 17 not employed 58 (63.7) 91 key: aor = adjusted odd ratio olusi et al.; aji, 2(1): 45-55, 2019; article no.aji.53020 52 table 5. univariate and multivariate analysis of participant’s adherence in relation to psychosocio factors factors adherence (%) non-adherence (%) total examined χ 2 p aor (95% ci) p family support 47.57 0.00 5.33 (3.22-8.82) 0.00 yes 231 (66.8) 115 (33.2) 346 no 26 (27.4) 69 (72.6) 95 adverse effect 3.05 0.08 0.70 (0.47-1.05) 0.08 yes 76 (52.4) 69 (47.6) 145 no 181 (61.1) 115 (38.9) 296 over all clinical satisfaction 0.39 0.54 1.13 (0.77-1.65) 0.54 excellent/very good 118 (59.9) 79 (40.1) 197 fair/poor 139 (57.0) 105 (43.0) 244 alcohol 5.41 0.02 2.62 (1.13-6.08) 0.02 no drinking 248 (59.6) 168 (40.4) 416 low drinking 9 (36.0) 16 (64.0) 25 key: aor = adjusted odd ratio table 6. univariate and multivariate analysis of participant’s adherence in relation to medication factors adherence (%) total examined χ 2 p aor (95% ci) p medication burden 9.07 0.01 1.49(1.11-2.01) 0.01 one 62 (63.9) 97 2-3 156(60.9) 256 4 or more 39 (44.3) 88 knowledge of medications 0.02 0.88 0.97 (0.61-1.53) 0.88 knows medications 201 (58.1) 346 does not know 56 (58.9) 95 type of art 19.09 0.00 0.54 (0.39-0.79) 0.00 free art 32 (50.8) 63 pay out of pocket 142 (52.8) 269 both 83 (76.1) 109 no of hospitalization due to hiv related illness 3.76 0.15 0.77 (0.53-1.11) 0.17 never 185 (55.9) 331 once 63 (67.0) 94 twice or more 9 (56.3) 16 key: aor = adjusted odd ratio a major cause of non-adherence especially for patients who completely rely on free supply of arv drugs from government established hiv clinics. paying for antiretroviral therapy may be a strong motivation for adherence but the financial implication may cause some setbacks. olusi et al.; aji, 2(1): 45-55, 2019; article no.aji.53020 53 the nature of participants employment was found to significantly (χ 2 = 9.55, p=0.05) influence their rate of adherence. patients who were not employed were recorded the highest (63.7%), followed by those who engaged in business or are self-employed (59.6%). civil servants had the least adherence rate of 38.8%. majority of them cited forgetfulness as barrier to adherence as they would have gotten to their places of work before remembering that they had not taken the drugs. the suggestion of keeping the drugs in office bags and inside office was rejected by the patients because their children often search their bags and could find the drugs, others do not want people in their offices to know that they are living on drugs. other reasons accounting for non-adherence in this study include pills stock out, busy schedules, religious fasting, and fear of experiencing side effects. factors such as gender, knowledge of medication, number of hospitalizations due to hiv related illnesses, adverse effect, overall clinic satisfaction and educational status do not vary significantly among study participants. 5. conclusion the overall 56% adherence to art among hiv infected people receiving care at the state specialist hospital akure is very poor and calls for strategies to improve adherence to treatment and consistency in attendance of the patients bearing in mind that adherence is central to the overall wellness of these patients and therefore the above suggested recommendations may be employed. 6. recommendations  setting up adherence counseling forum where patients are counseled on the importance of adherence  creating adherence monitoring team among the patients where they help remind each other through phone communications  effective management of depression where patients learn to see themselves as not lesser than those who are not infected  setting up of art alert in global system mobile (gsm) phones of patients  ensuring sustained availability and accessibility of drugs at no cost in public hiv care units consent and ethical approval permission to carry out the investigation was obtained from the ondo state ministry of health, after thorough scrutiny of the research proposal by the ethical approval committee of the ministry. patients’ consent were individually sought before their enrollment into the study. competing interests authors have declared that no competing interests exist. references 1. assefa y, jerene d, lulseged s, ooms g, van damme w. rapid scale-up of antiretroviral treatment in ethiopia: successes and system-wide effects. plos med. 2009;6(4):e1000056. 2. blower s, ma l, farmer p, koenig s. predicting the impact of antiretrovirals in resource-poor settings: prevention of hiv infection whilst controlling drug resistance. curr drug targets infect disord. 2003;3:345-53. 3. world health organization. global health observatory (gho) data hiv/aids; 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[issn: 2250-3013] _________________________________________________________________________________ © 2019 olusi et al.; this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: http://www.sdiarticle4.com/review-history/53020 http://creativecommons.org/licenses/by/4.0 _____________________________________________________________________________________________________ *corresponding author: e-mail: swagh.gene@gmail.com; asian journal of immunology 2(1): 78-88, 2019; article no.aji.53611 phytochemical analysis of simarouba glauca dc. and comparison of its bioactivity kiran r. pawar1, ganesh b. janavale1, sopan g. wagh1*, archana n. panche1, a. abhijit daspute2, manoj b. pohare3 and sanjay n. harke1 1 mahatma gandhi mission’s institute of biosciences and technology, n-6 cidco, main campus, aurangabad, maharashtra 431003, india. 2 laboratory of plant cell technology, faculty of applied biological sciences, gifu university, gifu 5011193, japan. 3 department of infection, immunity and cardiovascular diseases, university of sheffield, beech hill road, sheffield, s10 2rx, united kingdom. authors’ contributions this work was carried out in collaboration among all authors. authors krp, snh and gbj designed the study, performed the statistical analysis, wrote the protocol and wrote the first draft of the manuscript. authors anp, sgw and aad managed the analyses of the study. author mbp managed the literature searches. all authors read and approved the final manuscript. article information editor(s): (1) dr. darko nozic, professor, higher medical school in belgrade, university of belgrade, serbia. reviewers: (1) pipat chooto, prince of songkla university, thailand. (2) oluwatosin johnson, university of lagos, nigeria. complete peer review history: http://www.sdiarticle4.com/review-history/53611 received 20 october 2019 accepted 25 december 2019 published 26 december 2019 abstract medicinal implications of plants are well known to us since the time immemorial and are well documented in the ancient literature. according to who report, 80% of world populations count on herbal medicines to treat themselves in case of illness. and the main reason behind this could be the side effects caused by the drugs which are recommended now a days by the general practitioners. herbal medicines are known to produce negligible amount of side effects and that is why they are among the favorite candidates for health care. in the present study, we have attempted to rediscover the medical potential of the simarouba glauca. we have performed phytochemical analysis of simarouba glauca and compared its bioactivities (antibacterial, antifungal, anti-diabetic, hemolytic, antioxidant, and antimicrobial)with synthesized zinc oxide nanoparticles. original research article pawar et al.; aji, 2(1): 78-88, 2019; article no.aji.53611 79 keywords: simarouba glauca; antibacterial; antifungal; anti-diabetic; antioxidant. 1. introduction the potential of plant to serve human beings in wellness of health and overall immunity and range of aspects has been well documented since former times [1]. according to world health organization, 80% of world’spopulation mainly counts on traditional medicines for health care. plant products have been a part of phytomedicines since time immemorial [1,2]. there are about 2000 species of plants that are used for medicinal purposes and detected as having a therapeutic significance. many of these medicinal plants, which are known to the humans since ancient time, either are on the verge of extinction or already listed as endangered. lots of efforts are already in progress in the different corners of the world to conserve these plants and/or to enhancetheir ability to produce extra amount of medicinal compounds they are producing either by plant tissue culture techniques [3-7] or by the use of recently developed advanced techniques like clustered regularly interspaced short palindromic repeats with crispr associated protein 9(crispr/cas9) [8,9]. medicinal plants play a vital role in preventing various diseases [10] and their medicinal value lies in some bioactive constituents (like flavonoids and phenols) that produce a definite physiological action on the human body [1,11]. it has been estimated that 14 28% of higher plant species are used medicinally and that 74% of pharmacologically active plant derived components were discovered [12]. out of the several hundred thousand medicinal plant species around the globe, only a small portion has been investigated so far both phytochemically and pharmacologically [12]. plants are enriched with various phytochemicals such as vitamins, terpenoids, phenolic acids, lignins, stilbenes, tannins, flavonoids, quinones, coumarins, alkaloids, amines, betalains and other metabolites, which are rich in antioxidant activity [13]. studies have shown that many of these antioxidant compounds possess antiinflammatory, antiatherosclerotic, antitumor, antimutagenic, anticarcinogenic, antibacterial, and antiviral activities [14]. phytochemicals are chemicals, which are naturally present in plants. recently phyto chemicals have become more popular due to their countless medicinal uses. phytochemicals play a vital role against number of diseases such as asthma, arthritis, cancer etc. unlike pharmaceutical chemicals these phytochemicals do not have any side effects. phytochemicals are derived from different parts of plant such as leaves, flowers, roots, pulps, seeds, etc. [15]. phytochemicals are also known as “man friendly medicines” as they cure diseases without causing any harm to human beings [15]. to produce synthetic molecular analogs, a large proportion of plant based compounds are used as lead molecules in drug discovery which implies that phytochemicals play a crucial role in diversity oriented synthesis of natural product like pharma compounds [16]. it is a rain fed waste land evergreen edible oil tree. simarouba glauca belongs to the family simaroubaceae. it contains various groups of chemical components [17]. quassinoids is the main active group of chemicals in simarouba glauca which belongs to triterpine family [16]. pharmacological properties of simarouba glauca are haemostatic, antihelmenthic, antiparasitic, antidysentric, antipyretic and anticancerous. simarouba glauca is a native species to florida in the united states, southern florida, south america, and the lesser antilles [18]. simarouba glauca is indigenous to the rainforest and other tropical areas in mexico, cuba, haita and central america. its cultivation depends on rainfall distribution, water holding capacity of the soil and sub-soil moisture. simarouba glauca is found in several cultivated parts of india and was introduced in india during 1960s by plant genetic resources in the research station at amravati in maharashtra and university of agriculture science banglore in 1986 [19]. simarouba glauca is suited for temperature range of 10 to 50°c. it can grow at elevations from sea level to 1,000 m (3,300 ft). it grows 40 to 50 ft (12 to 15 m) tall and has a span of 25 to 30 ft (7.6 to 9.1 m) [18]. it has bright green leaves, small white flowers and small red fruits [16]. leaves of simarouba glauca are bluish oily green and smooth at apex and are pinnately compound. it has 3-21 leaflets. simarouba leaf decoction when taken in limited amounts can raise the immunity, reduce appetite loss and increase quality of life in cancer patients [18]. simarouba glauca has very good antibacterial, anti-tumorous properties so it is very effective in reducing the size of tumors and secondary infections in cancer patients and in curing cancer of first/second stages, whereas in later stages it can considerably increase the quality of life [18].nanotechnology involves the use of materials having nanoscale dimensions in pawar et al.; aji, 2(1): 78-88, 2019; article no.aji.53611 80 the range of 1–100 nm [20]. nanomaterials are particles having nanoscale dimension, and nanoparticles are very small sized particles with enhanced catalytic reactivity, thermal conductivity, non-linear optical performance and chemical steadiness owing to its large surface area to volume ratio [21,22]. the nanomaterials can be synthesized by different methods including chemical, physical, irradiation and biological methods. the development of new chemical or physical methods has resulted in environmental contaminations, since the chemical procedures involved in the synthesis of nanomaterials generate a large amount of hazardous byproducts [23,24]. hence the green synthesis methods for the nanoparticle synthesis are being used now a day’s which are clean, safe, ecofriendly and environmentally nontoxic. moreover, in this method there is no need to use high pressure, energy, temperature and toxic chemicals [24,25,26]. nanoparticles possess a wide array of application in the different fields’ viz., medicine, electronics, and therapeutics and as diagnostic agents [24]. nano-particles have started being considered as nano-antibiotic because of their antimicrobial activity [22]. they also have potential application in the field of medicine like drug discovery, biological activities such as anti-microbial, antioxidant etc. and diagnosis of diseases [20]. the zinc oxide nanoparticles have found fabulous applications in bimolecular, diagnostics and microelectronics [24], electromagnetic coupled sensors [20]. the zinc oxide nano particles have been used to remove arsenic and sulphur from water even through bulk zinc oxide cannot absorb arsenic. it is because nanoparticles have much larger surface areas than bulk particles [27]. they have fascinating properties and extensive applications. bioinspired synthesis of the zinc oxide nanoparticles has been achieved using environmentally and eco-friendly accepted systems. in this research work, we have performed phytochemical analysis of simarouba glauca and compared its bioactivities with synthesized zinc oxide nanoparticles. 2. materials and methods 2.1 collection and preparation of plant material simarouba glauca leaves were collected from local areas of aurangabad, ms, india. after collection of leaves they are cleanedwith distil waterto remove all the impurities and unwanted materials. dried to remove all the moisture content present in leaves so that it can be stored for further analysis. complete drying of leaves is followed by grinding to convert them in powder form for further use. 2.2 preparation of plant extract methanol extract: for the methanol extract, 200 ml of methanol and 20 gm of dry powder placed in soxhlet extractor. extraction was carried out for 3hrs and 9 cycles were performed. dark green colour extract was obtained. ethyl acetate extract: 200 ml of ethyl acetate and 20 gm of dry powder were placed in soxhlet extractor. extraction was carried out for 3 hrs and 9 cycles were performed. dark green color extract was obtained. methanol and ethyl acetate extract were evaporated using rotary evaporator and powder obtained was collected for further analysis. the powder was then kept in sterile bottles, under refrigerated conditions, until further use. preliminary phytochemical analysis: the extracts were subjected to preliminary phytochemical testing to detect for the presence of different chemicals or compounds. s. glauca leaves extract were screened for the presence of alkaloids, flavonoids, carbohydrates, glycosides, tannins, terpenoids, saponins and steroids. test for alkaloids: mayer’s test: in 1 ml of extract add 1 ml of mayer’s reagent. appearance of creamy white precipitation indicates the presence of alkaloids. test for flavonoids: in 5 mg of extract powdered 10 ml of sodium hydroxide. yellow fluorescence indicates the presence of flavonoids. test for terpenoids: salkowski’s test: in 5 ml of extract add 2 ml of chloroform and 3 ml of concentrated sulphuric acid. reddish brown colour indicates the presence of terpenoids. test for steroids: in 5 mg of extract add 2 ml of acetic acid unhydride and 2 ml of sulphuric acid. green colour indicates the presence of steroids. test for saponins: 1 mg of extract was mixed with 5 ml of distilled water and shaken well. appearance of creamy white miss bubbles indicates the presence of saponins. pawar et al.; aji, 2(1): 78-88, 2019; article no.aji.53611 81 test for tanins: 5 mg of extract was mixed with 3 ml of distilled water and heated on a water bath. the mixture was filtered and add 5 mg of ferric chloride to the filtrate. dark green colour indicates the presence of tanins. test for carbohydrates and glycosides: benedict’s test: in 2 ml of extract add 2 ml benedict’s reagent, heat in boiling water bath for 5 min, appearance of precipitation indicates the presence of glycosides. 2.3 qualitative analysis of phytochemicals by thin layer chromatography in the tlc analysis, 1 cm from the edge of the thin layer plate for a straight line across the plate. spot the extract in the line with the capillary tube to dry the spot and place the plate in a beaker with a mobile phase. layer the beaker when the solvent reaches more than 1 cm from the top of the plate. remove the beaker from the solvent line before the rf values have been calculated. 2.4 synthesis of zinc oxide nanoparticles for the synthesis of zinc oxide nanoparticles, prepare 1 mm of zinc acetate in 50 ml of sterile distilled water. 20 ml of 0.1 m sodium hydroxide and 25 ml plant leaf extract was then addedto the 1 mm zinc acetate solution. thissolution was then kept incubated on magnetic stirrer for 3 hrs. appearance of yellow color indicates the presence of zinc oxide nanoparticles. this mixture was then centrifuged to separate supernatant and pellete. supernatantwas discarded and pellete was dried in the hot air oven. dried powder was then preserved in an airtight bottle for further studies. characterization of zinc oxide nanoparticles was carried out by using uv visible spectrophotometry. 2.5 antibacterial activity in vitro antibacterial activities were examined for methanol, ethyl acetate extract and zinc oxide nanoparticles. antibacterial activities of plant leaf extracts and zinc oxide nanoparticles against three pathogenic bacteria (e. coli, s. auerus, and bacillus subtilis) were investigated by the well diffusion method. the petriplateswere poured with approximately 25 ml autoclaved nutrient agar media each. agar plates were inoculated with three different bacterial colonies and spread with sterile swab. four wells were punched on each plate using a sterile cork borer of 8 mm diameter. 100 μl of each extract was pipetted into respective wells. standard leaf extract was used as a control.the petri plates were incubated overnight at 37°c and the anti-bacterial activity was measured after 18 hrs of incubationby measuring the zone of inhibition. 2.6 antifungal activity in vitro antifungal activities were examined for methanol, ethyl acetate extract and zinc oxide nanoparticles. antifungal activities of plant leaf extracts and zinc oxide nanoparticles against pathogenic fungus (asperillusflavus) were investigated by the well diffusion method. all plates were autoclaved with pda agar media of approx. 25 ml. aspergillus flavus colonies were inoculated with agar plates and sterile swab spread. each plate was hit with four wells using a sterile 8 mm diameter cork borer. the pipetting into respective wells was 100 μl of each extract. standard leaf extract was used as a control. the petri plates were incubated overnight at 37°c and the antifungal activity was measured after 48 hrs of incubationby measuring the zone of inhibition. 2.7 haemolytic assay the haemolytic activity of the extract was determined using agar diffusion technique on blood agar plate. blood agar was prepared and well measuring 8 mm were made on the agar using cork borer. the wells were filled with 100 μl of different concentration of plant extracts solution and zinc oxide nanoparticles solution. standard leaf extract was used as a control. the plates were then incubated at 37ºc for 24 hrs and the haemolytic activity was measured after 24 hrs of incubationby measuring the zone of inhibition. 2.8 antioxidant activity scavenging of h2o2 radicals: the ability of the extracts to scavenge h2o2 was determined according to the reported method (reference). a solution of h2o2 (40 mm) was prepared in phosphate buffer (ph 7.4). extract of selected concentrations in distilled water were added to h2o2 solution (0.6 ml, 40 mm). the absorbance of h2o2 at 230 nm was determined after 10 minutes against a blank solution containing phosphate buffer without h2o2. ascorbic acid was used as a standard antioxidant. the % pawar et al.; aji, 2(1): 78-88, 2019; article no.aji.53611 82 of h2o2 scavenging of both extracts and standard compounds were calculated. % scavenged [h2o2] = [(a−a)/a] x 100 where a was the absorbance of the control and a was the absorbance in the presence of the sample of extract (rewrite this formula correctly again). dpph (1,1-diphenyl -2-picryl hydarzil) free radical scavenging activity: 0.1 mm solution of dpph in ethanol was prepared. 500 µg of sample was added in 1 ml of solution. the mixture was shaken vigorously and allowed to stand room temperature for 30 min. then the absorbance was measured at 517 nm by using uv visible spectrophotometer. lower absorbance of reaction mixture indicates higher free radical scavenging activity. the % dpph scavenging effect was calculated using following equation.the capability to scavenge the dpph radical was calculated using the following equation: dpph scavenging effect (%) = [(a0 – a1)/a0) × 100], where a0 is the absorbance of the control reaction, and a1 is the absorbance of presence of all of the extract samples and standard. antidiabetic activity: the antidiabetic activity of the extract was determined using starch agar plates. starch agar plates were prepared by using 0.1% starch and 1% nutrient agar. after that 5wells measuring 8 mm were made on the agar plates using cork borer. the wells were filled with given concentrationsof phosphate buffer and amylase enzyme and sample as indicated below. 1 st well – 40 ul amylase + 60ul phosphate buffer = 100 ul 2 nd well – 100 ul buffer 3 rd well – 40 ul amylase + 40 ul phosphate buffer + 20 ul sample = 100 ul 4 th well – 40 ul amylase + 20 ul phosphate buffer + 40 ul sample = 100 ul 5 th well – 40 ul amylase + 60 ul sample = 100 ul 3. results and discussion 3.1 preliminary phytochemical analysis phytochemical screening is a preliminary step in the characterization of plant products. preliminary screening for presence or absence of a particular class of compound is qualitative in nature and form a robust base for the quantitative estimation of bioactive components. phytochemicals were screened for alkaloids, carbohydrates, glycosides, coumarins, proteins & amino acids, phenolic compounds, tannins, flavonoids, terpenoids. qualitative phytochemical investigation of extract of simarouba glauca leaves showed the presence of saponins, tannins, flavonoids, glycosides, alkaloids, steroids, terpenoids. methanol and ethyl acetate extract of simarouba glauca leaves contain maximum number of phytoconstituents. the results of qualitative phytochemical analysis areshowed in the tables 1, 2, 3. table 1. preliminary phytochemical analysis of methanolic and ethyl acetate extracts sr no. phytochemicals methanol extract ethyl acetate extract 1 alkaloids + + 2 glycosides + + 3 flavonoids + + 4 terpenoids + + 5 steroids + + 6 saponins + 7 tannins + + 3.2 qualitative determination of bioactive compounds by thin layer chromatography methanol and ethyl acetate extract obtained from s. glauca leaves was used to perform thin layer chromatography in order to establish the purity and composition of materials. the purified components of methanolic and ethyl acetate extract of s. glauca leaves were identified by iodine test. for different components different bands were observed under ultraviolet light (figs. 1, 2). 3.3 synthesis of zinc oxide nanoparticles in visual observation colour of zinc acetate solution was changed to yellow after addition of simarouba leaf extract and yellowish precipitate indicates the formation of zinc oxide nanoparticles. for further confirmation uv visible spectroscopy was carried out. uv-visible spectroscopy is usually conducted to confirm the synthesis of zinc oxide nanoparticles. the absorption spectrum was recorded for the pawar et al.; aji, 2(1): 78-88, 2019; article no.aji.53611 83 sample in the range of 200 – 400 nm. the spectrum showed the absorbance peak at 310 nm corresponding to the characteristic band of zinc oxide nanoparticles. fig. 1. tlc for ethyl acetate, showing the separation of alkaloids, teroenoids and flavonoids fig. 2. tlc for methanol, showing the separation of alkaloids, teroenoids and flavonoids 3.4 antibacterial activity the results of antimicrobial activity of methanol, ethyl acetate extracts and zinc oxide nanoparticles of e. coli, s. auerus, and bacillus subtilis are given in table 4. from the results it was found that zinc oxide nanoparticles have widerinhibitory zone as compared tomethanol and ethyl acetate extracts. ethyl acetate extract have the least inhibitory zone. these results clearly indicate that zinc oxide nanoparticles have enhancedantibacterial activity as compared to methanol and ethyl acetate extracts(tables. 3, 4) (figs. 3, 4). 3.5 antifungal activity the antifungal assay showed that simarouba glauca has antifungal property against the tested fungi aspergillus niger. in this analysis, zinc oxide nanoparticles were found to be more effective as compared to methanolic and ethyl acetate extracts against the growth of fungi. the results of antifungal activity of methanolic, ethyl acetate extracts and zinc oxide nanoparticles of aspergillus flavusis given in table 5. 3.6 haemolytic assay the zone of haemolysis was directly proportional to concentration of the extract. from the results it was found that zinc oxide nanoparticles (table. 6) have enhancedzone of haemolysisas compared tomethanol and ethyl acetate extracts (fig. 5). 3.7 antioxidant activity antioxidant acitivity of the zinc oxide nanoparticles, methanolic and ethyl acetate extracts from the plant leaf ssimarouba glauca was carried out with hydroxyl radical scavenging assay and dpph scavenging activity. 3.8 antidiabetic activity amylase inhibition detection was carried out using starch agar plates technique. the amylase activity inhibition zone appeared around the wells having zinc oxide nanoparticles, methanolic and ethyl acetate extract (table. 7) (fig. 6). a large number of biologically active plant constituents have been found in the simarouba glauca extract in the phytochemical analysis. the result showed variations in the number of phytochemicals obtained from both extracts. more than 124 metabolites were detected in the ethyl acetate extract compared to both solvent extracts. this may be due to the high solubility of the active compounds of simarouba glauca with ethyl acetate by extraction compared to the petroleum ether solvent. in addition to these results, it is also documented that different light intensities have various effects on the efficiency of the metabolites produced by the plants as seen in case of glechoma longituba [28]. zang et al. (2015) explained, chloroplasts produces different metabolites, and chloroplasts development mainly depends on the different light intensities [29,30,31]. similar kind of effects can also be possible in case of simarouba glauca and appropriate light intensity management might be feasible to obtain higher yields of secondary metabolites in it, which might pawar et al.; aji, 2(1): 78-88, 2019; article no.aji.53611 84 be helpful in improving its either antibacterial, antifungal, anti-diabetic, hemolytic, antioxidant, and antimicrobial. moreover,s. glaucaand similar kind of medicinal plants’ specific antimicrobial activity isnowwell established, so one can also make the efforts to do the molecular studies, trace back the proteins and genes mainly responsible for these effects and clone them with easy, cheapand quick methods of cloning [32] for further studies by considering its commercialization in mind. producing these antimicrobial components in bacteria is easy and quick and additionally we do not have to destroy plants for this, which will be environment friendly and is the absolute need of the current time. table 2. ethyl acetate parameters stationary phase mobile phase no. of spots rf value alkaloids silica gel benzene : ethanol(45:5) 10 0.04, 0.22, 0.27, 0.31, 0.36, 0.47, 0.54, 0.61, 0.75, 1 glycosides silica gel ethyl acetate : butanol : water(20:20:15) 5 0.16, 0.30, 0.66, 0.76, 0.95 flavonoids silica gel ethyl acetate : formic acid : glacial acetic acid : water (20:2:2:5) 4 0.42, 0.81, 0.87, 0.93 terpenoids silica gel toluene : ethyl acetate 11 0.65, 0.09, 0.13, 0.21, 0.34, 0.44, 0.50, 0.59, 0.63, 0.73, 0.98 table 3. methanol parameters stationary phase mobile phase no. of spots rf value alkaloids silica gel benzene : ethanol(45:5) 7 0.29, 0.37, 0.72, 0.95, 0.78, 0.81, 0.89 glycosides silica gel ethyl acetate : butanol : water (20:20:15) 2 0.35, 0.91 flavonoids silica gel ethyl acetate : formic acid : glacial acetic acid : water (20:2:2:5) 5 0.10, 0.40, 0.75, 0.89, 0.95 terpenoids silica gel toluene : ethyl acetate (9:1) 7 0.27, 0.33, 0.41, 0.49, 0.56, 0.66, 1 table 4. antibacterial activity and zone of inhibition for respective sample sr. no. extracts conc. in mg/ml diameter of zone of inhibition in cm e. coli s. auerus b. subtilis 1 methanol 1 1.1 1.1 1.1 2 1.2 1.1 1.2 3 1.2 1.2 1.3 4 1.2 1.2 1.3 2 ethyl acetate 1 1.1 1.1 1.1 2 1.1 1.1 1.1 3 1.1 1.1 1.1 4 1.1 1.1 1.1 3 zinc oxide nanoparticles 1 1.2 1.1 1.3 2 1.3 1.3 1.4 3 1.4 1.5 1.5 4 1.5 1.5 1.5 pawar et al.; aji, 2(1): 78-88, 2019; article no.aji.53611 85 fig. 3. antibacterial activity, in against from left zinc oxide nanoparticals, methanol, ethyl acetate table 5. antifungal activity and zone of inhibition for respective sample sr. no. extracts conc. in mg/ml diameter of zone of inhibition in cm aspergillus flavus 1 methanol 1 1.1 2 1.2 3 1.2 2 ethyl acetate 1 1.1 2 1.1 3 1.1 3 zinc oxide nanoparticles 1 1.2 2 1.3 3 1.5 fig. 4. antifungal activity at different concentration level fig. 5. haemolytic activity in against from left zinc oxide nanoparticals, methanol, ethyl acetate pawar et al.; aji, 2(1): 78-88, 2019; article no.aji.53611 86 table 6. zones of haemolysis for respective sample extracts zones of haemolysis in mm conc. of extracts in mg/ml 1 mg/ml 2 mg/ml 3 mg/ml methanol 1.2 1.3 1.4 ethyl acetate 1.2 1.2 1.4 zinc oxide nanoparticles 1.3 1.3 1.4 table 7. zones of antidiabetic activity in against from left zinc oxide nanoparticals, methanol, and ethyl acetate extracts zones in cm well no. 1 2 3 4 5 methanol 1.2 1.1 1 ethyl acetate 1.5 1.2 1.2 zinc oxide nanoparticles 1.2 1.2 1.1 fig. 6. antidiabetic activity in against from left zinc oxide nanoparticals, methanol, and ethyl acetate the present study revealed that the s. glauca leaf posess antimicrobial activity, which is big plus of this study. currently the world is facing the problem of antimicrobial resistance and pathogenic bacteria are developing resistance to the commercially available antibiotics. therefore, it is an absolute necessity to keep looking for new antimicrobial solutions. and who know this simarouba glaucaextract might be a solution to nullify the effect of antibiotic resistance in bacteria and could become a most favored medicine in the near future. 4. conclusion based on our findings we conclude that simarouba glauca plant leaves have significant amount of phytochemical compounds. zinc oxide nanoparticles of simarouba glauca have shown enhanced in vitro antioxidant, antibacterial activity, haemolytic activity, antifungal activity compared to methanolic and ethyl acetate extracts of simarouba glauca. the present study revealed that the s. glauca leaf possessantimicrobial activity, which is big plus of this study. currently the world is facing the problem of antimicrobial resistance and pathogenic bacteria are developing resistance to the commercially available antibiotics. therefore, it is an absolute necessity to keep looking for new antimicrobial solutions. and who know this simarouba glaucaextract might be a solution to nullify the effect of antibiotic resistance in bacteria and could become a most favored medicine in the near future. consent it is not applicable. ethical approval it is not applicable. acknowledgement we are grateful to meander kumar gakula for checking the english in the manuscript. pawar et al.; 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asian journal of immunology 3(1): 298-304, 2020; article no.aji.60290 antibacterial activity of moringa oleifera root bark extract against some pathogenic organisms emmanuel k. amanze1*, ugonna d. nwankpa2, chinedu e. udekwu2, henry n. ogbonna2, chibuzo v. nwokafor1 and chukwuma g. udensi1 1 department of microbiology, michael okpara university of agriculture, umudike, abia state, nigeria. 2 department of biochemistry, michael okpara university of agriculture, umudike, abia state, nigeria. authors’ contributions this work was carried out in collaboration among all authors. author eka designed the study, wrote the protocol. author udn wrote the first draft of the manuscript. authors hno and ceu performed the statistical analysis. authors cvn and cgu helped with the analyses of the work. all authors read and approved the final manuscript. article information editor(s): (1) dr. wagner loyola, brazilian agricultural research corporation (embrapa), brazil. (2) dr. darko nozic, university of belgrade, serbia. reviewers: (1) r. b. basavaraj, bms institute of technology and management, india. (2) y. c. tripathi, forest research institute (fri), india. complete peer review history: http://www.sdiarticle4.com/review-history/60290 received 15 june 2020 accepted 21 august 2020 published 01 september 2020 abstract aim: the study is aimed at evaluating the antibacterial activity of moringa oleifera root bark extract against pathogenic organisms. method: the antibacterial activity of methanolic and aqueous extracts of moringa oleifera root bark was investigated against test organisms (staphylococcus aureus and escherichia coli) using agar well diffusion method. different extracts were prepared at different concentrations (200 mg/ml, 100 mg/ml, 50 mg/ml, 25 mg/ml, 12.5 mg/ml and 6.25 mg/ml). results: the methanol extracts showed a higher zone of inhibition than the aqueous extracts at all the extract concentrations and on both test organisms. also, the observed antibacterial activity was dose-dependent for both extract methods. conclusion: the present work showed that moringa oleifera root bark has antibacterial activity against staphylococcus aureus and escherichia coli suggesting its potential as an antibacterial agent against infections caused by the organisms. original research article amanze et al.; aji, 3(1): 298-304, 2020; article no.aji.60290 299 keywords: antibacterial; moringa oleifera; pathogenic organisms; plant extracts. 1. introduction medicinal plants contain substances that can be used for therapeutic purposes. plants have been a valuable source of natural products for the health of human beings, and they have excellent potential for producing new drugs [1]. therefore, such plants should be investigated for a better understanding of their properties and efficiency [2]. antibiotic resistance is a type of drug resistance where microorganisms can survive exposure to an antibiotic. the leading causes of antibiotic resistance are genetic mutation in bacteria [3]. inappropriate and irrational use of antimicrobial medicines provides a suitable environment for resistant microorganisms to emerge and persist. the higher the level of exposure to the antimicrobial agents, the higher the risk of the development of resistance. irrespective of the need for the antibiotic, as resistance toward antibiotics becomes more common, a greater need for alternative treatment arises. however, despite a push for new antibiotic therapies, there has been a continued decline in the number of a newly approved drugs. as regards this, there is a need for finding and investigating novel antimicrobial compounds. while most of the antibiotics have been developed from microorganisms, there are many reports on the antibacterial effectiveness of the traditional herbs against the gram-positive and gram-negative bacteria [4]. the plant materials remain an essential resource for finding the novel antimicrobial compounds. microbial cells are negatively affected by plant-derived substances via various mechanisms of actions, one of which is the attack of these substances on the phospholipid bilayer of the cell membrane [5]. the medicinal herbs have the bacteriostatic effects on the enzymatic activity associated with energy production, or they can cause denaturation of proteins, modifying cell wall permeability, or causing the loss of macromolecules. one such plant of medicinal value is moringa oleifera, commonly known as sahajan in hindi; belonging to the family moringaceae, a single genus family with 13 known species [6]. the different parts of the plant viz. leave, stem bark, root bark, flowers, fruits, and seeds are used in the indigenous systems of medicine for the treatment of a variety of human ailments. m. oleifera incredible medicinal value, which is claimed by many cultures and communities, is based on science. m. oleifera has been found to contain many essential nutrients, for instance, vitamins [7,8]. nutrition content of a plant plays an essential function in medicinal, nutritional, and therapeutic properties. m. oleifera leaves consist of high sources of vitamin c, calcium, and potassium as well as protein and also works as a useful source of natural antioxidants. due to the presence of several sorts of antioxidant compounds such as flavonoids, ascorbic acid, carotenoids, and phenolics, m. oleifera can extend the period of food containing fats [9]. despite the array of uses to which parts of moringa oleifera trees are put to, scanty literature is available on the uses of moringa oleifera bark as antimicrobial [10]. however, a critical step in the screening of plant material for antimicrobial activity is to evaluate its antibacterial activity against pathogenic microorganisms. hence, this study evaluates the antimicrobial activity of moringa oleifera root bark extract on bacterial pathogens. 2. materials and methods 2.1 sample collection dried samples of root barks of m. oleifera were collected at national root crop research institute umudike in ikwuano, abia state, nigeria. the root barks of m. oleifera sample was identified and authenticated at the herbarium of the department of plant sciences and biotechnology, michael okpara university of agriculture, umudike, abia state. 2.2 test organisms the test isolates of staphylococcus aureus (atcc 25923) and escherichia coli (atcc 25922) used in this study were obtained from the microbiology laboratory, michael okpara university of agriculture, umudike, and confirmed by morphological appearance color, shape elevation, pigmentation, opacity, and nature of edges of the colonies were observed and recorded for each isolate. gram test and motility test: a drop of an 18-20 hours’ peptone medium culture of the test organism was placed on a clean grease-free slide with the aid of pasteur pipette. the slide was then covered with a coverslip and viewed amanze et al.; aji, 3(1): 298-304, 2020; article no.aji.60290 300 under the microscope using an x40 objective lens. the movement of small motile bacteria is distinguished from the on-the-spot vibratory movement (brownian movement), which is shown by all microorganisms and particles when suspended in a fluid. true bacterial motility is the ability of an organism to move in different directions or a single direction [11]. biochemical test –isolated organisms were identified by standard microbiology identification techniques, including the catalase test, citrate utilization test, methyl-red test, vogesproskauer test, urease test, and indole test [11]. 2.3 preparation of crude extracts the method of mumtaz et al. [12] was adopted. the collected material was shade dried, made to a coarse powder, and then packed in polythene bags for further analysis. fifty grams of the plant material was soaked in 200 ml of solvents (aqueous and methanol) and left for 24 hrs. the fraction was separated using sterile muslin cloth and filter through sterile whatman filter paper (no. 2). the crude extracts after filtration stored in a refrigerator at 4⁰c. all the extracts were then concentrated with a rotary vacuum evaporator at 40⁰c. one gram (1 g) each of the methanol and aqueous extract was added to methanol and made up to 5 ml to give a concentration of 200 mg/ml. other concentrations of 100, 50, 25, 12.5, and 6.25 mg/ml were prepared by the double dilution method as described by cheesebrough [11]. 2.4 antimicrobial susceptibility testing the kirby-bauer method was applied based on zones of inhibition of agar plates [13]. exactly 1.0 g of the methanolic and aqueous extracts were dissolved and made up to 5 ml each with dimethyl sulfoxide (dmso) and water respectively to obtain 200 mg/ml of each extract. the concentration was diluted two folds to get 100 mg/ml and 50 mg/ml of each extract. mueller-hinton agar was prepared, and plates inoculated with the different isolates. the punched circular discs from whatman no. 2 filter paper were impregnated with 0.1 ml of different concentrations of each extract and allowed to airdry for a few minutes. each of the discs was pressed onto the surface of the inoculated medium to ensure contact with the medium. negative control was prepared by impregnating some discs with water and dmso, which was transferred and pressed unto the inoculated plates while standard antibiotics discs (ciprofloxacin, tetracycline, and ampicillin) served as positive controls. all the tests were carried out in duplicates. incubation was done at 37 o c for 24 hr and plates observed for zones of inhibition. the degree of sensitivity was expressed as a measure of the diameter of zones of inhibition in millimeters (mm). a diameter of 10 mm or higher was considered as an indication of the sensitivity of the test organism to the extracts. the mean inhibition zones (mm) were calculated as the difference between the disc diameter (6 mm) and the diameter of the inhibition zones [14]. 2.5 determination of minimum inhibitory concentration (mic) the inoculums of the microbial isolates used for the test were prepared by comparing with a standard of macfaland reagent in order to reduce microbial load or count. exactly 1.0 g each of both methanolic extracts was dissolved separately in 5 ml each of dmso and sterile water respectively to get a concentration of 200 mg/ml and labeled as solution 1.2 ml of solution 1. this was serially diluted into 8 folds to get corresponding concentrations of 100 mg/ml, 50 mg/ml, 25 mg/ml, 12.5 mg/ml, 6.75 mg/ml, 3.13 mg/ml, 1.56 mg/ml and 0.78 mg/ml each for both aqueous and ethanolic extract and labeled solutions 3 to 9. a previously prepared mueller-hinton broth containing various concentrations of the extract was inoculated with the standard inoculums of each test organism, followed by incubation of the tube at 37 0 c for 16-20 hr. thereafter, the tubes were observed for the presence or absence of growth in each tubes determined by turbidity of the test tubes. the lowest concentration of the extracts resulting in no growth after incubation was taken as the minimum inhibitory concentration (mic) of the melon fungus extracts [15]. 2.6 minimum bactericidal concentration (mbc) the minimum bactericidal concentration (mbc) of the plant extract on the bacterial isolates was carried out according to ajaiyeoba et al. [16]. one (1 ml) bacterial culture was pipetted from the mixture obtained in the determination of mic tubes, which did not show any growth and were sub-cultured onto nutrient agar and incubated at 37 0 c for 24 hours. after incubation, the concentration at which there was no single colony of bacteria was taken as mbc. amanze et al.; aji, 3(1): 298-304, 2020; article no.aji.60290 301 3. results the root bark extracts of moringa oleifera were analyzed for their antibacterial activity against staphylococcus aureus and escherichia coli. fig. 1. diameter zone of inhibition (mm) produced by methanol root bark extracts of moringa oleifera methanol root bark extract of moringa oleifera had a concentration-dependent inhibition on both bacteria with increased activity on e. coli than s. aureus. control drug = erythromycin 30 mcg fig. 2. diameter zone of inhibition (mm) produced by aqueous root bark extracts of moringa oleifera the diameter zone of inhibition (mm) produced by aqueous root bark extracts of moringa oleifera, there was a concentration-dependent inhibition on both bacteria with increased activity on e. coli than s. aureus. this activity, however, was much lower than the one exhibited by corresponding methanol extracts 0 5 10 15 20 25 30 200 100 50 25 12.5 6.25 control co n ce n tr at io n o f e xt ra ct zone of inhibition (mm) diameter zone of inhibition (mm) produced by methanol root bark extracts of moringa oleifera staphylococcus aureus escherichia coli 0 5 10 15 20 25 30 200 100 50 25 12.5 6.25 control c o n ce n tr at io n o f e xt ra ct zone of inhibition diameter zone of inhibition (mm) produced by aqueous root bark extracts of moringa oleifera staphylococcus aureus escherichia coli amanze et al.; aji, 3(1): 298-304, 2020; article no.aji.60290 302 table 1. mic and mbc values for the methanol and aqueous extracts against susceptible organisms test organisms methanol extract aqueous extract mic mbc mic mbc staphylococcus aureus 12.5 mg/ml 25 mg/ml 25 mg/ml 50 mg/ml escherichia coli 6.25 mg/ml 12.5 mg/ml 12.5 mg/ml 25 mg/ml methanol extracts exhibited lower mic and mbc on the test organisms than the aqueous extract. staphylococcus aureus also required a higher concentration of extract to be inhibited than did escherichia coli keys: mic = minimum inhibitory concentration mbc = minimum bactericidal concentration 4. discussion the place of medicinal plants as a natural remedy to diseases cannot be overemphasized [1]. while most antibiotics have been isolated from bacteria, many plants have exhibited antibacterial activities and have been used widely in the treatment of ailments caused by such organisms. different parts of moringa oleifera have been used in the treatment of different ailments, though with a few studies evaluating its antimicrobial potentials. the study evaluated the antibacterial activity of moringa oleifera root bark extract on clinical isolates. according to schillinger and lücke [17], inhibition was scored positive if the width of the clear zone around the antibacterial agent or colonies of the producer strain was 0.5 mm or larger. as the results indicate, at concentrations of 50 mg/ml and above, moringa oleifera root bark extract actually possesses inhibitory activities since the least zone of inhibition observed at these points is 12 mm. from this investigation, across the groups, the aqueous extracts of moringa oleifera root bark showed lower antibacterial activities compared to the methanol extracts. methanol extracts exhibited lower mic and mbc on the test organisms. this observed difference between the extracts may be attributed to the ability of methanol to extract more of the essential secondary plant metabolites and the insolubility of active compounds in water since these compounds are believed to exert antibacterial activity on the test organisms [18]. okigbo and ajale [19] reported that the inactivity of plant extracts might be due to the age of the plant, extracting solvent, method of extraction, and time of harvesting of plant materials. it can be inferred that methanol is a better extracting solution for this plant and purpose than water. this difference may also be due to the inhibitory ability of the methanol even without extract, whereas the ability of the aqueous extract to exhibit this inhibition may be primarily due to its penetrative ability. from the findings in this study, the methanol bark extract showed promising antibacterial activity the test pathogens at most of the concentrations. the antimicrobial activity of the leaf extracts of moringa oleifera root bark was evaluated against gram-positive and negative bacteria (staphylococcus aureus and escherichia coli). data presented in figs. 1 and 2 revealed that the leaf extracts were more effective in inhibiting escherichia coli (gram-negative) with a zone of inhibition ranging between 10 mm and 23 mm as compared to staphylococcus aureus (grampositive) with a zone of inhibition ranging between 9 mm to 19 mm. this result is contrary to that of agatemor [20] where it was reported that gram-negative bacteria are more resistant than gram-positive bacteria to antimicrobial plant extract. rao et al. [21] investigated the antibacterial activity of methanolic extract of m. oleifera by using well diffusion technique and reported that the most significant activity of this plant was seen against s. aureus. while working on the same plant species, devi et al. [22] investigated the antibacterial activity of methanolic extract of bark by agar well diffusion method against bacillus spp. and s. aureus. the results are also in agreement with ahmad et al. [23], who reported methanolic leaf extract of c. australis had the highest activity against s. aureus at 200 mg/ml concentration with 10.5mm zone of inhibition. the aqueous extract of moringa oleifera bark has shown strong antibacterial activity against the test organisms. the trends in this study are contrary to the reports by yagoub et al. [24] who, in their preliminary screening for antimicrobial activity of different plants against different organisms, methanolic extracts of a. indica produced zero zones of inhibition against e. coli. amanze et al.; aji, 3(1): 298-304, 2020; article no.aji.60290 303 the difference in the effect of this plant extracts within the organisms suggested that there are different antibacterial compounds in the plant extracts and that the compound that acted on one may not be the same as the one that acted on the others since antibacterial agents have different modes of action [25]. at concentrations of 200 mg/ml, the methanol extract showed an inhibitory activity (23 mm) on e. coli comparable to that of the standard drug erythromycin 30 mcg (25 mm). this further suggests its potential as an antibacterial agent. 5. conclusion based on these results, it could be concluded that methanol was the best extraction solvent for the antibacterial activity of moringa oleifera against all the tested organisms. the activity decreased with the decrease in the concentration of the extract. the varying degrees of sensitivity of the bacterial test organisms may be due to the intrinsic tolerance of microorganisms. the results of this study suggest that the moringa oleifera root bark can be used as an antibacterial agent against infections caused by staphylococcus aureus and escherichia coli consent it is not applicable. ethical approval it is not applicable. acknowledgements we acknowledge the support of the technical staff of the laboratory unit of the department of microbiology and the department of biochemistry, michael okpara university of agriculture, umudike. we sincerely appreciate the input of love and assistance competing interests authors have declared that no competing interests exist. references 1. nascimento gg, locatelli j, freitas pc, silva gl. antibacterial activity of plant extracts and phytochemicals on antibioticresistant bacteria. braz j micro. 2000;31: 247–256. 2. chintamunnee v, mohamoodally mf. herbal medicine commonly use against infectious disease in the tropic island of mauritius. j herb med. 2012;2:113-125. 3. fernando c, jose lm. phenotypic resistance to antibiotics. journal of pharmacology and pharmacy. 2013;2(2): 237-255. 4. alavijeh pk, alavijeh pk, sharma d. a study of antimicrobial activity of few medicinal herbs. asian j plant sci res. 2012;2(4):496-502. 5. ahmad s, sharma r, mahajan s, gupta a. antibacterial activity of celtis australis by in vitro study. inter j pharma sci. 2012; 4(2):629-631. 6. khawaja tm, tahira m, ikram uk. moringa oleifera: a natural gift a review. j pharma sci res. 2010;2:775-781. 7. fahey jw. moringa oleifera: a review of the medical evidence for its nutritional, therapeutic, and prophylactic properties part 1. trees life j. 2005;1:5-20. 8. kasolo jn, bimenya gs, ojok l. phytochemicals, and uses of moringa oleifera leave in ugandan rural communities. j med plants res. 2010;4: 753-757. 9. siddhuraju p, becker k. antioxidant properties of various solvent extracts of total phenolic constituents from three different agroclimatic origins of drumstick tree (moringa oleifera lam.) leaves. j agric food chem. 2003;51:2144-2150. 10. mudasser z, showkat a, rajendra s, surabhi m, ankur g, rajneesh ka. antibacterial activity of bark extracts of moringa oleifera lam. against some selected bacteria. pak j pharm sci. 2014; 27(6):1857-1862. 11. cheesbrough m. district laboratory practice in tropical countries. cambridge university press. 2006;62. 12. mumtaz j, mohiuddin k, warsi fk. studies on the antibacterial property of eucalyptus the aromatic plant. inter j pharm sci rev res. 2011;7(2):86-88. 13. prescott lm, harley jp, klein da. microbiology. 6th edn., mcgraw-hill co., new york, london; 2005. 14. akpaja eo, isikhuemhen os, okhuoya ja. ethnomycology and usage of edible and medicinal mushrooms among the igbo people of nigeria. int j med mushrooms. 2003;5:313-319. amanze et al.; aji, 3(1): 298-304, 2020; article no.aji.60290 304 15. alobo ap. proximate composition and functional properties of pleurotus tuberregium sclerotia flour and protein concentrate. plant foods hum nutr. 2003; 58:1–9. 16. ajaiyeoba eo, onocha pa, nwozo so, sama w. antimicrobial and cytotoxicity evaluation of buchholzia coriacea stem bark. fitoterapia. 2003;74(7-8):706709. 17. schillinger, v, lücke fk. antimicrobial activity of lactobacillus sake isolated from meat. applied environ micro. 1989;55: 1901-1906. 18. okigbo rn, ogbonnanya ou. antifungal effects of two tropical plants extracts ocimum gratissimum and afromaomum melegueta on post-harvest yam discorea spp rot. afr j biotech. 2006;5(9):727-731. 19. okigbo rn, ajale an. inhibition of some human pathogens with the tropical plant extracts chromolineena odorata and citrus aurantifolia and some antibiotics. int j mol med adv sci. 2005;1:34-40. 20. agatemor c. antimicrobial activity of aqueous and ethanol extracts of nine nigerian spices against four foodborne bacteria. j environ agric food chem. 2009;8(3):195-200. 21. rao pk, rao db, ravi ck, nadh mr, madhavi y, rao tr. in vitro antibacterial activity of moringa oleifera against dental plaque bacteria. j pharma res. 2011;4:39. 22. devi gs, priya v, abiramasundari p, jeyanthi pg. antibacterial activity of the leaves, bark, seed and flesh of moringa oleifera. inter j phar sci res. 2011;2(8): 2045-2049. 23. ahmad mm, salim-ur-rehman z, iqbalanjum fm, sultan ji. genetic variability to essential oil composition in four citrus fruit species. pak j botany. 2006;38(2):319324. 24. yaqoub so, shami ea, ahmed b. antimicrobial activity of some medicinal plants against some gram positive, gram negative and fungi. department of molecular biology, el neelain university, sudan; 2007. available:www.astf.net accessed on 20th august 2009. 25. bibi z, zainab a, mona sa, sajid k, humaira r, dina ws, asma a, arshad ma. in-silico elucoidation of moringa oleifera phytochemicals against diabetes millitus. saudi journal of biological sciences. 2020;issn:1319-562x. _________________________________________________________________________________ © 2020 amanze et al.; this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: http://www.sdiarticle4.com/review-history/60290 http://creativecommons.org/licenses/by/4.0 _____________________________________________________________________________________________________ *corresponding author: e-mail: elmafary@gmail.com; asian journal of immunology 3(1): 223-232, 2020; article no.aji.57637 molecular pathogenesis, clinical efficacy and safety of therapeutics used in the treatment of osteoarthritis shaayau shehu1*, abdulaziz umar kurya2, kamal murtala farouq3 and abdulhakim umar toro2 1 department of biochemistry, usmanu danfodiyo university, sokoto, nigeria. 2 school of life and allied health sciences, glocal university, saharanpur, uttar pradesh, india. 3 department of genetic engineering, sharda university, greater noida, uttar pradesh, india. authors’ contributions this work was carried out in collaboration among all authors. author ss designed and supervised the study, author auk wrote the first draft of the manuscript, outline the protocol and performed the statistical analysis while authors kmf and aut managed the literature search and analyses of the study. all authors read and approved the final manuscript. article information editor(s): (1) dr. cynthia aracely alvizo báez, autonomous university of nuevo leon, mexico. reviewers: (1) m.k. jayalakshmi, rajiv gandhi university of health science, india. (2) vanessa de melo cavalcanti dantas, instituto de pesquisa aggeu magalhães, brazil. (3) marcello zaia oliveira, federal university of parana, brazil. complete peer review history: http://www.sdiarticle4.com/review-history/57637 received 01 april 2020 accepted 07 june 2020 published 20 june 2020 abstract osteoarthritis (oa) also known as degenerative joint disease, is the most common form of arthritis which affects all the tissues of the joint, including the cartilage, bone, ligaments, and muscles. it can develop in any number of joints, but most commonly affects the knees, hands, and hips. oa is characterized by progressive cartilage deterioration, subchondral bone remodeling, loss of joint space, marginal osteophytosis, and loss of joint function. the prevalence rate is estimated to about 242 million people in the world. oa results from the disruption of the balance between synthesis and degradation of extracellular matrix components by the chondrocyte in combination with increased uncompensated chondrocyte apoptosis. it is increasingly understood that ageing contributes to the development of osteoarthritis by working in conjunction with a variety of other factors, both intrinsic and extrinsic to the joint. several abnormalities in components of the healthy review article shehu et al.; aji, 3(1): 223-232, 2020; article no.aji.57637 224 joints such as meniscus, articular cartilage, subchondral bone and synovial membrane results to manifestation the disease. in an attempt to discover new emerging therapeutic target, certain diagnostic strategies are applied such as x-ray, ultrasonography, anthroscopy and magnetic resonance imaging to have a deep insight on its effect and monitor the progression of the disease. interestingly, many clinical researches proved efficacy of therapeutics such as adamulimab which block tnf-α and plays significant role in the pathogenesis of the disease, diacerein which inhibit interleukin1 β, natural anti-inflammatory compound such as curcumin, bisphosphonate drugs such as alendronate and risedronate and anti-osteoporotic drugs such as strontium ranelate, chondroitin sulfate, intraarticular hyaluronic acids and glucosamine sulfate are reported to be effective and safe in the management of the disease. keywords: osteoarthritis; degenerative joint disease; pathogenesis and therapeutics. 1. introduction osteoarthritis (oa) also known as degenerative joint disease is the most common form of arthritis which affects all the tissues of the joint, including the cartilage, bone, ligaments, and muscles. it can develop in any number of joints, but most commonly affects the knees, hands, and hips [1]. oa typically occurs later in life, usually after age 50, although may start earlier in the case of joint injury but symptoms may vary in severity, whereby oa in its severe forms restricts mobility, interrupts sleep, and interferes with the sufferer’s enjoyment of life [2]. the disease has precisely no cure. however, some of the therapeutics mainly aimed at reducing the pain, improving the joint function, and in some instances delay the progression of the disease [3]. oa occurs when the protective cartilage that cushions the ends of two opposing bones wears down over time, although the articular cartilage and subchondral bone often show the most prominent changes [4]. oa is primarily characterized by progressive cartilage deterioration, marginal osteophytosis, subchondral bone remodeling, loss of joint space, and ultimately loss of joint function [5]. oa is also characterized by a degeneration of articular cartilage whereby the breakdown leads to matrix fibrillation, fissure appearance, gross ulceration, and full thickness loss of the joint surface which is accompanied by hypertrophic changes of bone with osteophyte formation and subchondral bone plate thickening. at the early stage of the disease, there are changes in the synovial membrane together with an inflammatory reactions while at advanced stages, joint contractures, muscles atrophy and limb deformity are commonly observed [6]. 2. materials and methods using online databases, we conducted a literature review of molecular pathogenesis and novel therapeutic potentials of osteoarthritis. the key articles used were retrieved predominantly from ncbi, google scholar, medline, uptodate, using the terms ‘osteoarthritis, degenerative joint disease, therapeutics of osteoarthritis as keywords for our search. we included scientific publications from 1 st january 2010 to 15 th december 2018 articles focusing on clinical characteristics, epidemiology, symptoms, diagnosis and treatments for osteoarthritis were eligible for selection. 3. epidemiology of osteoarthritis the prevalence of oa is break down based on different demographic criteria. the age-related demographics shows that primary osteoarthritis is mostly common in elderly and majority of the patients are often asymptomatic. approximately 80-90% of individuals older than 65 years have evidence of radiographic primary osteoarthritis. however looking at the gender variations women are at high risk of the infection than men, this also varies with age where as women older than 55 years have high prevalence rate. oa is a major cause of disability in older adults worldwide, according to world health organization (who) 9.6% of men and 18.0% of women aged 60 years and above has symptomatic osteoarthritis worldwide, this lead to restriction of movement in 80% of those with osteoarthritis and incapable of performing major daily activities of life in 20% of oa patients [7]. in the united states of america, knee oa accounts for more than 80% of the disease’s total burden and affects at least 19% of shehu et al.; aji, 3(1): 223-232, 2020; article no.aji.57637 225 american adults aged 45 years and older [8,9]. according to data produced by the dutch institute for public health, the prevalence of knee oa in individuals of 55 years and above is reported as 15.6% in men, 30.5% in women, and that of hips oa is 4.4% in men and 7.6% in women [10]. recent study of individual’s ≥60 years of age living in spain documented a prevalence of 6.7% in men and 8.0% in women having oa [9]. a review of several investigations in canada has reported the overall prevalence ranges between 7.5 and 14.7% [11]. in addition, the north and east regions of china had the lowest prevalence of symptomatic knee oa 5.4% and 5.5%, respectively, followed by the north-east 7.0%, south-central 7.8%, and north-west 10.8% regions. surprisingly, the prevalence was highest 13.7% in subjects living in the south-west region [12]. furthermore, prevalence of 22% to 39% was reported in india; however it is more common in women than men, whereby approximately 45% of women over the age of 65 years have symptoms while 70% of those over 65 years show radiological evidence of oa [7]. fig. 1. prevalence of oa in the united states of america showing the disease burden, adult infected, men and women [8,9] fig. 2. shows spain and canada infection rate in men and women [9,11] fig. 3. shows overall prevalence rate for different region in china regardless of gender variation [12] 80% 19% 7.6% 4.4% 0 20 40 60 80 100 disease burden adults affected women men knee oa hip oa 6.7% 8% 7.5% 14.7% 0 2 4 6 8 10 12 14 16 men women men women spain canada 5.4% 5.5% 7% 7.8% 10.8% 13.7% 0 2 4 6 8 10 12 14 16 china shehu et al.; aji, 3(1): 223-232, 2020; article no.aji.57637 226 4. development of osteoarthritis there is strong correlation between ageing and development of osteoarthritis where it is increasingly understood that ageing contributes to the development of osteoarthritis by working in conjunction with a variety of other factors, both intrinsic and extrinsic to the joint. it has become distinct that changes that results due to aging in the musculoskeletal system contribute to the development of osteoarthritis by working in conjunction with other factors, both intrinsic (e.g., alignment, overloading) and extrinsic (e.g., genetics) to the joint [13]. the primary changes with osteoarthritis occur in the articular cartilage, followed by associated changes in the subchondral bone [14]. recently, more focus has been placed on the subchondral bone as the primary cause of symptomatic disease. understanding of changes at early stages that lead to development of osteoarthritis is important, since these changes could still be reversible, and therefore, preventive treatment could be initiated to halt or reverse further progression of the disease. biomechanical factors in oa play an important role in the health of diarthrodial joints. altered joint loading associated to obesity, malalignment, trauma, or joint instability are most common critical risk factor for joint degeneration [15]. fig. 4. shows cartilage breakdown on healthy joint which progresses to several stages of oa development; stage 1 involves minimum disruption accompanied by 10% cartilage lost, stage 2 involve joint space narrowing where cartilage breakdown begins and characterized by osteophytes formation, in stage 3 there is moderate joint reduction where gaps in the cartilage expand until they reach bone and lastly stage four which involves 60% cartilage lost and is characterized by large osteophytes [16,17] shehu et al.; aji, 3(1): 223-232, 2020; article no.aji.57637 227 5. pathogenesis of osteoarthritis oa results from the disruption of the balance between synthesis and breakdown of extracellular matrix components by the chondrocyte in addition with increased uncompensated chondrocyte apoptosis [18]. in the young patient, the pathogenesis of knee osteoarthritis is predominantly related to an unfavorable biomechanical environment at the joint, which results in mechanical demand that exceeds the ability of a joint to repair and maintain itself thereby predisposing the articular cartilage to premature degeneration [19]. the pathophysiology of the process by which joint degeneration leads to the clinical syndrome of osteoarthritis remains poorly understood. however oa mostly involves degeneration of cartilage, abnormal bone remodeling, osteophyte formation and joint inflammation [20]. in healthy joint several components facilitate the proper functioning of joints such as, meniscus (shock absorber) a fibrocartilage which disperse the weight of the body and reduce friction during movement and mainly composed of water, type i collagen, and proteoglycans in its extra cellular matrix [21]. the articular cartilage a hyaline cartilage which decreases friction and distributes loads and mainly composed of water, 90% type ii collagen and proteoglycans in the matrix [22]. the subchondral bone which attenuates forces generated through locomotion, with the compact subchondral bone plate providing firm support to the joint and mainly composed of type i collagen. the synovium which produces synovial fluids that functions in reducing friction by lubricating the joint, absorbing shocks, supplying oxygen, nutrients and removing carbon dioxide and metabolic wastes from the chondrocytes within articular cartilage [23]. synovial membrane composed of two synoviocytes; synovial fibroblast and synovial macrophages. the synovial fibroblast produces the extracellular matrix components of the synovial fluid and thus is important for cartilage integrity and lubrication of the joint, while the synovial macrophages remain relatively quiescent in the healthy joint; they become activated in the inflamed joint and, along with infiltrating monocytes/macrophages, regulate secretion of pro-inflammatory cytokines [24,25]. several abnormalities such as mechanical abrasion in the knee have been found to promote oa, which is mostly seen in the older age and can ultimately leads to the progressive degenerative changes in the proper functioning of the joints. furthermore, the innate immune system plays a role in oa progression through the activation of both the complement and alternative pathways of inflammation, once activated; it leads to an inflammatory response that is a major driver of the disease [25]. 6. diagnosis of osteoarthritis  x-ray of affected joints will show a loss of the joint space. in more advanced cases, there may be bone spurs or evidence of worn-down ends of the bones in the affected joint [26].  magnetic resonance imaging (mri) has a tomographic viewing perspective and thus provides cross-sectional images of the anatomy free of the projectional limitations of radiography, the technique is uniquely able to depict all the components of the joint, their pathologies, articular cartilage, meniscus, intraarticular ligaments, synovium, effusion, bone attrition, bone marrow lesions, subchondral cysts, and intraand periarticular cystic lesions [27].  arthroscopy: is a is a surgical procedure orthopedic surgeons use to visualize, diagnose, and treat problems inside a joint by inserting a narrow tube attached to a fiberoptic video camera through a small incision which gives a clear view of the pathological changes inside the joint [28].  ultrasonography is a modern ultrasound technique which uses high-frequency sound waves to produce images of internal organs and other tissue [29]. 7. therapeutic targets of osteoarthritis 7.1 tnf-α blockers tnf-α is an inflammatory cytokine produced by monocytes/macrophages during acute inflammation, it plays significant role in the pathogenesis of oa. adalimumab is a human monoclonal antibody bioengineered to prevent the binding of tnf-α to its receptor and inhibit the progression of osteoarthritis. a 12-month randomised, double-blind, placebo-controlled trial evaluate subcutaneous administration of 40 mg adalimumab every two weeks in 60 patients with erosive hand oa, however the tolerability and safety profiles of adalimumab on those patients were very good and resulting to successful shehu et al.; aji, 3(1): 223-232, 2020; article no.aji.57637 228 neutralization of the tnfα there by slowing the progression of structural damage in erosive interphalangeal finger joint osteoarthritis [30]. anti-tnf-α therapy with infliximab (a chimeric monoclonal antibody) was reported to yield effective treatment and reduce the incident of secondary oa via other pathways [31]. 7.2 il-1β inhibitor il-1β also known as leukocytic pyrogen, is a key pathogenic factor in oa. hence use of diacerein which is an il-1β inhibitor, reduces the number of il-1 receptors, thereby resulting to reduction in functional il-1 heterodimer receptor complexes and ultimately lowers the disease progression [32]. a study of a symptomatic slow‐acting oa drug which accesses the primary outcome for 2 months after the end of a 3 month treatment period shows that diacerein is safe and has a significant effective for the treatment of knee oa [33]. another study confirmed that symptomatic benefit provided by diacerein in terms of pain reduction is minimal; the small changes observed in the joint space narrowing is of questionable clinical relevance and was observed only for oa of the hip [34]. in vitro and experimental models showed a reduction in cartilage destruction with il-1 by il-1 receptor antagonists [35]. one of recent articles shows a possible benefit in using higher doses of kineret (150-200 mg) in the treatment of osteoarthritis of large joints and suggest it alternativeness to ia steroid [36]. 7.3 curcumin clinical trials shows similar efficacy of curcuma formulation with nsaids and glucosamine for treatment of osteoporosis [37]. a multicenter study reveal the efficacy and safety of curcuma domestica extracts in pain reduction and functional improvement and further shows it effectiveness, similar to that of ibuprofen [38]. the adjuvant therapy of curcumin with diclofenac has more potential and beneficial effect than individual effect of diclofenac alone [39]. a randomized, double-blind, placebo-controlled, prospective clinical study of a highly bioavailable form of curcumin (theracurmin) in patients with osteoarthritis reveal significant effect of the compound in deceasing pain and its potential in the treatment of human knee osteoarthritis in the future [40]. meriva (a curcuminphosphatidylcholine complex) is also referred as effective and safe agent for the complementary management of osteoarthritis, leading to better disease control, a decreased use of nsaids, and overall improvement in the quality of life [41]. another study shows that formulation of 500 mg curcuma longa and boswellia serrate extract (cb) administered twice daily demonstrated a greater improvement in the treatment of oa than 100 mg of celecoxib administered twice a day in the scores for pain, walking distance and joint line tenderness. the cb formulation was equally effective as celecoxib in alleviating crepitus, and increasing the range of joint movements with no dose-related toxicity and ultimately the formulation was termed superior to celecoxib (nsaids) for the treatment of active oa [42]. 7.4 bisphosphonates bisphosphonates are a group of medicines that slow down or prevent bone loss, strengthening bones. they work by inhibiting osteoclasts which are responsible for breaking down and reabsorbing minerals such as calcium from bone. meta-analysis showed that bisphosphonates therapy is effective in relieving pain and accelerating functional recovery for patients with oa [43]. in another clinical studies, administering alendronate sodium (a bisphosphonate drug) for patients with oa has clinical efficacy in reducing joint complications with significant structural improvement of the joints, and may delay and prevent further disease progression probably through inhibition of leptin activity [44]. risedronate a bisphosphonate drug in comparison with placebo did not improve signs or symptoms of oa, nor did it alter progression of oa, only reduction in the level of marker of cartilage degradation was observed however sustained clinically relevant improvement in signs and symptoms was observed in all treatment and placebo groups [45]. in a one-year, placebocontrolled trial that included 59 patients with knee oa treated with zoledronic acid 5 mg intravenously as a single infusion, a significant reduction in visual analogue pain scores versus placebo was seen after six months [46]. 7.5 strontium ranelate strontium ranelate (sr) is an anti-osteoporotic drug responsible for balance between bone resorption and bone formation [47]. clinical trial reveals that treatment with 1 or 2 g of strontium ranelate per day is associated with significant structural changes in patients with knee osteoarthritis; furthermore, there is a beneficial effect on the symptoms specifically at dosage of 2 g/day [48]. another study shows that dose of 1800 mg/kg/day of sr significantly attenuated shehu et al.; aji, 3(1): 223-232, 2020; article no.aji.57637 229 cartilage matrix and chondrocyte loss, and decreased chondrocyte apoptosis, in a medial meniscal tear model using sprague–dawley rats [49]. 7.6 chondroitin sulfate a 1 year, randomized, double-blind, multicenter clinical study in japan, shows that treatment with sodium chondroitin sulfate at a dose of 1560 mg/d is more effective  than 260 mg/d more resultant to pain relief in patients with knee oa [50]. however, a 2-year multicentre exploratory study on efficacy of chondroitin sulfate versus celecoxib on knee osteoarthritis structural changes concluded that chondroitin sulphate is more superior over celecoxib at reducing cartilage volume loss in knee oa patients [51]. meta-analysis of randomized controlled trials demonstrated that oral chondroitin is more effective than placebo on relieving pain and improving physical function. although glucosamine showed positive effect on stiffness outcome, further studies are requested to investigate the accurate effectiveness of both the two drugs [52]. 7.7 intra articular hyaluronic acids a canadian evidence-based perspective demonstrated that treatment with intra articular hyaluronic acids is well tolerated, with significantly improved pain, function and stiffness outcomes compared with placebo or noninterventional controls in patients with mildto-moderate knee oa [53]. multi-center open perspective study suggests the clinical efficacy of a single intra-articular injection of 3 ml intraarticular hyaluronic acid solution containing 75 mg high molecular weight (>2 mda) native hyaluronic acid [54]. 7.8 glucosamine sulfate evidence from a real life setting trials and surveys shows that different therapeutic effect are obtained with different formulation of glucosamine. therefore, not all formulation of glucosamine should be afforded same level of recommendation [55]. glucosamine supplements composed of different chemical components and have become a mainstay in management of oa due to their symptom-relieving effects, cost effectiveness, important structure-preserving and relatively non-toxic adverse effect profiles. however, researches are required to fully understand the concept [56]. 8. conclusion many studies on osteoarthritis gave more insight on the inflammatory processes, biochemical changes and abnormalities in some of the component of healthy joint. in this review we have highlighted clinical efficacy and safety of some therapeutics used in the treatment of oa. however, extensive clinical trials should be taken to critically elucidate the efficacy of the therapeutics. consent it is not applicable. ethical approval it is not applicable. acknowledgements we appreciate the effort of usmanu dandodiyo university, glocal university, sharda university, and mewar university for their esteemed support and guidance towards completion of this review article. competing interests authors have declared that no competing interests exist. references 1. sharma l. osteoarthritis year in review 2015: clinical. osteoarthritis cartilage. 2016;24(1):36-48. doi: 10.1016/j.joca.2015.07.026 2. musumeci g, aiello fc, szychlinska ma, di rosa m, castrogiovanni p, mobasheri a. osteoarthritis in the xxist century: risk factors and behaviours that influence disease onset and progression. int j mol sci. 2015;16(3):6093-6112. doi: 10.3390/ijms16036093 3. kim jr, yoo jj, kim ha. therapeutics in osteoarthritis based on an understanding of its molecular pathogenesis. int j mol sci. 2018;19(3):674. doi: 10.3390/ijms19030674 4. hayashi d, roemer fw, guermazi a. imaging of osteoarthritis-recent research developments and future perspective. br j radiol. 2018;91(1085):20170349. doi: 10.1259/bjr.20170349 shehu et al.; 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article no.aji.56779 novel coronavirus covid-19: a general overview for emergency clinicians and current scenario md. bashir alam1* 1 department of food technology and nutrition science, noakhali science and technology university, sonapur-3814, bangladesh. author’s contribution the sole author designed, analysed, interpreted and prepared the manuscript. article information editor(s): (1) dr. wagner loyola, agricultural research corporation (embrapa) concordia, brazil. reviewers: (1) jorge castro bedriñana, universidad nacional del centro del perú, peru. (2) silke weber, unesp, brazil. (3) si thu aung, university of medicine mandalay, myanmar. complete peer review history: http://www.sdiarticle4.com/review-history/56779 received 19 april 2020 accepted 11 may 2020 published 25 may 2020 abstract a cluster of pneumonia cases linked to the novel coronavirus (2019-ncov) were announced by china at the end of december 2019. the current global situation, recent trends and ongoing progress in the containment and control of this epidemic, which has now spread 187 countries across the globe. a novel coronavirus virus (2019-ncov) has emerged in china, posing an international public health emergency in a few weeks, and has recently become a very high-risk group by the world health organization (who). a third introduction of a highly pathogenic and large-scale corona virus disease in humans was the sarscov-2 outbreak as a result of a severe acute respiratory syndrome coronavirus (sars-cov) in 2003 and the middle east respiratory syndrome coronavirus (mers-cov) in 2012. this virus was named coronavirus-2 severe acute respiratory syndrome (sars-cov-2) by the international committee on virus taxonomy (ictv) and coronavirus disease-19 (covid-19) disease. clinical symptoms of covid-19 patients include fever, cough, fatigue, and a small number of patients with signs of gastrointestinal infection. sarscov and mers-cov are particularly pathogenic in humans and are associated with high mortality. in this review, the epidemiology, pathophysiology, transmission, and clinical characteristics, and discussed current treatment and scientific developments and management of the recently discovered covid-19 are reviewed, with a focus on best practices and the public health complications. review article alam; aji, 3(1): 191-198, 2020; article no.aji.56779 192 keywords: sars-cov; sars-cov-2; covid-19; coronavirus; outbreak; current situation; prevention; control. abbreviations sars-cov : severe acute respiratory syndrome mars-cov : middle east respiratory syndrome covid-19 : coronavirus disease 2019 2019-ncov : novel corona virus 2019 who : world health organization ppe : personal protection equipment ffp3 : filtering face pieces (protection in three classes against dangers) n95 : respirators and surgical mask 1. introduction examples include the extreme acute respiratory syndrome (sars) and the middle east respiratory syndrome (mers) that caused earlier fatal outbreaks, and now the new 2019 coronavirus diseases (covid-19), triggered by sars2 coronavirus (sars-cov-2), originally known as unknown pneumonia, posing a serious threat to the public [1-3]. a wide group of viruses is the coronavirus family of single-strand rna viruses, which normally cause upper respiratory tract infection in humans. in 1960s, the first two members-229e and oc43-were recognized [4]. owing to the discovery of a new virus that triggered the pandemic serious acute respiratory syndrome (sars), this family of viruses gained attention only in 2003 [5,6]. two additional novel coronaviruses were later identified, nl63 and hku1, in 2004 and 2005. these two viruses cause infections in the respiratory tract and are widespread among humans [7,8]. therefore, with no major morbidity, the four known human corona viruses, 229e, oc43, nl63, and hku1, cause mild to moderate upper respiratory tract illness. a novel coronavirus has been found to cause sporadic cases of serious, acute respiratory infection (sari) since september 2012. the virus strain was previously unknown and genome sequencing suggested that this particular virus belongs to the genus beta coronavirus and is distinct from other known corona viruses and from sars [9]. in a small number of patients with sars, 7/28 (25%) had low titers of neutralizing antibodies for this newly discovered virus [10]. the newly identified virus was recently called middle east respiratory coronavirus syndrome (mers-cov) [11]. mers-cov was initially isolated from the respiratory tract of a patient in bisha, saudi arabia. the patient suffered serious pneumonia and acute renal failure in june 2012 [9]. the case was first released in promed in september 2012 [12]. apparently, the disease is going through a difficult path in the majority of patients. a continuum of disease incidence has been observed in a family cluster of four cases. from 6 april to 17 may 2013, 21 confirmed cases of mers-cov infection were reported in the alahsa district of the eastern province of saudi arabia (16 males and 5 females with a median age of 56 years) with nine deaths [13,14]. at the end of december 2019, a cluster of cases of pneumonia of unknown origin in wuhan, china, caused concern among health officials. the wuhan municipal health commission released warning on 31 december; a rapid response team was sent to wuhan by the chinese center for disease control and prevention (china cdc) and a report was sent to the world health organization (who) [15-18]. on 7 january 2020, the most recent coronavirus identified the causative pathogen, accompanied by the development of genomic characteristics and test methods [16,18]. now known as covid-19, both sars-cov and mers-cov are distinct but closely related [19]. with the recent detection of the 2019 novel coronavirus (covid-19), there are now a total of 7 coronaviruses known to infect humans: 1. human coronavirus 229e (hcov-229e) 2. human coronavirus oc43 (hcov-oc43) 3. human coronavirus nl63 (hcov-nl63) 4. human coronavirus hku1 5. severe acute respiratory syndrome-related coronavirus (sars-cov) 6. middle east respiratory syndrome-related coronavirus (mers-cov) 7. novel coronavirus (covid-19, also known informally as wuhan coronavirus) [20]. this analysis summarized the recent medical progress of the epidemiology, pathogenesis and clinical characteristics of covid-19 and addressed new treatment and scientific advances in the battle against a novel coronavirus outbreak. the targets of this review was to summarize the recent medical advancement of covid-19's epidemiology, transmission, and clinical features, and discussed current treatment and scientific method developments to fight the novel coronavirus outbreak. alam; aji, 3(1): 191-198, 2020; article no.aji.56779 193 2. current scenario (covid-19) after its initial appearance in wuhan, china, in the month of december 2019, the novel cov was included in the category of public health international emergency on 30 january 2020 [21,22]. besides affecting mainly china, sarscov-2/covid-19 has now spread 187 countries (fig. 1). out of the total 3,815,561 confirmed cases, 267,469 human deaths were reported as of 8 may 2020 (csse). as a result of such a fast leap in relation to the total number of confirmed sars-cov-2 infected persons and affecting many countries in a short period of time, it was classified by the who as a very high risk category and studies have classified pandemic risks and threats with the bloom. in increasingly growing cases of covid-19, it is worrying that we are making very high efforts to monitor the spread of this widely circulating virus among the world's population by taking effective prevention and control steps together with formulating global approaches and updated strategies with potential perspectives [23-30]. it is important to emphasize that there is an underreporting of these numbers due to the lack of diagnosis in many countries. 3. the disease (covid-19) covid-19 is the third cov outbreak in humans that occurred in last two decades, causing clinical manifestations of respiratory, digestive, and systematic affections, mainly expressed by pneumonia [31,32]. covid-19 is a modern, unexplained mutation of human beings. the covid-19 source is still unknown, although the initial cases concerned the huanan south china seafood market. although many of the early patients were working in or visiting the market, none of the exported cases came into contact with the market, indicating either human to human transmission or a more widespread source of animals. in addition to seafood, social media sources report that the huanan south china market has sold snakes, birds, and other small mammals, marmots and bats. the who confirmed that environmental samples from the marketplace were positive for the novel coronavirus but no clear association of animals was found [33]. because of close contact, few people can remain asymptomatic carriers without any clinical symptoms of cold, fever, fatigue or pathology of the lungs. as a professional carrier, they will shed virus to other people who come in contact with them up to 21 days [34]. initial signs include fever, mild chills, dry cough, tiredness and shortness of breath, moderate respiratory failure, pulmonary pneumonia, which deteriorate if not treated properly and are not sufficiently medicated [35]. more recent work has shown that many bat coronaviruses can infect human cells without having to adapt intermediately [36,37]. in addition, human serology data indicate bat coronavirus protein recognition and suggest that low-level zoonotic sars-like bat coronavirus transmission occurs outside recognized outbreaks [38]. mers-cov also has a zoonotic virus with possible bat origins [39]. in addition, human serology data indicate bat coronavirus protein recognition and suggest that low-level zoonotic sars-like bat coronavirus transmission occurs outside recognized outbreaks. 4. diagnosis of covid-19 serological testing at the height of the outbreak is of little use, though serum samples of recovered patients can be checked to know the igg titer. for highly infected patients, computed tomography (ct) and x-ray techniques may help detect pulmonary pneumonia lesions in the lungs in combination with clinical symptoms to demonstrate the covid-19 image [35]. the detection of viral nucleic acid (vna) is important for the diagnosis of exposed but asymptomatic carriers and it is possible to detect the viral rna using pharyngeal swab to prevent transmission and spread risk [40]. most popularly, rt-pcr (rrt-qpcr) is performed in real time over respiratory secretions so that viral rna can be identified within a short time [41]. researchers also developed a diagnostic technique for the rapid and colorimetric detection of covid-19 coronavirus by reverse transcriptional loopmediated isothermal amplification (rt-lamp). this isothermic lamp-based covid-19 detection system is called ilaco in this technique, six primers were used to amplify a fragment of the orf1ab gene, and phenol red is used as a ph indicator when the amplification changes color from pink to light yellow. at the same time, it remains rosy in negative cases [42]. moreover, several reference laboratories are advancing the sequencing of the full genome from the positive rrt-pcr isolates the full clinical outcome is not yet clear, as the symptoms reported range from mild to severe with even death in some cases [43]. serological test is generally used to measure immunity as well as acute infection to mumps virus, measles virus, rubella virus of these viruses. immunized patients will have persistent alam; aji, 3(1): 191-198, 2020; article no.aji.56779 194 fig. 1. corona virus (covid -19) update situation (according to 8 may 2020) globally, 8 may 2020, there have been 3,815,561 confirmed cases of covid-19, including 267,469 deaths, in 187 countries, reported to center for systems and engineering (csse) immunoglobulin g (igg) antibodies so detectable antibody levels antibody the patient is immune to infection. serological method is used for the diagnosis of primary ebv infection and for screening and monitoring of nasopharyngeal carcinoma whereas molecular methods is used for diagnose ebv associated lymphoma of the brain and ebv involvement in other organ [44]. 5. treatment of covid-19 when infected persons are hospitalized, symptomatic and supportive care should be provided to human patients immediately according to the severity of the symptoms [45,46]. therapy may include supplemental oxygen or high-flow nasal cannula (hfnc) oxygen therapy through the nasal route to relieve breathing stress if methylprednisolone is required intravenously to correct hypoxemia and, in extreme cases, intravenous adrenaline (iv), any repurposed medication such as lopinavir plus ritonavir as an anti-viral drug by oral way, moxifloxacin or any antibiotic to prevent secondary bacterial infection by im/iv route [35,44]. nevertheless, there are many drugs under review, including other antiretrovirals, such as remdesevir, but also antivirals such as oseltamivir and other treatments, including chloroquine and also indomethacin. researchers are making great efforts to design and grow effective covid-19 vaccines, which could take some time [47-49]. table 1. potential treatment options of covid-19 classes potential treatment option anti-viral > 85% of patients received antiviral agents, including oseltamivir (75 mg every 12 h orally), ganciclovir (0.25 g every 12 h intravenously) and lopinavir / ritonavir tablets (400/100 mg twice daily). remdesivir is currently being evaluated and known to prevent mars-cov in more than 10 medical establishments in wuhan [50]. anti-malarial an ancient antimalarial, chloroquine phosphate, has been effective in inhibiting the exacerbation of pneumonia due to its antiviral and inflammatory activity.[51] herbaltreatment traditional chinese medicine has been widely used during the last sars cov outbreak and is currently being used in china. the five most commonly used herbs were astragali radix(huanggi), glycyrrhyzae radix et rhozoma (gancao), sapposhnikovae radix (fangfeng), atractylodis macrocephalae rhizoma (baizhu) and lonicerae japonicaefflo [52]. alam; aji, 3(1): 191-198, 2020; article no.aji.56779 195 6. prevention of covid-19 preventing strategies focuses on patient isolation and diligent monitoring of pathogens, including appropriate screening and clinical treatment measures for infected patients to follow. for example, droplets, contact and airborne precautions should be taken during specimen processing, and incorporation of sputum should be avoided. the general guidelines of who and other organizations have issued the following general recommendations: 1. avoid close contact with people suffering from acute respiratory infections. 2. wash your hands frequently, particularly after contact with infected people. 3. avoid unprotected farm and wild animal contact. 4. people with acute airway symptoms should stay a little away, cover coughs or sneezes, and wash their hands with disposable tissues or shoes. 5. strengthen the implementation of appropriate hygiene procedures for the prevention and control of diseases, in particular in emergency medicine departments. 6. immuno suppressed people can prohibit public meetings the most effective method for the population is to frequently wash their hands and use portable hand sanitizing to prevent contact with their face and mouth after working in a potentially polluted environment. health workers who care for infected people should use touch and airborne measures to prevent pathogen transmission, including ppe helmets, eye protection, gowns and gloves such as n95 or ffp3 helmets. scientific work is going on to produce a coronavirus vaccine [53]. the governments should inspire travelers to postpone their travel to avoid exposure to covid-19 affected countries and those who are returning back from affected countries must be isolated and quarantined for health check-ups and evaluation of their health status [54]. people should wash their hands with soap-water or prefer to use sanitizers, should remain inside home, should refrain from gathering places and avoid contacts with livedead animals like wild animals, children, and older adults must take precautions as they are more prone to the severe respiratory distress syndrome of covid-19 [55]. 7. conclusion worldwide scientists, researchers and numerous health agencies work days and nights with a great deal of commitment to avoid further sarscov-2 transmission and spread by maintaining strict caution, preventive methods, improved prevention and control measures, and by developing adequate vaccines and treatments to avoid any pandemic situation that could otherwise occur if this virus could not be stopped. while many factors, resulting from the study, still need to be established, as is the case with many elements in other age groups facing the covid19 pandemic, as is not evident in pediatrics year after year. eventually, one approach to health will play an important role in the future battle against covid-19 as well as against other diseases. it is not a silver bullet, but still, the world needs global solutions to stop a pandemic either or minimize it [30]. in the light of increasingly growing covid-19 outbreaks, proactive steps by long-term care facilities are needed to identify and remove potentially infected staff and visitors, actively monitor potentially infected patients and implement successful prevention and control measures to avoid covid-19 infections. scientific researchers perform multiple medicine clinical trials and i believe these repositioning studies can help establish options for treatment with covid-19. consent it is not applicable. ethical approval it is not applicable. competing interests author has declared that no competing interests exist. references 1. deng sq, peng hj. characteristics of and public health responses to the coronavirus disease 2019 outbreak in china. j clin med. 2020;9(2). doi:https://doi.org/10.3390/jcm9020575 2. dhama k, sharun k, tiwari r, sircar s, bhat s, malik ys et al. coronavirus disease 2019 – covid19. preprints; 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(2) dr. jaffu othniel chilongola, tumaini university, tanzania. reviewers: (1) michele kimie sankako, state university of campinas, brazil. (2) anslem ajugwo, madonna university, nigeria. (3) mihaila romeo-gabriel, lucian blaga university of sibiu, romania. complete peer review history: http://www.sdiarticle4.com/review-history/55084 received 01 january 2020 accepted 06 march 2020 published 18 march 2020 abstract blood transfusion is an important component of patients management, as it saves millions of life each year, but it also serves as a vehicle for transmission of blood-pathogens such as hepatitis c virus and syphilis as a result world health organization has recommended that all blood samples should be screened for transfusion transmissible infections prior to transfusion. therefore this was conducted to determine the seroprevalence and socio-demographic distribution of hepatitis c virus and syphilis among blood donors in aminu kano teaching hospital, kano. this is a cross sectional study of blood donors presented at the donor clinic of aminu kano teaching hospital, kano original research article saleh et al.; aji, 3(1): 30-38, 2020; article no.aji.55084 31 between july and september 2018. a total of 90 blood samples were collected from voluntary and family replacement donors, aged between 18-57 years, the collected samples were screened for anti-treponema pallidum antibodies using a rapid diagnostic kits and were also screened for the presence of anti-hcv and anti-treponema pallidum antibodies using an enzyme-linked immunosorbent assay (elisa) kit. all of the total 90 (100%) donors screened were males. family replacement and voluntary donors were of 76 (84.4%) and 14(15.6%) donors respectively. the overall seroprevalence of hcv and syphilis was 7.8% (5.6% and 2.2% for hcv and syphilis respectively using elisa test). prevalence of 1.3% was found for syphilis using a rapid diagnostic kit. therefore critical screening of blood donors is of prime importance because they serve as an asymptomatic reservoir and a potential source of transmission of these infections. keywords: hepatitis c virus; syphilis; blood transfusion; elisa; blood donors. 1. introduction blood is one of the integral components of body constituents [1]. blood is composed of pale yellow fluid called plasma in which are suspended red cells (erythrocytes), white cells (leukocytes) and platelets (thrombocytes). plasma forms about 55% of the blood volume and contains about 95% of water, many solutes are dissolved in it including proteins, antibodies, salts, organic molecules, hormones, enzymes, products of digestion and wastes products of excretion [2]. blood flows through every organ of the body providing effective communication between tissues [1] there are almost 5-6 litres of blood in the circulatory system of an average adult and about 300 ml in the circulatory system of a new born infant [2]. blood has important transport, distributions regulatory and protective functions in the body such as the transport of oxygen from the lung to the tissues, nutrients absorbed from the digestive tract. example: monosaccharide and amino acids are transported to the cells of the body for use or storage; waste products of metabolism are transported from the tissues to sites of excretion; hormones are also carried from endocrine glands to the organs where they are needed; blood also assists in regulating the temperature of the body by absorbing and distributing heat throughout the body and skin surface where heat which is not required is dissipated; leukocytes are involved in body’s immune defenses, by producing antibodies in response to infection and protecting the body from damage by bacteria, viruses, parasites, toxins and tumour cells [2]. there are many medical conditions or problems that might require transfusion of blood or blood products some of these conditions include, severe anaemia, acute blood loss due to pregnancy, childbirth and accidents, types of cancer or cancer treatment that can affect blood cells and surgery, thus transfusion of blood and blood product has become a routine activity in clinical practices [3]. transfusion of blood and blood products saves millions of lives each year it can help patients suffering from threatening conditions and supports complex medical and surgical procedures it also plays an important role in maternal and child care and during man – made and natural disasters [4]. however blood transfusion serves as a vehicle for transmission of blood-borne pathogens including hepatitis c virus (hcv) and syphilis. transfusion transmissible infections are among the major public health problem worldwide affecting the quality of life and causing serious morbidity and mortality [5]. hepatitis c is a viral infection of the liver caused by hepatitis c virus, a member of family flaviviridae which has an enveloped linear single-stranded positive sense rna genome. hepatitis c virus is spread primarily by direct contact with contaminated blood or blood, use of infected sharp objects, transfusion of blood that are not screened for hcv infection intravenous drug –use, sexual exposure as well as vertical transmission that is from mother to her foetus during prenatal period. hepatitis c virus is one of a major public health problem worldwide, and is a frequent cause of acute hepatitis and chronic liver disease including cirrhosis and hepatocellular carcinoma [6]. globally an estimated 200 million people representing 3% of the population are chronically infected with the virus and 3 to 4 million are newly infected each year [7]. syphilis is a systemic bacterial infection caused by spirochete of genus treponema pallidum, which can be transmitted primarily through sexual contact via transfusion of blood and blood products and vertically from mother to her foetus saleh et al.; aji, 3(1): 30-38, 2020; article no.aji.55084 32 [8]. the causative agent of syphilis, treponema pallidum, is killed by storing blood for 48-72 hours at 4ºc. thus, storing of blood for at least three days in a refrigerator before transfusion can prevent the transmission of syphilis [9]. world health organization (who) estimated that approximately 12 million new cases of syphilis are reported each year in the world with more than 90% from developing countries. screening for transfusion transmissible infections is a critical part of the blood transfusion process as it ensures that transfusion is as safe as possible [10]. however, the world health organization recommended that all blood donations should be screened for evidence of infections prior to the release of blood for clinical or manufacturing use [11]. proper monitoring and selection of voluntary blood donors and routine screening of blood for syphilis, hcv and other transfusion transmissible infections has greatly reduced the risk of transmitting those infectious agents [12]. the improved screening and testing of blood donors has also significantly reduced transfusion-transmitted diseases in most developed countries. this has not been so in developing nations. poor health education and lack of awareness result in the reservoir of infections in the population. therefore estimating the prevalence of hepatitis c virus and syphilis among blood donors permits the assessment of occurrence in the blood donor population and consequently the safety of blood donations. it also provides data that is useful in formulating the strategies for improving the management of safe blood supply. in addition, it also gives an idea of the epidemiology of these diseases in a given time at a specific population or community [13]. this study aimed to determine the seroprevalence and sociodemographic distribution of hepatitis c virus and syphilis coinfection among blood donors at the study site. 2. materials and methods 2.1 study population this study was carried out at the department of haematology, aminu kano teaching hospital, kano state. a total of ninety (90) blood samples were collected from consented healthy family replacement and voluntary donors at the blood donor clinic of aminu kano teaching hospital, kano from july to september 2018. ethical clearance was obtained from the ethical committee of ministry of health kano. volunteered male blood donors of age >18 years who consented to participate in the study were recruited. while, those who refused to give their consents were excluded from the study. 2.2 study design an observational cross-sectional study of blood donors over the period of two months from july to september 2018 was carried out using a structured questionnaire; volunteers of age range between 18 to 57 years were screened for the determination of antibodies (igg and igm) to hcv and syphilis. 2.3 sample collection and processing five millilitres (5 ml) of blood sample was collected from each donor by standard venipuncture technique using a sterile syringe and a tube. the samples were distributed in such a way that one millilitre (1 ml) from each sample was transferred into plain tube for screening of syphilis using a rapid diagnostic kit (micropoint diagnostic®, nantong, china). a volume of 2 ml out of the total of 5 ml collected from each patient was aspirated in a test tube and centrifuged at 1000 rpm for 10 minutes to obtain the serum, which was used to carry out the hcsag screening [2]. a drop of the centrifuged blood serum was dispensed on the test side of the rapid test hcsag immunochromatographic testing strip (lab tech® hcsag d01, australia rapid test kit), which was placed on a flat, dry and nonabsorbent surface on the disinfected working bench and allowed to react for two minutes. after which the results were read according to manufacturers’ instructions. two (2) red lines which often appear on the control line and on the determinant (test) line of the test strip indicated a positive result, while a single line on the control line indicated a negative result. while, in the case where there is no red lines that appear on both the control and the determinant lines, the result was termed as invalid [14-16]. the remaining 3 ml of each sample was transferred into an appropriate edta container after which the plasma was extracted by centrifuging the blood at 3000 r/min for 5 minutes. the plasma was stored at -20ºc until ready for assay. screening of the plasma for hepatitis c virus and syphilis antibodies was conducted using an enzyme-linked immunosorbent assay (elisa) kit (dia. pro diagnostic®, bioprobes, italy) according to manufacturer’s standard operational procedure [17]. saleh et al.; aji, 3(1): 30-38, 2020; article no.aji.55084 33 3. results and discussion a total of 90 healthy blood donors were screened for syphilis infection using a rapid diagnostic test out of which 14(15.6%) were voluntary donors and 78(84.4) were family replacement and only 1 donor was positive to the test, therefore the prevalence is 1.3% as shown in table 1. the prevalence of hcv and syphilis pathogens tested by elisa were 5.6% and 2.2% respectively whereby the overall prevalence was 7.8% (table 2). table 3 showed the distribution of hcv and syphilis pathogens among healthy blood donors according to socio-demographic factors. all the 90 donors screened were males aged between 18-57years, among the age groups the donors within 48-57 years have the highest prevalence of 28.6% and 14.3% for hcv and syphilis respectively. out of 90 donors, 50(55.4%) were married and 40(44.4%) were single. high prevalence of hcv 6.0% was found among married donors, while single donors had higher prevalence of syphilis. majority of the donors were self-employed 57(63.3), followed by civil servants 20(22.2%), and the prevalence rate is high among the civil servants, family replacement donors constitutes the largest group of the donors screened 78(84.4%), hcv and syphilis infections were found only among the family replacement donors. table 4 shows the possible risk factors associated with hcv and syphilis seropositivity in blood donors, among which blood transfusion (33.3%), injection or skin cutting by a traditional healers (6.6%), family history of liver disease (25.0%) and local circumcision (5.5%) play a role in hcv seropositivity, while blood transfusion (6.7%), history of std (50.0%) and local circumcision (2.7%) play a role in syphilis seropositivity. data in most of the reported studies revealed that the prevalence of syphilis and hcv has greatly reduced as a result of the introduction of more sensitive and specific diagnostic assays, and more improvement in public health, especially in the developed countries. in this study, the prevalence of syphilis was 1.3% using rapid diagnostic test and 2.2% using elisa, which is in line with study of abdallah et al. [18]; olokoba et al. [19]; and butsashvili et al. [20], who reported 1.2%, 1.2% and 2.3%. also, a report of tessema et al. [21] had a rate of 1.3% which is similar to this study. in contrast to this study, higher prevalence of syphilis was reported from different african countries, prevalence of 3.7% in congo [22], 7.5% in ghana [23], 15% in sudan, 12.8% in ethiopia [4], 12.7% in tanzania [24], and 9.1% in cameroon [25,26]. these wide differences in the prevalence of syphilis infection among blood donors may be due to difference in sample size, high exposure to risk factors such as sexual behaviours and laboratory techniques used. lower rate of 0.5% and 0.7% syphilis prevalence were reported in some part of nigeria by fasola et al. [27] and benson et al. [28]. in other parts of the world like india 0.3% was found among healthy blood donors [29] and 0.75% in pakistan [30], 0.02% in turkey [31] in saudi arabia 0.028% [32] and 0.2% in south korea [33]. the low prevalence may be attributed to the development of effective treatment programmes, clinical selection of blood donor, and by selecting the low-risk donors. on the other hand, the seroprevalence of syphilis hcv in this study was 5.6% which is almost similar to prevalence reported by alao et al. [34] and buseri et al. [35], who reported 5.4% and 5.1% respectively. the prevalence rate of 5.6% hcv in this study is higher than 2.0%, 0.5%, 3.4% and 3.9% recorded by odenigbo et al. [36], erhabor et al. [37], bala et al. [38] and amiwero et al. [39] respectively. the prevalence of hcv antibodies in blood donors in developed countries ranges from 0.4% to 2%; seroprevalence studies in europe and united states documented a significant reduction in the risk of hcv [40]. lower prevalence of hcv was also found among blood donors in jordan [41] 0.2% in saudi arabia [42] 0.95% in syria [43] and 0.07% in turkey [44]. low prevalence of hcv among blood donors in some regions may be attributed to low exposure to some risk factors. in contrast to this study, higher prevalence 8.4% of hcv was recorded by ayolabi et al. [3]. also, egypt is reported to have a high prevalence 24.8% among healthy blood donors [13]. the highest prevalence of 14.3% was found among donors aged between 48-57years. this is almost similar to the work of alao et al. [34], who documented the highest age prevalence of hcv among blood donors in makurdi, nigeria, and dapus et al. [45], who reported the highest prevalence among the age group of 46-55 years. this may be attributed to the lower immunity in saleh et al.; aji, 3(1): 30-38, 2020; article no.aji.55084 34 older people. however, this fail to agree with 3.2% highest prevalence among donors aged between 21-30 years in eastern nigeria [36]. the result of this study showed that family replacement donors constituted the largest group of the donors, in which 85.4% were family replacement donors, while the voluntary donors constituted of 15. 6%. the seroprevalence rate of 5.6% and 2.2% for hcv and syphilis respectively were found among the family replacement donors. table 1. seroprevalence of syphilis among blood donors at aminu kano teaching hospital, kano using rapid diagnostic kit donor type number tested (%) syphilis number tested positive (%) voluntary donor 14(15.6) 0(0.0) family replacement donor 76(84.4) 1(1.3) total 90(100.0) 1(1.3) table 2. overall seroprevalence of hcv and syphilis among blood donors at aminu kano teaching hospital, kano serological marker number tested positive by elisa percentage (%) hcv 5 5.6 syphilis 2 2.2 total 7 7.8 table 3. seroprevalence of hcv and syphilis with regards to demographic factors of the blood donors demographic factors number donors screened (%) hcv no tested positive (%) syphilis no tested positive (%) age 18-27 46(51.1) 2(4.4) 1(2.2) 28-37 21(23.3) 1(4.8) 0(0.0) 38-47 16(17.8) 0(0.0) 0(0.0) 48-57 7(7.8) 2(28.6) 1(14.3) sex male 89(98.9) 5(5.6) 2(2.2) female 0(0.0) 0(0.0) 0(0.0) marital status married 50(55.6) 3(6.0) 1(2.0) single 40(44.4) 2(3.0) 1(2.5) occupation civil servant 20(22.2) 2(10.0) 1(5.0) student 13(14.4) 0(0.0) 0(0.0) self-employed 57(63.3) 3(5.3) 1(1.8) donor type voluntary donor 14(15.6) 0(0.0) 0(0.0) family replacement donor 76(84.4) 5(6.6) 2(2.6) saleh et al.; aji, 3(1): 30-38, 2020; article no.aji.55084 35 table 4. seroprevalence of hcv and syphilis with regards to possible risk factors risk factors number of donors screened (%) hcv number tested positive (%) syphilis number tested positive (%) received blood transfusion yes 6(6.7) 2(33.3) 1(16.7) no 84(93.3) 3(4.1) 1((1.2) received surgical procedure in health facility yes 9(10.0) 1(11.1) 0(0.0) no 81(70.0) 4(4.9) 2(2,5) ever given an injection or skin cutting by a traditional healer yes 30(33.3) 2(6.6) 1(0.0) no 60(66.7) 3(5.0) 2(3.3) history of jaundice yes 3(3.3) 1(33.3) 0(0.0) no 87(96.6) 4(4.5 2(2.3) family history of liver disease yes 8(8.9) 2(25.0) 0(0.0) no 82(91.1) 3(3.7) 2(2.4) history of std yes 2(2.2) 0(0.0) 1(50.0) no 88(97.8) 5(5.7) 1(1.1) 4. conclusion the present study clearly documented the seroprevalence of hepatitis c virus and syphilis pathogens among blood donors as 5.6% and 2.2% respectively. it can also be concluded that voluntary blood donors have been found to be safer than family replacement donors despite the low percentage of voluntary blood donors and there is no participation of female donors during this research; 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aji, 3(1): 30-38, 2020; article no.aji.55084 38 45. dapus od, piwuna to, joseph de, ogbenna aa, kut sd, chetle ld. hepatitis c virus antibody among blood donors: the experience in a nigerian blood transfusion service centre. global advanced research journal of medicine and medical sciences. 2013;2(5):108 113. ________________________________________________________________________________ © 2020 saleh et al.; this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: http://www.sdiarticle4.com/review-history/55084 _____________________________________________________________________________________________________ *corresponding author: e-mail: naser_elsawy@ymail.com; asian journal of immunology 2(1): 34-44, 2019; article no.aji.53258 therapeutic potential of the dual peroxisome proliferator activated receptor (ppar)/ agonist aleglitazar in attenuating tnf-mediated inflammation and insulin resistance in human adipocytes: a review naser a. elsawy1* 1 department of anatomy and embryology, faculty of medicine, zagazig university, egypt. author’s contribution the sole author designed, analysed, interpreted and prepared the manuscript. article information editor(s): (1) dr. cynthia aracely alvizo báez, professor, laboratory immunology and virology, faculty of biological sciences, autonomous university of nuevo leon, mexico. reviewers: (1) umezurike benedict chidozie, nigeria. (2) gayatri c. gawade, bharati vidyapeeth dental college and hospital, india. complete peer review history: http://www.sdiarticle4.com/review-history/53258 received 01 october 2019 accepted 05 december 2019 published 10 december 2019 abstract aleglitazar, is a novel promising drug, that related to peroxisome proliferator-activated receptor (pparα&γ) agonists which shows an ant-diabetic and anti-inflammatory characteristics. the goal of this study is to evaluate anti-inflammatory and anti-arthritic properties of aleglitazar in arthritis models induced in experimental animals and to conclude the comparative ulcerogenic potential of diclofenac in type 2 diabetes animals. the anti-inflammatory characteristics of aleglitazar will be investigated in carrageenan-induced hind paw edema as acute inflammation. the aim of the review: this study aims to assess anti-inflammatory and anti-arthritic activities of aleglitazar in arthritis models induced in experimental animals. it is to determine comparative ulcerogenic potential diclofenac in type 2 diabetic animals on gastric mucosa in rats. keywords: ppar; aleglitazar; tnf; inflammation insulin resistance; adipocytes. review article elsawy; aji, 2(1): 34-44, 2019; article no.aji.53258 35 abbreviations ang : angiotensin bmi : body mass index cox1 : cyclooxygenase-1 enzyme cox2 : cyclooxygenase-2 enzyme crp : c-reactive protein ffa : free fatty acids gcf : gingival crevicular fluid gdm : gestational diabetes mellitus gfr : glomerular filtration rate 1gir : glucose-insulin rate hba1c : glycated haemoglobin hdl : high-density lipoprotein cholesterol il-1β : interleukin 1β ldl : low-density lipoprotein cholesterol no : nitric oxide no/onoo : nitric oxide/peroxynitrite ratio nsaids : nonsteroidal antiinflammatory class of drugs pge2 : prostaglandin e2 pgs : prostaglandins pai-1 : plasminogen activator inhibitor-1 ppar α & γ : peroxisome proliferator activated receptor α & γ sc : serum creatinine stz : streptozotocin tg : triglycerides tnfα : tumor necrosis factor alpha t2dm : type 2 diabetes mellitus 1. introduction obesity is frequently associated with insulin resistance, and highly responsible for developing type 2 diabetes (t2d) [1,2], t2d is a metabolic disease that causes sugar to increase in blood circulation results from insulin resistance and the primary risk factor of t2d is inflammation [3]. diclofenac is a proven, commonly used nonsteroidal anti-inflammatory and anti-pyritic drug. diclofenac exerts its activity by inhibiting cyclooxygenase-1 (cox1) and cyclooxygenase2 (cox2) leading to inhibit prostaglandin synthases [4]. aleglitazar is a new drug that used as an antiinflammatory drug that improved insulin sensitivity, and dyslipidemia. aleglitazar is a single novel molecule that contains the both active synthetic peroxisome proliferator activated receptors (ppars α&γ) has potent and dual agonist action on ppars α&γ [5]. 2. obesity is a pathological condition result from an accumulation of excesses adipose tissue in a different site of the body including liver and skeletal muscle, and it's considered as a neurochemical imbalance can be measured by the body mass index (bmi), which is a ratio of weight to high. normal range of bmi (18.5-24.9 kg/m 2 ), overweight range (25-29.9 kg/m 2 ), obesity bmi 30 kg/m 2 and more [6] (fig. 1). weight gain and increased adipocyte, result in hypertrophic and loss of function of adipocyte [8]. free fatty acid (ffa) in this condition released in blood circulation, pro-inflammatory cytokines as tumor necrosis factor alpha (tnf-α), plasminogen activator inhibitor (pai)-1, and angiotensin (ang)), and adiponectin which is a specific protein in adipocyte that play a role in insulin resistance and atherosclerosis [9]. increased level of ffa and pro-inflammatory cytokines will lead to deposition in non-adipose tissues and decrease systemic inflammatory grade, resulting in activating the mechanism of insulin resistance in tissues (fig. 2) [8]. fig. 1. bmi: it is a method use for determination if a person weight within the normal range or at high risk for obesity [7] elsawy; aji, 2(1): 34-44, 2019; article no.aji.53258 36 fig. 2. adipose tissue expansion, insulin resistance, and the metabolic syndrome. macrophages are conducted, infiltrate and contributing further to pro-inflammatory m1 phenotype in the hypertrophic adipose tissue [8] 3. diabetes mellitus is a silent metabolic syndrome characteristic by hyperglycemia resulting from lowering or impaired insulin secretion or increase insulin resistance by the body tissue [6]. 3.1 types of diabetes insulin treatment is required at any type. most important types are: 1gestational diabetes mellitus (gdm) is known as any degree of glucose intolerance with onset or first recognition during pregnancy. diet modification is applied or insulin for treatment, this condition may not persistence after pregnancy [10]. 2type 1 diabetes (autoimmune diabetes mellitus): is an absolute insulin deficiency resulting from absolute destruction of β cells [11]. 3type 2 diabetes mellitus: is a relative insulin reduction that resists the action of insulin [11]. people suffers from obesity are more threatened to develop t2d, and metabolic syndrome [6]. 3.2 complications patients with t2d may present with characteristic symptoms such as thirst, polyuria, a defect of vision, and weight loss. in sever form, ketoacidosis or a non-ketotic hyperosmolar state may lead to loss of conscious. t2d can cause pathological and functional changes in the organ may be present for a long time before the diagnosis is made. the long-term effects of t2d lead to a gradual increase of the specific complications of retinopathy with a loss of vision, and nephropathy that may lead to renal failure [12]. 3.3 induction of t2d mellitus in experimental animals t2d induced by streptozotocin (stz), which is a molecule that produces a selective toxic effect in experimental animal [13]. doses should be fig. 3. induction of stz. intraperitoneally in rat model to induce t2dm [17] elsawy; aji, 2(1): 34-44, 2019; article no.aji.53258 37 calculated to perform a partial destruction of β cell mass to produce a non-ketotic and a mild insulin deficient state of t2d [14]. animals will be injected with a single dose of streptozotocin 65 mg/kg intraperitoneally (fig. 3) [15]. here are other ways to induce diabetes in a model rat by consuming of unbalanced high-fat diets that result in accumulation of fat in adipose tissue and increase body weight. the accumulation of fat will affect the releasing of deferent protein from the adipose tissue called adipokines, that plays an important role in insulin sensitivity and inflammation, which can be developed into some obesity-related disorder like type 2 diabetes [16]. 4. inflammation is a host response reflection to tissue injury, and indicates the occurrence by four signs (cardinal signs): redness, swelling, heat, and pain [18]. 4.1 carrageenan-induced hind paw edema in the mouse carrageenan induced inflammation by subcutaneous injunction in the right hind paw 100 μl (suspended in 1.5% vichele) of carrageenan that results in acute, non-immune, well reached, and highly reproducible inflammation, the action of pro-inflammatory agents, histamine, complement, and reactive oxygen will produce cardinal signs of inflammation develop subsequently to the injection. the traditional method to measure inflammatory response is quantified by increasing the size of paw oedema [18], which represent the magnitude of inflammation response effectively [19]. hind paw size can be measured either by plethysmometer's caliper which is a rough method (fig. 4) or by new the new technology to asses induced acute paw inflammation in the rats by epifluorescence imaging, a fluorescence intensity in the saline and carrageenan-treated hindpaw (fig. 5). carrageenan-induced hind paw edema as acute inflammation model in mice [18]. in addition, cotton pellet-induced granuloma as chronic inflammation model in rats [20]. 100 µl of l carrageenan suspended in normal saline 1.5% concentration will be subcutaneously injected into a hind paw of the animals on one side. 100 µl of saline will be injected into the contralateral hind paw and used as a control. using plethysmometer, the hind paw volumes will be measured before and after six hours of carrageenan injection. oedema is expressed as the variation between the change in carrageenan-treated hind paw volume and the control saline-treated (fig. 6) [18]. the results will be expressed as percentage of puffiness calculated as following the formula: alteration = (width of the hind paw at the end day of experiment− width of the hind paw prior carrageenan/width of the hind paw prior carrageenan × 100% ) [21]. fig. 4. hind paw measurement by caliper. the paw measure by placing the paw in the caliper paw touch either side of the caliper, measurements read in mm [19] elsawy; aji, 2(1): 34-44, 2019; article no.aji.53258 38 fig. 5. carrageenan-induced acute paw inflammation in the rat (fluorescence intensity in the saline and carrageenan) measured after injection by epifluorescence imaging 60 and 120 min [22] fig. 6. carrageenan control and injected hind paw (a) control hind paw, (b) injected hind paw by carrageenan 100 μl suspended in 1.5% vichele, show an edema and redness [23] 5. anti-inflammatory drugs 5.1 diclofenac diclofenac sodium is a nonsteroidal antiinflammatory class of drugs (nsaids), that used as anti-inflammatory and painkiller drug. diclofenac interposed by inhibiting the release ofcyclooxygenase-1 (cox1) and cyclooxygenase-2 (cox2), that convert arachidonic acid into prostaglandins (pgs). diclofenac should be used in minimal dose due to its toxicity. diclofenac at 5 mg/kg dose started to show an inhibition in hind paw edema after 2 hours [24]. diclofenac can increase the risk of heart attack, strokes, blood clots and gastroduodenal ulceration [25]. gastroduodenal ulceration and bleeding are the major limitations to the use of nsaids. it's lead to damage to the gastroduodenal mucosa via several mechanisms, including the topical irritant effect of these drugs on the epithelium, weakness of the barrier properties of the mucosa, inhibition synthesis of the gastric prostaglandin, decrees blood flow through the gastric mucosa and interference with the repair of superficial injury. the presence of acid in the lumen of the stomach also help in increase rate of the pathogenesis of nsaids induced ulcers and bleeding, through impairing the restitution process, interfering with haemostasis and inactivating several growth factors that are important in mucosal defence and repair [26] (fig. 7). 5.2 aleglitazar aleglitazar is a combination of pparα and γ agonist and designed to stimulate both α and γ receptors at minimum concentrations. ppars elsawy; aji, 2(1): 34-44, 2019; article no.aji.53258 39 fig. 7. morphological and histological examination of rats gastric mucosa in diclofenacinduced ulcer model (section was stained with haematoxylin and eosin and magnified by 20×). a. stomach of control rat; b. stomach of diclofenac-induced ulcer rat at a dose of (100 mg/kg) [27] are one of nuclear receptor superfamily, which participated in the transcriptional control of genes contributing in lipid and carbohydrate metabolism, and inflammation, especially in the setting of obesity, hypercholesterolemia, insulin resistance, and atherosclerosis. ppars are divided into 3 subtypes: pparα, pparγ, and ppar δ. ppar α found at high concentration in liver, kidney and the skeletal muscle and ppar γ found at high concentration in adipocytes, muscle cells, the liver and the kidney [5]. the synthetic agonists of ppar α e.g. fibrate used for lowering triglycerides (tg) and raising highdensity lipoprotein cholesterol (hdl) in plasma, while the synthetic ppar γ agonists e.g. thiazolidinediones pioglitazone used as antidiabetics [1]. 5.3 mechanism of action aleglitazar prepared to be an agonist for both ppar α&γ.pparα the agonistic action is controlled lipid levels, thus improving dyslipidemia. in addition, pparγ controls glucose level and resulting in reduce insulin resistance in diabetic patients [28]. 5.4 optimal dose aleglitazar (150 ug/kg/day) for 16 weeks was selected to explore any potential association of this dual ppar α/γ agonist with anti-arthritic and anti-inflammatory properties [29]. supratherapeutic dose (600 μg) of aleglitazar for 26 weeks in patients with normal to borderline impaired renal function with glomerular filtration rate (egfr 60 ml/min per 1.73 m2), raised serum creatinine by a mean 22% to a plateau after 4 weeks. then, serum creatinine resumed to baseline after 4 to 8 weeks of drug cessation [29]. 5.5 side effect in increase the incidence of the expected dose, will lead to increase in serum creatinine, decrease glomerular filtration rate, progressive nephropathy and bilateral oedema [29]. 5.6 ulcergenic effect aaleglitazar with a dose of (150 µg/kg) shows pharmacological effects on the 8 th day after pellets. the day before scarification, all rats prevented from food and liquid for 12 hours. gastric tissue will be removed and taken out, after cut open to the stomach the inner surface will be cleansed with saline 0.9% to clean any remains inside the stomach and hold it by bins to exposed its mucosa and stomach ulcer will examined under magnifying land lens to calculate ulcer index by the number and depth of the ulcers (gastric ulcer scoring), [30]. part of the stomach (fundus) fixed in formalin and exposed to histopathological study to compare it with diclofenac [27]. 5.7 drug-drug interaction aleglitazar used in combination with warfarin which used as an anticoagulant drug, it affects elsawy; aji, 2(1): 34-44, 2019; article no.aji.53258 40 the efficacy and safety by affecting the viscosity of the blood through angiotensin-2 receptor blocker so the blood vessels dilated that lead to decrease the velocity of the blood stream and increase the susceptibility of thrombosis [31]. in patients with normal to borderline impaired renal function and impaired glomerular filtration rate using aleglitazar in supratherapeutic dose will increase serum creatinine and decrease glomerular filtration rate that leads to progressive nephropathy and bilateral oedema so it interacts with diuretics [31]. 6 biochemical tests 6.1 fasting blood glucose a daily dose of aleglitazar150 μg observed to modulate the fasting plasma glucose (fbg) [5]. 6.2 insulin aleglitazar treatment helped to increase body's sensitivity to insulin significantly compared to placebo. 6.3 hba1c clinical trials of aleglitazar in patients with type 2 diabetes were conducted for 26 weeks (150 µg/day). the primary endpoint was significant reduction in glycated hemoglobin (hba1c) concentration from baseline to week 26 [32] (fig. 8). 6.4 serum creatinine serum creatinine (sc) is a routine test for kidney function assessment [33]. serum creatinine showed to increase and show to be reversible after cessation of aleglitazar within a follow-up period from 4 to 8 weeks [29]. 6.5 immuno-inflammatory reactions in obesity-related disorders c-reactive protein (crp) is a crucial inflammatory biomarker [34]. high sensitivity to aleglitazar shows a significant reduction in crp [35]. 6.6 free fatty acids (ffa) and triglycerides and – hdl pparα and pparδ enhance the movement of lipid out of circulation by increase uptake of free fatty acid and oxidation in tissue, while ppar γwill stimulate the process of lipid storage in adipose tissue. [36]. it has the most effective improvement in highdensity lipoprotein cholesterol, low-density lipoprotein cholesterol (ldl), and triglycerides. the result of lowering lipid profile reduce the risk of cardiovascular disease and particularly atherosclerosis [5]. 6.7 detection and estimation of serum level of tumor necrosis factor alpha (tnf-α) in the patient with diabetes the inflammatory mediators levels of gingival crevicular fluid (gcf), interleukin 1 beta (il-1beta) and monocytic secretion of prostaglandin e2 (pge2) are increased more than the non-diabetic patient [37]. tumor necrosis factor-alpha (tnf-α) synthesis in adipose tissue, the degree of adipose tissue related to the level of synthetase fig. 8. gradually reduction inhba1c. significant reduction in hba1c by aleglitazar was apparent by week [32] elsawy; aji, 2(1): 34-44, 2019; article no.aji.53258 41 fig. 9. tnf-α concentration in t2dm. (4.4+/-0.2 pg/ml) [41] of tnf-α, this is the bond between obesity and t2dm [38]. serum level of tnfα in obese patients with t2dm increased than normal or non-obese patient with t2dm, serum level of tnfα show positive correlation to degree of adipose tissue and negative correlation with glucose-insulin rate (gir),increase tnfα highly associated with increased insulin resistance (fig. 9) [39]. 6.8 nitric oxide (no) concentrations in serum obese rats were assigned to treatment by aleglitazar, the treatment was administered for nine weeks. nitric oxide (no) was measured. aleglitazar increased no release by 25% as the nitric oxide/peroxynitrite ratio (no/onoo-) which is an indicator of no synthase uncoupling increased by 33% with aleglitazar treatment [40]. 7. conclusion aleglitazar is a new promising drug, used as an anti-inflammatory, anti-diabetic, and antihyperlipidemic drug. in comparison with diclofenac the standard anti-inflammatory drug. inthe field of pharmacokinetics the auc of aleglitazar. increased with dose with values of (8.66 402) ng.h/ml while in diclofenac the auc is between (72.6 177.5) ng.h/ml. aleglitazar has more potent and has less side effect than diclofenac. but in some cases aleglitazar must be monitored under the supervision of a physician because it affects the safety and efficacy of warfarin and diuretics. consent it is not applicable. ethical approval as per international standard or university standard ethical approval has been collected and preserved by the authors. competing interests author has declared that no competing interests exist. references 1. massaro m, scoditti e, pellegrino m, carluccio m, calabriso n, wabitsch m, storelli c, wright m, de caterina r. therapeutic potential of the dual peroxisome proliferator activated receptor (ppar)î±/î³ agonist aleglitazar in attenuating tnf-î±-mediated inflammation and insulin resistance in human adipocytes. pharmacological research, [online]. 2016;107:125-136. available:http://dx.doi.org/10.1016/j.phrs.2 016.02.027 [accessed 20 mar. 2017] 2. type 2 diabetes: overview. institute for quality and efficiency in health care (iqwig). 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[online]. 2003;27(1):88-94. available:http://dx.doi.org/10.1038/sj.ijo.08 02187 [accessed 10 mar. 2017] _________________________________________________________________________________ © 2019 elsawy; this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: http://www.sdiarticle4.com/review-history/53258 http://creativecommons.org/licenses/by/4.0 _____________________________________________________________________________________________________ *corresponding author: e-mail: samiraalaani@hotmail.com; asian journal of immunology 3(1): 1-10, 2020; article no.aji.53605 high rates of birth defects in fallujah, iraq: radiological and chemical pollution of the affected children and their parents samira telfah abdulghani alaani1*, mohammad tafash2, abdulkadir abdulkareem murie1 and allawi muhyee al-esawi1 1 fallujah general hospital, fallujah, 00964, anbar province, iraq. 2medical college, anbar university, 00964, ramadi, iraq. authors’ contributions this work was carried out in collaboration among all authors. authors staa and mt designed the study, performed the statistical analysis, wrote the protocol and wrote the first draft of the manuscript. author aam managed the analyses of the study. author amae managed the literature searches. all authors read and approved the final manuscript. article information editor(s): (1) dr. jaffu othniel chilongola, department of biochemistry and molecular biology, kilimanjaro christian medical university college, tumaini university, tanzania. reviewers: (1) trupti pandit, wayne memorial hospital, usa. (2) einar arnbjörnsson, skane university hospital, lund university, sweden. complete peer review history: http://www.sdiarticle4.com/review-history/53605 received 22 october 2019 accepted 27 december 2019 published 09 january 2020 abstract background: lack of birth registers hindered knowledge of the frequency of birth defects (bd) in fallujah, iraq and comparison of changes in prevalence in time. one study found correlation between bd presentation and teratogenic metal load [1]. methods: we obtained reproductive history, kin health, environmental exposure and historical residence of families. hair was sampled from the nape of the neck from families and analyzed by icp/ms for metal content. findings: bd in newborn in 2010 were about 14.7% [2]. data of reproductive history from 56 couples with child, overcoming the lack of previous data, shows pattern of increase in bd presentation since 1991. information on parent's immediate kin in more than 1000 people, supports that bd are sporadic events. high residential stability of families allows to rule out local clusters for bd. focus on load for ten teratogenic and carcinogenic metals (v, cr, co, as, mo, cd, w, u, hg, original research article alaani et al.; aji, 3(1): 1-10, 2020; article no.aji.53605 2 pb) in hair of 43 bd children and 103 their parents, and of 11 healthy newborns and 16 their parents, compared with that of 10 children and 8 adults controls outside the war area, shows that metal contamination is diffuse in the whole fallujah population and is present already in newborn children hair. absolute levels of major teratogenic and carcinogenic contaminants (v, co, mo, u and pb) were significantly higher in fallujah people than in controls from other areas, with pb levels in bd children higher than other newborns in fallujah. interpretation: unusual high prevalence progressive in time and increased load of teratogenic metals even in new born hair are reported in fallujah population. the extensive metal contamination persistent in people's hair in post-war environment and the composite metal load might be a major factor in the increase in time of stochastic events that lead to bd presentation. keywords: fallujah; congenital anomalies. 1. introduction understanding potential long term effects of recent wars on civilian's health requires monitoring rates of birth defects, cancers and chronic pathologies. often, lack of pre-existing records constitutes hindrance. in fallujah, alarm on severely declining reproductive and children health come from doctors, reports from human right groups and political figures [3]. moreover, modalities of the presentation of bd, relative frequencies, and causes of their recent increase have not been investigated, nor teratogens have been searched for, and mechanistic aspects remain largely unknown till 2011 when the 1 st study on limited number of babies with birth defects & their parents done & showed strong correlation between the development of birth defects & the presence of metal load in the investigated sample. a first task is to collect recent data on prevalence of bd and to attempt a reconstruction of past events; recording reproductive history at registration, with specific questionnaire, is a valuable mean to achieve that aim [4]. together with the information on health status (bd presentation) within members of the immediate family of the parents of bd children, this information allows to dismiss that monogenetic, high penetrance genetic factors pre-exist in the parents of bd children [5]. candidate gene effectors are unknown for most structural bds, and for all a relevant component, generically called, of environmental and/or maternal effect is acknowledged [6]. it is unknown the number of genes contributing to anencephaly in humans, and, in mice. anencephaly is attributed to multifactorial combinations of hypomorphs and lowpenetrance heterozygotes [7]. chd have various phenotypes, sometimes compounded with different cardiac-unrelated features and cohort studies detected with very low frequency single genetic mutations putatively involved, while approximately 30% of chd and tetralogy of fallot (tof) are associated with wide genomic rearrangements (chromosome translocation, copy number variation and chromosome trisomies) [8]. combinations of genetic and nongenetic factors is associated to inheritance of clp in mice [9]. also in mice, synpolydactyly is linked to hoxd13 mutations and its manifestation is dependent upon environmental factors [10]. the traditional paradigm of prevalence data surveillance has been that detection of a particularly high prevalence of bd in one particular population, or a sudden increase in prevalence over time, can help identify possible environmental causes. presence of teratogenic metals in the war/post-war environment is expected because of the weapon systems used and proofs of fact of their presence in weaponry used in the middle east wars were reported [11]. metals persist in environment, are up taken via respiratory, skin contact and ingestion of contaminated water or food, and accumulate and persist in the hair of exposed people's [12]. metal exposure, acute and/or chronic has the potentials to induce reproductive and neoplastic damages, via epigenetic changes [13-16] by regulating genome stability, x chromosome inactivation, gene imprinting, and reprogramming gene expression. metals act as metalloestrogens [17], inhibit dna repair and alter dna methylation, change transcriptome and micrornas production [13], histone acetylation [18] and methylation [19,20] oxidative dna damage [21] all of which can lead to birth defects, whether translated into mutations or not [21,22]. alaani et al.; aji, 3(1): 1-10, 2020; article no.aji.53605 3 some metals can induce sporadic gene mutations because of internal radiation and is unclear the relative relevance of radiation versus chemical activities of du as a mutagen, while it was shown to induces epigenetic changes via hypomethylation of dna [20,21,22]. it was not previously attempted to conduct investigation of the association between presentation of bd families and metal load in post-war contexts, while studies of industrial or mine derived metals pollution have implied this correlation. the residential history of the parents, their exposure to major war events, the source of drinking water, all contribute to define the environmental context. here we report an observational investigation at fallujah general hospital of 56 families with bd and 11 controls inclusive of all the information above, and results of icp/ms analysis to determine load of ten teratogenic metals in hair of newborns and their parents, with or without bd and of children and adult controls from italy and jerusalem. 2. methods collection of data (supplementary fig. 1) was done by medical personnel. information included reproductive history, information on health of parent's brothers and sisters, historical residence and environmental exposure. classification of birth defects was according to definition of the primary phenotype. patients and controls were taken in order of arrival, among those that accepted in writing to answer to questionnaire, and consenting to the scientific use of data. pedigrees of families are as standard. clinical diagnosis was assisted by the use of instrumental diagnosis (xrays, echocardiography, ct scans, mri) as appropriate. samples of hair of about 2 cm length from the nape of the neck were used for metal load determination by icp/ms, as recommended by iaea [12]. hair samples treatments, digestions and analysis were as reported [23]. xseries 2 icp-ms (thermo fisher scientific, germany) was used in the standard configuration, with asx-510 auto-sampler (cetac, usa). additional details in supplementary file. for statistics, continuous variables were expressed as median and range, due to their non-normal distribution (assessed by shapiro-wilk test) and compared using mannwhitney u test. categorical variables are expressed as percent and compared with chisquare test for trend. two-tail p values <0.05 are considered significant. 3. results the frequency of birth defects was registered at the fallujah general hospital from november 2009 to september 2010. total number of deliveries were 5896 of which 869 birth defects, with a frequency of 14.7% [24]. in addition, as example, in the month of may 2010 with a total 547 deliveries, there were also 75 premature births of which 60(10.9%) late miscarriages (after 30 weeks) and 10 stillbirths (1.8%). we classified the bd according to the major primary defect. many of the bd children show also additional defects in other body compartments, as often the case. the frequency of the different classes of bd in these ten months was: chd congenital heart defects, include ads, vds, tof, 33%; nt-neural tube defects, include anencephaly (possibly underestimated, since it causes miscarriages which can be lost to hospitalization), spina bifida, hydrocephaly, cerebral palsy, 28%; clp-cleft lip/palate, 14%; "others"-22%. frequencies of relative prevalence are not significantly different from elsewhere [5,23]. data collection from 56 families with bd, 31 of them registered in the hospital for delivery or miscarriage between april and september 2010 and 25 that returned for check up in these months, and from 11 families with a newborn normal child, included reproductive history, health of collateral kin (brothers and sisters) and its progeny, environmental and residential information and was done using the questionnaire in fig. 1s. the population studied is highly stable in historic residence and converge to the hospital from different areas of town. the exposure to war chemicals in the environment is reported by subjective record: most of the parents in the study were present at the time of massive attacks to the town in 2004 and/or 2005, cleared rubble, assisted victims and families 116, 119, 120, 132, 142 rebuilt their own house on the remains a bombed one. all mothers declared good to optimum quality of nutrition during pregnancy; most families studied are of large size. these data are shown by examples in table 1 and in full in table i 1s for reproductive history of families with bd, table i 2s for kin and alaani et al.; aji, 3(1): 1-10, 2020; article no.aji.53605 4 environmental information of families with bd, and table i 3s, for pedigrees, kin and environmental information of iraqi control families. among the 56 families with bd, 41.36% were chd, 25.4% nt, 4.7%, clp, 14.3% skeletal defects (sk), and 14.3% "others", in similar trend with the prevalence in wider population, above. fig. 1. prevalence of miscarriage and birth defects in different time periods in fallujah. p< 0.001 for changes of bd prevalence and p< 0.062 for changes of miscarriage prevalence fig. 2. hair metal load in ppb, in children (panel a) and in adults (panel b). columns correspond to control from italy (black bars), controls from jerusalem (dark grey bars), controls from fallujah (light grey bars) and bd from fallujah (white bars). * p<0.05 vs controls from italy; § p<0.05 vs controls from jerusalem alaani et al.; aji, 3(1): 1-10, 2020; article no.aji.53605 5 table 1a birth defectsfamily history table 1b –birth defectskin and environmental exposure n/sex/age bd house/vicinity rescue/ clearing acute symptoms sibling number sibling+progen with bd residence2003 /10 bombed wp 145/m/32 chdca y2004/n n y fallujah,north 145/f/30 y2004/n n y fallujah.north 156/m/33 nt y2004/n n y n 7 11 fallujah,nazzal 156/f/33 y2004/n n y n 8 4 fallujah,alaskari 131/m/42 sk-o y2004/n n y n 3 26 fallujah,nazzal 101/f/23 o-nt y/y2004 y y y 7 2 albaghdadi/ 2007 fallujah, althubbadh 9/m/35 y/y y y n 10 25 fallujah, alandulus 9/f/32 y/y y y n 10 1 fallujah, alandulus a-reproductive history of families;a,b)with birth defect children,born in 2010. c,d)with birth defect children born before 2010.e) heathy child. b-corresponding data on exposure to war events,kin number and health,residence since empty= healthy filled in black= birth date filled in grey= dead male ██ female unknown sex twins still born miscarriage alaani et al.; aji, 3(1): 1-10, 2020; article no.aji.53605 6 table 2. progeny with bd of families with reproductive history starting before the year 2000 families were selected for one bd in their reproductive history after 2003, and because their reproduction spanned from before 2000 up to at least 2006. normal-healthy child; bd-birth defect; ms miscarriage. bd and ms are listed in order of presentation in time in each family: chd congenital hearth disease, nt neural tube, clp cleft lip/palate, sk skeletal malformation, kidney here stays for atrophic more than one case of same bd was present in some families: nt in 123 and 105, and chd in 131 and 145; families 101, 107, 106, 131,132 had children with major defects of different phenotypes. in all of these there was no bd in the immediate kin. given to incomplete responses, we have full information only of 50 families on kin, environmental exposure and residence. these include the health state with respect to bd of 552 siblings and 665 progeny (total 1217). the health information is incomplete by defect, since some parents were not informed fully of the health status of all of their kin. seven cases of birth defects were registered and two infant deaths. these involved 5 families, with families 136 and 138, with chd children, and a chd presented in the progeny of the father's siblings, potentially due the presence of a genetic mutation on the father side. different defects from those in the family of reference were reported in the progeny of the mother's sibling in family 1, and in the progeny of the father's sibling in family 114. for all, the whole extended family resided in the same neighbour, throughout the last 7 years. among the 56 families, there are 17 whose reproductive life started since at least 2002 and back up to 1992. the presentation of bd in time increased significantly stepwise during the observation period, from 0.017 in 19912000 to 0.615 in 2007-2010 (fig. 1 and table 2). if all the families with one bd in the last 6 years are considered, regardless of the extension backward of their reproductive life, similar results of distribution and same significance (p<0.001) for the trend in bd are obtained (supplementary table ii s). the frequency of miscarriages versus births at term also is significantly different, in both cases, comparing these periods of time (p<0.0062 for the 17 and p<0.0003 for the 56 families, respectively), but has a different pattern than that of bds. miscarriages were most frequent in all families in the time interval 20042006. we measured the load of teratogenic/mutagenic metals in the hair of parents and their children with birth defects delivered in fallujah hospital between april and september 2010, and of 25 families returning for control visits of children with birth defects born in the last 6 years. we also collected hair samples from 11 families with normal children in the same hospital, from families in italy and from arab children in jerusalem, without familiar records of bd. all participants gave their informed consent to use and delivered the data in scientific study. metal concentrations in hair was measured by icp/ms for ten toxic and carcinogenic metals (v, cr, co, as, mo, cd, w, u, hg, pb) also components of weapons, and are expressed in alaani et al.; aji, 3(1): 1-10, 2020; article no.aji.53605 7 ppb, in graphical form in fig. 2 and in full (supplementary table iii 1s for children and 2s for adults). bd children analyzed were 32 newborn and 13 between 6 months and 7 years of age, for a total of 45. the group of 11 iraqi children without bd was registered without any selective criteria among the newborns in the period of the study. the group of italian (from genoa and naples, both industrial and traffic polluted large towns) and jerusalem arab children, all between 6 months and 7 year of age, were taken randomly among families with no bd. the adults group analyzed include the corresponding parents for bd and all controls (with few exceptions where one of the parent refused to donate or was bold). while for as and hg there are no differences in all groups, and w is lower in all iraqi than outside iraq controls, the hair metal load in children is significantly higher in control subject from fallujah than in controls from ital y for co, mo, u, cr, v and pb. the same holds true for children with bd from fallujah relatively to co, mo and pb (fig. 2, a). differences in hair metal load is reported for adults in fig. 2, b. co, mo, u, v, and cr were significantly higher in parents of bd children than in controls from italy. in addition, cd and u were also greater in controls from falluhja with respect to italian controls. in fig 1, a, bd children were grouped regardless of their age (newborns and 6 months to 7 years), as there was no significant differences between the two groups in load for any of the ten metals considered (supplementary table iii 1s). subgroups comparison between chd and nt children for the load of each of the ten metals, showed no significant differences, suggesting that there is not specific metal association with one kind of these bd (not shown). we left out from the statistical analysis family 119 (table iii, s2), whit a much higher level of all the toxicants that all the other families. this family is one of four in this report living in an house rebuilt on the previous one bombed. outliners were also observed in other families, being more frequent for hg and pb. in terms of absolute amounts for all elements significantively higher in all the population in iraq, these are between 2-5 and more in median values than experimental controls, amounts available in the literature, and those previously obtained by our group for another group of ten italian healthy people of age from 2 to 60 years (not shown). 4. discussion in cases where a rapid increase in prevalence of birth defects is suspected/observed, there might be clustering of referrals to the structure where the investigation is undertaken. we have no reason to think that in this case since fallujah general hospital is the main and the largest public structure in the area, and people convey to it from different areas of town, as shown by the residence of parents. prenatal diagnosis and therapeutic abortions are not implemented in fallujah, determining a slight overestimation in comparison with data from countries where these are practiced. even taking this in consideration, the frequency of bd detected in fallujah is unusually high compared with those reported in the literature [4,23] usually not exceeding 6%. the data of prevalence are also susceptible of underestimation, due to limited availability of instrumental tools for diagnosis, with possible oversight of minor bd. nonetheless, the distribution for kinds of different major bd is similar to what reported in the world [4,23]. modality of the presentation in the families with a bd child is in the majority of cases sporadic and occasional, consistent with the possibility that bd are novel events due to dominant mutation and/or to epigenetic changes during gametogenesis or early embryogenesis. among the cases where more than one child with bd was born to the same parents, in three cases they differed in phenotypes and there was no bd in the immediate kin. this is most adequately interpreted on the ground of environmental/mother genetic background or epigenetic origin for the multiple events of bd [25,26]. in the few families with recurrence of the same bd in children, two nt and one with two cdh, a supplement of study may be required as to possibility of genetic changes. similar criteria apply in the cases of occasional recurrence of different or same bd in children in the kin of the family of reference. a persistent environmental effect in the siblings, associated to a generic "predisposition" and same historical residence can explain the recurrences as well as the occurrence of bd with different phenotypes. the historical reconstruction shows that frequency of bd in the last ten years of the last century was less that 2%, and that of miscarriages about at 5%, similar to that reported for other not heavily industrialized countries. the differences with the past in the alaani et al.; aji, 3(1): 1-10, 2020; article no.aji.53605 8 following time windows are highly significant. these also show the different pattern of increase of bd and ms. miscarriages increase, but with a different pattern than bd and the maximum is coincident with the years of major attacks to the city and decline from 2007, which may suggest that ms are an acute and shorter term response to the environmental changes. for bd, our data do not allow to know if and when the increase will stop and suggest that these are caused by accumulation of the environmental effectors. the fact that the presentation rate of birth defect is not declined in the last years suggest permanence in the environment of teratogenic substances. metal load in teratogens (v, co, mo, u and pb) is high in the whole population in fallujah. if we adopt, as suggested from the sporadic and random presentation of bd the epigenetic paradigm, then this observation, assumes major relevance. it is known that toxicant metals compared to other known war-contaminants have longer persistence in the environment, accumulate in the organs, act on multiple functions and are capable to induce sporadic epigenetic changes of great impact during gametogenesis and embryo development; interactive effects of multiple metal toxicant were suggested and are still largely undocumented, while is described their capability to unbalance the amounts of essential metals in the organism [25-28]. the toxicants which have increased load in the whole fallujah population are known for their capability to interfere with embryo development and may be relevant in the induction of ms as acute response, and of bd, as long time effect. here we show association of high load of co, mo and pb with all children, pb being specifically highest in bd children, and of u and v in control fallujah children. the load of these elements differs from that in adults who all have an u load higher in all controls outside iraq, and parents of bd children showing co, mo, v, and cr higher than that in all the adults groups. although the metal load in newborn children hair must derive from mother's exposure, these results show it does not reflects it immediately, suggesting the need of further study on the repartition of the load of different metals during embryo and foetal life, which may be relevant in understanding the mechanisms of metal's teratogenic action. notwithstanding the low mutagenic performance of teratogenic and carcinogenic metals, their effects are nonetheless important and transmittable to daughter cells within a lineage and transgenerationally in mammals [25-28]. via a mechanistic framework which suggests transmission of epigenetic modifications as a mechanism alternative to germ-line mutagenesis. by their multiple mechanisms of action metals can determine the alteration of functionality of cell lineages, organogenesis and homeostasis in the adult, and the type of cells in the body affected by metals range from somatic cells, also within the developing embryo, to germ cells, both of male and female [29]. the relevance of increase even of moderate amount in teratogenic metal load, and the possible combinatorial effects of their co presence is alluded to in many reports but need further studies to be fully understood. 5. conclusion we here reported mainly on two aspects related to the birth defects presentation, its frequency, much higher than in the world and of that retrospectively assessed in the place before the 2003 war, and the observational study that indicates that the metal load of fallujies, in general is unusually high for metals associated with weaponry. that is not enough to establish a cause-effect relationship, but is indicative of muted environmental that can condition differently ms and bd presentation and indicates a path to follow for future studies. the application of the historical method of recording births gave us a convenient tool for obtaining needed retrospective information and could be implemented for all registrations in countries where registration data have not been recorded in the past. we hope this knowledge will also help to propose studies directed to the management of the issue along lines of prevention and therapy. consent and ethical approval as per international standard guideline participant consent and ethical approval has been collected and preserved by the authors. competing interests authors have declared that no competing interests exist. alaani et al.; 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(2) dr. juandy jo, science analytical team leader, danone nutricia research, singapore. (3) dr. darko nozic, professor, higher medical school in belgrade, university of belgrade, serbia. reviewers: (1) giuseppe gregori, italy. (2) pooja sarmah, sapthagiri institute of medical sciences, rajiv gandhi university of health sciences, india. (3) amanda mcrae, university of the west indies, trinidad and tobago. (4) franco mantovan, university of verona, italy. complete peer review history: http://www.sciencedomain.org/review-history/27672 received 18 th september 2018 accepted 26 th november 2018 published 8 th december 2018 abstract background: high rate of childhood vaccination drop-outs is a significant public health problem in rural areas of developing countries especially in nigeria. the reasons for immunisation drop-out are poorly understood and little or no data is available to explain the phenomenon that could support the decision making. this study, therefore, determined the reasons for immunisation drop-out in the rural areas of abakaliki, nigeria. materials and methods: descriptive analytical crosssectional study design was used for the survey. data were collected using pretested, semi-structured interviewer administered questionnaire from 290 mothers/caregivers accessing childhood immunisation services in mile four and st. vincent hospitals selected using systematic random sampling technique. reasons for immunisation drop-out were gotten from caregivers when they brought their children for immunisation in subsequent immunisation dates after the drop-out interval. analysis was done using spss version 22 and level of statistical significance set at p< 0.05 and confidence level at 95%. ethical approval was obtained from the research and ethics committee (rec) of the federal teaching hospital abakaliki (fetha), ebonyi state, nigeria. results: the mean age of respondents in mile four hospital was 26.6 ±4.9 years while 27.1±4.2 years in the st.vincent hospital. the reasons for immunisation drop-out in mile four were no original research article eze; aji, 1(1): 30-35, 2018; article no.aji.44949 31 money for transport (2.1%), caregiver travelled (1.4%), baby was sick (1.4%), far distance to health facility (0.7%). in st.vincent reasons cited werefar distance to health facility (6.2%), no money for transport (4.8%), mother was sick (2.8%), caregiver travelled (2.1%) and baby was sick (2.1%).far distance to health facility was a predictor of immunisation drop-out in st. vincent group. it is 2.8 times more likely to cause immunisation drop-out than near distance to health facility. conclusion: high rate of immunisation drop-outs need to be addressed collectively both by individuals and government in order to achieve the targeted 95% immunisation coverage necessary for the sustained control of vaccine preventable diseases. keywords: immunisation drop-out; reasons; rural infants; abakaliki; nigeria. 1. introduction immunisation drop-out and coverage rates at primary health care (phc) level indicate the level at which communities utilise the preventive services and thus serve as a measure of the strength of public health system. they also measure the effectiveness of the immunisation programme [1]. immunisation drop-outs refer to infants who have used or missed immunisation services and do not return for subsequent vaccinations. when drop-outs exceed 10 percent, it indicates a problem of utilisation of services. in most settings where full immunisation coverage is low, most infants receive at least one dose of pentavalent vaccines, but the proportion that receive the needed second and third doses drops significantly [1]. however differences in the indicators of access and utilisation of immunisation services exist [2,3]. indicators of access to health services among other factors include level of education, sex, patriarchal social arrangement, rural residence, poverty, religious and cultural beliefs about certain diseases and location of health facilities etc. indicators of utilisation of immunisation services include quality of staff skills, protocols of treatment, availability of supplies and environment of health facilities, physical and financial accessibility of services, knowledge of what services exist, education about how to best utilise self and practitioner-provided services and cultural norms of treatment [2,3]. in previous studies most important reason given for immunisation dropout was distance to health facility [4]. however, reasons for childhood immunisation drop out are unknown in the study area, hence this study determined the reasons for dropout in rural areas of abakaliki, ebonyi state nigeria. 2. materials and methods descriptive analytical crosssectional study design was used for the survey. vaccines such as bcg, opv0 and hbv0 are given at birth, opv1, pcv1, penta1 are given at 6 th week of life, opv2, pcv2, penta2 are given at 10 th week of life, opv3, pcv3, penta3 are given at 14 th week of life while measles, yellow fever, meningitis vaccines are given at 9 th month of life. these vaccines are given free. this minimum sample size for the study was determined using the formula for comparing two proportions [5,6]. they were selected using systematic random sampling technique. data were collected using semi-structured interviewer administered questionnaire on the mothers/caregivers and immunisation registers of 290 infants who were brought for immunisation in mile four and st.vincent hospitals in ebonyi and izzi local government areas of ebonyi state after the drop-out interval. reasons for immunisation drop-out were gotten from caregivers when they brought their children for immunisation in subsequent immunisation dates after the dropout interval. the interval considered as drop-out period was 4 weeks after due date for the uptake of that vaccine. analysis was done using spss version 22.0. chi-squared test was used to determine association or differences between proportion of the variables and the level of statistical significance set at p< 0.05 and confidence level at 95%. logistic regression model was used to determine predictors of immunisation drop-out. ethical approval was obtained from the research and ethics committee (rec) of the federal teaching hospital abakaliki (fetha), ebonyi state, nigeria. 3. results the mean age of respondents in mile four hospital was 26.6 ±4.9 years while 27.1±4.2 years in the st. vincent hospital. fig. 1 showed the proportion of infants who dropped-out of immunisation in both groups. it also shows between group comparisons of eze; aji, 1(1): 30-35, 2018; article no.aji.44949 32 immunisation drop-outs which was found to be statistically significant (p<0.01). table 2 showed the reasons for immunisation drop-out among respondents in both groups. more caregivers (6.2%) in st. vincent cited distance to the health facility as the major reason for immunisation drop-out compared to those in mile four (0.7%), a difference that was significant (p=0.01). other reasons were not significant (p>0.05). in table 3, far distance to health facility was a predictor of immunisation drop-out in st.vincent hospital. it was 2.8 times more likely to cause immunisation drop-out than near distance to health facility. immunisation drop-outs refer to infants who have used or missed immunisation services and do not return for subsequent vaccinations. missed immunisation is when infants scheduled for immunisation did not come at that given date but returned later for uptake of the given vaccination. table 1. socio-demographic characteristics of respondents in both health facilities variables mile four (n=145) freq. (%) st. vincent (n=145) freq. (%) χ 2 p-value sex male 5 (3.4) 4 (2.8) ft 0.73 female 140 (96.6) 141 (97.2) age group (years) 15-19 11 (7.6) 9 (6.2) 6.38 0.16 20-24 50 (34.5) 37 (25.5) 25-29 48 (33.1) 68 (46.9) 30-39 36 (24.8) 31 (21.4) marital status married 137 (94.5) 134 (92.4) 2.44 0.69 single 8 (5.5) 11 (7.5 ) education primary 10 (6.8) 17 (11.7) 3.67 0.15 secondary 88 (60.7) 93 (64.1) tertiary 47 (32.4) 35 (24.1) employment paid employment 25 (17.2) 21 (14.5) 2.75 0.25 self employment 56 (38.6) 70 (48.3) unemployed 64 (44.1) 54 (37.2) religion christianity 142 (97.9) 143 (98.6) ft 1.00 others 3 (2.1) 2 (1.4) ft= fisher’s exact test table 2. respondents’ reasons for immunisation drop-out in each study group variables mile four group (n=145) freq. (%) st. vincent group (n=145) freq. (%) χ 2 p-value reasons for dropping-out yes yes distance to facility 1 (0.7) 9 (6.2) 6.6 0.01٭ no money for transport 3 (2.1) 7 (4.8) 1.6 0.33 caregiver travelled 2 (1.4) 3 (2.1) 0.2 1.00 baby was sick 2 (1.4) 3 (2.1) 0.2 1.00 mother was sick 0 (0.0) 4 (2.8) ft 0.12 ft= fisher’s exact test; ٭statistically significant eze; aji, 1(1): 30-35, 2018; article no.aji.44949 33 table 3. predictors of immunisation drop-out at st. vincent hospital variables immunisation drop-out aor 95% ci of aor p-value marital status not married 1 married 4.31 -0.89-10.51 0.09 educational status <secondary 1 ≥secondary 0.79 -0.39-1.61 0.59 far distance to health facility no 1 yes 2.78 1.47-6.76 0.05** **predictor fig. 1. showing proportion of missed immunisation and immunisation drop-outs in both facilities 4. discussion reasons for immunisation drop-out in most health facilities especially in rural areas of developing countries are not well understood. however, in similar settings, reasons may also be similar. reasons given for immunisation dropout in mile four were no money for transport (2.1%), caregiver travelled (1.4%), baby was sick (1.4%), far distance to health facility (0.7%) while in st.vincent hospital, reasons cited were far distance to health facility (6.2%), no money for transport (4.8%), mother was sick (2.8%), caregiver travelled (2.1%) and baby was sick (2.1%). there is a significant difference in the proportion of caregivers who cited far distance to health as reason for drop-out in the intervention and control groups. there is no difference in the proportion of respondents who gave other reasons in both groups. in this study, caregivers did not demonstrate lack of commitment to immunisation, rather they gave far distance to health facility and no money for transport as major reasons why they could not come back to study centres for further vaccines, 25.60% 5.50% 33.10% 22.10% 0.00% 5.00% 10.00% 15.00% 20.00% 25.00% 30.00% 35.00% missed immunisation immunisation drop-out p ro p o rt io n immunisation outcome mile four hospital st. vincent hospital eze; aji, 1(1): 30-35, 2018; article no.aji.44949 34 and they actually continued the immunisation in centers close to their residential areas. in a study by itimi et al. [7] mothers were not returning for immunisation due to lack of motivation, poor attitude of the health workers and relocation to a new environment. however the reason in this study could be that, the first doses which were received in the study center coincided with the mother’s post natal care checkup visits. when there was no longer a need for the mothers to return to the center for their own check-up, they now went to nearer centers for their children’s immunisation. the fact that immunisation was continued in nearby health centers was an indication that these group of parents actually are aware of the importance of this intervention. it is also interesting to note that this study demonstrated average drop-out in both males and females infants with that of the males being marginally lower though not statistically significant. the magnitude of its public health importance cannot be over-emphasised. for every child recalled for immunisation, so much has been done to prevent disease, morbidity and mortality in that child, prevent disabilities as well as disease transmission to many others who might have gotten infected through the child and also boosting herd immunity [4]. similar reasons found in umuahia and enugu, nigeria werebecause of far distance (49%) and 38% for no contact [4], health facility was far, health workers not always available, child was sick, mother was too busy, vaccination time inconvenient, caregiver not aware of another dose of vaccination among others [8]. other factors include mothers’ poor knowledge of immunisation against targeted diseases, parents’ concern about immunisation safety, long waiting time at the health facility and long distance from the hospital, false contraindications like catarrh and fever in the infant [9,10], immunisation awareness [11]. in benin, it was reported that cost of transportation and man hours lost in time spent attending the clinic were reasons for high drop-out rate [12]. these factors need to be addressed to reduce the drop-out rates. other factors are lack of political will, poor work attitude and mal-orientation of health workers, poor health infrastructure, religious insurgency/terrorism, ignorance, cultural/religious average aversion to vaccine acceptance or use, misconceptions about safety of the vaccines and lack of awareness about availability of vaccination services, inadequate cold chain facilities and vaccine stock-outs [13-15]. in that study, marital status was found as a predictor for immunisation completion compared to higher maternal educational status (secondary education and above) and occupation (government employees) found to be important independent predictors for immunisation completion among mothers/caregivers of infants in enugu [8]. 5. conclusion the reasons given for immunisation drop-outs in this study especially far distance to health facility and no money for transport call for more decentralised primary health care centres mostly in rural areas in order to achieve the targeted 95% immunisation coverage necessary for the sustained control of vaccine preventable diseases. consent as per international standard or university standard, patient’s written consent has been collected and preserved by the author(s). ethical approval as per international standard or university standard, written approval of ethics committee has been collected and preserved by the author(s). competing interests author has declared that no competing interests exist. references 1. immunization drop-out. health management information system news. sept 22 2011:1 2. nnonyelu an, nwankwo iu. social determinants of differential access to health services across five states southeast nigeria. europ. scientific j. 2014;3: 1857-7881. 3. ebonyi state ministry of health, health management information system unit; ebonyi state health systems development project. 2015;27. 4. nwokeukwu hi, emma-ukaegbu u, ajuogu e, osunkwo d, asinobi a. use of telephone calls in reduction of dropout rate of routine immunization in a tertiary health facility in southeastern nigeria. europ. j. preventive medicine. 2015;3(3):39-43. eze; aji, 1(1): 30-35, 2018; article no.aji.44949 35 5. araoye mo. research methodology with statistics for health and social sciences. ist edition. ilorin: natadex. 2003;69,107,118122. 6. onwasigwe cn. principles and methods of epidemiology. 2 nd edition. enugu: el demark publishers. 2010;147-148. 7. itimi k, paul od, ordinioha b. community participation and childhood immunization coverage: a comparative study of rural and urban communities of bayelsa state, south-south nigeria. nmj. 2012;53(1):2125 8. tagbo bn, eke cb, omotowo bi, onwuasigwe cn, onyeka eb, mildred uo. vaccination coverage and its determinants in children aged 11 23 months in an urban district of nigeria. world journal of vaccines. 2014;4:175-177. 9. ubajaka fc1, ukegbu au, okafor nj, ejiofor o. the prevalence of missed opportunities for immunization among children utilizing immunization services in nnamdi azikiwe university teaching hospital, nnewi, journal of biology, agriculture and healthcare. 2012;2(6):113114. 10. anah mu, etuk is, udo jj. opportunistic immunization with in-patient programme: eliminating a missed opportunity in calabar, nigeria. annals of afr. medicine. 2006;5(4):188-191. 11. hussey gd. strengthening the expanded programme on immunization in africa: looking beyond 2015. plos med. 2013;10:e1001405 12. sadoh ae, eregie co. continuing barriers to optimum immunization uptake in nigerian children: the role of missed immunization opportunities and inappropriately timed immunizations. niger j paed. 2007;34:57-61. 13. oluwadare c. the social determinants of routine immunization in ekiti state of nigeria. ethno-medicine. 2009;3:49-56. 14. etana b, deressa w. factors associated with complete immunization coverage in children aged 12-23 months in ambo woreda, central ethiopia. bmc public health. 2012;12:566. 15. unicef (2014) nigeria: low coverage of life-saving interventions. available:http://www.unicef.org/nigeria/chil dren_1927.html (accessed august 7, 2015) _________________________________________________________________________________ © 2018 eze; this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: http://www.sciencedomain.org/review-history/27672 http://www.unicef.org/nigeria/children_1927.html http://www.unicef.org/nigeria/children_1927.html http://creativecommons.org/licenses/by/4.0 _____________________________________________________________________________________________________ *corresponding author: e-mail: ibrahimshnawa3@gmail.com; cite as: shnawa, ibrahim m s, algebori hsh, and thewaini q n. 2024. “shared allergenicity in a lapin-model of delayed htpersensitivity to gram negative protoplasmic sonicate proteins psp”. asian journal of immunology 7 (1):181-84. https://journalaji.com/index.php/aji/article/view/142. asian journal of immunology volume 7, issue 1, page 181-184, 2024; article no.aji.123083 shared allergenicity in a lapin-model of delayed htpersensitivity to gram negative protoplasmic sonicate proteins psp ibrahim m s shnawa a,b*, algebori hsh c and thewaini q n d a department of medical biotechnology, college of biotechnology, al-qasim green university, qasim, iraq. b college of nursing technology, university of hilla, iraq. c department of basic sciences, college of dentistry, babylon university, iraq. d department of medical biotechnology, college of biotechnology, al qasim green university, iraq. authors’ contributions this work was carried out in collaboration among all authors. all authors read and approved the final manuscript. article information doi: https://doi.org/10.9734/aji/2024/v7i1142 open peer review history: this journal follows the advanced open peer review policy. identity of the reviewers, editor(s) and additional reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here: https://www.sdiarticle5.com/review-history/123083 received: 07/07/2024 accepted: 10/09/2024 published: 20/09/2024 abstract there were shared allergenicity in delayed type hypersensitivity between the protoplasmic sonicate proteins of pseudomonas aeruginosa with that of klebsiella oxytoca mild erythema was noted from 6 to72 hrs post to p. aeruginosa (psp) in klebsiella oxytoca psp primed rabbits (kopsp). mild, moderate to high erythema during 6 up to 72 hrs post to injecting kopsp in papsp primed rabbits. induration of 5 mm at 72 hrs of papsp intradermal id injecting in kopsp primed rabbits. while short research article https://doi.org/10.9734/aji/2024/v7i1142 https://www.sdiarticle5.com/review-history/123083 shnawa et al.; asian j. immunol., vol. 7, no. 1, pp. 181-184, 2024; article no.aji.123083 182 induration of 6 to 12 mm of kopsp id injected to papsp primed rabbits. consenquently there was a high quantitative and /or potency of the allergenic fraction of ko than that of pa. the shared fractions were characterized as delayed allergenicity of bilateral or reciprocal type delayed allergenic nature. such findings appeared to be novel contribution in bacterial protein allergens, with possible pan shared bacterial families. preserved protein fraction between these two different gram negative representatives of bacterial families. keywords: bacterial antigens; shared antigenicity; shared immunogenicity. 1. introduction bacterial antigens (bag) may express shared antigenicity (shag), shared immunogenicity (shi) and /or shared allergenicity (shall). these sharing fractions can be of quality, quantity and /or potencey. unilateral or bilateral and reciprocal or non-reciprocal natuere. shared immunogenicity showed a dual importance in clinical practice of human microbial infections. first it has cross-protection ability that is crucial in immunoprophylaction of infectious diseases. sceond it is problematic in the immunodiagnosis of these diseases. though, shared allergenicity stands as problematic issue in diagnosis and managment of human infectious diseases [1-8]. thus, the present short communication was aimed at presenting shared delayed skin hypersensitivity between the intracellular proteins of two different gram negative bacteria. 2. materials and methods psp from p. aeruginosa and k. oxytoca were prepared, partially purified, indentified and quantified as an intracellular bacterial proteins as in [9]. the concentration of papsp was 2.71 mg/ml. and that of kopsp was 1.81 mg/ml. the test immunogens were papsp+fca and kopsp+ cfa for p. aeruginosa and k. oxytoca respectively. specific immune priming of rabbits with test immunogens were made as in [10]. dth skin test was done and read as in [11]. homologous skin dth reactions scored as homologous sentisitins injected in rabbits primed with homologous protein. while heteologous shared dth skin reactions were checked when heterologous sensitins injected in rabbits primed with heterologous proteins [11]. 3. results and discussion the id injection of 0.1 ml. papsp sensitin in papsp specific immune primed rabbits was showing mild, moderate and high erythema reaction lasted from 6 up to 72 hrs. the induration reaction was evident at 48 hrs and 72 hrs post injection of the sensitin as 10 and 18 mm respectively. this accounts for the homologous delayed hypersensitivity reaction, table 1 a1. while the id injection of papsp to kopsp specific immune primed rabbits has showed mild erythema reaction lasted from 6 up to 72 hrs post-injection of the sensitin. the induration reaction was evident at 72 hrs post injection of the sensitin as 6 mm around the injection site. this accounts for the shared allergenicity in skin dth reaction, table 1 b-1. the id injection of 0.1 ml of kopsp in kopsp specific immune primed rabbits was showing an erythema reaction of mild nature as (+) for the duration of time lasted from 6 up to 72 hrs post injection of sensitin with nill induration reactions. this accounts for the homologous dth reactions table 1 /a-2. while the id injection of 0.1 ml of kopsp to papsp specific immune primed rabbits showed mild, moderate to high erythema reaction lasted from 6 up to 72 hrs respectively table 1/ b-2. induration reactions were apparent in 6 mm for 48 hrs and 12 mm for 72 hrs post injection of sensitins this accounts for shared dth reactions with nill necrosis reactions were evident . results tabulated in table 1 indicate that there was bilateral shared dth allergenicity between psp proteins of p. aeruginosa and k. oxytoca and the nature of this shared allergenicity be of quantitative rather than qualitative.in which k. oxytoca psp shared allergenicity was more in quantity than that of p. aeruginosa psp in rabbit models. changes in the conformation of the allergenic epitopes are mostly, paralleled by change in in the nature of their allergenic responses [2]. protein allergens expressed potential risk for cross reactivity [3]. modification of corticosteroid from their original core structure may frequently lead to cross-allergenicity to the new form of the corticosteroid [4]. three patterns of cross allergenicity to proton pump inhibitors were indicated [5]. t cells are taking part in the dth to quinolones reactions and cross-reactivity to other quinolones [6]. human adenovirus serotypes shnawa et al.; asian j. immunol., vol. 7, no. 1, pp. 181-184, 2024; article no.aji.123083 183 table 1. shared heterologous rabbit skin dth as compared to the homologous rabbit skin dth reactions nature of skin dth/reactions per hours erythema induration in mm necrosis a – 1/homologous pasp in papsp primed rabbits 6 48 72 + ++ +++ 10 18 a -2/homologous of kopsp in kopsp primed rabbits 6 48 72 + + + b-1./heterologous papsp in kopsp primed rabbits 6 48 72 + + + 6 b -2/heterologous kopsp in papsp primed rabbits 6 48 72 + ++ +++ 6 12 express cross-reactivity in inducing dth [7]. leukocyte migration inhibition to various cepham antibiotics displayed cepham shared allergeninicity in dth reactions [8]. bacterial antigenic epitopes can be with an array of immune potentials such as; immunogenic, autoreactive, immunosuppressive, and /or delayed type hypersensitivity inducing nature [12]. there were marked shared reactivity of burilin of m. ulcerans to tuberculin ppd of m tuberculosis as indicated by the induration upon intradermal injection. so that burilin positive patients when analysed in conjugation with either the presence of bcg scar or retesting of bcg vaccination, 12 of 14 bcg vaccinated burilin patients were burilin positive and 6 of the 12 were also ppd positive [13]. it had been reported that there were cell mediated immunity cross reactions of various species of mycobacteria that had been attributed to polymorphism of taget bacterial antigens [14]. 4. conclusion the present short communiction was focusing onto sharing in delayed hypersenitvity inducing epiopes from intracellular proteins of p. aeruginosa and k. oxytoca with rather difference in quantity of the allergenic fractions. both of which produce erythema and induration to variable degrees with absence of necrosis up to 72 hrs post to sensitin injection through id route. shared delayed hypersensitivity induced by intracellular bacterial protein that was functionally mapped in this short communication can be characteized as in the followings; 1. the shared allergenic epitope is in or on protoplasmic sonicate protein with an intracellular location with possible oligo amino acid sequence nature. 2. function as delayed type allergen. 3. response produces erythema and induration but not necrosis. 4. express quantitative differences among different protoplasm sonicate proteins 5. this shared allergenic epitope is of bilateral reciprocal nature. 6. attributed to t cell dependent hypersensitivity reactions, [1,12]. 7. such sharing delayed allergenic epitopes between bacteria that belongs to different gram negative families.it might be a pan shared preserved protein fraction,which may stands as a novel finiding. disclaimer (artificial intelligence) authors hereby declare that no generative technologies such as large language models (chatgpt, copilot, etc.) and text-to-image generators have been used during writing or editing of this manuscript. consent it is not applicable. ethical approval the care, housing, handing and experimentation on rabbits were done following the international acts regulating care, housing, hnadling and intervention. shnawa et al.; asian j. immunol., vol. 7, no. 1, pp. 181-184, 2024; article no.aji.123083 184 competing interests authors have declared that no competing interests exist. references 1. shnawa ims. serology of vibrio cholerae; revisting, sse, uk; 2023. 2. seider ca, zeindl r, fernandez-quintero m, et al. allergenicity and conformational diversity of allergens. allergies. 2024;4:116. doi: 10.330 3. gulec fem, caliskaner z, ozturk s, et al. is allergenic similarity predictable in respratory allergens. allergol. immunopathol. 2008;36(2):5-65. 4. allergen online. allegenic cross – reactivity. home of farrp allegen protein database. university of nebraska-lincoln. allegen online. 2023;22:1-6. 5. berbegal l, deleon fj, silvestre jf. hypersensitivity reactions to corticosteroids. acats dermosifitiogr. 2016;107(2):107-115. 6. lombardo c, ponadonna p. hypersensitivity reactions to proton pump inhibitors. drug allergy. 2015;2:110-123. 7. 7-schmid da, depta jph, pichler wj. t cell mediated hypersensitivity to quinolones; mechanisms and crossreactivity. cli. exp. aller. 2005;36(1):59-69. 8. bakay m, szalay k, beladi. crossreactivity bewtween human adenoviruses in delayed type hypersensitivty. apmis. 2005;113(3):197-202. 9. bjorn mj, iglewski bh, ives sk, et al. effect of iron on yields of exotoxin a in culture of pseudomonas aeruginosa pa103. infection and immunity. 1978;19(3): 785-791. 10. al shahery man, shnawa ims. immunological adjuvani city of sunflower oils; 1989. 11. burrell r. experimental immunology, burgess publishing co. amazon, usa. 1979;102. 12. uno k, yamasaku f. structural correlations with cross-reactivity of betalactam antibiotics in delayed type hypersensitivity: cross-allergenicity in hypersensitivity to cephems with a tetrazolyl group in the c3 side chain j. antimicrobial. chemotherap. 1989;24(2): 251-264. 13. dobos km, spotts ea, marston bj. serologic responses to culture filtrate antigen of mycobacterium ulcerans during burili ulcer disease. emergent infectious disease. 2000;6(2):158-164. 14. kulkarini s, hattikudur s, kamat rs. cell mediated immunity cross reactions of mycobacteria: polymorphism of target bacterial antigens. cli. exp. immunol. 1986;63:111-117. disclaimer/publisher’s note: the statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of the publisher and/or the editor(s). this publisher and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. _________________________________________________________________________________ © copyright (2024): author(s). the licensee is the journal publisher. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: https://www.sdiarticle5.com/review-history/123083 https://www.sdiarticle5.com/review-history/123083 _____________________________________________________________________________________________________ *corresponding author: e-mail: akeler@abuad.edu.ng; asian journal of immunology 3(1): 199-207, 2020; article no.aji.57099 seroprevalence of rubella igg and igm antibodies among pregnant women attending antenatal clinic in federal teaching hospital ido-ekiti, nigeria richard yomi akele1*, baget nerat pam1, bernard oluwapelumi oluboyo1, seyi samson enitan2, janet funmilayo akinseye1 and funmilayo ajoke adewumi3 1 department of medical laboratory science, college of medicine and health science, afe babalola university, ado-ekiti, nigeria. 2 department of medical laboratory science, babcock university, ilisan-remo, ogun state, nigeria. 3 department of microbiology and parasitology, college of medicine, ekiti state university, ado-ekiti, nigeria. authors’ contributions this work was carried out in collaboration among all authors. authors rya and bnp designed the study, wrote the protocol, collected data, performed analysis and wrote the first draft of the manuscript. authors boo and sse performed the statistical analysis and restructured the manuscript. authors jfa and faa managed literature searches. all authors read and approved the final manuscript. article information editor(s): (1) dr. darko nozic, professor, university of belgrade, serbia. (2) dr. jaffu othniel chilongola, tumaini university, tanzania. reviewers: (1) victor b. oti, nasarawa state university, nigeria. (2) simeon achunam nwabueze , namdi azikiwe university, nigeria. complete peer review history: http://www.sdiarticle4.com/review-history/57099 received 22 march 2020 accepted 29 may 2020 published 04 june 2020 abstract aim: though rubella is vaccine-preventable and enlisted on the expanded program on immunization (epi) list, vaccination and testing are not routinely practiced in ido-ekiti. there is also paucity of epidemiological data on the prevalence of rubella infection at ido-ekiti, hence the study aimed at carrying out a serosurvey to generate epidemiological data for this location. study design: this was a cross-sectional study. place and duration of study: the study was carried out between october 2018 and january 2019 at the antenatal clinic of federal teaching hospital ido-ekiti (fethi), ekiti state, nigeria. original research article akele et al.; aji, 3(1): 199-207, 2020; article no.aji.57099 200 methodology: one hundred and eighty four consenting pregnant women attending antenatal clinic at fethi were enrolled. structured questionnaire was administered to collect sociodemographic data and sera samples were also collected to determine seroprevalence of rubella igg and igm antibodies using the enzyme linked immunosorbent assay (elisa) method. prevalence rate was calculated and chi square value was determined. results: of the 184 sera samples analyzed 176 (95.7%) and 22 (12%) were seropositive for rubella igg and igm respectively. twenty (11%) of those seropositive for igm were also positive for igg and only 2(1%) were positive only for igm. prevalence rate for igg was highest among 2630years (98.3%) and 31-35years (18.8%) for igm. age had no-significant effect (p> 0.05) on seroprevalence distribution. in like manner, level of formal education, knowledge of the virus, and occupation had no significant effect (p> 0.05) on prevalence of the virus. however parity significantly (p< 0.05) influenced the pattern of serostatus for both igg and igm. conclusion: the high prevalence and similar distribution pattern irrespective of sociodemographic features of rubella virus in this study area suggests its endermicity and continuous transmission in the area. this emphasizes the need to implement routine immunization of children and susceptible women of child bearing age against rubella virus. keywords: seroprevalence; rubella igg and igm; pregnancy; congenital rubella syndrome; ido-ekiti; nigeria. 1. introduction rubella is a contagious viral infection characterized by distinctive macules of erythematous rash popularly called german measles. it is an enveloped positive-sense single-stranded ribonucleic acid (rna) virus belonging to the family togaviridae, genus rubivirus [1-3]. rubella is transmitted congenitally and by respiratory aerosols, transmission occurring mainly in children and young adults. the contagious period of the virus is approximately 5 to 7-days pre and 3 to 5-days post manifestation of clinical symptoms [4]. upon inhalation of virus loaded droplets, the virus infects cells of the mucosal membranes of the upper respiratory tract and replicates consequently spreading to regional lymph nodes. [4]. infection is mainly subclinical, alternatively causing a self-limiting illness with low-grade fever, lymphadenopathy, and skin rash. of major concern is the fact that infection in pregnancy often result in congenital rubella infection which causes miscarriages, stillbirth, abortion, or congenital rubella syndrome (crs) of the infants. congenital rubella syndrome encompasses cardiac, cerebral, ophthalmic, and auditory defects [5-7]. it is estimated that infection with the virus in the first trimester or shortly before pregnancy may result in approximately 90% of fetuses being infected and 100% of these infected fetuses will come down with congenital deformities, often resulting in miscarriage [4,8-9]. it has been reported that as pregnancy progresses, the risk of crs decreases [9]. in nigeria, a high prevalence rate of rubella has been reported in some areas. for example, 77% in lagos [10], 97.9% in zaria [11] and 93.1% in abakaliki [12]. seroepidermiological studies in ethiopia also has reported a similarly high prevalence, 91% in addis ababa [13] and 86.3% [14] in southern ethiopia. rubella is a vaccine-preventable viral infection [15] considered as an eradicable infection. based on this fact, some vaccination programs implemented between 1996 and 2010 in some high income, middle income and low-income countries have in the past been able to considerably reduce global crs cases [16,17]. this has stirred the world global assembly to endorse the global measles and rubella strategic plan in april 2012 [18] which is targeted at eliminating rubella in at least 5 who regions by 2020 [19]. however, routine administration of the rubella vaccine is yet to be implemented in many areas in nigeria most especially in ido-ekiti were this study was carried out. routine diagnosis for the rubella virus is also not practiced in this location just as the case is in many other locations in nigeria. thus, a paucity of empirical data on serosurvey of rubella. though some researchers have conducted serosurveillance in other locations, this is the first to be carried out in ido-ekiti and environs. this makes it pertinent to conduct this serosurvey in this location as findings may propel informed decision making and definition of the best interventional approach by those saddled with such responsibilities. findings of such investigation may be cardinal to the estimation of akele et al.; aji, 3(1): 199-207, 2020; article no.aji.57099 201 the incidence of crs and conversely crs burden at ido-ekiti and its environs. this study was therefore designed to find out the seroprevalence of anti-rubella igg antibodies (a marker of prevalence rate), anti-rubella igm (a marker of incidence rate), and some sociodemographic cardinals that may influence the distribution of the disease among pregnant women in ido-ekiti. 2. methodology 2.1 study area and population this cross-sectional study was conducted at the antenatal clinic of federal teaching hospital idoekiti (fethi). fethi is a referral hospital that receives patients from all over ekiti state and neighboring states like kwara and kogi state. it is sited in a sub-urban community located in idoosi local government area of ekiti state nigeria, on latitude 7.843093 and 5.182314 (find coordinates.https://www.distancesto.com/coordi nates/ng/ido-ekiti-latitude-longitude/history/759 44.htmllast accessed 04 april 2020) [20]. the study population comprised of 184 pregnant women at various stages of pregnancy that consented to participate in the study. 2.2 sample collection and processing a brief enlightenment on the basis, benefits and demerit of the study was given and then a written consent obtained from volunteers. a structured questionnaire was administered to those who indicated interest in participating in the study. the questionnaire was designed to collect sociodemographic data and data on possible risk factors that may predispose subjects to infection [14]. after duly filling the questionnaire, 3 mls of venous blood was collected into a labeled plain tube using standard aseptic procedures. blood samples were allowed to clot, aseptically, dislodged, and centrifuged at 2,500 rpm for 5 minutes [21]. sera were then separated into welllabeled cryovials, transported to the medical microbiology laboratory of afe babalola university, ado-ekiti on ice packs. the samples were stored at -20°c until ready for laboratory analysis. samples were collected over a period of four months. 2.3 sample analysis sera samples were analyzed for rubella igg and igm antibodies in the medical microbiology laboratory of afe babalola university ado-ekiti using the enzyme-linked immunosorbent assay (elisa) method. rubella igg and igm precoated elisa kits were obtained from dia pro. diagnostic bioprobes srl®, columella millano, italy. the analysis was carried out according to the manufacturer’s instructions. briefly, reagents provided were allowed to attain room temperature for 15 minutes before use. the 40x wash buffer was diluted with distilled water using a ratio of 1:40 before use. the micro-titer plate template was set up with 1 well as blank, 2 wells as negative control and 2 wells as the positive control. 10 µl of sera sample and 90µl sample diluent were dispensed into the respective wells except for the blank well, negative control well and positive control well. 100 µl of the negative and positive controls were dispensed into their wells respectively. the content was mixed by vibrating the plate gently. the microplate was covered with a sealing paper and incubated in a microplate incubator (marvotech plate incubator, china) at 37°c for 60 minutes [21]. after incubation, the microplate was washed five times using wash buffer. 100 µl of horseradish peroxidase enzyme (hrp) conjugate was added to each well except the blank; the microplate was covered with a sealing paper and also incubated in a microplate incubator at 37°c for 15 minutes. after incubation, the microplate was washed five times with the diluted wash buffer in an automatic plate washer (marvotech plate washer, china) [21]. 50 µl of substrate solution a and b were added to each well respectively and were mixed; the plate was covered and incubated at 37°c for 15 minutes. 50 µl of stop solution was added to each well and mixed. the absorbance was read in an elisa reader machine (marvotech elisa reader, china) at a wavelength of 450 nm [21]. 2.31 interpretation of rubella igg/igm result if the mean negative control o.d ≤ 0.1and the mean positive control o.d ≥ 0.8, the test is valid. cut-off o.d = the mean o.d value of the negative control × 2.1 positive results: sample o.d ≥ cut-off o.d negative results: sample o.d < cut-off o.d 2.4 statistical analysis the data obtained from questionnaires administered and those obtained from the laboratory analysis were managed and analyzed using the epi-info version 7.2 statistical akele et al.; aji, 3(1): 199-207, 2020; article no.aji.57099 202 package. results were reduced to percentiles and a pearson chi-square test was performed at a 95% confidence interval. p-values less than 0.05 (p< 0.05) was used to determine relationships between parameters evaluated and seroprevalence. 3. results and discussion a total of one hundred and eighty-four pregnant women attending antenatal clinic at the federal teaching hospital ado ekiti were enrolled in this study. their ages ranged between 16 to 41 years with a mean age of 28±13 years (table 1). one hundred and seventy-six (95.6%) of the subjects were seropositive for rubella igg and 22 (12.0%) for rubella igm. twenty (10.9%) were seropositive for both rubella igg and igm, 2 (1.1%) only for rubella igm; and 154 (83.7%) for rubella igg. seropositivity was higher among age group above 35 years: 12 (100%) for igg and age group 31-35 years, 6 (18.8) for igm (table 1). the distribution was however non-statistically significant (p = 0.50 and 0.85 respectively). higher but non-significant proportion of subjects in the third trimester 62 (98.4%) and second trimester 84 (96.6%) as against 30 (88.2%) in the first trimester were seropositive for igg. in contrast to this pattern, a higher proportion of the subjects in first trimester 8 (23.5%) and second trimester 12 (13.8%) as against 2 (3.2%) in the third trimester were seropositive for rubella igm. the highest rate of co-positivity for rubella igg and igm was observed in the second trimester 15 (17.2%) (table 2). the married ones among the subjects had a higher seroprevalence 152 (97.4%) for rubella igg while the singles had a higher seroprevalence 6 (26.1%) for rubella igm (table 3). the subjects with no form of formal education, zero number of parity and some level of knowledge of the virus had the least seroprevalence for rubella igg 3 (60.0%), 53 (91.3%), 32 (94.1%) respectively. on the other hand, those with zero number of parity had significant (p = 0.04) seronegativity for rubella igm 56 (93.6%). despite the global public health implication of rubella infection which has stimulated the endorsement of the global measles and rubella strategic plan in 2012, which was planned to cover 2012-2020, it is a thing of concern that there is still a paucity of epidemiological data on seroprevalence of rubella infection in nigeria. the few that exist are skewed to areas like zaria, jos, maiduguri, abia, and osogbo. this impact negatively on making rightful decisions on intervention approaches to curb the incidences of crs. rubella igg serostatus is an indicator of previous exposure to the virus or immunization, thus it is used as a reliable biomarker for the prevalence of the disease where vaccination history is known. on the other hand, igm is an indication of a fresh infection and as such used to estimate the incidence of the disease. when igg co-exist with igm, it could be an indicator of an infected person who is just recovering and rarely reinfection. the seronegative population for igg is the susceptible population which is considered high risk. in this present study, 95.6% of the pregnant women enrolled were seropositive for anti-rubella igg antibodies. this high seroprevalence is similar to 97.9% report by muhammad et al. [22], 93.1% by olajide et al. [5] and 90.2% by gubio et al. [23] all in zaria, nigeria. a lower seroprevalence was reported from a closer location to that of this present study by kolawole et al. [24] who reported 87.5% igg seroprevalence in osogbo, south-west of nigeria. this lower prevalence is likely due to the time lag between the two studies. lower igg seroprevalences have also been reported by hamdan et al. [25] in sudan (65.3%) and oyinloye et al. [26] in maiduguri, nigeria. this indicates variability in the epidemiology of the virus from one location to another. we also recorded 12% seropositivity for antirubella igm. this was a little higher than the findings of mengouo et al. [27] in cameroon who surveyed 2008 -2014. they, however, record 12.4% igm seroprevalence in 2012. the findings of getehun et al. [13] and junaid et al. [28] were higher (39.4 and 45.2% respectively) possibly because their studies were not restricted to pregnant women and time lag. from this present study, 11% of subjects positive for igm were also positive for igg while 1% was just igm. this could imply that the 12% got infected in the recent past but the 11% with igg and igm are already recovering while the remaining 1% was yet to produce igg (meaning they are still at the active stage of infection). incidentally, the 1% population with only igm were all in the first trimester; a high-risk period. the other 12% thought to have recovered were all distributed through the first, second, and third trimester of pregnancy. these imply that most of them especially those in the first and early part of the akele et al.; aji, 3(1): 199-207, 2020; article no.aji.57099 203 table 1. seroprevalence of rubella igg and igm based on the age group age group (years) number rubella igg rubella igm rubella igg and igm positive (%) p-value positive (%) p-value positive (%) p-value 16-20 8 5 (62.5) 1 (12.5) 1 (12.5) 21-25 72 69 (95.8) 8 (11.1) 7 (9.7) 26-30 60 59 (98.3) 0.50 2.34 6 (10.0) 0.89 0.58 5 (8.3) 0.15 3.76 31-35 32 31 (96.9) 6 (18.8) 6 (18.8) above 35 12 12 (100) 1 (8.3) 1 (8.3) total 184 176 (95.6) 22 (12.0) 20 (11.0) table 2. seroprevalence of rubella igg and igm in relation to stages of pregnancy trimester number rubella igg rubella igm rubella igg and igm positive (%) p-value positive (%) p-value positive (%) p-value first 34 30 (88.2) 8 (23.5) 6(17.6) second 87 84 (96.6) 0.17 2.52 12 (13.8) 0.07 5.12 12 (13.8) 0.06 1.55 third 63 62 (98.4) 2 (3.2) 2 (3.2) total 184 176 (95.6) 22 (12.0) 20 (10.9) akele et al.; aji, 3(1): 199-207, 2020; article no.aji.57099 204 table 3. seroprevalence of rubella igg and igm antibodies in relation to some sociodemographic data sociodemographic data number rubella igg rubella igm positive (%) p-value positive (%) p-value marital status married 156 152 (97.4) 15 (9.6) single 23 21 (91.3) 0.76 1.56 6 (26.1) 0.56 0.85 separated 5 3 (60.0) 1 (20) educational status primary 52 49 (94.2) 6 (11.5) secondary 86 85 (98.8) 0.10 2.59 10 (11.6) 0.30 0.99 tertiary 41 39 (95.1) 5 (12.2) none 5 3 (60.0) 1 (20) religion christianity 143 140 (97.9) 17 (11.9) islam 37 34 (91.9) 0.71 0.91 5 (13.5) 0.32 2.12 others 4 2 (50.0) nil occupation student 15 11 (73.3) nil housewife 49 47 (95.9) 8 (16.3) public servant 41 40 (97.6) 0.07 3.10 5 (12.2) 0.07 3.46 trader 42 40 (95.2) 6 (14.3) farmer 37 34 (91.9) 3 (8.1) parity 0 58 53 (91.3) 2 (3.4) 1-2 79 76 (96.2) 0.05 3.14 14 (18.7) 0.04 3.75 3-4 29 29 (100) 6 (20.7) above 4 18 18 (100) nil knowledge of the virus yes 34 32 (94.1) 0.48 2.01 6 (17.6) 0.42 0.22 no 150 144 (96.0) 16 (10.7) akele et al.; aji, 3(1): 199-207, 2020; article no.aji.57099 205 second trimester must have been going through the active phase of infection during the first trimester which might put the fetus at the risk of crs. the susceptibility of these populations is 4% going by the igg seroprevalence, but considering that the remaining 11% which had both igg and igm are likely to have had a recent infection and were just recovering; the susceptibility could be placed at 15.2%. this is comparable with 13.7% susceptibility reported by tamirat et al. [14] in southern ethiopia. rubella infectivity is not associated with age (p > 0.05) though ages 16 – 20 had the least prevalence for igg likewise igm. this agrees with the report of muhammad et al. [22] but contrary to some others within the country. this was probably due to the high endemic nature of the virus in zaria, nigeria. the virus is said to be in constant circulation among the population thus downplaying the effect of some of the risk factors like age [22]. a similar situation is observed in this present study. higher rubella igm seroprevalence rates have also been documented among children and adolescents than in adults with the trend seen to decrease with an increase in age [23,28-32]. antibodies were found at every trimester which did not differ significantly from one another (p > 0.05), a finding that agreed with that of olajide et al. [5]. igg was highest in the third trimester and igm higher in the first trimester. this is contrary to fokunang et al. [33] who reported the highest prevalence in the second trimester. this difference may be accrued to cultural variation. some ethnic groups are more predisposed to presenting early at antenatal clinics during pregnancy than the others. formal educational status, religion, and occupation had no significant effect (p > 0.05) on the distribution of seroprevalence of igg and igm in this present study. this finding is similar to the findings of mohammed et al. [22]. it was observed that though a high percentage of the subjects had a tertiary or at least a secondary school level formal education, it did not cumulate to adequate knowledge about the virus. even the few who indicated knowledge of the virus did not request to be vaccinated as all subjects in this study claimed not to have been vaccinated against the virus. parity was however associated with the risk of rubella infection (p < 0.05). its effect was seen both on igg and igm serostatus. the nulliparous (zero parity) had a 93.1% igg prevalence and 3.4% for igm as compared to the multiparous that had at least 18.7% and more igm prevalence which aligns with the repost of onakewhor and chiwuzie [31] but contrary to that of olajide et al. [5] who reported no association between reproductive character and risk of rubella infection. parity may be associated with the risk of infection since mothers who have a number of children may stand a risk of being infected from the children [12]. the study is limited by the unwillingness of some of the participants to provide genuine information and inability to recall past experiences, hence, possibly introducing some biases to the study. the accuracy generalizations about pregnant women based on this study may also be limited because it is an institution based study. a subsequent community based longitudinal study may resolve these limitations. 4. conclusion this study reveals a high rate of exposure of pregnant women to rubella virus in this study area. this suggests continuous transmission and endermicity of rubella in the area. prevalence showed similar distribution pattern irrespective of sociodemographic features examined. however, infection is significant with respect to parity. the findings emphasize the need to implement routine childhood immunization against rubella virus as well as vaccinating susceptible women of childbearing age. consent the authors declare that written informed consent was obtained from the patients for the publication of this report. a copy of the written consent is available for review by the editorial office/chief editor/editorial board members of this journal. ethical approval the authors hereby declare that ethical approval for the study was obtained from the ethics and research committee, federal teaching hospital ido-ekiti, reference number: erc/2017/02/20/ 50b. subjects were fully informed of their right to opt-out of the study at any point they so desire. they were informed about investigations carried out with their samples, potential risk of the study, and were assured of confidentiality of the results. acknowledgements the authors are grateful to the authority of federal medical teaching hospital ido-ekiti who permitted us to carry out this study in their facility through the ethical committee. the cooperation akele et al.; 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asian journal of immunology 3(1): 145-159, 2020; article no.aji.56272 structure and function of covid-19 encode proteins in the transcription and replication mechanism with its preventive measures and propose efficacy treatments: a critical systematic review oguh c. e.1*, obiwulu e. n. o.2, oniwon w. o.3, okekeaji u.4, ugwu c. v.1, umezinwa o. j.5 and osuji c. a.1 1 department of biochemistry, university of nigeria, nsukka, enugu state, nigeria. 2 department of integrated science, delta state college of education, agbor, delta state, nigeria. 3 department of biochemistry, kogi state university, anyigba, nigeria. 4 department of pharmaceutical microbiology and biotechnology, university of nigeria, nsukka, enugu state, nigeria. 5 department of science laboratory technology, university of nigeria, nsukka, enugu state, nigeria. authors’ contributions this work was carried out in collaboration among all authors. author oce designed the study, wrote the protocol and manage the analyses of the study. author oeno wrote the first draft of the manuscript. authors owo, ou, ucv and uoj managed the literature searches. author oca read and edit the final manuscript. all authors read and approved the final manuscript. article information editor(s): (1) dr. cynthia aracely alvizo báez, autonomous university of nuevo leon, mexico. reviewers: (1) loc nguyen, vietnam. (2) lívia garcia bertolacci-rocha, universidade federal de goiás, brasil. (3) mujeeb shittu, michigan technological university, usa. complete peer review history: http://www.sdiarticle4.com/review-history/56272 received 03 april 2020 accepted 25 april 2020 published 02 may 2020 abstract the sudden occurrence outbreak of coronavirus disease in 2019 (covid-19) by the severe acute respiratory syndrome coronavirus2 (sars-cov-2) poses a serious harm worldwide and local economies. due to high numbers of infection and death, the pandemic calls for an urgent demand of active, effective, affordable and available drugs to control and diminish the pandemic. coronavirus disease 2019 is a public health unexpected and sudden crisis which required action of review article oguh et al.; aji, 3(1): 145-159, 2020; article no.aji.56272 146 international concern. at present there is no generally recognized effective pharmaceutical treatment to the disease, although it is to a great extent for patient contracting the severe form of the disease. the development of new strategies to prevent or control the spread of covid-19 infections and the understanding of the virus replication, and pathogenesis required immediate action. therefore, this systematic review was to investigate the biochemical effect of the virus in human, symptoms, prevention, statistics cases and summarize the evidence regarding chloroquine and hydroxychloroquine for the treatment of covid-19. keywords: coronavirus; mechanism; prevention; structure; treatment. abbreviations covid-19: coronavirus disease 2019; sars-cov2: severe acute respiratory syndrome coronavirus 2; s: spike; m: membrane; e: envelope glycoproteins; he: hemagglutinin esterase: n: nucleocapsid protein; rdrp: rnadependent rna polymerase; mhv: mouse hepatitis virus; ace2: angiotensin-converting enzyme 2; 3clpro: coronavirus main protease; plpro: papain-like protease; fecv: feline enteric coronavirus; fipv: feline infectious peritonitis virus; tgev: transmissible gastro enteritis virus; pedv: porcine epidemic diarrhea virus; prcov: porcine respiratory coronavirus; ccov: canine coronavirus; bcov: bovine coronavirus; grna: rna genome; dmv: double-membrane vesicles; rtc: replication and transcription complex; nsp3: non-structural protein3; cq: chloroquine; hcq: hydroxychloroquine; ees: early endosomes; els: endolysosomes; cryo em: cryo electron microscopy; ifa: immune fluorescence analysis; ergic: endoplasmic reticulum-golgi apparatus intermediate compartment. 1. introduction coronavirus is a novel disease that had not been previously identified in human of recent. coronavirus disease 2019 is a respiratory disease illness that is transitive from one individual to the other. the type of virus that causes covid-19 is a new inventive disease that was firstly recognize during a survey outbreak in december. 2019 at wuhan, china, this disease has spread to more than 213 countries with 2,931,787 confirmed cases and over 203,596 confirmed deaths worldwide as of april 26, 2020 [1] with u.s, spain, italy and france as the most affected countries with 960,896, 223,759, 195,351 and 161,488 confirm cases respectively. the name coronavirus, coined in 1968, is derived from the corona-like or crown-like morphology observed for these viruses in the electron microscope. the virus is highly contagious and thousands of new cases are reported around the world every day. the risk of infection from the virus that causes covid-19 is higher for people who have close contacts with people with the disease, such as healthcare workers and household members and other people at higher risk for this infection are those who are living in an area with ongoing spread of covid-19 or those who traveled to effected place. risk of death is only higher in older people (above an age of 60 years) and people with pre-existing health conditions. coronaviruses infect both animals and humans, especially animals such as bats, this animal host the largest variety of corona viruses shows to be immune to corona virus-induced illness in human [2]. researchers have been working to trace the achievable treatments in order to save lives and produce vaccines for future prevention and control. the viruses infect a variety of organisms such as human and animal host cells, and also carry out their infection and replication on the host. also, many proteins located on the virus have most important function in the replication mechanism, although the role is yet to be defined. in this case, it is important to investigate the role and importance of these proteins during mechanism. therefore; this review explains the structure, classification, symptoms, prevention, statistics cases and summarize the evidence regarding chloroquine and hydroxychloroquine for the treatment of covid-19. 1.1 structure of covid-19 coronaviruses are enveloped viruses with round and sometimes pleiomorphic virions of approximately 80 to 120 nm in diameter (fig. 1). the coronaviruses also contain positive-strand rna, which have the largest rna genome (approximately 30 kb) reported to date (178, 196). corona viruses (covid-19) are mainly large viruses containing a single-stranded positive-sense rna genome protected by membrane within a membrane envelope within the virus. the viral membrane is covered with oguh et al.; aji, 3(1): 145-159, 2020; article no.aji.56272 147 glycoprotein spikes that give coronaviruses their crown like appearance. covid-19 virus encode five structural proteins in their genomes. these are the spike (s), membrane (m), envelope (e) glycoproteins, hemagglutinin esterase he and nucleocapsid n protein, (fig. 1). the virus envelope proteins and n protein is embedded in all virion but he is only present in some beta coronaviruses. in addition to that, it is thought the virus particles are huddled together owing to interaction between these proteins [3,4]. 1. s glycoproteins: they are located outside the virion which gives the virion the typical shape. the s proteins form homotrimers, which allow the formation of sun-like morphologies that give the name of coronaviruses [5]. s proteins bind to the virion membrane via the c-terminal transmembrane regions, also they interact with m proteins [6]. the virions is attach to a specific surface receptors in the plasma membrane of the host cell through the nterminus of the s proteins. this s glycoprotein initiate host cell invasion by both sars-cov and sars-cov-2 via binding to a receptor protein called angiotensin-converting enzyme 2 (ace2) which is located on the surface membrane of host cells [7,8]. a study conducted by [9, 10], shows that the invasion process requires s protein priming which is accompany by the host cell produced serine protease. also the viral genome also encodes several nonstructural proteins which include the rna-dependent rna polymerase (rdrp), coronavirus main protease (3clpro) and papain-like protease (plpro) [9,10]. 2. m glycoproteins: the m glycoproteins on the virus have three transmembrane regions. the proteins are glycosylated in the organelle called golgi apparatus [11]. this reformation of the m protein is important for the virion to fuse into the cell and to make protein antigenic. the m protein plays a key important role in stimulate virions in the cell. the virus n protein also forms a complex by binding to genomic rna and the m protein elicit the production and formation of interacting virions in this endoplasmic reticulum-golgi apparatus intermediate compartment (ergic) with this complex [12,13]. 3. e glycoproteins: this are small proteins that are constitute of just about 76 to 109 different amino acids. approximately 30 different amino acids in the n-terminus of the e proteins permit attachment to the membrane of viruses [14]. in addition, coronavirus e proteins play a critical important role in pulling together and morphogenesis of virions within the cell. a study of coronavirus e and m proteins were expressed together with mammalian expression vectors to form virus-like structures within the cell [15]. in a study, there was a great decrease in the ability of the recombinant mouse hepatitis virus (mhv) and sars viruses to elicit e protein expression in the genome to support this status [16,17]. 4. n proteins: they are phosphor proteins that are capable of binding to helix and have elastic structure of viral genomic rna. it plays a crucial role in virion structure, replication and transcription of coronaviruses, because the n protein focus in both the replication and the transcriptional region of the coronaviruses and the ergic area where the virus is collected [16,17]. (fig. 2). 1.2 classification of coronaviruses the classification of coronaviruses is based on genomic organization, resemblance in genomic sequence, antigenic properties of viral proteins, replication strategies and structural characteristics of virions, pathogenic, cytopathogenic and physicochemical properties [18]. the coronaviruses are species of virus belonging to the nidovirales order, which comprises coronaviridae, arteriviridae, roniviridae and mesoniviridae families [19]. the arteviridae family consist of swine and equine pathogens, and the roniviridae family is composed of invertebrate viruses. the coronaviridae family is the largest among the four families, range from 26 to 32 kb by its genomic sizes of coronaviridae [20]. coronaviridae virus family subdivided into two subfamilies, coronavirinae and torovirinae. coronavirinaeis gulf into four genera, alpha coronavirus, beta coronavirus, gamma coronavirus and delta coronavirus (fig. 3). alpha coronaviruses type 1 species are classified into the porcine tgev (transmissible gastro enteritis virus), feline fcov, fecv (feline enteric coronavirus) and fipv (feline infectious peritonitis virus), porcine pedv (epidemic diarrhea virus), prcov (porcine respiratory coronavirus) and the canine ccov. alpha coronaviruses also agree with human covs such oguh et al.; aji, 3(1): 145-159, 2020; article no.aji.56272 148 as hcov-229e and hcovnl63, but various bat coronaviruses. beta coronaviruses infect a wide varieties of mammalians, with species such as human with sars-cov, mice, hcov-oc43, hcov-hku1, and mers-cov, bovine coronavirus (bcov), and murine coronavirus (mhv). likewise to sars-cov and mers-cov, sars-cov-2 violence the lower respiratory system to cause viral pneumonia in human, and may also affect the heart, kidney, gastrointestinal system, liver, and central nervous system leading to multiple organ failure [21,22]. current information indicates that sarscov-2 is more transmissible and very contagious than sars-cov [23].the beta coronavirus genome encrypt more than a few or several structural proteins, including the glycosylated spike (s) protein that functions as a major inducer of host immune responses. gamma coronaviruses are exact of birds, with one exclusion of a beluga whale coronavirus. the delta coronavirus genus was generated in 2012 and with various groups (hku11, hku12, hku13) coronavirus from mammals to birds [24] fig. 3. fig. 1. coronavirus virion. (a) electron micrograph of mouse hepatitis virus mhv particles. (b) schematic of virion fig. 2. cartoon illustration of the coronavirus structure and viral receptor ace2 on the host cell surface oguh et al.; aji, 3(1): 145-159, 2020; article no.aji.56272 149 fig. 3. classification of covid-19 2. covid-19 mechanism of action (replication and transcription) coronavirus entry starts with the s protein binding to a target receptor on the cell surface, where after fusion is mediated at the cell membrane, delivering the viral nucleocapsid inside the cell for subsequent replication. the action of coronaviruses replication occurs in the cytoplasm of the host cell. the viruses mainly bind to the host receptor cell surface through the spike (s) protein. a conformational structure occurs in the structure and the process of entry into the virus cell start when s protein is bound to the receptor [25]. this process with endocytosis is reliant of ph through the receptor. the virus particle releases the rna genome after going into the cytoplasm. this genome is a nonsegmented rna virus with the largest known rna genome (grna), single-stranded, which is approximately 26-32 kb. the genome consists of seven several genes. it is prearranged into 5’ non-structural protein coding regions include the replicase genes (gene 1), which are two-thirds of the genome, and 3’ structural and nonessential accessory protein coding regions including the gene 2-7 [26]. the replicase gene 1 products are encoded two very large open reading frames orf1a and 1b, which are interpreted or translated into two large polypeptides pp1a and pp1b, which are produce and synthesized directly from the 5’ two-thirds of the genomic rna of cov (fig. 4). after the protein have been synthesized, which is made up of 16 units, non-structural protein (nsp1 to nsp16) is change with the influence of viral proteases pp1a and pp1b [27]. same time, the double-membrane vesicles (dmv) is virus replication and transcription complex (rtc) [28,29]. the 16 unit’s proteins form dmv. these nsp proteins, particularly non-structural protein3 (nsp3), have an important function in the virion structure, and the replication and transcription of cov [30,31]. genes 2 to 7 are transform from sub genomic mrna. sub genomics rnas encode the main viral envelope protein (e), structural proteins (s), membrane protein (m), nucleocapsid protein (n), and the accessory proteins, which are vital for virus-cell receptor binding. the recently structural synthesized proteins are released into the endoplasmic reticulum. all of these proteins, along with the n protein, are connected to the viral genomic rna and localized in the ergic region [28,32] as shown in fig. 4. although, n protein is known to be very essential for coronavirus replication, the exact function or role that this protein plays in this process remains unidentified. but, several research suggested that n protein interact with nsp3, which plays a critical and important role in the virus replication early in infection. the communication between viral s protein and angiotensin-converting enzyme 2 (ace2) on the host cell surface is of important interest since it initiates the infection process. cryoelectron microscopy (cryo-em) structure analysis has make known that the binding affinity of sarscov-2 s protein to ace2 is approximately 10−20 times higher than that of sars-cov s protein binding to ace2 [33,34]. it is consider that the binding affinity may contribute to the reported oguh et al.; aji, 3(1): 145-159, 2020; article no.aji.56272 150 fig. 4. mhv genome organization and replicase proteins fig. 5. life cycle of coronavirus in a host cell higher transmissibility spread and contagiousness of sars-cov-2 as compared to sars-cov [23]. the search also exists for discovery a breakthrough of therapeutic agents such as drugs and vaccine for targeting the highly preserved proteins related with both sars-cov and sars-cov-2 proteins [34-37]. rnadependent rna polymerase (rdrp) and coronavirus main protease (3clpro) of sarscov-2 share over 95 % of sequence similarity with those of sars-cov notwithstanding the fact that these two viruses shows or demonstrate only 79 % sequence similarity at the genome level proteins [34-37]. on the foundation of oguh et al.; aji, 3(1): 145-159, 2020; article no.aji.56272 151 sequence arrangement and homology modeling, sars-cov and sars-cov-2 share an extremely preserved receptor-binding domain (rbd), a domain of s protein and 76% of sequence similarity in their s proteins [34-37]. although the papain-like protease (plpro) sequences of sars-cov-2 and sarscov are only 83% similar, they share the same active sites [35]. 3. transmission of covids-19 the virus that causes covid-19 most likely emerged or come out from an animal source, which is now spreading from one person to the other. via respiratory droplets generate when an infected individual sneezes or coughs can extend the spread of the virus to close contact (within about 6 feet length). the covid-19 can also be contracted by touching a surface or object that has been contaminated with the virus from an infected person and then touching their own mouth, nose, or perhaps their eyes. 3.1 symptoms of covid-19 patients with covid-19 will eventually have slight to severe respiratory illness with symptoms of:  fever  cough  running nose  sore throat  body ache  shortness or difficulty breathing  at severe complications from this virus such as pneumonia in the lungs, multi-organ failure and in some cases lead to death. fig. 6. common and uncommon symptoms of covid-19 oguh et al.; aji, 3(1): 145-159, 2020; article no.aji.56272 152 3.2 preventive actions and spreading of covid-19 to others  people should avoid close by contact with people sneezing and coughing and people that are sick.  avoid touching face, eyes, nose, and mouth with unwashed hands.  washing of hands regularly with soap and running water for at least 30 seconds. also the use of alcohol-based hand sanitizer that contains at least 65% alcohol if soap and water are not available.  stay home when you are sick.  people with covid-19 symptoms produce a respiratory droplets when they cough or sneezes so, should practice cough manners or etiquette i.e maintaining distance when coughing or sneezing, cover coughs, wear face mask, sneezes with disposable tissues or clothing, and wash hands regularly.  clean and disinfect frequently all touched objects and surfaces.  limit person-to-person transmission which include reducing secondary infections amidst close by contacts and health care workers, and preventing transmission amplification.  identify, isolate and be concerned for patients timely, including providing enhance care for infected patients to avoid spread of the disease.  avoid unprotected contact with wild animals to identify and reduce spread of virus from the animal source.  converse critical risk and event information to all communities and counter misinformation.  address all crucial unknowns regarding clinical harshness, extent of transmission and infection treatment options, and accelerate the development of diagnostics, therapeutics and vaccines.  minimize social and economic impact through multispectral partnerships.  people that have traveled from an affected area, should be isolated and restrictions for 2 weeks. if the patient develop symptoms during the isolation period with fever, cough, and trouble breathing, then seek for medical advice or call the health care provider for covid-19 in the community before telling them about the travel and the symptoms. fig. 7. countries areas with reported confirmed cases of covid-19, 26 april 2020 by ecdc oguh et al.; aji, 3(1): 145-159, 2020; article no.aji.56272 153 fig. 8. epidemic curve of confirmed covid-19, by date of report and who region through 25 march 2020 fig. 9. distribution of covid-19 cases worldwide, as at 26 april, 2020 by ecdc 3.3 summary situation worldwide as at 26 april 2020 reported by ecdc [38]  globally: 2,931,787 confirmed cases worldwide and 203,596 deaths worldwide.  asia: 456350 confirmed, and most case were reported in turkey, iran, china, india and saudi arabia with 107773, 89328, 83909, 26496, and 16299 respectively and 16865 deaths, most death are in iran, oguh et al.; aji, 3(1): 145-159, 2020; article no.aji.56272 154 fig. 10. distribution of covid-19 death cases worldwide, as at 26 april, 2020 by ecdc china, turkey, india and indonesia with 5650, 4636, 2706, 824 and 720 death respectively.  america: 1134686 confirmed, most reported countries are us, brazil, canada, peru and mexico with 939053, 58509, 45341, 25331 and 13842 respectively and 63649 deaths, most death were recorded in us, brazil, canada, mexico and peru with 53189, 4016, 2465, 1305 and 700 death respectively.  europe: 1214584 confirmed, five most confirm case were in spain, italy, germany, united kingdom and france with 219764, 195351, 154175, 148377 and 124114 respectively and 119306 deaths, most death were recorded in italy, france, spain, united kingdom and belgium with 26384, 22614, 22524, 20319 and 6917 death respectively.  oceania: 8080 confirmed, five most cases were in australia, new zealand, guam, frence polynesia and fiji with 6703, 1121, 141, 57, 18 death respectively and 106 deaths, with the most death case recorded in australia, new zealand, guam and northern mariana islands with 81, 18, 5, 2 death respectively.  african region: 30316 confirmed cases, five most countries are south africa, egypt, morocco, algeria and cameroon with 4361, 4319, 3897, 3256 and 1518 respectively and 1382 deaths, the top most countries are algeria, egypt, morocco, south africa and cameroon with 419, 307, 159, 86 and 53 death cases respectively  others: 696 confirmed and 7 deaths 4. treatment 4.1 efficacy and safety of chloroquine, remdesivir (gs-5734) and hydroxychloroquine for the treatment of covid-19 coronavirus disease-2019 (covid-19) is a public health emergency of international concern. as of this time there is no recognize exact, effectual, proven, pharmacological treatment for the cure of covid-19. invitro studies have suggested that chloroquine, an immune modulant drug traditionally used to cure malaria, is effective in reducing viral replication in other contagion, as well as the sars-associated coronavirus (cov) and mers-cov [39,40]. chloroquine (n4-(7-chloro-4-quinolinyl)-n1,n1diethyl-1,4-pentanediamine) (cq) has been used worldwide for above 80 years, and it is part of the world health organization (who) model list of vital medicines. the drug is also inexpensive and has a set up clinical safety profile. however, the effectiveness and safety of the drug chloroquine for treatment of sars-cov-2 (the novel virus causing covid-19) pneumonia remains indistinct. oguh et al.; aji, 3(1): 145-159, 2020; article no.aji.56272 155 a group of chinese researchers, from literature have studied the impact of chloroquine in vitro, using vero e6 cells contaminated by sars-cov2 at an array of contagion (moi) of 0.05. the study established the fact that chloroquine was extremely effectual in decrease viral n protein replication, with an effectual dosage (ec) 90 of 6.90 μm that can be easily attainable with normal standard dosing, due to its advantageous penetration in tissues, including in the lung. the authors described that chloroquine is known to obstruct virus contagion by cumulating endosomial ph and by meddling with the glycosylation of cellular receptor of sarscov. also guesswork on the possibility that the known immunomodulant effect of the drug may improve the antiviral effect in vivo [41]. a description letter written by chinese authors give an account that a news meeting from the state council of china had stated that chloroquine phosphate had established marked effectiveness and satisfactory safety in treating covid-19 related pneumonia in multi-center clinical trials conducted in china. the authors also specified that these discovery came from more than 100 patients included in the trials [42]. evidence of such data was sought in the trial registries to review and was found none. so far cases had been reported in 213 countries as at 26th april 2020, the low cost of chloroquine is a main advantage for both the extreme stressed healthcare systems of involved elevated-income countries and none funded healthcare systems of mid-and low-income counties. the skilled agreement was in print on 20th february by a multicentre cooperation group of the department of science and technology of guangdong province and health commission of guangdong province paper and have to do with specifically to the use of chloroquine phosphate. no information was provided on condition that the method used to attain agreement [43]. based on the in vitro evince and still not published clinical experience, the board recommended and endorsed chloroquine phosphate tablet, at a dose of 500 mg twice per day for 10 days, for patients diagnosed as slight mild and severe cases of sars-cov-2 pneumonia, on condition that there were no opposition or contraindication to the drug. research have shown that chloroquine (cq) and remdesivir (gs-5734), efficiently inhibited sarscov-2 contagious in vitro. remdesivir is a nucleoside analog pro drug technologically advanced by gilead sciences (usa). a clinical report in united states showed that treatment with remdesivir improved and better the clinical state of the first patient infected by sars-cov-2 [44] and a stage iii clinical trial of remdesivir in opposition to sarscov-2 was open in wuhan on february 4, 2020. although, as an experimental drug, remdesivir is not likely to be largely obtainable for treating a very large number of patients in a timely way. however, of the two potential drugs for treating of covid-19, cq happen to be the best drug of choice for large-scale use because of its obtainability and availability, a safety record proven, and a moderately low in cost. chloroquine cq (n4-(7-chloro-4-quinolinyl)-n1, n1-diethyl-1,4-pentanediamine) have been in used for long in the treatment of malaria and amebiasis. however, plasmodium falciparum technologically advanced extensive resistance to it, and with the advancement of new anti-malarial drugs, cq has become a choice for the prophylaxis of malaria. furthermore, over dose of cq can cause acute or severe poisoning and death [45]. in the past years, due to occasional use of cq in clinical practice, its manufacture and market supply was very much reduced, at least in china. hydroxychloroquine (hcq) sulfate, is a derivative of cq, and was first synthesized in 1946 by initiating a hydroxyl group oh into cq. hcq establish and demonstrated to be much less (~40%) toxic than cq in animals [46]. hcq is still extensively obtainable and available to treat autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus. since cq and hcq have similar chemical structures and mechanisms of acting as a feeble base and immune modulator, hcq have shown to be more effective and potent drugs to treat sars-cov-2. in fact, as of february 23, 2020, seven clinical trial records were found in chinese clinical trial registry for using hcq to treat sars-cov-2. what makes hcq to be more efficacious than cq in treating sars-cov-2 infection still be short of experimental prove. liu et al. [47] assess the antiviral impact of hcq against sars-cov-2 infection in comparison to cq in vitro. initially, the cytotoxicity of hcq and cq in african green monkey kidney veroe6 cells (atcc-1586) was determined by standard cck8 assay. the result demonstrated that the 50% cytotoxic concentration (cc50) values of cq and hcq were 273.20 and 249.50 μm, respectively, oguh et al.; aji, 3(1): 145-159, 2020; article no.aji.56272 156 which shows a no significant different from each other. the antiviral activity of cq versus hcq was compared, and the dose–response curves of the two compounds against sars-cov-2 were evaluated at four several multiplicities of infection (mois) by enumerate of viral rna copy numbers in the cell supernatant at 48 h post infection (p.i.). the result showed that, at all mois (0.01, 0.02, 0.2, and 0.8), the 50% maximal effectiual concentration (ec50) for cq were 2.71, 3.81, 7.14 and 7.36 μm respectively, and hcq were 4.51, 4.06, 17.31, and 12.96 μm respectively at all mois. cq values was lower than that of hcq. the differences in ec50 values were statistically significant at an moi of 0.01 (p < 0.05) and moi of 0.2 (p < 0.001). consequently, the selectivity index (si = cc50/ec50) of cq (100.81, 71.71, 38.26, and 37.12) was higher than that of hcq (55.32, 61.45, 14.41, 19.25) at mois of 0.01, 0.02, 0.2, and 0.8 respectively. these results were verify by immune fluorescence microscopy as indication by several expression stage of the virus nucleoprotein (np) at the indicated drug concentrations at 48 h p.i. this study suggest that the anti-sars-cov-2 activity of hcq appear to be less strong or potent compared to cq, at slightest at certain mois. the two drugs cq and hcq are feeble bases that are known to raise the ph of acidic intracellular organelles, such as endosomes/lysosomes, vital for membrane synthesis. furthermore, cq could inhibit sars-cov entry via altering the glycosylation of ace2 receptor and spike protein [48]. extra additional experiment confirmed that hcq efficiently inhibited the entry step, in addition to the postentry stages of sars-cov-2, which was also found upon cq treatment. to further discover the full mechanism of action of cq and hcq in inhibiting the covid-19 entry, co-localization of virions with early endosomes (ees) or endo lysosomes (els) was examine by immunofluorescence analysis (ifa) and confocal microscopy. quantification examination showed that, at 90 min p.i. in untreated cells, 16.2% of adopt virions (anti-np, red) were detected in early endosome antigen 1 (eea1)-positive ees (green), whereas more virions (34.3%) were transfer into the late endosomal–lysosomal protein lamp1+ els (green) (n > 30 cells for each group). by difference, in the presence of cq or hcq, significantly more virions (35.3% for cq and 29.2% for hcq; shows a significant p <0.001) were detected in the ees, while only very few virions (2.4% for cq and 0.03% for hcq; p < 0.001) were found to be co-localized with lamp1+ els (n > 30 cells). this suggested that both cq and hcq obstruct the transport of sars-cov-2 from ees to els, which happen to be a condition to release the viral genome as in the case of sars-cov [49]. however, cq and hcq treatment caused obvious changes in the number and size/morphology of ees and els. in cq and hcq-treated cells, unusually increase ee vesicles were seen, arrows in the upper panels), many of which are even larger than els in the untreated cells. in the untreated cells, most ees were much smaller than els. previous report study have showed that treatment with cq induced the formation of expanded cytoplasmic vesicles [50]. within the ee vesicles, virions (red) were contained around the membrane (green) of the vesicle. the treatment with cq did not cause noticeable changes in the number and size of els; however, the regular vesicle structure seemed to be disrupted, at least partially. by difference, in hcq-treated cells, the size and number of els raised significantly, arrows in the lower panels. since acidification is important and vital for endosome maturation and function, then surmise that endosome maturation might be blocked at midway stages of endocytosis, resulting in failure of additional transport of virions to the ultimate releasing site. cq was reported to elevate the ph of lysosome from about 4.5 to 6.5 at 100 μm [51]. research have shown that oral absorption of cq and hcq in humans is very effective in the treatment of sars-cov-2. in animals, both drugs share similar tissue distribution patterns, with high concentrations in the liver, spleen, kidney, and lung spread levels of 200–700 times higher than those in the plasma [52]. it was reported that safe dosage (6–6.5 mg/kg per day) of hcq sulfate could generate serum levels of 1.4–1.5 μm in humans. therefore, with a safe recommended dosage, hcq concentration in the above tissues is likely to be achieved to inhibit sars-cov-2 infection. 5. conclusion coronaviruses are diverse family of viruses that interact at various levels with constituent of host cells taking this benefit of some of the cellular oguh et al.; aji, 3(1): 145-159, 2020; article no.aji.56272 157 machineries for replication and proliferation. in conclusion, cq and hcq can efficiently inhibit sars-cov-2 infection in vitro and it is more effective with hcq. in combination with its antiinflammatory function, and was predict that the drug has a good effect to combat the virus disease. this possibility awaits confirmation by clinical trials. hcq is less toxic than cq, prolonged and overdose usage of hcq can still cause poisoning. although the use of chloroquine can also support expert opinion, clinical use of this drug in patients with covid-19 should stick to the ethical approval as a trial as stated by the who. consent it is not applicable. ethical approval it is not applicable. competing interests authors have declared that no competing interests exist. references 1. coronavirus disease (covid-2019) situation reports 1−45; world health organization; 2020. available:https://www.who.int/emergencies /diseases/novel-coronavirus2019/situation-reports 2. anthony sj, johnson ck, greig dj, kramer s, che x, wells h, hicks al, joly do, wolfe nd, daszak p, karesh w, lipkin wi, morse ss, mazet jak, goldstein t. global patterns in coronavirus diversity. virus evol. 2017;3(1):vex012. 3. de haan ca, rottier pj. molecular interactions in the assembly of coronaviruses. adv virus res. 2005;64: 165-230. 4. masters ps. the molecular biology of coronaviruses. adv virus res. 2006;66: 193-292. 5. tan yj, lim sg, hong w. characterization of viral proteins encoded by the sarscoronavirus genome. antiviral res. 2005; 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this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: http://www.sdiarticle4.com/review-history/56272 https://doi.org/10.1038/s41422–020–0282–0 https://doi.org/10.1038/s41422–020–0282–0 https://doi.org/10.5582/bst.2020.01047 https://doi.org/10.5582/bst.2020.01047 https://doi.org/10.1038/s41421-020-0156-0 https://doi.org/10.1038/s41421-020-0156-0 http://creativecommons.org/licenses/by/4.0 _____________________________________________________________________________________________________ *corresponding author: e-mail: nazmul_pharmacy@yahoo.com; (†)authors contributed equally asian journal of immunology 3(1): 173-181, 2020; article no.aji.56428 investigation of neuropharmacological effects of flemingia stricta (roxb.) leaves md. shahrear biozid†1, mohammad nazmul alam†1*, md. jainul abeden†1, md. masudur rahman1 and md. rafikul islam1 1 department of pharmacy, international islamic university, chittagong, kumira, chittagong, bangladesh. authors’ contributions this work was carried out in collaboration among all authors. authors msb, mmr, mna and mri proposed and designed the study. authors msb, mna and mja conducted all laboratory experiments. authors msb, mmr, mna and mri analyzed and interpreted experimental results as well as participated in manuscript preparations. all authors read and approved the final manuscript. article information editor(s): (1) dr. darko nozic, university of belgrade, serbia. reviewers: (1) k. d. p. p. gunathilake, wayamba university, sri lanka. (2) dra poliana guerino marson, federal university of tocantins, brazil. (3) isabel c. gomez-betancur, university of antioquia, colombia. (4) john ogedengbe, university of abuja, nigeria. complete peer review history: http://www.sdiarticle4.com/review-history/56428 received 02 march 2020 accepted 07 may 2020 published 18 may 2020 abstract background: traditional preparation of the leaf of flemingia stricta (fabaceae) roxb. , a medicinal plant of the indian subcontinent, has been used for the treatment of different diseases as a herbal preparation. our purpose was to analyze the neuropharmacological effects of different chemical extracts of flemingia stricta roxb. in mice. methods: in present study, the anxiogenic activity of crude extracts of flemingia stricta leaves was determined using standard animal behavioral models, such as hole cross and open field; sedative and anxiolytic potential were evaluated by performing thiopental sodium induced sleeping times tests and elevated plus maze test respectively. results: the crude extracts at the doses of 200 and 400mg/kg exhibited a significant dosedependent suppression of movement of mice in both open field and hole cross test. in the anxiolytic and sedative study, extracts displayed an increased percentage of entry of mice into open arm at both doses which are 200 and 400mg/kg. plant extracts produced a significant increase in sleeping original research article biozid et al.; aji, 3(1): 173-181, 2020; article no.aji.56428 174 duration and reduction of onset of sleep compared to control at the both doses of 200 and 400 mg/kg. conclusion: this study clearly showed that the treated extracts have promising anxiolytic and sedative effect. further studies on the prime constituent of this plant extract may provide lead compound in future. keywords: flemingia stricta; sedative; elevated plus maze; anxiolytic; locomotor activity; neuropharmacology. abbreviations f. stricta: flemingia stricta; epm: elevated plus maze; gaba: gamma-aminobutyric acid; cns: central nervous system, fsm: f. stricta methanol, fsh: f. stricta n-hexane, fsc: f. stricta chloroform, fsa: f. stricta aqueous, fse: f. stricta ethanol. 1. background from ancient times herbal drugs are called as green medicine for their safe and trustworthy health care paradigms. furthermore, herbal drugs are cost effective than chemically manufactured drugs. due to these benefits, public as well as industrial interests to the traditional herbal medicines are rising day by day [1]. people are leading a hectic life in this 21 st century. too much pressure in daily life increases anxiety and depression in people which results in psychiatric disorders around 20% of the adult population [2-4]. this is an alarming situation for our future generation. therefore, there is an increasing demand for the discovery of anxiolytic, sedative drugs. anxiolytic and sedative drugs act on central nervous systems (cns) which are extensively used as effective pharmacological agents to treat psychological disorders [5]. benzodiazepines are among the most prescribed and effective antianxiety drugs used worldwide [6]. barbiturates and ethanol are also frequently used. both barbiturates and benzodiazepines demonstrate their effect by binding with gamma aminobutyric acid receptor (gabaa receptor) [7]. day to day use of benzodiazepines and barbiturates might cause psychological and physiological dependence which leads to drug tolerance. as a result, repeated uses of this type of drugs can decrease the effectiveness. safety is the main issue of barbiturates as depressant because only ten times of their pharmacological dose might be lethal [8-12]. according to the statistical data, alcohol addiction in american society for men and women is 5% to 10% and 3% to 5% respectively. hence, a natural cns depressant with minimum or no toxicity is therefore, essential [13]. flemingia stricta (fabaceae) roxb. is distributed in southeast asia which includes bangladesh, bhutan, china, india, indonesia, laos, myanmar, philippines, thailand and vietnam [14,15]. it is an erect subshrub. it is found in hilly areas of bangladesh and it has various traditional names given by local tribes [16]. chakma healers traditionally use this plant for the treatment of polio. it has also several traditional benefits which consist of treat rheumatism followed by bone fracture, cough, asthma, goiter, urinary problems, snake bite, insect bite, leprosy, tumor and cancer, caries, hysteria, tuberculosis, insomnia and intestinal worms [17-19]. according to the literature review on this plant, it showed that the plant has been used as traditional medicine for the treatment of various diseases for many years. furthermore, we also performed in-vitro antioxidant and phytochemical studies on this plant extract which paved our way to go for animal study [20]. therefore, we undertook the study to assess the neuropharmacological potential of f. stricta leaves, by using animal models and studying the effect of the different chemical plant extracts on their exploratory behavior. 2. methods 2.1 plant collection and identification whole plants of f. stricta were collected from bhatiary, chittagong region, bangladesh. the plants were identified by dr. shaikh bokhtear uddin, taxonomist and professor, department of botany, university of chittagong, chittagong, bangladesh. 2.2 preparation and extraction of leaf extract the collected leaves were thoroughly washed with distilled water and dried under the shade. biozid et al.; aji, 3(1): 173-181, 2020; article no.aji.56428 175 the dried sample was coarsely powdered (500 g) and extracted with methanol for 3 days to allow the total extraction process. after that the plant extract was filtered with sterilized cotton filter and the filtrate was gathered in a beaker. the plant extracts then kept in a water bath at 60 °c to evaporate the solvent from the solution. the container allowed to airtight for 72 h and filtrate thus obtained was concentrated by using a rotary evaporator. the extract was divided into two portions. one portion (2.5 g) was poured into glass vials to be tested as crude methanol extract, whereas the second portion (8 g) was dissolved in concentrated methanol and partitioned successively into four different extracts [21]. the fractions were then concentrated using a rotary evaporator. 2.3 animal male swiss albino mice, 3-4 weeks old, weighing between 20-25 g, were collected from the international center for diarrheal disease and research, bangladesh. animals were maintained under standard environmental conditions [temperature: (24±1)°c, relative humidity: 55%65% and 12 h light/12 h dark cycle] and had free access to feed and water ad libitum. prior to experimentation, animals were familiarized in laboratory conditions for one week. 2.4 acute toxicity study mice were divided into control and test groups (n = 5). the test groups received the extract per orally at the doses of 400, 600, 800 and 1000 mg/kg. then the animals were kept in separate cages and were allowed to food and ad libitum. the animals were observed for possible behavioral changes, allergic reactions and mortality for the next 72 h [22]. 2.5 neuropharmacological tests the study was done to find out if extracts had any effect on the central nervous system. effect on the exploratory behavior of mice was evaluated by hole cross test and open field test. elevated plus maze test was conducted for determination of anxiolytic activity whereas thiopental sodium induced sleeping time test was for sedative activity. 2.6 open field test the method was adopted as described by gupta et al. [22]. in the open field test, the animals were divided into control, positive control and test groups containing 5 mice each. the test groups received extract of f. stricta at the doses of 200 and 400 mg/kg body weight orally whereas the control group received the vehicle (1% tween 80 in water) and standard group received diazepam at the dose of 1 mg/kg (i.p). the floor of half square meter open field was divided into a series of squares each alternatively colored black and white. the apparatus had a 40cm height walls. the number of squares traveled by the animals was counted for 5 min at 0, 30, 60, 90, 120 min after oral administration of both doses of the extract. 2.7 hole cross test the apparatus was a cage of 30 cm × 20 cm × 14 cm with a steel partition fixed in the middle, dividing the cage into two chambers. a hole of 3.5 cm diameters was made at a height of 7.5 cm in the center of the cage. animals were randomly divided into control, positive control and test groups containing 5 mice each. the test groups were treated with extract of f. stricta at the doses of 200 and 400 mg/kg body weight orally whereas the positive control group with diazepam (1 mg/kg) and control group with vehicle (1% tween 80 in water). number of passages of the animals through the hole from one chamber to the other was counted for 5 min at 0, 30, 60, 90 and 120 min after oral administration of the extract as well as diazepam and vehicle [23]. the apparatus was thoroughly cleaned after each trial. 2.8 thiopental sodium induced sleeping time test for the experiment, the animals were randomly allocated to four groups, each with 5 mice. the test groups were given the leaf extract of f. stricta at doses of 200 and 400 mg/kg body weight, while the positive control was treated with diazepam (1 mg/kg) and control group with vehicle (1% tween 80 in water). thirty minutes later, thiopental sodium (40mg/kg) was administered to each mouse to induce sleep. the animals were observed by placing them on separate chambers for the latent period (time between thiopental administrations to loss of righting reflex) and duration of sleep i.e. time between the loss and recovery of righting reflex. the onset of sleep and total sleeping time was recorded for control, positive control and test groups [24]. 2.9 elevated plus maze test the method initially suggested by handley and mithani was employed with minor modifications biozid et al.; aji, 3(1): 173-181, 2020; article no.aji.56428 176 c ontr ol d ia ze pam fsm 2 00 fsm 4 00 fsh 2 00 fsh 4 00 fsc 2 00 fsc 4 00 fsa 2 00 fsa 4 00 fse 2 00 fse 4 00 0 50 100 150 0 min 30 min 60 min 90 min 120 min n u m b e r o f s q u a re s t ra v e le d [25]. the apparatus consists of two open arms (5 × 10) cm and two closed arms (5 × 10 × l5) cm radiating from a platform (5 × 5) cm to form a plus-sign figure. the apparatus was situated 40cm above the floor. the open arms edges were 0.5 cm in height to keep the mice from falling and the closed-arms edges were 15 cm in height. sixty minutes after administration of the test drugs, each animal was individually placed in the center of the epm and was allowed 5 min for free exploration. next, the number of open and enclosed arm entries, and time spent on open arms was manually registered [26]. entry into an arm was defined as the point when the animal placed all four paws onto the arm. the percentage of open arm entries (100 × open/total entries) and the percentage of time spent in the open arms (100 × open/(open + enclosed)) were calculated for each animal. observations made from an adjacent corner produced significant (p< 0.05, p < 0.01) decreases of locomotion from its initial value during the period of the experiment. maximum suppression of locomotor activity was displayed at the dose of 400 mg/kg body weight, which was comparable to the reference drug diazepam. 2.10 statistical analysis statistical comparisons were performed using one-way anova followed by post-hoc dunnett’s test with the spss program (spss 20.0, usa). the data were calculated as the mean ± standard error of mean (s.e.m.) and data of this graphs are deposited to the authors. the values obtained from plant extracts were compared with the control group and were considered statistically significant when p<0.05. graphs were represented by graph pad prism software. 3. results 3.1 neuropharmacological tests 3.1.1 open field test the open field test of f. stricta treated groups represented significant and dose dependent decrease of number of movements of mice from 0 minutes to 120 minutes. according to the study, 400 mg/kg body weight treated groups showed more significant reduction of movements than 200 mg/kg body weight. according to the chemical fractions, from 30 to 120 minutes, all the chemical extracts decreased the number of squares traveled by mice significantly at both 200 mg/kg and 400 mg/kg treated groups, but aqueous and n-hexane extract at 200 mg/kg showed the lowest effect. (fig. 1). 3.1.2 hole cross test hole cross test of f. stricta treated groups showed a decrease of movement of mice. data represented in fig. 2 suggests that the number of fig. 1. effect of extracts of f. stricta on exploratory behavior on mice (open field test) (n=5); (p<0.05) dunnett’s test as compared to control biozid et al.; aji, 3(1): 173-181, 2020; article no.aji.56428 177 c ontr ol d ia ze pam fsm 2 00 fsm 4 00 fsh 2 00 fsh 4 00 fsc 2 00 fsc 4 00 fsa 2 00 fsa 4 00 fse 2 00 fse 4 00 0 10 20 30 0 min 30 min 60 min 90 min 120 min n u m b e r o f h o le s c ro s s e d c ontr ol d ia ze pam fsm 2 00 fsm 4 00 fsh 2 00 fsh 4 00 fsc 2 00 fsc 4 00 fsa 2 00 fsa 4 00 fse 2 00 fse 4 00 0 50 100 150 200 onset of sleep duration of sleep t im e ( m in ) holes crossed from one chamber to another chamber by mice was decreased significantly for all chemical extracts of f. stricta leaves compared to control at doses of both 200 and 400 mg/kg. maximum suppression of locomotor activity was found at doses of 400 mg/kg (fig. 2). among all the fractions, chloroform (fsc), methanol (fsm) and ethanol (fse) extract showed comparatively better dose dependent reduction of movement of mice at both doses than other extracts after 60 minutes. 3.1.3 thiopental sodium induced sleeping time test in the thiopental induced hypnosis test, both 200 and 400 mg/kg showed a significant reduction in the time of onset of sleep. moreover, 200 and 400 mg/kg treated groups also increase the duration of thiopental sodium induced sleeping time in mice in the case of methanolic (fsm) and ethanolic (fse) plant extract. though chloroform (fsc) and n-hexane (fsh) demonstrated promising effect (fig. 3). fig. 2. effect of extracts of f. stricta on exploratory behavior on mice. (hole cross test) (n=5); (p<0.05) dunnett’s test as compared to control fig. 3. effect of extracts of f. stricta on thiopental sodium induced sleeping time (n=5); (p<0.05) dunnett’s test as compared to control biozid et al.; aji, 3(1): 173-181, 2020; article no.aji.56428 178 c ontr ol d ia ze pam fsm 2 00 fsm 4 00 fsh 2 00 fsh 4 00 fsc 2 00 fsc 4 00 fsa 2 00 fsa 4 00 fse 2 00 fse 4 00 0 20 40 60 80 % of entry into open arm % of time spent in open arm % o f e n tr y i n to o p e n a rm , % o f t im e s p e n t in o p e n a rm 3.1.4 elevated plus maze test this test is used to detect the anxiolytic activity. the extracts of f. stricta at the dose of 200 mg/kg and 400 mg/kg significantly increased the percentage of entries of mice into the open arms, and the percentage of time spent in the open arms which are shown in fig. 4. but, 400 mg/kg showed more significant effect than 200 mg/kg dose. ethanol (fse) extract showed comparatively more promising results than other extracts. but other extracts also showed promising effects (fig. 4). 4. discussion the result of open field and hole cross test demonstrated that this plant extracts reduced the frequency of movements of mice. locomotor activity was dose dependently decreased from 30 minutes to 120 minutes which proved its sedative activity. hence, it can be stated that both doses of chemical extracts of f. stricta leaves decreased the frequency and the amplitude of movements. the above result also showed that crude extracts of f. stricta plant had strong sedative and hypnotic action that mainly mediated in the cns by the gabaa receptor complex. thus, it can be said that the plant has promising sedative effects. thiopental sodium induced sleeping time test represented the depressant activity of central nervous system by decreasing the onset time of sleep as well as increasing the duration of sleep [27-29]. chemical constituents of plant extracts might be the reason for its benzodiazepine like sedative activity. previous literature review on this plant showed that this plant has several traditional uses. furthermore, it also contains several chemical constituents such as tannins, glycosides, alkaloids, saponins, phytosterols, flavonoids. elevated plus maze test is extensively used to determine the anxiolytic activity of drugs which is related to binding of drugs to gabaa receptor complex [30-32]. in this experiment, we observed that f. stricta increased the open arm entries along with time spent in open arms which precisely showed the anxiolytic effect of this plant extract in mice. generally, anxiolytic and hypnotic drugs such as benzodiazepines and their metabolites are highly lipid soluble which helps these drugs to reach the central nervous system readily. but this beneficial effect might turn into nightmare due to its high lipid solubility which can increase the risk of amnesia. amnesia is a disease related with memory deficit which is caused by not only brain damage or disease but also by the use of several anxiolytic and hypnotic drugs [33-35]. fig. 4. effect of extracts of f. stricta on epm test during 5 min test session (n=5); (p<0.05) dunnett’s test as compared to control biozid et al.; aji, 3(1): 173-181, 2020; article no.aji.56428 179 fig. 5. mechanism action of anxiolytic and hypnotic drugs therefore, optimization of lipid solubility is essential to decrease this type of side effect. thus, excessive lipid soluble property of anxiolytic drugs might be harmful for consume. hence, we need a compound which can make a great balance between lipophilicity and hydrophilicity means not excessive lipophilic or hydrophilic. polarity of water is highest among five chemicals. polarity of methanol and ethanol is less comparably to water. on the contrary, nhexane and chloroform are more lipids soluble. according to our study, it could be said that, this plant might possess molecules which results in significant effects of f. stricta extracts made by different chemicals. here, f. stricta extracts made by chemicals such as methanol, ethanol and chloroform extracts showed better effects than n-hexane and .aqueous extracts. however, further studies are needed to investigate the underlying mechanisms of this plant extract. there is a great need of the discovery of new anxiolytic drugs with fewer side effects. hence, our experiment might provide a good drug candidate for future. 5. conclusion all of the results were dose-dependent and statistically significant. according to the results of this experiment, it can be conclude that the crude extracts of f. stricta possess significant neuropharmacological activity. therefore, we can suggest that the extract may fulfill the therapeutic need for the treatment of anxiety and related neuropsychiatric disorders. nevertheless, further exploration is required to discover the exact mechanisms that are responsible for this anxiolytic activity of this plant extract. consent it is not applicable. ethical approval the set of rules followed for animal experiment were approved by the institutional animal ethics committee, department of pharmacy, international islamic university chittagong, bangladesh according to governmental guidelines. acknowledgement the authors wish to thank botanist dr. shaikh bokhtear uddin, professor, department of botany, university of chittagong, bangladesh, who helped to identify the plant. we would like to express our gratitude to the authority of the biozid et al.; aji, 3(1): 173-181, 2020; article no.aji.56428 180 international centre for diarrheal disease and research, bangladesh (icddrb) for providing the experimental mice. the authors are grateful to the department of pharmacy, international islamic university chittagong, chittagong, bangladesh, for providing research facilities. the authors are also grateful to their parents. competing interests authors have declared that no competing interests exist. references 1. chew al, jessica jja, sasidharan s. antioxidant and antibacterial activity of different parts of leucas aspera. asian pac j trop biomed. 2012;2(3):176–180. 2. buller r, legrand v. novel treatments for anxiety and depression: hurdles in bringing them to the market. drug discov today. 2001;6:1220-1230. 3. yadav av, kawale la, nade vs. effect of morus alba l. 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asian journal of immunology 3(1): 114-123, 2020; article no.aji.56168 general overview of coronavirus disease 2019 (covid19): a summary of evidence mohammad asadul habib1* 1 department of food technology and nutrition science, noakhali science and technology university, sonapur-3814, bangladesh. author’s contribution the sole author designed, analyzed and interpreted and prepared the manuscript. article information editor(s): (1) dr. jaffu othniel chilongola, tumaini university, tanzania. reviewers: (1) pichon maxime, university hospital of poitiers, france. (2) karen miyuki asano, instituto pasteur, brazil. complete peer review history: http://www.sdiarticle4.com/review-history/56168 received 30 march 2020 accepted 11 april 2020 published 15 april 2020 abstract coronavirus disease 2019 (covid-19) has spread throughout china and gained world-wide attention as a result of acute respiratory illness due to a novel coronavirus (sars-cov-2), traditionally known as covid-19. on 30 january 2020, the world health organization (who) officially announced the outbreak of covid-19 a public health emergency of international concern.a third introduction of a highly pathogenic and large-scale coronavirus disease in humans was the sars-cov-2 outbreak as a result of a severe acute respiratory syndrome coronavirus (sars-cov) in 2003 and the middle east respiratory syndrome coronavirus (mers-cov) in 2012. medical signs of covid-19 patients include fever, cough, exhaustion and a limited number of patients with signs of gastrointestinal infection. elderly and people with underlying diseases are susceptible to infection and prone to adverse results that could be associated with acute respiratory distress syndrome. there are currently few specific antiviral approaches available but many effective antiviral and repurposed drug candidates are under urgent investigation. the aim of this review was to summarize the recent medical advancement of covid-19's epidemiology, transmission, and clinical characteristics, and discussed current treatment and scientific developments to fight the novel coronavirus outbreak. mini-review article habib; aji, 3(1): 114-123, 2020; article no.aji.56168 115 keywords: sars-cov; sars-cov-2; mers-cov; covid-19; outbreak; coronavirus. abbreviations sars-cov : severe acute respiratory syndrome mars-cov : middle east respiratory syndrome china cdc : chinese center for disease control and prevention covid-19 : coronavirus disease 2019 2019-ncov : novel coronavirus 2019 tcm : traditional chinese medicine who : world health organization ppe : personal protection equipment ffp3 : filtering face pieces (protection in three classes against dangers) n95 : respirators and surgical mask 1. introduction in late december 2019, a cluster of pneumonia cases of unknown origin in wuhan, china was creating alarm among health officials. the wuhan municipal health commission issued an alert on 31 december, a rapid response team was sent to wuhan by the chinese center for disease control and prevention (china cdc) and a notification was sent to the world health organization (who) [1-4]. potential influenza, avian influenza, adenovirus, acute coronavirus syndrome (sars-cov), and middle east coronavirus (mers-cov) were all potential causes excluded. wuhan's huanan seafood wholesale market was involved in epidemiological testing, which has been shut down and disinfected and active cases have been identified [2,4,5]. on january 7, 2020, the latest coronavirus identified the causative pathogen, followed by the development of genomic characteristics and test methods [2,4-7]. now known as the covid-19, both sars-cov and mers-cov are distinct, but they are closely related [5,8]. early cases suggested that covid19 (i.e. a new name for a disease caused by a novel coronavirus) may be less severe than sars and mers. neither sars-cov nor mers-cov can be more infectious in 2019, but the incidence of disease among rapidly expanding numbers of people and evidence of transmission from human to human [3,9-12]. this review summarized the recent medical advancement of covid-19's epidemiology, pathogenesis, and clinical characteristics, and discussed current treatment and scientific developments to fight the novel coronavirus outbreak. 2. sars-cov 16 years ago in 2003, severe acute respiratory coronavirus syndrome (sars-cov) stunned the world by its high virulence and efficient transmissibility among humans [6,13,14], triggering the first large-scale epidemic of the 21st century. globally, there were over 8,000 patients infected and 774 deaths. sars harmed the economy with a decline in domestic demands and foreign travel, in addition to the major burden on the health system [15]. 2.1 transmission of sars-cov sars-cov is believed to be an animal virus that spread to other species (civet cats) and first infected humans in southern china's guangdong province in 2002, from an as-yet-uncertain animal source, possibly bats [16]. sars-cov seemed to be the main mode of transmission via respiratory droplets [16] as well as fecal-oral transmission may be [14]. apart from the superspreaders, two to four secondary cases were estimated to infect every person [17]. it was estimated that the median incubation duration was four to seven days [18] and that peak viral load was reached on the 10th day of illness [14]. sars-cov may affect all age groupings. particularly at risk were health care staff and immune-compromised patients [19]. 3. mers-cov in september 2012, saudi arabia first reported mers-cov infection, but in april 2012 the retrospective outbreak was confirmed in jordan. almost 2500 cases of mers cov (with at least 850 related deaths) from 27 countries around the world have been recorded worldwide by 2019; all mers cases have been linked to travel or residence, with more than 80 percent involving saudi arabia in and around the arab peninsula [20]. in the republic of korea in 2015, the biggest known mers outbreak outside the arab peninsula was caused. the outbreak was linked to the return of a traveler from the arab peninsula. countries across europe, asia, north africa, the middle east, and the us have also reported cases of patients who have been either moved to treatment or who have become ill after their return from the middle east [21]. habib; aji, 3(1): 114-123, 2020; article no.aji.56168 116 3.1 transmission of mers-cov mers-cov may also be transferred through direct contact, respiratory droplets (particles > 5 micrometers) or aerosols (particles < 5 micrometers) from one person to another. the development of infection in people whose only risk was close contact with people who had mers has been identified as a person-to-person transmission [21]. the mers-cov reservoir is supposed to be dromedary camels, but the mechanism of transmission from camels to humans is unknown. many cases were identified as direct transmission between people and humans in health facilities. if mers is suspected in a patient, steps to prevent the transmission of infection in the healthcare sector must be undertaken promptly [21]. 4. covid-19 coronaviruses are a broad family of viruses that cause disease from the common cold to more severe diseases like middle east respiratory syndrome and severe acute respiratory syndrome. covid-19 is an unexplained modern mutation of human beings. the covid-19 source is still unknown although the initial cases were related to the huanan south china seafood market. while many of the early patients worked in or visited the market, none of the exported cases came into contact with the market, suggesting either human to human transmission or a more widespread animal source [22]. in addition to seafood, social media sources report that the huanan south china market has sold snakes, birds, and other small mammals, marmot, and bat. the who announced that environmental samples taken from the marketplace have reported positive for the novel coronavirus but no specific animal association has been identified [22]. an initial report indicated that snakes may be the potential source based on codon use [23], but others rejected the claim [24]. researchers are currently working on defining the covid-19 source, including possible animal intermediate vectors. the appearance of sras and mers-cov is the product of a zoonotic reservoir. in 2002, sarscov, the first highly pathogenic human coronavirus, was introduced with the animal-tohuman transmission in wet markets. sars-cov viral rna was found in both palm civets and raccoon dogs sold in these wet markets [25]. sars-cov was however not present in animals and it appeared that the intermediate reservoirs of those species were an adapted virus for infecting humans more efficiently. further surveillance efforts in bat species have reported closely associated coronaviruses [26]. more recent research has shown that many bat coronaviruses can infect human cells without needing intermediate adaptation [27,28]. furthermore, human serology data show bat coronavirus protein recognition and suggest that low-level zoonotic transmission of sars-like bat coronavirus occurs beyond recognized outbreaks [29]. mers-cov also has a zoonotic virus with possible bat origins [30,31], but camel contact and endemic infection are commonly recorded in the primary mers-cov cases [32]. the epidemic was significantly reduced by strict quarantine and elimination in se asia of live markets for sarscov. a similar solution to mers-cov was not feasible due to the cultural value of camels, and occasional outbreaks in the middle east remain. these sars and mers lessons highlight the importance of the ongoing outbreak to rapidly determine the source of the covid-19 disease. 4.1 update situation of covid-19 as of 9 april 2020, 79,385 people had died so far from the covid-19 coronavirus outbreak.1, 356,780 confirmed cases occurred in 206 countries and territories (fig. 1) [33]. it is important to emphasize that there is an underreporting of these numbers due to the lack of diagnosis in many countries. 4.2 transmission of covid-19 many domestic and wild animals may serve as coronavirus hosts, including camels, cattle, cats, and bats [34]. animal coronaviruses usually are not known to spread among humans [35]. there are exceptions, however, such as sars and mers, which are transmitted by cough or sneezing primarily by close contact with infected people by respiratory droplets. regarding covid-19, early patients were confirmed to have some link to the huanan seafood market in wuhan, china, indicating that these early infections were due to transmission from animal to human. nonetheless, later cases among medical staff and others with no history of exposure to that market or visiting wuhan were recorded, which was taken as an indication of human-to-human transmission [11,36-39]. habib; aji, 3(1): 114-123, 2020; article no.aji.56168 117 fig. 1. coronavirus disease (covid19) update situation (according to 9 april 2020) table 1. potential treatment options of covid-19 classes potential treatment options reference anti-viral > 85% of patients received antiviral agents, including oseltamivir (75 mg every 12 h orally), ganciclovir (0.25 g every 12 h intravenously) and lopinavir / ritonavir tablets (400/100 mg twice daily). remdesivir is currently being evaluated and known to prevent mers-cov in more than 10 medical establishments in wuhan. [44] antimalarial an ancient antimalarial, chloroquine phosphate, has been effective in inhibiting the exacerbation of pneumonia due to its anti-viral and antiinflammatory activity. [45] herbal treatments traditional chinese medicine has been widely used during the last sarscov outbreak and is currently being used in china. the five most commonly used herbs were astragali radix (huangqi), glycyrrhizae radix et rhizoma (gancao), saposhnikoviae radix (fangfeng), atractylodis macrocephalae rhizoma (baizhu), and lonicerae japonicae flo. [46] chinese health authorities 'new guidelines outlined three primary transmission routes for covid-19: 1) transmission of droplets, 2) transmission of contacts and 3) transmission of aerosols. transmission of droplets has been documented when respiratory droplets (as formed when an infected person coughs or sneezes) are ingested or inhaled in close proximity by people nearby; the transmission of contact can occur when an individual touches a virussurface or object and then touches his or her mouth, nose, or eyes; and transmission of aerosols can occur when inhaled high dose of aerosols into the lungs in a relatively closed environment [34,40]. 4.3 clinical manifestation and diagnosis of covid-19 the complete clinical consequence is not yet apparent, because the symptoms recorded vary from mild to extreme with even death in some [35]. the complete clinical consequence is not yet apparent, because the symptoms recorded vary from mild to extreme with even death in some reported include headache, diarrhea, habib; aji, 3(1): 114-123, 2020; article no.aji.56168 118 table 2. clinical trials identified at clinicaltrials.gov related to drug repositioning for covid-19 treatment [47] intervention clinical condition sponsor n o test/status beginning/ estimated end phase hydroxychloroquine 30 participants with pneumonia caused by 2019ncov shanghai public health clinical center nct04261517 /recruiting patients 6-2-2020 /31-122020 3 chloroquine 10000 participants in a prophylaxis study for covid19 university of oxford nct04303507 / not yet recruiting may 2020 / may 2022 n/a human immunoglobulin pneumonia caused by 2019ncov with 80 participants peking union medical college hospital nct04261426 / not yet recruiting patients 10-2-2020 / 30-062020 2 and 3 remdesivi severe respiratory infection caused by 2019-ncov with 452 participants capital medical university nct04257656 / recruiting patients 6-2-2020 / 31-052020 3 remdesivir 308 participants with mild/moderate respiratory infection caused by 2019ncov capital medical university nct04252664/ recruiting patient 05-02-2020 / 27-042020 3 arbidol (umifenovir) pneumonia caused by 2019ncov with 380 participants jieming qu, ruijin hospital nct04260594 / not yet recruiting patients 7-02-2020 / 30-122020 4 arbidol or lopinavir-ritonavir or oseltamivir 400 participants infected with2019-ncov tongji hospital nct04255017 / recruiting patients 01-02-2020 / 01-072020 4 arbidol or lopinavir-ritonavir 125 participants infected with 2019-ncov guangzhou 8th people's hospital nct04252885 / recruiting patients 28-01-2020 / 31-072020 4 darunavir-cobicistat combination pneumonia caused by 2019ncov with 30 participants shanghai public health clinical center nct04252274 / recruiting patients 30-01-2020 / 31-122020 3 tcm combination with lopinavir-ritonavir, α-interferon via aerosol 150 participants infected with 2019-ncov beijing 302 hospital nct04251871 / recruiting patients 22-01-2020 / 22-012021 n/a recombinant human interferon α2β 328 participants with covid19 tongji hospital nct04293887 / not yet recruiting 01-03-2020 / 30-062020 1 carrimycin or lopinavir-ritonavir or arbidol or chloroquine phosphate 520 participants with covid19 beijing youan hospital nct04286503 / not yet recruiting 23-02-2020 / 28/022021 4 habib; aji, 3(1): 114-123, 2020; article no.aji.56168 119 intervention clinical condition sponsor n o test/status beginning/ estimated end phase danoprevir-ritonavir and interferon inhalation or lopinavir-ritonavir or tcm plus interferon inhalation 50 participants with pneumonia caused by 2019ncov the ninth hospital of nanchang nct04291729 / recruiting 14-02-2020 / 30-042020 4 xiyanping or lopinavir-ritonavir-interferon inhalation 384 participants with pneumonia caused by 2019ncov jiangxi qingfeng pharmaceutical co. ltd. nct04275388/not yet recruiting 19-02-2020 / 14-122020 n/a xiyanping combined with lopinavir-ritonavi 80 participants with covid19 jiangxi qingfeng pharmaceutical nct04295551 / not yet recruiting 14-03-2020 / 14-042021 n/a combinations of oseltamivir, favipiravir, and chloroquine 80 participants with covid19 rajavithi hospital nct04303299 /not yet recruiting 15-03-2020 / 30-112020 3 thalidomide 40 participants with covid19 first affiliated hospital of wenzhou medical university nct04273581 / not yet recruiting 18-02-2020 / 30-052020 2 thalidomide 100 participants with pneumonia caused by 2019ncov first affiliated hospital of wenzhou medical university nct04273529 / not yet recruiting 20-02-2020 / 30-062020 2 vitamin c 140 participants with severe pneumonia caused by 2019ncov zhi yong peng nct04264533 / recruiting 14-02-2020 / 30-092020 2 methylprednisolone 80 participants infected with2019-ncov peking union medical college hospital nct04244591 / recruiting patients 26-01-2020 / 25-122020 2 pirfenidone 294 participants with severe pneumonia caused by 2019ncov huilan zhang nct04282902 / recruiting 04-02-2020 / 01-062020 3 bromhexine hydrochloride 60 participants with suspected and mild pneumonia caused by 2019ncov second affiliated hospital of wenzhou medical university nct04273763 / enrolling by invitation 16-02-2020 / 30-042020 n/a bevacizumab 20 participants with severe covid-19 pneumonia qilu hospital of shandong university nct04275414 / recruiting february 2020 / may 2020 2 and 3 fingolimod 30 participants with covid19 1° affiliated hospital of wenzhou medical university nct04280588 / recruiting 22-02-2020 / 01-062020 2 habib; aji, 3(1): 114-123, 2020; article no.aji.56168 120 hemoptysis, runny nose, and phlegm-producing cough [35,41]. patients with mild symptoms have been reported to recovery after 1 week and serious cases of alveolar damage to the virus have been reported to lead to death. the cases of death were mainly patients of medium age and older adults with preexisting conditions (tumors, cirrhosis, hypertension, heart artery disease, diabetes, and parkinson's) [42]. the recommendations for case identification list the following symptoms: fever, drop in lymphocytes and white blood cells, new pulmonary infiltration of chest x-rays and no progress in symptoms after 3 days of antibiotic treatment [36]. 4.4 old tricks and new challenges in treating covid-19 no vaccine or special antiviral drug protocol is currently used to treat seriously ill patients. patient management focuses primarily on providing treatment, e.g. oxygenation, ventilation, and fluid control. as part of a vital management phase of covid-19, combined treatment of low dose systemic corticosteroids and anti-viral and interferon atomization was encouraged [43]. table 1 also included other recorded therapeutic drugs used for treating seriously ill patients. 4.5 clinical trials for covid-19 treatment on drug repositioning a study listed 24 clinical trials (table 2), in which 19 clinical phase 2, 3, or 4 studies were performed.the eligibility requirements for the retrieved studies were: include a base identification number for clinicaltrials.gov; describe the number of participants and the timeframe for study; describe the clinical conditions of the participants, and use treatments with medicines already tested or approved for any other disease in patients infected with the novel coronavirus sars-cov-2 (2019-ncov). clinical trials may be an effective technique as they promote the development of new groups of medicines; they are cost-effective and require less time to market; and pharmaceutical supply chains exist for manufacturing and distribution [47]. 4.6 prevention of covid-19 preventive approaches concentrate on patient isolation and careful infection management, including effective screening and clinical care steps to be followed for infected patients. for example, during specimen collection droplets, touch and airborne precautions should be taken and sputum induction avoided. the general guidelines of who and other organizations have issued the following general recommendations [48]:  avoid close contact with people suffering from acute respiratory infections.  wash your hands frequently, particularly after contact with infected people.  avoid unprotected farm and wild animal contact.  people with acute airway symptoms should stay a little away, cover their coughs or sneezes with disposable tissues, and wash their hands with sanitizer or soaps.  strengthen the implementation of appropriate hygiene procedures for the prevention and control of diseases, in particular in emergency medicine departments.  immunosuppressed people can prohibit public meetings. the most effective technique for the population is to wash their hands regularly and use portable hand sanitizing to avoid contact with their face and mouth after engaging with an area that is possibly polluted. health workers caring for infected people should make use of contact and airborne precautions which include ppe masks, eye protection, gowns and gloves, such as n95 or ffp3 masks, to avoid pathogen transmission. scientific research is going to develop a vaccine for coronavirus [48]. 5. conclusion in the context of rapidly increasing outbreaks of covid-19, proactive steps by long-term care facilities are required to recognize and eliminate potentially infected staff and visitors, actively track potentially infected patients and enforce effective infection prevention and control measures to prevent covid-19. scientific researchers conduct numerous clinical trials related to drugs that can be an important strategy because they promote the development of new classes of medicines and i hope these repositioning trials will help to identify alternatives for covid-19 treatment. consent it is not applicable. habib; 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(2) festus otajevwo, western delta university, nigeria. (3) m. zamri saad, universiti putra malaysia, malaysia. complete peer review history: http://www.sciencedomain.org/review-history/24615 received 19 th february 2018 accepted 4th may 2018 published 16th may 2018 abstract bacteria and viruses use an array of evasion mechanisms to escape from the host immune system. due to antigenic variation, pathogenic micro-organisms can escape the immune system. microorganisms can occur in different types, such as the 97 serotypes of streptococcus pneumoniae. influenza viruses change their antigenic make-up, in particular, the hemagglutinin molecule by antigenic drift and antigenic shift. trypanosomes and malaria parasites use dna programmed expression of highly variable surface antigens. micro-organisms can also produce proteins that degrade (iga protease) or inactivate antibody molecules (protein a and protein g). some bacteria and viruses produce proteins that inhibit complement activation. virus can become invisible for recognition by t-lymphocytes by interference with antigen presentation. antiviral immunity can be review article nguyen et al.; aji, 1(1): 1-13, 2018; article no.aji.41198 2 suppressed by viral homologues of cytokines and cytokine receptors and other proteins. despite the extensive immune evasion strategies used by viruses, bacteria and other micro-organisms, the immune system in most cases is ultimately able to control an infection. keywords: evasion mechanisms; iga proteases; capsular polysaccharides; antigenic drift; antigenic shift; complement inhibitors; antigen presentation; cytokine homologues. 1. introduction micro-organisms and parasites use a number of different ways to escape the immune system. the christian religious history has the legend of saint julia, who tried to escape from her future husband. the story of this legend is that in the 14 th century, julia, the daughter of a heathen king in portugal, was promised by her father to be the bride of the king of sicily. julia refused because she wanted to remain a virgin and in order to prevent she had to marry, she prayed to god for help. soon thereafter she grew a beard and her husband-to-be then refused her. unfortunately, julia’s father became so mad that this prearranged marriage was cancelled that he had her crucified. saint julia has been popular through the ages and her crucifixion is depicted in many works of art, including statues, drawings and paintings [1]. the scene of her crucifixion is also depicted by jheronimus bosch in the martyrdom of saint julia (fig. 1). for the occasion of the 500 th anniversary of jheronimus bosch in 2016, the painting was loaned by the gallerie dell'accademia, venice, italy to the noord-brabants museum in ‘s hertogenbosch, the netherlands, the home town of jheronimus bosch. as a part of the deal the painting was fully restored and only then the beard of saint julia became clearly visible. growing a beard as a strategy to escape marriage. various micro-organisms and parasites have evolved different strategies to escape the immune system of the host. this strategy is called evasion. evasive mechanisms contribute strongly to the virulence and pathogenicity of these organisms. different categories of evasive mechanisms can be distinguished, each with different targets on the immune system, which will be discussed in this review. fig. 1. detail of the painting the martyrdom of saint julia by jheronimus bosch (around 1497). the painting is alternatively named saint wilgefortis triptych, because saint julia had such as strong (fortis) will (wilge). gallerie dell'accademia, venice, italy. (http://boschproject.org/#/artworks/saint_wilgefortis_triptych) nguyen et al.; aji, 1(1): 1-13, 2018; article no.aji.41198 3 2. immune evasion mechanisms 2.1 due to antigenic variation pathogenic micro-organisms can escape the immune system one of the ways in which a micro-organism can escape elimination by the immune system is by altering its antigenic make up [2]. such a makeover can occur in three different ways. first, a micro-organism can occur in different types. for example, the bacterium streptococcus pneumoniae has ninety seven serotypes that differ in the structure of the capsular polysaccharide (fig. 2) [3]. infection with a given serotype leads to type-specific immunity, which, however, does not protect against infection with any of the other pneumococcal serotypes [4]. for the acquired immune system, every pneumococcal serotype is therefore a separate micro-organism. this means that streptococcus pneumoniae can cause a primary infection several times in the same individual. the second way of antigenic variation is more dynamic and is found among others in the influenza virus, the cause of influenza. there are three different types of influenza virus, a, b and c, of which influenza a causes the most serious disease symptoms [5]. most infections that occur worldwide during the influenza season (autumn and winter) are caused by a single type of the influenza a virus. over time, protective immunity arises in the population, which mainly consists of antibodies and cytotoxic tlymphocytes directed against the viral hemagglutinin protein [6]. the hemagglutinin is involved in attachment to target cells and antibodies against hemagglutinin can (thereby) prevent the spread of the virus in the body [7,8]. due to changes in the hemagglutinin protein (see below), a virus type is created against which the accumulated immunity in the population does not work or does not function properly [9]. such a changed virus can, therefore, cause a new infection. the influenza virus can alter the antigenic makeup of the hemagglutinin in two ways: antigenic drift and antigenic shift (fig. 3) [10]. mutations in the gene coding for the hemagglutinin (and for the second important virus surface protein neuraminidase) produce a new variant of the influenza virus (antigenic drift) every two or three years [11]. this variant is less well recognized by the antibodies and cytotoxic t lymphocytes present. this allows the influenza virus to cause a generally mild flu epidemic [12]. such an epidemic is mild because although some epitopes of the hemagglutinin and/or neuraminidase have changed, not all of them have. so there is still a certain amount of residual immunity in the population. antigenic shift is a much rarer event, but with far greater consequences [13]. an antigenic shift can occur when a (human) influenza a virus ends up in a secondary host (e.g. a bird). the influenza rna genome is segmented into eight genes, one of which is coding for hemagglutinin and one for neuraminidase [14]. in a secondary host, in a cell that is infected with two different influenza viruses, exchange of a complete rna segment can take place [15]. thus, in a host cell infected with both the human and avian influenza virus, exchanges between both viruses can occur. fig. 2. streptococcus pneumoniae, a gram-positive facultative anaerobic bacterium is encapsulated by a thick layer of polysaccharides (arrow in left panel). the capsule is made up of one of 93 different types of polysaccharides; the structural composition of four common occurring serotypes is shown in the right-hand panel nguyen et al.; aji, 1(1): 1-13, 2018; article no.aji.41198 4 fig. 3. antigenic shift and antigenic drift of influenza a virus. the major surface antigens of the influenza a virus are hemagglutinin and neuraminidase. by point mutations in the rna encoding hemagglutinin, the antigenic make-up of the molecule can change somewhat. this is called antigenic drift. this allows original antibodies to bind less well or not at all and the mutated virus has a better chance of survival. in an antigenic shift, two different influenza a virus particles exchange a complete rna segment, allowing a completely different hemagglutinin molecule to be expressed. accumulated immunological memory from previous influenza contacts is then no longer effective because antibodies (and memory t lymphocytes) no longer recognize the altered hemagglutinin molecule. such an altered influenza virus is, therefore, more easily able to cause an epidemic from this, a (human) virus variant can emerge with an avian hemagglutinin (fig. 3). at least 18 subtypes of the hemagglutinin occur (h1 to h18), of neuraminidase 11 (n1 to n11) [16]. the most common influenza a types in humans are h1n1, h2n2 and h3n2 [17]. h5, h6, h7 and h8 are especially common in birds [18]. due to antigenic shift, the h5n1 variant originated in which the avian 76 h5 ended up in a human influenza a virus [19,20]. the differences between the human and avian influenza hemagglutinin are so great that antibodies and cytotoxic t lymphocytes formed during previous infections do not give any cross protection. influenza strains in which such an antigenic shift has occurred occur once every 15 to 20 years [10]. the so-called hong kong influenza pandemic in 1968, with world-wide one million deaths, was caused by a virus variant due to antigenic shift [19,21]. the most recent influenza pandemic started in mexico in 2009 and was initially called swine flu. later, under pressure from mexico, this name was changed to new influenza a (n1h1) (fig. 4). what was special was that this variant particularly affected young children, while normally older people are particularly susceptible to influenza [22,23]. in retrospect, many people aged about 50 years and older were already found to have (cross-reactive and protective) antibodies against this virus, due to exposure to a similar influenza in their youth [24]. the n1h1 spread rapidly around the world, and initially, there was fear that millions of people would be killed. a vaccine against h1n1 has been accelerated and offered to major risk groups i.e. children between 6 months and 4 years, household members of younger children, and adults with chronic disease [25]. in retrospect, the h1n1 pandemic was mild, probably mainly because the elderly in which the mortality is concentrated during the annual flu season were barely susceptible to the new influenza a (n1h1) [24]. an estimated 300,000 people worldwide have died directly or indirectly from the virus [26]. a total of 65,600 deaths was confirmed in africa, 29,700 in the americas, 31,000 in europe, and 78,600 in asia [26]. at the moment the h1n1 vaccine became available, the peak of the pandemic might already have passed. the third way in which antigenic variation can occur is due to programmed changes in the dna of the micro-organism or parasite [27]. in its most nguyen et al.; aji, 1(1): 1-13, 2018; article no.aji.41198 5 fig. 4. a worldwide outbreak of new n1h1 influenza virus in 2009, as reported in the press and communicated to travelers extreme form, this mechanism is used by trypanosomes. trypanosomes are protozoans that are transmitted by insects and cause sleeping sickness [28,29]. the trypanosome is surrounded by a single protein, the variantspecific glycoprotein (vsg) [30]. after infection, this vsg generates a powerful antibody response that neutralizes the parasite. however, trypanosomes have a thousand different vsg genes of which only one is expressed each time. the le trypanosome that has been altered from vsg expression thus escapes the immune system and leads to renewed growth and flareup of the disease [30]. this will result in a chronic cycle of trypanosome degradation with immune complex formation and inflammation, followed by renewed disease activity. ultimately, this leads to severe neurological age and coma. the malaria parasite also uses this mechanism of antigenic variation to protect itself against the immune system [31]. in the erythrocyte stage of malaria, there is an expression of parasite proteins on the membrane of the red blood cell, especially of the pfemp1 protein [32,33]. the pfemp1 protein suppresses the production of ifn- and thus a cellular immune response [34]. via pfemp1 an infected erythrocyte adheres to vascular wall tissue and can thus prevent phagocytosis by spleen macrophages. pfemp1 does elicit an antibody response and these antibodies can bind to infected erythrocytes. antibody-loaded erythrocytes are captured in the spleen and phagocytosed. the malaria parasite has sixty variants of pfemp1, of which only one is expressed each time [35]. switching to another variant of pfemp1 means that the already produced antibodies can no longer bind and that infected erythrocytes are no longer trapped. 2.2 micro-organisms produce proteins that can degrade or inactivate antibody molecules micro-organisms can protect against antibodymediated complement lysis or phagocytosis by enzymatic degradation of the antibodies. a number of bacteria, including neisseria species, haemophilus influenzae and streptococcus pneumoniae form proteolytic enzymes that can split secretory iga (siga) antibodies into two monomeric fab fragments and an fc fragment [36,37]. this iga protease is capable of cleaving both free siga and bound siga antibodies. the fab fragments remain on the surface of the micro-organism but are unable to activate effector mechanisms (complement, phagocytosis) [38]. infections with the above bacteria occur on mucous membranes and iga is the most important isotype of the antibodies present [39]. the bacterial iga proteases are especially capable of splitting siga1 while siga2 is relatively resistant to iga proteases [36,37]. but because the iga1 fab fragments remain bound on the surface of the micro-organism, binding of iga2 antibodies can be inhibited thereby [40,41]. nguyen et al.; aji, 1(1): 1-13, 2018; article no.aji.41198 6 igg antibodies can also be broken down by bacterial enzymes. pseudomonas aeruginosa and other bacteria produce cysteine proteases that can cleave igg molecules at the hinge region. besides proteolytic cleavage of the molecule, igg can also be functionally inactivated by certain bacterial proteins [42-44]. staphylococcus aureus expresses a protein on its surface called protein a, which can bind to the fc portion of igg. binding of protein a to igg blocks fc receptormediated phagocytosis [45,46]. moreover, it inhibits the binding of c1q to igg and thus complement activation [47]. in other bacteria, proteins with similar functions are found: groupg streptococci produce protein g and peptostreptococcus produces protein-l. these proteins can also bind to igg [48-50]. 2.3 some bacteria and viruses produce proteins that inhibit complement activation many bacteria produce n-formyl peptides such as fmlp [51]. these peptides are very potent chemoattractants for phagocytes [52]. fmlp is bound to phagocytes via specific receptors: formyl peptide receptor (fpr) and the related fpr-like-1 receptor (fprl1) [53]. the fmlp is not only an chemoattractant but also stimulates phagocytosis [54,55]. staphylococcus aureus has developed a strategy to prevent the attraction of phagocytes to the site of the infection by producing the protein chips (chemotaxis inhibiting protein of s. aureus) [56]. chips binds to fprl1 and thus blocks the functioning of this receptor [57]. chips also binds to the c5a receptor on phagocytes and thereby blocks the function of another chemotactic peptide, the complement fragment c5a [58]. another staphylococcal protein that interferes with the complement system is scin (staphylococcal complement inhibitor) [59]. scin blocks the c3 converter activity of c4b2a and c3bbb [60-62]. in total, s. aureus possesses about ten different proteins that can all inhibit complement activation. together, this will disrupt all functions mediated by the complement system (chemotaxis and lysis and opsonization) [62-64]. these and other proteins that are used to escape the immune system of the host lie encoded on the bacterial genome together in a so-called immune vascular cluster (iec), of which s. aureus possesses two [65,66]. not only s. aureus and other bacteria use proteins to prevent activation of the complement system (fig. 5) but also certain viruses. vaccinia virus encodes a strong complement inhibitor, vaccinia complement control protein (vcp). vcp strengthens the split of c3b and c4b by factor i and thus inhibits both the classic and alternative complement activation path [67-70]. 2.4 interference with antigen presentation makes viruses invisible for recognition by t-lymphocytes viruses have developed different ways to escape the immune system. it is, of course, important that virus replication occurs only in host cells, where the virus is not immediately accessible to the immune system. during viral replication, components of viral proteins are presented to the immune system by mhc class i and class ii proteins. in that way, the virus would betray its presence in an infected cell. however, if the virus does not replicate, but remains latent, it is invisible. herpes simplex virus type i infects epithelial cells and sensory neurons [71]. after a cellular immune response, the infection is under control, but the virus can still remain latent in the nerve cells [72]. reactivation of the virus can if the antiviral immunity is reduced or temporarily disturbed, lead to a re-infection of the skin [73]. another herpes virus, the previously discussed epstein-barr virus, can remain latent in b lymphocytes [74]. for this, it must express a certain viral protein, ebna-1, since this is necessary to maintain the viral genome. ebna-1 cannot be presented in the context of mhc class i, because it cannot be broken down by the proteasome. this keeps the virus invisible to the immune system [75-77]. other viruses also have proteins that interfere with antigen presentation and thus try to prevent a cellular immune response from getting under way. for example, the cytomegalovirus (cmv) has at least twelve different proteins that block the presentation of cmv peptides in the mhc at different sites [78]. these cmv proteins are encoded on the unique long (ul), or unique short (us) part of the cmv genome [79]. us3 and us10 proteins prevent mhc class i molecules from leaving the endoplasmic reticulum [80,81]. if nonetheless mhc class i molecules are formed, us2 and us11 proteins bind to this, after which the mhc molecules are degraded by proteasomes [82,83]. disabling mhc class i expression prevents recognition by cytotoxic t lymphocytes but makes the cell susceptible to nguyen et al.; aji, 1(1): 1-13, 2018; article no.aji.41198 7 killing by nk cells [84]. the cmv protein ul16, however, blocks the activating nk cell receptor nkd2d and ul18 stimulates the inhibitory nk cell receptors [85,86]. cmv, therefore, has an extensive package of viral proteins at its disposal to combat killing by cd8+ t lymphocytes or by nk cells. 2.5 viral homologues of cytokines and cytokine receptors and other proteins suppress antiviral immunity if a virus, despite its attempts to prevent recognition by the immune system, would still evoke an immune response, it can try to suppress that response. one of the strategies employed is that the viral genome encodes homologues of suppressive cytokines and/or soluble cytokine receptors. [87-90]. ebv encodes a viral homolog of il-10, which is very similar to human il-10 but has only its immunosuppressive properties [91,92]. ebv also encodes an il-12p40 related protein [93]. pox viruses use soluble cytokine receptor homologous proteins and cytokine binding proteins to neutralize proinflammatory cytokines [94]. these viruses also code for a soluble chemokine antagonist that binds with high affinity to cc-chemokines. fungi also use inhibition of cytokines to escape the immune response of the host. virulent cryptococcal strains secrete proteins with anti-tnf-α and anti-il-12 activity while stimulating the il-10 production of the host [95]. in addition to blockade of the cytokine function, viruses can also neutralize the action of antibodies by synthesis of viral fc receptors (herpes simplex and cytomegalovirus) [96,97]. finally, viruses can also resist apoptosis in order to escape cytotoxic t lymphocytes and nk cells. the most successful is the adenovirus, which possesses a protein that is very similar to the anti-apoptotic bcl-2. ebv also has two proteins that resemble bcl-2 [98]. inhibition of caspase activity and reduction of the expression of apoptosis receptors such as fasl are other ways in which viruses prevent apoptosis [99-101]. fig. 5. complement evasion by bacterial proteins. figures show examples of bacterial proteins which can interfere with specific pathways of the complement system. further explanation is given in the text nguyen et al.; aji, 1(1): 1-13, 2018; article no.aji.41198 8 despite the extensive immune evasion strategies used by viruses, bacteria and other microorganisms, the immune system in most cases is ultimately able to control an infection. however, when components of the immune system do not function adequately, such as with congenital or acquired immune deficiencies, even seemingly innocent micro-organisms can lead to serious infections. 3. conclusion saint julia, by changing her antigenic makeup, tried to evade from her husband to be. this relief was only temporary, because another man, notably her own father, had her crucified. the analogy with micro-organisms that try to escape the immune system partly holds true. escape from complement mediated killing does not prevent phagocytosis and subsequent intracellular killing. consent it is not applicable. ethical approval it is not applicable. acknowledgements no external funding for this study was used. the university college roosevelt covered all costs associated with the development and the publishing of the present manuscript. the corresponding author had full access to the data and final responsibility for the submission of the publication. competing interests thuc anh nguyen, angkarina angkasuwan, claudia rossi, dr frans j. van overveld, and prof. dr ger t. rijkers declare that they have no relevant affiliations or financial involvements with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. this includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. no writing assistance was utilized in the production of this manuscript. references 1. friesen ie. the female crucifix: images of st. wilgefortis. waterloo, ontario, canada: wilfried laurier university press; 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this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: http://www.sciencedomain.org/review-history/24615 _____________________________________________________________________________________________________ *corresponding author: e-mail: celso@alergoimuno.med.br; asian j. immunol., vol. 7, no. 1, pp. 63-70, 2024 asian journal of immunology volume 7, issue 1, page 63-70, 2024; article no.aji.115657 exploring the role of leukocyte adherence inhibition test in assessing non-ige mediated immunoreactivity to benzoic acid in allergic patients celso eduardo olivier a*, daiana guedes pinto a, ana paula monezzi teixeira a, jhéssica letícia santos santana a, raquel acácia pereira gonçalves santos a, and regiane patussi santos lima b a instituto alergoimuno de americana, brazil. b lavoisier’s laboratories, são paulo, brazil. authors’ contributions this work was carried out in collaboration among all authors. all authors read and approved the final manuscript. article information doi: https://doi.org/10.9734/aji/2024/v7i1130 open peer review history: this journal follows the advanced open peer review policy. identity of the reviewers, editor(s) and additional reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here: https://www.sdiarticle5.com/review-history/115657 received: 02/04/2024 accepted: 13/04/2024 published: 17/04/2024 abstract background: several publications report benzoic acid as responsible for non–ige-mediated allergic reactions. no standardized lab exam identifies these reactions besides in vivo provocation tests. aim: to evaluate the potential of the leukocyte adherence inhibition test (lait) to discriminate non–ige-mediated immunoreactivity against benzoic acid in patients with non–ige-mediated allergic phenotypes. original research article https://doi.org/10.9734/aji/2024/v7i1130 olivier et al.; asian j. immunol., vol. 7, no. 1, pp. 63-70, 2024; article no.aji.115657 64 study design: we retrospectively examined the medical charts of 100 patients diagnosed with allergic rhinitis, allergic bronchitis, asthma, sinus headache, atopic dermatitis, and/or urticaria with clinical suspicion of non–ige-mediated benzoic acid hypersensitivity who were investigated with ex vivo challenge test monitored by lait against benzoic acid. the cohort counted 30 males; the mean age was 41.9 years, the sd was 20.4 years, and the range was 2 to 90 years. place and duration of study: instituto alergoimuno de americana – são paulo – brazil – between january 2018 and march 2024. methodology: the percentage of leukocyte adherence inhibition (lai) promoted by the ex vivo challenges against 1 mg/ml benzoic acid was distributed in ranges through a cascade distribution chart to outline the variability of the results. results: the lai ranged from 0% to 97%; the mean was 41.1%; the median was 40.5%; the standard deviation was 24.2%; the mode was 0 and 59 (each appeared four times). the cascade distribution demonstrates a wide distribution of lai results. this extensive distribution of lai results suggests that some patients had mild, moderate, or severe non–ige-mediated immunoreactivity against benzoic acid, while others did not present any immunoreactivity against it. conclusion: our preliminary results support that the lait performed with benzoic acid may discriminate diverse degrees of ex vivo immunoreactivity in patients suffering from diversified allergic phenotypes. it is worth carrying out more in-depth studies to evaluate the usefulness of lait in diagnosing non–ige-mediated benzoic acid allergies. keywords: allergy; asthma; atopic dermatitis; bronchitis; diagnosis; exposome-wide association study; hypersensitivity; leukocyte adherence inhibition test; non–ige-mediated immunoreactivity; precision medicine; rhinitis; sinus headache; urticaria. abbreviations lai : leukocyte adherence inhibition lait : leukocyte adherence inhibition test 1. introduction benzoic acid is an organic compound formed by an aromatic ring and a carboxyl group [1]. benzoic acid is a parent and a metabolic common pathway of a large group of structurally related substances (aromatic salts, alcohols, aldehydes, esters, and acetals) legally regulated to be used as additives to industrialized foods, cosmetics, and medicaments [2]. used as an antimicrobial, the legally acceptable daily intake uppermost limit for benzoic acid (or the benzyl/benzoic moiety) is 5.0 mg/kg body weight [3]. “there is evidence that benzyl benzoate is hydrolyzed to benzyl alcohol and benzoic acid; as well, benzyl alcohol and benzaldehyde suffer in vivo oxidation to benzoic acid” [4]. “in plants and animals, benzoic acid is produced endogenously through the phenylalanine– tyrosine pathway” [5]. “several of its derivatives occur naturally in foods, such as fruits (apple, avocado, blackberry, blueberry, cherry, cranberry, melon, papaya, plum, raspberry, strawberry, tomato), vegetables (artichokes, asparagus, beans, cabbage, corn, leek, mushroom, potatoes), meats (beef, chicken, pork, shellfish), cheeses, teas and wines” [6]. food additives (among them benzoic acid) have long been described as sensitizer agents responsible for human allergic reactions [7]. a double-blinded provocation study done with benzoic acid produced objective reactions in 7% of patients with urticaria [8]. there is a report of a child who developed chronic cheilitis when ingesting daily benzoates-preserved industrialized foods [9]. there are reports of asthmatic patients who had a crisis of bronchospasm after the intake of benzoatecontaining antiasthmatic medicines [10]. reports of cross-reactivity among benzoates, azo dyes, and aspirin in patients with urticaria are also common [11]. most provocation tests performed with benzoic acid do not elicit immediate reactions; instead, the reactions appear within 14 hours after the challenge [12]. the immunoreactivity elicited against benzoic acid is non–ige-mediated and is not yet acknowledged explicitly among the recently classified hypersensitivity mechanisms [13]. the main knowledge about the mechanism of hypersensitivity against benzoate (and similar food additives) was brought by an ex vivo leukocyte challenge test determining the olivier et al.; asian j. immunol., vol. 7, no. 1, pp. 63-70, 2024; article no.aji.115657 65 increasing sulfidoleukotriene production [14]. since leukotrienes are known mediators of the leukocyte adherence inhibition phenomenon, we hypothesize that the employ of the leukocyte adherence inhibition test (lait) could help identify the endotype responsible for benzoic acid hypersensitivity [15-17]. “we routinely employ the lait in our facilities to evaluate non–ige-mediated immunoreactivity against suspected allergens, previously engaging in exhaustive provocation tests” [18-24]. to evaluate the potential of the lait to discriminate non–ige-mediated immunoreactivity against benzoic acid, we retrospectively compiled the electronic medical charts of patients with non– ige-mediated allergic rhinitis, allergic bronchitis, asthma, sinus headache, atopic dermatitis, and/or urticaria who were investigated with this procedure. the present study hypothesizes that the lait may differentiate diverse degrees of immunoreactivity against benzoic acid among patients suffering from allergic phenotypes. 2. materials and methods 2.1 subjects after receiving institutional review board approval from the instituto alergoimuno de americana (brazil; 03/2024), we proceeded with the electronic chart review of 8,500 outpatients who attended our facility from january 2018 to march 2024. a cohort of 100 outside patients had been submitted to an ex vivo allergen challenge test with benzoic acid 1mg/ml monitored with lait for presenting non–ige-mediated allergic rhinitis, allergic bronchitis, asthma, sinus headache, atopic dermatitis, and/or urticaria. this study did not include pregnant women, breastfeeding, and patients under biological and/or systemic anti-inflammatory therapy (corticoids, cyclosporin). “the cohort counted 30 males; mean age 41.9 years; sd 20.4 years; range 2 to 90 years; median 43 years; modes = 26; 28; 43; 43 and 53 (each appeared four times); geometric mean = 34.4 years. this procedure was offered to patients with clinical suspicion of benzoate hypersensitivity who demonstrated a non-reactive or inconclusive skin test against benzoic acid” [25]. 2.2 ex vivo investigation: leukocyte adherence inhibition test “we performed the lait as previously described” [26-34]. shortly, each donor's fresh plasma was divided into two parts and used in paralleled ex vivo challenging tests with benzoic acid 1 mg/ml and the unchallenged plasma assay. we collected the plasma with high leukocyte content (buffy coat) from the heparinized tube after one hour of sedimentation at 37 °c. then we distributed aliquots of 100 μl into eppendorf tubes kept under agitation for 30 minutes (200 rpm at 37 °c) with benzoic acid (10μl of a solution with 1mg/ml and ph 7.5) or without benzoic acid (when used as control). after incubation, the plasma was allocated into a standard neubauer hemocytometer counting chamber with a plain, non-metallic glass surface and left to stand for 2 hours at 37 °c in the humidified atmosphere of the covered water bath to allow leukocytes to adhere to the glass. next, we counted the leukocytes, removed the coverslip, and washed the chamber by immersion in a beaker with pbs at 37 °c. then, we added a drop of pbs to the hemocytometer's chamber and allocated a clean coverslip over it. the remaining cells were counted in the same squares as previously examined. the percentage of leukocyte adherence (la) of each assay was estimated as: (the number of leukocytes observed on the hemocytometry chamber after washing divided by the number of leukocytes observed on the hemocytometry chamber before washing) and multiplied by 100 (%). the leukocyte adherence ratio (lar) was estimated based on the ratio between the la from the antigen-specific challenged plasma and the la from the unchallenged control plasma: lar = la of the challenged sample divided by la of unchallenged control plasma multiplied by 100 (%). to further calculate the leukocyte adherence inhibition (lai), we subtracted the lar from 100 (%). we employed the lai results for the cascade distribution chart and the statistics calculations, both performed with the help of the microsoft excel® statistical package. 3. results as a retrospective survey, there was no research protocol; therefore, we report the incidentally immune investigation as registered in the digital medical charts. the lai ranged from 0% to 97%; the mean was 41.1%; the median was 40.5%; the standard deviation was 24.2%; the mode was 0 and 59 (each appeared four times). olivier et al.; asian j. immunol., vol. 7, no. 1, pp. 63-70, 2024; article no.aji.115657 66 the cascade distribution demonstrates a wide range of distribution of lai results (fig.1). four patients ignored the presence of the allergen on the plasma and presented no inhibition of leukocyte adherence (lai = 0%) after contact with benzoic acid (4% of the tests). some patients showed low or moderate immunoreactivity during the ex vivo challenge test. in contrast, others displayed strong immunoreactivity, which could possibly reflect the participation of benzoic acid in a theoretical non– ige-mediated hypersensitivity condition to be further corroborated by in vivo provocation tests. 4. discussion the non-ige-mediated hypersensitivities are characterized by a challenging technical diagnosis due to the lack of standardized immunoassays [35]. diagnosing these conditions among allergic patients is based on individual laborious medical work accomplishing anamnesis, cutaneous tests, and challenge in vivo provocation tests performed after meticulous exclusion diets [36]. “as an increaser of the sulfidoleukotriene production, hypersensitivity to benzoic acid may be, in a certain way, similar (and potentially an enhancer) of the hypersensitivity produced by non-steroidal anti-inflammatory drugs (nsaids). the principal pharmacological action of nsaids is the inhibition of the cyclooxygenase enzymes, which catalyze the synthesis of prostaglandins and thromboxanes” [37]. “cyclooxygenase enzymes catalyze the conversion of arachidonic acid released from the cellular membrane by cytosolic phospholipases activated by nociceptive mechanisms” [38]. “the cyclooxygenases and the lipoxygenases oxidize the arachidonic acid liberated into the cytosol. the cyclooxygenases pathway generates proinflammatory autacoids such as prostaglandins and thromboxanes. the lipoxygenase pathway generates leukotrienes” [39]. the pharmacologic inhibition of the cyclooxygenases increases the lipoxygenases' activity, increasing the leukotrienes' production. any substance that (pharmacologically or immunologically) increases the production of leukotrienes affects the autacoid balance, adversely producing allergic symptoms. the lait theoretically explores every immune pathway as an ex vivo challenge test with a viable leukocyte buffy coat, allowing the interaction of all immune-circulating participants [40]. however, as an observant of the final phenomenon, the lait did not indicate which pathways were involved in inhibiting the adherence (or increasing the production fig. 1. cascade distribution chart of the range groups of leukocyte adherence inhibition (lai) results (x-axis %) of ex vivo benzoic acid challenges monitored by the leukocyte adherence inhibition test (lait), according to the respective number of outcomes over 100 tests/subjects (y-axis) olivier et al.; asian j. immunol., vol. 7, no. 1, pp. 63-70, 2024; article no.aji.115657 67 of leukotrienes), whether pharmacological or immunological [41-44]. as proposed by the exposome-wide association study, the lait also configures itself as an exposome measurement, qualifying itself as an immune marker of the contact and the response to a specific antigen instead of being associated with a specific phenotype [45]. this preliminary retrospective survey demonstrated an extensive range of results from the ex vivo challenge test with benzoic acid monitored by lait in a cohort of patients with various allergic phenotypes. we routinely employ the lait as a complementary triage test to select worthwhile antigens to proceed with more laborious in vivo provocation tests when the specific ige is undetectable. none of our patients presented an exclusive reaction to benzoic acid. every patient was simultaneously tested with protein allergens (mites, fungi, food allergens), demonstrating positive results for some of them [46]. our results may suggest that allergic patients may impair their symptoms by a pharmacological or an immune additional action of benzoic acid over the hypersensitivity response. 5. conclusions our preliminary results show that the lait may differentiate diverse degrees of ex vivo immunoreactivity against benzoic acid in patients clinically diagnosed with non–ige-mediated allergies. the propaedeutic meaning of these results, however, must be established. more studies with prospective larger double-blind cohorts need to evaluate the potential contribution of lait for the etiologic diagnosis of patients suspected of symptomatic hypersensitivity against benzoic acid and other similar food, cosmetic, and pharmaceutical additives. 6. limitations this study is a retrospective analysis of data collected over six years. there was no protocol research, and the subject's data were limited to the essentials available on our electronic sheets. the number of subjects is appropriate for a preliminary study; 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asian journal of immunology 4(1): 125-134, 2021; article no.aji.77165 fresh raffia palm wine, fermented soya bean milk supplementation, enzymatic antioxidant and lipid peroxidation biomarkers in lactating female wistar rats forwah jacques ndeh1*, ebot walter ojong2, uwem okon akpan1 and immaculate ihuoma ekeagba3 1 chemical pathology unit, department of medical laboratory science, faculty of allied medical sciences, university of calabar, nigeria. 2 department of medical laboratory sciences, faculty of health sciences, university of buea, south west region, cameroon. 3 worcaccce group integrated healthcare services, technology ,training and innovative research foundation (wgihstairf), p.o.box 45, bamenda, north west region, cameroon. authors’ contributions this work was carried out in collaboration among all authors. all authors read and approved the final manuscript. article information editor(s): (1) prof. cynthia aracely alvizo báez, autonomous university of nuevo leon, mexico. reviewers: (1) asmarani kusumawati, gadjah mada university, indonesia. (2) isaac john umaru, federal university wukari, nigeria. (3) julio cesar mendes soares, federal university of santa maria, brazil. complete peer review history, details of the editor(s), reviewers and additional reviewers are available here: https://www.sdiarticle5.com/review-history/77165 received 04 october 2021 accepted 08 december 2021 published 29 december 2021 abstract aim: lactation is the most crucial period characterized by intensive caloric requirements to boast up female’s nutritive system and breastfeeding life. current study evaluated the effects of fermented soya bean milk (fsbm) and fresh palm wine (fpw) supplements on endogenous antioxidant enzymes activities and lipid peroxidation biomarker in thirty five (35) lactating wistars rats. methods: animals were obtained from the experimental research animal house of university of calabar, nigeria. at parturition, the animals were randomly divided into five groups containing five original research article ndeh et al.; aji, 4(1): 125-134, 2021; article no.aji.77165 126 (5) rats (n=5). oral treatment done as follow: group i: control group, was given normal feed of protein content 20mg/kg and distilled water (1 ml/kg), group ii: metoclopramide (5 mg/kg), group iii: 10ml /kg of fpw. group iv: three (3) sub-groups of group four (4) received 10%, 20% and 40% of fsbm respectively, group v: co-administration of 40% fsbm plus fpw (10ml /kg) supplements .treatment was done every six hour daily for a duration of ten (10) days. results: serum malondialdehyde concentration, a biomarker of lipid peroxidation was significantly increased (p < 0.05) in metoclopramide treated group than control group, fsbm and fpw supplemented groups. there was a significant decrease (p < 0.05) in serum activities of four endogenous antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase) in metoclopramide treated group when compared to control group, fsbm and fpw supplemented groups. conclusion: while fermented soya bean milk and fresh palm wine supplementation increased lactation and endogenous enzymatic antioxidant activities without lipid peroxidation, metoclopramide at 5 mg/kg concentration increased lactation with lipid peroxidation. keywords: soya bean; palm wine; lactation; lipid peroxidation; endogenous antioxidant enzymes. 1. introduction according to the international society of antioxidant in nutrition and health (isanh) and medical dictionaries, the term antioxidant was initially utilized to refer specifically to chemical products that prevent the consumption of oxygen [13]. endogenous antioxidants are agents which scavenge free radicals and prevent oxidative stress damage caused by excess free radicals in the human body. they are also known as antiradicals [4]. endogenous enzymatic antioxidants can be defined as enzymes or molecules existing within the cellular fluid compartment of the body that are capable of preventing, inhibiting or delaying harmful oxidative reactions caused by high levels of free radicals or oxidants, thus playing an important role in forming antioxidant defensive mechanism and protective system of the body [5]. in recent times, antioxidants in the body in required, acceptable and appropriate levels have been reputed to help the body in repairing, equipping and preventing the formation of and scavenging of free radicals and other potentially toxic oxidizing species in vivo [6-8]. the fundamental classes of antioxidants that form the total antioxidant status of an individual include the primary or chain breaking antioxidant enzymatic systems, secondary and tertiary antioxidants. the primary or chain breaking antioxidant systems include superoxide dismutase, catalase, glutathione peroxidase and reductases. the secondary or preventive antioxidants include glutathione, vitamin c or ascorbate, vitamin e, vitamin a, uric acid, etc .the tertiary or repairing antioxidants include small molecules and plasma proteins such as albumin, transferrin, ceruloplasmin and antioxidant micronutrients such as iron, copper, selenium and manganese [9]. a number of these compounds are of exogenous nature and are obtained from certain food substances namely: antioxidants like αtocopherol, β-carotene, and ascorbic acid, and some micro-nutrient elements such as zinc and selenium [10, 11]. lactogenesis and lactation are essential for optimal feeding of infants during their first one year of dependable life and this has a direct impact on growth, development, immunity and health aspect in the neonatal period [12]. mammalian lactating cells are therefore equipped with some sort of complex defiance mechanisms for radical detoxification [13].oxidative stress (os) is considered a metabolic disturbance that affects organ systems and its presence will affect not only the health status of the animals but also the quality and quantity of the final products, such as milk [14]. there is evidence that oxidative damage increases during lactation in some domesticated and laboratory animals [15, 16]. the soya bean is native to the korean peninsula and the manchurian area and has been one of the major sources of protein in korean food [17]. numerous foods are made by from soybeans, including doenjang (soya bean paste), ganjang (soya bean source), cheonggukjang (fast-fermented bean paste), bean curd, soybean milk, and bean-curd dregs [18]. soybean consumption is effective for the prevention of osteoporosis, arteriosclerosis, strokes and dementia, and can reduce the risk of cancer and obesity [19, 20]. palm wine is a whitish sap collected in vessels attached to the base of the palm tree from where some leaves have been removed. fresh raffia palm wine (raphia hooker) from these sources is sweet and contains little alcohol but, with fermentation, the alcohol content increases over time. unbottled ndeh et al.; aji, 4(1): 125-134, 2021; article no.aji.77165 127 palm wine has a slightly lower alcohol content (around 3%) than bottled palm wine (around 4%) [21,22]. biochemical constituents of palm wine include carbohydrates (sugars), protein, amino acid, lipid, lactic acid, alcohol, vitamins such as vitamin b 6 , b1, b2, vitamin c and some antioxidants, mineral and trace elements like iron and selenium. the mean sugar content of fresh palm wine ranges between 0.10 in maltose and 8.74 mg/100ml in sucrose [2327]. the present study was aimed at evaluating the effects of fermented soya bean and fresh palm wine supplement on lipid peroxidation biomarker and activities of four endogenous antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase) in lactating wistar rats in vivo. 2. methods this experimental study was carried out from january 2018 to december 2018 at the department of physiology, faculty of basic medical sciences, college of medical science, university of calabar, and the laboratory analysis of samples was carried out at the chemical pathology, and haemalogy and blood transfusion units, department of medical laboratory science, university of calabar teaching hospital (ucth) calabar, cross river state, nigeria. 2.1 animal grouping and drug administration adult female apparently healthy wistar rats weighing 180-200g were obtained from biochemistry department experimental research animal house of university of calabar, calabar,nigeria .oral treatment was administered as follow: group i: (control group) was given normal vital feed pellets and distilled water, orally (1 ml/kg). group ii: metoclopramide (5 mg/kg), group iii: an oral treatment (through orogastric tube) of 10 ml/kg body weight per day of fresh raffia palm wine for 10 days. the dosage was calculated as 10 (ml)/1000 (g) × the body weight of the animal. fresh undiluted palm wine from the sap of oil palm trees was collected at every 3 days interval from a local palm wine tapper. the palm wine on collection was administered to the animals and then conserved in a refrigerator for 3 days following which it was often replaced with a fresh collection. group iv: the three (3) sub groups of group four (4) received 10%, 20% and 40% soya bean, respectively. group v: was co-administered 20% soya bean supplement and fresh palm wine (10ml/kg). administration was carried out orally for a period of ten (10) days. 2.2 sample collection after ten days, the rats were anaesthetized by chloroform inhalation in a closed chamber and blood samples were obtained via cardiac puncture into specimen bottles and allowed to clot, and separated by centrifugation at 4000 rev/per minutes for 10 minutes using centrifuge hettich (universal 32, made in germany) at an average room temperature of 23˚c. the supernatant obtained was used for biochemical assays. sample haemolysis was avoided. 2.3 biochemical assay of malondialdehyde in serum malondialdehyde (mda) is one of many low molecular weight end-products of lipid hydroperoxide decomposition and is most often measured as a biomarker and index of lipid peroxidation. mda activity was estimated using the nwlss™ malondialdehyde commercial assay kits purchased from northwest life sciences specialized product nwk-mda01, vancouver wa, specificity: malondialdehyde, sensitivity: 0.08 µ) based on the method adapted by janero, [28]. the test principle is based on the reaction of mda with thiobarbituric acid (tba); forming an mda-tba2 adduct that absorbs strongly at 532 nm. 2.4 biochemical assay of serum superoxide dismutase activities in the serum superoxidase dismutase (sod) activity was assayed using the north west life science specialties (nwlss™) sod assays kit (product nwk-sod02, specificity: cu/zn, mn and fe superoxide dismutase, sensitivity: 5 u/ml). the method is based on the principle of superoxide inhibition of auto-oxidation rate of hematoxylin as originally described by, martin, [29], with modifications to increase robustness and reliability. about 920 μl of assay buffer was added to each cuvette. this was followed by addition of 40 μl of assay buffer (for blank) and 40 μl of sample. the mixture was incubated for two (2) minutes. after which 40 μl hematoxylin ndeh et al.; aji, 4(1): 125-134, 2021; article no.aji.77165 128 reagent was added and mixed quickly to start the auto-oxidation reaction. an absorbance was measured at 560 nm for every 10 seconds for 5 minutes. 2.5 biochemical assay of catalase activities in the serum catalase (cat) activity was estimated using the north west life science specialties (nwlss™) nwlss tm catalase assay kits (product nwkcato1, specificity: 6.0 u catalase/ml) based on the method of beers, (1952) [30], with modifications to increase robustness and convenience. the test is based on monitoring the consumption of h2o2 substrate. the ultraviolet absorption by hydrogen peroxide which is considered as a function of time and was used to follow the catalase-peroxide reaction. the absorption spectrum of hydrogen peroxide was measured spectrophotometrically at the wavelength of 240nm. the optical density as a measure of peroxide concentration, increases linearly with peroxide concentration and directly proportional to catalase in the sample in accordance with the beer-lambert law. the reaction products, oxygen and water, do not absorb light in this spectral region nor does catalase. 2.6 biochemical assay of glutathione peroxidase activity (gpx) in serum glutathione peroxidase (gpx) activity was assessed using the north west life science specialties (nwlss™) cgpx (gpx1) elisa kit (product nwk-gpx02, specificity: glutathione peroxidase, sensitivity: 12.5 pg/ml) based on the method described by avissar, [31]. the assay is based on a sandwich enzyme-linked immunosorbent assay, where sample gpx concentration was determined by comparing the 450 nm absorbance sample wells to the absorbance of known standards. 2.7 statistical analysis the analysis of data was computed using spss software version 20 (spss inc., chicago, usa) and microsoft excel (2007). the results of our study were analyzed with the aid of cross tabulations to explore proportional associations between variables and chi square test was used to explore proportional association between groups. all data were expressed as mean ± standard error of the mean (mean ± sem). statistical significance was carried out using one way analysis of variance (anova) followed by tukey’s post-hoc test. values of p < 0.05 were considered significant. 3. results the results of the current study are shown in the following tables below. table 1 shows the effects of ten (10) days administration of fermented soya bean milk and fresh palm wine supplementation on lipid peroxidation biomarker (serum malondialdehyde (mda) concentration in female lactating wistar rats. these results show that the mean values of mda ( in µmol/l) after 10 days from group i to group v .the mtcl treated group had a higher level of mda (p < 0.05) when compared to the control group, while group v had the lowest level of mda indicating the different degree of lipid peroxidation and lactation. as shown in table 1, the mtcl treated group had a highest level of mda (p < 0.05) compared to the control indicating a high degree of lipid peroxidation and lactation, while group v had the lowest level of mda among the test groups, indicating a lowest degree of lipid peroxidation and lactation. group iii and iv had moderate levels of mda. table 2 shows the effects of ten (10) days administration of fermented soya bean milk and fresh palm wine supplements on the serum activities of four endogenous antioxidant enzymes in female lactating wistar rats namely: 3.1 superoxide dismutase (sod) these results show that the mean values of sod (iu/l) after 10 days from group i to group v. the mtcl treated group had the lowest level of sod while group v had the highest level of sod indicating the degree of elevation of endogenous antioxidants enzymes and less oxidative stress. catalase (cat): these results show that the mean values of catalase -cat (iu/l) after 10 days from group i to group v . the mtcl treated group had the lowest level of catalase while group v had the highest level of catalase indicating the degree of elevation of endogenous antioxidant enzymes and less oxidative stress. ndeh et al.; aji, 4(1): 125-134, 2021; article no.aji.77165 129 table 1. lipid peroxidation biomarker (serum mda levels ) in female lactating wistar rats treated with fermented soya bean milk and fresh palm wine supplements, metoclopramide, distilled water and normal feed groups animal handling and treatment of groups i ii iii iv v control treated with treated with treated with treated with treated with treated with p-value substances administered distilled water metcl fpw fsbm fsbm fsbm fsbm quantity 1ml/kg 5mg/kg 10ml/kg 10% 20% 40% 40%+fpw mean±sem mda after 10 days (µmol/l) 1.33± 0.11 1.79± 0.15 1.53 ± 0.09 1.43 ± 0.11 1.61 ± 0.07 1.44 ± 0.05 1.42 ± 0.07 p< 0.05* metcl = metoclopramide, mda = malondialdehyde, fpw =fresh palm wine, fsbm= fermented soya bean milk mean ± sem= mean plus or minus standard error of the mean. * there was a significant difference in mda levels in fsbm, and fpw treated groups (p < 0.05) compared to the control group, fsbm and fpw supplemented groups table 2. serum activities of four endogenous antioxidant enzymes (sod , cat and gpx ) in female lactating wistar rats treated with soya bean milk and fresh palm wine supplements, metoclopramide, distilled water and normal feed group animal handling and treatment of groups i ii iii iv v p-value control treated with treated with treated with treated with treated with treated with substances administered distilled water metcl fpw fsbm fsbm fsbm fsbm quantity 1mg/kg 5mg/kg 10ml/kg 10% 20% 40% 40% +fpw mean±sem 2.25± 0.09 1.69±0.07 2.18 ± 0.10 1.76± 0.10 1.85±0.05 2.34±0.13 2.98 ± 0.12 p<0.05 α activity (iu/l) mean ± sem catalase activity (iu/l)after 10 days 55.00 ± 0.71 48.20±1.55 66.80±1.59 65.00 ±1.14 67.20± 1.28 70.40± 1.17 78.20±1.02 p<0.05 β mean ± sem activity gpx(iu/l) after 10 days 49.40±0.93), 45.20± 1.56 43.60±0.93 42.60 ±1.50 43.60± 1.50 49.00± 0.94 50.80 ± 0.86 p<0.05 γ sod= superoxide dismutase, cat= catalase, gpx =glutathione peroxidase, metcl = metoclopramide, fpw =fresh palm wine, fsbm= fermented soya bean milk , mean ± sem= mean plus or minus standard error of the mean ndeh et al.; aji, 4(1): 125-134, 2021; article no.aji.77165 130 glutathione peroxidase (gpx): these results show that the mean values of glutathione peroxidase (gpx) (iu/l) after 10 days from group i to group v. the mtcl treated group had the lowest level of glutathione peroxidase (gpx) while group v had the highest level of glutathione peroxidase (gpx) indicating the degree of elevation antioxidants and less oxidative stress. α there was a significant increase in sod activities in fsbm and fpw treated group (p < 0.05) when compared to the control group and mtcl treated group. in table 2 above it is seen that the mtcl treated group had a statistically significant decrease (p < 0.05) activity of sod when compared to the control group and the treated group with fsbm 10%, 20%, 40% and fpw while group v had the highest activity of sod (p < 0.05) indicating the different degree of elevation in the activities of endogenous antioxidant enzymes and lactation with less lipid peroxidation. β there was a significant increase in cat activities in fsbm and fpw treated group (p < 0.05) when compared to the control group and mtcl treated groups. in table 2 as shown above, the mtcl treated group had a significant decrease in (p < 0.05) the activity of catalase when compared to the control, fsbm 10% , fsbm 20% and fpw treated groups, while group v had the highest activity of catalase indicating different degrees in the elevation of endogenous antioxidant enzymes activities and lactation with less lipid peroxidation. γ there was a statistically significant increase in gpx levels in fsbm, and fpw treated group (p < 0.05) when compared to the control group and mtcl treated groups. in table 2 as shown, the mtcl treated group had significant decrease (p < 0.05) in the activity of glutathione peroxidase (gpx) when compared to the control, fsbm 10%, 20%, 40% and fpw treated groups respectively, while group v had a significantly higher (p <0.05) activity of glutathione peroxidase (gpx) indicating different degrees of elevation in endogenous antioxidant enzymes and lactation with less lipid peroxidation. 4. discussion the mean ± sem of serum mda level after 10 days of the current study was displayed in table 1.the results showed a statistically significant increase (p < 0.05) in serum mda levels. in group ii subjects, which was treated with metoclopramide as compared to group i subjects, the control group which was treated only with distilled water and normal feed of protein content 20mg/kg metoclopramide is a medication used to treat stomach disorders such as reflux, nausea, and vomiting (anti–emetic) [31], but in lactating subjects it can cause lactation induction because of its galactagogue properties and lactating augmentative action. these properties could be attributed to the mechanism of action of metoclopramide on its receptors located in the gastro-intestinal, gastroesophageal mucosal, membranes of hepatocytes, breasts and central nervous system including others target sites, where poly unsaturated fatty acids are broken down with the resultant increase of lipid peroxidation which can subsequently assessed by the biomarker called malondialdehyde (mda) formed and released as a by-product [32]. the common side effects of metoclopramide are sleepiness, headache, or restlessness, depression and tardive dyskinesia which are signs of increase oxidative stress and increased lipid peroxidation [33]. the subgroup of group iv which was treated with 20% soya bean showed an increased in the level of serum mda compared to the other treated groups. this could also be due to a possible pro-activity of the supplements on oxidants resulting in increased lipid peroxidation of cells. it could have also been due to the inability of the supplement at this concentration that effectively break down the lipid peroxidation chain reaction since at higher concentrations it helps to reduce oxidative stress because of its high antioxidant content, daidzein , genistein and aglycone composition.these are inline with [34,35]. however, the increase was not statistically significant compared to metoclopramide treated and the normal control groups (p > 0.05). superoxide dismutase (sod) activities as shown in table 2 was decreases in the metoclopramide treated group compared to the control group although the results were not statistically significant. (p > 0.05). this result could be due to the depletion of the endogenous antioxidant as a result of its utilization in combating the existing oxidative stress as suggested by the level of increased mda in metoclopramide treated group. the result also could have been due to the inability of metoclopramide to stimulate the release of endogenous antioxidants as well as its tendency to cause increase in reactive oxygen species (ros) within the system. there was however, a remarkable increase in sod activities , in the fresh palm wine treated group as shown in table ndeh et al.; aji, 4(1): 125-134, 2021; article no.aji.77165 131 2 which could have been be due to its antioxidant activity in fresh palm wine .these results agreed with [36]. the increase was however, rivaled by that of the soya bean 40% group, which showed the highest increase in serum sod activities. this result is suggestive of sb 40% group as having an antioxidant activity more than the standard anti-oxidant or possibly a more lasting effect than that of fresh palm wine and agreed with [37]. this anti-oxidant activity of the supplement on serum sod activities could be attributed to the presence of isoflavones [38]. coadministration of sb and fresh palm wine increased the activities of sod more than when the supplement was given singlely in the 10% and 20% treated groups as shown in table 2. this suggests that the co-administration of the supplement and fresh palm wine could be more potent than the supplement alone at lower doses of 10% and 20%. in table 2 there was a statistically significant decrease in the level of catalase observed in the metoclopramide, and the supplement treated groups at 10% and 20%. this result suggests the inability of metoclopramide and the supplement at 10% and 20% to specifically augment the release of endogenous catalase enzyme when compared to the activity of the supplement at 40%.this is expected because catalase and seleniumcontaining glutathione peroxidase have been demonstrated in human milk but their levels in colostrum is higher than that in corresponding mature milk and this agreed with [39]. however, this highest levels in colostrum usually decline during early lactation, despite the fact that total lipids increase as reported by yuksel (2015) [40]. as shown on table 2, there was a statistically significant decrease in the activities of gpx in the supplement treated groups at 10% and 20% and also that of fresh palm wine. this result suggests that the supplement at these concentrations also do not seem to have antioxidant activity, specifically on gpx release or synthesis [41]. 5. conclusion metoclopramide at 5 mg/kg concentration is capable of increasing lipid peroxidation according to this study and the fsbm supplement at 40% and fresh palm wine mixture showed an increase in endogenous enzymatic antioxidant activities with more lactation and less lipid peroxidation when compared to the control groups. what is known about this topic in many african cultures and tradition there is long practice that fermented soya beam milk and fresh raffia palm wine and is capable of augmenting lactation and clinically, metoclopramide is known to be a good galactogogue and side effects .what is not known till the time of this is the effect of the fermented soya bean milk and fresh raffia palm wine on the oxidative stress and the endogenous antioxidant enzymes and lipid peroxidation. what this study adds findings from this study have help highlighted the role of the combine effects of fresh raffia palm wine and fermented soya bean milk supplementation during lactation in viviparous mammals. in addition the long standing traditional dilemma and the superstitious belief about the usage of fresh palm wine fermented soya bean milk in humans have been elucidated by the findings of the current study . availability of data and materials data sets generated and analyzed in this study are available from the corresponding author on request. disclaimer the products used for this research are commonly and predominantly use products in our area of research and country. there is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. also, the research was not funded by the producing company rather it was funded by personal efforts of the authors. ethical approval ethical approval was obtained from the ethical committee of university of calabar on animal handling, consistent with standard animal welfare guidelines. competing interests authors have declared that no competing interests exist. references 1. 18th isanh middle east antioxidants world congress, beirut antioxidants 2017. 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aji, 4(1): 125-134, 2021; article no.aji.77165 134 41. kazuhiro suzuki, hidekazu koike, hiroshi matsui, yoshihiro ono, masaru hasumi, haruki nakazato hironobu okugi, yoshitaka sekine, kazuya oki, kazuto ito takumi yamamot ,genistein, a soy isoflavone, induces glutathione peroxidase in the human prostate cancer cell lines lncap and pc‐ 3. international journal of cancers. 20 june 2002;99(6):846-852. _________________________________________________________________________________ © 2021 ndeh et al.; this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: https://www.sdiarticle5.com/review-history/77165 https://onlinelibrary.wiley.com/action/dosearch?contribauthorstored=suzuki%2c+kazuhiro https://onlinelibrary.wiley.com/action/dosearch?contribauthorstored=koike%2c+hidekazu https://onlinelibrary.wiley.com/action/dosearch?contribauthorstored=matsui%2c+hiroshi https://onlinelibrary.wiley.com/action/dosearch?contribauthorstored=matsui%2c+hiroshi https://onlinelibrary.wiley.com/action/dosearch?contribauthorstored=ono%2c+yoshihiro https://onlinelibrary.wiley.com/action/dosearch?contribauthorstored=hasumi%2c+masaru https://onlinelibrary.wiley.com/action/dosearch?contribauthorstored=nakazato%2c+haruki https://onlinelibrary.wiley.com/action/dosearch?contribauthorstored=okugi%2c+hironobu https://onlinelibrary.wiley.com/action/dosearch?contribauthorstored=sekine%2c+yoshitaka https://onlinelibrary.wiley.com/action/dosearch?contribauthorstored=oki%2c+kazuya https://onlinelibrary.wiley.com/action/dosearch?contribauthorstored=ito%2c+kazuto https://onlinelibrary.wiley.com/toc/10970215/2002/99/6 http://creativecommons.org/licenses/by/4.0 _____________________________________________________________________________________________________ *corresponding author: e-mail: eeyeboah@uew.edu.gh; asian journal of immunology 4(1): 180-189, 2021; article no.aji.76491 effects of copper and zinc supplementation on haematological, renal and liver function in healthy wistar rats emmanuel effah-yeboah a*, emmanuel agyapong asare b, janice dwomoh abraham a, papa kofi amissah reynolds a, joshua dwomoh a, eldith adongo a, stephen appiah a and gadafi iddrisu balali c, d a department of biological sciences education, college of agriculture education, akenten appiahmenka university of skills training and entrepreneurial development, mampong-ashanti, ghana. b department of chemistry education, college of agriculture education, akenten appiah-menka university of skills training and entrepreneurial development, mampong-ashanti, ghana. c department of theoretical and applied biology, kwame nkrumah university of science and technology, kumasi, ghana. d department of science, sda college of education, agona-ashanti, ghana. authors’ contributions this work was carried out in collaboration among all authors. all authors read and approved the final manuscript. article information open peer review history: this journal follows the advanced open peer review policy. identity of the reviewers, editor(s) and additional reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here: https://www.sdiarticle5.com/review-history/76491 received 20 september 2021 accepted 27 november 2021 published 30 december 2021 abstract copper and zinc are essential elements that aid in various physiological and biochemical functions. nevertheless, exposure to these heavy metals could also be detrimental to some vital organs such as the liver and kidney of the body. increased usage of agrochemicals in crop production has the potential of increasing bioaccumulation of trace elements in both humans and animals as they feed on these plants. the present study, therefore, investigated the effects of copper and zinc supplementation on haematological, renal, and liver functions in healthy wistar rats. a total of twenty-five wistar rats of five weeks old (weighing 185g-250g) were recruited for the study. animals were grouped into five namely control, zinc low, zinc high, copper low and copper high as groups 1, 2, 3, 4, and 5 respectively and administered feeds containing low and high original research article effah-yeboah et al.; aji, 4(1): 180-189, 2021; article no. aji.76491 181 doses of copper and zinc two weeks post-acclimatization. laboratory investigations on haematological, renal, and liver markers were assessed after three weeks of feeding. both copper and zinc doses significantly (p <0.05) elevated the counts for red blood cell (rbc), hemoglobin (hb), hematocrit (hct), white blood cells (wbcs), and platelets with no significant effect on their weight gain compared to the control group of rats. zinc doses elevated the levels of alanine transaminase (alt), with a low dosage having a significant effect on ast and alp. the effect of copper and zinc doses significantly (p <0.05) reduced creatinine levels, with no significant effect on urea concentrations. the elevation of alt and platelets as a result of copper and zinc exposure suggests their deteriorating effect on the liver and other organs of the body. long-term exposure to these trace elements can lead to a lot of pathologies. keywords: copper; zinc; haematological; renal; liver function. 1. introduction copper and zinc play functional roles in diverse physiological and biochemical activities in the body [1,2]. their effects on the improvement of enzymatic activities, protein synthesis, and stabilization of dna and rna have made them ideal for consumption [3,4,5]. nonetheless, extreme exposure could be detrimental to vital organs of the human body [6,5]. long-term exposure to these metals has been associated with the slow progression of physical, muscular, and neurological degenerative processes that mimic diseases like parkinson’s disease and alzheimer’s disease [7,8]. anthropogenic activities such as mining, smelting, and the use of agrochemicals in farming have immensely contributed to the release of these metals into the environment [9,10]. although chemical fertilizers, pesticides, and weedicides increase plant yield, their application has been associated with the accumulation of trace elements such as copper and zinc in the leaves, stems, and roots of crops [11,12]. consumption of leafy vegetables may therefore expose humans to these heavy metals [13,14]. available reports suggest increased human exposure to heavy metals, a result of rapid growth and applications in agriculture, industrial, domestic, and technology [15]. while there have been studies on heavy metal exposure, these have largely been limited to assessing their levels and concentrations in environmental matrices (soil, water, and vegetables) [1,16,17,18]. the few available reports however evaluated the concentration of heavy metals in the animal rearing system and the administration of copper sulphate on markers of renal functions and feeding patterns of wistar rats [19,20]. recent studies point to increasing concentrations of heavy metals in both medicinal plants and herbal distillates, leading to the production of reactive oxygen species upon human consumption and exposure [21,22,23]. based on these reports, we suspect that exposure to heavy metals (copper and zinc) may result in pathophysiological effects in humans. therefore, we qualitatively, investigated the effects of copper and zinc supplementation on haematological, renal, and liver function in healthy wistar rats. 2. materials and methods 2.1 study design twenty-five (25) male and female wistar rats (185g – 250g) aged five weeks old were randomly selected for the experiment. these rats were obtained from the pharmacological farm of kwame nkrumah university of science and technology (knust), ghana. 2.2 study site animal housing and treatment were carried out at the laboratory of the department of science education, university of education, winneba, asante mampong campus. hematological, renal, and liver tests were done at edep laboratory service, santasi –kumasi, ghana. 2.3 maintenance of animals rats were housed in a one-tier hutch during the study. they were kept in animal rooms at the effah-yeboah et al.; aji, 4(1): 180-189, 2021; article no. aji.76491 182 laboratory where room temperature was maintained at 26±3∘c under a 12-hour light-dark cycle. the rats were thoroughly examined and any abnormalities and/or ailments were recorded before the research began. two hundred and twenty-five grams (225 g) of concentrate feed was weighed with a camry dial spring scale (iso9001 approved) every morning and given to the animals as their daily feed. feeding and water troughs were cleaned every morning before feeding. 2.4 preparation of feed after two weeks of acclimatization, experimental rats were purely subjected to concentrate feed. the feed was formulated and prepared at the university of education, winneba, mampong campus (uew-m) laboratory. ingredients were made up of, wheat brand (5 kg/30 kg); maize (15 kg/30 kg); concentrate (10 kg/30 kg); copper sulphate and zinc tablet. they were weighed according to their required quantities with the camry dial spring scale (iso9001 approved). the measured quantities were thoroughly mixed by spade, bagged, and then stored in the animals’ feed room. 2.5 preparations and treatments a total of 25 experimental rats were used to assess the effect of copper and zinc on the haematological, renal, and liver markers. animals were divided into five (5) groups and housed under controlled environmental conditions. copper sulphate (cuso4) and zinc tablet used for the study were obtained from uew-m laboratory and laugh pharmacy (mampongashanti) respectively. in the preparation of the wheat brand maize concentrate with cuso4 treatment, an amount of 3.929 g and 0.982 g of cuso4 powder was weighed for the high and low concentrations respectively using an electronic scale. it was then dissolved in 200ml distilled water and was uniformly mixed with 5 kg of the feed concentrate. also, for the zinc treatment, 200 g and 50 g of zinc tablets were dissolved in 200 ml distilled water for the high and low doses respectively. it was uniformly mixed with 5 kg of the feed concentrate. the mixture was bagged and stored in the animals’ feed room. each group was given an equal amount of feed throughout the experimental period. weekly measurements of weight were recorded. the five treatments used for the study were; group 1 (control) fed on wheat brown maize concentrate and water. group 2 (zinc low) fed on wheat brand maize concentrate with low zinc ppm group 3 (zinc high) fed on wheat brand maize concentrate with high zinc ppm group 4 (copper high) fed on wheat brand maize concentrate with high cuso4 ppm group 5 (copper low) fed on wheat brand maize concentrate with low cuso4 ppm. 2.6 haematological and biochemical parameters experimental rats were sacrificed and blood was dispensed into edta tubes and then analyzed for haematological markers such as red blood cells (rbcs), haemoglobin (hb), hematocrit (hct), mean corpuscular volume (mcv), mean corpuscular haemoglobin (mch), mean corpuscular haemoglobin concentration (mchc) using an automated haematology analyzer (mslab 12, china). another portion of blood was collected in empty vacutainer tubes and centrifuged at 13000 rpm for 5 mins. the serum was then retrieved and biochemical parameters such as ast, alt, alp, tb, tp, creatinine, and urea concentrations in serum were analyzed using commercially available kits and a standard bs-120 mindray chemistry analyzer. 2.7 statistical analysis graphpad prism version 5.0 for windows (graphpad software, san diego, ca, usa) was used for data analysis. data were presented as mean ± sem online graphs and tables. for comparing the control and treatment groups, independent samples -tests for the significance of differences were used. to compare the biological effects of the treatment, an analysis of variance (anova) was used. newman keuls posthoc analysis was also used to identify the source of the variation. values of less than 0.05 were considered statistically significant. 3. results 3.1 effect of copper and zinc doses on the weight of wistar rats treated groups showed a steady weight gain after two weeks of acclimatization, although it was not statistically significant (p <0.05) from the control group (fig. 1). effah-yeboah et al.; aji, 4(1): 180-189, 2021; article no. aji.76491 183 fig. 1. effect of cu high, cu low, zn high, and zn low on weight as compared with the weight of the ctl group 3.2 effect of copper and zinc doses on haematological parameters the effects of copper and zinc dosages on haematological parameters were investigated and compared with similar parameters of normal control rats. following the feed administration with variously defined dosages, haematological parameters were significantly increased (p <0.05) compared to the control group. 3.3 effect of copper and zinc doses on liver enzymes zinc low doses significantly (p <0.05) elevated the levels of the liver enzymes (ast, alt, and alp) whereas zinc high doses only showed a significant (p <0.001), increase in alt compared to the control. comparing the different doses of copper, though not significant, the elevation of the biochemical markers was higher in cu low than in cu high as compared to the control group (table 2). 3.4 effect of copper and zinc doses on renal function doses of all treatment groups showed a significant (p <0.05) reduction of creatinine levels compared to the control. zinc high doses showed an increase in urea levels, however, urea levels in copper doses (low and high) and zinc low doses were statistically not significant (fig. 2). fig. 2. effect of cuh, cul, znh, and znl on creatinine and urea levels; each column represents the mean±sem. (n= 5). •p <0.05, •p <0.001 compared to control group (one way anova followed by newman-keuls test) weights of rats initial week 1 week 2 week 3 0 100 200 300 400 ctl cuh cul znh znl creatinine levels c tl c uh c ul z nh z nl 0 50 100 150     urea levels c tl c uh c ul zn h zn l 0 2 4 6 8 10 effah-yeboah et al.; aji, 4(1): 180-189, 2021; article no. aji.76491 184 table 1. effect of cu high, cu low, zn high, and zn low on levels of haematological markers as compared with the control rbc (10 6 /µl) hb (g/dl) hct (%) mcv(fl) mch(pq) mchc(g/dl) mpv(fl) platelets (10 3 / µl) wbc (103/dl) ctl 5.6± 0.1 10.6±0.1 35.00±1.0 59.1±0.7 19.32±0.3 31.78±0.3 9.06±0.4 26.18±2.9 2.36±0.2 cuh 8.3±0.2••• 13.98±0.3••• 51.46±1.3••• 62.96±1.2 17.12±0.3•• 27.2±0.5••• 9.8±0.2 897.6±111.3••• 8.82±0.2••• cul 8.1±0.2••• 14.04±0.3••• 50.52±1.5••• 62.4±1.6 17.34±0.3•• 27.9±0.2••• 9.42±0.3 779.2±67.2••• 5.69±0.7•• znh 9.0± 0.5••• 15.22±0.6••• 56.3±2.2••• 60.78±0.8 16.88±0.4••• 27.64±0.4••• 9.20±0.2 805.3±53.6••• 13.25±1.1••• znl 7.4±0.2••• 13.06±0.2••• 44.82±1.6••• 60.64±1.1 17.70±0.4•• 29.2±0.9•• 8.70±0.3 767.8±45.9••• 11.71±0.6••• each column represents the mean±sem. (n= 5). •p <0.05, ••p <0.001, •••p <0.0001 compared to control group (one way anova followed by newman-keuls test) table 2. effect of cu high, cu low, zn high, zn low on levels of enzymes and proteins associated with the liver (alt, ast, alp, glb, alb, tp) alt (u/l) ast (u/l) alp (u/l) globulin (g/l) albumin(g/l) total protein (g/l) ctl 100.5± 1.99 172.4±25.7 235.9±20.3 50.2±1.8 28.4±1.7 5.5±0.1 cuh 79.6±4.56 195.0±21.7 339.5±72.9 56.24±1.3 28.8±1.4 5.8±0.3 cul 102.6±7.6 235.8±9.4 388.5±52.4 48.74±1.2 29.2±0.4 6.2±0.2 znh 157.6±26.9•• 227.1±12.6 299.1±55.8 57.94±6.6 28.12±0.6 4.4±0.6 znl 147.3±12.3• 259.8±25.1• 783.2±201• 63.8±6.2 27.36±1.8 5.6±0.7 each column represents the mean±sem. (n= 5). •p <0.05, ••p <0.001 compared to control group (one way anova followed by newman-keuls test) effah-yeboah et al.; aji, 4(1): 180-189, 2021; article no. aji.76491 185 4. discussion the present study investigated the effects of copper and zinc supplementation on haematological, renal, and liver markers in healthy wistar rats. the effects of these trace elements administered as feeds significantly (p <0.05) elevated the haematological markers of the treated rats compared to the control. this agrees with [24] who reported significant (p <0.05) changes in haematological parameters, especially white blood cells (wbc) following the administration of copper oxide and/or zinc oxide nano-particles on rats. also, a study by [25] showed a significant (p <0.05) increase in red blood cells (rbcs), haemoglobin (hb), and hematocrit (hct) following zinc supplements. the significant elevation of the haematological parameters such as rbc, hb, wbc, and platelets counts, as shown in table 1 following the supplementation of these trace elements may be a result of bone marrow deficiency leading to left shift production of these markers [26]. also, high platelets count suggests reactive thrombocythemia as a result of bone marrow suppression. this is in line with studies that suggest that persistent hyperglycemia predisposes individuals to thrombogenesis with platelet aggregation as a result of acute zinc supplements thereby prolonging bleeding time [27,28]. the high elevation of rbc and hb following copper sulphate exposure suggest vascular oxidative stress which can lead to impaired oxygen delivery hence the increment in its count for homeostatic balance [24]. this is supported by findings from lucas and rifkinds [29] who reported a pronounced increase in rbc and hb as a result of vascular oxidative stress following copper bound amyloid-β peptide (cuaβ) exposure. the observed significant difference between treatment and control groups in some of the enzymes associated with liver function (alt) following zinc doses (high and low) administration suggests organ damage, especially in the liver. low levels of alt are generally considered a good marker of liver health; however, low levels of alt occur as a result of certain underlying medical conditions such as deficiency in vitamin b6 or chronic kidney disease. also, high levels of alt are a good indicator of damage caused to liver cells such as liver inflammation and cirrhosis [30]. serum alanine transaminases (alt) are not only measured to determine possible liver damage but also useful in monitoring the general health status [31, 32, 33]. this is an enzyme that is mainly found in the cytosol of the hepatocytes and plays a role in gluconeogenesis [34]. alt is released as a result of liver injury from the injured liver cells into the serum, which causes the levels to increase significantly in the serum [35, 36]. also, only low doses of zinc treatment significantly (p <0.05) affected most of the liver enzymes (alt, ast, alp) indicating the essential role of zinc and hence it's right proportional supply to the body). zinc deficiency may induce oxidative stress, compromise the function of oxidative-sensitive transcription factors that can affect cell function, proliferation and survival. again, it can also result in cell and tissue damage by modulating specific signal cascades with resulting damage to enzymes, mitochondria and ribosomal structures. hepatic damage following zinc deficiency sides with other studies that report on zinc deficiency as a predisposing factor for acute and chronic liver disease [37, 38]. copper doses, both high and low did not have any significant effect on the enzymes associated with liver function. this can be correlated to the significant reduction of creatinine levels following copper treatment. the observable insignificant effect of copper doses on renal function may be due to the body’s homeostatic mechanism of regulating excess copper via bile secretion by the liver [39, 40]. urea is a principal nitrogenous waste product of metabolism as a result of protein deamination [41], its physiological role in the detoxification of ammonia and conservation of water in the kidney has made it ideal as a biomarker for renal function [42]. even though statistically, no significant differences were observed in the treatment groups compared to control regarding blood urea levels, its slight increment following high doses of zinc treatment cannot be overlooked. the insignificant differences observed may be because blood urea levels are only affected following an extreme reduction of the glomerular filtration rate [43]. albumin and direct bilirubin levels were found to be very low as compared to their normal ranges. bilirubin acts as an antioxidant that protects tissues throughout the body from damage by substances that can cause the breakdown of effah-yeboah et al.; aji, 4(1): 180-189, 2021; article no. aji.76491 186 cells. given this, lower levels of bilirubin indicate a possibility of certain body parts being vulnerable to damage. several medical conditions are associated with lower levels of bilirubin in the body. these conditions include ulcerative colitis, brain lesions and diabetic retinopathy [44, 33, 24, 45, 25]. on the other hand, lower levels of albumin are associated with inflammation, shock and the inability of the body to absorb and digest protein. 5. conclusion results from the study reviewed that, copper and zinc at both high and low doses caused high elevation of haematological markers. this affirms the essential role of cu and zn in blood formation and immunity in the body. however, low levels of zn yielded low liver and renal markers, this suggest that long-term exposure to zinc can have a deteriorating physiological and pathological effects on the liver and other organs of the body. 6. recommendations based on the findings of this current study, it is recommended that;  further studies should be carried out to ascertain the histopathological effects of copper and zinc on the liver and kidney.  whenever cu and zn treatments are administered orally to rats, there may be a high propensity of the gut of the rats being affected, it is therefore recommended that further investigation be made to study the effects of cu and zn on the gut of rats as well as possible microbiota present in the gut.  the food and agriculture organization (fao) and the ministry of food and agriculture (mofa) must ensure strict compliance to permissible levels of trace elements in pesticides used in crop production.  farmers must be educated on the dangers of breaching the permissible levels of trace elements on-farm produce as well as in the supplementation of animal diets. disclaimer the products used for this research are commonly and predominantly use products in our area of research and country. there is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. also, the research was not funded by the producing company rather it was funded by personal efforts of the authors. ethical approval all animal experiments, procedures, and techniques used in this study were conducted in compliance with the national institute of health guidelines for care and use of laboratory animals, with ethical approval from the institutional review board, university of cape coast, ghana (ethical clearance number: ucc irb/chas/2017). competing interests authors have declared that no competing interests exist. references 1. engwa ga, ferdinand pu, nwalo fn, unachukwu mn. mechanism and health effects of heavy metal toxicity in humans poisoning in the modern world-new tricks for an old dog? : intechopen; 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229:18-24. 43. elwir s, rahimi rs. hepatic encephalopathy: an update on the pathophysiology and therapeutic options. journal of clinical and translational hepatology. 2017;5(2):142. 44. schieffer km, bruffy sm, rauscher r, koltun wa, yochum gs, gallagher cj. reduced total serum bilirubin levels are associated with ulcerative colitis. plos one 2017;12(6):e0179267. available:https://doi.org/10.1371/journal.po ne.0179267ture 45. tsukada n. low bilirubin levels indicate a high risk of cerebral deep white matter effah-yeboah et al.; aji, 4(1): 180-189, 2021; article no. aji.76491 189 lesions in apparently healthy subjects; 2018. accessed on: 24 th may 2020 available: www.nature/scientific report.com _________________________________________________________________________________ © 2021 effah-yeboah et al.; this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: https://www.sdiarticle5.com/review-history/76491 http://creativecommons.org/licenses/by/4.0 _____________________________________________________________________________________________________ *corresponding author: e-mail: ashraf.alaqqad@gmail.com; asian journal of immunology 4(1): 120-124, 2021; article no.aji.77408 henoch-schonlein purpura successfully treated with oral steroids: a case report mohamed eltaieb ali1, ashraf alakkad1* and naglaa mohamed hamed2 1 internal medicine department, madinat zayed hospital, uae. 2 dermatology department, madinat zayed hospital, uae. authors’ contributions this work was carried out in collaboration among all authors. all authors read and approved the final manuscript. article information editor(s): (1) dr. darko nozic, university of belgrade, serbia. (2) prof. cynthia aracely alvizo báez, autonomous university of nuevo leon, mexico. reviewers: (1) rosario eça, centro hospitalar lisboa central, portugal. (2) simin dokht shoaei, sbmu, iran. complete peer review history, details of the editor(s), reviewers and additional reviewers are available here: https://www.sdiarticle5.com/review-history/77408 received 20 september 2021 accepted 30 november 2021 published 01 december 2021 abstract a 38-years old male patient, documented case of irritable bowel syndrome (ibs), presented to the clinic with complaints of severe central abdominal pain and severe rash. the day before presenting to the clinic, the patient went to the emergency department for the same complaints. according to the patient, the abdominal pain was colicky in nature. there were no associated complaints of nausea, vomiting, or gi bleeding, but did have an upper respiratory tract infection three weeks ago, after which he had started experiencing these symptoms. his infection was treated with oral antibiotics and was soon got resolved. the staff at the ed had investigated the cause, including a ct abdomen with contrast, but the reports had been all clear. he had mildly raised serum creatinine at that time. on clinical examination, the patient had severe tenderness in his abdominal region. the rash was extensive, reddish, raised, and purpuric in nature and had spread all over his lower limbs and hips on both sides. the upper limbs were also involved up to the forearms. all the lab investigations conducted on the patient were normal, including cbc, creatinine, serum c3, c4, and the rheumatoid factor. crp was found to be elevated and positive occult blood was seen in the stool of the patient. the combination of the above medical symptoms and investigations revealed that the patient was suffering from abdominal angina. the symptoms had a classic presentation pattern: positive occult case study ali et al.; aji, 4(1): 120-124, 2021; article no.aji.77408 121 blood in stool, a purpuric rash, abdominal pain, and renal involvement that was preceded by a documented case of upper respiratory tract infection (urti). later, the patient was found to have all findings consistent with immune-mediated iga vasculitis (henoch schonlein purpura) and was then treated accordingly. the above-mentioned case was an interesting presentation: the condition is not often presented to the hospital with such clear manifestations, leading to an absolute diagnosis. the following sections of this case study will explore how the patient was diagnosed, treated, and managed accordingly to save his life. keywords: henoch schonlein purpura; irritable bowel syndrome; gi bleeding; abdominal angina. 1. introduction abdominal angina is a type of pain related to the gastrointestinal tract that is seen to occur mostly after meals when there is an insufficient supply of blood to the colon and it is unable to meet the demands of the rest of the viscera of the gi tract. abdominal angina is most commonly seen to occur in cases of mesenteric occlusive vascular disease [1]. abdominal angina gets its name from the intermittent claudication that occurs in similar cases of myocardial angina or vascular disease [2]. however, abdominal angina is a rare phenomenon, both in the united states as well as elsewhere in the world. this is because most cases of mesenteric occlusive disease get diagnosed early and so urgent evaluation and timely treatment ensure that no further complications develop in the patient [3]. however, since there were obvious signs of what the patient was suffering from, it was later on that it was concluded that abdominal angina might just be a part of his presenting symptoms. the spectrum of the symptoms were more than indicated by the diagnosis and that is why the patient underwent further examination and investigation to gain a more accurate indication of the condition that he was suffering from.later on, the presence of abnormally elevated protein levels in the patient’s blood pointed to a condition similar to henoch schonlein purpura, but the normalcy of the rheumatoid factor and c4 tests lead to the exclusion of this diagnosis as well. another trip to the dermatologist helped in confirm the diagnosis as henoch schonlein purpura or iga vasculitis. henoch schonlein purpura is an iga-mediated immune vasculitis that involves the small vessels of the gastrointestinal tract, kidneys, joints, skin, respiratory system and lungs. the presentation of the patient was considered to be classic of this condition which allowed for timely diagnosis and appropriate management to avoid complications in the long run [4]. 2. case study a 38-year old male patient, who appeared to be quite anxious and restless, presented to the clinic. this patient complained of having severe central abdominal pain and a skin rash. he had been to many hospitals and clinics, and nearly all of them had failed to diagnose what was wrong with him, despite there being obvious signs of illness, owing to his deteriorating state. earlier, the patient was diagnosed with irritable bowel syndrome (ibs). the day before he went to the emergency department for the same complaints. the ed had promptly ordered lab investigations, along with a ct abdomen with contrast to rule out possible warning signs that might have developed insidiously inside the patient. the ct scan was completely normal. the same applies to the lab reports, except for a small rise in the levels of serum creatinine. 2.1 examination when the patient was thoroughly examined, the nature of his pain turned out to be colicky. however, the pain was not associated with any kind of nausea, vomiting, gi loss, or bleeding. the patient reported suffering from an upper respiratory tract infection about three weeks ago, for which he was treated successfully with oral antibiotics. the physical examination of the patient revealed that he was tearful and anxious because of his condition. he had obvious signs of central abdominal tenderness and there was an extensive rash which was raised, reddish and purpuric, across both the lower limbs and hips of the patient. the upper limb was also involved, but only up to the forearms of the patient. ali et al.; aji, 4(1): 120-124, 2021; article no.aji.77408 122 the following is an image of the patient’s rash, which has been used with the patient’s consent. image 1 image 2 image 1 and 2. purpuric, reddish-purple rash involving the lower limbs of the patient 2.2 investigations at the clinic, the patient again had some of investigations done for a better picture of his condition. here is a summary of what was found: chart 1. cbc investigations cbc normal creatinine abnormal in the earlier reports but had normalized. crp mildly elevated serum c3 and c4 normal levels rheumatoid factor normal other important findings positive occult blood in stool 2.3 diagnosis and management from the above reports, it was clear that the patient was suffering from abdominal angina as he had the classic triad of purpuric rash, abdominal pain, and renal involvement, all of which had begun after the patient had an upper respiratory tract infection. it was also suspected as an ideal differential that the patient might go on to develop henoch schonlein purpura (iga vasculitis) with cryoglobulinemia. however, since this profile was not being favored due to the normal rheumatoid factor and c4 levels, the possibility was excluded. as a final resort, the patient was sent to the dermatology clinic for another opinion and to have a skin biopsy. the biopsy without immunofluorescence supported a diagnosis of iga vasculitis and so he was treated accordingly. this biopsy led to the patient being administered a course of daily oral prednisolone (20 mg, po) along with oral ppi for his abdominal symptoms. the response of the patient to steroids was dramatic. his rash regressed spontaneously and, within 48 hours of starting this treatment, the patient’s abdominal pain had completely resolved as well. after this, a skin biopsy was taken without immunofluorescence again, and the following image, used with consent, was visible at that time. image 3. a skin biopsy without immunofluorescence there is an obvious resolution of the rash when compared with the patient’s earlier images. immunofluorescence was not ordered for this patient because there was no fresh rash present at the time of biopsy, so it was omitted to avoid confusion. this is how this case was managed successfully in this patient with an absolute resolution of all the symptoms that the patient had presented with and had been suffering from for a long time. ali et al.; aji, 4(1): 120-124, 2021; article no.aji.77408 123 3. discussion the iga present in the human body has a unique way of dealing with the microorganisms it encounters. it sometimes causes an autoreactive response, which can become an autoimmune mediated response, resulting in the development of conditions such as iga vasculitis, which is also known as henoch schonlein purpura [5]. bacterial and viral diseases have been seen to trigger iga vasculitis and covid19 has also been seen to cause similar responses in the human host body. these bacteria and viruses are seen to elicit an autoimmune response which results in further insults and, finally, the appearance of iga vasculitis in affected individuals. some of the most well-known triggers that cause iga vasculitis include streptococcus, s.aureus, varicella zoster virus, human immunodeficiency virus (hiv), cytomegalovirus, and so on [6][7]. iga vasculitis can affect both children and adults. in adults, it mostly takes the appearance of palpable purpura which are markedly present in the lower limbs of the patient. at times, the upper limbs and trunk of the patient can also be involved [8]. approximately 70% of patients who suffer from iga vasculitis experience abdominal pain. of these, 30% also experience an episode or multiple episodes of gastrointestinal bleeding. however, this bleeding presents around a week after the rash has manifested on the skin of the patient [9]. when a patient’s kidneys become involved, which is an infrequent occurrence, the options for an appropriate medication or treatment regimen become narrower to avoid further damage to the kidneys. almost all the affected patients have shown significant improvement as soon as a corticosteroid management plan is started. prednisolone especially favors the renal profile without causing any further damage to the patient [10]. apart from corticosteroids, the other available treatment options include dapsone, rituximab, intravenous immunoglobulin, and ace inhibitors, depending on the underlying causes of the symptoms and the severity of the iga vasculitis that the patient is suffering from. 4. conclusion iga vasculitis, also known as henoch schonlein purpura, is an inflammatory disease of the blood vessels that mostly affects children, although it can also occur in adults. people who suffer from iga vasculitis are seen to have a raised red or purple rash marked predominantly on the lower limbs. later in the progression of the disease, an affected person may develop problems of the gastrointestinal tract, joints, or even the renal system, although the severity and intensity of all these symptoms differ from patient to patient. this patient also developed these symptoms in a similar pattern – at first, he only developed an upper respiratory tract infection, which was successfully treated and therefore, not of any importance. later on, he developed abdominal pain along with severe rash on his lower extremities that became the cause of his presenting complaints. the labs were all almost normal revealing no underlying disease, but later on, it was revealed that the patient was suffering from iga vasculitis. once diagnosed, it was easily managed with oral steroids and the condition resolve in real time, leaving behind no room for complications. there have been reports of recurrence of the disease, but since it is rare and the symptoms controlled, the same treatment protocol can help to control these flare-ups easily. consent as per international standard or university standard, patient’s written consent has been collected and preserved by the author(s). ethical approval as per international standard or university standard written ethical approval has been collected and preserved by the author(s). competing interests authors have declared that no competing interests exist. references 1. mahajan k, osueni a, haseeb m. abdominal angina. [updated 2021 may 24]. in: stat pearls [internet]. treasure ali et al.; aji, 4(1): 120-124, 2021; article no.aji.77408 124 island (fl): stat pearls publishing; 2021. available:https://www.ncbi.nlm.nih.gov/boo ks/nbk441943/ 2. mosselveld p, van werkum mh, menting tp, pieters ammj, müller metm, bosch fh. a case of progressive abdominal angina. neth. j. med. 2018;76(5):255. 3. mensink pb, moons lm, kuipers ej. chronic gastrointestinal ischaemia: shifting paradigms. gut. 2011;60(5):722-37. 4. roache-robinson p, hotwagner dt. henoch schönlein purpura. [updated 2021 aug 11]. in: stat pearls [internet]. treasure island (fl): stat pearls publishing; 2021. available:https://www.ncbi.nlm.nih.gov/boo ks/nbk537252/ 5. sugino h, sawada y, nakamura m. iga vasculitis: etiology, treatment, biomarkers and epigenetic changes. international journal of molecular sciences. 2021;22 (14):7538. available:https://doi.org/10.3390/ijms22147 538. 6. nakatsuka k. serum anti-streptococcal iga, igg and igm antibodies in iga associated diseases. acta paediatr. jpn. 1993;35:118–123. doi: 10.1111/j.1442-200x.1993.tb03020. x. 7. ushigome y, yamazaki y, shiohara t. iga vasculitis with severe gastrointestinal symptoms may be an unusual manifestation of varicella zoster virus reactivation. br. j. dermato, 2017;176: 1103–1105. doi: 10.1111/bjd.14948 8. jennette jc, falk rj, bacon pa, basu n, cid mc, ferrario f, flores-suarez lf, gross wl, guillevin l, hagen ec, et al. 2012 revised international chapel hill consensus conference nomenclature of vasculitides. arthritis rheum, 2013;65:1– 11. doi: 10.1002/art.37715. 9. saulsbury ft. clinical update: henochschönlein purpura. lancet. 2007;369:976– 978. doi: 10.1016/s0140-6736(07)60474-7 10. pozzi c, bolasco pg, fogazzi gb, andrulli s, altieri p, ponticelli c, locatelli f. corticosteroids in iga nephropathy: a randomised controlled trial. lancet. 1999; 353: 883–887. doi: 10.1016/s0140-6736(98)03563-6. _________________________________________________________________________________ © 2021 ali et al.; this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: https://www.sdiarticle5.com/review-history/77408 https://www.ncbi.nlm.nih.gov/books/nbk441943/ https://www.ncbi.nlm.nih.gov/books/nbk441943/ https://www.ncbi.nlm.nih.gov/books/nbk537252/ https://www.ncbi.nlm.nih.gov/books/nbk537252/ https://doi.org/10.3390/ijms22147538 https://doi.org/10.3390/ijms22147538 http://creativecommons.org/licenses/by/4.0 _____________________________________________________________________________________________________ *corresponding author: e-mail: celso@alergoimuno.med.br; cite as: olivier, celso eduardo, daiana guedes pinto, ana paula monezzi teixeira, cibele silva miguel, raquel acácia pereira gonçalves santos, jhéssica letícia santos santana, and regiane patussi santos lima. 2024. “cellular and humoral immunoreactivity against hen’s egg white: relevance in allergic patients”. asian journal of immunology 7 (1):274-84. https://doi.org/10.9734/aji/2024/v7i1150. asian journal of immunology volume 7, issue 1, page 274-284, 2024; article no.aji.127742 cellular and humoral immunoreactivity against hen’s egg white: relevance in allergic patients celso eduardo olivier a*, daiana guedes pinto a, ana paula monezzi teixeira a, cibele silva miguel a, raquel acácia pereira gonçalves santos a, jhéssica letícia santos santana b and regiane patussi santos lima c a instituto alergoimuno de americana, brazil. b instituto de ensino e pesquisa do hospital de amor de barretos, brazil. c lavoisier laboratórios, são paulo, brazil. authors’ contributions this work was carried out in collaboration among all authors. author ceo is responsible for the conceptualization, data curation, formal analysis, literature review, and wrote the original draft. authors dgp, apmt, csm, jlss and rpsl performed the laboratory procedures. author rapgs performed cutaneous tests. all authors read and approved the final manuscript. article information doi: https://doi.org/10.9734/aji/2024/v7i1150 open peer review history: this journal follows the advanced open peer review policy. identity of the reviewers, editor(s) and additional reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here: https://www.sdiarticle5.com/review-history/127742 received: 04/10/2024 accepted: 06/12/2024 published: 09/12/2024 abstract background: hypersensitivity to chicken eggs is the second most common food allergy, presenting ige-mediated and non-ige-mediated hypersensitivity mechanisms. while ige-mediated hypersensitivity is easily detected, non-ige-mediated endotypes present challenging obstacles to diagnosis. original research article https://doi.org/10.9734/aji/2024/v7i1150 https://www.sdiarticle5.com/review-history/127742 olivier et al.; asian j. immunol., vol. 7, no. 1, pp. 274-284, 2024; article no.aji.127742 275 aim: to evaluate the potential of the tube titration of precipitins (ttp) and the leukocyte adherence inhibition test (lait) to discriminate cellular and humoral immunoreactivity against hen's egg white (albumen). study design: we retrospectively examined the medical charts of two cohorts of patients clinically diagnosed with diverse allergic phenotypes with clinical suspicion of non–ige-mediated hypersensitivity against hen's egg white, who were investigated with the help of ttp or lait. methodology: the registered results of the semi-quantitative serum ttp against 1 mg/ml albumen extract (ttp cohort) as well as the registered results of the leukocyte adherence inhibition (lai) percentage promoted by the ex vivo challenges against 1 mg/ml albumen extract (lait cohort) were distributed through a cascade distribution chart to outline the variability of the results. the statistical characteristics inside each cohort were calculated. results: the cascade distribution for ttp with hen's egg extract showed a relatively uniform distribution. there were eight negative results. the mean was estimated at 1:156; the median was 1:64; the standard deviation was estimated at 1:185; the mode was 1:512 (appeared eighteen times). the cascade distribution for lait with hen's egg extract showed a relatively uniform distribution. there were twenty and one negative results. the lai ranged from 0% to 98%. the mean was 44.2%; the median was 48.5%; the standard deviation was 32.2%; the mode was 0% (appeared 21 times). conclusion: our preliminary results support that the ttp and lait performed with hen's albumen extract may discriminate diverse humoral and cellular immunoreactivity degrees in patients suffering from diversified allergic phenotypes. keywords: albumen; endotype; hen's egg white; hypersensitivity; leukocyte adherence inhibition test; non–ige-mediated immunoreactivity; phenotype; precipitins. abbreviations lai : leukocyte adherence inhibition lait : leukocyte adherence inhibition test ttp : tube titration of precipitins 1. introduction the domestic chicken gallus domesticus resulted from hybridizing and domesticating several asiatic wild gallus species that started around eight thousand years ago [1]. the extensive creation of chicken for broilers or the production of eggs destined for human nutrition is nowadays one of the many contributors to the reconfiguration of the biosphere [2]. hens are female adult chickens that lay eggs even if not fertilized. eggs are organic vessels oviparous animals produce to incubate an embryo outside the animal's body [3]. after being laid, eggs may be a target of predators in nature for their high nutritional content [4]. chicken eggs are constituted by a protective shell, a yolk (or vitellus), an egg white (or albumen), and an embryo (when fertilized) [5]. egg white mainly consists of water (88%), protein (10.5%), carbohydrate (0.5%), ash (0.8%), and lipids (0.2%) [6]. the most abundant protein in albumen is ovalbumin (54%), a phosphoglycoprotein that converts to sovalbumin (a less reactive form of albumin) during storage [7]. ovotransferrin (also known as conalbumin, an iron-binding glycoprotein) is the second most abundant protein (12%), followed by ovomucoid (11%), a heat-resistant major allergen which functions as a trypsin inhibitor [8,9]. other proteins were also quantified: ovomucin (3.5%), lysozyme (3.5%), ovoinhibitor (1.5%), ovoglycoprotein (1.0%), ovoflavoprotein (0.8%), ovomacroglobulin (0.5%), avidin (0.5%) and about more thirty proteins found in decrescent proportions [10,11]. in addition to their nutritional values, albumen's proteins present several functional properties explored by the artisanal culinary and food industry [12]. usually referred to in cosmetic ingredients as albumen, egg white proteins are used to enhance skin and hair properties, enhance skin tone, and reduce wrinkles. it is incorporated into face masks, cleansers, and skincare formulations [13]. hypersensitivity to egg proteins is the second most common food allergy, with a self-report prevalence of up to 7% of the inquired population olivier et al.; asian j. immunol., vol. 7, no. 1, pp. 274-284, 2024; article no.aji.127742 276 [14]. allergy to egg white proteins was one of the first thoroughly investigated reported cases of food allergy, successfully treated with desensitization in 1912 [15]. patients may present cutaneous manifestations (urticaria, angioedema, atopic and contact dermatitis), gastrointestinal symptoms (allergic proctocolitis, eosinophilic esophagitis, food-protein induced enterocolitis syndrome), respiratory inflammations (rhinitis, bronchitis) and anaphylaxis [16,17]. the allergen nomenclature sub-committee of the world health organization and international union of immunological societies (who/iuis) has recognized ten allergens related to gallus domesticus, from which four are abundant in albumen according to their official nomenclature: gal d 1 (ovomucoid), gal d 2 (ovalbumin), gal d 3 (ovotransferrin) and gal d 4 (lysozyme c) [18]. bird-egg syndromes are symptomatic allergic cross-reactivities among egg proteins, poultry meat, and aeroallergens from feathers [19]. egg white proteins may also function as aeroallergens, producing respiratory allergic phenotypes [20]. non-ige-mediated cellular immunoreactivity against food allergens had already been reported by our group with the help of the leukocyte adherence inhibition test (lait), as well as humoral immunoreactivity against other food allergens with the help of tube titration of precipitins (ttp) [21-23]. these tools are evaluated as laboratory tools to endotype immunoreactivity conditions as a personalized medicine strategy [24]. we routinely employ the lait and the ttp in our facilities as a triage to evaluate non–igemediated immunoreactivity against suspected allergens before performing more exhaustive in vivo provocation tests [25-31]. to evaluate the potential of the lait and ttp to endotyping non– ige-mediated cellular and humoral immunoreactivity against hen's eggs albumen extracts, we retrospectively compiled the electronic medical charts of patients investigated by one of these assays. the present study is a proof-of-concept that hypothesizes that lait and the ttp may differentiate diverse degrees of cellular and humoral immunoreactivity against hen's egg white proteins in allergic patients. 2. materials and methods 2.1 subjects after receiving institutional review board approval from the instituto alergoimuno de americana (brazil; 9/2024), we reviewed the electronic chart of 9,600 outpatients who attended our facility from january 2018 to november 2024. among these we found 100 patients submitted to ttp with hen's egg white and 100 patients submitted to lait with hen's egg white. a cohort of 100 consecutive outside patients (ttp cohort) had been submitted to ttp with hen's egg white for presenting in patients with non–ige-mediated allergic phenotypes (urticaria, angioedema, atopic and contact dermatitis, allergic proctocolitis, eosinophilic esophagitis, food-protein induced enterocolitis, rhinitis, and/or bronchitis). this cohort counted 23 males; mean age 42.7 years; sd 20.3 years; range 4 to 92 years; median 42.5 years; modes = 35 and 38 years (each appeared five times). a cohort of 100 consecutive outside patients (lait cohort) had been submitted to tial with hen's egg white for presenting in patients with non–ige-mediated allergic phenotypes (urticaria, angioedema, atopic and contact dermatitis, allergic proctocolitis, eosinophilic esophagitis, food-protein induced enterocolitis, rhinitis, and/or bronchitis). this cohort counted 27 males; mean age 39.9 years; sd 23.4 years; range 0 to 85 years; median 41.5 years; mode = 1 (appeared five times). this study did not include patients under biological and/or systemic anti-inflammatory therapy. these procedures were offered to patients with clinical suspicion of hen's egg white hypersensitivity who demonstrated a nonreactive or inconclusive skin test against hen's egg white extract, as well with total ige inside reference levels, and non-detectable levels of specific ige against any allergen, including hen’s egg white proteins [32,64]. 2.2 hen's egg white extract hen's egg white was homogenized and then left for 48 hours in a coca-based extractor solution (propylparaben 0.5g, methylparaben 1g, sorbitol 30g, nacl 5g, nahco3 2.5g, 1,000ml h2o) at 4 °c for protein extraction before centrifugation and separation of the water-soluble fraction from olivier et al.; asian j. immunol., vol. 7, no. 1, pp. 274-284, 2024; article no.aji.127742 277 solid particles and oily fraction [33]. the protein quantification of the allergen extract was done according to bradford's protein-dye binding methodology [34]. the solution was diluted in antigen dilution solution (nacl 10g; kh2po4 0.72g; na3po4 2.86g; methylparaben 1g; propylparaben 0.5g; glycerin 400ml; h2o 600ml) to an estimated protein concentration of 1 mg/ml and stored at 4 °c into amber opaque glass vials. the hen's egg white extract solution was used for allergic skin tests, ttp, and lait. all relevant and mandatory laboratory health and safety measures have been complied with during the experiments. 2.3 lait: ex vivo investigation: leukocyte adherence inhibition test 2.3.1 lait: procedure for allergen ex vivo challenging we performed lait as previously described [3541]. shortly, each donor's fresh plasma was divided into two parts and used in parallel ex vivo challenging tests with the hen's egg white extract and the unchallenged plasma (added with antigen dilution solution as a control). we collected plasma with high leukocyte content (buffy coat) from the heparinized tube after one hour of sedimentation at 37 °c. then, we distributed aliquots of 100 μl into eppendorf tubes with (or without) the challenging extract and kept them under agitation for 30 minutes (200 rpm at 37°c). 2.3.2 lait: procedure for adherence assay after incubation, the plasma was allocated into a standard neubauer hemocytometer counting chamber with a plain, non-metallic glass surface and left to stand for 2 hours at 37°c in the humidified atmosphere of the covered water bath to allow leukocytes to adhere to the glass. next, we counted the leukocytes, removed the coverslip, and washed the chamber by immersion in a beaker with phosphate buffer saline (pbs) at 37°c. then, we added a drop of pbs to the hemocytometer's chamber and allocated a clean coverslip over it. the remaining cells were counted in the same squares as previously examined. 2.3.3 lait: procedure for calculation the percentage of leukocyte adherence (la) of each assay was estimated as: (the number of leukocytes observed on the hemocytometry chamber after washing divided by the number of leukocytes observed on the hemocytometry chamber before washing) and multiplied by 100 (%). the leukocyte adherence ratio (lar) was estimated based on the ratio between the la from the antigen-specific challenged plasma and the la from the unchallenged control plasma: lar = la of the challenged sample divided by la of unchallenged control plasma multiplied by 100 (%). to further calculate the leukocyte adherence inhibition (lai), we subtracted the lar from 100 (%). we employed the lai results for the cascade distribution chart and the statistics calculations, both performed with the help of the microsoft excel® statistical package. 2.4 ttp: in vitro investigation: tube titration of precipitins as previously reported, the semi-quantitative titration of precipitins (ttp) against the allergen extract was performed in a transparent vitreous tube array [42-44]. shortly, the patient's blood was collected in a clot-activator collecting tube. after separation, the serum was centrifugated at 2,000 rpm for 10 minutes. each allergen extract was allocated in sets of eleven glass tubes at progressive duplicated serum dilutions. the progressive dilutions were combined with the 15 μl of the hen's egg extract with 250 μl of the patient's serum, progressively diluted into physiological saline solution (nacl 0,9%) in the dilution ratios of 1:1; 1:2; 1:4; 1:8; 1:16; 1:32; 1:64; 1:128; 1:256; and 1:512. one tube was a blank control done with the water and serum to observe occasional spontaneous precipitation (sia test). after 24 hours, the tubes were examined, and the titers (the highest dilution factor that yields a positive reading) were recorded [45]. 3. results as a retrospective survey, there was no research protocol; therefore, we report the incidental immune investigation as registered in the digital medical charts. the cascade distribution graph (cascade distribution graph is the designation of the standard graph derived from the excell® software) for the ttp cohort with hen's egg extract showed a relatively uniform distribution (fig. 1). there were eight negative results. the mean was estimated at 1:156; the median was 1:64; the standard deviation was estimated olivier et al.; asian j. immunol., vol. 7, no. 1, pp. 274-284, 2024; article no.aji.127742 278 at 1:185; the mode was 1:512 (appeared eighteen times). some patients showed low or moderate concentrations of precipitins against hen's egg white. in contrast, others showed high concentrations of precipitins against the hen's egg white, which could reflect the participation of hen's egg white allergens in these patients' non–ige-mediated hypersensitivity conditions. the cascade distribution for the lait cohort with hen's egg extract showed a relatively uniform distribution (fig. 2). there were twenty and one negative results. the lai ranged from 0% to 98%. the mean was 44.2%; the median was 48.5%; the standard deviation was 32.2%; the mode was 0% (appeared 21 times). some patients showed low or moderate immunoreactivity during the ex vivo challenge test. in contrast, others displayed strong immunoreactivity, which could reflect the participation of hen's egg white allergens in these patients' non–ige-mediated hypersensitivity conditions. fig. 1. cascade distribution chart of the tube titration of precipitins (x-axis %) resulting from the hen's egg white extract against the serum of the ttp cohort of 100 tests/subjects (y-axis). fig. 2. cascade distribution chart of the range groups of leukocyte adherence inhibition (lai) results (x-axis %) of the ex vivo challenge test against hen's egg white extract monitored by the leukocyte adherence inhibition test (lait), according to the respective number of outcomes over the lait cohort with 100 tests/subjects (y-axis) olivier et al.; asian j. immunol., vol. 7, no. 1, pp. 274-284, 2024; article no.aji.127742 279 4. discussion when scientists need an animal model of chronic allergy to egg proteins, they administer mice subcutaneous injections of 10 μg of ovalbumin with an adjuvant such as aluminum hydroxide [46]. in 1931, woodruff and goodpasture reported the first technique employing chicken eggs to propagate viruses [47]. nowadays, several immunization vaccines (such as for measles, mumps, rubella, influenza, yellow fever, q fever, and rabies) are prepared from viruses propagated in eggs and present residual or significative contents of egg proteins, originating controversy about vaccination in patients with egg allergy [48-50]. most concerns and safety evaluations about human parenteral egg components are always taken looking for immediate anaphylactic responses. however, no effective surveillance has ever been done about the possibility of worsening of the immunoreactivity after further immunogenic stimulus, or ever the risk presented by the parenteral administration of a food allergen protein to a non-yet-sensitized individual and the risk of developing further hypersensitization. laboratory allergy diagnosis to hen's egg white proteins is yet far from ideal, and continuous research is being done to improve the laboratory tools by developing new techniques [51]. lait and ttp performed with egg proteins seem to be promising tools for evaluating cellular and humoral responses after immunization with eggpropagated virus vaccines to evaluate the risk of development of eventual hypersensitivity reactions. the primary strategy of personalized medicine is to use endotype biomarkers for cellular and humoral immunoreactivity to personalize treatments for allergic patients [52]. the semiquantitative titration of precipitins is a pioneering laboratory exam upon which the fundamental bases of immunology were constructed [53]. precipitating antibodies suggest the presence of a humoral immune response against the tested antigens [54]. before the discovery of ige, researching precipitins against food allergens was the primary way to realize in vitro diagnosis of humoral immunoreactivity [55,56]. the lait is an ex vivo challenge test performed with a viable leukocyte buffy coat that can theoretically explore most known immune pathways as it allows the interaction of all immune-circulating participants with the allergens [57]. several immune pathways can produce the final leukocyte adherence inhibition [58-61]. the present study is a proof-of-concept that hypothesizes that lait and the ttp may endotype or differentiate diverse degrees of cellular and humoral immunoreactivity against hen's egg albumen among patients suffering from several non–ige-mediated allergic phenotypes (urticaria, angioedema, atopic and contact dermatitis, allergic proctocolitis, eosinophilic esophagitis, food-protein induced enterocolitis, rhinitis, and/or bronchitis. the retrospective compilation of our data showed a large distribution of results when we ascertained the results of ttp and tial to explore humoral and cellular immunoreactivity against the albumen extract. these immunoassays provide evidence about cellular and humoral immunoreactivity distributed into an extensive spectral range that may suggest immune tolerance or hypersensitivity. this preliminary retrospective survey demonstrated extensive results from the ttp, and the ex vivo challenge test monitored by lait against hen's eggs albumen in two cohorts of patients with diverse non–ige-mediated allergic phenotypes. ttp and lait are complementary triage tests used at our facilities to select worthwhile antigens to proceed with more laborious in vivo provocation tests when the specific ige is undetectable. none of our patients presented an exclusive reaction to these allergens. when we perform these assays, we perform them within a battery of several other allergens to choose the more reactive ones to prioritize the in vivo provocation tests. every patient was simultaneously evaluated for immunoreactivity against several chemical and biological allergens, demonstrating positive results for some of them. our results suggest that some of our patients may impair their allergic symptoms by additional immunoreactivity against hen's egg albumen allergens. 5. limitations this study is a retrospective analysis of data collected over six years and ten months. there was no protocol research, and the subject's data was limited to the essentials available on our electronic sheets. therefore, we could not establish a cross-comparison between positive olivier et al.; asian j. immunol., vol. 7, no. 1, pp. 274-284, 2024; article no.aji.127742 280 and negative controls to validate the results. the number of subjects is appropriate for a preliminary study; however, future studies must be more comprehensive. the lack of a research protocol implies the possibility of a bias produced by the point of view of the physician who indicated the exam based on a clinical suspicion led by anamnesis, physical examination, undetectable specific ige results, and nonreactive or inconclusive skin tests. the purpose of ttp and lait was not to discriminate reactive patients from healthy people. this was done with in vivo provocation tests. these assays aim to prioritize allergens to be evaluated by further in vivo provocation tests. the study lost the followup of many of these patients, making it impossible to establish a relationship between the immunoassays' results and the patient's clinical outcome. unfortunately, it was not possible to compare the two procedures with paired tests because they were taken from distinct groups of patients. the methodology of ttp and lait are standardized in our laboratory, and there is no significant operator-dependent variability every time they are submitted to quality control, however, as any laboratory assay, the results depends of the sample and the immune conditions of the patient at the moment of the collection, what explain possible variations of the results along different samples took at different moments of life, what justify the statistical analyses of a great group pf patients. 6. conclusion our preliminary results show that the lait and ttp may differentiate diverse degrees of immunoreactivity against hen's egg albumen in patients clinically diagnosed with diverse non– ige-mediated allergic phenotypes (rhinoconjunctivitis, rhinosinusitis, bronchitis, atopic dermatitis, and urticaria). this methodology may be easily incorporated into specialized centers since the technologies to perform tial and ttp are relatively inexpensive and can be performed with minimum laboratory equipment. however, the technique depends on trained biomedical personnel performing artisanal and time-consuming laboratory procedures. as preliminary results, the propaedeutic meaning of these results and the possibility of interferents must be yet established [62]. more studies focused on the quality-bydesign approach with prospective larger doubleblind cohorts need to evaluate the potential contribution of lait and ttp for endotyping cellular and humoral immunoreactivity in patients suspected of hypersensitivity against hen's egg white allergens [63]. 7. future directions and recommendations for clinical practice the primary intended use of in vitro or ex vivo allergen challenge tests is to spare the patients from being submitted to exhaustive and dangerous in vivo challenge tests. exploring the humoral and the cellular arms of immune systems, the ttp and tial alone or combined may represent, in the near future, a tool for allergists to construct an etiologic diagnosis from their patients, as well as determine the endotypes (mechanisms) of hypersensitivity, in order to choose more convenient and personalized therapies for them. future studies with prospective planning and control groups will be necessary to avoid the characteristic biases of retrospective studies. disclaimer (artificial intelligence) author(s) hereby declare that no generative ai technologies such as large language models (chatgpt, copilot, etc.) and text-to-image generators have been used during the writing or editing of this manuscript. consent as per international standards or university standards, patient(s) written consent has been collected and preserved by the author(s). ethical approval as per international standards or university standards written ethical approval has been collected and preserved by the author(s). acknowledgements the instituto alergoimuno de americana funded this work. we want to thank the laboratory technicians alessandra vieira de oliveira and luciana 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author(s) and contributor(s) and not of the publisher and/or the editor(s). this publisher and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. _________________________________________________________________________________ © copyright (2024): author(s). the licensee is the journal publisher. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: https://www.sdiarticle5.com/review-history/127742 https://www.sdiarticle5.com/review-history/127742 _____________________________________________________________________________________________________ *corresponding author: e-mail: raafath2001@yahoo.com; asian journal of immunology 3(1): 106-113, 2020; article no.aji.56084 a case of furuncular myiasis due to cordylobia anthropophaga in infant in makkah, saudi arabia raafat a. hassanein1,2*, amal m. almatary3,4 and mohammad o. alkurbi1 1 department of laboratory medicine, faculty of applied medical sciences, umm al-qura university, saudi arabia. 2 department of zoonoses, faculty of veterinary medicine, assiut university, assiut, egypt. 3 parasitology unit, maternity and children hospital, makkah, saudi arabia. 4 department of parasitology, faculty of medicine, assiut university, egypt. authors’ contributions this work was carried out in collaboration among all authors. author rah designed the study, wrote the protocol and wrote the first draft of the manuscript. authors ama and moa managed the analyses of the study. author moa managed the literature searches. all authors read and approved the final manuscript. article information editor(s): (1) cynthia aracely alvizo báez, autonomous university of nuevo leon, mexico. reviewers: (1) s. k. kuria, walter sisulu university, south africa. (2) papa kofi amissah-reynolds, university of education, winneba, ghana. complete peer review history: http://www.sdiarticle4.com/review-history/56084 received 02 february 2020 accepted 09 april 2020 published 14 april 2020 abstract the cutaneous myiasis has been rarely reported in saudi arabia. we intended to describe here a case of furuncular cutaneous myiasis caused by cordylobia anthropophaga larvae in infant from south area in makkah, saudi arabia. a female infant, 17-day-old, had lived with her family in area closed to animal houses. physical examinations showed 2 tender erythematous nodules with small central ulceration on the infant back and hand. during investigation, 2 larvae came out from the lesion. c. anthropophaga was identified by paired mouth hooks (toothed, spade-like, oral hooks) and 2 posterior spiracles, which lack a distinct chitinous rim. although rarely described in saudi arabia until now, cutaneous myiasis should be expected in people living nearby such livestock, with flies and poor hygienic conditions. case study hassanein et al.; aji, 3(1): 106-113, 2020; article no.aji.56084 107 keywords: cordylobia anthropophagi; cutaneous; myiasis; furuncular myiasis; saudi arabia. 1. introduction myiasis is defined as the infestation of live human and vertebrate animals with dipterous larvae, which, at least for a certain period, feed on the host’s dead or living tissue, liquid bodysubstances, or ingested food. it can involve cutaneous, enteric, ophthalmic, nasopharyngeal, auricular, and urogenital systems by larvae of the order of diptera (true flies). it is widespread in tropical and subtropical areas, including central america, south america, and tropical africa [1,2]. in addition, myiasis is continuously recorded throughout the world due to travelers returning from endemic areas. it has been reported in several countries, including france [3] china [4], us [5], japan [6], saudi arabia [7,8]. the cutaneous myiasis is the most frequent and impressive form and it can be divided into 3 types by clinical features and type of larvae: furuncular, creeping (migratory) and wound (traumatic) myiasis [9]. furuncular myiasis is defined as the penetration of the larvae inside the healthy skin and development of a boil-like nodule. it is more common in tropical countries and can be caused by dermatobia hominis, c. anthropophaga, cuterebra sp. and wohlfahrtia vigil [10,1,11,12, 13,14]. gasterophilus and hypoderma are two flies that produce a creeping myiasis. flies that cause wound myiasis include screwworm flies such as cochliomyia hominivorax and chrysomya bezziana, and wohlfahrtia magnifica [9]. the human botfly, dermatobia hominis (linnaeus, 1781), causes obligatory myiasis and this parasite depends on the host to complete its life cycle [15]. humans can be infested through direct exposure to environments contaminated with eggs of the fly [16]. in this study, we present the case of furuncular myiasis in an infant living with her family nearby livestock’s area, with flies and poor hygienic conditions south of makkah, ksa. the larva was removed from a boil-like lesion of infant back and hand and identified as c. anthropophaga from morphology. 2. case report a 17-day-old female infant presented to pediatric department at maternity and children hospital, makkah, saudi arabia on 10 february 2020, with 1-week history of 5 painful erythematous nodules boil-like lesions with a small central punctum appeared on her back and developed as an abscess on the back and hand (fig. 1). a third instar larva 8×3 mm in size (fig. 2), yellowish color with cylindrical body was removed from two of the lesions and identified in the parasitology unit, maternity and children hospital. the larvae were first washed in distilled water and then cleared in 10% sodium hydroxide (naoh) solution for 1 h. the larvae were washed again in distilled water, transferred to 10% acetic acid for 30 min and washed again in distilled water. the specimens were then dehydrated in ascending series of ethanol (30%, 50%, 70% and 90%) for 30 min each. the larvae were then soaked in absolute alcohol for at least 1 h and then transferred into xylene for one hour. at this stage, all the internal organs of the maggot were removed and the posterior spiracles were cut transversely, also the anterior end cut transversely and the medium portion longitudinally, then specimens were mounted onto a glass slide using dpx and left to dry to the next day. the slides were examined under a light microscope for taxonomy studies and identification [17]. the morphology of the larvae body was almost completely covered by conic small black, scattered spines with a brown apex, pointing towards the posterior end (fig. 3). two coppercolored posterior spiracles, each bearing 3 sinuous spiracular slits which lack a distinct chitinous rim (fig. 4). in the anterior end of larva, the mouth with a pair of spade-like stout hooks with curved and sharp tips without cluster of conic denticles and anterior spiracles were characteristically observed (fig. 5). these morphological characteristics were consistent with those described for c. anthropophaga [18]. laboratory tests demonstrated high monocytes 2.54 x 10 3 /ul, high eosinophils 1.40 x 10 3 /ul, high mean corpuscular haemoglobin 33.1 pg, high bilirubin (5.1 umol/l), high potassium-k (5.7 mmol/l), high creatinine (41.0 umol/l), high calcium (2.8 mmol/l), low sgot/ast (30) indicating renal impairment. skin lesions were markedly improved after the remove of larvae and 2-week antibiotic treatment, and there was no recurrence. hassanein et al.; aji, 3(1): 106-113, 2020; article no.aji.56084 108 fig. 1a. boil-like lesions caused by cordylobia anthropophaga on the back of the patient fig. 1b. boil-like lesions caused by cordylobia anthropophaga on the abdomen of the patient fig. 1c. boil-like lesions caused by cordylobia anthropophaga on the hand of the patient hassanein et al.; aji, 3(1): 106-113, 2020; article no.aji.56084 109 fig. 2. barrel-shaped larvae were about 8 mm in length and 4 mm in width fig. 3. conic spines on the body with a brown apex, pointing towards the posterior end (magnification 20x) hassanein et al.; aji, 3(1): 106-113, 2020; article no.aji.56084 110 fig. 4. two copper-colored posterior spiracles, each carrying 3 sinuous spiracular slits which lack a distinct chitinous rim (magnification 20x) fig. 5. anterior end of c. anthropophaga showing paired mouth hooks (toothed, spade-like, oral hook) protruded ventrally from anterior 2 black mouth-hooks (magnification 20x) 3. discussion the definitive study on the tumbu fly (c. anthropophaga) was carried out in sierra leone in 1923 [10]. in africa, furuncular myiasis is caused by 3 species of the genus cordylobia: c. anthropophaga (endemic throughout africa), c. rodhaini (parts of africa with rainforests) and c. ruandae (zaire border and rwanda), whereas d. hominisis common in central and south america [3]. among cordylobia spp., c. anthropophaga is the dominant causative agent compared with the other species [19]. the larvae can penetrate the unbroken skin of the host, who is usually lying on the ground or by the contaminated clothes. interestingly, the host usually feels no symptoms at the time of skin penetration by larvae. therefore, most patients do not think maggots as a cause of their skin problems. within 1 to 2 weeks, the larvae develop into the second and third in stars and the hassanein et al.; aji, 3(1): 106-113, 2020; article no.aji.56084 111 8×3mm sized mature larvae that can emerge from the central pore of the skin lesions [20]. in this report, morphological characteristics of larvae of c. anthropophaga (ex, a pair of spadelike hooks, anterior spiracles, and posterior spiracles) are well observed in figs. 3, 4 and 5. these morphological characteristics were consistent with those described for c. anthropophaga [4,13,14,18]. in the posterior spiracles, the 3 spiracular openings are slightly sinuous slits in c. anthropophaga, whereas tortuously sinuous in c. rodhaini. c. anthropophaga has mouth hooks with curved and sharp tips, but there is neither cluster of conic denticles nor oral grooves. on the other hand, c. rodhaini has mouth-hooks with broad and rounded tips and cluster of conic denticles as typical feature. therefore, sufficient knowledge and experience for diagnosis is needed to avoid misdiagnosis, such as pyoderma, impetigo, staphylococcal furunculosis, and cutaneous leishmaniasis [1]. early lesions may resemble other reactions due to insect bite, but furuncular lesions with an intense inflammatory reaction in the surrounding tissue rapidly develop [20,21]. myiasis has a greater economic impact as an infestation of animals than of humans; there are no species of diptera which are restricted to humans for their development but there are many that have a very restricted host range on animals [22,23]. the infestation for our case may be occurs through a fly itself where the infant family lived near animal houses such as sheep, horses and cattle in south area in makkah (malikan area), saudi arabia. this case report again highlights this disease should be expected in people living nearby such livestock, with flies and poor hygienic conditions. these people should be taught hygienic practices and methods to control fly population. the “tumbu fly”, c. anthropophaga, is an agent of obligatory cutaneous (furuncular) myiasis, widely distributed in the sub-saharan region [24], although at least one autochthonous case has been recently reported in saudi arabia [25]. this species, with rodents as natural hosts, has secondarily adapted to other wild and domestic animals and to humans [26]. myiasis is more common in summer and spring seasons of tropical countries [27]. our case occurs in february where the end of winter and atmospheric temperature in makkah area is slightly high especially at daytime like spring temperature. the goal of treatment is removal of the larva and prevention of the secondary infection. occlusion, larvicides such as ivermectin, or manual squeezing can be used to remove the larva. occlusion deprives the larva of oxygen and either kills the larva or induces it to move upward in search of air [1]. manually squeezing out the larva is therapeutic option in all forms of furuncular myiasis. sometimes, surgical removal is needed for removing the larvae or their fragments [28]. 4. conclusion in conclusion, we report a case of furuncular cutaneous myiasis on the back and hand in an infant by c. anthropophaga, living nearby such livestock, with flies and poor hygienic conditions, which has been rarely described in saudi arabia. considering the increasing close association with animal houses, physicians should be more concerned about clinical manifestations of cutaneous myiasis and morphologic characteristics of dipterous larvae. consent as per international standard informed and written parental consent has been collected and preserved by the authors. ethical approval as per international standard written ethical permission has been collected and preserved by the author(s). acknowledgement the authors would like to express sincere appreciation to staff members and technicians of the maternity and children hospital, makkah, saudi arabia for their assistance in parasitological identification and data collection. competing interests authors have declared that no competing interests exist. hassanein et al.; 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our opinion in response to political activists’ arguments against compulsory vaccination raphael kumah abolasom a, albert opoku b,c*, prince twene d and nicholas amoah owusu e a bono regional hospital, ghana. b nursing and midwifery training college, tepa, trinity hospital pankrono, kumasi, ghana. c adesh university bathinda, india. d aberdeen university, uk. e food and drugs authority, ghana. authors’ contributions this work was carried out in collaboration among all authors. author rkb designed the study and contributed to literature search with author ao and pt. authors ao, rkb and nao wrote the first draft of the manuscript. all authors read and approved the final manuscript for publication. article information editor(s): (1) dr. wagner loyola, brazilian agricultural research corporation, brazil. (2) prof. cynthia aracely alvizo báez, autonomous university of nuevo leon, mexico. reviewers: (1) m. zamri saad, universiti putra malaysia, malaysia. (2) eeritus ibrahim mohamadsaeed shnawa, university of qasim and hilla university, iraq. (3) amandeep goyal, university of kansas medical center, usa. (4) mulugeta berhanu feyisa, ethiopia. complete peer review history, details of the editor(s), reviewers and additional reviewers are available here: https://www.sdiarticle5.com/review-history/80740 received 20 november 2021 accepted 27 december 2021 published 30 december 2021 abstract some political actors and activists have waged war against compulsory vaccination in ghana by insinuating that such decision by government is an affront to the rights and freedoms of citizens as enshrined in the 1992 republican constitution. this paper scrutinizes this position and supports the fact that government did not err in its decision to impose compulsory vaccination on persons travelling into and out of ghana. in doing this, relevant materials were reviewed. the world health organization at its thirteenth world health assembly on 25 th april 1960 recommended compulsory opinion article abolasom et al.; aji, 4(1): 154-159, 2021; article no.aji.80740 155 vaccination. chapter five (5), article twelve (12) section two (2) of the 1992 constitution makes it possible for citizens to enjoy their rights and freedoms but subject to respect for the rights and freedoms of others and for public interest”. the public health act, 2012 (act 851), part ii (vaccination), section 22, subsection (1), gives power to the minister of health, through a legislative instrument (li) to order compulsory vaccination for public interest. the vaccination act 1919, cap.76 in section 3, subsections (1) and (2) also give powers to minister of health to issue such directives. again, articles 31 and 32 of the 1992 republican constitution, give room for the declaration of state of emergencies making it possible for one to be “legally deprived” of the enjoyment of the fundamental human rights and freedoms enshrined in chapter five (5) of the 1992 constitution in times of natural disasters such as covid-19. keywords: covid 19; vaccination; mandatory; ghana and political activists. 1. introduction countries have had to adopt several strategies to control the spread of the covid-19 virus. these measures include social distancing (physical distancing), suspension or modification of working activities, restricted movement and obligatory use of facial masks [1]. although necessary, yet these measures have had both direct and indirect negative consequences on national economies and personal health [2], leading to a rise in mental disorders and unhealthy behaviors [3]. in 2020, efforts were made across the world to rapidly develop vaccines for covid-19. in this process, scientific and medical communities worked together with governmental institutions to produce a vaccine in record time [4]. the covid-19 vaccination rollout encountered challenges at the early stages [5]. a study conducted among 2,734 people in all sixteen regions of ghana showed that some citizens felt that the vaccine was unsafe, they were not sure of the effects. others too for cultural, social and religious reasons opposed the vaccination exercise; people were generally uncomfortable [6]. unfortunately our dear country is politically polarized to the extent that people listen more often to the opinions of their party people than facts and experts [7]. it is against this backdrop that i find the comments made by other stalwarts and particularly, sammi gyamfi, the national communication’s officer of the ndc as not only unfortunate, but dangerous and has the potential to do great disservice to the good people of this country . for him to posit that, “vaccination must be by choice and not by force. the imposition of compulsory covid vaccination requirement on all ghanaians traveling into and out of ghana by the ghana health service is reprehensible and an affront to the 1992 constitution” [8], is nauseating and an unhealthy position to say the least. 2. is compulsory vaccination ethical? foremost, healthcare, for that matter clinical practice, is guided by both law and ethics [9]. for purposes of education, it is imperative to state that, to consider an action as ethically right or wrong, there is the need to make reference to some basic ethical perspectives in healthcare practice [10]. using deontology theory, the end result is not the focus but the act or action being taken [11]. in essence, deontologists are of the view that the rightness of an action does not depend primarily or entirely on the value of the consequences of that action [12], but rather on the action or intent of the action: whether good or bad. on the other hand is consequentialism or the consequentialist’s perspective of ethics. with consequentialism, the effects of the vaccines administered are that on which premium is placed [13]. the intent to vaccinate may be a good one but if it yields negative consequences then it becomes problematic in the sight of the consequentialist [14]. explicitly, the primary focus of the consequentialist is the results that will be realized after vaccinating citizens [15]. thus, experts are of the view that vaccinating citizens will reduce the level of covid-19 infection [16], which in turn is expected to lead to a reduction in covid-19 related deaths and complications. in addition to the above, another perspective of ethics in healthcare practice is “virtue ethics”. it places emphasis on the character of a person or rests on the person of character [17]. there exists a strong relationship between patients and care professionals [18]. it is on the basis of this perspective that healthcare professionals were among the first sect to have received the vaccines. this was a deliberate policy advocacy abolasom et al.; aji, 4(1): 154-159, 2021; article no.aji.80740 156 strategy. the idea is that, when health care professionals take the vaccine, because they are professionals, citizens will be encouraged to get vaccinated [19] thereby helping the government to achieve its goal of “vaccination for all. therefore, it is not right and not in the best interest of this country for people to advocate against compulsory vaccination by looking at only one side of the argument. 3. is compulsory vaccination legal? the world health organization at its thirteenth world health assembly on 25 th april 1960 considered the issue of compulsory vaccination versus voluntary vaccination and concluded as follows: “it is understandable, therefore, that it can be very difficult for the health authorities to adopt a position on this matter. it is all the more so in that to protect the community effectively from diphtheria and smallpox it is known that a sizeable proportion of the population 70 per cent or even more must be immunized. furthermore, it is essential that the immunity thus obtained should be consolidated by means of booster doses or revaccination. now, to cover such a large proportion of the population there is no other solution but compulsory vaccination and or persuasion through an expensive campaign of health education” [20]. in effect the world health organization approved of compulsory vaccination. in ghana, the public health act, 2012 (act 851), part ii (vaccination), section 22, subsection (1), gives power to the minister of health, through a legislative instrument (li) to give orders to whoever the li may be applicable to be vaccinated by the public vaccinator unless in cases where the public vaccinator is of the view that the vaccination could result in injury and dire consequences to the health of the populace. apart from the reasons supra, all health workers and citizens if ordered by the minister to be vaccinated, will have no choice than to be vaccinated. sanctions will be visited upon any professional or citizen who fails to comply with the order according to section 22; subsection (2). one may argue that the patient charter gives the patient a right, yes that is true, but such demands must be made within the right context. a patient’s right should not cause other citizens to be exposed to the virus. this position is supported by chapter five (5), article twelve (12) section two (2) of the 1992 constitution. it does state that, “ every person in ghana, whatever his race, place of origin, political opinion, color, religion, creed or gender shall be entitled to the fundamental human rights and freedoms of the individual contained in this chapter but subject to respect for the rights and freedoms of others and for public interest”. the vaccination act 1919, cap.76 also maintains in section 3 “compulsory vaccination” subsections (1) and (2) as follows: (1) the minister may, by executive instrument, order generally or with reference to a particular area, district or place, or with respect to a particular class or classes of persons, that the persons to whom the instrument applies who do not produce satisfactory evidence of successful vaccination or of having suffered from smallpox shall be vaccinated by a public vaccinator, unless in the opinion of the public vaccinator vaccination would be injurious to health. (2) the instrument may prescribe at what age a person is liable to a penalty for non-compliance with the terms of the instrument, and who is liable in cases where the persons failing to be vaccinated have not attained that age. from the above provisions of the vaccination act, it is clear that the ministry acting through its agencies is vested with powers to order for compulsory vaccination and also be able to visit sanctions upon whoever defies the order. again, articles 31 and 32 of the 1992 republican constitution, give room for the declaration of state of emergencies. this provision makes it possible for one to be “legally deprived” of the enjoyment of the fundamental human rights and freedoms enshrined in chapter five (5) of the 1992 constitution. this further means that, the president in consultation with the appropriate authorities could declare a state of emergency denying citizens of the freedom of movement among other rights and hence equally has the locus to operate through the ministry of health and the ghana health services to institute measures such as compulsory vaccinations in times of natural disasters (with reference to articles 31 and 32 of the 1992 constitution). since the list of natural disasters under article thirty (31) is in-exhaustive, natural disasters could be likened to this era of the covid-19 pandemic. of course, section 22 of act 851 gives room for the use of discretion by the public vaccinator to administer the vaccine or otherwise. logically the decision of a public vaccinator to determine the safety or otherwise of abolasom et al.; aji, 4(1): 154-159, 2021; article no.aji.80740 157 the vaccine must be based on science and empirical evidence. checks conducted reveals that no adverse findings against vaccinating ghanaians has been documented or known yet. admittedly, the fact that compulsory vaccination is both legal and ethical does not mean inferior, sub-standard vaccines should be used on the people. vaccine potency, its side effects and its ability to yield the desired scientific results must be paramount. vaccination must serve its purpose, compulsory vaccination is a fruitless venture should it have no positive impact on the immunity of the people [21,22]. it is totally needless if its unable get populations to reach herd immunity and eventually reducing and preventing subsequent infections. in the case of covid-19, taking the vaccine is not enough, people must continue masking, handwashing, social and physical distancing among others. 4. conclusion voluntary vaccination lacks compulsion, it is therefore most often a time poorly executed, people skip doses and it is administered in a haphazard manner resulting in an increase of disease incidence. again, patients suffering from tuberculosis and chicken pox are isolated from other patients so that they do not infect others. one would therefore ask, have the rights and freedoms of such patients being infringed upon? the answer is “yes”, but the isolation is being done to promote the common good of the society. some other anti-vaccine movements cite religious reasons for their opposition to compulsory vaccination. however, many law courts in the united states of america have ruled contrary to this position and that religion must not be used as a medium to be exempted from decisions arising from health legislations. to buttress this, the supreme court of the state of florida ruled that “religious freedom cannot be used as a cloak for any person with contagious or infectious disease to spread such disease because of his religion”. for the avoidance of doubt, the focus of this article is not on whether the covid-19 vaccines are efficacious or not, but as to whether the government of ghana has the mandate to embark on compulsory vaccination amidst covid-19 pandemic. of course, although antivaccine movements exist anytime, anywhere, yet vaccination programs get rolled out and instituted. therefore, without mincing words, appropriate institutions must subject populations to compulsory vaccination in order to prevent a further spread of the pandemic as long as the vaccines are safe, yield the desired results and have been approved for use by the relevant international and state institutions. the world health organization at its thirteenth world health assembly on 25 th april 1960 recommended compulsory vaccination. chapter five (5), article twelve (12) section two (2) of the 1992 constitution makes it possible for citizens to enjoy their rights and freedoms but subject to respect for the rights and freedoms of others and for public interest”. the public health act, 2012 (act 851), part ii (vaccination), section 22, subsection (1), gives power to the minister of health, through a legislative instrument (li) to order compulsory vaccination for public interest. the vaccination act 1919, cap.76 in section 3, subsections (1) and (2) also give powers to minister of health to issue such directives. again, articles 31 and 32 of the 1992 republican constitution, give room for the declaration of state of emergencies making it possible for one to be “legally deprived” of the enjoyment of the fundamental human rights and freedoms enshrined in chapter five (5) of the 1992 constitution in times of natural disasters such as covid-19. the above clearly demonstrates that government did not err in ordering compulsory vaccination. consent it is not applicable. ethical approval it is not applicable. competing interests authors have declared that no competing interests exist. references 1. roma p, monaro m, muzi l, colasanti m, ricci e, biondi s, napoli c, ferracuti s, mazza c. how to improve compliance with protective health measures during the covid-19 outbreak: testing a moderated mediation model and machine learning algorithms. international journal of environmental research and public health. 2020;17(19):7252. abolasom et al.; aji, 4(1): 154-159, 2021; article no.aji.80740 158 available:https://doi.org/10.3390/ijerph1 7197252 2. mazza c, ricci e, biondi s, colasanti m, ferracuti s, napoli c, roma p. a nationwide survey of psychological distress among italian people during the covid-19 pandemic: immediate psychological responses and associated factors. international journal of environmental research and public health. 2020;17(9):3165. available:https://doi.org/10.3390/ijerph1 7093165 3. fontanesi, l, marchetti d, mazza c, di giandomenico s, roma p, verrocchio mc. the effect of the covid-19 lockdown on parents: a call to adopt urgent measures. psychological trauma: theory, research, practice, and policy. 2020;12:s79– s81. available:https://doi.org/10.1037/tra0000 672 4. garcía-montero c, fraile-martínez o, vaccines cb. undefined. 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cite as: olivier, celso eduardo, daiana guedes pinto, ana paula monezzi teixeira, jhéssica letícia santos santana, raquel acácia pereira gonçalves santos, and regiane patussi santos lima. 2024. “endotyping cellular and humoral immunoreactivity against aluminum in allergic patients: a retrospective study”. asian journal of immunology 7 (1):149-58. https://journalaji.com/index.php/aji/article/view/139. asian journal of immunology volume 7, issue 1, page 149-158, 2024; article no.aji.122945 endotyping cellular and humoral immunoreactivity against aluminum in allergic patients: a retrospective study celso eduardo olivier a*, daiana guedes pinto a, ana paula monezzi teixeira a, jhéssica letícia santos santana b, raquel acácia pereira gonçalves santos a and regiane patussi santos lima c a instituto alergoimuno de americana, brazil. b instituto de ensino e pesquisa do hospital de amor de barretos, brazil. c lavoisier laboratórios, são paulo, brazil. authors’ contributions this work was carried out in collaboration among all authors. all authors read and approved the final manuscript. article information doi: https://doi.org/10.9734/aji/2024/v7i1139 open peer review history: this journal follows the advanced open peer review policy. identity of the reviewers, editor(s) and additional reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here: https://www.sdiarticle5.com/review-history/122945 received: 09/07/2024 accepted: 11/09/2024 published: 14/09/2024 abstract background: several publications report that aluminum is responsible for hypersensitivity reactions. there is no standardized lab exam that can endotype (determine the pathophysiology responsible for the phenotype) hypersensitivity to aluminum. original research article https://doi.org/10.9734/aji/2024/v7i1139 https://www.sdiarticle5.com/review-history/122945 olivier et al.; asian j. immunol., vol. 7, no. 1, pp. 149-158, 2024; article no.aji.122945 150 aim: to evaluate the potential of the tube titration of precipitins (ttp) and the leukocyte adherence inhibition test (lait) to endotype humoral and cellular immunoreactivity against aluminum in patients clinically diagnosed with allergic contact dermatitis, intrinsic atopic dermatitis, and/or non–ige-mediated urticaria. study design: we retrospectively examined the medical charts of two cohorts of 100 patients each; range 6 to 87 years; diagnosed with allergic phenotipes, who were investigated with the help of ttp (first cohort) or ex vivo challenge tests monitored by lait (second cohort) against aluminum. methodology: the registered results of ttp against 1 mg/ml aluminum solution were distributed in ranges through a cascade distribution chart to outline the variability of the results inside the first cohort. the registered results of the leukocyte adherence inhibition (lai) percentage promoted by the ex vivo challenges with 1 mg/ml aluminum solution were distributed in ranges through a cascade distribution chart to outline the variability of results inside the second cohort. results: most ttp results concentrated on higher dilutions. the mean was estimated at 1:301; the median was 1:256; the standard deviation was estimated at 1:204; the mode was 1:512 (appeared 46 times). the lai ranged from 0% to 88%. the mean was 52%; the median was 49%; the standard deviation was 23.9%; the mode was 60% (appeared five times). the cascade graphs demonstrate a wide range of distribution of ttp and lai results. conclusion: our preliminary results support that the ttp and lait performed with 1 mg/ml aluminum solution may discriminate diverse humoral and cellular immunoreactivity degrees in patients suffering from allergic contact dermatitis, intrinsic atopic dermatitis, and/or non–igemediated urticaria. keywords: aluminum; endotype; hypersensitivity; leukocyte adherence inhibition; leukocyte adherence inhibition test; phenotype; precipitins; precision medicine. abbreviations lai : leukocyte adherence inhibition lait : leukocyte adherence inhibition test ttp :tube titration of precipitins 1. introduction aluminum (or aluminium) is a post-transition metal with the symbol al, atomic number 13, and a relative atomic mass of 26.98, which has a great affinity to oxygen [1]. the american contact dermatitis society elected aluminum as the "allergen of the year 2022" [2]. aluminum may be present naturally in jewelry, piercings, cosmetics, tubes of toothpaste, medicines, tattoo inks, foods (at shallow levels), or due to using aluminum cooking utensils and food additives [3]. some foods, such as potatoes, spinach, and some teas, may contain naturally higher levels of aluminum [4]. remarkably, the mean concentration of aluminum in soy-based infant formulas is remarkably higher than in milk-based and corn-based infant formulas [5]. the sixtyseventh meeting of the joint fao/who expert committee on food additives held in rome in 2006 established the provisional tolerable weekly aluminum intake as 1 mg/kg body weight [6]. aluminum in its metal presentation (e 173) and aluminum salts are bactericides frequently used as preservatives for foods and cosmetics [7]. aluminum sulfates (e 520 to e 523) are mainly used in the food industry as thickeners and emulsifiers [8]. aluminum-containing additives are also used for specific purposes: a) as raising agent in baking powder, such as aluminum sodium sulfate (e 521) and sodium aluminum phosphate-acidic (e 541); b) as firming agents during the processing of jellyfish and pickles such as aluminum potassium sulfate (e 522); c) as emulsifiers in processed cheese, such as sodium aluminum phosphate-basic (e 541); d) as anti-caking agents in powder mixes for beverage mixers and non-dairy creams such as sodium aluminosilicate (e 554); e) as anti-caking agents for table salt and vanilla powder such as calcium aluminum silicate (e 556); f) as carriers for pigments (titanium dioxide and iron oxides) such as the potassium aluminum silicate (e 555) and aluminum powder (e 173) in decorating sugar-coated flour confectionery candy coatings, also known as lakes [9-14]. aseptic aluminum-containing plastic packages and cartons prevent oxidation and light damage to perishable food [15]. elemental aluminum and its salts (such as sulfate, phosphate, hydroxide, and silicate) have a wide variety of uses, such as food additives, cooking utensils, food packaging (beverage cans and foil), water treatment, olivier et al.; asian j. immunol., vol. 7, no. 1, pp. 149-158, 2024; article no.aji.122945 151 cosmetics (sunscreens), toiletry products (antiperspirants, tubes of toothpaste and sun blockers), antiacids with aluminum hydroxide, anti-diarrheic with kaolin (hydrated aluminum silicate or e 559), producing contact dermatitis, granuloma, and urticaria in sensitized people [1619]. pool water flocculants are made with aluminum salts and may produce occupational allergies [20]. aluminum-containing compounds have been employed as adjuvants in vaccines to amplify antigen-specific th2 responses [21]. there is a positive dose-response relationship between plasmatic aluminum concentrations and cognitive impairment among workers with occupational exposure to aluminum [22]. aluminum nanoparticles are associated with memory impairment and hippocampal inflammation induced by microglial activation, following upregulation of the inflammatory cytokine il-1β (interleukin-1β) in mice [23]. current lab models to study alzheimer's disease are established through intraperitoneal injection of aluminum chloride (alcl3) in rats or aluminum nanoparticles in mice [24,25]. diagnosis of delayed hypersensitivity to aluminum is usually clinically made in patients with contact dermatitis with the help of cutaneous path tests performed with aluminum salts dispersed in petrolatum [26]. however, systemic aluminum immunoreactivity has already been documented by systemic immunoassay markers, such as eosinophilia [27]. the clinical suspicion of immediate and delayed hypersensitivity against aluminum is usually confirmed through skin allergy tests and patch tests, respectively. until now, no widespread lab exam has been able to document specific immunoreactivity against aluminum to help with clinical diagnosis. the leukocyte adherence inhibition test (lait) and the tube titration of precipitins (ttp) are performed in our facilities as triage tests for suspected allergens prescribed before the performance of more exhaustive in vivo provocation tests [28-34]. the present study hypothesizes that the lait and the ttp may differentiate endotypes and degrees of immunoreactivity against aluminum among patients suffering from common allergic phenotypes. to evaluate the potential of the lait and the ttp to discriminate humoral and cellular immunoreactivity against aluminum, we retrospectively compiled the electronic medical charts of patients clinically diagnosed with allergic contact dermatitis, intrinsic atopic dermatitis, and/or non–ige-mediated urticaria who were investigated with these procedures. 2. materials and methods 2.1 subjects after receiving institutional review board approval from the instituto alergoimuno de americana (brazil; 06/2024), we proceeded with the electronic chart review of 9,200 outpatients who attended our facility from january 2018 to august 2024. the first cohort of 100 outside patients had been submitted to ttp with 1 mg/ml of aluminum solution for presenting allergic contact dermatitis, intrinsic atopic dermatitis, and/or non–igemediated urticaria. this cohort counted 24 males and 76 females; mean age 38.4 years; sd 20.4 years; range 6 to 87 years; median 36.5 years; mode = 46 (appeared five times); geometric mean = 32 years. the second cohort of 100 outside patients had been submitted to an ex vivo allergen challenge test with aluminum solution 1mg/ml monitored with lait for presenting allergic contact dermatitis, intrinsic atopic dermatitis, and/or non– ige-mediated urticaria. this cohort counted 28 males and 72 females; mean age 40.3 years; sd 17.5 years; range 8 to 75 years; median 42 years; mode = 42 (appeared six times); geometric mean = 35.2 years. this study did not include patients under biological and/or systemic anti-inflammatory therapy. these procedures were offered to patients with clinical suspicion of aluminum hypersensitivity who demonstrated a nonreactive or inconclusive skin test against aluminum 1 mg/ml solution [35]. the cascade graphs were mounted using the functionalities of the spreadsheet editor microsoft excell® software. 2.2 aluminum solution the aluminum solution was prepared with powdered acetate aluminum diluted with distilled water at 1 mg/ml to perform the allergic skin tests, ttp, and lait. olivier et al.; asian j. immunol., vol. 7, no. 1, pp. 149-158, 2024; article no.aji.122945 152 2.3 ex vivo investigation: leukocyte adherence inhibition test 2.3.1 procedure for allergen ex vivo challenging we performed the lait as previously described [29,30,36-45]. shortly, each donor's fresh plasma was divided into two parts and used in paralleled ex vivo challenging tests with aluminum acetate solution 1 mg/ml and the unchallenged plasma assay. we collected the plasma with high leukocyte content (buffy coat) from the heparinized tube after one hour of sedimentation at 37 °c. then, we distributed aliquots of 100 μl into eppendorf tubes kept under agitation for 30 minutes (200 rpm at 37 °c) with aluminum solution (10μl of a solution with 1mg/ml) or without aluminum solution (when used as control). 2.3.2 procedure for adherence assay after incubation, the plasma was allocated into a standard neubauer hemocytometer counting chamber with a plain, non-metallic glass surface and left to stand for 2 hours at 37 °c in the humidified atmosphere of the covered water bath to allow leukocytes to adhere to the glass. next, we counted the leukocytes, removed the coverslip, and washed the chamber by immersion in a beaker with pbs at 37 °c. then, we added a drop of pbs to the hemocytometer's chamber and allocated a clean coverslip over it. the remaining cells were counted in the same squares as previously examined. 2.3.3 procedure for calculation the percentage of leukocyte adherence (la) of each assay was estimated as: (the number of leukocytes observed on the hemocytometry chamber after washing divided by the number of leukocytes observed on the hemocytometry chamber before washing) and multiplied by 100 (%). the leukocyte adherence ratio (lar) was estimated based on the ratio between the la from the antigen-specific challenged plasma and the la from the unchallenged control plasma: lar = la of the challenged sample divided by la of unchallenged control plasma multiplied by 100 (%). to further calculate the leukocyte adherence inhibition (lai), we subtracted the lar from 100 (%). we employed the lai results for the cascade distribution chart and the statistics calculations, both performed with the help of the microsoft excel® statistical package. 2.3.4 in vitro investigation: tube titration of precipitins (ttp) as previously reported, the semi-quantitative ttp against the aluminum solution was performed in a transparent vitreous tube [46]. shortly, the patient's blood was collected in a clot-activator collecting tube. after separation, the serum was centrifugated at 2,000 rpm for 10 minutes. the allergen extracts were allocated in sets of eleven glass tubes at progressive duplicated serum dilutions. the progressive dilutions were combined with the 15 μl of the antigen (1 mg/ml) with 250 μl of the patient's serum, progressively diluted into physiological saline solution (nacl 0,9%) in the dilution ratios of 1:1; 1:2; 1:4; 1:8; 1:16; 1:32; 1:64; 1:128; 1:256; and 1:512. one tube was a blank control done with the water and serum to observe occasional spontaneous precipitation (sia test). after 24 hours, the tubes were examined, and the titers (the highest dilution factor that yields a positive reading) were recorded [47]. 3. results as a retrospective survey, there was no research protocol; therefore, we report the incidental immune investigation as registered in the digital medical charts. the cascade distribution graph showed a wide distribution range of ttp results. there was one negative result. most positive results concentrated on the higher dilutions (fig. 1). the mean was estimated at 1:301; the median was 1:256; the standard deviation was estimated at 1:204; the mode was 1:512 (appeared 46 times). all sia tests were negative. the lai ranged from 0% to 88%. the mean was 52%; the median was 49%; the standard deviation was 23.9%; the mode was 60% (appeared five times). the cascade distribution graph demonstrates a wide range of distribution of lai results (fig. 2). three patients ignored the presence of the allergen on the plasma and presented no inhibition of leukocyte adherence (lai = 0%) after contact with the aluminum solution. some patients showed low or moderate immunoreactivity during the ex vivo challenge test, while most displayed strong immunoreactivity, suggesting aluminum's participation in the hypersensitivity condition. 4. discussion aluminum was one of the first inorganic adjuvants envisaged to boost the immune olivier et al.; asian j. immunol., vol. 7, no. 1, pp. 149-158, 2024; article no.aji.122945 153 response of infectious vaccines and subcutaneous allergen immunotherapies [48]. the first description of using aluminum salts to boost immune response was published in 1924 when used as an adjuvant to tetanus and diphtheria toxoids [49]. further (1938), aluminum salts were presumed as adjuvants to subcutaneous allergen immunotherapies for their depot effect, increasing the permanence of the immunotherapeutic agent on the site of the injection, allowing more time for the innate immune system to process the antigens (adsorptive adjuvant) [50]. later (1985), it was established that the presence of aluminum salts increased the uptake of allergens by antigenpresenting cells, stimulating antigen-induced tcell proliferation [51]. aluminum-containing adjuvants also activate innate immune response by stimulating dendritic cells, inducing cd4+ t cell differentiation [52]. aluminum acts as a vaccine adjuvant by producing cellular necrosis that releases inflammatory cytokines (such as il33), stimulating innate and adaptive immunity [53]. it is a corollary to think that ingestion, contact, and parenteral administration of this metal may produce residual asymptomatic or even symptomatic conditions in a society "drowned" in aluminum through food additives, cosmetic preservatives, medicines, vaccine adjuvants, and others [54]. aluminum hypersensitivity might represent just the iceberg's tip of an organism saturated by its effects, alerting the patient's conscious mind to avoid contact and ingestion. personalized medicine is an approach dedicated to diagnosing the endotypes responsible for disease phenotypes. [55]. while phenotypes are defined by the visible clinical manifestations of the conditions (some of these induced by multiple underlying mechanisms), an endotype is a subtype of a given phenotype defined by its pathophysiological mechanism [56]. the classical four hypersensitivity mechanisms described by gell and coombs in the sixties are now amplified to seven types with several subtypes, implying an increased responsibility of the clinical caretakers to investigate the mechanisms responsible for the disease phenotypes in order to recommend better strategies to avoid the allergens and to prescribe tailored treatments for these infirmities [57,58]. endotyping the hypersensitivity mechanisms may also help distinguish superimposable phenotypes presenting similar symptoms that may hamper establishing a precise diagnosis [59]. to detect immune responses against aluminum, we spreadsheet a retrospective compilation of data fig. 1. cascade distribution chart of the tube titration of precipitins (ttp on the x-axis) resulting from the aluminum solution against the serum of a cohort of 100 tests/subjects (y-axis) olivier et al.; asian j. immunol., vol. 7, no. 1, pp. 149-158, 2024; article no.aji.122945 154 fig. 2. cascade distribution chart of the range groups of leukocyte adherence inhibition (lai) results (x-axis %) of ex vivo aluminum solution monitored by the leukocyte adherence inhibition test (lait), according to the respective number of outcomes over a cohort with 100 tests/subjects (y-axis) produced at our facilities by ttp and tial, exploring humoral and cellular immunoreactivities against aluminum. ttp and lait are complementary triage tests used at our facilities to select worthwhile antigens to proceed with more laborious in vivo provocation tests when the specific ige is undetectable and skin tests are inconclusive or unfeasible due to the patient's skin conditions. these assays do not identify the exact immune mechanisms responsible for the clinical condition. instead, they provide clues about sensitization and immunoreactivity distributed into an extensive spectral range between immune tolerance and symptomatic hypersensitivity. ttp and lait must be interpreted as triage immune markers of the humoral and cellular responses after contact with a specific antigen, configuring themselves as techniques to quantify an exposome measurement, as proposed by the exposomewide association study [60]. this preliminary retrospective survey demonstrated extensive results from the ttp and the ex vivo challenge test monitored by lait against aluminum in two cohorts of patients with various allergic symptoms. none of our patients presented an exclusive reaction to aluminum. every patient was simultaneously tested with several chemical and biological allergens, demonstrating positive results for some of them. our results suggest that reactive allergic patients may impair their symptoms by an additional immunoreactivity against environmental or ingested aluminum. 5. conclusion our preliminary results show that the ttp and lait may differentiate diverse degrees of immunoreactivity against aluminum in patients clinically diagnosed with non–ige-mediated cutaneous allergies. this methodology can provide a socioeconomic impact since the methodologies to perform ttp and lait are inexpensive and can be performed in a single lab attached to the facilities with minimum laboratory equipment. however, the propaedeutic meaning of these results and the possibility of interferents must be better established [61]. more studies focused on the quality-by-design approach with prospective larger double-blind cohorts need to evaluate the potential contribution of ttp and lait for endotyping immunoreactivity of patients olivier et al.; asian j. immunol., vol. 7, no. 1, pp. 149-158, 2024; article no.aji.122945 155 suspected of symptomatic hypersensitivity against aluminum and other similar food processing additives [62,63]. 6. limitations this study is a retrospective analysis of data collected over six years. there was no protocol research, and the subject's data were limited to the essentials available on our electronic sheets. therefore, we could not establish a crosscomparison between positive and negative controls to validate the results. the number of subjects is appropriate for a preliminary study; however, future studies must be more comprehensive. the lack of a research protocol implies the possibility of a bias produced by the physician's point of view who indicated the exam (ceo) based on a clinical suspicion led purely by the anamnesis, physical examination, routine lab exams, and allergic skin tests. the study lost many of these patients to follow-up, so assuring the relationship between the immunoassays' results and the patient's clinical outcome is impossible. disclaimer (artificial intelligence) author(s) hereby declare that no generative ai technologies such as large language models (chatgpt, copilot, etc.) and text-to-image generators have been used during the writing or editing of this manuscript. ethical approvals the authors have collected and preserved written ethical approval per international standards. acknowledgements the instituto alergoimuno de americana funded this work. we want to thank laboratory technician alessandra vieira de oliveira for her help with the exams. competing interests authors have declared that no competing interests exist. references 1. laeter jr, böhlke jk, bièvre p, hidaka h, peiser hs, rosman kjr, taylor pdp. atomic weights of the elements. review 2000 (iupac technical report). pure appl. chem. 2003;75(6):683-800. 2. novack de, yu j, adler bl. aluminum: the 2022 american contact dermatitis society allergen of the year. cutis. 2022; 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asian journal of immunology 3(1): 46-60, 2020; article no.aji.54889 immunomodulatory potentials of probiotics: a review david chinemerem nwobodo1,2* and malachy chigozie ugwu2 1 department of microbiology, renaissance university, ugbawka, enugu state, nigeria. 2 department of pharmaceutical microbiology and biotechnology, faculty of pharmaceutical sciences, nnamdi azikiwe university, awka, anambra state, nigeria. authors’ contributions this work was carried out in collaboration between both authors. author dcn designed the study, searched the literature and wrote the draft versions of the manuscript. author mcu supervised the literature search and edited the lay-out. both authors read and approved the final manuscript. article information editor(s): (1) dr. darko nozic, higher medical school in belgrade, university of belgrade, serbia. reviewers: (1) alessandro poggi, irccs ospedale policlinico, san martino, italy. (2) ekaterini simões goudouris, universidade federal do rio de janeiro, brazil. complete peer review history: http://www.sdiarticle4.com/review-history/54889 received 18 december 2019 accepted 21 february 2020 published 31 march 2020 abstract in recent years, research has focused on natural mechanisms for the management, treatment, and curing of human infections and diseases. one of such natural methods is the application of probiotics, which are live microorganisms which when administered in adequate amounts confer a health benefit on the host. the beneficial effects associated with probiotics were originally thought to be a result of improvements in the intestinal microbial balance, however, there are shred evidence that probiotics can also provide benefits by modulating the immune functions. the ability of these probiotics, majorly the lactobacillus and bifidobacterium species to boost the immune system is proposed to be a result of their interactions with the cells of the immune system. they have been reported to stimulate various parts of the immune system, through several mechanisms enhancing their functions. it has also been established that the effects of probiotic bacteria may also result from soluble factors from these microbes that alter epithelial permeability or mediate activation, maturation or survival of dendritic cells, b and t-cells. probiotic bacteria, their cell wall components, and other stimulating molecules have been shown to have significant effects on the functionality of the immune systems through the activation of multiple immune mechanisms. this study is aimed at describing the immunological mechanisms of probiotics and their beneficial effects on the host immune system. review article nwobodo and ugwu; aji, 3(1): 46-60, 2020; article no.aji.54889 47 keywords: immunomodulatory potentials; probiotics; lactobacillus; human infections; bifidobacterium. 1. introduction the word probiotic is derived from a greek word which means “pro-life” [1]. in 1965, lilly and stillwell first used the term ‘probiotic’ to describe substances secreted by one organism which stimulates the growth of another [2]. in 2002, the food and agricultural organization of the united nations/world health organization proposed that probiotics are live microorganisms which when administered in adequate amounts confer a health benefit on the host [3]. presently, research is focused on natural mechanisms for managing, treating and curing human infections and diseases as a result of the several side effects associated with the use of chemotherapeutic agents and synthetic drugs. probiotics known as a group of beneficial microbes are currently emerging as one of such natural mechanisms. most commonly used probiotic microorganisms belong to the genera lactobacillus and bifidobacteria, however, some strains of escherichia coli, bacillus species, and the yeast saccharomyces are also used [4-6]. recently, clostridium butyricum was also approved for probiotic use in the european union [7]. some of the reported probiotic microorganisms include; lactobacillus delbruecki subsp.bulgaricus, l. rhamnosus gg, l. plantarum, bifidobacterium bifidum and b. infantis [8,9]. b. adolescentis bbmn23 and b. longumbbmn68 [10]. others include streptococcus thermophilus, escherichia coli, saccharomyces bourladii [6,11], saccharomyces cereviciae [12], some species of lactococcus and enterococcus [13]. reported benefits of probiotics include: maintaining the balance of the intestinal flora, increasing lactose tolerance and synthesis of b complex vitamins, absorption of calcium thus maintenance of intestinal homeostasis [1]. others are anti-mutagenic/anticancer activity, cholesterol-lowering effect, secretion of antipathogen substances and enhancement of the immune system/responses [14-16]. the integrity of any eukaryotic organism depends not only on the proper expression of its genes but also on its freedom from /and defense against invading microorganisms [17]. the human body is equipped with defense mechanisms which serve to protect against harmful agents and invading pathogens. it is because of the effectiveness of these defensive mechanisms that both man and animals have survived for so many years. a good example of this is the phagocytosis of bacteria by macrophages and other phagocytic cells of the immune system. however, the host does not depend solely on its immune system to protect it from these agents of diseases. one of the most important characteristics of probiotics is the regulation of host immune response by manipulating the host immune response towards the infectious microbes and can be useful in the treatment of infectious diseases [18]. innovative approaches had been attempted as an alternative to vaccines as immune boosters, and these include the use of live biotherapeutic agents (probiotics) such as yeasts and bacteria. probiotics have been used successfully to improve the host immune response in different health conditions [19]. it is, therefore, the aim of this review to explore the effect of probiotics on the immune system and describe the immunological mechanisms in modulating the host immune system. 2. brief history of probiotics the concept of probiotics evolved in the late 1800s and early 1900s [20]. different microorganisms have been used for their supposed ability to prevent and cure diseases, which made lilly and stillwell derive the word probiotics in 1965 [21]. the original observation of the beneficial role played by probiotic bacteria was first reported at the beginning of the 20th century by russian scientist and nobel laureate elie metchnikoff. he suggested that it would be possible to modify the gut flora and replace harmful microbes with useful ones and thus proposed that the consumption of yogurt containing lactobacillus would result in a decrease in toxin-producing bacteria in the gut and an increase in the life span of the host [8,20]. henry tissier, a french pediatrician in the year 1900 observed that children with diarrhea had in their stool a low number of bacteria characterized by a particular y shaped morphology. these were named bacillus bifiduscommunis and were later assigned to the genus bifidobacterium (bifid). the “bifid” bacteria were on the contrary abundant in healthy children [22]. in 1906, he suggested that these bacteria could be administered to patients with diarrhea to help restore a healthy gut flora [8,20,22]. thus the works of metchnikoff and tissier were the first to nwobodo and ugwu; aji, 3(1): 46-60, 2020; article no.aji.54889 48 make scientific suggestions about the probiotic uses of bacteria. the first reported clinical trials were done in the 1930s on the effect of probiotics on constipation [3]. the associated effect of probiotics on the immune system came into play in 1984, when baalmear et al. [23] observed that animals with complete gut flora have increased phagocytic activity compared with germ-free animals. similarly, perdigon et al. [24] discovered that a particular probiotic species lactobacillus casei was active in stimulating phagocytic activity when administered to mice. in 1987, brandtzaeg et al. suggested that the effect of lactic acid bacteria could be a result of the absorption of the cells or their products by macrophages and transportation to deeper lying lymphatic follicles where they interact with immunocompetent cells [25]. in an in vitro experiment carried out in 1989, fuller found that lactobacillus was successful in modulating inflammatory diseases, enhanced barrier functions and stimulated immunity [26]. nussler and thomson in 1992 reported that lactic acid bacteria and their products can interact with immune cells, leading to the production of cytokines that have a manifold effect on immune and non-immune cells [27]. to date, several types of research relating probiotics to the immune system have been carried out with several mechanisms being proposed to elucidate such immuno-stimulating relationships. 3. safety assessment of probiotics for human use strains of lactic acid bacteria (lab) have a long history of safe use. different species of lactobacillus and enterococcus, has been consumed frequently since the consumption of fermented milk as food [8]. probiotic species such as lactobacillus acidophilus have been safely used for several years [8]. nevertheless, there is a need for the safety aspects to be always considered and possible adverse effects should be continuously evaluated [28]. members of the genera lactococcus, bifidobacteria, and lactobacilli are most commonly given the “generally regarded as safe” (gras) status, while members of the genera streptococcus, enterococcus and some other genera of lab are considered opportunistic pathogens [8,9,29]. the safety of probiotics has been considered in reviews and clinical reports which have recorded low cases of human bacteremia [29]. in france, it was estimated that the risk of lactobacillus infection is about one case per 10 million people over a century of probiotic consumption [8,30]. surveillance studies by adams and marteau in 1995 supported the safety of commercial lab [31]. in 1998, salminen et al. reported that no harmful effects were observed in controlled clinical studies with lactobacilli and bifidobacteria [32]. further evidence of poor opportunistic pathogenicity of probiotics were provided in clinical studies where certain probiotics was safely administered to immunocompromised patients, premature infants, elderly and patients with crohn’s disease and there were no recorded side-effects [31]. in a recent randomized human study involving the elderly, lefevre et al. reported that the probiotic product was safe and well-tolerated [16]. different probiotic formulations have been administered to a considerable large number of individuals suffering from different conditions, under controlled conditions and have been proven to be without associated risks [9]. considering their widespread use, documented correlations between adverse events and probiotic consumption are very few [8,31]. 3.1 properties of probiotic organisms to be able to exhibit health benefits, probiotics should be able to survive the harsh conditions of the stomach and gi tract of humans after consumption. some of the properties of an ideal probiotic microorganism include [9,33,34]: i. ability to survive the passage through the digestive system. ii. ability to adhere to the epithelial cells of the mucosa. iii. non-pathogenicity and non-toxicity. iv. tolerance to food additives and stable in the food matrix. v. excluding or reducing pathogenic adherence. vi. multiply and produce acids, hydrogen peroxide and bacteriocins antagonistic to pathogen’s growth. vii. maintenance of viability in large numbers. 4. probiotics and the innate immune system consumption of probiotics initiates a host response due to the interaction with intestinal enterocytes, as intestinal cells are known to produce various immunomodulatory molecules when stimulated by bacteria [35]. oral introduction of lactobacilli can enhance nonspecific host resistance to microbial pathogens nwobodo and ugwu; aji, 3(1): 46-60, 2020; article no.aji.54889 49 and thereby facilitate the exclusion of pathogens in the gut. lactobacilli modulate the immune response of the host by its interaction with the immune cells and the intestinal epithelium [36]. several strains of live lactic acid bacteria have been shown to induce in vitro the release of the pro-inflammatory cytokines, tumor necrosis factor α, and interleukin 6, reflecting stimulation of nonspecific immunity, [37]. studies have shown that the lactobacillus casei dn114001 strain induces mucosal immune stimulation, reinforces the non-specific barrier and modulates the innate immune response in the gut, thereby maintaining the intestinal homeostasis [38]. 4.1 effect of probiotics on phagocytic cells it has been discovered that animals with complete gut flora have increased phagocytic activity compared with germ-free animals [11,39]. presumably, the normal flora prevents invading organisms from adhering to host cells by covering binding sites, thereby easily exposing them to phagocytic cells (neutrophils, monocytes, macrophages) [2]. oral introduction of lactobacillus casei and lactobacillus bulgaricus activates the production of macrophages and administration of l. casei and lactobacillus acidophilus activates phagocytosis in mice [40]. similarly, enhanced phagocytosis had been long reported in humans by l. acidophilus lal [41]. phagocytosis is responsible for early activation of the inflammatory response before antibody production [17]. phagocytic activity results in the further recruitment of immunocompetent cells and the generation of an inflammatory response [5]. the phagocytic activity of raw264.7 macrophages murine model is enhanced after inoculation with bifidobacterium adolescentis bbmn23 or b. longum bbmn68 [5]. immune stimulation by these probiotics on the macrophages could be attributed to the enhanced macrophage activity on the components of the immune system. it has been observed that there is an increase in the phagocytic activity of peritoneal macrophage after days of feeding with probiotic cultures [2,42]. ordinarily, at the earlier stage of infection, the macrophages are recruited to the infected site to engulf the microbes, and when macrophages are activated after bacterial recognition by toll-like receptors (tlrs), they consequently produce high level of proinflammatory cytokines such as il-1β, tnf-α, il8, il-6 and chemokines which recruits more macrophages and other immune cells e.g. neutrophils and basophils [43]. studies show that probiotics mostly belong to gram-positive bacteria, and contain lipoteichoic acids and thick peptidoglycan cell wall components (fig. 1). these cellular components can activate macrophages to secrete cytokines or important mediators which could as well trigger other immune components leading to the stimulation of the immune system [43,44]. there is also a report that oral delivery of l. casei probiotic strains to mice could activate mononuclear phagocytes for increased phagocytic activity and lysozyme production [45]. they express nonspecific esterase, lysosomal hydrolases, and ectoenzymes, thereby contributing to nonspecific uptake of invading materials [43,45,46]. human studies have confirmed these effects in circulating phagocytes of adult subjects including the elderly (table 1.) continuous engulfment by the phagocytes strengthens the immune cells and keeps them at alert [5]. 4.2 probiotic effects on inflammatory response it has been shown that the administration of lactobacillus plantarumat the dose of 10 10 cfu/day significantly increased neutrophils, macrophages, and fibroblasts [48], (table 1). in the inflammatory response, the neutrophils are the first cells to be lured, followed by the macrophages for the engulfment of the infecting pathogen. the fibroblast halts and prevents further spread of the infection [48]. this has also been observed and reported that in vitro, lactobacillus has been used to modulate inflammatory diseases, enhance barriers functions and stimulate immunity [26]. some probiotic bacteria have been reported to induce a pattern of dendritic cell (dc) maturation, characterized by the release of small amounts of tumor necrotic factor α and il-12, with increased levels of il-10, and inhibit the generation of proinflammatory th1 cells [49]. the most patent anti-inflammatory effects by a probiotic is produced by bifidobacteria, which upregulated il10 production by dendritic cells in a dose dependent manner [45]. nwobodo and ugwu; aji, 3(1): 46-60, 2020; article no.aji.54889 50 fig. 1. mechanisms of immunomodulatory actions of probiotic bacteria key: ; increase/ enhance, ; activate/ stimulate, ; induce, ; release table 1. clinical evidences of immune stimulation by probiotics [47,48] probiotics immunological functions subjects lactobacillus acidophilus la1, l. rahmnosushn001, b. bifidum bb12, b. lactishn019 phagocytic activity of blood mononuclear and polymorphonuclear cells healthy adults and elderly volunteers lactobacillus caseishirota, b. lactishn019 the tumoricidal activity of blood mononuclear cells healthy adults and elderly volunteers; patients with colorectal cancer lactobacillus brevis labre, b. lactishn019 production of interferons by peripheral blood mononuclear cells healthy adults and elderly volunteers lactobacillus plantarum increased neutrophils, macrophages, and fibroblast adult volunteers lactobacillus rhamnosus gg anti-rotavirus antibody responses children with rotavirus l. rahmnosusgg antibody responses following vaccination adult volunteers bacillus subtilis cu1 increased the levels of secretory iga in stools and saliva, high serum ifn-gamma elderly during common infectious disease according to a report by nikolov in 2012, the inflammation-suppressing properties of probiotics may be able to: (i) counteract some of the inflammationaggravating bacteria. this will decrease the inflammatory response; (ii) improve the barrier effect of the mucosa, which will inhibit the translocation of inflammation-inducing luminal contents into the body; (iii) directly interact with pro-inflammatory processes [50]. 4.3 stimulation of cytokines by probiotics cytokines are chemical messengers used by both innate and adaptive immunity. research has shown that live probiotic strains induce the production of protective cytokines that enhance epithelial cell regeneration and inhibit epithelial nwobodo and ugwu; aji, 3(1): 46-60, 2020; article no.aji.54889 51 cell apoptosis [51]. cytokine-induced apoptosis was prevented in intestinal epithelial cells in the presence of l. rhamnosus gg [48]. apoptosis which is programmed cell death was prevented by l. rhamnosus gg, but the mechanism is not yet fully known. the inhibition of apoptosis enhances the survival of intestinal cells and promotes proliferation during recovery from epithelial injury [52]. probiotics induce pro-inflammatory cytokine release in vitro, such as il-6 and tumor necrosis factors (tnfs) [53]. this is in agreement with reports by huang et al. [54] that bacillus strains could stimulate systemic and intestinal ifnγ production in mice. zhu et al. [5], who reported that l. casei had been shown to induce the cytokine il-6 production, also demonstrated that b. adolescentis bbmn23 is a strong stimulator for tnf production. il-6 and tnf are cytokines usually secreted by macrophages when activated [43]. stimulation of these pro-inflammatory and other cytokines by probiotic may be as a result of the immunological responses their presence caused in the body. in the mucosa, probiotic bacteria induce the secretion of cytokines from intestinal epithelial cells (iec) in a strain-specific manner. the strain specificity could be due to soluble factors produced by probiotic that might modulate cytokine production by peripheral blood mononuclear cells (pbmc) [2]. the iecs serve as the initial point of contact between the host and intestinal microbes and they communicate expansively with commensal bacteria and probiotics. this interaction influences the inflammatory signaling pathway in iecs. the il-6 and il-8 released by the epithelial cells are proinflammatory, so an intense epithelial stimulation can favor an inflammatory immune response [55]. this cytokine produced by iec, macrophages and t cells, can induce the terminal development of b cells in plasmatic cells, which express iga. the cytokines released by th2 cells are involved in the induction of the iga immune response [56]. findings based on the use of cell lines as experimental models reported that the quality and dose of probiotic preparations could impact the il-8 production by enterocytes [57]. il-8 appears to be a major cytokine produced by enterocytes following an encounter with probiotics. the il-8 cytokine primarily functions as a neutrophil chemoattractant [35]. the macrophages and other immune cells stimulate the production of these cytokines when they come in contact with the probiotic microorganisms, thereby enhancing the immune system for the elimination of any available pathogen in the case of infection or invasion by pathogens. 5. probiotics and the adaptive immune cells the majority of the studies concerning probiotic effects on lymphocytes function has utilized animal models. according to aattouri et al. [58], oral ingestion of lactic acid bacteria such as lactobacilli and bifidobacteria strains by rats and mice increases lymphocyte proliferation and interferon production. 5.1 stimulation of b-lymphocytes by probiotics earlier studies using rat models by naidu et al., [59] reveals that lactic acid bacteria administered orally increased the number of antibodysecreting cells, including those in the intestinal mucosa with enhanced b-cells proliferation and antibody production (iga and igg). this could be a result of the cell wall components of these bacteria leading to the stimulation of antibodies and cytokines for the proliferation of b-cells (fig. 1). it has been reported that interactions between host cells and bacteria or their structural components may lead to modulation of tor bcell-mediated immune responses, either locally or systemically [60]. when these b-cells are proliferated in response to probiotic, they differentiate into plasma cells producing more antibodies [17]. early animal studies showed that probiotics were able to enhance systemic antibody responses to parenterally delivered foreign antigen in mice [47,57]. in a study on rats co-colonized with l. plantarum and escherichia coli, herias et al. [61], reported a higher circulating concentration of total iga and e. coli specific iga and igm compared with rats which were colonized with e. coli alone. if in this study rats were colonized with e. coli and it stimulated the production of iga and igm. then when it was co-colonized with l. plantarum, and an increase in circulating iga was recorded, then it could probably be that l. plantarum also trigger the production of iga. also indicating that synergistic use of probiotics may yield an increased immune effect. probiotics have also been reported to boost overall siga antibody responses [16]. this results in a significant enhancement of systemic antibody response and thereby triggering intestinal immunity and subsequent elimination of the evading pathogens. this is because most siga recognizes and opsonizes bacteria in the lumen, thus preventing their access to the lamina propria (lp) [60,62]. siga is important in the nwobodo and ugwu; aji, 3(1): 46-60, 2020; article no.aji.54889 52 maintenance of gut microbiota homeostasis and the protection of the gastrointestinal and respiratory tracts against pathogens, as it is the main immunoglobulin class in human external secretions [16]. the mechanisms whereby probiotics modulate immune responses leading to tolerance or siga activation appear to be highly dependent on the strains [49]. in human studies, l. rhamnosus gg and b. breve yit4046 were shown to stimulate anti-rotavirus antibodies in response to rotavirus diarrhea in children (table 1). 5.2 stimulation of t-lymphocytes by probiotics research has shown that t-helper lymphocyte (cd4 + ) numbers are increased in the gutassociated lymphoid tissue (galt) following oral delivery of l. casei [11,13]. providing evidence that probiotic stimulation can increase the size of the lymphocyte population. in a report, orally administered lactic acid bacteria in rats revealed increased numbers of tlymphocytes, cd4+ cells and also enhanced lymphocyte proliferation [59]. the biological property of probiotic bacteria involved in lymphocyte proliferation is their capacity to affect immune cell redistribution by improving the competence of lymphatic endothelial cells to trap t lymphocytes [63]. 5.3 stimulation of natural killer cells by probiotics oral delivery of l. rhamnosus hn001 or l. casei shirota to mice has been shown to increase ex vivo natural killer cell tumoricidal activity [45,47]. this proposes that the consumption of these probiotics enhances the destruction of tumor cells by stimulating natural killer (nk) cells, thereby reducing tumor risks. in human studies, b. lactishn019 has been demonstrated to upregulate peripheral blood nk cells mediated cytotoxicity against tumor cells (table 1). 6. mechanisms of immune modulation by probiotics studies have demonstrated that specific chemical compounds isolated from bacteria can induce specific immune responses (figs. 1, 2 & table 2) and thus provide the scientific basis for a molecular description of the immunological effects observed after the administration of probiotics. excluding the extracellular bacteria products, a major role in immunomodulatory activity should be mediated by the structural components of the cell, particularly the cell envelope. this is the outermost structures that the immune cells come into contact with first and includes the cell wall or s-layer proteins, capsules and pellicle [64]. fig. 2. molecules or parts of probiotic bacterialcells demonstrated to modulate host immune and epithelial cells nwobodo and ugwu; aji, 3(1): 46-60, 2020; article no.aji.54889 53 findings indicated that both cell wall and cytoplasm can be recognized and stimulate the immune system [33]. because of the limitation that bacteria cannot pass through the epithelial cells, it can be uptaken by microfold cells (mcells), and only the antigenic particles or products of degradation of the bacteria can make contact with the immune cells by pattern recognition receptors (prr) that recognize pathogen-associated molecular patterns (pamp) [65]. therefore, pamp can explain the different production of cytokines induced by bacteria, demonstrating the different components of the cell wall causing the distinct immune-boosting effects. in a study, raw264.7 macrophages were exposed to heat-killed probiotic bifidobacterium spp., l. acidophilus, l. bulgaricus, l. casie, l. gasseri, s. thermophilus including the cell envelope components and cytoplasmic extracts of these bacteria and they stimulated macrophages to produce tnf-α, il-6 and nitric oxide (no) [45], suggesting that bioactive compounds are potentially located everywhere in the bacteria cell. in the intestinal fluid, probiotics influence the production of antimicrobial peptides (amps) by paneth cells, the production of mucus by goblets cells as well as the level of secretory iga that is under the dependence of a proliferation-inducing ligand (april) produced by iec. probiotics affect the expression level of tlrs on dc as wells as immune cells in peyer’s patches, leading to a production of a wide range of cytokine regulating the evolvement of the immune response. 7. immunomodulatory molecules of probiotic bacteria 7.1 bacterial cell wall components cell wall components of l. caseiare antiinflammatory [48]. peptidoglycan (pgn) and lipopolysaccharide (lps) are well known potent activators of immune responses (fig. 2). peptidoglycan (pgn) is the main constituent of gram-positive bacterial cell wall, accounting for up to 90% of their weight, whereas it constitutes only 15-20% of the cell wall of gram-negative bacteria [69]. specialized conserved pattern recognition receptors (prr) on the host cell membranes, such as toll-like receptors (tlrs) and the nucleotide-binding domain (nod) proteins are the primary sensors of the innate immune system and recognize microbeassociated molecular patterns, including pgn and lps [37,50,69]. in particular, tlr4 is a specialized receptor for lps, whereas both nod1 and nod2 recognize muramyl peptides released by pgn [37]. the different immune stimulation by cell wall components is shown in fig. 2 and table 2. in gram-positive bacteria cell walls, molecules are protruding from the external surface of the pgn layer known as teichoic acids (tas). tas are phosphodiester polymers of glycol or ribitol and can be covalently linked to either peptidoglycan (wall teichoic acid, wtas) or the cytoplasmic membrane (lipoteichoic acid, ltas). ltas purified from l. casei yit 9029 and l. fermentumyit 0159 were demonstrated to induce elevated levels of tnf-α in mouse raw264.7 macrophages mediated by tlr2 (fig. 2) [45]. 7.2 other immunomodulatory molecules of probiotic bacteria it has been established that the effects of probiotic bacteria may also result from soluble factors that alter epithelial permeability or mediate activation, maturation or survival of dendritic cells [70]. 7.2.1 surface layer (s-layer) probiotics can interact with the host immune system through their surface layer, a monomolecular crystalline envelope produced by the self-assembly of protein or glycoprotein subunits on the outer cell surface [45]. s-layers are commonly found in prokaryotes and makeup to 10-15% of the total protein content of a cell [71]. s-layer protein a (slpa) released from l. acidophilus ncfm cells has been demonstrated to be recognized and bound by dendritic cellspecific intercellular adhesion molecule-3grabbing non-intergrin (dc-sign), a c-type lectin receptor presents on both macrophages and dendritic cells [66]. it was observed that l. acidophilus ncfm expressing slpa was captured by dc-sign on dc, and thus activated the il-4 producing t-cells. these data were confirmed by an experiment performed with purified slpa protein, which ligated to dc-sign and induced il-10 expression by dcs in the presence of lipopolysaccharide [66]. nuclear factor kappa-light chain enhancing activated bcells (nf-kb), is triggered in the presence of slayer by human epithelial colorectal adenocarcinoma cell line (fig. 2). nf-kb is a protein complex that is found in almost all animal cells, which controls the transcription of dna. it is also involved in cellular responses to stimuli nwobodo and ugwu; aji, 3(1): 46-60, 2020; article no.aji.54889 54 table 2. immunomodulatory molecules of probiotic microorganisms probiotics molecules effects lactobacillus casei lactobacillus plantarum peptidoglycan general immune stimulation [33,48] lactobacillus casei lactobacillus fermentum lipoteichoic acid increased levels of tnf-α in mouse macrophage cell [45]. bifidobacteriumlongum dna increased cytokine il-10 [45]. lactobacillusacidophilus surface layer activation of il-4 producing t-cells [66]. b. bifidummimbb75 bopa protein induce production of il-8 by colorectal cell line [67,68]. escherichia colinissle 1917 flagellin induce production of il-8 by colorectal cell line [68]. s. thermophiles l. plantarum cell wall-associated polysaccharide (caps) general immune stimulation [33,48]. such as bacterial and viral antigen with the production of cytokines. nf-kb is the key signaling channel in the inflammatory signaling pathway [2,45]. 7.2.2 bopa protein and flagellin bopa is a cell surface-associated lipoprotein of b. bifidum mimbb75 that mediates adhesion to the human caco-2 intestinal epithelial cells. upon purification from b. bifidummimbb75 strain, bopa has been demonstrated to induce the production of il-8 by caco-2 cells in a dosedependent manner [67]. flagellin, a structural protein released from e. colinissle 1917 strain, has been reported to increase the production of the pro-inflammatory cytokine il-8 in caco-2 cells [68]. 7.2.3 genomic dna it has been shown that prokaryotic dna contains an unmethylatedcpg motif that can activate immune responses in vitro and in vivo [45]. dna mixture isolated from the probiotic mixture vsl#3 containing 8 lyophilized lactic acid bacteria strains generated non-inflammatory responses from epithelial and immune cells [37,72]. in an earlier study, lammers et al. [73] reported that bacterial dna extracted from bifidobacterial cultures of the commercial product, vsl#3 influenced cytokine production by peripheral blood mononuclear cells (pbmcs), increasing il10. the anti-inflammatory effect of genomic dna from vsl#3 bacteria was also confirmed in an in vivo murine study, which demonstrated that tlr9 signaling was essential in mediating this anti-inflammatory effect [74]. it has been suggested that the immunological effect observed with bifidobacterial genomic dna is favored by the high guanine-cytosine (gc) content of the bifidobacterium genes (58-61%), which explains the availability of different cpg motifs in the genomes of these bacteria [73]. 7.2.4 cytoplasmic extracts cytoplasmic extracts from different probiotic bacteria have demonstrated to stimulate several cytokines involved in immune functions, including nitric oxide (no) (fig. 2) [45]. it is very important to note that the potential bioactivity of specific bacterial components can be masked by other cell structures, and the effect of a single molecule can be influenced by the presence of additional bioactive substances. in support of this concept, a study by kaji et al. [75] identified tas as a key factor for triggering the synergism of inducing il-10 production. they demonstrated that tas alone weakly induced il10 production, but when macrophages sensed wtas or ltas in the presence of l. caseishirota strain, these stimuli cooperatively induced potent production of il-10. 8. the role of microbiota and probiotics in regulating immune responses in cancer patients the susceptibility and development of cancer is a result of a complex interaction between gene regulation and the environment [76,77]. gut microbiota is critical for intestinal immune maturation, protecting the host against pathogens and damaging inflammatory reactions [78], and probiotics (and prebiotics) present more common ways to establish and maintain healthy microbiomes. lactic acid bacteria (lab) species constitute members of healthy human gut microbiota. recent studies have reported that nwobodo and ugwu; aji, 3(1): 46-60, 2020; article no.aji.54889 55 certain lab strains are capable of inhibiting tumor progression [79,80]. generally, the antitumor mechanisms of lab appear to be by the modulation of the immune response and the induction of cellular apoptosis. in two independent studies by konishi et al. [79] and baldwin et al. [81], they reported that two strains of l. casei decreased tumor cell proliferation and enhance apoptosis in allograft models of colorectal cancer. in a more recent study, the oral administration of the probiotic strain l. casei bl23 reduced the onset of chemically induced tumors by the stimulation of il-12 (fig. 2) or nkcell cytotoxicity (fig. 1) mechanisms [82]. in the same study, they demonstrated the protective effects of l. casei bl23 in different mouse models of cancer, including colorectal-associated cancer (cac) and the tc-1 allograft model. similarly, the intratumoral inoculation of 3 mg of heat-killed propionibacterium acnes in subcutaneous melanoma promoted local and systemic th1 and tc1 responses associated with in situ granuloma formation and tumor regression [83]. p. acnes is recognized by tlr2 on monocytes, macrophages, and dcs, leading to the activation of il-12 promotor [84]. according to delia et al. [85] and touchefeu et al. [86], preparations containing b. bifidum, l. acidophilus, l. casei, and the vsl#3 formulation containing streptococcus, lactobacillus, and bifidobacterium spp. have been proven to reduce radiation-induced gut toxicity, such as diarrhea. reports are suggesting that genetically modified probiotic microorganisms may have better effects. a l. acidophilus strain harboring a deletion in the phosphoglycerol transferase gene and unable to synthetize lta prevented the progression of colonic polyps in apc dflox mice [87]. elafin-overexpressing l. casei and l. lactis reduced colitis in mice and ex vivo in inflamed epithelial cells from human colitis [88]. l. gasseri genetically modified to overexpress superoxide dismutase was reported to decrease colitis in il10-deficient hosts [89]. these findings and reports further raise the possibility that probiotics, if properly considered could become an adjuvant therapy for cancer treatment. 9. special observation in a very recent study carried out in 2019, it was reported that probiotics can have different effects on the immune system in males, compared with female piglets [90]. it is important to understand that immunity differs considerably with sex. accurate development of the immune system is crucial in guaranteeing it responds properly to both harmful and harmless stimulation throughout life, and this development, even during the early days of life, is dependent on sex [91]. in the study, the immune cells, antibodies, and other immune-related molecules were different in males and females in response to probiotic supplementation. female pigs produced more of the immunoglobulins iga and igm in their lymph tissue, while in male pigs, the process occurred in the large intestine [90]. this proposes that, during infancy, females may have greater potential for local immune regulation than their male counterparts. this finding implies that specific probiotics may be more beneficial for girls, whilst others could produce improved health outcomes for boys. given the primary differences in immune development between males and females, taking sex into account could provide a means to progress the effectiveness of probiotics for pharmaceutics and other therapies that act on the immune system. 10. conclusion and future perspectives accumulating researches have shown that probiotics have effects on the immune system. these effects can be seen in their ability to trigger or stimulate the immune cells and suppress certain immune processes. meantime, research is still ongoing to completely understand the mechanisms involved in immune modulation by probiotics. a probiotic-induced immune stimulation is a complex interplay of the host-microbe interactions. the immune responses (innate and adaptive) can be modulated by probiotic bacteria in a strainand dose-dependent manner. however, there is limited knowledge for in vivo use, safety and effect in immunocompromised individuals and newborns. thus, improved knowledge of probiotics and its effect on the host immune system by various mechanisms will promote proper strain selection for a specific prophylactic or therapeutic use, ultimately leading to more personalized therapy. there is also a need for more controlled trials with sufficient numbers of volunteers. consent it is not applicable. ethical approval it is not applicable. nwobodo and ugwu; aji, 3(1): 46-60, 2020; article no.aji.54889 56 competing interests authors have declared that no competing interests exist. references 1. ukeyima mt, enujiugha vn, sanni ta. current applications of probiotic foods in africa. african journal of biotechnology. 2010;9(4):394-401. 2. kemgang ts, kapila s, shanmugam vp, kapila r. cross-talk between probiotic lactobacilli and host immune system. journal of applied microbiology. 2014; 117:303–319. 3. food and agriculture organization of the united nations/world health organization. guidelines for evaluation of probiotics in food. london, ontario: canada; 2002. 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this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: http://www.sdiarticle4.com/review-history/54889 http://creativecommons.org/licenses/by/4.0 _____________________________________________________________________________________________________ *corresponding author: e-mail: med.anouar.saber@gmail.com; asian j. immunol., vol. 7, no. 1, pp. 71-78, 2024 asian journal of immunology volume 7, issue 1, page 71-78, 2024; article no.aji.116487 interferences in immunological assays: causes, detection, and prevention saber anouar a*, rajae hazim a and admou brahim a a laboratory of immunology, university hospital mohammed vi, faculty of medicine and pharmacy cadi ayad, marrakech, morocco. authors’ contributions this work was carried out in collaboration among all authors. author sa designed the work, wrote the first draft of the manuscript and managed the literature searches. author ab revised the work. all authors read and approved the final manuscript. article information doi: https://doi.org/10.9734/aji/2024/v7i1131 open peer review history: this journal follows the advanced open peer review policy. identity of the reviewers, editor(s) and additional reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here: https://www.sdiarticle5.com/review-history/116487 received: 28/02/2024 accepted: 02/05/2024 published: 10/05/2024 abstract immunological assays are fundamental tools in modern diagnostics, providing indispensable insights vital for clinical decisions and scientific inquiries. however, the reliability of these assays is at times compromised by various interferences, casting doubt on the authenticity of results. this abstract explores the origins, detection methods, and potential solutions concerning interferences in immunological assays. various factors, such as cross-reactivity, sample matrix effects, endogenous interferences, and test-specific nuances, can introduce deviations in immunological assessments. understanding these interference mechanisms is crucial for devising effective countermeasures. a range of approaches, from implementing interference controls and serial dilution analyses to utilizing specific tests susceptible to interference, have been employed for detection. additionally, advancements in technology have enhanced detection capabilities by introducing tools resistant to interference. to address anomalies in immunological assays, a comprehensive approach is essential. review article https://doi.org/10.9734/aji/2024/v7i1131 anouar et al.; asian j. immunol., vol. 7, no. 1, pp. 71-78, 2024; article no.aji.116487 72 implementing rigorous quality standards during assay design and execution is paramount. furthermore, documenting interference incidents and establishing guidelines for disclosing such occurrences promote transparency in academic and clinical settings. collaborative efforts involving researchers, assay manufacturers, and regulatory agencies are integral to driving progress and ensuring result accuracy. this paper emphasizes the importance of identifying, characterizing, and mitigating the diverse interferences present in immunological assays. tackling the complexities of these interferences and embracing innovative strategies are central to refining the precision and utility of diagnostic immunology. keywords: interferences; immunological tests; cross reactivity; antibody-related interferences; binding proteins; matrix; contrary effect. 1. introduction alterations in immunological assay outcomes, known as interferences, significantly impact the accuracy and precision of diagnostic results. such interferences can either enhance or diminish the perceived concentration of a targeted molecule, often skewing clinical interpretations and research conclusions. specifically, when an interference leads to an outcome that appears greater than the genuine value, it's designated as a 'positive' interference [1,2]. on the contrary, 'negative' interferences denote values that are perceived to be lower than they truly are. the biomedical domain, inclusive of diagnostic and research sectors, holds deep concerns regarding these interferences. essentially, they represent potential pitfalls that might distort the perceived levels of crucial biomolecules, including antigens, antibodies, and cytokines, among others. these quantifications play indispensable roles in diagnosing ailments, gauging therapeutic impacts, and navigating the realms of basic research. interferences, with their dualistic influence, either amplifying or diminishing results, accentuate the need for a comprehensive understanding of their origins and manifestations. it is paramount for professionals in the field to both anticipate and address these interferences adeptly to safeguard the integrity of assay outcomes. this exposition delves into the multifaceted nature of interference sources, their mechanistic underpinnings, detection techniques, and counter-strategies. by navigating these challenges, we aspire to bolster the confidence in immunological assay results, ensuring their clinical and research relevance remain uncompromised 2. types of interferences in immunologcal assays interferences in immunological assays encompass a diverse range of factors and phenomena that can affect the accuracy and reliability of results. these interferences can be categorized into several types, each with its own unique characteristics [3,4]. one common type of interference is crossreactivity. this occurs when antibodies, designed to target specific antigens, exhibit a lack of specificity, leading them to interact with unintended molecules. such cross-reactivity can arise due to structural similarities between the target antigen and other molecules, resulting in a false signal. another type of interference involves the presence of co-reactants and the improper specificity of antibodies. co-reactants present in the sample can interfere with the assay, leading to erroneous results. additionally, antibodies used in the assay may not be perfectly specific, causing them to interact with molecules other than the intended target. interference can also occur through the presence of anti-analyte antibodies, anti-reagent antibodies, or anti-animal protein antibodies. these antibodies can cross-react with the assay components, affecting the assay's performance. proteins that bind specifically to hormones, such as albumin and pre-albumin, can introduce interference in hormone assays. these binding proteins may compete with the antibodies used in the assay, leading to inaccurate results. anouar et al.; asian j. immunol., vol. 7, no. 1, pp. 71-78, 2024; article no.aji.116487 73 the effect of the sample matrix, known as the matrix effect, can also be a source of interference. conditions such as hemolysis, lipemia, icterus, and others can alter the properties of the sample, potentially affecting the assay outcome. lastly, the "hook effect" is an interference type that occurs when there is an excess of antigen relative to antibodies in the sample. this excess can saturate the binding sites of the antibodies, preventing accurate measurement. understanding these various types of interferences is crucial for assay development, optimization, and interpretation, as they can impact the reliability of immunological assays in clinical diagnostics and research. researchers and healthcare professionals must carefully consider and address these interferences to ensure accurate and meaningful results. 2.1 cross-reactivity the phenomenon of interference occurs when an antibody fails to accurately distinguish its intended target antigen, resulting in a lack of specificity. this inherent limitation in antibody specificity can have notable implications, especially in competitive assays, where only a single type of antibody is employed. in such cases, interference tends to be more frequent. this interference often takes the form of what is referred to as a cross-reactant, which competes with the marked antigen (ag) for antibody binding sites [5,6]. as a consequence, interference in competitive assays typically leads to a positive interference effect, causing a distortion in the assay results [1]. in non-competitive assays, such as sandwich assays, interference can occur due to the neutralization of one of the antibodies, whether it's the capture antibody or the labeled antibody. however, this type of interference is less common in non-competitive assays [7]. this is because two different epitopes are generally recognized by excess monoclonal antibodies, providing a higher degree of specificity and reducing the likelihood of interference. consider, for instance, the context of steroid assays where these interferences can originate from various sources. physiologically, conditions like pregnancy can introduce interference, altering the accuracy of steroid measurements. pathological factors, such as renal insufficiency, can also contribute to interference, posing challenges in diagnostic settings. additionally, therapeutic interventions like corticosteroid or estrogen therapy can introduce exogenous elements that interact with the assay components, further complicating the interpretation of results. diagnostic considerations should also be taken into account when addressing interferences, as they can vary widely in origin and impact. in conclusion, the lack of specificity in antibodyantigen interactions is a key factor contributing to interference in immunoassays, whether they are competitive or non-competitive. understanding the origins and implications of these interferences is crucial for researchers and healthcare professionals alike. it underscores the importance of rigorous assay design, optimization, and the need to consider potential interfering factors, whether they are of physiological, pathological, therapeutic, or diagnostic origin, in order to ensure the accuracy and reliability of immunological assay results. 2.2 antibody-related interferences in assay 2.2.1 anti analytes antibodies antibodies against analytes, referred to as antianalyte antibodies, can be found in the serum of patients with autoimmune disorders, such as anti-thyroglobulin antibodies in thyroid disorders or anti-insulin antibodies in diabetes. these autoantibodies arise as a result of the immune system mistakenly targeting the body's own molecules, including hormones or proteins, and can interfere with diagnostic assays designed to measure these molecules accurately [8,9]. furthermore, in some cases, anti-analyte antibodies can develop in response to specific treatments, such as the presence of anti-insulin antibodies in patients receiving insulin therapy. additionally, there are instances where antibodies against certain analytes are present without a known etiology, with one common example being macro-prolactin, a complex formed between antibodies and prolactin. depending on the specific assay used, these interferences caused by anti-analyte antibodies can lead to results that are either overestimated or underestimated. this underscores the importance of recognizing and addressing such interferences in clinical diagnostics, as they can impact the accuracy of test results and, consequently, patient care and treatment decisions. anouar et al.; asian j. immunol., vol. 7, no. 1, pp. 71-78, 2024; article no.aji.116487 74 2.2.2 heterophilic antibodies the serum of certain individuals may contain heterophilic antibodies, which are antibodies directed against animal immunoglobulins (ig) known as anti-idiotypes or anti-isotypes (igg or igm). these antibodies can develop as a result of various factors, including contact with animals, accidental exposure, medical treatments, or certain diseases [10]. one example of heterophilic antibodies is rheumatoid factor (rf), an autoantibody that targets the fc portion of igg antibodies. rf is commonly found in patients with rheumatoid arthritis and in approximately 5% of healthy individuals. its presence in serum can lead to falsely elevated test results [11-13]. heterophilic antibodies like rf can introduce significant challenges in diagnostic testing, potentially leading to misinterpretation of results and clinical decisions. therefore, their detection and appropriate management are crucial for accurate laboratory analyses and reliable healthcare outcomes. 2.2.3 anti-reagent antibodies interference caused by antibodies targeting specific reagents used in assays has also been documented. these antibodies, known as antireagent antibodies, can pose challenges in immunoassays. one example is the presence of anti-avidin or anti-streptavidin antibodies, particularly relevant when the assay methodology relies on the interaction between biotin and avidin or streptavidin [14]. in such cases, these antibodies can disrupt the binding between biotin and the respective protein, potentially leading to inaccurate test results. detecting and addressing anti-reagent antibodies is critical in the field of diagnostic testing, as they have the potential to introduce variability and errors into the assay outcomes. developing strategies to mitigate the impact of such interference is essential to ensure the reliability and precision of immunoassays, which play a pivotal role in clinical diagnostics and biomedical research. 2.3 interferences caused by binding proteins some analytes exist in the serum in both free (active) and bound forms, with the latter typically binding to transport proteins. this phenomenon is particularly relevant for molecules like steroid hormones, thyroid hormones, and vitamin d. the transport proteins involved often include albumin, pre-albumin, and specific hormonebinding proteins. the combination of the free and protein-bound forms constitutes the total form of the analyte. in many cases, the measurement of the free (active) fractions, such as free t3 and free t4, is crucial for obtaining accurate results. maintaining consistent in vitro (laboratory) conditions with those found in vivo (within the organism) is essential for precision. it's also important to consider potential sources of interference, such as free fatty acids, which can displace t4 from its binding protein [15-16]. to measure these analytes accurately, indirect methods can be employed to isolate the free form through processes like equilibrium dialysis or ultrafiltration. these methods are known for their precision and are not affected by anomalies in transport proteins, whether quantitative or qualitative. they are also unaffected by the presence of autoantibodies in the serum or by abnormal forms of albumin with increased affinity for t4, such as familial dysalbuminemic hyperthyroxinemia. when analyzing total forms, it is necessary to completely displace the bound form and prevent the tracer from binding to the binding proteins. this can be achieved through solvent extraction, denaturation of the transport protein, or the addition of a competitor that displaces the analyte without being recognized by the assay's antibodies [17]. these considerations are critical for obtaining accurate and reliable measurements of analytes present in both free and bound forms in serum. 2.4 matrix effect the majority of immunoassays are conducted on serum, and these tests are typically minimally impacted by hemolysis, lipemia, or icterus. biochemical interferences significant hemolysis, characterized by the release of proteases, can have pronounced effects on analytes sensitive to proteolysis, such as insulin, acth, pth, glucagon, or calcitonin, leading to an underestimation of results [18]. hemolysis, the breakdown of red blood cells and release of their anouar et al.; asian j. immunol., vol. 7, no. 1, pp. 71-78, 2024; article no.aji.116487 75 contents into the serum, can disrupt the integrity of these analytes, thus highlighting the importance of maintaining sample quality for accurate immunoassay results. 2.4.1 immunological interferences excessive lipid presence can interfere with and alter the affinity of certain antibodies in assays that rely on antigen-antibody interactions, consequently leading to inaccurate results. one such example is the measurement of free thyroxine. when lipids are in excess in a blood sample, they can disrupt the reaction between antibodies and antigens, especially in immunoassays. in the measurement of free thyroxine, for instance, an excessive amount of lipids can alter the binding between the specific antibody and thyroxine, resulting in erroneous results. this immunological interference underscores the importance of carefully preparing serum samples to ensure the reliability of assays and to avoid biases stemming from external factors such as lipemia. therefore, it is crucial to follow proper sample collection and preparation procedures to minimize these interferences and obtain accurate laboratory data. 2.4.2 optical interferences many assays rely on a colorimetric principle: measuring a specific color at a defined wavelength corresponding to the parameter being assayed. any alteration in the color of plasma or serum, such as that caused by hemolysis, lipemia, or icterus, can potentially interfere with these measurements. colorimetry, a widely used analytical technique, is based on the absorption of light by a particular substance, resulting in a distinct color. in clinical diagnostics, colorimetric assays are employed to quantify a wide range of analytes, from glucose and cholesterol to enzymes and hormones. the accuracy of these assays depends on the precise measurement of the color produced, typically at a specific wavelength, which is then correlated with the concentration of the target substance. however, the presence of optical interferences, such as hemolysis (the breakdown of red blood cells), lipemia (excess fat in the blood), or icterus (jaundice), can significantly affect the color of the serum or plasma. this alteration can lead to inaccurate colorimetric measurements, thereby compromising the reliability of the assay results. therefore, it is crucial to carefully assess and address potential optical interferences when conducting colorimetric assays, particularly in clinical laboratories where precise and dependable results are essential for patient care and medical decision-making. 2.4.2 other factors the interaction between an antigen and an antibody can be influenced by various other factors beyond those previously discussed. these additional factors include ph, protein concentration, and ionic strength of the sample. ph: the ph level of the sample can significantly impact the binding affinity between antigens and antibodies. different antibodies may have optimal binding capabilities within specific ph ranges, and deviations from these ranges can alter the binding kinetics. researchers and assay developers must carefully consider and control the ph conditions to ensure accurate and reproducible results. protein concentration: the concentration of proteins in a sample, particularly those unrelated to the target antigen, can affect the availability of binding sites and, consequently, the assay's sensitivity and specificity. high concentrations of non-specific proteins can lead to increased background noise and reduced assay precision. proper sample preparation techniques, including protein removal or dilution, are essential to mitigate these interferences. ionic strength: the ionic strength, determined by the concentration of ions in the sample, can influence the electrostatic interactions between antigens and antibodies. changes in ionic strength can affect the stability of antibodyantigen complexes. researchers often use buffer solutions to control and maintain the ionic strength within an optimal range for the assay. considering these factors is vital when designing, optimizing, and performing immunoassays to ensure reliable and meaningful results. by carefully managing ph, protein concentration, and ionic strength, researchers can minimize potential interferences and enhance the accuracy and reproducibility of immunological measurements." anouar et al.; asian j. immunol., vol. 7, no. 1, pp. 71-78, 2024; article no.aji.116487 76 2.5 the hook effect the 'hook effect,' a phenomenon observed in certain immunoassays, occurs when there is an excessive amount of the target antigen present in the sample [1]. this excess antigen saturates the binding sites of both the capture and detection antibodies, preventing the formation of a stable three-dimensional network in the assay. in typical immunoassays, the concentration of antigens is expected to fall within a linear range, allowing for a proportional response in the test signal. however, when the antigen concentration exceeds this linear range, the 'hook effect' comes into play. in this situation, the signal produced by the assay plateaus or decreases as the excess antigen interferes with the binding of antibodies, particularly in sandwich-type assays. this interference can lead to a significant underestimation of results because the assay no longer accurately reflects the actual concentration of the analyte in the sample. researchers and clinical laboratory professionals must be aware of the 'hook effect' and take measures to address it when designing and performing immunoassays. strategies for mitigating this effect may include sample dilution or modifying the assay procedure to accommodate high antigen concentrations. properly managing the 'hook effect' is crucial for obtaining accurate and reliable results, particularly in clinical diagnostics and research applications. 3. implication of interferences medical consequences for the patient: an erroneously abnormal immunoassay result, whether it is falsely elevated or lowered, may suggest a pathological condition and can have regrettable consequences for the patient. similarly, a falsely normal result can be detrimental to the proper management of a medical condition [19]. unjustified therapeutic decisions: initiating, adjusting, or discontinuing drug therapy; surgical procedures; initiating or excluding a medication protocol (such as renal transplantation, chemotherapy, etc.) can all be influenced by these erroneous results. consequences in clinical research: as insidious as they may be, interferences in immunoassays often eventually come to light. in some cases, they have only been recognized after the results they influenced were already published." interferences in immunoassays can have farreaching implications, affecting both individual patient care and the reliability of scientific research. falsely abnormal results can lead to unnecessary medical interventions or the omission of necessary treatments, potentially impacting patient health. in clinical research, the discovery of interferences, although often subtle, is essential to maintain the integrity and validity of research findings. researchers must remain vigilant in identifying and addressing these interferences to ensure the accuracy of both diagnostic tests and scientific studies [20-21]. 4. interference detection and prevention biologists play a critical role in ensuring the accuracy of laboratory test results, particularly when there is a discrepancy between expected clinical outcomes and laboratory findings. in such cases, several strategies can be employed to detect and mitigate potential interferences [22]. one approach is to repeat tests using different sample dilutions. if the results vary depending on the dilution level, it may suggest the presence of interference. additionally, employing different assay methods can be a valuable tool. if different results are obtained using various methods for the same sample, this can signal potential interference [23-25]. maintaining effective communication between biologists and clinicians is equally essential. clinicians must provide relevant clinical information to aid in the interpretation of results. biologists, in turn, have a responsibility to educate clinicians about the limitations of immunoassays and the risks associated with obtaining false results. collaboration between these two professional groups is crucial, especially when reported results do not align with the clinical context. furthermore, manufacturers have a role to play in ensuring the quality of reagents used in laboratory tests. rushing new immunoassays to market without thorough evaluation of their sensitivity to heterophilic antibodies on a sufficient sample size can lead to issues. continual improvement and diligence in assessing and addressing potential anouar et al.; asian j. immunol., vol. 7, no. 1, pp. 71-78, 2024; article no.aji.116487 77 interferences in assay development are essential [26]. 5. conclusion in the pursuit of accurate and reliable laboratory results, the vigilance of biologists stands as a crucial safeguard. when discrepancies arise between expected clinical outcomes and laboratory findings, biologists employ various strategies to detect and prevent potential interferences. the use of different sample dilutions and assay methods aids in uncovering anomalies, ensuring that results truly reflect the underlying biological reality. equally vital is the seamless communication between biologists and clinicians. clinicians' provision of pertinent clinical information facilitates a more precise interpretation of results. meanwhile, biologists must educate clinicians about the limitations of immunoassays and the risks associated with obtaining false results. this collaborative effort ensures that laboratory findings align harmoniously with the clinical context, thereby enhancing patient care. manufacturers, too, bear responsibility in this quest for accuracy. the rushed introduction of new immunoassays without rigorous sensitivity testing can introduce unwanted complications. to maintain the integrity of laboratory tests, manufacturers must continuously strive for improvement and diligently assess potential interferences. in conclusion, the triad of biologists' vigilance, effective communication among healthcare professionals, and the commitment of manufacturers is paramount to ensuring the accurate and reliable interpretation of laboratory results. the collective effort of these stakeholders not only safeguards patient wellbeing but also upholds the integrity of scientific research, marking a resounding commitment to the pursuit of knowledge and healthcare excellence. consent it is not applicable. ethical approval it is not applicable. competing interests authors have declared that no competing interests exist. references 1. sapin r. interférences dans les immunodosages : mécanismes et conséquences en endocrinologie. annales d’endocrinologie. 2008;69(5):415‑25. 2. ghazal k, brabant s, prie d, piketty ml. hormone immunoassay interference: a 2021 update. ann lab med. 1 janv 2022;42(1):3‑23. 3. baudin b, pilon a. interférences et pièges en immuno-analyse. revue francophone des laboratoires. mars 2019;2019(510): 60‑6. 4. caruso b, bovo c, guidi gc. causes of preanalytical interferences on laboratory immunoassays a critical review. ejifcc. mars 2020;31(1):70‑84. 5. boscato lm, egan gm, stuart mc. specificity of two-site immunoassays. journal of immunological methods. févr 1989;117(2):221‑9. 6. cobo a, martín-suarez a, calvo mv, domínguez-gil a, de gatta mmf. clinical repercussions of analytical interferences due to aldosterone antagonists in digoxin immunoassays: an assessment. therapeutic drug monitoring. avr. 2010;32 (2):169‑76. 7. hainque b, baudin b, lefebvre p. appareils et méthodes en biochimie et biologie moléculaire. éd. revue et augmentée. paris: flammarion médecinesciences. 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clin chem. oct. 1998;44(10):2178‑82. _________________________________________________________________________________ © copyright (2024): author(s). the licensee is the journal publisher. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: https://www.sdiarticle5.com/review-history/116487 _____________________________________________________________________________________________________ *corresponding author: e-mail: ikeokwu.anderson@gmail.com; asian journal of immunology 4(1): 59-76, 2021; article no.aji.66498 trends and socio-demographic distribution of immunological diseases in saint vincent and the grenadines adedeji okikiade1, ikeokwu anderson1*, twanna browne-caesar1, kevin brown1, rebecca lawrence1, daniel e. osieme1 and o. funmilayo janet 1 1 all saints university college of medicine, saint vincent and the grenadines. authors’ contributions this work was carried out in collaboration among all authors. authors ao and ia designed the study, performed the statistical analysis, wrote the protocol and wrote the first draft of the manuscript. ao and ia managed the analyses of the study. all authors managed the literature searches. all authors read and approved the final manuscript. article information editor(s): (1) dr. tania mara pinto dabés guimarães, federal university of minas gerais, brazil. (2) prof. darko nozic, university of belgrade, serbia. (3) prof. cynthia aracely alvizo báez, autonomous university of nuevo leon, mexico. reviewers: (1) irina a. pashnina, regional clinical children’s hospital, russia. (2) leonid p. churilov, saint petersburg state university, russia. complete peer review history: http://www.sdiarticle4.com/review-history/66498 received 14 april 2021 accepted 19 june 2021 published 24 june 2021 abstract background: more than 100 human diseases are due at least in part to an inappropriate immune system response that results in damage to an individual’s organs, tissues, or cells. immunological diseases can affect any part of the body, and have myriad clinical manifestations that can be difficult to diagnose. at the same time, immunological diseases share many features related to their onset and progression. in addition, overlapping genetic traits enhance susceptibility to many of the diseases, so that a patient may suffer from more than one immunological disorder, or multiple immunological diseases may occur in the same family. aim: the study aims is to explore the trends and socio-demographic distribution of immunological diseases in saint vincent and the grenadines from 2014-2018 in milton cato memorial hospital saint vincent and the grenadines, including temporal trends and variations in age and sex from original research article okikiade et al.; aji, 4(1): 59-76, 2021; article no.aji.66498 60 2014 to 2018 by using routinely collected administrative health data/patient records. methods: from 2014 to 2018, individuals with immunological diseases were identified from the hospital records of milton cato memorial hospital, which records information on all patient coming in for healthcare services. a structured data extraction tool was employed to extract the data from the hospital record using the open data kit (odk). data was analysed using statistical package for social sciences (spss) version 23 and r studio statistical software for analysis. the chi-square test was used to test for association. all statistical tests were two-tailed and level of confidence was set at 95%, and p values < 0.05 was considered to be statistically significant. results: the mean age of patient with sle was 35.65 ± 21.16 yrs. old and the median age was 34 years old, almost two-third 218(62.6%) were females. yearly, women showed a significantly higher incidence of immunological disease than men except in 2017 where the incidence for males were slightly higher than that of the females, there was an annual decrease in the incidence from 2014 to 2018, with a peak incidence in 2016 (0.94/1000 person-years). the lowest incidence was noted in 2018 (0.17/1000 person-years). among sex, there was an annual decrease in the incidence from 2014 to 2018, with a peak incidence in 2014 for male (0.71/1000 person-years) and in 2016 for females (1.34/1000 person-years). the lowest incidence was noted in 2018 (0.14/1000 personyears) and (0.20/1000 person-years) for both male and female respectively. conclusions: the study showed that the incidence of immunological disease, type 1 diabetes mellitus myopathy/myositis and sle in saint vincent have decreased in the last decade, whereas the mortality rates of both sle and type 1 diabetes mellitus have increased. this finding of increased mortality of sle and t1d suggests that this disease is no longer rare and will have implications for future healthcare planning. age and sex were found to be risk factors for sle. our data confirmed the known predilection of sle in women. the peak age of diagnosis is middle age, contrary to the generally held belief that lupus mainly targets young people. keywords: immunological disease; incidence; saint vincent and the grenadines. 1. introduction more than 100 human diseases are due at least in part to an inappropriate immune system response that results in damage to an individual’s organs, tissues, or cells. immunological diseases are a spectrum of diseases affecting multiple organs and tissues [1]. immunological disease could be classified as either autoinflammatory or autoimmunity. autoinflammatory is defined as self-regulated inflammation which is when local mediators at site of cell injury leads to activation of innate immunity cell, and this includes; macrophages and neutrophils, in which its final outcome is to target tissue damage [1]. autoimmunity is selfregulated inflammation, where there is an anomaly in dendritic cell, b and t cell, response responses in primary and secondary lymphoid organs lead to breaking of tolerance, with development of immune reactivity towards native antigens. the adaptive immune response plays the predominant role in the eventual clinical expression of disease. organ-specific autoantibodies may predate clinical disease expression by years and manifest before target organ damage is discernible [1]. immunological diseases can affect any part of the body, and have myriad clinical manifestations that can be difficult to diagnose. at the same time, immunological diseases share many features related to their onset and progression. in addition, overlapping genetic traits enhance susceptibility to many of the diseases, so that a patient may suffer from more than one immunological disorder, or multiple immunological diseases may occur in the same family [2]. treatments are available for many immunological diseases, cures have yet to be discovered. for these and other reasons, the immunological diseases are best recognized as a family of related disorders that must be studied collectively as well as individually. while many of these diseases are rare, collectively they affect 14.7 to 23.5 million people in a country like the usa and for reasons unknown their prevalence is rising [2]. since cures are not yet available for most immunological diseases, patients face a lifetime of illness and treatment. this therefore affects qaly and daly, qalys (quality-adjusted life year) and dalys (disability-adjusted life year) are common terms used to evaluate and compare health interventions using costeffectiveness analysis to measure the impact on both the length and the quality of life. qalys are okikiade et al.; aji, 4(1): 59-76, 2021; article no.aji.66498 61 a measure of years lived in perfect health gained whereas dalys are a measure of years in perfect health lost [3]. they often endure debilitating symptoms, loss of organ function, reduced productivity at work, high medical expenses. most of these diseases disproportionately affect both sexes, and are among the leading causes of death for young and middle-aged individuals on which they impose a heavy burden on patients’ families and on society [3]. there are gaps in evident literature for the incidence rates of immunological in the caribbean and saint vincent and the grenadines in specific. hence, this gap gave a recognition that more needs to be done to highlight evidence needed to close the gaps in our knowledge and achieve our overall goal of reducing the rising toll of immunological disease. for example, we need to gain a better understanding of the distribution of these diseases through epidemiologic studies, and of the environmental triggers that contribute to their onset. this research would give more insight about the genetic and environmental factors contributing to these diseases, and also set a platform to develop effective prevention strategies that arrest the immunological process before it can irreversibly damage the body. this research sets forth an ambitious and comprehensive research agenda aimed at generating more accurate epidemiologic profiles of immunological diseases; developing a greater understanding of the fundamental biologic principles underlying disease onset and progression; devising improved diagnostic tools; creating more effective interventions; and producing public and professional education and training programs. the study aims are to explore the trends and socio-demographic distribution of immunological diseases in saint vincent and the grenadines from 2014-2018. 2. research methodology 2.1 study area saint vincent and the grenadines (svg) is an upper-middle-income multi-island state in the eastern caribbean, located in the windward island chain of the lesser antilles. it consists of 32 islands, inlets, and cays, but only 7 of these beyond the main island of saint vincent are inhabited (bequia, canouan, mayreau, union, mustique, palm island, and petit saint vincent) [4]. the main island of saint vincent is the largest island in size at 340 square kilometers and in population with over 90 percent of the population. the islands are connected by sea ferries and air charters through four grenadine airports. the country is divided into six administrative units, or parishes: charlotte, grenadines, saint andrew, saint david, saint george, and saint patrick. five of these parishes are located on the island of saint vincent. kingstown, located in the saint george parish on saint vincent island, is the capital of the country and largest urban centre [5]. fig. 1. map and parishes of saint vincent and the grenadines okikiade et al.; aji, 4(1): 59-76, 2021; article no.aji.66498 62 in 2020, svg had an estimated population of 110172, the male population (56,052) outnumbered the female (54,120). the population is young, with almost 25 per cent under the age of 15 and 41.7 per cent under the age of 35. although this under-35 age group has decreased since the 2001 census by 6.1 per cent, it remains the largest proportion of the total population. the 2020 census determined the population aged under 5 years to be 8,723. a little over 9 per cent of the population is over the age of 65.5. according to the saint vincent 2001 housing and population census, 24.2 percent of the population lived in and around the capital, kingstown, in 2011 [6]. since then, the urban population has increased by 2 percentage points [7]. saint vincent, like the rest of the countries of the organization of eastern caribbean states (oecs), is experiencing an increase in elderly population and a decline in the fertility rate. this shift is largely the result of long-term successes in increasing access to care and treatment for infectious diseases [8]. the aging population contributes to the increased burden of chronic diseases. life expectancy for vincentians averages 72 years of age overall, 74 for females and 70 for males. chronic noncommunicable diseases (ncds) account for 70 percent of visits to outpatient services and are among the top five causes of death [9]. in 2004 the top five cause of death, in rank order, were diabetes, malignant neoplasms, cerebrovascular disease, heart disease, and hypertension [8]. as with many of its neighbouring countries, primary care service coverage indicators are extremely strong, with universal coverage of vaccines for key childhood illnesses and skilled attendance at delivery. the country is experiencing epidemiological transitions, as seen in the increasing burden of non-communicable diseases (ncds), which accounts for the top five causes of death, and in the increasing average age of the population [5]. the estimated prevalence of hiv in saint vincent is 1%, but stigma against individuals with hiv and aids continues to persist across the islands [5]. health care service delivery in saint vincent is largely provided by the public sector, but the private sector has grown in recent years to complement the limited specialty services and alleviate some of the burden on the public sector [5]. the private commercial sector is not well documented but is known to be concentrated in kingstown. data on the division of health services between the public and private sectors are not available. specialized health services are also concentrated in kingstown. ngos provide limited care, mostly through service delivery [5]. at the primary care level, the public sector is divided into nine health districts with 39 health clinics spread throughout the country. on average, each health clinic is equipped to cater to a population of 2,900 with no patient required to travel more than three miles to access care. at the secondary level, milton cato memorial hospital (mcmh) which is a 215-bed hospital, is the country’s only governmental acute care referral hospital providing specialist care. the private sector is active at the primary care level with private providers offering generalist and/or obstetric services [4]. tertiary care is limited on the island in both sectors. the private sector offers more long-term care facilities for the elderly with five facilities, while the one public sector facility primarily serves the impoverished populations. the private sector also offers advanced diagnostics, which are limited in the public sector to the lab at mcmh [5]. like many other caribbean countries, many citizens of saint vincent travel abroad for tertiary care. though the majority of health service providers are in the public sector, the private sector also plays a prominent and growing role; physicians in saint vincent commonly practice in both the public and private sectors [5]. financing for the health sector is provided through the ministry of health’s (mohe) portion of the consolidated fund, the national insurance service (nis), and private expenditures. available data on private expenditures are limited. public health services are primarily covered most through the mohe budget. primary care services are free of charge and all other services are highly subsidized. nis covers the costs of hospital services for its members [5]. 2.2 study design the study utilized a retrospective populationbased study which consists of secondary data derived from milton cato memorial hospital of patients with immunological disease from 20142018. 2.2.1 inclusion criteria data on immunological disease from 2014-2018 (for the purpose of this study, immunological consisted of both autoinflammatory disease okikiade et al.; aji, 4(1): 59-76, 2021; article no.aji.66498 63 cases and autoimmune disease cases. cases of autoinflammatory disease was identified as cases in which: (1) there is an idiopathic recurrent fever with systemic inflammation, (2) an inflammatory finding for which the trigger is not clear is present, (3) there is no association with a high titre of autoantibodies or self-responsive t cells and (4) an abnormality of innate immunity is observed [1]. cases of autoinflammatory disease was identified cases in which; [1] t an abnormality of adaptive immunity is observed, [2] there is an association with a high titre of autoantibodies or self-responsive t cells [1]. 2.2.2 exclusion criteria incomplete data on immunological disease from 2014-2018. 2.3 data collection procedure a structured data extraction tool was employed to extract the data from the hospital record with the aid of an android mobile device using the open data kit (odk). the data extraction tool was developed and modified with reference to existing tools used in similar studies. the data extraction tool comprises of information on sociodemographic (age, sex), year of diagnosis and diagnosis. 2.4 outcome measures and data analysis for annual incidence, the year-specific numerator included subjects with incident cases of immunological disease in the specific calendar year, and the denominator included the mid-year population from the population and demographic health survey (dhs) from 2013-2019 which are cross-sectional surveys conducted every year, compiled by the statistical office ministry of finance, economic planning, sustainable development and information technology of the government of saint vincent and the grenadines population this nationally representative survey involved a multi-stage sampling design up to the household level with enumeration areas distributed by region and type of residence using the most recent national census as its sampling frame. crude rates, sex and age-specific rates, standardized rates adjusted for sex and age using the 2014-2018 mid-year population, and their 95% confidence intervals (cis) were calculated. incident cases of immunological disease were defined as those without immunological disease, disease in a particular year (e.g., 2014) and the preceding year (e.g., 2012 to 2013) that met the algorithm in that year (e.g., 2014) and the following year (e.g., 2015). subgroup analyses were performed according to age and sex. data was edited, collated and entered into the 2019 microsoft excel data sheet, after which it was exported into the international business machine (ibm) statistical package for social sciences (spss) version 23.0 and r studio statistical software for analysis. the analysis involved the calculation of descriptive statistics (such as frequency distributions, percentages and means) and inferential statistics. continuous variables were expressed as means ± standard deviation while categorical variables were expressed as absolute frequencies. parametric analysis was used after tests for normality confirmed that continuous variables were normally distributed. the chi-square test was used to test for association. all statistical tests were two-tailed and level of confidence was set at 95%, and p values < 0.05 was considered to be statistically significant. test of normality was done to check for normal distribution of data using the shapiro-wilk test and kolmogorovsmirnov test with significance level set at 0.05. assumptions were set that if the sig. value of both test (p>0.05), the data is normal. if it is below 0.05, the data significantly deviates from a normal distribution and non-parametric testing was employed such as the median will be used instead of the mean to represent summative statistics due to the median is not affected by outliers or extreme values. the information provided by the probability value (p-value) does not provide an estimate for the magnitude of the effect of interest and the precision of this magnitude [10]. as a result of this, most of the inferential statistics reported in this report, did not only provide information on the p-value but also on the magnitude of the effect (effect size statistics) in the form of correlation coefficient, regression coefficient and also their confidence intervals (cis). confidence intervals (cis) were interpreted as the value that encompasses the population or ‘true’ value. this style of reporting both the effect sizes and their cis gave a clear understanding of the relationships between the variables [10]. 3. results table 1, shows the socio-demographics distribution of patient with immunological diseases in respect to age and sex. from 2014 to 2018, the total number of immunological cases seen in milton cato general hospital was okikiade et al.; aji, 4(1): 59-76, 2021; article no.aji.66498 64 347, with almost one-third 104(30%) occurring in the year 2016. among the cases of immunological diseases, the mean age was 35.65 ± 21.16 yrs old and the median age= 34 yrs old, almost two-third 218(62.6%) were females. table 2 shows that a greater percentage 125(36.0%) had diabetes mellitus type 1, 111(32.0%) had unspecified myopathies, 26(7.5%) had sle, only a few rare diseases were identified, this includes charge disease 1(0.3%), iridocyclitis 17(4.9%) and arpkd 2(0.6%) table 3 shows that among patients with diabetes mellitus, more than two-thirds 81(64.8%) were females. in patient with sle, only few 2(7.7%) were males with almost all 24(92.3%) females. females with myositis more than half 62(55.9%) compared to the males 49(44.1%) with myositis. table 4 shows that among patients with diabetes mellitus, all of the patients 125(100%) are within 21-40 years of age. fig. 2 shows the trend in incidence by year. yearly, women showed a significantly higher incidence of immunological disease than men except in 2017 where the incidence for males were slightly higher than that of the females, there was an annual decrease in the incidence from 2014 to 2018, with a peak incidence in 2016 (0.94/1000 person-years). the lowest incidence was noted in 2018 (0.17/1000 person-years). among sex, there was an annual decrease in the incidence from 2014 to 2018, with a peak incidence in 2014 for males (0.71/1000 personyears) and in 2016 for females (1.34/1000 person-years). the lowest incidence was noted in 2018 (0.14/1000 person-years) and (0.20/1000 person-years) for both male and female respectively. fig. 3 shows that the overall peak age of incidence was 31 to 35 years in 2014. in 2014, the peak age incidence among men was different > 70 years. however, the peak age of prevalence among women was similar to the overall incidence graph 31 to 35 years of age. table 1. socio-demographics characteristics of patients variable frequency (n=347) percentage (%) age ≤5 23 6.6 6-10 18 5.2 11-15 22 6.3 16-20 32 9.2 21-25 28 8.1 26-30 18 5.2 31-35 45 13.0 36-40 46 13.3 41-45 20 5.8 46-50 13 3.7 51-55 12 3.5 56-60 20 5.8 61-65 11 3.2 66-70 16 4.6 >70 23 6.6 sex male 129 37.2 female 218 62.8 year 2014 103 29.7 2015 62 17.9 2016 104 30.0 2017 59 17.0 2018 19 5.4 mean ± s.d (35.65 ± 21.16 yrs. old), 95% c.i for mean (33.41-37.88), median age= 34 yrs. old s.d =standard deviation, c. i= confidence interval okikiade et al.; aji, 4(1): 59-76, 2021; article no.aji.66498 65 table 2. type of immunological diseases/rare disease variable frequency (n=347) percentage (%) disease type adpkd 9 2.6 arpkd 2 0.6 autoimmune hemolytic anemia 2 0.6 calciphylaxis 1 0.3 charge syndrome 1 0.3 crohn disease 1 0.3 crohn disease/diabetes type 2 1 0.3 cryoglobulinemia 1 0.3 diabetes type 1 125 36.0 diabetes type 2/pseudogout 1 0.3 gout 11 3.2 gout /diabetes type 2 1 0.3 gout/iridocyclitis 1 0.3 iridocyclitis 16 4.6 iridocyclitis/diabetes type 2 2 0.6 juvenile arthritis 1 0.3 kawasaki 5 1.4 leukemia 2 0.6 myasthenia gravis 1 0.3 unspecified myopathy/myositis 111 32.0 unspecified myopathy/myositis/diabetes type 2 7 2.0 pernicious anemia 1 0.3 idopathic hypophysitis 2 0.6 rheumatoid arthritis 5 1.4 sarcoidosis 1 0.3 sjogren syndrome 3 0.9 systemic lupus erythematous (sle) 26 7.5 sle/diabetes mellitus/type 2 1 0.3 systemic sclerosis 3 0.9 ulcerative colitis 3 0.9 adpkd= autosomal dominant polycystic kidney disease, arpkd= autosomal recessive polycystic fig. 4 shows that the overall peak age of incidence was 31 to 35 years in 2015. in 2015, the peak age incidence among men was similar to the overall incidence graph 31 to 35 years of age. however, the peak age of prevalence among women was different (56-70) years of age to the overall incidence graph. fig. 5 shows that the overall peak age of incidence was 66-70 years in 2016. in 2016, the peak age incidence among men and women was similar to the overall incidence graph 66-70years of age. fig. 6 shows that the overall peak age of incidence was 36-40 to years in 2017. in 2017, the peak age incidence among men and women was similar to the overall incidence graph 36-40 years of age. fig. 7 shows that the overall peak age of incidence was 66-70 years in 2018. in 2014, the peak age incidence among men was different > 70 years. however, the peak age of prevalence among women was similar to the overall incidence graph 66-70 years of age. in the table 5, among sex, the females had higher proportions across the years (2014-2018) compared to that of the male we hereby fail to reject the null hypothesis which postulates that, there is no significant higher proportion of females to males who have a immunological disease from 2014 -2018 in saint vincent and the grenadines due to there was no statistically significant association observed (p>0.05). among the age groups, those within the age group of 31-40 years had significantly higher proportions across the years (2014-2018) compared to that of other age groups, this difference was statistically significant (p<0.05). okikiade et al.; aji, 4(1): 59-76, 2021; article no.aji.66498 66 table 3. distribution of immunological diseases by sex variable sex male female rare disease freq (%) freq (%) total (%) adpkd 5(55.6) 4(44.4) 9(100) arpkd 2(100) 0(0) 2(100) charge syndrome 0(0) 1(100) 1(100) calciphylaxis 1(100) 0(0) 1(100) pituitary adenoma 0(0) 1(100) 1(100) immunological disease autoimmune haemolytic anaemia 0(0) 2(100) 2(100) crohn disease 0(0) 1(100) 2(100) cryoglobulinemia 0(0) 1(100) 1(100) diabetes type 1 44(35.2) 81(64.8) 125(100) gout 7(63.6) 4(36.4) 11(100) iridocyclitis 7(43.8) 9(56.2) 16(100) juvenile arthritis 0(0) 1(100) 1(100) kawasaki 1(20) 4(80) 5(100) leukaemia 0(0) 2(100) 1(100) myasthenia gravis 0(0) 1(100) 1(100) unspecified myopathies/myositis 49(44.1) 62(55.9) 111(100) pernicious anaemia 0(0) 1(100) 1(100) rheumatoid arthritis 2(40) 3(60) 5(100) sarcoidosis 1(100) 0(0) 1(100) sjogren syndrome 0(0) 3(100) 3(100) systemic lupus erythematous (sle) 2(7.7) 24(92.3) 26(100) systemic sclerosis 1(33.3) 2(66.7) 3(100) ulcerative colitis 0(0) 3(100) 3(100) immunological disease with comorbidity (diabetes type 2) crohn disease & diabetes type 2 0(0) 1(100) 1(100) unspecified myopathies/myositis & diabetes type 2 5(60) 2(40) 7(100) pseudogout & diabetes type 2 0(0) 1(100) 1(100) gout & diabetes type 2 1(100) 0(0) 1(100) okikiade et al.; aji, 4(1): 59-76, 2021; article no.aji.66498 67 variable sex male female rare disease freq (%) freq (%) total (%) gout & iridocyclitis 1(100) 0(0) 1(100) iridocyclitis& diabetes type 2 0(0) 2(100) 2(100) sle & diabetes type 2 0(0) 1(100) 1(100) adpkd= autosomal dominant polycystic kidney disease, arpkd= autosomal recessive polycystic n.b: unspecified myopathy/myositis are collection of inflammatory and non-inflammatory muscle disorders with clinical and /or laboratory evidence table 4. distribution of immunological disease by age variable age ≤10 11-20 21-30 31-40 41-50 51-60 61-70 >70 rare disease freq (%) freq (%) freq (%) freq (%) freq (%) freq (%) freq (%) freq (%) total (%) adpkd 0(0) 0(0) 1(11.) 1(11.1) 2(22.) 1(11.1) 3(33.3) 1(11.1) 9(100) arpkd 1(50) 0(0) 0(0) 0(0) 0(0) 0(0) 0(0) 1(50) 2(100) charge syndrome 1(100) 0(0) 0(0) 0(0) 0(0) 0(0) 0(0) 0(0) 1(100) calciphylaxis 0(0) 0(0) 0(0) 0(0) 0(0) 0(0) 1(100) 0(0) 1(100) pituitary adenoma 0(0) 0(0) 0(0) 0(0) 1(50) 1(50) 0(0) 0(0) 2(100) immunological disease autoimmune haemolytic anaemia 0(0) 2(100) 0(0) 0(0) 0(0) 0(0) 0(0) 0(0) 2(100) crohn disease 0(0) 0(0) 0(0) 0(0) 1(0) 0(0) 0(0) 0(0) 1(100) cryoglobulinemia 0(0) 0(0) 0(0) 0(0) 0(0) 1(100) 0(0) 0(0) 1(100) diabetes type 1 0(0) 0(0) 0(0) 125(100) 0(0) 0(0) 0(0) 0(0) 125(100) gout 0(0) 2(18.2) 0(0) 0(0) 0(0) 3(273) 2(18.2) 1(9.1) 11(100) iridocyclitis 0(0) 1(6.2) 2(12.5) 4(25.0) 5(31.2) 3(18.8) 1(6.2) 0(0) 16(100) juvenile arthritis 1(100) 0(0) 0(0) 0(0) 0(0) 0(0) 0(0) 0(0) 1(100) kawasaki 5(100) 0(0) 0(0) 0(0) 0(0) 0(0) 0(0) 0(0) 5(100) leukaemia 1(50) 0(0) 0(0) 0(0) 0(0) 0(0) 0(0) 1(50) 2(100) myasthenia gravis 0(0) 0(0) 0(0) 0(0) 0(0) 0(0) 0(0) 1(100) 1(100) unspecified myopathies/myositis 13(11) 20(18.0) 11(9.9) 14(12.6) 11(9.9) 17(15.3) 11(12.6) 14(12.6) 111(100) pernicious anaemia 1(100) 0(0) 0(0) 0(0) 0(0) 0(0) 0(0) 0(0) 1(100) rheumatoid arthritis 0(0) 0(0) 0(0) 0(0) 1(20) 3(60) 0(0) 1(20) 5(100) okikiade et al.; aji, 4(1): 59-76, 2021; article no.aji.66498 68 variable age ≤10 11-20 21-30 31-40 41-50 51-60 61-70 >70 rare disease freq (%) freq (%) freq (%) freq (%) freq (%) freq (%) freq (%) freq (%) total (%) sarcoidosis 0(0) 0(0) 1(100) 0(0) 0(0) 0(0) 0(0) 0(0) 1(100) sjogren syndrome 0(0) 0(0) 1(33.3) 2(66.7) 0(0) 0(0) 0(0) 0(0) 3(100) systemic lupus erythematous (sle) 1(3.8) 7(26.9) 6(23.1) 8(30.8) 3(11.5) 1(3.8) 0(0) 0(0) 26(100) systemic sclerosis 0(0) 0(0) 0(0) 0(0) 2(66.7) 0(0) 0(0) 1(33.3) 3(100) ulcerative colitis 0(0) 0(0) 1(33.3) 0(0) 0(0) 0(0) 2(66.7) 0(0) 3(100) immunological disease with comorbidity (diabetes type 2) crohn disease & diabetes type 2 0(0) 0(0) 0(0) 0(0) 0(0) 0(0) 1(100) 0(0) 1(100) unspecified myopathies/myositis & diabetes type 2 1(14.3) 0(0) 0(0) 0(0) 0(0) 1(14.3) 3(42.8) 2(28.6) 7(100) pseudogout & diabetes type 2 0(0) 0(0) 0(0) 0(0) 0(0) 1(100) 0(0) 0(0) 1(100) gout & diabetes type 2 0(0) 0(0) 0(0) 0(0) 0(0) 0(0) 0(0) 1(100) 1(100) gout & iridocyclitis 0(0) 0(0) 0(0) 0(0) 0(0) 0(0) 1(100) 0(0) 1(100) iridocyclitis& diabetes type 2 0(0) 0(0) 0(0) 0(0) 0(0) 0(0) 2(100) 0(0) 2(100) sle & diabetes type 2 0(0) 0(0) 0(0) 0(0) 1(100) 0(0) 0(0) 0(0) 1(100) adpkd= autosomal dominant polycystic kidney disease, arpkd= autosomal recessive polycystic n.b: unspecified myopathy/myositis are collection of inflammatory and non-inflammatory muscle disorders with clinical and /or laboratory evidence okikiade et al.; aji, 4(1): 59-76, 2021; article no.aji.66498 69 table 5. trend analysis by age group and sex variable df χ2 (p-value) 95% confidence interval (pvalue) sex 2014 2015 2016 2017 2018 freq (%) freq (%) freq (%) freq (%) freq (%) total (%) lower limit upper limit male 40(38.8) 28(45.2) 32(30.8) 21(35.6) 8(42.1) 129(37.2) female 63(61.2) 34(54.8) 72(69.2) 38(64.4) 11(57.9) 218(62.8) 4 3.971 (0.423) f 0.371 0.475 total 103(100) 62(100) 104(100) 59(100) 19(100) 347(100 age ≤5 8(7.8) 5(8.1) 5(4.8) 4(6.8) 1(5.3) 23(6.6) 4 80.026 (0.000) f* 0.00 0.09 6-10 2(1.9) 5(8.1) 9(8.7) 0(0.0) 2(10.5) 18(5.2) 11-15 9(8.7) 3(4.8) 2(1.9) 5(8.5) 3(15.8) 22(6.3) 16-20 10(9.7) 4(6.5) 13(12.5) 4(6.8) 1(5.3) 32(9.2) 21-25 7(25.0) 4(6.5) 7(6.7) 9(15.3) 1(5.3) 28(8.1) 26-30 4(22.2) 2(3.2) 8(7.7) 4(6.8) 0(0) 18(5.2) 31-35 19(42.2) 11(17.7) 9(8.7) 4(6.8) 2(10.5) 45(13.0) 36-40 7(6.8) 6(9.7) 15(14.4) 18(30.5) 0(0.0) 46(13.3) 41-45 9(8.7) 5(38.5) 3(2.9) 2(3.4) 1(5.3) 20(5.8) 46-50 3(2.9) 2(3.2) 5(4.8) 2(3.4) 1(5.3) 13(3.7) 51-55 2(1.9) 4(33.3) 2(1.9) 2(3.4) 2(10.5) 12(3.5) 56-60 7(6.8) 5(25.0) 6(5.8) 1(1.7) 1(5.3) 20(5.8) 61-65 2(1.9) 3(27.3) 3(2.9) 2(3.4) 1(5.3) 11(3.2) 66-70 4(3.9) 0(0.0) 10(9.6) 1(1.7) 1(5.3) 16(4.6) >70 10(9.7) 3(4.8) 7(6.7) 1(1.7) 2(10.5) 23(6.6) total 103(100) 62(100) 104(100) 59(100) 19(100) 347(100 *statistically significant (p<0.05). f (fisher’s exact test) ci = confidence interval) χ2= chi-square test statistics, df= degree of freedom okikiade et al.; aji, 4(1): 59-76, 2021; article no.aji.66498 70 table 6. association between social demographic characteristics variable sex df χ2 (p-value) 95% confidence interval (p-value) sex male female freq (%) freq (%) total (%) lower limit upper limit age ≤5 4(3.1) 19(8.7) 23(6.6) 14 24.861 (0.017) f* 0.04 0.31 6-10 5(3.9) 13(6.0) 18(5.2) 11-15 2(1.6) 20(9.2) 22(6.3) 16-20 14(10.9) 18(8.3) 32(9.2) 21-25 13(10.1) 15(6.9) 28(8.1) 26-30 5(3.9) 13(6.0) 18(5.2) 31-35 15(11.6) 30(13.8) 45(13.0) 36-40 20(15.5) 26(11.9) 46(13.3) 41-45 6(4.7) 14(6.4) 20(5.8) 46-50 4(3.1) 9(4.1) 13(3.7) 51-55 4(3.1) 8(3.7) 12(3.5) 56-60 11(8.5) 9(4.1) 20(5.8) 61-65 6(4.7) 5(2.3) 11(3.2) 66-70 7(5.4) 9(4.1) 16(4.6) >70 13(10.1) 10(4.6) 23(6.6) total 129(100) 218(100) 347(100) *statistically significant (p<0.05). f (fisher’s exact test) ci = confidence interval) χ2= chi-square test statistics, df= degree of freedom okikiade et al.; aji, 4(1): 59-76, 2021; article no.aji.66498 71 fig. 2. incidence of immunological diseases from 2014 -2018 fig. 3. peak age of incidence of immunological diseases in 2014 in the table 6, a statistically significant association was observed between age and sex, those within the age group of 36-40 years had significantly higher proportions of both male and female compared to that of other age group, this difference was statistically significant (p<0.05). 4. discussion findings from the study showed that type i diabetes was the leading immunological disease in the country, which was followed by myopathies and myositis. in contrast to the study findings, a systematic review conducted reported that celiac disease increased the most and the highest increase in incidence, comparing old to new surveys is allocated to myasthenia gravis [11]. however, the study also indicated that between the countries, celiac disease, type 1 diabetes and myasthenia gravis frequencies increased the most in canada, israel and denmark, respectively. 2014 2015 2016 2017 2018 total population 0.93 0.56 0.94 0.53 0.17 male 0.71 0.50 0.57 0.67 0.14 female 1.17 0.63 1.34 0.39 0.20 0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 in c id en c e r a te ( p er 1 0 0 0 p er so n ) incidence of immunological diseases from 2014 -2018 <5 6_1 0 11_ 15 16_ 20 2125 26_ 30 31_ 35 36_ 40 4145 4650 5155 5660 6165 6670 >70 male 0.00 0.00 0.19 0.97 0.90 0.47 1.01 1.02 1.30 0.25 0.29 1.12 1.00 2.02 1.45 female 1.85 0.47 1.69 1.03 0.71 0.49 3.77 0.81 1.18 0.55 0.32 1.67 0.00 0.72 1.33 total population 0.92 0.23 0.91 1.00 0.81 0.48 2.40 0.92 1.25 0.40 0.30 1.38 0.52 1.40 1.39 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 in c id en c e r a te ( p er 1 0 0 0 p er so n s) peak age of incidence of immunological diseases in 2014 okikiade et al.; aji, 4(1): 59-76, 2021; article no.aji.66498 72 fig. 4. peak age of incidence of immunological diseases in 2015 fig. 5. peak age of incidence of immunological diseases in 2016 in this study we observed a decreasing trend in the incidence of immunological diseases, also observed was an annual decrease in the incidence from 2014 to 2018, with a peak incidence in 2016 (0.94/1000 person-years). the lowest incidence was noted in 2018 (0.17/1000 person-years). the decrease in trend reported in the present study might be explained due to the use of the hospital database could stem from the lack of accurate diagnosis which could lead to missed or undiagnosed cases. another explanation of the findings is that data analysed <5 6_1 0 11_ 15 16_ 20 2125 26_ 30 31_ 35 36_ 40 4145 4650 5155 5660 6165 6670 >70 total population 0.57 0.58 0.30 0.40 0.46 0.24 1.39 0.79 0.69 0.27 0.60 0.98 0.78 0.00 0.42 male 0.00 0.68 0.00 0.39 0.45 0.00 1.77 0.77 0.26 0.25 0.57 0.74 1.00 0.00 0.87 female 1.15 0.47 0.64 0.41 0.47 0.49 1.00 0.81 1.18 0.28 0.64 1.25 0.54 0.00 0.00 0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 in c id en c e r a te ( p er 1 0 0 0 p er so n s peak age of incidence of immunological diseases in 2015 <5 6_1 0 11_ 15 16_ 20 2125 26_ 30 31_ 35 36_ 40 4145 4650 5155 5660 6165 6670 >70 total population 0.57 1.05 0.20 1.30 0.80 0.95 1.13 1.97 0.41 0.66 0.30 1.18 0.78 3.48 0.97 male 0.68 0.23 0.00 0.97 0.23 0.70 0.76 1.02 0.00 0.25 0.00 1.49 0.00 2.69 0.87 female 0.46 1.90 0.42 1.64 1.41 1.22 1.51 2.96 0.89 1.11 0.64 0.84 1.62 4.33 1.06 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 in c id en c e r a te ( p er 1 0 0 0 p er so n s peak age of incidence of immunological diseases in 2016 okikiade et al.; aji, 4(1): 59-76, 2021; article no.aji.66498 73 in this study were obtained when subjects visited healthcare institutions. therefore, no information was available for immunological diseases patients who did not visit a healthcare institution, which could underestimate the immunological diseases burden. the incidence rate of immunological diseases reported was lower compared to the findings in a systematic review which reported a mean ± s.d of the net % increased /year incidence of autoimmune diseases worldwide were 19.1±43.1 [12]. fig. 6. peak age of incidence of immunological diseases in 2017 fig. 7. peak age of incidence of immunological diseases in 2018 <5 6_10 11_1 5 16_2 0 2125 26_3 0 31_3 5 36_4 0 4145 4650 5155 5660 6165 6670 >70 total population 0.46 0.00 0.51 0.40 1.03 0.48 0.50 2.36 0.28 0.26 0.30 0.20 0.52 0.35 0.14 male 0.23 0.00 0.19 0.39 1.13 0.00 0.25 2.30 0.00 0.00 0.00 0.37 0.50 0.00 0.00 female 0.69 0.00 0.85 0.41 0.94 0.98 0.75 2.42 0.59 0.55 0.64 0.00 0.54 0.72 0.27 0.00 0.50 1.00 1.50 2.00 2.50 3.00 in c id en c e r a te ( p er 1 0 0 0 p er so n s peak age of incidence of immunological diseases in 2017 <5 6_1 0 11_ 15 16_ 20 2125 26_ 30 31_ 35 36_ 40 4145 4650 5155 5660 6165 6670 >70 total population 0.11 0.23 0.30 0.10 0.11 0.00 0.25 0.00 0.14 0.13 0.30 0.20 0.26 0.35 0.28 male 0.00 0.23 0.00 0.00 0.23 0.00 0.00 0.00 0.00 0.25 0.29 0.37 0.50 0.00 0.58 female 0.23 0.24 0.63 0.21 0.00 0.00 0.50 0.00 0.29 0.00 0.32 0.00 0.00 0.72 0.00 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 in c id en c e r a te ( p er 1 0 0 0 p er so n s peak age of incidence of immunological diseases in 2018 okikiade et al.; aji, 4(1): 59-76, 2021; article no.aji.66498 74 this difference between the incidence rate of both studies can be overestimated or underestimated due to various population size of both countries and the difference in multiplier rate (1000 vs 100000) by both studies which impairs the basis of comparison. the population size in the caribbean was approximately 120,000 people as at the time of the study. 4.1 social demographic characteristics of individuals who have immuno logical disease the incidence of immunological disease peaked within the age group of 31-40 years, after which it declined slowly. the incidence of immunological disease among female was higher compared to that of males with a peak age occurring at 31-35 years. findings from the study was in conformity with that of a systematic review which reported most immunological diseases were more common in women and a disproportionate occurrence of these diseases among these women [12]. 5. conclusion sequel to the findings of this study, this study showed that the incidence of immunological disease, type 1 diabetes mellitus myopathy/myositis and sle in saint vincent have decreased in the last decade, whereas the mortality rates of both sle and type 1 diabetes mellitus have increased. this finding of increased mortality of sle and t1d suggests that this disease is no longer rare and will have implications for future healthcare planning. age and sex were found to be risk factors for sle. our data confirmed the known predilection of sle in women. the peak age of diagnosis is middle age, contrary to the generally held belief that lupus mainly targets young people. 6. recommendations disease registries should be expanded to a population-based multidisciplinary immunological diseases registry to enhance collection and analysis of data over time on causation, natural history, morbidity and mortality of immunological diseases. utilizing a multidisciplinary, integrated approach with collection of data on multiple diseases. support research on the feasibility and optimal design of the registry to allow collation of data at the state and national levels. provide epidemiology, statistical, clinical disease, and bioinformatics expertise, incorporate biomarker data in registries and provide infrastructure for long-term support of registries and epidemiology studies. provide long-term support for existing genetic repositories; establish genetic repositories for additional immunological disorders; ensure adequate representation of disease phenotypes and races. develop high throughput, standardized, specific, and sensitive laboratory assays for infectious and non-infectious environmental factors that can be used in large epidemiologic studies. identify new opportunities and continue support for training and career development for new and established basic science and clinical investigators in immunological disease research. include specialized training in epidemiology and bioinformatics. provide increased training opportunities for health care professionals by establishing collaborative training programs between professional and non-profit health organizations and clinical programs for research in immunological disease. develop and promote the use of a wide range of educational programs and continuing medical education materials in immunological disease for health care professionals, incorporating the latest research advances on autoimmunity and autoimmune diseases. establish a centralized, consolidated immunological disease information centre accessible to professionals and the public via the internet where there is provision of information about clinical trials to evaluate prevention and treatment regimens that will enable patients and their physicians to make informed choices. the present literature survey is not aiming to investigate etiologies or environmental factors affecting immunological induction or progression. it is expected that an improved knowledge of the worldwide distribution of immunological disorders will help to understand the role of different genetic factors and different environmental influences involved in auto-immunogenesis. support research on gene/environment interactions important in development and manifestation of immunological diseases. provide resources for production, storage, and distribution of materials and probes for genetic research to the research community centrally. support research to develop novel assays to identify prior exposures to environmental agents, including chemicals, toxins, and infectious agents. support basic and clinical research on okikiade et al.; aji, 4(1): 59-76, 2021; article no.aji.66498 75 mechanisms by which infectious agents or other environmental factors may trigger or modulate immunological diseases. support basic research on mechanisms and loss of self-tolerance, including mechanisms to control autoreactive cells. support basic research on tissue specificity, target organ recognition, and immune injury and pathogenesis among different immunological diseases. support core facilities for production and distribution of specialized reagents for research, including mhc-tetramers, antibodies, and microarrays. 7. strength and limitation of study despite our important findings, this study had a few strengths and limitations. the strengths to this study include; the data being population based, recall bias was not an issue to any misclassification errors on the side of providing conservative estimates, and it is the first of its kind to estimate the incidence of immunological diseases in saint vincent and the grenadines. some apparent limitations of using the patient records, which signifies our prevalence and incidence estimate were based on use of health services; stem from, implication from this signifies that data analysed in this study were obtained when subjects visited healthcare institutions. therefore, no information was available for patients with immunological diseases who did not visit a healthcare institution, which could underestimate the autoimmune burden. however, this may not have had a substantial impact on our findings, as the milton cato general hospital provides diverse healthcare delivery services that is accessible and affordable compared to other public and private healthcare providers. consent it is not applicable ethical approval ethical approval was gotten to access medical information of patients from the ministry of health and wellness and hospital administrator at milton cato memorial hospital in saint vincent and the grenadines. competing interests authors have declared that no competing interests exist. references 1. mc dermott mf, aksentijevich i, galon j, et al. germline mutations in the extracellular domains of the 55 kda tnf receptor, tnfr1, define a family of dominantly inherited autoinflammatory syndromes. cell. 1999;97:133–144. 2. progress in autoimmune diseases research. [cited 2020 jul 18]. available:https://www.niaid.nih.gov/sites/de fault/files/adccfinal.pdf. 3. gold mr, stevenson d, fryback dg. halys and qalys and dalys, oh my: similarities and differences in summary measures of population health. annual review of public health. 2002; 23:115–34. available:https://www.ncbi.nlm.nih.gov/pub med/11910057 4. world health organization (who). the top 10 causes of death. who.int. who; 2020. available:https://www.who.int/newsroom/fact-sheets/detail/the-top-10-causesof-death 5. rodriguez m, williamson t, vogus a, macgregor-skinner e, pena d-l, wilson a, et al. saint vincent and the grenadines health system and private sector assessment. 2012;(february):67–79. 6. statistical office, government of saint vincent and the grenadines. [cited 2021 feb 17]. available from: http://stats.gov.vc. 7. world bank. world development indicators. washington, dc: ibrd/world bank. accessed september 15, 2011. available:http://data.worldbank.org/. 8. ministry of health, wellness and the environment (mohe). 2007. strategic plan for health 2007–2012. kingstown, st. vincent. 9. gillespie jj, o. f. n. short-term technical assistance for feasibility study and drafting of financial proposal for the health sector improvement and reform programme in st. vincent and the grenadines. ecorys health consortium. rotterdam, netherlands; 2010. 10. nakagawa s, cuthill ic. effect size, confidence interval and statistical significance: a practical guide for biologists. biological reviews. 2007; 82(4):591–605. 11. lerner a, jeremias p, matthias t. the world incidence and prevalence of autoimmune diseases is increasing. okikiade et al.; aji, 4(1): 59-76, 2021; article no.aji.66498 76 international journal of celiac disease. 2016;3(4):151–5. available:http://pubs.sciepub.com/ijcd/3/4/ 8/ 12. jacobson dl, gange sj, rose nr, graham nmh. epidemiology and estimated population burden of selected autoimmune diseases in the united states. clinical immunology and immunopathology; 1997. [cited 2020 may 14];84(3):223–43. available:https://www.sciencedirect.com/sc ience/article/abs/pii/s0090122997944123? via%3dihub _________________________________________________________________________________ © 2021 okikiade et al.; this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: http://www.sdiarticle4.com/review-history/66498 http://creativecommons.org/licenses/by/4.0 _____________________________________________________________________________________________________ *corresponding author: e-mail: doyinsolajayeju90@gmail.com; asian journal of immunology 2(1): 11-17, 2019; article no.aji.52193 haematological assessments of cocos nucifera linn. water and oil on apparently healthy wistar albino rats d. o. akinjayeju1* and t. t. adebolu1 1 department of microbiology, the federal university of technology, akure, ondo state, nigeria. authors’ contributions this work was carried out in collaboration between both authors. both authors read and approved the final manuscript. article information editor(s): (1)dr. darko nozic, professor, higher medical school in belgrade, university of belgrade, serbia. reviewers: (1) karen cordovil, brazil. (2)camilo torres-serna, universidad santiago de cali, colombia. (3)debarshi kar mahapatra, dadasaheb balpande college of pharmacy, india. complete peer review history: http://www.sdiarticle4.com/review-history/52193 received 11 august 2019 accepted 21 october 2019 published 28 october 2019 abstract in this study, the effects of the consumption of cocos nucifera l. water and oil on the haematological parameters of apparently healthy wistar albino rats were investigated using standard methods. the albino rats (n= 100) were divided into 8 groups (a-h) of 12 rats each while the remaining four rats were put in another group to serve as control. the first four groups(a-d) were daily orogastrically administered different volumes of coconut oil, 0.5 ml, 1.0 ml, 1.5 ml, 2.0 ml respectively for 4 weeks while groups e-h were daily orogastrically administered different volumes of coconut water, 0.5 ml, 1.0 ml, 1.5 ml, 2.0 ml respectively for four weeks. the control rats (group l) however were not administered either coconut water or coconut oil. all the rats were allowed free access to rat chow and water. the administration of either of the coconut water or oil caused a significant increase (p≤0.05) in the pcv, wbc and the differential leukocytes counts within the four weeks of this study with coconut water having higher effect. it is conceivable that the consumption of either coconut water or coconut oil could exert both hematinic and immuno-stimulatory effects in the consumers to checkmate low pcv and also boost innate immunity. original research article akinjayeju and adebolu; aji, 2(1): 11-17, 2019; article no.aji.52193 12 keywords: coconut water, coconut oil; immuno-stimulatory potential; haematological parameters; differential leukocytic count; haematinic potential. 1. introduction coconut (cocos nucifera linn.) fruit, is eggshaped or elliptic. it consists of a fibrous outer layer called coconut husk (mesocarp), which covers a hard layer called shell (endocarp). inside the shell is a kernel (endosperm), which is considered the most important part of the fruit. coconut water is sterile, does not produce heat, does not destroy red blood cells and is readily accepted by the body. it makes the absorption of drugs easier by its electrolytic effect, urinary antiseptic and eliminates poisons in case of mineral poisoning [1]. small amounts of amino acids are also present in coconut water. the percentage of arginine, alanine, cystine and serine in the protein of tender coconut water are higher than those in cow’s milk. trace amount of amino acid such as aspartic acid, glutamic acid, histidine, leucine, lysine, proline, phenylalanine and tyrosine are also reported in tender coconut water [2]. coconut water is readily available, economically reasonable, and traditionally tolerable besides having various general and oral health benefits without any side effects with numerous amino acids needed by the human immune system to boost immunity. coconut oil is an edible oil extracted from the “meat” of matured coconuts [3]. coconut oil is a mild flavored, mostly colorless oil produced from mature coconuts. it is a clear liquid above 76°f (24°c) and a white or light brown solid at lower temperatures. it has several applications in the food industry. it is used in processed foods because it is relatively inexpensive and can provide crisp texture to foods. coconut oil is a safe and effective moisturizer, produces a highlather soap due to is solubility in hard water, and can be used in cosmetics, toothpaste, lotions, sunscreens, laundry detergents, as well as several other self-care and household items [4]. it has a high smoke point, the temperature where it starts to break down, which makes it shelf stable and ideal for foods that need a longer shelf-life. coconut oil is considered a saturated fat and contains 9 calories per gram. therefore it should be consumed only in small amounts because it can raise the ldl (bad) cholesterol alongside hdl (good) cholesterol level. one tablespoon of coconut oil contains 117 calories, 14 grams of fat, 12 grams of saturated fat and therefore must be taken in moderation [5]. in addition to its culinary uses, inclusion in cosmetics and soaps, this oil is also consumed in folklore medicine to treat various ailments. this study therefore is to assess the effects of consumption of coconut water and oil on the haematological parameters of wistar albino rats as a guide to the possible effects in humans when consumed for a considerable period of time for health promoting purposes. 2. materials and methods 2.1 coconut water fresh coconut water was collected from cleaned coconuts bought from the commercial fruit market in akure, nigeria. 2.2 coconut oil coconut oil (aquilla 100% virgin coconut oil with nafdac reg no. 08-1725l) used in this study was purchased from a pharmaceutical shop in akure, nigeria. coconut oil fat is a fat consisting of about 90% saturated fat. the oil contains predominantly triglycerides with 86.5% saturated fatty acids, 5.8% monounsaturated fatty acids and 1.8% polyunsaturated fatty acids. 2.3 experimental design wistar albino rats were used for this study because of their close genetic structural makeup to humans. a total of hundred wistar albino rats were used in this study and divided into were divided into8 groups (a-h) of 12 rats each whilethe remaining four rats were put in another group to serve as control. the first four groups (a-d) were daily orogastrically administered different volumes of coconut water, 0.5 ml, 1.0 ml, 1.5 ml, 2.0 ml respectively for 4 weeks while groups e-h were daily orogastrically administered different volumes of coconut water, 0.5 ml, 1.0 ml, 1.5 ml, 2.0 ml respectively for four weeks.the control rats (group l) however were not administered either coconut water or coconut oil. all the rats were allowed free access to rat chow and water. at intervals of 1 week, the blood of 3 rats randomly picked from each group was collected through venepuncture for the haematological assay. akinjayeju and adebolu; aji, 2(1): 11-17, 2019; article no.aji.52193 13 2.4 haematological assay the blood of apparently healthy wistar albino rats was collected weekly into edta bottles after which the packed cell volume (pcv), total white blood cell counts and differential leukocytes counts of the collected blood samples were evaluated according to nwangwa & chukwuemeka [6]. 2.5 statistical analysis data generated from the study were subjected to two-way analysis of variance (anova). treatment means were compared using duncan’s new multiple range test (dnmrt) at 5% level of significance using spss version 26. 3. results and discussion main characteristics of the study population group title number of rats treatment a – d coconut oil 48 0.5 ml, 1.0 ml, 1.5 ml, 2.0 ml + distilled water and feed e – h coconut water 48 0.5 ml, 1.0 ml, 1.5 ml, 2.0 ml + distilled water and feed i control 4 distilled water and feed. administration of coconut oil to apparently healthy albino rats caused a significant increase (p<0.05) in pcv from an initial value of 48.00±0.50% to 51.00±0.00% after 28days of administering the rats with the oil at a 0.5ml dose. in the same vein, administration of coconut water also caused a significant increase (p<0.05) in the pcv from the initial value of 48.00±0.50% to 56.50±0.50% after 28 days of administering the rats with the coconut water at a dose of 0.5 ml. however, the percentage increase in pcv was observed to be higher in the rats administered coconut water than those administered coconut oil at all doses and the higher the dose of the different component of coconut used, the higher the pcv of the rats except in a few cases (table 1). effects of coconut oil or coconut water on the total white blood cell counts of apparently healthy wistar albino rats showed the total white blood cell count of healthy rats fed with coconut oil increased from an average initial value of 11.00±0.50 to 11.00±0.50, 11.60±0.90, 12.60±0.60 and 13.50±0.50 after 7,14,21,28 days of feeding the rats with coconut oil at a dose of 0.5 ml. in the same vein, rats fed with coconut water which had an initial average value of 11.00±0.50, increased to 11.95±0.95, 11.15±0.25, 12.15±0.25 and 11.05±0.10 by day 7, 14, 21 and 28 of the administration of the water the rats at a dose of 0.5 ml. percentage increase in the total white blood cell counts was higher in rats administered coconut water than those administered coconut oil at all doses and the higher the dose of the different coconut product, the higher the wbc of the animals (table 2). the administration of coconut oil to the apparently healthy rats at a dose of 0.5ml had no significant effect on the initial neutrophil value of 55.50±0.50 to 56.50±0.50, 55.00±0.50, 55.00±0.33 and 53.00±0.20% after 7, 14, 21 and 28 days. however, the administration of the same proportion of coconut water had a significant increase in the neutrophil count of the rats from 55.50±0.50 to 57.00±0.40, 57.00±0.33, 58.50±0.50 and 56.00±0.40 after days 7, 14, 21 and 28 (table 3). the eosinophil count of the apparently healthy rats administered coconut oil had no significant increase from the intial value of 2.50±0.00 to 2.00±0.00, 2.00±0.00, 2.50±0.50 and 2.50±0.50 at a dose of 0.5 ml at days 7, 14, 21 and 28. at the same proportion of coconut water, the eosinophil count had a slight increase to 3.00±0.00 and 3.00±0.00 at days 21 and 28 respectively. an increase in the doses however, had slight effect on the eosinophil counts (table 4). effects of coconut oil or coconut water on the differential leukocytic counts of apparently healthy wistar albino rats showed the lymphocyte counts of healthy wistar rats fed with coconut oil had no significant effect from the initial value of 32.00±0.50 to 32.50±0.50, 30.00±0.00, 31.00±0.00 and 30.00±0.00 after 7, 14, 21 and 28 days of administration of the oil to the ratsat a dose of 0.5 ml. the administration of coconut water to the rats however, recorded a significant increase in the lymphocyte count from 32.00±0.50 to 34.50±0.50 at day 28 at same proportion (table 5). akinjayeju and adebolu; aji, 2(1): 11-17, 2019; article no.aji.52193 14 table 1.effects of administration of coconut oil and coconut water on pcv of apparently healthy wistar albino rats (%) sample days of assay doses of sample administered 0.0ml 0.5ml 1ml 1.5ml 2.0ml coconut oil day 0 48.50±0.50 a 48.50±0.50 a 48.50±0.50 b 48.50±0.50 b 48.50±0.50 a day 7 48.50±0.50 51.50±0.50 c 51.00±0.33 c 53.50±0.50 c 54.00±0.30 c day 14 48.50±0.50 49.50±0.50 b 49.00±0.00 b 62.50±0.50 e 56.50±0.50 e day 21 day 28 48.50±0.50 48.50±0.50 52.50±0.50 d 51.00±0.00 c 50.50±0.50 c 46.50±0.50 a 46.00±0.00 a 56.00±0.00 d 55.00±0.00 d 53.00±0.20 b coconut water day 0 48.50±0.50 a 48.50±0.50 a 48.50±0.50 a 48.50±0.50 a 48.50±0.50 a day 7 48.50±0.50 56.50±0.50 c 56.00±0.50 cd 58.00±0.00 d 58.50±0.50 d day 14 48.50±0.50 56.50±0.50 c 55.50±0.00 c 56.50±0.50 c 57.50±0.43 c day 21 day 28 48.50±0.50 48.50±0.50 54.50±0.50 b 56.50±0.50 c 54.00±0.50 b 56.50±0.50 d 55.50±0.50 b 57.50±0.33 d 56.50±0.50 b 58.50±0.33 d data represent the average for 3 replicates + s.e.m and those with different alphabet superscripts along columns for each treatment are statistically significant at p<0.05 table 2. effects of administration of coconut oil and coconut water on the wbc counts of apparently healthy wistar albino rats (%) sample days of assay doses of sample administered 0.0ml 0.5ml 1ml 1.5ml 2.0ml coconut oil day 0 11.00±0.00 a 11.00±0.00 a 11.00±0.00 ab 11.00±0.00 a 11.00±0.00 a day 7 11.00±0.00 11.00±0.50 a 10.70±0.70 a 14.50±1.50 c 16.55±0.50 c day 14 11.00±0.00 11.60±0.40 a 12.50±1.50 bc 12.00±1.00 ab 15.95±0.65 bc day 21 day 28 11.00±0.00 11.00±0.00 12.60±0.60 b 13.50±0.50 c 11.50±0.50 abc 12.65±0.65 c 12.35±0.95 ab 13.00±1.00 bc 13.70±0.70 b 16.05±0 . 95 c coconut water day 0 11.00±0.00 a 11.00±0.00 a 11.00±0.00 a 11.00±0.00 a 11.00±0.00 a day 7 11.00±0.00 11.95±0.95 c 11.20±0.20 a 11.05±0.15 a 15.60±0.40 b day 14 11.00±0.00 11.15±0.25 ab 11.00±1.00 a 14.00±1.00 c 19.15±0.25 d day 21 day 28 11.00±0.00 11.00±0.00 12.15±0.25 c 11.05±0.15 a 13.10±0.20 b 13.00±0.20 b 13.15±0.25 bc 12.80±0.70 b 17.50±0.40 c 17.00±0.20 c data represent the average for 3 replicates + s.e.m and those with different alphabet superscripts along columns for each treatment are statistically significant at p<0.05 table 3. effects of administration of coconut oil and coconut water on the neutrophil counts of apparently healthy wistar albino rats (%) sample days of assay doses of sample administered 0.0ml 0.5ml 1ml 1.5ml 2.0ml coconut oil day 0 55.50±0.50 a 55.50±0.50 b 55.50±0.50 b 55.50±0.50 a 55.50±0.50 a day 7 55.50±0.50 56.50±0.50 c 60.00±0.20 d 55.50±0.50 a 57.50±0.50 a day 14 55.50±0.50 55.00±0.50 b 56.50±0.50 c 55.50±0.50 a 56.00±0.50 a day 21 day 28 55.50±0.50 55.50±0.50 55.00±0.33 b 53.00±0.20 a 54.00±0.20 a 59.50±0.50 d 55.00±0.20 a 57.00±0.20 b 55.00±0.40 a 60.00±0.33 b coconut water day 0 55.50±0.50 a 55.50±0.50 a 55.50±0.50 ab 55.50±0.50 b 55.50±0.50 a day 7 55.50±0.50 57.00±0.40 b 57.00±0.33 c 56.50±0.25 c 60.00±0.33 c day 14 55.50±0.50 57.00±0.33 b 55.00±0.33 a 55.50±0.50 b 58.00±0.33 b day 21 day 28 55.50±0.50 55.50±0.50 58.50±0.50 c 56.00±0.40 a 56.00±0.40 b 55.00±0.40 a 59.00±0.33 d 54.00±0.33 a 59.50±0.50 c 61.50±0.50 d data represent the average for 3 replicates + s.e.m and those with different alphabet superscripts along columns for each treatment are statistically significant at p<0.05 3.1 discussion in this study, the effect of coconut oil and coconut water on the haematological profile of apparently healthy albino rats was investigated. the administration of either coconut oil or coconut water caused a significant increase p(<0.05) on the haematological parameters such akinjayeju and adebolu; aji, 2(1): 11-17, 2019; article no.aji.52193 15 as pcv, total wbc, lymphocytes and neutrophil counts at a dose which ranged from 0.5ml-2.0ml administered to the rats but no effects on the eosinophil counts.these findings are in agreement with the reports of zakaria et al. [7] who claimed that this improvement is due to the presence of medium-chain fatty acids in coconut oil and majorly the presence of electrolytes, vitamins, amino acids in coconut water. kalman et al. [8] postulates that “coconut water can be used intravenously in humans and as a rehydrating drink for athletes”. packed cell volume also known as hematocrit is a measurement of the percentage of red blood cells present in the blood. pcv levels that are too high or too low can indicate a blood disorder, dehydration or other medical condition. a normal level of pcv indicates the capability of the red blood cells to deliver oxygen. the values of pcv obtained from this study showed that apparently healthy rats administered coconut water and coconut oil had an increase in the pcv which indicates an increase in the viable red blood cell that are capable of delivering oxygen for use in the body. the superior increase in pcv of rats administered coconut water suggests that coconut water has the ability to stimulate viable red blood cells. the significant increase in the packed cell volume of the rats may indicate that coconut water and coconut oil contain several compounds, such as proteins, peptides lipopolysaccharides and glycoproteins [6]. table 4. effects of administration of coconut oil and coconut water on the eosinophil counts of apparently healthy wistar albino rats (%) sample days of assay doses of sample administered 0.0ml 0.5ml 1ml 1.5ml 2.0ml coconut oil day 0 2.50±0.50 a 2.50±0.50 a 2.50±0.50 a 2.50±0.50 a 2.50±0.50 b day 7 2.50±0.50 2.00±0.00 a 2.00±0.00 b 2.00±0.00 b 1.00±0.00 a day 14 2.50±0.50 2.00±0.00 a 2.00±0.00 b 3.00±0.00 c 2.00±0.00 a day 21 day 28 2.50±0.50 2.50±0.50 2.50±0.50 a 2.50±0.50 a 1.00±0.00 a 3.00±0.00 c 2.00±0.00 b 1.00±0.00 a 3.00±0.00 c 3.00±0.00 c coconut water day 0 2.50±0.50 a 2.50±0.50 a 2.50±0.50 a 2.50±0.50 b 2.50±0.50 a day 7 2.50±0.50 1.00±0.00 a 2.00±0.00 b 1.00±0.00 a 1.00±0.00 a day 14 2.50±0.50 2.00±0.00 a 2.00±0.00 b 1.00±0.00 a 2.00±0.00 b day 21 day 28 2.50±0.50 2.50±0.50 3.00±0.00 a 3.00±0.00 b 1.00±0.00 a 3.50±0.50 c 0.00±0.00 a 3.50±0.50 c 1.00±0.00 a 2.00±0.00 ab data represent the average for 3 replicates + s.e.m and those with different alphabet superscripts along columns for each treatment are statistically significant at p<0.05 table 5. effects of administration of coconut oil and coconut water on the lymphocyte counts of apparently healthy wistar albino rats (%) sample days of assay doses of sample administered 0.0ml 0.5ml 1ml 1.5ml 2.0ml coconut oil day 0 32.00±0.00 a 32.00±0.00 c 32.00±0.00 c 32.00±0.00 b 32.00±0.00 a day 7 32.00±0.00 32.50±0.50 d 31.50±0.50 c 32.00±0.00 b 33.00±0.00 b day 14 32.00±0.00 30.00±0.00 a 29.50±0.50 b 32.00±0.00 b 36.50±0.50 d day 21 day 28 32.00±0.00 32.00±0.00 31.00±0.00 b 30.00±0.00 a 27.50±0.50 a 29.00±0.00 b 29.50±0.00 a 29.50±0.50 a 35.00±0.00 c 37.00±0.00 e coconut water day 0 32.00±0.00 a 32.00±0.00 a 32.00±0.00 a 32.00±0.00 ab 32.00±0.00 a day 7 32.00±0.00 31.50±0.50 a 33.00±0.00 b 32.50±0.50 b 35.50±0.50 c day 14 32.00±0.00 32.00±0.00 a 34.00±0.00 c 36.50±0.50 c 37.50±0.50 d day 21 day 28 32.00±0.00 32.00±0.00 31.50±0.50 a 34.50±0.50 b 30.00±0.00 a 35.00±0.00 c 31.50±0.50 a 36.00±0.00 c 34.50±0.50 b 38.00±0.00 d data represent the average for 3 replicates + s.e.m and those with different alphabet superscripts along columns for each treatment are statistically significant at p<0.05 akinjayeju and adebolu; aji, 2(1): 11-17, 2019; article no.aji.52193 16 white blood cells (wbc) are known to be key actors in immune responses, as they are the first line of defense against invading microorganisms. an increase in wbc values usually indicates a boost/ increase in immune strength and a decrease indicates fall in immune strength [5]. the values of wbc obtained from this study showed that apparently healthy rats administered coconut water and coconut oil had an increase in wbc count which indicates an increase in immune strength. the greater increase observed with the administration of coconut water suggests that coconut water has the ability to stimulate the production of more immune cells than coconut oil. the significant increase in total white blood cell count of the rats indicate that coconut water and coconut oil contain several compounds, such as proteins, peptides lipopolysaccharides and glycoproteins that have immunostimulatory potential on the immune system [6]. lymphocytes are non-phagocytic mononuclear leucocytes that have the ability to recognize foreign cells and initiate a chain of responses to get rid of foreign cells. however, the observed increase in lymphocyte counts in this study following exposure to coconut water and coconut oil may be one of the mechanisms used to defend the body against the toxic effect of saturated fatty acids [9]. neutrophils usually makes up about 65% of total white blood cell count. they make the first line of action against infections and are non-specific in their activity. the percentage neutrophils counts obtained in this study was significantly higher when compared with the control group. this increase may provide better level of immunity in the rats [10]. eosinophils are responsible for protection against parasitic infections. the eosinophils values of the rats fed with both coconut water and coconut oil were not significantly affected when compared to the control group. this suggests that the administration of either may not confer protection against parasitic infections [9]. this research work has broadened the fact that further work has to be carried out on discovering the mode of action of coconut oil and coconut water and the active components in them. this could possibly be used to determine the proportion of the coconut products, especially coconut oil to be consumed so as to avoid the reduction of beneficial bacterial flora in the gut which can lead to the upset in the microbial balance in the gut 4. conclusion in conclusion, the consumption of coconut water and coconut oil has haematinic potential and immunostimulatory potential with coconut water having a higher effect. the consumption of both especially coconut water is therefore advocated because it increased pcv which is a function of viable red cells in the blood and wbc, lymphocytes and neutrophils which play key roles in immunity. consent it is not applicable. ethical approval as per international standard written ethical approval has been collected and preserved by the author(s). competing interests authors have declared that no competing interests exist. references 1. prades a, dornier m, diop n, pain jp. coconut water uses, composition and properties: a review. fruits. 2011;67:87– 107. 2. kathiravan t, kumar r, lakshmana jh. pulsed electric field processing of functional drink based on tender coconut water (cocos nucifera l.) (hemidesmus indicus) blended beverage/croat. journal of food science technology. 2014;6(2): 84–96. 3. adolf ka, edna dd, rebecca a. potential bacterial health risk posed to consumers of fresh coconut (cocos nucifera l.) water. food and nutrition sciences. 2012; 3:1136–1143. 4. lima ebc, sousa cns, meneses ln, ximenes nc, santos júnior ma, vasconcelos gs. cocos nucifera l. arecaceae: a phytochemical and pharmacological review. brazilian journal of medical and biological research. 2015;48(11):953–964. 5. st-onge mp, mayrsohn b, o’keefe m, kissileff hr, choudhury ar, laferrere b. impact of medium and long chain triglycerides consumption on appetite and food intake in overweight men. european akinjayeju and adebolu; aji, 2(1): 11-17, 2019; article no.aji.52193 17 journal of clinical nutrition. 2014;68(10): 1134-1140. 6. nwangwa ek, chukwuemeka pa. regenerative effects of coconut water and coconut milk on the pancreatic b-cells and cyto-architecture in alloxan induced diabetic wistar albino 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_________________________________________________________________________________ © 2019 akinjayeju and adebolu; this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: http://www.sdiarticle4.com/review-history/52193 http://creativecommons.org/licenses/by/4.0 _____________________________________________________________________________________________________ *corresponding author: e-mail: hafr@sund.ku.dk; asian journal of immunology 4(1): 160-174, 2021; article no.aji.83640 high fat diet triggers a prompt and transient increase in adipose tissue granulocyte colony stimulating factor and circulating myeloid cells in mice helene m. s. eld a, louise madsen a, christina h. lund a, stine broeng metzdorff a and hanne frøkiær a* a department of veterinary and animal sciences, faculty of health and medical sciences, university of copenhagen, ridebanevej 9 st, 1870 frederiksberg c, denmark. authors’ contributions this work was carried out in collaboration among all authors. all authors read and approved the final manuscript. article information open peer review history: this journal follows the advanced open peer review policy. identity of the reviewers, editor(s) and additional reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here: https://www.sdiarticle5.com/review-history/83640 received 25 october 2021 accepted 28 december 2021 published 30 december 2021 abstract scope: the short-term effects of feeding high fat diet (hfd) to mice was investigated with focus on the effect on myelopoesis, circulating neutrophils and the induction of granulocyte colony stimulating factor (g-csf). methods: male mice were fed hfd (45%) during a period of 5 weeks with samples taken after 3 days and 1, 3, 4 and 5 weeks. blood was analyzed for neutrophils and monocytes, for g-csf and granulocyte-macrophage (gm)-csf, and for cytokine expression. visceral adipose tissue (vat) expression of various genes and production of g-gsf and gm-csf in cultured vat was determined. results: three days after commencement of hfd, the number of circulatory neutrophils and monocytes increased but returned to baseline-level at day 8. this transient increase coincided with an increased blood concentration of g-csf and a transient increase in bone marrow and spleen neutrophils. in supernatant from cultivated visceral adipose tissue isolated from hfd fed mice on day 3 and 8, g-csf was increased. the expression of toll-like receptor 4 in adipose tissue was down-regulated from week 4. in vitro, lipopolysaccharide (lps) was a poor stimulator of g-csf, original research article eld et al.; aji, 4(1): 160-174, 2021; article no.aji.83640 161 while g-csf or lps together with g-csf or gm-csf induced increased g-csf production. gcsf suppressed production of lps-induced tnf and increased il-10 production in dendritic cells suggesting that g-csf down-regulates lps-induced inflammation. conclusion: hfd induces a transient increase in adipose tissue g-gsf and circulating myeloid cells in mice. we suggest g-csf induces increased myelopoiesis and simultaneously downregulates lps-induced inflammation. keywords: adipose tissue; granulocyte-coloni stimulating factor (g-csf) production; high fat diet; monocytes; neutrophils. 1. introduction daily or regular intake of high fat meals is common in large parts of the world and may lead to overweight. alongside the weight gain, a state of systemic low-grade inflammation (lgsi) often emerges, which in the long run may lead to the development of metabolic syndrome and further on, to chronic diseases such as type 2 diabetes (t2d) and cardiovascular diseases [1]. however, the precise relationship between high fat diet (hfd) consumption, inflammation and obesity, is still not fully understood. also consumption of a single high fat meal has been reported to induce an inflammatory response and has been suggested to jumpstart the low-grade inflammation [2]. a systematic review on the postprandial inflammatory response to a high fat meal in healthy adults did only reveal an effect on blood il-6 but not on other pro-inflammatory markers and the 47 studies included in the review revealed large variations in the postprandial inflammatory response [2]. the reason for this variation remains obscure. very little has been described regarding the effects of the early course of a hfd before the onset of diet-induced weight gain and obesity as regard the inflammatory events taking place in bone marrow, adipose tissue and blood, with potency to jumpstart the development of lgsi and comorbidities. microbiota has been suggested to play a role in hfd-induced inflammation via its production of endotoxin [3, 4]. increased dietary lipid increases the lymphatic flow and leads with it an increased amount of endotoxin from the gut [5, 6]. the full consequences of increased endotoxin influx are however not fully understood. the effect of an lps injection on granulopoiesis and increased g-csf levels is well established [7, 8], but the consequences of a continuous lps influx from the gut as expected from a permanent or regular hfd have only been sparsely reported. cani et al. showed that a hfd fed to mice for four weeks lead to an increase in the circulatory endotoxin level and provided evidence that lps was responsible for the onset of metabolic disease leading to t2d in mice [9]. apart from lps, also free fatty acids (ffa), in particular saturated ffa stimulate inflammation through the binding to tlr4 [10]. hence, as both ffa and lps influxes are presumed to be increased in hfd fed mice both may contribute in the stimulation of an inflammatory response [11, 12] indicating that solely measuring the lps concentration in e.g. blood, may not directly correlate with an inflammatory response. mice fed a diet rich in fat (45-60%) serve as a frequently used model for studying the physiological effects of an excess energy intake and obesity. obesity and firmly established lgsi typically emerge after hfd feeding for eight weeks or more whereas comorbidities such as t2d establish even later [13]. the inflammatory response is complex and studies of the relationship between obesity and physiological changes have revealed that a connection between visceral adipose tissue (vat), hematopoiesis (bone marrow) and blood exists [14, 15]. whether shorter periods of feeding hfd and inflammation exhibit similar relationships is unclear. talukdar et al. showed that as early as three days after the onset of feeding a hfd, neutrophil infiltration in vat, which remained high during 90 days of feeding was seen [16]. others have reported a transient neutrophil infiltration in the vat ceasing one week after the onset of feeding a hfd [17]. hence, neutrophil influx in vat does take place, however the length of this event and how it is effectuated and perhaps abrogated remains obscure. due to the short lifespan of neutrophils, a constant infiltration into adipose tissue requires a constant supply of neutrophils from the bone marrow. an increase in circulatory neutrophils may thus require a steady mobilization signal and an increased production in the bone marrow. here, we investigated the effect of short-term feeding a hfd to young mice on bone marrow eld et al.; aji, 4(1): 160-174, 2021; article no.aji.83640 162 and blood cell composition and further assessed the circulatory and vat level of g-csf and gmcsf as well as the expression of genes involved in lps-induced inflammation in vat. we found that a transient neutrophil infiltration in vat coincided with a transiently increased vat production of g-csf. we suggest that g-csf holds dual activity stimulating neutrophil recruitment while simultaneously suppressesing inflammatory responses of other myeloid cells. this may explain the transient nature of myelopoiesis and advocates against lps as a key initiator of hfd-induced lgsi [9]. 2. materials and methods 2.1 description of the study area 2.1.1 animals and feeding regime used in the study male c57bl/6 mice (taconic, lille skensved, denmark) 6 weeks of age, were housed with 4 mice per cage and fed ad libitum either a high-fat diet (hfd) (altromin c-1000 rodent diet modified as hfd with 45% energy from fat, brogaarden, denmark) or a standard rodent diet with 5% kcal from fat (altromin c-1000, brogaarden). at study day 1, all mice were weighed and allocated to hfd (40 mice) or the standard diet (20 mice). the mice were euthanized by cervical dislocation and tissue samples collected under sterile conditions. the experiment was carried out in accordance with the council of europe convention european treaty series 123 on the protection of vertebrate animals used for experimental and other scientific purposes, and the danish animal experimentation act (lbk 1306 from november 23, 2007) approved by the animal experimentation inspectorate, ministry of justice, denmark (license number: 2012-152934-00256 c1-6). 2.2 method of sample and data collection at day 3, 8, 23, 30 and 37, eight mice fed hfd and four fed standard diet were weighed and anesthetized. after blood sampling, the mice were euthanatized. whole blood was collected from the retro-orbital vein with heparin-coated capillaries into edta-coated eppendorf tubes. thirty μl blood was transferred to cryotubes containing 180 μl lysis buffer (50% lysis/binding solution concentrate, 50% isopropanol, thermo fisher, waltham, ma, usa) and stored at -80°c until rna extraction. the remaining blood was used for complete blood count (cbc) and leukocyte differentiation and isolated plasma was transferred and frozen at -80°c until elisa was performed. immediately after euthanasia, spleen, epididymal (visceral) adipose tissue (vat) and femur were collected. the spleen was cut in two and one part was placed in rnalater (ambion by life technologies, thermo fisher) for qpcr and the other part was placed in cold sterile pbs for cell isolation and flow cytometry. bone marrow (bm) cells were isolated from the femur by flushing them out from cleaned bones with cold pbs followed by flow cytometry analysis [18]. the vat was isolated and placed in a preweighed tray with ice-cold pbs and weighed. hereafter, a small part of the vat was placed in rnalater for qpcr and the rest used for culturing. 2.3 cell isolation and flow cytometry analyses analyses on full blood was performed without any purification. bone marrow and spleen cells were isolated as described previously [18]. blood neutrophilic granulocytes and monocytes proportions were assessed by blood leukocyte counting by an automatic cell counter (advia 2120i hematology system, siemens, germany). to analyze single cell suspensions of spleen and bm cells by flow cytometry, cells were counted, washed and suspended in facs wash buffer (pbs, 1% fetal bovine serum). for the identification of neutrophils and mature monocytes, a cocktail of anti-mouse ly6g (1a8), ly6c (al-21), cd11b (m1/70) and cd115 (t38320) antibodies were used (bd pharmingen, bd biosciences, san jose, ca, usa) [18]. for the identification of mature myeloid cells, fcreceptors were first blocked with fc-block (bd pharmingen, bd biosciences) for 10 min at 4°c, washed in facs wash buffer and incubated with the antibody cocktail at 4°c for 30 min. after washing with facs wash buffer, cells were analyzed using a bd facs canto ii (bd biosciences). data were analyzed with flowjo tm software (bd biosciences) and the number of neutrophils and monocytes was calculated from total number of cells for each sample. 2.4 rna isolation and qpcr adipose tissue was homogenized in lysis buffer (magmax-96 rna isolation kit; ambion, thermo fisher) by using glass beads and the the fastprep®-24 instrument (mp biomedicals, thermo fisher). total rna from homogenized adipose tissue, bm cells and blood cells was eld et al.; aji, 4(1): 160-174, 2021; article no.aji.83640 163 extracted by magmax express (applied biosystems, foster city, ca, usa) using the magmax-96 rna isolation kit (ambion, thermo fisher) for tissues and the magmax-96 blood rna isolation kit (ambion, thermo fisher) for blood cells, as previously described [19] and according to manufacturer’s instructions. cdna was produced from ~200 ng total rna using high-capacity cdna reverse transcriptase kit (applied biosystems) according to the manufacturer’s instructions. gene expression of selected genes listed in table 1 was analyzed on the steponeplus instrument (applied biosystems) using universal fast thermal cycling parameters (applied biosystems) and taqman fast universal pcr mastermix (applied biosystems). relative quantification of the gene expression was calculated by the comparative cycle threshold (ct) method. the expression of target genes was normalized to the gene expression of actb (beta-actin) as the reference gene: [∆ct =ct (target) –ct (reference)]. relative quantification of gene expression was calculated as 2 -∆∆ct , where ∆∆ct = [∆ct (sample)-∆ct (calibrator)]. mean ∆ct of samples from control mice was used as the calibrator. 2.5 culturing of adipose tissue the epidydimal fat pads (vat) were washed in pbs and two 50 mg pieces were placed in two different wells of a 6-well-plate and cut into 5 smaller pieces and cultured for 24 hours in 2 ml m199 media with glutamine, 25 mm hepes and 1% pen/strep [20]. after culturing the adipose tissue supernatant from each mouse was frozen at -80°c until cytokine quantification. 2.6 cytokine quantification plasma g-csf and g-csf, gm-csf, cxcl-2, tnf, il-10 and il-1β in the supernatant of cultured adipose tissue and of stimulated cells were quantified by duoset elisa kits from r&d systems (mn, usa) according to manufacturer’s instructions. 2.7 in vitro treatment of murine bone marrow cells bone marrow cells were isolated as described elsewhere [21]. briefly, bone marrow from c57bl/6 mice was flushed from the femur and tibia and washed twice in sterile pbs. cells (2.9×10 6 ) were seeded in 12-well-plates in 1 ml rpmi 1640 (sigma-aldrich, st. louis, mo), containing 10% (v/v) heat-inactivated fetal calf serum, penicillin (100 u/ml), streptomycin (100 µg/ml), glutamine (4 mm) and 50 µm 2mercaptoethanol. cells were then treated with lps (sigma-aldrich), mouse g-csf (r&d systems) and/or gm-csf (from a gm-csftransfected myeloma cell line [21] in a final concentration of 0.1 µg/ml lps, 10 ng/ml g-csf and gm-csf. the cells were incubated for 1 or 4 days at 37°c in a 5% co2 humidified atmosphere. bm cells were harvested and examined for neutrophils and mature monocytes using flow cytometry and the concentration of cytokines in supernatant was determined by elisa. dendritic cells were prepared as described [21]. 2.8 statistical analysis statistical analysis was performed in graphpad prism version 5.03 (graphpad software). significance was evaluated by 2-way anovas, 1-way anovas or t-tests to determine differences between treatment groups and time points. relative quantification (rq) is plotted in gene expression experiments and statistical analysis was performed on ∆ct values. in experiments where control groups showed constant expression level all data from control mice were pooled. 3. results 3.1 high fat diet-increased body and epididymal fat pad weight is preceded by a transient increase in blood myeloid cells in mice male mice aged 6 weeks at the start of the experiment, were fed either a 45% hfd or a control diet with 4% fat. the mice were weighed regularly during the 37 days feeding period. both groups gained weight during the first three weeks, indicating that the mice were not fully outgrown at the beginning of the experiment [22]. at day 30 and 37, the hfd mice had increased their body weight and the weight of the epididymal fatpads significantly compared to the control mice (figs. 1a-b). percentages of blood monocytes and neutrophils from the mice as determined by cbc revealed a constant percentage of monocytes and granulocytes in the control group (0.6% and 10.5%, respectively), (figs. 1c-d). in the hfd group, at day 3, the level of monocytes increased more than twofold, but decreased to the base-level at day 8 (fig. 1c). also the level of granulocytes showed an eld et al.; aji, 4(1): 160-174, 2021; article no.aji.83640 164 indication of an increase at day 3, this was however not significant (p=0.16). when analyzing the concentration of the growth factor g-csf in the plasma, the control groups showed a fairly constant level around 75 pg/ml. in the hfd group at day 3, the g-csf concentration was increased but dropped thereafter to that of the control mice (figs. 1e). table 1. taqman assays used for gene expression analysis gene gene name assay id actb actin, beta mn00607939_s1 elane arg1 ccl2 neutrophil elastase arginase 1 chemokine (c-c motif) ligand 2 mm01168929_g1 mm00475988 mm00441242_m1 cxcl2 chemokine (c-x-c motif) ligand 2 mm00436450_m1 hp haptoglobin mm00516884_m1 stfa2l1 stefin a2 like 1 mm04212095_mh foxp3 forkhead box p3 mm00475162_m1 tlr4 toll-like receptor 4 mm00445273_m1 s100a8 itgam/cd11b cd14 cd8a f4/80 s100 calcium binding protein a8 (calgranulin a) integrin alpha m cluster of differentiation 14 cluster of differentiation 8 a egf-like module-containing mucin-like hormone receptor-like 1 mm00496696_g1 mm00434455 mm00438094 mm01182107-g1 mm00802529_ml fig. 1. hfd induces a prompt transient rise in myeloid cells in the blood which is absent at the first signs of weight gain. a: body weight and b: vat weight in mice fed high fat diet (hfd) or chow for 37 days (hfd n=8, control n=4 for each time point). two-way anova performed. percentages of c: neutrophils and d: monocytes in blood as determined by cbc. all control groups pooled n=20. one-way anovas performed. e: concentration of g-csf in blood determined by elisa. one-way anova performed. all data is shown as mean +/-sem, ***<0.001, **<0.01, *p<0.05 eld et al.; aji, 4(1): 160-174, 2021; article no.aji.83640 165 3.2 myelopoiesis is transiently increased early in the high fat fed feeding period we found no differences between the hfd and the control group in the proportions of myeloid progenitor cells (supplementary fig. 1). the fully differentiated myeloid cells (gating strategy in fig. 2a), however, exhibited increased level of neutrophils (ly6g + ly6c + cd11b + ) at day 8 (1.9·10 7 vs 1.4·10 7 per bm, p=0.012) but returned to that of the control mice at day 23 (fig. 2b). at day 37, a significant increase in neutrophils was seen in the hfd group compared to control (1.6·10 7 vs. 1.3·10 7 , p=0.03). also the monocyte population (ly6g ly6c + cd11b + cd115 + ) in the hfd group showed a trend towards increased numbers at day 8 (p=0.18) and day 23 (p=0.09) but not at later time points. of note, from day 30 the proportion of monocytes increased markedly in both groups, presumably due to the outgrowth of the mice at this age. a number of genes including elane, arg1, haptoglobin (hp) and itgam (cd11b), are expressed in neutrophils at specific differentiation stages (fig. 2d) [23]. the expression of these genes in the bm cells was analyzed. in line with the increased neutrophil population detected in bm at day 8, a significant increase in the expression of hp and cd11b indicative of neutrophils in the late differentiation state or mature neutrophils was detected at day 8 (fig. 2e). no differences between the two groups were detected in the expression of arg1 or elane. thus, hfd causes a prompt but transient increase in mature myeloid cells in the bm. 3.3 spleen myeloid cells are transiently increased early in the hfd feeding period when analyzing the myeloid population in the spleen during the course of hfd, we saw a nonsignificant increase in neutrophils (ly6g + ly6c + cd11b + ) at day 8 (p=0.081) compared to control group (fig. 3b). mature monocytes (ly6g ly6c + cd11b + cd115 + ) showed a trend towards increased numbers in the hfd group at day 3 and day 23 (p=0.11 and p=0.12, respectively) (fig. 3c). these data support the finding from the blood leukocyte counting (fig. 1c and d) showing that an early increase in blood neutrophils is induced upon hfd feeding. expression analysis of blood was performed on hp to identify immature neutrophils, on il1b and stfa2l1 to identify mature neutrophils, and cd14 to identify monocytes. the expression of hp showed a tendency to be increased on day 3 (p=0.15) in hfd fed mice while on day 8 (p=0.061) and day 23 (p=0.019) the expression dropped to become lower than the chow fed mice and then increased again on day 37 to the level of the control group (fig. 3d). in the hfd group, the expression of il1b and stfa2l1 was significantly increased on day 8 compared to the control group, while from day 23 the expression of these genes was not different from the control group. the expression of cd14 showed a significantly higher level at day 23 compared to control group, while the expression did not significantly differ from the control group at any other day. hence, during the first week of hfd feeding, the number of mature neutrophils in circulation transiently increases but drops subsequently to the level of the chow fed control group. these data indicate an early but transient recruitment of neutrophilic granulocytes from the bone marrow to the blood upon the onset of hfd feeding. 3.4 cultured adipose tissue from mice at the onset of high fat feeding secretes elevated g-csf to investigate if adipose tissue could contribute to the enhanced g-csf level measured in the blood at day 3, vat isolated from mice fed hfd or control diet were cultured for 24 hours and the concentration of released g-csf and gm-csf was measured in the supernatant (fig. 4a-b). adipose tissue from mice on control diet showed a constant production of g-csf and gm-csf and data from different time points was therefore pooled. the g-csf concentration from cultured hfd vat showed on day 3 a trend to be increased (p=0.08) and at day 8 a significantly increased g-csf production (p=0.01) (fig. 4a). adipose tissue from 23, 30 and 37 days did not show increased g-csf production. the hfd did not result in a changed gm-csf production, which showed a stable level around 0.5 ng per gram cultured adipose tissue during the entire experiment (fig. 4b). also the concentration of il-1 was analyzed but the concentration in all samples was at or below the detection limits (data not shown). 3.5 pro-inflammatory as well as antiinflammatory events occur in adipose tissue of high-fat fed mice to investigate which factors were induced in adipose tissue during the five weeks of hfd eld et al.; aji, 4(1): 160-174, 2021; article no.aji.83640 166 fig. 2. high fat feeding reveals an early and transient up-regulation of neutrophils in bone marrow. a: gating strategy for identification of neutrophils and monocytes in isolated bone marrow cells. b: the number of neutrophils (ly6c + ly6g + cd11b + ) and c: monocytes (ly6c + ly6g cd11b + cd115 + ) in bone marrow from hfd fed mice and control mice, hfd n=8, c n=4, individual unpaired t-tests performed. d: schematic overview over the expression of selected genes in various differentiation stages of neutrophil granulocytes. e: expression of genes at different time points in the hfd fed group (n=8) compared to control mice (n=4) by unpaired t-tests performed on dct values. mean+/-sem, **p<0.01, *p<0.05 eld et al.; aji, 4(1): 160-174, 2021; article no.aji.83640 167 feeding, the expression of different genes was analyzed in the vat of hfd fed and control mice (fig. 5). for the control group, all the genes tested showed a stable expression during the entire experiment, accordingly expression data from the various days were pooled. in the hfd group, the expression of ccl2 encoding the monocyte-recruiting chemokine ccl2 [24] was slightly increased at day 8 (p=0.065) compared to control and was increased at day 23, day 30 and 37 (fig. 4). the expression of cxcl2 encoding the neutrophil-recruiting chemokine cxcl2 first showed a decreased expression on day 3 (p=0.08) and then returned to the control level from day 8 though with an increased expression at day 30. hence, the chemokines exhibited distinct expression profiles indicating a gradual increase of the monocyte-recruiting ccl2 and an initial reduction of the neutrophilrecruiting cxcl2. the expression of hp was transiently up-regulated at day 3 and day 8, while the expression of stfa2l1 transiently showed a tendency to be increased on day 8 (not significant). the expression of f4/80 signifying the presence of macrophages [25] did not at any time point deviate from the expression of the control group, while s1008a expressed constitutively by monocytes and neutrophils [26] showed an up-regulation at day 30 and 37. while the expression of cd8a did not at any time point differ from the control group, the expression of foxp3 increased at the end of the study (day 37). also the expression of tlr4 was analyzed and showed a significantly decreased expression at day 30 and 37 compared to the control group. 3.6 g-csf stimulates the production of g-csf in bm cells but inhibits an inflammatory response in monocytederived cells to investigate how g-csf, alone or in concert with lps affected the development of myeloid cells, lps was added to freshly isolated bm cells, with or without concomitantly added g-csf or gm-csf, and incubated for 1 or 4 days, where after the cellular composition was assessed by flow cytometry (fig. 6a-b). after 1 day of stimulation with g-csf or gm-csf, the number of neutrophils doubled compared to cells cultured in media only, and after 4 days neutrophils were almost tripled compared to media-grown cells at day 1 (fig. 6a). the number of monocytes also increased in the presence of g-csf or gm-csf, after 1 day to 1.5-2 fold and after 4 days to 4-6 fold the number in media. in the presence of lps, the number of neutrophils at day 1 and 4 gradually dropped to around 60 and 20% of the media day 1 control. likewise, when lps was added together with either g-csf or gm-csf, the levels of neutrophils reached about 75% of the level obtained without lps at day 1 and, at day 4, the number of granulocytes dropped to below the day 1 media control level. in contrast, lps stimulated the development of monocytes leading to more than the doubled number of monocytes at day 4 compared to media controls at day 1 and 4 (fig. 6b). lps added together with g-csf or gm-csf did not lead to further increase in the number of monocytes as compared to stimulation with g-csf or gm-csf alone. neutrophils have a short lifespan, thus it is not possible from this experiment to establish whether lps stimulates a faster development and thereby deplete the number of cells or halts the differentiation of these cells. to establish whether lps stimulation affected the production of g-csf or gm-csf in the isolated bone marrow cells, which could represent an indirect way to stimulate myelopoiesis, we tested the concentration of g-csf and gm-csf in the supernatant of the stimulated bone marrow cells. lps alone did not induce production of g-csf, but addition of g-csf led a strong production of g-csf, and g-csf together with lps further increased the concentration (fig. 6c). compared to day 1, the g-csf level at day 4 measured after addition of g-csf alone or in combination with lps decreased indicating that the g-csf is spend during the development of neutrophils. gm-csf did not induce significant g-csf production, but together gm-csf and lps induced a modest level of g-csf (fig. 6c). neither lps nor g-csf or gm-csf induced production of gm-csf in the cells (fig. 6c). we also tested the production of cxcl-2 known to be induced by lps (fig. 6c). lps induced a clear increase in cxcl-2, while g-csf or gm-csf did not stimulate cxcl2 production. interestingly, in the presence of gm-csf, the lps-induced cxcl2 production doubled. prestimulating bm-derived dendritic cells with gcsf prior to lps stimulation resulted in reduced tnf induction while the production of il-10 was increased (fig. 6d). together, these results demonstrate that the presence of lps influences the g-csf and gmcsf-induced development of myeloid cells. moreover the production of g-csf depends on the presence of g-csf and is enhanced by lps eld et al.; aji, 4(1): 160-174, 2021; article no.aji.83640 168 and affects the inflammatory response through its anti-inflammatory effect on monocyte-derived cells. 4. discussion it is well-established that a constant or regular intake of an energy surplus in the form of fat may lead to overweight or obesity and in turn systemic low-grade inflammation, which includes increased numbers of circulating neutrophils and monocytes [1]. however, how such diets influence the hematopoiesis over shorter periods in young and normal weight individuals has only been sparsely studied. in the present study we found that in young mice, a moderate high-fat (45%) diet induced increased weight after 4-5 weeks of hfd feeding. this was preceded by a prompt and transient increase in circulatory gcsf, neutrophils and monocytes, in g-csf production by vat and in increased production of neutrophils and monocytes in bone marrow. of note, at the time where weight gain became significant, no signs of elevated circulatory neutrophils were evident in the mice. fig. 3. high fat feeding induces an early transient up-regulation of neutrophils and monocytes in spleen. a: gating strategy for identification of neutrophils and monocytes in spleen. the proportion of b: neutrophils (ly6c + ly6g + cd11b + ) and c: monocytes (ly6c + ly6g cd11b + cd115 + ) in spleen from hfd fed mice and control mice. d: expression of genes at different time points in the hfd fed group compared to control mice by unpaired t-tests on dct values. mean+/-sem, **p<0.01, * p<0.05 eld et al.; aji, 4(1): 160-174, 2021; article no.aji.83640 169 fig. 4. hfd induces a prompt but transient increase in the production of g-csf from adipose tissue. the concentration of a: g-csf and b: gm-csf in supernatant from cultured isolated vat as measured by elisa. concentration in the hfd fed group compared to the average of all control mice by one-way anovas. mean+/-sem, * p<0.05 fig. 5. hfd increases simultaneous expression of pro-inflammatory and anti-inflammatory genes in adipose tissue. expression of genes in vat at different time points in the hfd fed group compared to the average of all control mice by one-way anova on dct values. mean+/sem, ***<0.001, **p<0.01, * p<0.05 eld et al.; aji, 4(1): 160-174, 2021; article no.aji.83640 170 fig. 6. lps enhances the g–csf and gm-csf-induced g-csf, which holds anti-inflammatory properties. the number of a: neutrophils (ly6g + ly6c + cd11b + ) and b: monocytes (ly6c + ly6g cd11b + cd115 + ) after stimulation with lps alone or together with g-csf or gm-csf of bone marrow for 1 or 4 days relative to the number measured in unstimulated bone marrow cells on day 1. c: the concentration of g-csf, gm-csf and cxcl-2 in the supernatant of bone marrow cells after stimulation with lps alone or together with g-csf or gm-csf for 1 or 4 days. d: the production of tnfα and il-10 in lps stimulated dendritic cells +/-preincubation with gcsf a transient increase in circulating neutrophils has been reported previously in mice fed a high-fat (60%) diet [15, 27]. our data shows that also a considerably lower fat content in the diet (45%) eld et al.; aji, 4(1): 160-174, 2021; article no.aji.83640 171 may lead to this transient increase in neutrophils and further makes it probable that an elevated transient level of circulating g-csf preceding the increase in neutrophils is the cause. g-csf is the prime signaling molecule for mobilizing neutrophilic granulocytes from the bone marrow into circulation [28]. the early and transient upregulation of g-csf in blood coincided with the transient increase in granulocytes measured in blood and in spleen during the first week of hfd feeding and may explain the increased level of neutrophils in circulation. like others, [27] we did not find any changes in the proportions of myeloid progenitor cells upon hfd feeding. this is in contrast to nagareddy et al. (2014) and singer et al. (2014) who found increased numbers of myeloid progenitors concomitantly with increased number of neutrophils and monocyte in bone marrow of obese mice [14, 15]. of note, these studies investigated either obese mice or ob/ob mice, where the increased number of circulating neutrophils and monocytes seem to be caused by metabolic conditions rather than a change in diet able to increase the endotoxin absorption. the cause of the increased blood g-csf concentration was not established. however, one possibility is that the high content of dietary fat in the gut leads to an increased lymphatic flow, in turn leading to an increased influx of lps from the intestinal lumen through the lymphatic system. an increased lipid content in the diet is reported to increase the lymphatic flow [29] and may also increase the absorption of lps [1, 30]. lps may stimulate an increased production of gcsf in various tissue [31] in turn leading to recruitment of granulocytes into circulation. we demonstrated here that vat from mice fed hfd for 3-8 days had an increased production of gcsf while at later time points, the production was reduced to the same level as vat from mice on a standard diet. other tissues than vat may also induce increased production of g-csf and thus contribute to a transiently increased g-csf level in the blood. the most potent g-csfproducing cells comprise monocytes and macrophages, but g-csf is also produced by fibroblasts and endothelial cells [32], all being ubiquitously present in the various tissues of the body. in the adipose tissue, we found a transient increase in the expression of hp and stfa2l1 during the first week of hfd feeding indicating an increased number of neutrophils in the last differentiation stages [23]. this corresponds to the findings in earlier studies showing an early transient influx of neutrophils upon feeding a hfd [17, 27]. after this initial stage and after the g-csf peak, we found a down-regulation of tlr4 expression in the hfd fed group at the time where mice on hfd had increased their weight compared to control mice, which might indicate a state of lps tolerance, however such direct relation is purely speculative. apart from playing a key role in the mobilization of neutrophils from the bone marrow into circulation, g-csf is important in the promotion of the granulocytic lineage, mainly neutrophils [33]. we found an increase in both neutrophils and monocytes in bm from hfd fed mice during the first week(s), and speculated that the increased g-csf could stimulate the promotion of these cells. to investigate this, we stimulated freshly isolated bm cells with g-csf and gmcsf, alone or together with lps, and found that in contrast to g-csf and gm-csf, lps was a poor stimulator of neutrophils and monocytes; rather it seemed to halt the generation of neutrophils and had only modest effect on the generation of monocytes. of note, the addition of g-csf induced significant production of g-csf in the bone marrow cells. lps alone did not induce g-csf production but slightly enhanced the production induced by g-csf. isolated bm cells are primarily comprised by hematopoietic cells but the presence of some endothelial cells cannot be excluded. the endothelial cells were previously identified as the only cells in bm producing g-csf in response to lps [7]. this is in agreement with our data showing only modest effect of lps on g-csf production. however, the epithelial cell-produced g-csf might induce g-csf production in the myeloid cells thus leading to an enhanced effect and probably increased generation of neutrophils and monocytes as indicated from our results. in contrast to g-csf, gm-csf did not stimulate gcsf production but together with lps, the induction of both g-csf and cxcl2 was enhanced. this illustrates the difference between the two growth factors; while gm-csf is purely pro-inflammatory, g-csf holds pro-inflammatory (through its neutrophil mobilizing and promoting property) as well as immune-regulating properties [34]. the increased production of g-csf readily ceased within the first week of hfd feeding indicating the action of immune-regulating properties. we did not demonstrate direct eld et al.; aji, 4(1): 160-174, 2021; article no.aji.83640 172 causality between the g-csf and immune regulation in vivo, but we could show that g-csf inhibited tnf production while increasing il-10 production in vitro. especially monocytes and their derivatives expressing the g-csf receptor are influenced by g-csf in an anti-inflammatory way leading to attenuated production of proinflammatory cytokines upon lps stimulation [35]. ligation of the g-csf receptor induces internalization and degradation of the receptor resulting in down-regulation of the responsiveness to g-csf. mice challenged with lps after preteatment with g-csf, were protected against the dose of lps that killed nonpretreated mice through a mechanism involving inhibition of tnfα [36]. blood taken from healthy humans injected with g-csf responded with less cytokine production when stimulated in vitro with lps [37] and volunteers pretreated with g-csf before injection of minute doses of lps exhibited lower levels of the pro-inflammatory cytokines il6 and tnf [38]. together, this points towards a strong anti-inflammatory activity of g-csf, however, for how long the elevated serum gcsf found in mice fed a hfd may prevent inflammation remains to be investigated as does the possibility of other mechanisms taking over in preventing hfd-induced inflammatory responses. to this end, il-10 produced by monocytes and macrophages holds even stronger anti-inflammatory activity and influences monocytes as well as adipocytes [39, 40] and il10 from monocytes was recently demonstrated to induce epigenetic chances in adipocytes [40]. whether il-10 is involved in mechanisms causing establishment of long-term endotoxin tolerance is however purely speculative and the presented data calls for new studies that investigate a possible long term antiinflammatory effect of g-csf. 5. conclusion in conclusion, the presented data confirm previous reports showing that hfd fed to mice induces a transient increase in circulatory neutrophils and monocytes. this is paralleled with a transient peak in g-csf. the vat contributes to the transient production of g-csf, which is followed by down-regulation of tlr4 expression. as g-csf inhibits production of tnf and up-regulates il-10 production we suggest that the transient g-csf peak stimulates endotoxin tolerance. this may confer an antiinflammatory state to the body upon high-fat meals that may influence post-prandial responses and might contribute to the high variations seen in the inflammatory response to a single meal. further studies are warranted to pursue the possible importance of g-csf in post-prandial inflammatory response. disclaimer the products used for this research are commonly and predominantly use products in our area of research and country. there is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. also, the research was not funded by the producing company rather it was funded by personal efforts of the authors. supplementary materials supplementary materials available in this link: https://www.journalaji.com/index.php/aji/libraryfi les/downloadpublic/3 competing interests authors have declared that no competing interests exist. references 1. duan y, zeng l, zheng c, song b, li f, kong, xu k. inflammatory links x between high fat diets and diseases, frontiers in immunology. 2018;9:2649. 2. emerson sr, kurti sp, harms ca, haub md, melgarejo t, logan c, rosenkranz sk, magnitude and timing of the postprandial inflammatory response to a high-fat meal in healthy adults: a systematic review, adv nutr. 2017;8(2): 213. 3. mohammad s, thiemermann c. role of metabolic endotoxemia in systemic inflammation and potential interventions, front immunol. 2020;11:594150. 4. hersoug lg, møller p, loft s. gut microbiota-derived lipopolysaccharide uptake and trafficking to adipose tissue: implications for inflammation and obesity, obesity reviews. 2016;17(4):297. 5. ghoshal s, witta j, zhong j, de villiers w, eckhardt e. chylomicrons promote intestinal absorption of lipopolysaccharides, j lipid res. 2009; 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this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: https://www.sdiarticle5.com/review-history/83640 http://creativecommons.org/licenses/by/4.0 _____________________________________________________________________________________________________ *corresponding author: e-mail: celso@alergoimuno.med.br; cite as: olivier, c. e., pinto, d. g., teixeira, a. p. m., santana, j. l. s., santos, r. a. p. g., lima, r. p. s., & monteiro, e. s. (2024). endotyping non ige mediated immunoreactivity to dermatophagoides farinae: implications for allergic patients: a retrospective study. asian journal of immunology, 7(1), 90–99. retrieved from https://journalaji.com/index.php/aji/article/view/134 asian journal of immunology volume 7, issue 1, page 90-99, 2024; article no.aji.117841 endotyping non ige mediated immunoreactivity to dermatophagoides farinae: implications for allergic patients: a retrospective study celso eduardo olivier a*, daiana guedes pinto a, ana paula monezzi teixeira a, jhéssica letícia santos santana a, raquel acácia pereira gonçalves santos a, regiane patussi santos lima b and everton salgado monteiro c a instituto alergoimuno de americana, brazil. b lavoisier’s laboratories, são paulo, brazil. c department of allergy and immunopathology, faculty of medicine, são paulo university, brazil. authors’ contributions this work was carried out in collaboration among all authors. all authors read and approved the final manuscript. article information doi: https://doi.org/10.9734/aji/2024/v7i1134 open peer review history: this journal follows the advanced open peer review policy. identity of the reviewers, editor(s) and additional reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here: https://www.sdiarticle5.com/review-history/117841 received: 19/05/2024 accepted: 28/05/2004 published: 31/05/2024 original research article https://doi.org/10.9734/aji/2024/v7i1134 https://www.sdiarticle5.com/review-history/117841 olivier et al.; asian j. immunol., vol. 7, no. 1, pp. 90-99, 2024; article no.aji.117841 91 abstract background: several publications report dermatophagoides farinae allergens as responsible for several types of non–ige-mediated allergic reactions. there is no standardized lab exam to endotype (quantify the participation of these mechanisms) in the context of allergic disease pathophysiology. aims: to evaluate the potential of the leukocyte adherence inhibition test (lait) and the tube titration of precipitins (ttp) on serum to discriminate non–ige-mediated immunoreactivity against d. farinae in patients with non–ige-mediated allergic phenotype with clinically suspected hypersensitivity to their allergens. study design: we retrospectively examined the medical charts of 134 allergic patients investigated with lait and 100 allergic patients investigated with ttp against an extract of d. farinae. place and duration of study: instituto alergoimuno de americana – são paulo – brazil – between january 2018 and may 2024. methodology: the registered results of the semi-quantitative serum ttp against d. farinae extract were distributed in ranges through a cascade distribution chart to outline the variability of the results. the registered results of the percentage of leukocyte adherence inhibition (lai) promoted by the ex vivo challenges with d. farinae extract were distributed in ranges through a cascade distribution chart to outline the variability of results. the statistical characteristics of these cohorts were calculated. results: the ttp showed a wide distribution range, with four negative and most positive results concentrating on the higher dilutions. the mean was estimated at 1:242; the median was 1:128; the standard deviation was estimated at 1:210; the mode was 1:512 (appeared 36 times). the lai ranged from 0% to 100%. the mean was 52%; the median was 55.5%; the standard deviation was 23.6%; the mode was 63% (appeared seven times). the cascade distribution demonstrates a wide range of lai results. conclusion: our preliminary results support that the semi-quantitative ttp and the lait have the potential to endotype and clinically discriminate diverse degrees of cellular and humoral non–igemediated immunoreactivity against d. farinae in allergic patients. keywords: allergy; asthma; bronchitis; dermatophagoides farinae; diagnosis; hypersensitivity; leukocyte adherence inhibition test; non–ige-mediated immunoreactivity; precipitins; rhinitis. abbreviations lai : leukocyte adherence inhibition lait : leukocyte adherence inhibition test ttp : tube titration of precipitins 1. introduction storing food and sharing mattresses with mites exposed humanity to commensalism, which was only noticed about a hundred years ago [1]. storage mites have been reported as causes of allergic diseases since 1924 when willem storm van leeuwen described several cases of asthma in patients exposed to mite-infested wheat and oats [2]. storage mites are eight-legged members of the arachnid class and a significant cause of allergic diseases through atopic sensitization to their multiple allergens [3]. dermatophagoides farinae is a storage mite belonging to the phylum arthropoda, subphylum chelicerata, class arachnida; subclass acari; superorder acariformes; order sarcoptiformes; suborder psoroptidia; family pyroglyphidae; subfamily dermatophagoidinae [4]. the allergic reactions produced by the ingestion of storage mites are frequently reported as "oral mite anaphylaxis" or "pancake syndrome" [5]. several ige-mediated and non–ige-mediated hypersensitivity mechanisms were associated with these conditions [6]. besides a typical storage mite, d. farinae is also a house dust mite found in mattresses and pillows, which provides food, moisture, and a thermal source [7]. besides corporal allergens, several allergens are identified in the feces, eggs, and excretions of d. farinae, which accumulate in the environment and are recognized as significant immunogens [8]. the allergen nomenclature sub-committee from the world health organization and the international olivier et al.; asian j. immunol., vol. 7, no. 1, pp. 90-99, 2024; article no.aji.117841 92 union of immunological societies (who/iuis) had already classified dozens of allergens belonging to d. farinae [9]. most d. farinae allergens were identified by proteomics, using ige-based techniques such as enzyme-linked immunosorbent assay inhibition tests, immunoblots, basophil activation tests, and skin prick tests [10]. besides the ige-mediated allergenicity, the immunoreactivity of the d. farinae allergens on the innate immune system and its contribution to the production of symptoms in allergic patients have also been studied [11]. several igg epitopes of d. farinae allergens were already identified by peptide microarray immunoassay [12]. specific d. farinae allergens, such as the der f 38, stimulate innate immunoreactivity by binding to toll-like receptors (such as tlr4), acting as allergy inducers [13]. d. farinae also stimulates the transcription of long noncoding rnas (non-protein-coding rna that exert transcriptional and post-transcriptional regulation on messenger rnas and micrornas), causing cell dysfunction and dysregulation of circulating cd8+ t cells of patients with asthma [14]. some d. farinae allergens, besides acting as ige ligands, also stimulate innate immune cells, such as the α-tubulin (der f 33), which increases the expression of interleukin-4 and upregulates cd80 and tnf-α levels in dendritic cells [15]. exosomes from d. farinae induce immunogenic inflammation by stimulating epithelial cells and macrophages to release inflammatory-related cytokines such as interleukin-33, thymic stromal lymphopoietin, tnf-alpha, and il-6 [16]. d. farinae induces innate inflammation via interleukin-33 via receptor-interacting protein kinase signaling [17]. besides their allergens, mites also harbor a microbiome that produces toxins and a diversified set of allergens that can trigger innate and adaptive allergic responses [18]. environmental allergens such as mites and microorganisms may produce innate immune dysregulation, resulting in epithelial damage, inadequate adaptive response, and a persistent inflammatory state [19]. the mite's microbiome is rich in pathogen-associated molecular patterns (pamps), which are also involved in mast cell activation and non–ige-mediated allergic responses [20]. the microbial damageassociated molecular patterns (damps) promote type ivc hypersensitivity reactions through th17 cell cytokines and group 3 innate lymphoid cells, leading to the liberation of neutrophil extracellular traps (nets), stimulating the innate immune response and inflammation [21,22]. damps trigger the type vii hypersensitivity reaction through pattern recognition receptors (prrs) [23]. non–ige-mediated hypersensitivity to mite bacterial microbiome is documented in patients with acne rosacea [24,25]. to endotype non–ige-mediated immunoreactivity against suspected allergens, we routinely employ the semi-quantitative tube titration of precipitins (ttp) and the leukocyte adherence inhibition test (lait), an ex vivo challenge immunoassay made with viable leukocytes already reported to demonstrate allergen-specific immunoreactivity against d. farinae in allergic patients [26]. to evaluate the potential of these procedures to discriminate non–ige-mediated immunoreactivity against d. farinae, we retrospectively compiled the electronic medical charts of patients with non–ige-mediated allergic rhinitis, allergic bronchitis, allergic conjunctivitis, and/or atopic dermatitis who were investigated with them. patients diagnosed with these allergic conditions were eligible for this investigation after demonstrating non-reactive or inconclusive skin tests against d. farinae extract, a normal range total ige, and undetectable specific ige for d. farinae. 2. materials and methods 2.1 subjects after receiving institutional review board approval from the instituto alergoimuno de americana (brazil; 04/2024), we proceeded with the electronic chart review of 8,800 allergic patients who attended our outpatient facility from january 2018 to may 2024. a cohort of 134 patients had been submitted to an ex vivo allergen challenge test with d. farinae extract monitored with lait for presenting non– ige-mediated allergic conditions. the lait cohort counted 44 males; mean age 42.9 years; sd 17.8 years; range 17 to 89 years; modes = 19 and 25 (each appeared seven times); geometric mean = 39.3 years. a cohort of 100 patients had been submitted to ttp with d. farinae extract for presenting non– ige-mediated allergic conditions. the ttp cohort counted 35 males; mean age 40.8 years; sd 20.5 years; range 3 to 86 years; modes = 94 (appeared five times); geometric mean = 33 years. olivier et al.; asian j. immunol., vol. 7, no. 1, pp. 90-99, 2024; article no.aji.117841 93 “these procedures were offered to patients with allergic rhinitis, allergic bronchitis, allergic conjunctivitis, atopic dermatitis, and/or urticaria with a normal range total ige, undetectable specific ige against d. farinae (investigated through immunocap®), and a non-reagent or inconclusive investigation performed with allergic skin tests done with the d. farinae extract” [27]. 2.2 antigen preparation “the d. farinae extracts were obtained from frozen cultures. the contents of the bottles containing mites (adults, nymphs, larvae, feces, and eggs) and culture medium were weighed and left at a rate of 10 ml of pbs buffer per gram of material (10%) by gentle magnetic stirring (1,000 rpm) for four hours at 4ºc. the material was centrifuged at 5,000 rpm for 30 minutes. the supernatant was kept apart. the sediment was resuspended in the same conditions as the previous step and stirred at four °c for 24 hours. the centrifugation was repeated, and the supernatants were mixed into an erlenmeyer flask. the solution was filtered through a double paper filter and later through a 0.2 μm pore-size filter with the help of a vacuum flask and a vacuum pump. the extract was dialyzed with the aid of the thermo fisher scientific snakeskin™ 88244 dialysis tubing in distilled water (1:50 ratio) with three water changes for 24 hours to eliminate molecules of low molecular mass (< 5,000 da). after dialysis, the extract was centrifuged at 10,000 rpm for 30 minutes at 4ºc and frozen at -40 ºc. the protein concentration was estimated spectrophotometrically and diluted to 1 mg/ml in saline (nacl 0.9%) to perform the allergic skin tests, lait, and ttp” [28]. 2.3 ex vivo investigation: leukocyte adherence inhibition test (lait) 2.3.1 procedure for allergen ex vivo challenging lait was performed as previously described [2939]. shortly, each donor's fresh plasma was divided into two parts and used in paralleled ex vivo challenging tests with d, farinae extract, and the unchallenged plasma assay. we collected the plasma with high leukocyte content (buffy coat) from the heparinized tube after one hour of sedimentation at 37 °c. then we distributed aliquots of 100 μl into eppendorf tubes kept under agitation for 30 minutes (200 rpm at 37 °c) with antigen extract (10μl of a solution with 1mg/ml and ph 7.5) or with the antigen dilution solution (when used as control). 2.3.2 procedure for adherence assay after incubation, the plasma was allocated into a standard neubauer hemocytometer counting chamber with a plain, non-metallic glass surface and left to stand for 2 hours at 37 °c in the humidified atmosphere of the covered water bath to allow leukocytes to adhere to the glass. next, we counted the leukocytes, removed the coverslip, and washed the chamber by immersion in a beaker with pbs at 37 °c. then, we added a drop of pbs to the hemocytometer's chamber and allocated a clean coverslip over it. the remaining cells were counted in the same squares as previously examined. 2.3.3 procedure for calculation the percentage of leukocyte adherence (la) of each assay was estimated as: (the number of leukocytes observed on the hemocytometry chamber after washing divided by the number of leukocytes observed on the hemocytometry chamber before washing) and multiplied by 100 (%). the leukocyte adherence ratio (lar) was estimated based on the ratio between the la from the antigen-specific challenged plasma and the la from the unchallenged control plasma: lar = la of the challenged sample divided by la of unchallenged control plasma multiplied by 100 (%). to further calculate the leukocyte adherence inhibition (lai), we subtracted the lar from 100 (%). we employed the lai results for the cascade distribution chart and the statistics calculations, both performed with the help of the microsoft excel® statistical package. 2.4 in vitro investigation: tube titration of precipitins (ttp) as previously reported, the semi-quantitative tube titration of precipitins (ttp) against d. farinae extract was performed in a transparent vitreous tube [40]. shortly, the patient's blood was collected in a clot-activator collecting tube. after separation, the serum was centrifugated at 2,000 rpm for 10 minutes. the allergen extracts were allocated in sets of eleven glass tubes at progressive duplicated serum dilutions. the progressive dilutions were combined with the 15 μl of the antigen (1 mg/ml) with 250 μl of the patient's serum, progressively diluted into physiological saline solution (nacl 0,9%) in the dilution ratios of 1:1; 1:2; 1:4; 1:8; 1:16; 1:32; 1:64; 1:128; 1:256; and 1:512. one tube was a blank control done with the water and serum to observe occasional spontaneous precipitation olivier et al.; asian j. immunol., vol. 7, no. 1, pp. 90-99, 2024; article no.aji.117841 94 (sia test) to detect circulating immune complexes [41]. after 24 hours, one of us examined the tubes, and the titers (the highest dilution factor yielding a positive reading) were recorded [42]. 3. results as a retrospective survey, there was no research protocol; therefore, we report the incidental immune investigation as registered in the digital medical charts. therefore, we could not establish a cross-comparison between positive and negative controls to validate the results. the ttp showed a wide distribution range, with four negative results and most positive results concentrated on the higher dilutions (fig. 1). the mean was estimated at 1:242; the median was 1:128; the standard deviation was estimated at 1:210; the mode was 1:512 (appeared 36 times). all sia tests were negative. the lai ranged from 0% to 100%. the mean was 52%; the median was 55.5%; the standard deviation was 23.6%; the mode was 63% (appeared seven times). the cascade distribution demonstrates a wide range of distribution of lai results (fig. 2). six patients ignored the presence of the allergen on the plasma and presented no inhibition of leukocyte adherence (lai = 0%) after contact with d. farinae extract (4.5% of the tests). some patients showed low or moderate immunoreactivity during the ex vivo challenge test, while others displayed strong immunoreactivity, which could reflect the participation of d. farinae allergens in a non–igemediated hypersensitivity condition. fig. 1. cascade distribution chart of the tube titration of precipitins (x-axis %) resulting from the d. farinae extract against the serum of a cohort of 100 tests/subjects (y-axis) fig. 2. cascade distribution chart of the range groups of leukocyte adherence inhibition (lai) results (x-axis %) of ex vivo d. farinae extract challenges monitored by the leukocyte adherence inhibition test (lait), according to the respective number of outcomes over a cohort with 134 tests/subjects (y-axis) olivier et al.; asian j. immunol., vol. 7, no. 1, pp. 90-99, 2024; article no.aji.117841 95 4. discussion diagnosing hypersensitivity reactions is essential in increasing patients' awareness of their sensitivities and commitment to avoiding allergens. the development of several lab methods to detect specific ige has raised concerns about the eviction of house dust mite allergens, mainly by chemical treatment and encasement methods for pillows and bed mattresses [43]. however, non-ige-mediated food-storage mite hypersensitivity may be a challenging diagnosis in the field of food allergies [44]. significant mite mortality and reduction of der f 1 fecal allergen were observed in carpets and mattresses after the treatment with hard surface steam cleaners [45]. some studies demonstrated that negative ions produced by ionizers kill dust mites and can be used to reduce mite populations on surfaces such as floors and clothes [46]. sublingual immunotherapy drops are a highly effective treatment for d. farinae allergy by increasing the subsets of t immune cells, specifically th17 cells and cd4+cd25+ regulatory t cells (treg cells), in peripheral blood [47]. before the discovery and dissemination of lab methods to detect allergen-specific ige, endotyping of allergic diseases was performed with the help of allergic skin tests, complement fixation assays, and the research of precipitins [48]. however, diagnosing non-ige-mediated hypersensitivity is difficult in medical practice since clinical analysis diagnostic laboratories do not offer specific tests to support this diagnosis, which is only possible in academic research institutions. an ex vivo challenge test with a viable leukocyte buffy coat, the lait allows the living interaction of all immune-circulating participants with the tested allergen, such as the innate and adaptive immune cells, cytokines, alarmins, and antibodies [49]. however, as an observant of the final phenomenon, the lait does not indicate which pathways were involved [26,50-52]. the ttp classically proves that patients produce circulating antibodies against the specific allergen added to the serum in sufficient quantities to produce immune complexes large enough to precipitate visibly at the bottom of the tubes. ttp is the most basic laboratory exam upon which immunology was established as a science [53,54]. this preliminary retrospective survey demonstrated extensive results from the ex vivo challenge test monitored by lait and the ttp with d. farinae in two heterogeneous cohorts of allergic patients. these exams mostly demonstrate that ige-mediated hypersensitivity is not responsible for the allergic diseases in these patients. these allergic diseases should instead be viewed as a conjoint of intricate immune hypersensitivity mechanisms that act conjointly to produce damage. endotyping these mechanisms helps to clarify the individual's immune response to better prescribe the management and the treatment in personalized medical care. we routinely employ the lait and the research of tube precipitins as a complementary triage test to select worthwhile antigens to proceed with more laborious in vivo provocation tests when the specific ige is undetectable. our results suggest that most allergic patients present some immunoreactivity against d. farinae allergens, while some do not. however, the complete significance of these results is not yet fully established. as an isolated piece of information, the lait immunoreactivity or the presence of precipitins do not prove that the tested antigens explain the allergic symptoms exactly. indeed, the clinical diagnosis must be accomplished by the in vivo challenges, the degree of colonization of the patient's environment, and the benefits of a change of ambient and an occasional desensitization treatment [55-57]. more studies with prospective larger double-blind cohorts need to evaluate the potential contribution of these methods to diagnosing patients suspected of d. farinae non–ige-mediated hypersensitivity. 5. conclusion our preliminary results support that the semiquantitative ttp and the lait have the potential to endotype and clinically discriminate diverse degrees of cellular and humoral non–igemediated immunoreactivity against d. farinae in allergic patients. this methodology can provide a socioeconomic impact since the technology to perform tial and ttp is inexpensive and can be performed in a single room attached to the facilities with minimum laboratory equipment. however, the propaedeutic meaning of these results must be better established, as well as the effects of possible interferents [58]. more studies focused on the quality-by-design approach, including prospective larger double-blind cohorts, need to evaluate the potential contribution of olivier et al.; asian j. immunol., vol. 7, no. 1, pp. 90-99, 2024; article no.aji.117841 96 lait and ttp for endotyping immunoreactivity of patients with allergic phenotypes suspected of presenting d. farinae non-ige-mediated hypersensitivity [59]. consent as a retrospective survey of results recorded incognito, consent was given collectively by the institution's ethics committee following the principles of the declaration of helsinki [60]. ethical approvals the authors have collected and preserved written ethical approval per international standards. acknowledgements the instituto alergoimuno de americana funded this work. we want to thank our new laboratory technician, alessandra vieira de oliveira, for participating in the exams. competing interests authors have declared that no competing interests exist. references 1. vackova t, pekar s, klimov pb, hubert j. sharing a bed 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immunological assays: causes, detection, and prevention. asian j immunol. 2024;7(1):71-78. 59. chiarentin l, gonçalves c, augusto c, miranda m, cardoso c, vitorino c. drilling into "quality by design" approach for analytical methods. crit rev anal chem. 2023;4:1-42. 60. world medical association declaration of helsinki: ethical principles for medical research involving human subjects. jama. 2013; 310(20):2191-4. _________________________________________________________________________________ © copyright (2024): author(s). the licensee is the journal publisher. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: https://www.sdiarticle5.com/review-history/117841 https://doi.org/10.9734/jammr/2021/v33i2231172 https://doi.org/10.9734/jammr/2021/v33i2231172 https://www.sdiarticle5.com/review-history/117841 _____________________________________________________________________________________________________ *corresponding author: e-mail: drasv@ymail.com; asian journal of immunology 3(1): 233-252, 2020; article no.aji.58422 acute immune mediated lung injury in covid 19: a review a. s. v. prasad1* 1 department of internal medicine, gandhi institute of technology and management, rishikonda, visakhapatnam, india. author’s contribution the sole author designed, analysed, interpreted and prepared the manuscript. article information editor(s): (1) dr. cynthia aracely alvizo báez, autonomous university of nuevo leon, mexico. reviewers: (1) ritika gupta, india. (2) abioye, adesoye idowu, university of ilorin, nigeria. complete peer review history: http://www.sdiarticle4.com/review-history/58422 received 01 june 2020 accepted 16 june 2020 published 23 june 2020 abstract there are many gaps in our present understanding of the of the sars cov 2 related matters like its pamps,(pathogen associated molecular patterns),antigenic profile, immune evasive mechanisms and also other host related matters, like prr s(pattern recognizing receptors) and the deranged host defense mechanisms, that cause self-damage. these constraints come in way of accurately delineating the pathogenesis of covid19 lung disease. hence is the speculative nature of any concept trying to explain the same. an integrated approach is embarked upon, taking into account the known clinical, radiological, laboratory, and autopsy findings, in search of clues that may suggest a possible mechanism, that explains the underlying lung damage in covid 19. it is seen that no single mechanism or syndrome could explain fully the pathology and pathogenesis of lung damage in covid19. hence, multiple mechanisms consistent with each known facet of the pathology are explored. thus the inflammatory damage of the alveolar tissue is sought to be explained by the3 complement activation pathways i.e. the alternative pathway, the mbl/lectin pathway/ and tissue factor/extrinsic pathway(of the classical complement activation), the contact cascade involving the kallikrein-kinin pathway, and the cytokine mediated pro and anti inflammatory mechanisms. the vascular pathology like hemorrhages and small blood vessel micro-thrombi as observed at autopsy , are viewed from the point of view of simple activation of the coagulation cascade to small vessel vasculitis (leucocytoclastic vasculitis) and coagulative micro angiopathy. besides, the role of tm-pc-epcr system (thrombomodulin-protein c-epcr system) is explored. the points in favour and against of each of the above are discussed.the central role played by the macrophage polymorphism is focused in the context of the simultaneous presence of review article prasad; aji, 3(1): 233-252, 2020; article no.aji.58422 234 active inflammation in the lung tissue and the interstetium and healing by interstitial fibrosis, seen in the lungs of covid 19 patients. the role played by the other humoral and cellular elements of both innate and adaptive immunity is briefly reviewed. the uniqueness and diversified features of covid 19 lung pathology, suggests two things that the immune mediated damage seems more probable than could be explained by the viral infectivity and that the pathology seems to stem from a mixture of different underlying and overlapping syndromes. hence, the author prefers to call all the covid 19 related features of lung pathology as “acute immune mediated lung injury". (aili) than trying to bunch them under a single syndrome. keywords: pathogen associated proteins (pamps); pathogen recognizing receptors (prrs) complement; cytokines; phagocytosis; antibody; ards; immune evasion; capillaritis; microangiopathy. 1. introduction the lung pathology and pathogenesis of covid 19, is elusive and hence remained speculative, till date. described originally as a ‘pneumonialike’ condition, similar to one caused by sars cov 1 (2003) and mers cov, (2011), which are members of the same family of corona virus. the former was less severe but caused pandemic and the later was 34% more severe,mortality-wise , but remained confined to middle east mostly. the covid 19, thus resembled more of sars 1 and hence the virus is named sars cov 2. in due course of time, the postscript, ‘like’ is dropped and the syndrome has come to be called ‘covid 19 pneumonia’, contrary to the facts. the opinions as to the cause of covid 19 induced lung damage varied from ards, shlh (secondary hemophagocytic lymphohistiocytosis) and cytokine storm from the clinical point of view and ‘interstitial pneumonia’ from radiological point of view. the autopsy findings raise suspicion of capillarites (small blood vessel vasculitis) to coagulative microangiopathy. it is felt that the clinical as well as laboratory, radiological and autopsy data if taken together sheds some light as to the exact lung pathology and pathogenesis of covid 19 lung disease. hence these aspects are briefly reviewed in search of clues to the lung pathology/pathogenesis of covid 19. 2. clinical data 2.1 pneumonialike condition of covid 19 vs ards li, x., ma, x [1] has elaborately discussed the matter and could make out some differences between the two conditions. peter g gibson et al. 2020) [2] have to say " we are familiar with ards, however when it occurs as part of covid-19, it has different features and there remain, unanswered questions". wu c, chen x, cai y, xia j, zhou x, xu s (2020) suggest that among the patients affected, the pneumonia-like process of covid 19. unto 20% only develop pneumonia of which, about 3 to 4% develop severe form necessitating ventilator support. ~50% develop hypoxemia by day 8. severe illness and cytokine release syndrome appear to develop mostly within 5–10 days after the onset of symptoms in susceptible patients. ardslike clinical picture is suggested to develop in 42% of patients presenting with covid-19 pneumonia, and 61-81% of those requiring icu care [3]. puah sh et al. report that covid-19 ards follows a predictable time course over days, with median time to incubation period of 8.5 days after onset of symptom in singaporean patients [4]. not all covid 19 patients suffer pneumonia like syndrome. even though ali of those who develop this very few of them develop severe respiratory distress, necessitating ventilator support. most of them have comorbid diseases and constitute to the 2 to 3% mortality observed. the autopsy findings which suggested ards are the firm, heavy lung filled with fluid and presence on bits of hyaline membrane in the lungs. severe illness and cytokine release syndrome appear to develop mostly within 5–10 days after symptom onset in susceptible patients. not all covid 19 patients suffer pneumonia like syndrome. even though those who develop this very few develop severe respiratory distress, necessitating ventilator support. most of them have comorbid diseases and constitute the 2 to 3% mortality observed. the autopsy findings which suggested ards are the firm, heavy lung filled with fluid and presence on bits of hyaline membrane in the lungs. peter g gibson et al opine that most of the covid-19 patients, meet the berlin definition (2012) for ards. all of them have present with an acute clinical emergency, with bilateral opacities in the lung imaging, gas exchange abnormalities, and their disease can't be explained by heart failure or volume overload. the ards berlin criteria defined that for a prasad; aji, 3(1): 233-252, 2020; article no.aji.58422 235 patient to be diagnosed as having ards, the onset must be within 1 week of a known clinical insult or new or worsening respiratory symptoms. [5], wang et al. [6] reported median timed onset of ards, as 8.0 days, zhou et al. [7] as 12 days. chen et al. [8] and guan et al. [9] did not report the onset of ards. the lowest median time reported is 4 days from the onset of first symptom. the main departure from ards and sars cov2 pneumonia is the preserved lung compliance in face of severe hypoxia, and poor oxygenation without increased work of breathing, which are not seen in ards. it is also seen that ards developed in some covid 19 patients after they have been put on ventilator, sparking speculation as to whether such development is due to natural progression of the covid 19 or ventilator induced. robba c, battaglini d patroniti n [10] et al. suggested 3 phenotypes of patients, basing on their ct findings of lungs in covid 19 (discussed under imaging features, below). they opine that not all cases of covid lung involvement resemble ards. gattinoni et al. [11] described two distinct phenotypes, type l and type h (type 1 and type 2). type l disease, is characterized by normal lung compliance and gas volume in the presence of hypoxemia. these patients may improve, or they may worsen. about 20% to 30% of patients had or evolved to type h disease, characterized by worse lung compliance and increased edema and lung weight. "the transition from type l to type h may be due to the evolution of the covid-19 pneumonia on one hand and the injury attributable to high-stress ventilation on the other". sars of covid 2 vs (shlh)/cytokine storm: macrophage activation syndrome (mas), also known as secondary haemo phagocytic lympho histocytosis (shlh) or cytokine storm is distinguished by: 1. absent haemophagocytosis 2. lower ferritin levels. 3. coagulopathy not being due to liver synthesized factors. 4. dic is seen as a terminal event in some covid 19 patients only even though hypercytokinemia. 2.2 radiology / imaging data the imaging technics have high sensitivity of about 97% when tested within 5 days of rt pcr became positive and still can detect the changes as early as 3 days, but 56% are found to be normal when imaged in 2days of patient becoming covid 19 positive. thus the sensitivity of imaging the lungs in covid 19, is a function of time of doing the test. c. hani m.-p. revel et al. (2020) extensively reviewed not only the ct findings of covid 19, but other causes of differential diagnosis of the typical ground glass shadows seen in imaging of covid 19 lung [12]. some of the important radiological findings are presented below. for differential diagnosis the readers may refer to the reference suggested. 1. bilateral ground glass appearance which are round in 50% cases. they are called covid balls 2. areas of focal consolidation. 3. crazy paving pattern resulting from intralobular reticulations. 4. late signs: signs suggesting organizing pneumonia. 1. linear consolidations 2. reverse halo sign i.e., areas of groundglass surrounded by peripheral consolidation. study by salehi et al. revealed that the frequencies of the different ct abnormalities were as follows: ggo (central ground glass opacity) was observed in 88.0% of patients, consolidation in 31.8%, bilateral involvement in 87.5% and peripheral distribution in 76.0% of patients [13]. guan cs, et al. (2020) assert that “ground-glass opacities may be due to mild oedema of the alveolar septi, hyperplasia of the interstitium, partial filling of airspaces, or a combination of these features. besides, the crazy-paving pattern may correlate with hyperplasia of interlobular and intralobular interstitia” [14]. the two radiological features still alluding explanation are 1. peripheral and subpleural distribution 2. bilateral lower lobe involvement autopsy findings: xu, l. shi, y. wang, et al. [15] described in detail, the post-mortem findings of covid 19. gross pathology: the lungs are firm and increased in weight and laden with secretions and exudate. both the surface of lung and the cut section showed, patchy areas of haemorrhage. microscopic findings: these can be grouped as cellular, vascular and other features. prasad; aji, 3(1): 233-252, 2020; article no.aji.58422 236 the cellular exudate consisted of cd4+ and cd8+ lymphocytes in the lung tissue and interstetium and with some perivascular collection. also present are platelet cells, 65+ atypical megakaryocytes and desquamated type 2 pneumatocytes showing cytopathic effect. neutrophils are absent except in the immunosuppressed case. vascular features: small blood vessels (capillaries) are found to be thickened, with oedematous walls, dilated and showed increased permeability with consequent seepage of plasma into the lung tissue. there is lymphocytic accumulation around the capillaries and micro throbi inside the capillaries. the bigger pulmonary vessels near hilum are not affected. 2.3 other autopsy findings 1. focal presence of haemorrhagic. patches, both in gross appearance and cut section of lung. 2. focal greyare as of consolidation 3. presence of clots in the lung tissue. 4. scattered hyaline membrane and fibrin threads. 3. laboratory data the laboratory tests found to be positive in covid 19 also give some clues as to the underlying mechanisms involved in the pathology and parthenogenesis of covid 19, if interpreted properly. the involvement of liver, kidney, and heart is indicated by the increase in lab findings pointing in that direction. probably they represent subclinical involvement of the organs concerned, as overt manifestation of their involvement is seen only in a few of the patients and some in the later phase than earlier phage of the disease evolution. leuko-erythroblastic reaction reported by some is a finding that needs to be investigated.the same is explained as probable involvement of bone marrowis the increased prothrombin time (pt) indicates the involvement of the extrinsic coagulative pathway as the cause of thrombotic phenomena observed in the small blood vessels at autopsy. the presence of d dimers, fdp, to the underlying thrombolytic (fibrinolytic) process involving capillaries of the lungs. of interest, is the observation of lymphopenia whereas lymphocytosis is commonly seen in all other viral infections. the lymphopenia is connected to increased severity / mortality in covid 19 patients. the lab parameters also vary as the disease progresses. the platelets, the neutrophils and cd8+t cells are found to be normal in patients not admitted to icu, where as neutrophilia and low platelets and cd 8+ t cells counts are seen in icu pts. the icu patients have increased risk of complications and mortality. the risk stratification of covid 19 pneumonia patients has been analyzed by janusz jankowski t al. the lab tests found positive in covid 19 patients irrespective of their stage of disease, and what they suggest are tabulated and presented in appendix 3. 4. discussion any envisaged pathogenic mechanism should explain all the clues available from the clinicopathological data reviewed above. to put it succinctly, such concept has a clear beginning (in the form of virus-host interaction) a clear destination (the pathology elucidated by autopsy findings), a clear source (the normal physiological mechanisms). what is to be clinched is the means by which the normal physiology is turned into pathology in the context of covid 19. the gaps in the present understanding of the issues involved, leaves no alternative than to ‘hazard a guess ‘as to the actual pathogenesis of the disease. the viral factors: the genome of sars cov 2 encodes four main structural proteins: spike (s), (which has s1 and s2 sub-units) envelope (e), nucleocapsid (n) and membrane (m) proteins .it is known that the sars cov 2 attaches through spike protein to the cell surface negotiate the ace 2 receptor of the host cell, to gain entry into the body. it is even suggested that by internalizing the ace 2 receptor down regulates its protective lung function, prevailing under normal physiological conditions the difference in degree of severity and infectivity seen between the different countries and within certain states of a country, lead to the conclusion that there are different strains of the covid 19 virus and that the virus suffered nearly 200 mutations so far. the worldwide distribution belonged to strain types a2 a, a3, b and bb4. the ccmb (centre for cellular and molecular biology), hyderabad, india, found that a different 'cluster of strains’ , named as “clade a3i", responsible for infection in different states in india, some of which registered more cases / mortality ( maharashtra, gujarat , tamil nadu and delhi) than others. (vide the pre-publication press note released on 3 rd june 2020). the host factors: the fight between the virus and the defence begins with the virus presenting prasad; aji, 3(1): 233-252, 2020; article no.aji.58422 237 its conserved molecular signature expressed on its surface (pamps) which the defence cells recognize by special receptors located on their surface (prrs) pprs and pamps: pprs (pattern recognizing receptors) are proteins which recognize pamps (pattern associated molecular patterns), a conserved specific sequence which the pathogens display on their surface (epitopes) for the recognition by the prrs of the effector immune cells the details of the pamps and prrs in case of covid 19 are still not fully elucidated. some authors dealing with this aspect, quote the information available on the sars cov 1, which holds about 79% homology in antigenic structure with sars cov 2. the sofar known prrs and pamps are listed in appendix 1, with the assumption that any of these might be found relevant to the context of caved 19, by future research. the possible interactive pathway between the pamps and prrs is depicted in appendix 2. the recognized viral surface antigens are presented by the antigen presenting cells (arc) of the effect or immune cellular elements after being processed by mhc class 2 elements. here also clarity as to details are lacking. this is followed by the humoral or cellular response by both innate and adaptive immunity systems, the sole purpose being destruction and elimination of the virus. here also gaps in current knowledge is considered as hindrance to the understanding of actual happening in the context of covid 19 pathogenicity. the virus probably and ingeniously turns the host’s defense, against the host itself by various subversive actions, to far then their own survival and multiplication, resulting in immune mediated injury to host tissues the covid 19 lung pathology should be considered in these lines, as a fallout of the aberrant immune response. the reported damage to the lungs is belied to start within a week or so of the patient becoming symptomatic, the role played by the innate immune mechanisms assume importance, as they first encounter the virus. it is only after a week or so, that the adaptive immune mechanisms come to fore. so, in the later course of the disease, both the immunity mechanisms may supplement or compliment or act independently to sustain the immunological damage. the humoral and cellular components of both system play important roles but some play greater role than the others. so the more important elements are discussed in detail while with due consideration to the subsidiary role played by the other elements, discussion is limited to a brief consideration about them. the discussion is in accordance with the importance of the role each element of both the systems. the discussion is limited only to the destructive immune mechanisms of both the immunity system and nothing will be referred to the protective role they play. 4.1 the role of innate immune system 4.1.1 role of complement: the alternative pathway: joshua m. thurman and v. michael holers, et al. [16] has reviewed this matter: 1. the alternative pathway is capable of auto activation because of a process termed “tick over” of c3 [17]. 2. tick over generates a conformationally altered c3, designated c3(h2o). 3. this is capable of binding, the factor b 4. factor d cleaves factor b into ba and bb. 5. bb remains bound to c3(h2o) to form c3(h2o)bb.( fluid-phase c3-convertase). 6. this alternative pathway c3-convertase, although only produced in small amounts, can cleave multiple c3 proteins into c3a and c3b. 7. the complex is believed to be unstable until it binds properd in, a serum protein. 8. the addition of properd in forms the complex c3bbbp, a stable compound. 9. this binds an additional c3b to form alternative pathway c5-convertase. 10. the c5-convertase of the alternative pathway consists of (c3b)2bbp (sometimes referred to as c3b2bb). 11. after this step, the complement system follows the same path regardless of the means of activation (alternative, classical, or lectin). 12. c5-convertase cleaves c5 into c5a and c5b. 13. c5b binds sequentially to c6, c7, c8 and then to multiple molecules of c9 to form membrane attack complex. (mac). regulatory proteins that disrupt the complement activation process: since c3b is free and abundant in the plasma, it can bind to either a host cell or a pathogen surface antigen. thus, the complement activation can kill a virus infected cell or even the healthy host cells. to prevent the host cell damage, there are several inbuilt inhibitory mechanisms operating under the prasad; aji, 3(1): 233-252, 2020; article no.aji.58422 238 physiological conditions. conversely the virus may subvert the very same mechanisms to sustain its own survival as seen in case of some the virus (vide infra). 4.1.2 physiological inhibitors of the alternative complement cascade o complement receptor 1 (cr1) or cd35) and daf (decay accelerating factor also known as cd55) completes with factor b in binding with c3b on the cell surface and can even remove bb from an already formed c3bbb complex. o complement factor(cf), a plasma protease, prevents the formation of a c3 convertasewhich cleaves c3b into its inactive form, ic3b. factor i requires a c3bbinding protein cofactor such as complement factor h, cr1, or membrane cofactor of proteolysis (mcp or cd46) [18]. o complement factor h can inhibit the formation of the c3 convertase by competing with factor b for binding to c3c3bl [19] accelerate the decay of the c3 convertase. [20] and act as a cofactor for factor i-mediated cleavage of c3b [21]. complement factor h preferentially binds to vertebrate cells (because of affinity for sialic acid residues), allowing preferential protection of host (as opposed to bacterial) cells from complement-mediated damage. o cfhr5 (complement factor h-related protein [22]. immune evasion by the virus: the lipid bi-layer membrane of covid 19 is cited as the cause of failure of host’s immune system. beyond this there is no data as to how actually sazrscov2 evades the immune system. other others have narrowed the analogy with sars cov 1 and mers cov. the author cites few examples of other virus whose immune evading mechanism was studied and reported. the future research is expected to throw more light on this aspect. 4.1.3 illustrations of how some virus subvert the defence against self cell destruction 1. synthesis of new regulatory proteins illustration: west nile virus synthesizes two isoforms of ns1 protein to regulate c3. soluble ns1 increases factor i-mediated cleavage of c3b to ic3b while the cell surface-bound ns1 decreases deposition of c3b and mac [23]. 2. incorporation of the host complement regulatory proteins to virions: to evade complement-mediated destruction , human immunodeficiency virus-1 (hiv-1), human t-lymphotropic virus-1 (htlv-1) and human cytomegalovirus (hcmv) incorporate the complement controlled proteins cd55 and cd59 into their virions to circumvent the complement response [24,25]. 3. prevention of the cell lysis by inhibiting the remainder of the complement cascade illustration: hiv enters human cd4+ t cells through complement receptors. hiv gp41 and gp120 proteins activate complement through the classical and lectin pathways, respectively. at the same time, the above two proteins inhibit mac formation by recruiting factor h and cd59 to the surface of the virally-infected host cell to abolish complement-mediated lysis [26]. 4. modulate cytokine expression to induce a pro-coagulant state illustration: mediated by il-1(interleukin) , tnfα,(tumour necrosis factor) and il-6, marburg virus, ebola virus and hanta virus induce tissue factor expression on the endothelial surface [27,28]. 5. use coagulation factors to enhance viral binding and replication illustration: human species a adenovirus-18 (hadv-18) and 31(hadv-31). it is known that the innate immune reaction recruits the alternate complement activation pathway and the adaptive immune response involves the classical complement activation pathway. there is a third, the lectin pathway. the alternate complementary pathway starts with cleaving of c3. the classical pathway and the lectin pathway both converge on production of c3 by a loop. 4.1.4 complement mediated immune damage 4.1.4.1 explanation for interstitial pneumonia in lung pathology 1. c3 is cleaved into c3b(catalytic fragment) and c3bi (non catalytic fragment). further events may follow either of the following pathways. 2. the c3i pathway: c3bi is bound to cr 3 (cd11b/cd18), integrin αmβ2) receptor of the macrophage. 3. affinity modulation of macrophage integrins is sufficient to allow binding of prasad; aji, 3(1): 233-252, 2020; article no.aji.58422 239 opsonized particles, but increased diffusion to allow clustering is required in order to activate phagocytosis, which is facilitated by cytokines il-4, m-csf, tnf-α and gmcsf. c3b pathway: this catalytic fragments continues the cascade of alternate complement activation pathway with ultimate production of mac complex which cause the lysis and death of the infected cell including the virus. the complement receptor activated stimulation will not elicited inflammatory reaction as it cannot recruit the pro-inflammatory cytokines and chemokines. it could be inferred from the polymorphism (see below) exhibited by the alveolar macrophages that the complement mediated perhaps stimulates the alternatively activated m2 type macrophages which are known to be anti inflammatory in response. this could come as handicap to the host because, m2 type stimulation also stimulates the extracellular matrix protein, collagen, which produces interstitial fibrosis as seen in the covid 19 lung. thus one of the pathological / radiological feature of interstitial pneumonia/ fibrosis is explained. secondly since this inhibits the expression of the inflammatory mediated m1 type macrophages. absence of inflammatory stimulus may make the pt in whom this pathway acts asymptomatic but yet the lung damage in the form of interstitial fibrosis progresses. it is known that the symptoms of covid 19 are due to inflammatory response mounted by the body of the hostpatient. role of tissue factor(tf)/(extrinsic) pathway in the pathogenesis of covid 19:  the increase pt (prothrombin time) and injury to subendothelial tissues by cytokine induced vascular damage implicate a role of this pathway in the coagulopathy seen in covid 19. tissue factor(extrinsic) path way:  tissue factor/plateletthromboplastin/ factor3/ cd 135. it is produced by sub endothelial cells and leukocytes. injury to sub endothelial cells/ platelets releases this factor which initiates the coagulation process. in combination with factor v11, it activates factor x to factor xa. the (tissue factor pathway inhibitor (tfpi) is a protease present in the ecs inhibits the tf and is a natural anticoagulant. tf, produced by sub-endothelial cells and leukocytesinjury to sub-endothelial cells/ platelets initiates the coagulation process. in combination with factor v11, it activates factor x to factor xa. the (tissue factor pathway inhibitor (tfpi) is a protease present in the ecs inhibits the tf and is a natural anticoagulant. the tf pathway may be recruited by the cytokine induced damage of the vasculature orinjury to the platelets which consequently release the tf. this is supported by the immature megakaryocytes(indicating rapid turnover/distruction of the platlets) found in the microscopic findings of autopsy. the role of mbl (mannose binding leptin) / lectin pathway: the activation of complement via the mannan-binding lectin (mbl) pathway is initiated by the mbl complex consisting of the carbohydrate binding molecule, mbl, two associated serine proteases, masp-1(mannoseassociated serine protease) and masp-2, and a third protein, masp-19. when the carbohydraterecognising heads of mbl bind to specifically arranged mannose residues on the surface of a pathogen, masp-1 and masp-2 are activated to cleave complement components c4 and c2 into c4a, c4b, c2a, and c2b. in f, two smaller mblassociated proteins (maps) are found in complex with mblc4b2a3b, the c3 esterase so formed carries onwards the complement cascade in the same way as the alternative pathway described in detail above. the involvement of the lectin pathology is supported by the finding reported by cynthia magro (2020)of deposits of terminal complement components c5b-9 (membrane attack complex), c4d, and mannose binding lectin (mbl)-associated serine protease (masp)2, in the microvasculature, leptin is a glycoprotein and the sars cov has glycoprotein antigens which may be recognized by the c lectin receptors (pprs) present on the surface of the macrophages and other effector cells ,leading to their activation leptin can modulate the response to an inflammatory challenge by altering production of proinflammatory and antiinflammatory cytokines and may also affect cytokine signalling by a variety of mechanisms, including induction of socs-3.(suppressor of cytokine signalling). 4.1.5 explanation for vascular changes in lung pathology: these can be explained by activation of the contact cascade or by cytokine induced damage. https://en.m.wikipedia.org/wiki/complement_component_4 https://en.m.wikipedia.org/wiki/complement_component_2 prasad; aji, 3(1): 233-252, 2020; article no.aji.58422 240  contact cascade 1) factor xii (fxii; hageman factor) of the contact system is proteolyticallycleaved to fxiia by negatively charged surfaces of damaged cells, and also activated platelet plasma membrane. 2) fxiiainitiates the coagulation cascade leading to clot formation. 3) cleaves prekallikrein to kallikreinfor subsequent release of bradykinin. 4) through an endothelial g-coupled receptor (bradykinin receptor 1; bkr1), bradykinin induces vasodilation, neutrophil chemotaxis and vascular permeability [29].  the cytokine induced vascular changes in the lungs: alexander h. sprague and raouf a. khalil et al. [30] and. marlies van de, wouwer désiré colle, edward m. conway metal [31] have extensively reviewed this subject matter. interested readers may refer these articles for full details. cytokines are a diverse group of soluble short acting proteins, glycoproteins and peptides produced by various immune cells and vascular cells, activate specific receptors and modulate the functions of many cells and tissues.  cytokines include tumour necrosis factors, interleukins, lymphokines, monokines, chemokines, interferons, colony stimulating factors, and transforming growth factors.  cytokines are produced by macrophages, t cells and monocytes, platelets, endothelial cells (ecs) and vascular smooth muscle cells (vsmcs).  cytokines elicit inflammatory response by interacting with specific receptors on various cell types and activate jak-stat, (janus kinase/signal transducers and activators of transcription nf-κb, and smad (suppressor of mothers against decapentaplegic. miscellaneous)signalling pathways leading to cell adhesion, permeability and apoptosis  cytokines also interact with mitochondria to increase the production of reactive oxygen species.  cytokine-induced activation of these pathways in ecs modifies the production/ activity of vasodilator mediators such nitric oxide, prostacyclin, endotheliumderived hyperpolarizing factor, bradykinin, vasocontractile mediators: endothelin , angiotensin ii.  cytokines interact with vsmcs(vascular smooth muscle cells) to activate ca2+, protein kinase c, rho-kinase, and mapk pathways, which promote cell growth and migration, and vsm reactivity. cytokines are either proinflammatory or inflammatory.  proinflammatory cytokines: produced by activated macrophages, mediate the following effectsup regulation of inflammatory reaction by tnf-α, il-1, il6, il-12, il-19, and ifn-β.stimulation of acute phase reactants tnf-α, il-1, il-6, il-11, ifn-γ, tgf-β.chemoattractant such as il-8, mip-1α (macrophage inflammatory proteins) ,mip-1β, rantes(regulated on activation, normal t cell expressed and secreted) pf-4, mcp-1, -2, -3(monocyte chemoattractant protein)  anti-inflammatory cytokines are involved in the down-regulation of inflammatory reactions. they include il-4, il-10, il-13, ifn-α, and tgfβ. (transforming growth factor) the antiinflammation induced vascular injury results from interaction of the inflammatory cells, the endothelial cells(ec), vascular smooth muscle cells(vsmc) and extra-cellular matrix (ecm).  ecs are major determinant of vascular tone, leukocyte adhesion, and smc proliferation. il1activates t cells;il-2, which stimulates proliferation of antigenactivated t and b cells; il-4, il-5, and il-6, which stimulate proliferation and differentiation of b cells;  ifnγ, which activates macrophages; and il-3, il-7 tnfα, ifn-γ, il-8, and mcp-1 influenced tissue factors which initiated coagulation cascade and down-regulated anticoagulant thrombomodulin.  tnf-α enhanced the endothelial cells to produce anti-fibrinolysis pai-1 [32]. gmcsf, which stimulate haematopoiesis. platelet derived growth factors were the main inducers of mcp-1 gene [33].  the activated platelets stimulated nf-κb in endothelial cells and enhanced the expression of leukocyte receptors which induced the secretion of mcp-1 and il-8. besides, the significant increased expression of lung par-1 on pulmonary cells, as fibroblasts, macrophages, epithelial and endothelial cells might prasad; aji, 3(1): 233-252, 2020; article no.aji.58422 241 represent another cause for elevated mcp-1 (monocyte chemoattractant protein and il-8 chemotaxis molecules [34]. role of endothelial cells: under normal conditions, the endothelium maintains a vasodilator, antithrombotic and anti-inflammatory state. for vascular homeostasis, endothelial cells are of utmost importance and they produce a variety of mediators, surface proteins, and autocoids involved in vasomotion, coagulation, and inflammation [35]. endothelium separates blood clotting factors from exposure to subendothelial prothrombotic extracellular matrix components. endothelium also expresses vasoactive factors that modulate platelet reactivity, coagulation, fibrinolysis and vascular contractility, all of which contribute to thrombotic formation. such factors include nitric oxide, prostacyclin, von willebrand factor (vwf), thrombomodulin, endothelin, etc. endothelial cells counteract coagulation by providing tissue factor and thrombin inhibitors and receptors for protein c activation. role of tnf alfa and c3: when tnf-α is upregulated, it contributes to changes in coagulation and c3 induction [36]. tnf-α plays a pivotal role in the disruption of macrovascular and microvascular circulation both in vivo and in vitro [37] and is an important cytokine that can induce both apoptosis and inflammation [38]. in the presence of ros, there is an increased production of tnf-α and, in turn, tnf-α signalling accentuates oxidative stress [39]. tnfα up regulation is also associated with a changed coagulation propensity [40]. in short, tnf-α participates in vasodilatation and oedema formation, as well as leukocyte adhesion to the epithelium through expression of adhesion molecules. furthermore, it regulates blood coagulation, contributes to oxidative stress at sites of inflammation, and indirectly induces fever [41]. tnf-α also plays a central role in the pathogenesis of insulin-resistant metabolic derangements. when tnf-α is upregulated, it contributes to changes in coagulation and c3 induction. tnf-α plays a pivotal role in the disruption of macro vascular and microvascular circulation both in vivo and in vitro and is an important cytokine that can induce both apoptosis and inflammation. in the presence of ros, there is an increased production of tnf-α and, in turn, tnf-α signalling accentuates oxidative stress. tnf-α upregulation is also associated with a changed coagulation propensity. in short, tnf-α participates in vasodilatation and oedema formation, as well as leukocyte adhesion to the epithelium through expression of adhesion molecules. furthermore, it regulates blood coagulation, contributes to oxidative stress at sites of inflammation, and indirectly induces fever. tnf-α also plays a central role in the pathogenesis of insulin-resistant metabolic derangements. tnf can induce platelet consumption, and platelets do express tnfr1 and tnfr2 [42,43]. tnfr1 expressed on other cells also causes the release of factors with agonist activity for platelets and tnf-α is able to activate platelets through stimulation of the arachidonic acid pathway. complement c3: rbcs carry the complement receptor 1 (cr1), also known as c3b/c4b receptor or cd35, on its membrane [44]. immune complexes, which have reacted with complement and bear c3b fragments also bind to the cr1 on human rbcs, and cr1 on rbcs serves as a transport system for immune complexes in the circulation to prevent immune complex deposition outside the fixed macrophage system [45,46]. complement also interacts with the surface of activated platelets as well as with other components of the complement system including, c1q, c4, c3 and c9, which bind to activated platelets, [47]. furthermore, thrombinactivated platelets can actually initiate the complement cascade, [48] and c3a and its derivative c3a-des-arg, induce platelet activation and aggregation in vitro [49]. platelets express complement receptors c3ar, cr4, as well as a receptor for ic3b and c5a, and the c1q receptors gc1qr and cc1qr on their membranes. cc1qr, in particular, was shown to mediate platelet aggregating and activating effects. of importance is that platelets may also interact with the complement system via proteins that are not considered classical complement receptors, such as p-selectin [50] or gp1bα [51]. tm-ps-epcr system: marlies van de wouwdésiré collen and edward m. conway et al extensively reviewed this aspect [52] to which interested readers may refer for more details. thrombin-mediates activation of protein c (pc), with thrombomodulin (tm), acting as a cofaactor. thrombomodulin, a cell surfaceexpressed glycoprotein, synthesized by vascular endothelial cells, is critical for pc activation y the thrombin–tm complex is further enhanced ≈20 prasad; aji, 3(1): 233-252, 2020; article no.aji.58422 242 fold in vivo when pc is bound to the endothelial cell protein c receptor (epcr). platelet factor 4 (pf4) accelerates pc activation by inducing a conformational change that increases its affinity for thrombin–tm complex .activated pc (apc) is a known natural anticoagulant. apc and epcr have a role not only in coagulation but also in inflammation also apc suppress further thrombin formation by proteolytic inactivation of the coagulation factorsva and viiia. along with protein s (ps), apc may also increase fibrinolytic activity, by neutralizing plasminogen activator inhibitor 1 (pai-1). this results in a hypercoagulable state. pro-inflammatory cellular effects of coagulation proteases as well as the anti-inflammatory effects of apc/epcr are mediated by signalling via protease activated receptors par on mononuclear cells, endothelial cells, platelets, fibroblast, and smooth muscle cells. the beneficial effects of apc in sepsis are mainly dependent on the par-mediated cellprotective properties rather than the anticoagulant protease function on coagulation cofactors fv/va and fviii/viiia. protein c, which is activated by thrombin, complexes with endothelial protein c receptor and thrombomodulin and together with protein s forms the activated protein c complex that inactivates activated coagulation factors v and viii. the receptor par-1 is differentially activated by thrombin and the activated protein c/epcr complex, resulting in antithrombotic and anti-inflammatory effects. thrombin and vasoactive agents release von willebr and factor as ultra-large platelet-binding multimers, which are cleaved by adamts13. platelets can also facilitate leukocyte-endothelium interaction. platelet activation is prevented by nitric oxide, prostacyclin and exonucleotidases. thrombin-cleaved adamts disintegration of platelet aggregates while tissue-type plasminogen activator initiates fibrinolysis. fibrin and products of platelets and inflammatory cells modulate the angiogenic response of endothelial cells and contribute to tissue repair. endothelial pars (protease activated receptors) participate in the regulation of vascular tone and permeability. in endothelial cells, pars play a key role in promotion vascular barrier function as they provide a positive signals for endothelial adhesion molecules (vascular cell adhesion molecule-1 (vcam-1), intercellular adhesion molecule-1(icam-1), and e-selectin [53]. pars contribute to the pro-inflammatory response. for example par4 induces leukocyte migration and par2 helps macrophages to produce cytokines such as interleukin-8 (il-8). activation of pars alternatively lead to the transactivation of and signalling through receptors such as colocalized pars, ion channels, and toll-like receptors. 4.1.6 role of other elements of innate immunity 4.1.6.1 role of the interferons interferons(ifns) are a group of signalling proteins made and released by host cells in response to the presence of several viruses. type i interferons (ifn-alpha and ifnbeta) are secreted by virus-infected cells while type ii, immune or gamma interferon (ifn-gamma) is mainly secreted by components of both innate and adaptive immunity t cells (of adaptive immunity), natural killer (nk) cells and macrophages (of innate immunity). ifnγ, a cytokine which is crucial for innate and adaptive immunity against many pathogens is produced: 1. as a part of the innate immune response: natural killer (nk) natural killer t (nkt) cells mucosal epithelial cells, macrophages innate lymphoid cells(ilc) produce it . 2. as a part of adaptive immunity: cd4 th1 cells, cd8 cytotoxic t lymphocyte (ctl) produce it. 4.1.7 functions it serves  macrophage activation increases their antigen presentation and lysosome activity.  increased expression of class i and class ii mhc molecules.  increased expression of apcs (antigenpresenting cells) through induction of antigen processing genes, including subunits of the immunoproteasome (mecl1, lmp2, lmp7), tap and eraap and direct upregulation of mhc heavy chains and b2-microglobulin itself.  role in macrophagepolymorphism: m1 macrophages are stimulated by interferon (ifn)-γ which secrete proinflammatory cytokines (like tnf-α, il-1β, il-12, il-18) prasad; aji, 3(1): 233-252, 2020; article no.aji.58422 243 and the chemokines. (like ccl15, ccl20, cxcl8-11 and cxcl13).  antiviral (replicatory), immuno-regulatory, and anti-tumour properties [19].  aberrant ifnγ expression causes number or autoimmune diseases.  activates inducible nitric oxide synthase (inos).  induces production of igg2a and igg3 from activated plasma b cell.  promotes adhesion and binding required for leukocyte migration.  primes alveolar macrophages against secondary bacterial infections. how ifnγ exerts its cellular responses? 1. it interacts with interferon gamma receptor 1 (ifngr1) and interferon gamma receptor 2(ifngr2). 2. binding activates jak-stat pathway. 3. ifnγ also binds to the glycosaminogly can heparan sulphate (hs) and inhibits its biological activity. 4. promotes nk cell activity. ifn-α has a general inflammatory action which skews the immune response towards a th1 profile, which leads to induction of classically activated m1 macrophages.(see macrophage polymorphism below) 1. ifn α functions 1. ifn-α8 enhances the proliferation of human b cells, and activates nk cells. the subtypes α10 and α2, and α8, are the most efficient nk cell activators. 2. subtypes α21 and α2 enhance the expression of ifn-gamma inducible protein-10 (ip10),a chemokine, that promotes th1 inflammatory response. in dendritic cells. 3. ifn-α1 causes increased hla-ii expression and can directly inhibit tumour cell growth in vitro. 4. subtype α2 increases the expression of hla-i molecules, which correlates with ifn-α-mediated activation of memory cd8 cells and increased catalytic action against virally infected cells and tumour cells (via cytotoxic cd8 cells). interferon beta: it is released at the end of an immune attack, blocks the action of gamma interferon and helps to reduce inflammation and the body's immune reaction. role of other important innate cellular elements: these are presented in appendix 4  role of adoptive immunity in covid lung pathology: cd8+ t cells and cd 4+ at cells: the autopsy findings considered above, showed that cd8+and cd4+ cells in the lung tissue interstetium. cd 8+ cd8+ t cells directly kill the virus upon stimulation. cd 4+ cells helps to secrete antibodies which have diverse actions. ali ganji et al. [53] have shown that cd8 mfi increased significantly in covid-19 infected patients (p < 0.05), implying increased expression of cd 8+ t cells. zheng et al. [54] found that the total numbers of t cells, nk cells and ctls were reduced in all patients, with severe cases of covid 19 having significantly lower proportions than those seen in mild cases. cd8+ t and nk cells from covid-19 patients had increased expression of the inhibitory receptor nkg2a. furthermore, cells expressing nkg2a had diminished production of cd107a, ifn-γ, il-2, tnf-α and granzyme b. these findings suggest functional exhaustion of nk and cd8+ t cells and inhibition of antiviral immunity during sars-cov-2 infection. it was opined that down regulation of nkg2a may be crucial for disease control.  helper t cells: type 1 helper (th1), cells produce interleukin (il)-2, gammainterferon (ifn-gamma) and tumour necrosis factor-beta, (pro-inflammatory). th1 activate classically activated m1 macrophages. cells are cytotoxic and hence kill the virus laden cells, when activated. the zcd4+cells helps to stimulate b-cell function. type 2 helper (th2) cells express il-4, il-5, il-6 and il-10 (anti-inflammatory). th2 cells activate alternately activate m2 macrophages. role of complement in adaptive immunity: the classical complement pathway is activated by the virus in adaptive immune cells .it takes part in mac induced cell lysis by itself. also it acts through potentiation loop in producing 3cb and classical 3c esterase. its role in complement mediated phagocytosis and complement mediated cytotoxicity, as already seen above. prasad; aji, 3(1): 233-252, 2020; article no.aji.58422 244 the central role of alveolar macrophages (am):  under physiological conditions the expression of the phagocytic receptor macrophage 1 antigen (mac-1) keeps the ams down-regulated, to prevent damage of body tissues from the activated ams.  the adaptive immunity is suppressed through am's effects on interstitial dendritic cells, b-cells and t-cells.  the macrophage performs the phagocytic function, both igg mediated(through fcy .receptor)and complement mediated,(through cr1,cr2 and cr3 receptors) .  can cause tissue damage through pr inflammatory cytokines (mediated by m1 macrophages) can exert anti inflammatory effect with antiinflammatory cytokines as well as healing of damaged tissues by fibrosis (through m2 type macrophages.)  play role as antigen presentation cells (arc) and recognizes pamps of the virus with the prr,on its surface, in conjunction with mhc class 2 molecules .  it controls through, inos(inducible nitric oxide synthase), the differentiation and maturation of dendritic cells through. inhibition of the granulocyte-macrophage colony-stimulating factor (gm-csf)and tnf-alpha-mediated mechanisms.  through il 4 and il10, it causes the reduced production of metalloproteinases (endopeptidases which break down collagen and other extracellular proteins) by human ams.  causes differentiation of naïve cd4-t cells into mature th2 type cells.  il4 enhances mhc class ii antigen and mac-1(surface receptor as part of innate complement system) expression, thus promoting phagocytosis.  il10 inhibits the secretion of proinflammatory cytokines tnf-alpha and inf-gamma, thus suppressing the proliferation of t-cells, nk cells, and am.  by similar immunomodulation mechanisms to tgf-β. il-10reduces the rate of apoptosis. indirectly enhancing alveolar macrophage-mediated inhibition of t-cell proliferation. alveolar macrophages induce expression of the αvβ6 integrins, the cell-surface receptors, activate tgf-β.  tgfβ tightly regulates antiinflammatory activity by suppressing proinflammatory cytokine production, thereby inhibiting t-lymphocyte function.  this induces a downstream signalling cascade leading to transcription factors, regulating the expression of tgf-β target genes  important inhibitor receptors of ams include tim-3, pd-1, cd32b, and cd200r. the pathways by which macrophages are activated and deactivated are shown in table 1 below. macrophage polymorphism: the alveolar macrophages (am) exist as two phenotypes, m1 and m2 macrophages, which have different means of stimulation, express different kinds of cytokines and chemokines and have opposite physiological actions. the m1 macrophages are pr inflammatory and m2 are not only anti inflammatory in function, but also have action on extracellular matrix promoting healing by stimulating collagen fibres, leading to fibrosis. m2 macrophages have 3, m2b and m2c. the salient points of both the phenotypes and the sub-types are shown in table 3. table 1. activation of amsequence of events 1. binding of pamps to tlrs.(prps) 2. actin polymerization (in alveolar macrophages). 3. suppression of integrin expression 4. deactivation of tgf-β and the down regulation of the basal phosphorylation level of smad 2/3; 5. activation and detachment of alveolar macrophages from the alveolar epithelial cells 6. macrophages become primed (by ifn-γ and tnf-α) 7. phagocytosis and secretion of proinflammatory cytokines (tnf-α and il-6) 8. the ros (reactive oxygen species) produced by respiratory burst. 9. oxidative damage to lung tissue. 10. positive feedback effect -enhancement of production of tnf-α by macrophages (step 7). prasad; aji, 3(1): 233-252, 2020; article no.aji.58422 245 table 2. deactivation of ams sequence of events 1. secretion of ifnγ by activated lymphocytes. 2. stimulation of the production of matrix metalloproteinase mmp-9 by macrophages. 3. mmp-9 activates latent tgf-β. 4. reduced expression of αvβ6 integrins on alveolar epithelial cells. 5. return of the alveolar macrophage to a resting state * ams have been reported to produce mmp-9 partly via pge2-dependent pka signaling pathways, which are the pathways involved in the inhibition of phagocytosis. **activation of tgf-β is also advantageous because its production stimulates collagen synthesis in interstitial fibroblasts, which is necessary for restoring alveolar wall architecture. table 3. different polymorphic types of macrophage type of macrophage stimulated by express functions m1 macrophages (classically activated) 1.lps 2. th1 cytokines (ifn-γ, il-2, il-12, il18 and tnf-β) 3.gmcgo (tnf-) α, il-1, il-6, and il-12, and type i interferons (ifn) th1 cell-attracting chemokines (ccl5 cxcl9 and cxcl10) ( (rantes) pro-inflammatory m2 macrophages (alternatively activated macrophages (aams) 1.th2 cytokines including (il-4, il-5, il-6, and il-10) 2. m-csf 1. scavenging receptors 2. mannose and galactose receptors. 3. secrete high amount of il-10 4. express higher levels of the il-1 decoy receptor and il-1ra 5. express the chemokines ccl17, ccl22 and ccl24 1. anti-inflammatory action 2. higher phagocytic activity 3.promotetissue remodelling, 4.vasculogenesis 5. tumour progression m2 a macrophages il-4 and il-13 1. upregulate expression of arginase-1, mannose receptor mrc1 (cd206), 2. antigen presentation by mhc ii systemof il-10 and tgf. anti-inflammatory m2 b macrophages immune complexes  lps il-1, il-6, il-10, tnf anti-inflammatory m2 c macrophages il-10,  transforming growth factor beta (tgf) and glucocorticoids, il-10 and tgfβ suppression of inflammatory response 5. summary the elusive pathology of covid 19 lung disease is dueto its protean and varied manifestations. thus it might resemble from consolidation /pulmonary oedema/ ards or cytokinestorm ;yetit is distinct from each of these specific syndromes. in fact covid lung pathology resemblesa mixture of all theseindividual entities.alveolitis due to inflammatory cytokines explains the consolidation finding. damage to capillaries with consequent seepage of translate explains the pulmonary oedema like component. this in combination with interstitial fibrosis formed due to m2 macrophage 2, with resultant hypoxia might explain the ards-like picture. the terminal events of disseminated intravascular coagulation (dic) and multi organ dysfunction prasad; aji, 3(1): 233-252, 2020; article no.aji.58422 246 (mod), which is a common pathway for severe sepsis or cytokine storm like picture seen in a few covid 19 patients which contributed to the overall mortality due to covid 19.the failure to pin point to single pathology, perhaps is due to this fact. further the simultaneous occurrence of thrombotic and haemorrhage, seen in covid lung, suggests a range of diseases such as activation of simple coagulation cascadeand subsequent fibrinolysis, small vessel vasculitis to thrombotic thrombocytopenia. simple coagulation cascade activation/ fibrinolysis, cannot explain either the presence of immune cells like cd8+ and cd4+ cells nor the consolidation/fibrosis component. small vessel vasculitis(capillaritis) / luecocytoclastic / hypersensitive angiitis is a systemic disease and the specific histological features like leukocyteclasia (a process in which the neutrophils are destroyed leading to debris collection) is not demonstrated, in the autopsy findings of covid 19. further it is either due to drug induced allergy or idiopathic. thethhrombotic phenomena are not a feature of small vessel vasculitis. the acute nature and non involvement of medium sized blood vessels and absent fibroid necrosis are points wagnersgranulomatosis a non possibility. the absence of haemoptysis even though, patchyhaemorrhages are seen on gross as well as microscopic autopsy findings indicates that the bleeding is not substantially enough to give rise to clinically haemoptysis which distinguishes covid lung disease from good. pasture's syndrome the haemorrhagic part is perhaps due to purpura as supported by low platelet counts and presence of atypical megakaryocytes seen in the autopsy findings. this takes us to the possibility of thrombotic thrombocytopenia or coagulative microangiopathy which is a systemic syndrome unlike the localised pathology as seen in covid 19. however all coagulative microangiopathy is described in literature in case of fall bladder, but not in case of lung. if it is true, covid 19 is the first ever disease to cause such "localised coagulative microangiopathy. disseminated intravascular coagulation is a serious condition that may explain both the observed pathological entities, but its protean manifestation are not discernible in routine cases except the seriously terminally ill patients distained for mod(multiorgan dysfunction). the elusiveness of covid 19 lung pathogenesis could be due the culmination of more than one pathogenic pathway. the different pathways explaining the interstitial fibrosis and vascular changes already seen bear testimony to this fact. while the effector cells (macrophages, and other phagocytes, nkcells, nak cells and dendritic cells etc)do deliver the final blow, nevertheless the role played by other elements of both innate as well as acquired immunity(like antibody classes, opsonins and complement etc) cannot be ignored. it must be emphasized that immunity, innate or acquired can be no less damaging than the protection they offer against the invading pathogens. the system has inbuilt checks and controls, so that the destructive machinery is not directed against the healthy host cells, but are specifically directed against the pathogen or pathogen infected cell.the ingenuity of the pathogen to sabotage these inbuilt safeguards in immune system to farthen its interests, in which process, the fallout of deranged defence mechanisms, trains its guns against host's own tissues, as already discussed above. thus, foundation for immune mediated injury, in covid 19 induced lung damage is laid on a firm footing. 6. conclusion an integrated approach, taking into consideration, all the available clinical, laboratory, radiological and autopsy data lead to arriving at a possible mechanism underlying the lung damage in covid 19.the pathway of complement activation, contact cascade, the role of the cytokines and the role of various elements of the innate and adaptive immunity are reviewed. the central role played by the macrophage polymorphism, in the pathogenesis and pathology of the covid19 is stressed. the relevance of ards cytokine storm, the small vessel vasculitis, coagulative microangiopathy, disseminated intravascular coagulation and multi organ disfunction in relation to covid 19 pathology are discussed. it is also shown that no single mechanism could explain the whole gamut of the pathology and pathogenesis of covid 19 lung damage. the immune mediated damage seems to be more relevant ,rather than the infectivity of the virus. it is obvious that no single entity could explain the inflamatory,fibrotic and vascular features observed in the lung of covid 19 patients, it seems to be due to a mixture of different overlapping immune mechanisms, resulting in evolution of the unique pathogenic mechanism of covid 19. it is perhaps justifiable, from the author’s point of view to call the covid 19 related lung damage as “acute immune mediated lung injury (aili). prasad; aji, 3(1): 233-252, 2020; article no.aji.58422 247 consent it is not applicable. ethical approval it is not applicable. 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93:10212– 10216. 53. ali ganji, et al. increased expression of cd8 marker on t-cells in covid-19 patients.blood cells mol dis. 2020; 83:102437. 54. zheng et al. functional exhaustion of antiviral lymphocytes in covid-19 patients. cellular & molecular immunology; 2020. https://www.ahajournals.org/doi/10.1161/01.atv.0000134298.25489.92#r8-014928r prasad; aji, 3(1): 233-252, 2020; article no.aji.58422 250 appendix 1 microbial pattern recognition proteins --------------------------------------------------------------------- 1. mannose receptor. 2. complement receptors. 3. dc-sign. 4.toll-like receptors(tlrs). 5. scavenger receptors cd14, and mac-1 the prrs are divided into four families: 1. toll-like receptors (tlr) 2. nucleotide-binding oligomerization domain-like receptors (nlr) 3. rig-1 like receptors (rlr) 4. c-type lectin receptors (clr) pamps 1. glycans lipoglycans such as lipopolysaccharide, a component of the grambacteria outer membrane peptidoglycans such as bacterial muramyl dipeptide b-1,3-glucans from the cell wall of various fungi species 2. proteins bacteria flagellin -------------------------------------------------------------------------- appendix 2 interaction of prrs and pamps: step 1. ligand recognition/ binding: pamps are recognized and are attached to prrs. step 2. activation of the kinases and antiviral signalling cascades: 1. tbk1 (tank-binding kinase 1) 2. mapk (mitogen-activated protein kinases) 3. ikkα and ikkβ. κb kinase α and β step 3. these kinases phosphorylate and activate interferon(ifn)-regulatory factors 3 and 7 (irf3/7), ap-1, nf-κb, step 4.these proteins transcriptionally induce the gene expression of 1.type-i ifns (mainly ifn-α subtypes and ifn-β), prasad; aji, 3(1): 233-252, 2020; article no.aji.58422 251 appendix 3. laboratory tests reported in covid 19. their significance laboratory test significance increased esr crp (c-reactive protein) ferritin ac. phase reactants/markers of ac inflammation ldh. (lactic dehydrogenase) ast(aspartate transaminase) fibrinogen prothrombin liver cell dysfunction hs-ctnt(high sensitivity cardiac troponin ) maker of cardiocyte damage. ac mi, myocarditis pt(prothrombine time) (both indicate clotting disorder causing bleed) extrinsic coagulation pathway involvement. bu/bun (blood urea nitrogen) creatinine renal function impairment. t8+/t4+ cells immune reaction. leucopenia /lymphopenia thrombocytopenia neutrophilia leucoerythroblasticpicture. bone marrow insult purpura secondary infection or complication bone marrow involvement in covid 19. appendix 4 role of other important innate cellular elements 1. natural killer cells: they are the counterparts of cells of adaptive immunity. functions: they can directly kill the pathogen or through the antibody mediated cell mediated cytotoxicity. nk cell dependent antibody induced cytotoxicity: 1. nk cell expresses fcγ receptors cd16 or fcγriii. 2. these receptors recognize and bind to the reciprocal portion of antibody, (such as igg,) which binds to the surface of a pathogen-infected target cell. 3. the nk cell releases cytotoxic factors that cause the death of the target cell through perforin granzyme pathway. 2. mait cells (mucosal associated invariant t cells) a subset of t cells they display innate, effector-like qualities. ait cells secrete pro-inflammatory cytokines and alsolyse bacterially-infected cells. supports the adaptive immune response . they have memory like phenotype. 3. natural killer t (nkt) cells a group of t cells that share properties of both t cells and natural killer cells. . recognize foreign lipids and glycolipid antigens. 4. gammadelta t cells (γδ t cells) subset of t cells -express a unique t-cell receptor (tcr) -initiation and propagation of immune responses prasad; aji, 3(1): 233-252, 2020; article no.aji.58422 252 5. innate lymphoid cells (ilcs) 1. lcs contribute to immunity via a. secretion of signalling molecules, b. regulation of both innate and adaptive immune cells 2) mucus production in the respiratory tract. 3) restoration and maintenance of epithelial integrity. 4) secrete ifn-γ in response to viral infection in the lungs . 6. dendritic cells: these are professional antigen processing cells. present antigens to t cells. mhc class ii molecules thereby are critical for the initiation of the antigen-specific immune response.2. role of nab (natural antibodies) 1. opsonisation 2. activation of the complement. mhc class ii molecules: the main function of major histocompatibility complex (mhc) class ii molecules is to present processed antigens, which are derived primarily from exogenous sources, to cd4(+)tlymphocytes. _________________________________________________________________________________ © 2020 prasad; this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: http://www.sdiarticle4.com/review-history/58422 http://creativecommons.org/licenses/by/4.0 _____________________________________________________________________________________________________ *corresponding author: e-mail: joy_chikwendu@yahoo.com; asian journal of immunology 2(1): 56-63, 2019; article no.aji.52961 epidemiological study of intestinal parasites in school children in vandeikya lga, benue state, nigeria t. s. atsuwe1, v. u. obisike2, j. i. chikwendu1*, c. k. kondo1, c. t. tyoakoso1 and e. u. amuta1 1 federal university of agriculture, makurdi, nigeria. 2 abia state university, uturu, abia, nigeria. authors’ contributions this work was carried out in collaboration among all authors. authors tsa, ckk and ctt designed the study and carried out the field research. author eua wrote the protocol and supervised the research. author vuo performed the statistical analysis. author jic wrote the first draft of the manuscript and managed the literature searches. all authors read and approved the final manuscript. article information editor(s): (1) dr. jaffu othniel chilongola, department of biochemistry and molecular biology, kilimanjaro christian medical university college, tumaini university, tanzania. reviewers: (1) cristina hegedüs, university of agricultural sciences and veterinary medicine cluj-napoca, românia. (2) shamala moodley, mangosuthu university of technology, south africa. complete peer review history: http://www.sdiarticle4.com/review-history/52961 received 01 october 2019 accepted 04 december 2019 published 12 december 2019 abstract a study was conducted to determine the epidemiology of intestinal parasites in school children in vandeikya lga, benue state, nigeria. two hundred and ninety three (293) stool samples from school children were examined in selected schools across the local government area. 22 of 293 children were infected with parasites which include: ascaris lumbricoides, entamoeba histolytica, hookworm, strongyloides stercularis and taenia spp. hookworm had the highest prevalence rate of 9(3.1%), entamoeba histolytica 7(2.3%), taenia spp had 3(1.0%), strongyloide stercolaris had 2(0.7%) while ascaris lumbricoides recorded the least prevalence rate of 1(0.3%). for mixed infections, entamoeba histolytica and ascaris lumbricoides recorded the highest prevalence of 5(1.7%); entamoeba histolytica, hookworm and strongyloides stercolaris recorded prevalence of 2(0.7%); and entamoeba histolytica, ascaris lumbricoides and hookworm recorded prevalence of 2(0.7%); there was however, no significant difference (p>0.05) in prevalence of mixed infections. original research article atsuwe et al.; aji, 2(1): 56-63, 2019; article no.aji.52961 57 for prevalence based on age, there was significant difference (p<0.05) between the age groups, 2 calculated (10.117), 2 tabulated (4.891), df = 2. there was no significant difference (p>0.05) in the infection rate based on sex, 2 calculated (3.245), 2 tabulated (5.991), df = 2. intestinal parasites are prevalent in vandeikya lga, benue state. risk factors like open defecation, use of stream and well water should be minimized in order to prevent infection. keywords: prevalence; intestinal parasites; mixed infection. 1. background intestinal parasites are helminthes (worms) and protozoa that reside in the intestines of their hosts; disease burden is higher in children and women [1]. at least 880 million children are in need of treatment for intestinal worms [1]. according to world health organization estimates, 500 million people in the world are infected with entamoeba histolytica; this protozoon causes symptomatic illness in about 50 million people and is responsible for 100,000 mortalities [2]. intestinal parasites may cause morbidities like: weakness, inflammation of the intestines, abdominal pains, nausea and dysentery in those infected [3]. in addition, infection in children may result in reduced ability to learn, reduced food absorption leading to malnutrition, anaemia, stunted growth, and may lead to death [4]. intestinal parasites are endemic in the tropics where favorable climatic, environmental and sociocultural factors permit transmission [5]. the infection rate for these parasites has primarily been attributed to poverty, unhygienic environmental conditions and over-dispersion of parasites [4]. significantly higher prevalence of intestinal parasites was reported in children from homes where livestock are reared and in children of farmers [6]. according to a study conducted in ethiopia, hygiene factors like the inconsistent use of shoes is strongly associated with prevalence of hookworm infection, and long untidy finger nails and drinking of well water are important risk factor for e. histolytica [7]. drinking of rainwater is a possible risk factor for intestinal parasites [8]. in contrast, however, no association was found between household drinking water source and prevalence of intestinal parasites in school aged children in burkina faso [9]. proper understanding of prevalence, distribution and associated risk factors for intestinal parasites could help in development of strategy aimed at transmission interruption and control of the diseases they cause. this study was therefore aimed at: 1) investigating the prevalence and distribution of intestinal parasites in school aged children in vandeikya lga and (2) determining the association between prevalence of parasite species in children and three (3) risk factors: drinking water source, occupation of parents and type of toilet used. 2. materials and methods 2.1 study area the research was carried out in select public and privates schools across vandeikya local government area of benue state. 2.2 samples collection school children that participated in the study were aged 3 – 15 years. school children were given 20 ml universal bottles the previous day and asked to return it the next morning with their early morning stool sample in it. questionnaires were used to obtain data on age, sex, drinking water source, type of toilet system used at home and occupation of parents. after collecting the stool samples from the children at school, the specimen were then taken to the science laboratory at general hospital gboko where they were analyzed. 2.3 method of identification of intestinal parasites the method that was used to identify the presence of intestinal parasites was direct microscopy of wet preparation, and stool concentration. stool sample was emulsified in saturated salt solution in the test tube, the supernatant was poured out and the concentrate was centrifuge for 5 minutes. a drop or two of saline solution was placed on the microscopic slide with a cover slip and viewed under a microscope with ×10 and ×40 objective lens. positive stool samples were identified by: presence of characteristic eggs for taenia species, hookworm and ascaris species. for strongyloides species, the presence of larvae was used for identification, while presence of characteristic trophozoites in stool was used of diagnose e. histolytica. atsuwe et al.; aji, 2(1): 56-63, 2019; article no.aji.52961 58 2.4 statistical analysis chi-square test was used to determine the homogeneity of the disease in the different schools. 3. results table 1 shows age related prevalences of a range of intestinal parasites present in stool samples of research subjects: the total prevalences of the intestinal parasites were as follows: ascaris lumbricoides 1 (0.34%), entamoeba histolytica 7 (2.39%), hookworms 9 (3.07%), strongyloides stercularis 2 (0.68%), taenia spp. 3 (1.02%). among children aged 0-4 years, hookworm was the most prevalent intestinal parasite with prevalence of 2 (2.99%), and no intestinal parasite was observed among children aged 15-19 years. there was no significant difference (p>0.05) in prevalence between the different age groups. table 2 shows sex related prevalences of intestinal parasites in stool samples of research subjects. in males, prevalences of ascaris lumbricoides was 1 (0.6%), entamoeba histolytica 4 (2.55%), hookworms 4 (2.55%), strongyloides stercularis 1 (0.64%) and taenia spp 0 (0.0%). prevalences in females were as follows: ascaris lumbricoides 0 (0.0%), entamoeba histolytica 3(2.21%), hookworms 5 (3.68%), strongyloides stercularis 1 (0.74%), taenia spp. 3 (2.21%). females had higher total prevalences of intestinal parasites 12(14%) than males 10(8.3%). table 3 shows the prevalence of mixed intestinal parasite infections in school children with respect to age. entamoeba histolytica, ascaris lumbricoides and hookworm had prevalence of 2 (0.68%); entamoeba histolytica and ascaris lumbricoides had the highest prevalence rate of 5(1.71%); and entamoeba histolytica, hook worm and strongyloides stercolaris had prevalence of 2(0.68%). table 4 shows the prevalence of mixed intestinal parasites in school children with respect to sex. prevalence of mixed infections was higher among males 4(2.55%) than among females 5(3.68%), giving a 9(3.07%) total prevalence of mixed infections. fig. 1 represents the prevalence of intestinal parasites in relation to the type of toilet used by the subjects. those that used nearby bush were more infected accounting for 45% of total infections with intestinal parasites, while those that used “pit” and water system toilets accounted for 40% and 15% total infection respectively. fig. 2 shows the relationship between source of drinking water and the prevalence of intestinal parasites. school children who obtain drinking water from streams were most infected accounting for 60% of total infections; children who obtain drinking water from wells had the second highest infection rates accounting for (30%) of total infection; while children who obtain drinking water from borehole were the least infected accounting for (10%) of the total infection. table 1. age related prevalence of intestinal parasites age no examined a. lumbricoides e. histolytica hookworms s. stercoralis taenia spp 0-4 67 0(0.00) 1(1.49) 2(2.99) 1(1.49) 0(0.00) 5-9 146 1(0.68) 3(2.05) 4(2.74) 1(0.68) 3(2.05) 10-14 77 0(0.00) 3(3.90) 3(3.90) 0(0.00) 0(0.00) 15-19 3 0(0.00) 0(0.00) 0(0.00) 0(0.00) 0(0.00) total 293 1(0.34) 7(2.39) 9(3.07) 2(0.68) 3(1.02) 2 cal = 10.117, df = 12, p>0.05 table 2. sex related prevalence of intestinal parasites sex no examined a. lumbricoides (%) e. histolytica hookworm (%) s. stercoralis (%) taenia spp (%) total (%) male 157 1(0.64) 4(2.55) 4(2.55) 1(0.64) 0(0.00) 10(6.37) female 136 0(0.00) 3(2.21) 5(3.68) 1(0.74) 3(2.21) 12(8.82) total 293 1(0.34) 7(2.39) 9(3.07) 2(0.68) 3(1.02) 22(7.51) 2 cal = 3.245, df = 4, p>0.05 atsuwe et al.; aji, 2(1): 56-63, 2019; article no.aji.52961 59 table 3. prevalence of mixed infections with intestinal parasites in different age groups age no examined no infected (prevalence) total e. histolytica a. lumbricoides hookworm (%) e. histolytica a. lumbricoides (%) e. histolytica hookworm s. stercoralis (%) 0-4 67 0(0.00) 1(1.49) 1(1.49) 2(2.99) 5-9 146 1(0.68) 2(1.4) 1(0.68) 4(2.74) 10-14 80 1(1.25) 2(2.50) 0(0.00) 3(3.75) total 293 2(0.68) 5(1.71) 2(0.68) 9(3.07) 2 cal = 2,948, df = 4, p>0.05 table 4. prevalence of mixed infections with intestinal parasites in different sexes sex no examined no infected (prevalence) total (%) e. histolytica a. lumbricoides hookworm (%) e. histolytica a. lumbricoides (%) e. histolytica hookworm s. stercoralis (%) male 157 1(0.64) 2(1.27) 1(0.64) 4(2.55) female 136 1(0.74) 3(2.21) 1(0.74) 5(3.68) total 293 2(0.68) 5(1.71) 2(0.68) 9(3.07) 2 cal = 0.052, df = 2, p>0.05 atsuwe et al.; aji, 2(1): 56-63, 2019; article no.aji.52961 60 fig. 1. relationship between toilet facility and parasitic infection in pupils fig. 2. relationship between source of drinking water of the pupils and parasitic infection fig. 3 shows the relationship between parental occupation of school children and infection with intestinal parasites. children whose parents were farmers accounted for (50%) of those infected, children whose parents were traders accounted for (30%) of those infected, while children whose parents were civil-servants accounted for 20% of those infected. 4. discussion the study revealed the presence of a. lumbricoides, e. histolytica, hookworms, s. stercoralis, and taenia spp infections in school aged children. hookworms and e. histolytica had the highest prevalences. the temperature in the tropics is known to favour the prevalence of a range of intestinal parasites and could be the reason for their presence in the study area. seasonal variations in abiotic factors like temperature, humidity and rainfall may affect intensity of intestinal parasites [10], particularly soil transmitted helminthes [11]. hookworms and e. histolytica had significantly higher prevalences than the other parasites. although the trophozoite stage of e. histolytica which is used to diagnose the parasite is fragile and cannot survive outside the host for longer than 24 hours, the infective stage of the parasite which is the cyst stage can withstand desiccation. inspite of harsh temperature, cysts may linger in the environment for months, and could be the reason for high e. histolytica prevalence. in addition, the cysts of e. histolytica can be transmitted through water and food. 0 5 10 15 20 25 30 35 40 45 50 type of toilet in te st in al p ar as it e s in p u p ils ( % ) bush pit water system 60% 30% 10% stream well borehole atsuwe et al.; aji, 2(1): 56-63, 2019; article no.aji.52961 61 fig. 3. relationship between parental occupation of the pupil and parasitic infection hookworms are transmitted primarily by walking barefoot on soil containing parasite larvae, and by ingestion of larvae in the case of ancylostoma duodenale. multiple modes of infection could have contributed to high prevalence of hookworms in this study. in agreement with our findings, entamoeba histolytica also had the highest prevalence among intestinal parasites infecting food vendors in wolaita sodo, ethiopia, while tapeworms and hookworms had equal prevalences [12]. the author opined that untrimmed fingernails of researcher subjects could be contributing to high prevalence of intestinal parasites in the study. in contrast however, e. histolytica had second highest prevalence and a. lumbricoides had the highest prevalences among tuberculosis suspected patients in gondar, northwest ethiopia, however, among protozoan infections, e. histolytica had the highest prevalence [13]. the researchers opined that unsafe water sources, economic status and environmental sanitation could be contributing to high prevalence of parasitic diseases in the study area. intestinal parasite prevalences were highest in children aged 5-9 years, as the children grew older (15-19 years), zero (0%) prevalences were recorded for all intestinal parasites. children aged 5-9 may be more prone to playing with soil than other age groups and are likely to move around bare-foot thus exposing themselves to parasite infections. in addition this age group may have lower immunity than their older counterparts which may be an additional factor responsible for observed higher prevalences with intestinal parasites. aging has an effect of immunity, and this could be a reason for variations in prevalences among different age groups with similar levels of exposure to infection [14]. in a research carried out in ethiopia, prevalences of intestinal parasite as high 15.5%, in children under 5 years were reported; the author noted that poor hand washing habits of this age group could be responsible for the high prevalence [15]. higher prevalence of intestinal parasites in older age groups than in younger age groups may be attributed to possible increased exposure to contaminated soil in older age groups [16]. mixed infection (polyparasitism) was recorded among pupils examined; infection with as many as three different intestinal parasites was recorded in children. mixed infections could be as a result of the fact that factors such as high temperature, poor sanitation and the use of unsafe water favour a range of intestinal parasites, and as such, children are exposed to infection with more than one parasite. there were three (3) groups of mixed infections, and e. histolytica was present in all three (3) groups of mixed infections. this could be as a result of the ability of e. histolytica cysts to survive harsh environmental conditions leading to a buildup of the cysts in the environment. 4.1 toilet system children who defecate in the bush had highest prevalence with intestinal parasite. this could be because after defecating in the open, children 0 10 20 30 40 50 60 occupation of pupils' parents in te st in al p ar as it es in p u p ils ( % ) farmers traders civil servants atsuwe et al.; aji, 2(1): 56-63, 2019; article no.aji.52961 62 tend to wipe their anuses with leaves of plants or paper and may not wash their hand afterwards. children who use pit toilet had the second highest prevalence of intestinal parasites, this could be because children tend to defecate on the mouth of the pit toilet which is sometimes unroofed and in bad conditions. untidy pit toilets could attract houseflies causing spread of intestinal parasite eggs and cysts. higher prevalence of intestinal parasite in people who practice open defecation was also reported in benue state, nigeria [17]. 4.2 source of drinking water infection rate relating to the source of drinking water showed that pupils that obtain their drinking water from the streams recorded the highest number of parasitic infection; this could be as a result of run off of parasite infected soil into streams during rainfall and run off water from gutters, drainages and sewages. the ova of intestinal parasites are easily transported by water and other means thus contaminating the source of drinking water. high prevalence was reported among people using stream and well as primary source of water in ebonyi, abakaliki, nigeria [18]. 4.3 parental occupation/socioeconomic factors children are active and playful, the children of farmers in particular because of the nature of the occupation of their parents , stay for long hours on the farms with their parents, thereby making them highly prone to contact with soil contaminated with intestinal parasites. 5. conclusion intestinal parasites are prevalent in vandeikya lga, benue state. risk factors like open defecation, use of stream and well water should be minimized in order to prevent infection. consent and ethical approval ethical clearance was obtained from research ethics board. informed consent was obtained from parents and school authorities, and informed accent was obtained from research subjects. competing interests authors have declared that no competing interests exist. references 1. world health organization. regional strategy on neglected tropical diseases in the who african region 2014-2020. who regional office for africa 2017; 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7:140-147. 13. tegegne y, wondmagegn t, worku l, zeleke aj. prevalence of intestinal parasites and associated factors among pulmonary tuberculosis suspected patients attending university of gondar hospital, gondar, northwest ethiopia. journal of parasitology research. 2018;article id 9372145. 14. valiathan r, ashman m, asthana d. effects of age on the immune system: infants to elderly. scandinavian journal of immunology. 2016;83(4):255266. 15. gebretsadik d, metaferia y, seid a, fenta gm, gedefie a. prevalence of intestinal parasitic infection among children under 5 years of age at dessie referal hospital: cross sectional study. biomedical central research notes. 2018;11(1):771. 16. suntaravitun p, dokmakaw a. prevalence of intestinal parasites and associated risk factors for infection among rural communities of chachoengsao province, thailand. korean journal of parasitolgy. 2018;56(1):33-39. 17. akor jo, obisike vu, omudu ea, imandeh gn. the prevalence of polyparasitism in oju local government area of benue state, nigeria. international journal of tropical disease & health. 2019;39(2):1 6. 18. ani oc, itiba ol. evaluation of parasitic contamination from local sources of drinking-water in abakaliki, ebonyi state, nigeria. nigerian journal of parasitology. 2015;36(2):153-158. _________________________________________________________________________________ © 2019 atsuwe et al.; this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: http://www.sdiarticle4.com/review-history/52961 http://creativecommons.org/licenses/by/4.0 _____________________________________________________________________________________________________ *corresponding author: e-mail: drdjuraev@mail.ru; asian journal of immunology 3(1): 124-130, 2020; article no.aji.55718 results of allergological and immunological research in patients with polipoid rhinosinusitis j. a. djuraev1*, u. s. khasanov1, u. n. vohidov2 and s. s. sharipov2 1 tashkent medical academy, uzbekistan. 2 tashkent state dental institute, uzbekistan. authors’ contributions this work was carried out in collaboration among all authors. authors jad and usk designed the study, performed the statistical analysis, wrote the protocol and wrote the first draft of the manuscript. author unv managed the analyses of the study. author sss managed the literature searches. all authors read and approved the final manuscript. article information editor(s): (1) dr. wagner loyola, brazilian agricultural research corporation, brazil. (2) dr. darko nozic, university of belgrade, serbia. reviewers: (1) ekaterini goudouris, universidade federal do rio de janeiro, brazil. (2) gilbert batieka bonsaana, university for development studies, ghana. complete peer review history: http://www.sdiarticle4.com/review-history/55718 received 10 february 2020 accepted 16 april 2020 published 22 april 2020 abstract an exceptional role in protecting against pathogenic effects of various pathogenic and opportunistic microorganisms is played by immunoglobulins of the main classes. as our studies showed, before treatment, the igg content in patients did not differ significantly from the control. the average iga level in patients of both groups before surgery was slightly reduced (p> 0.05). given the important role of iga in protecting the body and, above all, the mucous membranes from infection, it can be concluded that such a disturbance of the biosynthesis of this class of immunoglobulin may be one of the reasons for the decrease in immune reactivity and frequent infectious processes. a sharp increase in serum ige levels (239 ± 19.1 iu/ml) was observed in patients with eosinophilic polyposis rhinosinusitis, which we associate with an increase in il-4 content and increased allergization of the body. this relative difference between patients of the two groups proves the need for appropriate diagnosis and treatment of chronic polyposis of rhinosinusitis. the aim of our research work was study allergically and immunologically results in patients with polipoidrhinosinusitis. original research article djuraev et al.; aji, 3(1): 124-130, 2020; article no.aji.55718 125 keywords: nasal polyposis; osteomeatal complex; rhinosinusitis; nasal septum; allergen; immune system. 1. introduction the second half of the last century was marked by the transition of the center of gravity from otiatrics to rhinology. the reason for this is not only an increase in the load on the upper respiratory tract from the outside in the human environment, but also significant changes in the population, expressed in the accumulation of negative genetic deficiency and, above all, in the immune system. particularly complex and unclear are the causes of nasal polyposis [1,2]. today, many theories have been proposed for the etiology and pathogenesis of chronic polyposis rhinosinusitis (cid), unfortunately, many causative factors, as well as their relationship and role in the formation of nasal polyps, are not fully understood [3]. a number of theories of the etiology of nasal polyposis are known, and they are not mutually exclusive: bacterial and fungal infections, superantigenic stimulation of the immune system, the formation of biofilms, anatomical abnormalities of the osteomeatal complex, ciliary dysfunction, allergies, secondary immunedeficiency [4]. in this regard, some foreign authors, according to which diseases accompanied by the formation of nasal polyps are divided into 5 groups. the first group consists of systemic genetically caused diseases in the form of kartagener syndrome and cystic fibrosis, the second group is chronic polypous-purulent rhinosinusitis (neutrophilic polyps), the third group is a local pathology in the osteomeatal complex with impaired mucociliary clearance in the paranasal sinuses and the formation of a productive process in thechoanal polyps, the fourth group is a chronic infectiousallergic rhinosinusitis with a final stage of development in the form of a chronic polypous allergy rhinosinusitis (eosinophilic polyps), the fifth group is the “asthmatic triad” as a pseudoallergic disease caused by a metabolic disorder of arachidonic acid: aspirin-induced nasal polyps [5,6]. s. v. ryazantsev et al. (2006) consider nasal polyposis and snps as a multifactorial etiological syndrome that occurs in individuals predisposed to a specific tissue reaction. according to this theory, the formation of nasal polyps is associated with an innate predisposition and exposure to environmental factors, such as mechanical, physical, chemical, biological factors (viruses, bacteria, fungi, allergens). in this case, a disturbance of the reactivity of the parasympathetic nervous system, immunity, mucociliary transport, mucosal hypersensitivity, defects in membranes and dna in the nuclei of cells areconsidered a congenital predisposition [7,8]. the combination of these two factors includes pathogenetic mechanisms, that is, they lead to a disturbance of neuro-trophic innervation, mast cell degranulation, the release of biologically active substances, which in turn increase vascular permeability, followed by tissue edema and the formation of polyps. according to another theory proposed by m. yu. korkmazov (2010), forpolypous sinusitis, microcirculation in the middle nasal passage changes in the form of vasomotor reactions, narrowing of the arterioles and a decrease in the number of functioning capillaries, which lead to the accumulation of metabolic products and retention of tissue fluid, which contributes to the development of edema [9,10]. n. n. naumenko et al. (2005) suggest that forpolypousrhinosinusitis, a disturbance of the adaptive-trophic function of the autonomic nervous system is important, and contributes to the development of neurodystrophic changes in the nasal mucosa, leading to the formation of polyps. v. s. piskunov (2006) believes that a disturbance of the aerodynamics of the nose during deformities of the nasal septum leads to a slowly developing inflammatory process of the nasal mucosa, which manifests itself as the formation of a polyp in the absence of pathological changes in the paranasal sinuses. in recent years, the role of metabolic disorders of arachidonic acid, which leads to "aspirin intolerance" [11,12], has been intensively studied in patients with cid. the classical clinical picture of the “aspirin” triad implies the presence of bronchial asthma in combination with eosinophilic rhinitis or cprs, the manifestations of which sharply increase after taking nonsteroidal anti-inflammatory drugs (nsaids) [13]. in this regard, the importance of leukotrienes in the development of allergic inflammation is very djuraev et al.; aji, 3(1): 124-130, 2020; article no.aji.55718 126 significant. they increase vascular permeability, promote the mobilization and activation of proinflammatory cells in the airways, participate in the release of other pro-inflammatory agents, increase the secretory activity of the glands, can enhance the action of other allergy mediators, for example, histamine [14] and as a result "aspirininduced" polyps [15] develop. in addition, recently, many authors have been paying attention to the role of toll-nodreceptors in the occurrence of nasal polyps and the genetic aspects of the development of cprs [16,17]. unfortunately, the results of these studies have not yet led to a complete disclosure of the pathogenesis of the polyposis process. thus, chronic polypousrhinosinusitis should be considered amultiple etiologies disease, in the development of which the influence of infectious factors and allergens plays the main role, which leads to the development of a chronic inflammatory process and allergization of the body, which affect different body systems, which aggravates the main pathology and leads to a decrease in the quality ofpatient’s life, therefore this problem is urgent, and needs a solution which is urgent. 2. materials and methods in accordance with the purpose of the study and to achieve its objectives, clinical studies were conducted in 150 patients with chronic kidney disease who were examined and treated in the ent department of the 3rd clinic of the tashkent medical academy in 2018-2019. all patients with chronic kidney disease underwent specific allergological examination, which included collecting an allergic history and staging skin tests with allergens. allergological examination was carried out according to a special scheme developed by the allergy research laboratory of the 2 nd clinic of the tashkent medical academy. 3. results and discussion for skin tests, allergens from timothy grass pollen, team hedgehog, fescue, ryegrass, bonfire, birch, wormwood, ragweed, corn, sunflower, ash, walnut, oak and dandelion were used (table 1). table 1 shows that the reaction to each allergen was different. so, in 92 patients there was a positive reaction of varying degrees to histamine. however, no reaction was observed on fescue, ragweed, and hedgehog. in addition, a negative reaction to an allergen was recorded in 58 patients. in 92 patients, a scarification test gave a positive result. this indicates the undoubted participation of the allergic factor in the development of polypousrhinosinusitis. these data correlated with blood test results. so, in a blood test in 92 (33.3%) patients, eosinophilia was noted. peripheral blood eosinophils were determined in a general blood test in all patients. in 92 patients with chronic polypousrhinosinusitis, the number of eosinophils was increased. in particular, eosinophilia was observed in 88 examined with a table 1. allergy data in patients with crs pollenallergen patients with crs (n=150) results + ++ +++ dandelion 18 2 2 14 fescue sunflower 16 3 13 sagebrush 6 4 2 ash 8 2 2 4 bonfire 10 2 2 6 birch 2 1 1 ragweed corn 9 3 6 oak 8 4 4 cocksfoot histamine 92 38 25 29 neg. 58 djuraev et al.; aji, 3(1): 124-130, 2020; article no.aji.55718 127 positive result for the allergen, however, in the remaining 62 patients the number of eosinophils remained within the normal range (table 2). peripheral blood eosinophilia indicates the continued role of allergies in the development of crs. allergic background was found in 58.67% of the examined patients. based on the results of the study, it should be noted that the presence of a positive allergic reaction in patients with eosinophilia suggests the appointment of topical cs for the purpose of pathogenetic treatment, as well as 2-3 generation antihistamines for the symptomatic treatment and removal of symptoms of the disease. studies of the immune status in crs patients were carried out in the laboratory of immunocytokines of the institute of immunology of the academy of sciences of the republic of uzbekistan (head of laboratory, md ismailova a.a.). the determination of cellular, humoral immunity units was carried out using monoclonal antibodies by the method of indirect rosette formation, to determine the cytokines used test systems produced by vector-best of the russian federation, based on the sandwich method of enzyme-linked immunosorbent assay using horseradish peroxidase as an indicator enzyme. the results will be analyzed in the following subchapters. given the important pathogenetic role of immune system disorders in the mechanisms of crs development, as well as unsatisfactory treatment results, allergization of the body, and the possibility of developing toxic and side effects, we studied the immune status of patients. an immunological study of cellular and humoral immunity was performed in 79 (52.7%) patients who were hospitalized in the ent department of the 3rd clinic of the tashkent medical academy in 2012-2013. (table 2). as can be seen from table 3, in patients there was a significant decrease in the content of tlymphocytes (cd3 cells), which averaged 47.56 ± 6.57 and 46.84 ± 4.52, significantly differing from the control data (p <0, 05). the absolute value of cd3 cells was also below normal. the level of immunoregulatory subpopulations of t-lymphocytes of t-helper cells (t4) and tsuppressors/cytotoxic cells (t8) was lower than the control values. for t-helpers, the cd4 cell count averaged 26.96 ± 2.58 and 26.19 ± 2.18, which was significantly lower than the control (p <0.05). in addition, there was a significant decrease in the number of t-helpers (p <0.05). both the absolute and relative contents of tsuppressors cytotoxic cells (cd8) were also significantly lower than the control (p <0.05). an equivalent decrease in the number of both thelpers and t-suppressors caused significant changes in the immunoregulatory index. the ratio of cd4/cd8 lymphocytes was below the control. these data indicate impaired immunity as a result of a prolonged inflammatory process that caused secondary immunodeficiency in the body of patients with various forms of chronic polypousrhinosinusitis. thus, in patients with crs, there were significant disturbances of the t-cell immunity indices, which is typical for chronic respiratory diseases. indicators of the humoral immunity are presented in table 4. an exceptional role in protecting against pathogenic effects of various pathogenic and opportunistic microorganisms is played by immunoglobulins of the main classes. as our studies showed, before treatment, the igg content in patients did not differ significantly from the control. the average iga level in patients of both groups before surgery was slightly reduced (p> 0.05). given the important role of iga in protecting the body and, above all, the mucous membranes from infection, it can be concluded that such a disturbance of the biosynthesis of this class of immunoglobulin may be one of the reasons for the decrease in immune reactivity and frequent infectious processes. table 2. the content of eosinophils in peripheral blood in patients with crs eosinophils in the peripheral blood patients with eprs, (n=90),% patients with nprs, (n=60),% control group, (n=20),% 6,7±1,51* 2,1±1,09 2,2±1,07 note:* differences regarding the group up to 5 years are significant (* p<0,05) djuraev et al.; aji, 3(1): 124-130, 2020; article no.aji.55718 128 table 3. indicators of cellular immunity in patients with crs indicator patients with eprs, (n = 48) patients with nprs, (n=31) control group, (n=20) whitebloodcells, μl 6187,5±943,88 6035,5±1759,84 6295,0±737,33 lymphocytes, % 30,7±9,33 29,7±4,19 29,4±4,15 lymphocytes, μl 1717,0±440,38 1790,3±328,99 1945,0±225,89 cd3+, % 47,2±6,05 47,2±5,24 57,9±4,26 cd3+, μl 814,2±281,30 758,8±135,84 1009,1±214,33 cd4+, % 26,9±2,43** 26,5±2,28** 36,4±1,90 cd4+, μl 466,4±140,06* 443,0±92,37*** 856,7±71,23 cd8+, % 19,6±4,60 20,4±3,30 22,6±1,90 cd8+, μl 330,4±125,28 339,4±45,45* 498,4±60,84 iri 1,44±0,21 1,3±0,13** 1,75±0,10 cd16+, % 19,7±3,60 16,1±4,89 14,2±1,84 cd20+, % 22,3±2,00 21,6±2,71 21,9±2,02 cd20+, μl 383,7±121,50 366,0±91,66 412,4±95,94 cd23+, % 24,2±2,25 19,8±1,47 20,0±1,34 cd38+, % 31,6±7,02 31,4±6,80 20,9±5,35 cd95+, % 24,0±3,15 19,7±1,55 19,8±1,51 note: * differences relative to control group data are significant (*** p<0,001) table 4. indicators of cellular immunity in patients with crs indicator patients with eprs, (n=48) patients with nprs, (n=31) control group, (n=20) igg, mg% 1144,2±168,93 1184,3±99,74 1176,2±99,17 iga, mg% 141,9±14,15 167,8±17,88*** 128,4±16,94 igm, mg% 118,3±18,22 120,3±15,22 121,7±15,16 cic large, cu 17,8±6,03 19,8±5,09 11,8±3,14 cic small, cu 28,1±9,17*** 22,3±8,80 21,8±6,92 note: * differences relative to control group data are significant (*** p<0,001) the igm content showed a tendency to increase, but both the average values and its individual values did not differ significantly from the norm. the disturbances identified by us were manifested: disturbance of the cellular component of immunity with insufficiency of immunoregulatory subpopulations of thelpers and t-suppressors (cd4 and cd8 cells); inhibition of humoral immunity (iga); a decrease in natural cytotoxicity (cd16 cells). this allows us to consider patients with crs as patients with prevailing secondary immunodeficiency, which requires appropriate treatment using immunomodulators. an immunological study of cytokines and total ige in serum was carried out in 79 (52.7%) patients who were hospitalized in the ent department of the 3 rd clinic of the tashkent medical academy in 2012-2013 (table 5). as our study showed, elevated levels of il-2 in serum were observed in patients of both groups, but especially in patients with chronic "neutrophilic" polypousrhinosinusitis. such an increase in the level of cytokine indicates a disturbance of the immune state of the body, since il-2 is responsible for the immune response. at the same time, the content of il-4, which is responsible for the allergization of the body, was also increased in patients of both groups, but more in patients with eosinophilic polypousrhinosinusitis. the content of il-8 increased (9.9 ± 3.72 pg/ml) in patients with “neutrophilic” polyposis rhinosinusitis, remaining within the normal range in patients with “eosinophilic” polyposis rhinosinusitis. djuraev et al.; aji, 3(1): 124-130, 2020; article no.aji.55718 129 table 5. the content of cytokines and their ratio with total ige in serum in patients with crs indicator patients with eprs, (n=48) patients with nprs, (n=31) control group, (n=20) 2-2, pg / ml 10,6±3,53 12,5±4,81 5,5±0,44 2-4, pg / ml 6,4±2,31 7,3±1,13 4,7±1,15 2-8, pg / ml 8,3±4,58 9,9±3,72 5,7±0,84 ige,iu / ml 239±19,1*** 43,0±30,26 14,6±8,57 note: * differences relative to control group data are significant (* p<0,05, ** p<0,01, *** p<0,001) a sharp increase in serum ige levels (239 ± 19.1 iu/ml) was observed in patients with eosinophilic polyposis rhinosinusitis, which we associate with an increase in il-4 content and increased allergization of the body. this relative difference between patients in the two groups proves the need for appropriate diagnosis and treatment of crs. 4. conclusions thus, we did not find a clear deficit in the content of igg and igm. only an increase in the level of iga was observed, indicating the activation of the body's immune systems, which can serve as a factor in the development of a long inflammatory process, and in the subsequent relapse of the disease. our data confirmed that multistep surgical intervention and inadequate conservative treatment adversely affect the immunological reactivity of crs patients. in these patients, disturbances of immune homeostasis and a deficiency of cellular and humoral immunity are detected. a study of cellular and humoral immunity revealed that “eosinophilic” polyposis rhinosinusitis is characterized by an increase in the number of natural killers, allergization factor and apoptosis, il-2, il-4 and ige, while “neutrophilic” polyposis rhinosinusitis indicates a significant increase in iga and il8, which indicates the presence of a long-lasting chronic inflammatory process. consent as per international standard informed and written participant consent has been collected and preserved by the authors. ethical approval as per international standard written ethical permission has been collected and preserved by the author(s). competing interests authors have declared that no competing interests exist. references 1. voxidov un, xasanov us, dƶuraev ƶa, sultonov dm, soʙirov şş .estimation of data of specific allergic research in chronic polypoid-rhinosinusitis.2016;(9):374-376. 2. xasanov us, voxidov un, dƶuraev ƶa. the condition of lumbar sinus in chronic inflammatory diseases of the nasal and paranasal sinuses in patients with myocarditis. europeanscience. 2018;9(41). 3. xasanov us, dƶuraev ƶa, toşpulatov ƶ. peculiarities of diseases of the nose and paranasal sinuses in patients with myocarditis. molodoyuchenyy. 2016;10: 547-550. 4. abdukhahharovich dj, saidakramovich ku, nuridinovich vu. the prevalence of chronic inflammatory diseases of the nose and paranasal sinuses in patients with myocarditis. european science review. 2018;5-6. 5. bellavite p, marzotto m, chirumbolo s, conforti a. advances in homeopathy and immunology: a review of clinical research. frontbiosci (scholed). 2011;3: 1363-1389. 6. bousquet j, heinzerling l, bachert c, papadopoulos ng, bousquet pj, burney pg, lodrup carlsen kc. practical guide to skin prick tests in allergy to aeroallergens. allergy. 2012;67(1):18-24. 7. bousquet pj, combescure c, neukirch f, klossek jm, mechin h, daures jp, bousquet j. visual analog scales can assess the severity of rhinitis graded according to aria guidelines. allergy. 2007;62(4):367-372. 8. chen ys, langhammer t, westhofen m, lorenzen j. relationship between matrix metalloproteinases mmp‐2, mmp‐9, tissue inhibitor of matrix metalloproteinases ‐1 and il‐5, il‐8 in nasal polyps. allergy. 2007;62(1):66-72. 9. compalati e, passalacqua g, bonini m, canonica gw. the efficacy of sublingual immunotherapy for house dust mites djuraev et al.; 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2012. _________________________________________________________________________________ © 2020 djuraev et al.; this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: http://www.sdiarticle4.com/review-history/55718 http://creativecommons.org/licenses/by/4.0 _____________________________________________________________________________________________________ *corresponding author: e-mail: ibrahimshnawa3@gmail.com; cite as: i m s, shnawa, and al-janabi aaj. 2024. “immune characterisation of human local pneumotropic isolates of streptococcus pneumoniae serotype 1 and 6 as local vaccine strains”. asian journal of immunology 7 (1):285-91. https://doi.org/10.9734/aji/2024/v7i1151. asian journal of immunology volume 7, issue 1, page 285-291, 2024; article no.aji.127471 immune characterisation of human local pneumotropic isolates of streptococcus pneumoniae serotype 1 and 6 as local vaccine strains shnawa i m s a,b* and al-janabi aaj c a department of medical biotechnology, college of biotechnology, al qasim green university, qasim, babylon, iraq. b college of nursing, university of hilla, babylon, iraq. c department of biology, college of science, university of kufa, najaf, iraq. authors’ contributions this work was carried out in collaboration between both authors. both authors read and approved the final manuscript. article information doi: https://doi.org/10.9734/aji/2024/v7i1151 open peer review history: this journal follows the advanced open peer review policy. identity of the reviewers, editor(s) and additional reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here: https://www.sdiarticle5.com/review-history/127471 received: 10/10/2024 accepted: 10/12/2024 published: 17/12/2024 abstract s. pneumoniae is one of the principle human pneumopathogen. such pathogenicity features in man can be delineated by severity, capsule nature and yield of pure growth onto primary plate cultures. while in laboratory animal models, it accounts for; the nature of capsule serotype, shortness of time for the death end point and extent of tissue pathology. the virulent serotypes c1 and c 6 of human pneumotropic s. pneumoniae were immune characterized as vaccine strains. capsules were separated, identified and quantified. then dispensed as 10mg deried amounts in ampoules. on reaching specific immune priming of rabbits the ampoules dissolved in 10 mls sterile saline and original research article https://doi.org/10.9734/aji/2024/v7i1151 https://www.sdiarticle5.com/review-history/127471 shnawa and al-janabi; asian j. immunol., vol. 7, no. 1, pp. 285-291, 2024; article no.aji.127471 286 each ml admixed with 1ml lanoline and injected intramuscularly per each rabbit's thigh muscles. the immune primed rabbits left for 28 days then test bleed to check for an evident immune conversion then, at the day 35 after immunization the immune rabbits were challenged with live s. pneumoniae 1x 10 to six cfu/ml., in a rate of 2 mls. the carbohydrate based capsule prototype monvalent vaccines were found as; pure, safe, antigenic, immunogenic and immune protective to the rat of 80% for c1 and 60% for c6 serotype in rabbit models. they induced systemic humoral immune precipitins responses higher than mucosal pricipitin responses. proinflammatory cytokine responses tnf alpha, tnf beta and il6 and anti-inflammatory cytokine il10 were mounted as mucosal responses higher than the systemic responses. the proved immune characteristics of these capsule serotypes were suggestive for use of these strain as vaccine strains along with other serotypes in development of the local prototype multiserotype vaccines of this pathogen. keywords: antigenicity; cytokine; immunogenicity; immune efficacy; proinflammatory precipitins; vaccine strain. 1. introduction the nature of capsule serotype in s. pneumoniae played as a determinal factor in pathogenesis, pathogenicity and immunogenicity [1-10]. s. pnenumoniae in our previous work in this area, have shown inter and intraserotype variations in pathogenicity to mice and rabbits [10]. so far the immune characterisation of the sertype 1 and 6 is concerned as a vaccine strains [10]. there were few bacterial specific carbohydrate based vaccine that are approved for mass vaccination in human clinical practice of bacterial infectious diseases. these vaccines are avialable in use for those at risk human beings by the vaccine approval authorities [1]. such as that of multiple carbohydrate based vaccine of s. pneumoniae [2-10]. though at times human pneumococcal pneumonia reported every now and then at diferent part of the developing world countries [10]. so local human pneumotropic s. pneumoniae isolates gained prevelence may express different pathogenic and immunogenic potentials. such differences may be from the same serotypes present in the current internationally approved vaccine [10,11]. the present paper was aims at tempts to immune characterize human pneumotropic local serotype 1 and 6 s.pneumoniae isolates as local vaccine strains. 2. materials and methods seed strains: a collection of eight serotypes of s. pneumoniae that were recovered from clinical human cases of pneumonia. the study strains were elected following certain criteria both in man and laboratory animals. in man, serotype nature, severity while in animal model, serotype nature, shortness in death end point time and extent of tissue pathology. the most virulent of which were serotype 1 and 6 so they were eleted for immune characterisation as vaccine strains [10]. laboratory development: the 24 hr freshly revived original isolates of each serotype 1 and 6 [author collection [10] of the s. pneumoniae serotype 1 and 6 growth were made onto blood agar plates. sterile heavy swab inocula were made onto 20 trypicase soy agar plates for each serotype. the inoculated plates were incubated at 5-10% co2 tension at 37c for 24 hrs. pure growth were harvested by 5 ml sterile saline solution per each plate. the suspensions were brought to ph 8.5 by the addition of 10% koh. the alkalinized suspensions were heated at 90c for 1hr in water bath. these heated suspensions let stand to cool and acified with 5n acetic acid. the acidified suspensions then centrifuged at 5000 rpm for 10min.pellets were discarded and supernatant s were mixed with two volumes of 1% sodium acetate in 95% ethanol solution then kept at 4c for 24 hrs. supernate and pellets were formed. the formed precipitates were removed by centrifugation at 2500rpm for 10 min. pellets were discarded and supernatants were mixed with 5% sodium actate in 0.5 n acetic acid for 24 hrs at 4c. these mixed solutions were centrifuged at 5000 for 30 min. supernatant discarded and pellets were washed with 85%,95% absolute ethanol. then dried at 37c. the 20 plate yeilds 200 mg powder materials. these materials were cautiousely dispensed in 10 mg per ampoule under aceptic precautions and kept till use. each ampoule dissolved in 10 mls., sterile saline representing the immungenic doses for 10 rabbits and stands as prototype monovalent capsular carbohydrate based experimental vaccine in rabbits [12]. purity: a purity checks were done by quadrate streak method onto trypticase soy agar plates in shnawa and al-janabi; asian j. immunol., vol. 7, no. 1, pp. 285-291, 2024; article no.aji.127471 287 each step of the laboratory developmennt of the prototype monovalent pneumococcal vaccine [13]. safety: a 0.1 ml of the capsule experimental prototype vaccines were im injected into rabbits through thigh muscles three replicates for each serotype. the injected animals were followed for up to five days. then eviscerated to test for gross and histology changes [13]. adjuvant: lanolin solution [13]. immunogens: one volume of lanolin was admixed with one volume of 1mg/ml. capsular polysaccharide. the mixture stands as the test immunogens [13]. immunization protocol: the immunogen 2 ml mixture was im injected into the thigh mscle of rabbit and left for 15 days then test bleed [13]. laboratory animals: a group of newzeland rabbits were acclimatzed for housing conditions for two weeks and kept ad libitum cnodition during specific immune priming and challenge experiments. these rabbits were categorized into the following groups; safety group........................................6 rabbits serotype 1 specific immune priming...10rabbits serotype 6 specific immune priming...10 rabbits sham control .......................................10 rabbits challenge models: the specific immune primed rabbit groups at the day 22 postpriming were challenged with live s. pneumoniae in strenght of 1x10 to six /m, survivivors were scored 21 days post challenge [14] blood sampling: test and control rabbits groups were subjected to blood collection by cardiac puncture. sera were saved, dispensed at 0.5 ml alqouts in appendroph tubes and kept at 20 till test for antibody and cytoikine levels [15] tracheal mucosal globulins: parts of the challenged and control rabbits trachea were incised and open up into sterile petri –plates. tracheal mucasa were scrapped into the plates then 5 ml formal normal 0.5% saline were added to the scraps and mixed thorughly and tubbed into centrifuge tubes. scraps saline tubes were centrifuge at 5000 rpm for 10 min. supernatants were kept for processing of mucosal globulin separations in accordance with the method [16]. pellets were discarded immune investigations: determinations of antibody response levels were done by precipitation tests as in [17,18]. cytokine response measurements were done as the manufaturer instrutions. efficacy percent difference statistics: let the efficacy percent of serotype 1 is a and serotype 6 is b .so the efficacy difference percetage is: [a-b]/[a+b/2] x 100 3. results purity: all of the laborsatory development purity checks onto trypticase soy agar plates were found of negative growths. safety: neither gross nor histologic changes were noted in the inoculated safety check rabbits. antigenicity: one from each of the vaccine lot ampoules were used for check of antigenicity test by precipitation tests. 3.1 immunogenicity humoral antibody responses: the mean of serum and mucosal antibody titres for the challenged rabbits were 400 and 20 for serotype 1 and 213.33 and 13.33 for serotype 6. systemic responses were higher than mucosal responses, table 1. cytokine responses: mucosal cytokine responses were higher than systemic cytokine responses both for the serotypes 1 and 6., table 2. cytokine imbalance: cytokine imbalance were noted in serotype 1 vaccine at mucosal response only, table 3. immune efficacy: the immune efficacy of sertype 1 was 80% and for serotype 6 was 60%, table 4. the efficacy percenatge difference is equal to 28.571%.since practically good vaccine is that gaving around 90% protection and the efficacy percetage difference approaching one third .thus serotype c 1 is more immune protective than serotype 6 in local multivalent candidate pneumococcal vaccine taking in consideration the limits of immune interference. prototype carbohydrate based vaccine features: s. pneumoniae serotype 1 prototype shnawa and al-janabi; asian j. immunol., vol. 7, no. 1, pp. 285-291, 2024; article no.aji.127471 288 vaccine was found; pure, safe, antigenic, weak immunogenic needs exogenous adjuvant, induce humoral systemic precipitin responses higher than mucosal responses. mucosal cytokine responses were higher than systemic responses. induce cytokine imbalance at mucosal surfaces but not at systemic compartment. it was immune efficus to 80% in a lapin challenge model. serotype 6 prototype vaccine was found; pure, safe, antigenic, week immungenic need exogenous adjuvant. induce systemic humoral immune resoponses higher than mucosal responses. mucosal cytokine responses were higher than the systemic responses. it neither induce cytokine imbalance at mucosa nor at systemic compartment. it was immune efficus to 60% in lapin challenge model, table 5. table 1. specific immune precipitins respnses of the immne challenged rabbits groups titres serotype 1 specific immune primed rabbits mucosal response systemic response 20 400 serotype 6 specific immune primed rabbits mucosal response systemic response 13.33 213.33 control rabbits mucosal response systemic response 3.33 6.3 table 2. cytokine responses of immune primed challenged rabbits rabbit groups tnf alpha tnf beta il6 il10 serotype 1 specific immune primed challnged rabbits mucosa systemic 473.11-+29 250-+30.7 387.21 -+1.39 250.11-+7.6 360.59-+4.3 223.5-+20.2 253.36-+23.5 202.6 -+14.5 serotype 6 specific immune primed challenged rabbits mucosa systemic 233.55-+1.4 245.2-+35 225.25-+7.3 236.32-+27.8 238.8-+4.9 219.51-+59.6 199.68-+13.5 200.76-+8.1 control mucosa systemic 125.12-+55 115.29-+11.7 110.35 -+11.5 101 -+11.5 93.54 -+2.5 100.62-+0.75 110.25 -+ 11.5 101.15 -+11.5 table 3. s. pneumoniae capsular 1 and 6 primed challenged rabbits cytokine imbalance rabbit groups tnf alpha tnf b il6 il10 sertype 1 specific immune primed challenged rabbits mucosa systemic 4:1* 2:1 3:1 2:1 3:1 2:1 2:1** 2:1 serotype 6 specific immune primed challenged rabbits mucosa systemic 1.5:1 1:1 2:1 1:1 2:1 2:1 2:1 1:1 control mucosa/ systemic 1:1 1:1 1:1 1:1 • the number of folds of cytokine concentrations for the test to control rabbits ** imbalance is the number of concentration folds for the proinflammatory to the anti-inflammatory cytokines. shnawa and al-janabi; asian j. immunol., vol. 7, no. 1, pp. 285-291, 2024; article no.aji.127471 289 table 4. the immune efficacy of the developed vaccines in rabbits prototype vaccine infective dose number of rabbits servive to total percentages type 1 1x10 to 6 10 8:10 80% type 6 1x10 to 6 10 6:10 60% control 1x10 to 6 10 0:10 0% table 5. features of the carbohydrate based s. pneumoniae serotype 1 and 6 monotypic prototype vaccines in rabbit challenge model features protype c1 prototype c6 approved pneumococcal vaccine understanding disease understaning causal pneumonia s. pneumoniae pneumonia s. pneumoniae pneumonia s. pneumoniae virulence factor serotype 1 serotype 6 23 serotypes purity pure pure pure safety safe safe safe antigenicity antigenic antigenic antigenic immunogenicity immungen* immunogen* immunogen* immune efficacy in rabbit immune efficacy in man 80% ? 60% ? 60% • amplified on combination with lanlin 4. disscussion the subunit bacterins of the bacterial pathogens when introduced to the body of a small mammal laboratory animals like rabbits, tables 1 – 5, may triggers th1, th2 and /or b cells to grow, proliferate, expand and activated as an effector and/ or memory cells. as a consiquences, humoral mucosal and systemic antibody responses as well as cytokine network activations both for innate and adaptive cytokine types [19,20] the systemic humoral immune responses were higher than mucosal responses [16]. while the mucosal cytokine responses were higher than the systemic responses [4]. cytokine imbalance was noted betwen pro and antiinflammatory cytokines serotype 1 at mucosal compartment but not systemic. serotype 6 does not express imbalance at both mucosal and systemic compartments [21]. the immune effcacy of the present prototype carbohydrate based capsular bacterins were ranging between 6080% [11,22]. the vaccinologic features of the prepared candidates capsular bacterins were; antigenic, weak immunogenic need an exogenous adjuvant leading to antibody and cytokine responses. such responses were both at mucosal and systemic compartments with an immune efficacy ranging betwen 60 to 80% in rabbit models. the proposed s. pneumoniae vaccine strains may be of help in development of an eight local pneumococcal vaccine with the other reported six serotypes [10] which may be more efficieous in this area than the internationally approved 23 pneumococcal vaccine [22]. the vaccine strains form an integral part in vaccine development, since it forms the first step in vaccine development processes. s. pneumoniae serotype 1 and 6 are among the known serotypes ensembled in various up date and approve vaccine formulations [23,24]. 5. conclusion local pneumotropic isolates of s. pneumoniae serotype 1 and 6 were found valid as vaccine strains based on lapin immune challenged models. the proved immune characteristics of these capsule serotypes were suggestive for use of these strain as vaccine strains along with other serotypes in development of the local prototype multiserotype vaccines of this pathogen. disclaimer (artificial intelligence) authors hereby declare that no generative ai technology models (chat gpt, coilot, etc) and text-to-image generator have been used during writing or editing of this manuscript. shnawa and al-janabi; asian j. immunol., vol. 7, no. 1, pp. 285-291, 2024; article no.aji.127471 290 consent it’s not applicable. ethical approval care, housing, handling and managment done on rabbits were following the international acts regulating care, housing, handling and intervensions. competing interests authors have declared that no competing interests exist. references 1. shnawa ims. vaccines for biomedics: a second revised edition of vaccinology at a aglance. bpi, india-uk; 2022. 2. sukhova ev, yashunky dv, tsuetkov ye, nifanticv ne. high cytokine and opsonosing antibody production induced by bovine serum albumin bsa-conjugate tetrasaccharide relte to streptococcus pneumoniae type 3 capsulr polysaccharide. front. immunol. 2020;11: 578019. doi.10.3389/fimmu/2020.578019 3. maddux jt, stomberg zr, curtiss r iii et al. evaluation of recommbinant attenuated salmonella vaccine strains for broad protection against extraintestinal pathogenic eschrichia coli. front. immunol. 2017;8:1280. doi.10.3389/fimmu.2017.01-1280. 4. kashiwagi y, miyata a, kumagai t et al. production of inflammatory cytokines in response to dipheria-pertusses dpt, hemophilic infleunzae type b and 7 vaccine pnemococcal pcv vaccines. vaccines and immunotherpeutics. 2013;14(3): 677-685. doi.10. 4161/ hv.23264. 5. dos. san, tw, goncalves pa, rodriguez d et al. a fusion protein comprising pneumococcal surace protein a and pneumolysin derivate confers protection in murine model of pneumococcal pneumonia. plos. one. 2022;17(12): e0277304. doi.10.1371/jpone.0277304. 6. malley r, anderson pw. serotype dependent pneumococcal experimental vaccine that induce cellular as well as humoral immunity. biol. sci. 2011;109(10): 3623-3627. doi.10.1073. 112383109. 7. turner a k, generation of a live attenuated entertoxigenic eschrichia coli. combination vaccine express six antigens. clin. vacc. immunol. 2011;18(12) doi.1128/cvi.05345-11. 8. kamboj k k. significant variations in serotype specific immunity of seven valent s pneumoniae capsular polysaccharide crm197 conjugate vaccine occurs deposite vigorous t cell help induced by carrier protein. j. infect. dis. 2003;187 (10):1629-1638. doi.10.1086/374785. 9. masomian m, ahmed z, gew l ti, puh cl. development of next generation streptococcus pneumoniae vaccines conferring broad protection. vaccines. 2020;8:132. doi. 103390/vaccines8010132. 10. al-janabi a a j,shnawa ims,alsadi am. inter and intraserotype pathogenicity of streptococcus pneumoniae in mice and rabbit. j. pharmacuetical research international. 2024;36(10):49-54. 11. kayhty h, ahan h. bridging phase 2 and phase 3 pneumococcal immunogenic data for future combination vaccines clin. infect. dis. 200133(supl.4):s292-298. 12. linker a, russel s. a new polysacchraide resembling alginic acid isolating. j. clin. chem. 1966;241(16):3845-3851. 13. shnawa ims.vaccine technology at a glance.boffin access.uk; 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2024. disclaimer/publisher’s note: the statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of the publisher and/or the editor(s). this publisher and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. _________________________________________________________________________________ © copyright (2024): author(s). the licensee is the journal publisher. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: https://www.sdiarticle5.com/review-history/127471 https://www.sdiarticle5.com/review-history/127471 _____________________________________________________________________________________________________ *corresponding author: e-mail: shadrackkimenju@gmail.com; asian journal of immunology 4(1): 77-84, 2021; article no.aji.69854 advances and challenges in transmission-blocking malaria vaccine development. a systematic review laura nyawira wangai1, shadrack kimenju kahiro2*, kenny kimani kamau1, david waweru nderu1, immaculate marwa nyaiseba1, david butto amarch1, mark kilongosi webale1 and elly munde1 1 school of health sciences, kirinyaga university, kutus, kenya. 2 department of biochemistry, jomo kenyatta university of agriculture and technology, p.o. box 62000-00200, nairobi, kenya. authors’ contributions this work was carried out in collaboration among all authors. all authors read and approved the final manuscript. article information editor(s): (1) dr. cynthia aracely alvizo báez, autonomous university of nuevo leon, mexico. reviewers: (1) g-halli r. rajasekariah, biofirm pty ltd, australia. (2) wagner quintilio, instituto butantan, brazil. complete peer review history: https://www.sdiarticle4.com/review-history/69854 received 15 may 2021 accepted 19 july 2021 published 03 august 2021 abstract malaria continues to cause enormous human suffering throughout most of the tropics and subtropics. in sub-saharan africa alone, it is estimated that about two million children die each year due to malaria. vector control and malaria chemotherapy strategies that were previously effective in controlling and treating malaria, respectively, are now largely ineffective owing to the spread of insecticide-resistant mosquitoes and drug-resistant parasites. a vaccine targeting the sexual stages of the parasite and block transmission is needed to reinforce current malaria control and eradication efforts. here, we review the status of malaria transmission-blocking vaccines. we focused on the efficacy, progress and development challenges of transmission-blocking vaccines. pfs25 and pfs48/45 are essentially the lead-candidates malaria transmission-blocking vaccine and should be studied further in clinical trials. our review highlights the need to develop novel malaria elimination interventions, particularly an effective malaria vaccine with transmission-blocking activity. review article wangai et al.; aji, 4(1): 77-84, 2021; article no.aji.69854 78 keywords: malaria mosquitoes pfs25 pfs48/45 vaccines; drug-resistant parasites plasmodium. 1. background information malaria is caused by unicellular plasmodium protozoan parasites that are injected by female anopheles mosquitoes. p. falciparum, which causes the most severe form of malaria, is a substantial threat today, causing more than 200 million clinical cases and 409,000 deaths in 2019 [1]. while the level of malaria has dropped over the last 10 years, this decline has recently stalled, with some regions experiencing a resurgence in the number of malaria cases [2]. unfortunately, resistance to artemisinin-based combination therapy (act), the current frontline treatment for malaria, is present in southeast asia and now spreading in africa [3]. this highlights the need for novel strategies in malaria prophylaxis and treatment, especially an effective vaccine that can induce a protective antimalaria immune response. even though the development of a malaria vaccine is still a challenging task, the development of several vaccine candidates is ongoing. malaria vaccines can thus be broadly categorised into three groups based on the parasite stage being targeted, namely sporozoite, liver-stage and asexual blood-stage vaccines. liver-stage and asexual blood-stage malaria vaccines (a transmission-blocking vaccine), unlike an anti-sporozoite vaccine, do not either directly protect a vaccinee against malaria infection, reduce parasitaemia or prevent disease; but rather employs all these four strategies. anti-sporozoite vaccines would help by preventing malaria-infected travellers from transferring malaria parasite into a malaria-free zone [4]. the malaria parasite is particularly vulnerable to immune intervention during the sexual and sporogony stages because (1) parasites pass through a substantial numerical bottleneck-with about 100 parasites of the billions of asexual blood-stage parasites developing into gametocytes, get ingested by mosquitoes and subsequently developing into oocysts; (2) the sexual and sporogony stages persist extracellularly for hours unlike other parasite stages; (3) many of the candidate antigens tend to be highly immunogenic owing to their low antigenic diversity as a result of no immune selection in host since parasites do not express vaccine candidate antigens while in the human host [5]. although antibodies targeting gametocytes (sexual stages) are not able to block human infection or disease, they can potentially stop malaria transmission. recently, there has been increasing interest in the use of gametocytespecific antigens as candidates for a transmission-blocking vaccine (tbv) [6]. such a vaccine would induce antibody production that is taken up alongside the gametocytes during a mosquito blood meal and neutralize the parasites in the mosquito midgut upon expression of the antigens. key sexual-stage antigens that have been considered as candidate tbv include pfs48/45, pfs230, pfs25, and pfs47 [7,8]. it has been reported that antibodies from malariaexposed individuals or vaccinated animals against these antigens could block malaria parasite development in mosquitoes [9]. these findings offer an impetus to test human monoclonal antibodies against tbv antigens, such as those generated recently against pfs25, for the ability to block malaria transmission. 2. role of transmission-blocking vaccine the main goal of transmission-blocking vaccine development programs is to eradicate malaria parasites using an inexpensively produced, stably formulated, and easily administered vaccine. it is unlikely that a single antigen subunit vaccine will be enough. it noteworthy however that transmission-blocking vaccines will have a great impact on malaria control when they are combined with other vaccines or control modalities. these may include bed nets, protective vaccines, and massive chemotherapy administration, in controlling or even perhaps eradicating malaria in geographically isolated areas e.g. islands or isolated human and mosquito populations) [10]. in instances where malaria is eradicated, long-term control will be necessary to prevent the re-introduction of the parasite. this could be accomplished by enforcement of mandatory tbv vaccination for persons returning from areas where malaria persists and/or demonstrate evidence of prior vaccination with tbv. the adoption of tbv combined with other malaria control strategies may boost the effectiveness of existing malaria control efforts. firstly, a combination of tbv and multistage malaria vaccines could contribute substantially in curtailing the spread of’ mutant parasites that may be resistant against the protective components of the vaccines by reducing malaria wangai et al.; aji, 4(1): 77-84, 2021; article no.aji.69854 79 transmission to a rate that would make an otherwise partially effective protective component highly effective [11]. secondly, co-administration of tbv with effective anti-malarial drugs will help prolong malaria drug efficacy and delay the emergence or spread of drug-resistant parasites. finally, tbv could reduce morbidity/mortality caused by the spread of the virulent strains of the parasite. previous studies have demonstrated that the more an individual is exposed to new strains of parasites, the more likely they are to develop severe clinical disease [12–14]. however, it is still unclear whether this phenomenon is caused by a new “virulent” parasite strain that is biologically different (e.g., has a different cytoadherence profile) from other strains circulating in a geographical area [13] or it is simply caused by an immunologically new strain to the host. 3. candidate vaccines prior fertilization, extracellular gamete (male and female)-specific antigens are the earliest point during sexual development to which an antibodymediated transmission-blocking effect has been described. the primary function of gametes is to find and fuse with a gamete of the opposite sex, fertilization, in a process that occurs within minutes after a mosquito ingests the sexual stages [15]. since the components of the complement cascade are present in the blood meal are still active for minutes to hours after ingestion, the parasite has developed protective mechanisms against complement-mediated lysis [16]. it is therefore not surprising that the two major mechanisms implicated in antibodymediated transmission blockade are directed towards gamete surface proteins that interfere with fertilization and sensitization of gametes to complement-mediated lysis [17]. other mechanisms that contribute to transmission blockage may involve a cellular component, such as antibody-dependent cell cytotoxicity (adcc) and opsonization have not yet been described, but may occur as well. the major advantage of transmission-blocking vaccines that target pre-fertilization antigens is that immune responses are boosted by each subsequent natural infection. whether boost will increase the titer of transmission-blocking antibodies or extend the longevity of transmission-blocking immunity is yet to be determined. furthermore, antigen immunogenicity also needs to be studied since seroepidemiological studies indicate that less than 50% of humans carrying gametocytes contain detectable antibodies to either of the two lead pre-fertilization vaccine candidates, namely pfs230 and pfs48/45 [17]. other pre-fertilization vaccine candidates are pfs2400, pfs40 and pfg27/25 [18]. 3.1 immune response to pfs25 malaria vaccines that target both human and mosquito infections have the potential to impact malaria control. the importance of developing such a vaccine is underscored in a who world malaria report, which indicates stagnation in the progress towards reducing global malaria cases since 2015 [19]. the malaria vaccine rts, s, which targets human infections as indicated by its impact on clinical disease, has completed phase 3 testing and is in pilot implementation studies in three african countries, namely kenya, ghana and malawi [20,21]. efforts to develop a vaccine that disrupts mosquito infection, known as a malaria transmission-blocking vaccine (tbv), have been ongoing since the reporting of induced tbvinduced immunity in chickens against plasmodium gallinaceum in 1976 [22]. the pace of malaria tbv development, until recently, has been hindered by the lack of capacity to produce candidate antigens for clinical testing [22]. currently, clinical testing is are limited to pfs25, a p. falciparum-specific antigen. pfs25 is a 25 kda sexual-stage protein present on the p. falciparum zygote and ookinete surface in the mosquito midgut. the leading pfs25 tbv is a chemically conjugated vaccine comprising of a pichia pastoris expressed pfs25 and the carrier protein exoprotein a (epa) or a recombinant detoxified form of pseudomonas aeruginosa exotoxin a9. the pfs25-epa conjugate has the biophysical characteristics of a nanoparticle with a size similar to the hepatitis b virus-like-particle used in rts, s [23]. ex vivo standard membrane feeding assays (smfa) during phase 1 trials of pfs25-epa conjugates formulated with alhydrogel™, an aluminium-based adjuvant conducted in the united states of america, and mali revealed that four doses are required to generate antibody titers that significantly reduce parasite transmission [22]. however, poor clinical trial results have halted the development of pfs25epa as a stand-alone tbv. which can probably be tackled by using the correct carrier protein. wangai et al.; aji, 4(1): 77-84, 2021; article no.aji.69854 80 3.2 immune response to pfs230 and pfs48/45 another family of sexual-stage proteins with cysteine-rich domains includes the antigens pfs230 and pfs48/45 that have been targeted for tbv development. however, until recently, there was no recombinant antigen preparation available with the identity, purity, and quality necessary for human clinical trials [24]. of the two, pfs230 cysteine-rich domain was the first antigen to be produced. it consists of a 230 kda sexual-stage protein that is composed of fourteen 6-cysteine-rich domains that suitable for human clinical testing [25]. recombinant pfs230 domain 1 (pfs230d1m) is wellcharacterized and has shown to induce transmission-blocking antibodies in small animals using the smfa16 [24]. parasite-derived antigen (pfs23016a) has been shown to induce the production of transmission-blocking monoclonal antibodies (4f12). a phase 1 safety and immunogenicity study evaluating pfs230 d1epa nanoparticles formulated on alhydrogel™ has been completed [24]. pfs230d1m is conjugated chemically to epa forming nanoparticles with a similar size as hepatitis b virus10. 3.3 post fertilization target antigens several post-fertilization target antigens have been described, including pfs28 (d&y& kaslow. submitted), pfs25. chitinase and mosquito midgut late trypsin [26,27]. the two major mechanisms mediating blockade post-fertilization involve arresting the morphological transformation of the round, sedentary zygote to the oblong, motile ookinete and preventing the egress of the ookinete from the blood meal to the midgut epithelium basal lamina [11]. preliminary evidence suggests that transmission-blocking antibodies may also interfere with normal sporozoite development. however, the exact mechanism(s) by which this occurs is not yet known though there is increased gametocyte clearance and impaired development in later stages [28]. the major advantages of postfertilization target antigens are immunogenicity and limited pre-existing antigenic diversity, unequivocally reflecting the lack of prior immune selection on these antigens. since these target antigens are expressed late in sexual sporogonic development, high antibody titers may be required to account for antibody degradation from proteolysis in the blood meal and boosting after a natural infection may not occur. 4. first generation transmissionblocking vaccine, tbv25h one of the most advanced candidate transmission-blocking vaccine in development is a 6 histidine-tagged protein tbv25h (transmission-blocking vaccine based on pfs25 with a histidine tag) secreted from recombinant succharomvces cerevisiae [29]. a modified fedbatch fermentation protocol was developed that optimizes recombinant protein production using the simplest and least expensive fermentors available. the 6 histidine-tag is used to purify the protein with ease from the yeast culture supernatant by affinity chromatography with nickel-nta agarose. the protein is further purified by simple gel filtration chromatography and then adsorbed to alum. thus, in keeping with the goal of technology transfer to newly industrialized countries, a simple fermentation and post-fermentation process was developed that should allow the ultimate end-users to produce the vaccine themselves [30]. however, tbv25h/alum is not an ideal transmissionblocking vaccine for three reasons; it may require refrigeration, in vivo studies have demonstrated that multiple injections may be required to induce the production of transmission-blocking antibodies and immunity is relatively short-lived. in its current formulation, tbv25h will be invaluable tool for determining the safety of tbv25h/alum and help improve our understanding on how antibodies work to block infectivity in vitro and how it can be elicited in humans. a series of human clinical trials will be required [5]. assessing the efficacy and the impact of a tbv poses some unique challenges [11]. the lack of reliable in vitro correlates of protective efficacy has limited the early phases of protective malaria vaccine development; the latter field-testing phases of protective vaccine development are straightforward. in contrast, the early phases of development of a tbv have the advantage of a reliable (but tedious) in vitro assay for assessing transmission-blocking activity, the membrane feeding assay in which in vitro cultured gametocytes are mixed with test serum and fed through an artificial membrane to starved mosquitoes. the degree to which the test serum inhibits infectivity is determined 1 week after an infectious blood meal by scoring mercurochromestained mosquito midguts for oocysts [31]. nevertheless, in vivo efficacy for transmissionblocking vaccines will require labour-intensive, expensive field testing [21]. wangai et al.; aji, 4(1): 77-84, 2021; article no.aji.69854 81 the assessment of the actual efficacy of a tbv requires field testing unlike any vaccine tested to date, malaria or otherwise. although a phase i trial designed to establish safety and immunogenicity has been used to assess efficacy in vitro (using the membrane feeding assay) and a phase iib (naturally acquired parasite infection rather than experimental parasite challenge) can be used to assess in vivo efficacy by feeding laboratory-reared mosquitoes directly on infected volunteers, designing and executing robust phase iii trials (i.e. testing efficacy in the field) that determines the impact of the vaccine on natural transmission and on preventing morbidity and mortality is difficult [8]. this is because, in phase iii trials, the statistical or study unit is not individual-based but requires a well-defined and, at least partially, isolated population of sufficient size in a malaria-endemic or seasonal malaria area. the setting must be such that the rate of transmission is not so high that a possible positive effect is missed, but not so low that the observed effect would have little consequence as a public health measure. the control and test study units, or communities, must have little interchange between vaccinated and unvaccinated populations but must be similar to allow comparative analyses between among sites [22]. depending on the rate of transmission, the endpoints that are chosen, the similarity in malaria transmission and endemicity, the existence of a malaria transmission control programme (e.g., the presence of p. vivax transmission), and the cooperation of all individuals in each community, as few as one or two test villages and an equal or greater number of control villages may be all that is necessary to preliminarily evaluate efficacy [28]. several ethical issues, however, must be adequately addressed before undertaking these human clinical trials. despite the enormity of the health problems that malaria poses worldwide. an effective malaria vaccine has eluded us. lack of understanding as to what the mechanism of protective immunity is, limited research funding, and perhaps lack of the appropriate technologies necessary to develop an effective vaccine has probably contributed to the delayed development of effective malaria vaccines. novel approaches and technologies may be required such as tbvs. 4.1 sequence polymorphisms of vaccine candidates considering that amino acid sequence polymorphisms present a plausible bottleneck for malaria subunit vaccine development [32], there is a major concern that b-cell epitopes targeted by transmission antibodies might be highly variable [13]. to assess whether any of the polymorphisms affect the antibody affinity towards pfs48/45, serum samples from rats immunized with r0.10c were tested in the smfa with three distinct p. falciparum isolates of asian and african origin, i.e. nf54, nf166 and nf135 [33]. taken together, available data from sequencing and antibody binding studies of field isolates suggest that parasite diversity is unlikely to become an obstacle for the clinical development of pfs48/45-based vaccines [13]. expression of a recombinant vaccine has been hindered by the inability to produce sufficiently high yield recombinant proteins that refolds into the native structure required for the induction of tbv antibody production [34]. proper folding of many cysteine-rich proteins, including pfs48/45, depends on the correct formation of disulphide bridges [35]. in eukaryotes, the oxidizing environment of the endoplasmic reticulum (er) provides a milieu for disulphide bonds formation, a process that is mediated by er oxidoreductase 1 (ero1) and protein disulfide isomerase (pdi) [36]. p. falciparum possesses four protein disulfide isomerases (pfpdis) of which, pfpdi-8 and pfpdi-11 have been expressed in gametocytes [37]. pfs48/45 and pfs25 have been expressed in different eukaryotic expression systems possessing ero1 and pdi analogous but some systems have a track record for production of recombinant malaria antigens the reported yields of properly folded recombinant pfs48/45 have been disappointingly low [38,39]. 4.2 preclinical studies numerous animal studies have shown that pfs48/45, pfs25 and other candidates vaccination elicits antibodies with the capacity to inhibit parasite fertilization in mosquitoes as determined in the smfa [28]. in general, a recombinant protein which assumes a proper fold, as determined by the reactivity with mabs against conformational epitopes, elicits high levels of transmission-blocking antibodies [21]. in contrast, miss-folded protein does not elicit transmission-blocking antibodies suggesting that correct folding of critical epitopes involved in transmission-blocking immunity is essential for an efficacious tbv [21]. adjuvants are vital for an efficacious subunit-vaccine by enhancing seroconversion rates and concentrations of functional antibodies [40]. in the preclinical wangai et al.; aji, 4(1): 77-84, 2021; article no.aji.69854 82 adjuvant selection, it is important to focus on those with a human clinical development path with records of both safety and manufacturability [28]. whether this level of vaccine-specific antibodies is comparable to naturally occurring antibodies against pfs48/45 is unknown. this is mainly because most studies of naturally acquired immunity have reported antibody data as categorical variables (either positive or negative) rather than continuous variable. the availability of new recombinant proteins will help future studies to estimate the level of vaccinespecific as well as naturally acquired antibodies. the ability of newly designed adjuvants to enhance levels of functional antibodies against pfs48/45 has been investigated in small rodents using recombinant proteins produced in the l. lactis expression system [6]. formulations containing the synthetic tlr4 agonist glucopyranosyl lipid adjuvant (gla) or a combination of synthetic lipid adjuvant (sla) and saponin containing qs21 in a liposome formulation (sla-lsq) induce the highest titers of antibodies to sexual stage antigens. both the gla and sla agonists have been assessed in human clinical trials as a part of vaccines against several infectious diseases suggesting they might be useful for the development of a tbv[35]. viral vectors are considered an attractive alternative to protein-based vaccines because they circumvent the need for production and purification of properly folded protein and because of their ability to effectively induce both humoral and cell-mediated immune responses [9]. in recent years this approach has been explored extensively [41]. for instance, pfs48/45 has been tested in a chad63-mva heterologous prime-boost regime as a glycosylated and nonglycosylated recombinant protein [42]. mouse igg against non-glycosylated pfs48/45 shows transmission-blocking activity in smfa using homologous nf54 parasites and to a lesser extent also in the direct membrane feeding assay (dmfa) using p. falciparum parasites collected from naturally exposed gametocyte donors [6]. collectively, these findings suggest that pfs25 and pfs48/45-based subunit vaccines have the capacity to 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vaccines. vaccine. 2015 ;33(52):7433–43. _________________________________________________________________________________ © 2021 wangai et al.; this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: https://www.sdiarticle4.com/review-history/69854 http://creativecommons.org/licenses/by/4.0 _____________________________________________________________________________________________________ *corresponding author: e-mail: celso@alergoimuno.med.br; cite as: olivier, celso eduardo, daiana guedes pinto, ana paula monezzi teixeira, jhéssica letícia santos santana, raquel acácia pereira gonçalves santos, and regiane patussi santos lima. 2024. “endotyping non–ige-mediated immunoreactivity to polyethylene glycol: implications for allergic patients”. asian journal of immunology 7 (1):100-111. https://journalaji.com/index.php/aji/article/view/135. asian journal of immunology volume 7, issue 1, page 100-111, 2024; article no.aji.119914 endotyping non–ige-mediated immunoreactivity to polyethylene glycol: implications for allergic patients celso eduardo olivier a*, daiana guedes pinto a, ana paula monezzi teixeira a, jhéssica letícia santos santana a, raquel acácia pereira gonçalves santos a and regiane patussi santos lima b a instituto alergoimuno de americana, brazil. b lavoisier’s laboratories, são paulo, brazil. authors’ contributions this work was carried out in collaboration among all authors. author ceo designed the study, wrote the protocol and the manuscript’s first draft, and managed the literature searches. authors dgp, apmt, jlss and rpsl performed laboratory research. author rapgs performed cutaneous tests. all authors read and approved the final manuscript. article information doi: https://doi.org/10.9734/aji/2024/v7i1135 open peer review history: this journal follows the advanced open peer review policy. identity of the reviewers, editor(s) and additional reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here: https://www.sdiarticle5.com/review-history/119914 received: 01/07/2024 accepted: 16/07/2024 published: 17/07/2024 original research article https://doi.org/10.9734/aji/2024/v7i1135 https://www.sdiarticle5.com/review-history/119914 olivier et al.; asian j. immunol., vol. 7, no. 1, pp. 100-111, 2024; article no.aji.119914 101 abstract background: several publications report polyethylene glycols (pegs) and pegylated therapeutics as responsible for non–ige-mediated allergic reactions. there is no standardized lab exam that can endotype (quantify the participation of these mechanisms inside each patient’s pattern of symptoms) besides in vivo provocation tests. objective and aim: to evaluate the tube titration of precipitins (ttp) and the leukocyte adherence inhibition test (lait) to discriminate and endotype non–ige-mediated immunoreactivity against a pegs solution in patients with non–ige-mediated allergic phenotypes. research design and research protocol: to retrospectively examine the medical charts of two cohorts (with 100 patients each) diagnosed with non–ige-mediated allergic rhinitis, allergic bronchitis, asthma, allergic sinus headache, atopic dermatitis, and/or urticaria who were investigated with the help of ttp or ex vivo challenge tests monitored by lait against pegs. methodology: we identified the registered results of the semi-quantitative serum ttp against 1 mg/ml pegs 4000 solution, which were distributed in ranges through a cascade distribution chart to outline the variability of the results inside the first cohort. the lai done with the ex vivo challenges with 1 mg/ml pegs 4000 solution were distributed in ranges through a cascade distribution chart the statistical characteristics of the two cohorts were calculated. results: the ttp showed a wide distribution range of results. the mean was estimated at 1:246; the median at 1:192; the standard deviation at 1:188. the lait showed a wide distribution range of results. the lai ranged from 0% to 98%. the mean was 41.9%; the median was 45%; the standard deviation was 28.5%. the cascade distribution demonstrates a wide range of lai results. conclusion: some patients showed any or low immunoreactivity during the ex vivo challenge test, while most displayed moderate or strong immunoreactivity, which could reflect the participation of pegs antibodies in a non–ige-mediated hypersensitivity condition. keywords: allergy; asthma; atopic dermatitis; bronchitis; covid-19 vaccination; diagnosis; exposome-wide association study; endotype; hypersensitivity; leukocyte adherence inhibition test; macrogol; non–ige-mediated immunoreactivity; precision medicine; polyethylene glycol; e1521; rhinitis; sinus headache; urticaria. abbreviations lai : leukocyte adherence inhibition lait : leukocyte adherence inhibition test ttp : tube titration of precipitins pegs : polyethylene glycol polymers 1. introduction for a long time, petroleum-derived polymeric products were discarded as tars. the first polymerized structures to be identified from tars were the polyethylene glycol polymers (pegs) after continued condensation studies in 1860 [1]. pegs are polymers of parental ether monomers such as ethylene glycol, ethylene oxide, or oxyethylene, commonly available as compounds together with their anionic or nonionic derivatives, in mixtures of different chain lengths polymers, indicated by their average molecular weight (200 to over 10,000 da) [2]. pegs are used as solvents and emulsifying agents in processed food, cosmetics, medicines, cleansing, and personal care products. they may also be involved in the production of fabrics, plastics, resins, papers, ceramics, glasses, rubber, metals, wood preservatives, et cetera [3]. “chemically designated as alpha-hydro-omegahydroxypoly(oxy-1,2-ethanediol) or polyethylene oxides, pegs are regulated additives, allowed to be used as emulsifiers in industrialized food and food supplements (e 1521) and as plasticizers in film coating formulations for food supplement tablets and capsules” [4]. “pegs are amphipathic substances soluble in water and in many organic solvents, including aliphatic ketones, alcohols, chloroform, glycol ethers, esters, and aromatic hydrocarbons. acting as emulsifiers, they prevent the separation of unstable water/oil emulsions in industrialized food such as mayonnaise, dairy-based ice cream, frozen yogurts, margarine, blended spreads, cakes, pastries, and frozen desserts” [5]. when used as dietary emulsifiers, pegs may increase intestinal permeability by interfering with the physical properties of the mucus layer, facilitating the growth of pro-inflammatory intestinal microbiota and olivier et al.; asian j. immunol., vol. 7, no. 1, pp. 100-111, 2024; article no.aji.119914 102 contributing to the impairment of inflammatory intestinal diseases and food allergies [6,7]. pegs (also designated as macrogol in pharmacy) are used as vehicles for drugs and ointment bases, capsules, tablet and pill binders, suppositories, and liquid prescriptions, including parenteral, topical, ophthalmic, oral, and rectal preparations. they also prevent protein aggregation and inactivation of pharmaceutical proteins encapsulated in bioerodible polyester microspheres, acting as stabilizers [8]. pegylation (or pegylation) is a process developed in the 1970s to covalent or noncovalently attach peg polymers to molecules, such as drugs, therapeutic proteins, peptides, antibody fragments, and vesicles, then described as “pegylated” [9]. “pegylation changes the physical and chemical properties of the biomedical molecule, such as its conformation, electrostatic binding, and hydrophobicity, resulting in an improvement of the pharmacokinetic behavior of the drug, reducing coalescence, degradation and in vivo elimination of the active pharmacological principles” [10-12]. “pegylated products can induce anti-peg antibody production associated with type iii complement-activated immunoreactivity” [13]. “lower molecular weight pegs are absorbed by the digestive tract and excreted in the urine; however, high molecular weight pegs are not absorbed and form hydrogen bonds with water in the gastrointestinal tract, where they hydrate stools” [14]. long-chain pegs are laxative medications in isosmotic balanced electrolyte preparations [15]. pegs are also used in topical dermatological preparations as penetration enhancers [16]. pegs and their derivatives (laureth, ceteth, ceteareth, steareth, oleth, laurate, dilaurate, stearate, distearate) do not readily penetrate intact skin and are added to a great variety of cosmetic applications as surfactants, cleansing agents, emulsifiers, skin conditioners, and humectants [17]. however, absorption through damaged skin of pegs present in burn creams was associated with fatal intoxication in burned patients characterized by acute renal failure and metabolic acidosis with increased calcium, anion, and osmolar gap [18]. allergic reactions due to pegs used as excipients have already been reported [19]. pharmaceutical excipients are a hidden potential cause of drug and vaccine hypersensitivity reactions [20]. allergic reactions to pegs were put into evidence, particularly after pfizerbiontech® and moderna® mrna covid-19 vaccines were approved for mass vaccination [21,22]. endotyping pharmaceutical excipient immunoreactivity has become a herculean task due to the variety and diversity of formulations, mainly after the appearance of the pegylated lipid nanoparticle vaccines [23]. pegs are the most popular nanomedicine components due to their chemical properties [24]. the ige-mediated anaphylaxis elicited by pegs excipients was the first and easiest endotype to diagnose [25,26]. skin and basophil activation tests are the most available assays to identify immediate and/or ige-mediate allergy to pegs [27]. however, intradermal skin tests performed with pegs may produce immediate anaphylactic reactions, which makes the percutaneous route a more secure way to perform in vivo challenges [28]. at our facilities, we had performed hundreds of skin tests with pegs employing the skin scrape test technique with no systemic reaction, despite dozens of positive wheal-andflare local diagnostic reactions elicited by pegs [29]. a dual cytometric bead assay (dcba) was recently described for detecting anti-peg igg, igm, and ige in patient sera [30]. besides the type i ige-mediated hypersensitivity reaction, several other mechanisms have been proposed, such as igg-mediated anaphylaxis (complement-related or not) and direct mast cell activation [31]. the production of anti-poly (ethylene glycol) antibodies, besides provoking hypersensitivity reactions, can also be problematic when a pegylated drug is administered, accelerating drug clearance and decreasing its therapeutic efficacy [32,33]. covid-19 mrna vaccination is associated with elevated levels of vaccine-induced anti-pegs antibodies and increased systemic reactogenicity [34]. pegs excipients are also suspected to elicit life-threatening immuneinduced organ-specific reactions such as acute pancreatitis, acute interstitial nephritis, and liver injury [35]. we routinely employ the leukocyte adherence inhibition test (lait) and the tube titration of precipitins (ttp) in our facilities as a triage to evaluate non–ige-mediated immunoreactivity against suspected allergens before the performance of more exhaustive in vivo provocation tests [36-42]. the present study hypothesizes that the lait and the ttp may olivier et al.; asian j. immunol., vol. 7, no. 1, pp. 100-111, 2024; article no.aji.119914 103 differentiate diverse endotypes and degrees of immunoreactivity against pegs among patients suffering from the most common allergic phenotypes. to demonstrate diversity in immune responses and to evaluate the potential of the lait and the research of precipitins to endotyping non–ige-mediated immunoreactivity against pegs, we retrospectively compiled the electronic medical charts of patients with non– ige-mediated allergic rhinitis, allergic bronchitis, asthma, sinus headache, atopic dermatitis, and/or urticaria who were investigated with these procedures into our institution. 2. materials and methods 2.1 subjects after receiving institutional review board approval from the instituto alergoimuno de americana (brazil; 05/2024), we reviewed the electronic chart of 9.000 outpatients who attended our facility from january 2018 to june 2024. the sample selection was performed by retrieving the results from the electronic sheets. a cohort of 100 outside patients had been submitted to ttp with pegs solution 1mg/ml for presenting non–ige-mediated allergic rhinitis, allergic bronchitis, asthma, allergic sinus headache, atopic dermatitis, and/or urticaria. this cohort counted 31 males; mean age 34.8 years; standard deviation (sd) 21.3 years; range 0 to 80 years; median 32.5 years; mode = 28 years (appeared five times). a different cohort of 100 outside patients had been submitted to an ex vivo allergen challenge test with pegs solution 1mg/ml monitored with lait for presenting non–ige-mediated allergic rhinitis, allergic bronchitis, asthma, allergic sinus headache, atopic dermatitis, and/or urticaria. this cohort counted 27 males; mean age 39.9 years; sd 20.5 years; range 3 to 84 years; median 39.5 years; modes = 24 and 41 years (each appeared four times). this study did not include patients under biological and/or systemic anti-inflammatory therapy. these procedures were offered to patients with clinical suspicion of pegs’ hypersensitivity who demonstrated a nonreactive or inconclusive allergic skin scrape test against pegs’ solution [29]. 2.2 pegs solution the pegs solution was prepared with powdered peg 4000 (acquired from acs científica) diluted with isotonic saline solution at 1 mg/ml to perform the allergic skin scrape tests, ttp, and lait. 2.3 ex vivo investigation: leukocyte adherence inhibition test 2.3.1 procedure for allergen ex vivo challenging we performed the lait as previously described [43-51]. shortly, each donor’s fresh plasma was divided into two parts and used in paralleled ex vivo challenging tests with pegs solution 1 mg/ml and the unchallenged plasma assay. we collected the plasma with high leukocyte content (buffy coat) from the heparinized tube after one hour of sedimentation at 37°c. then we distributed aliquots of 100 μl into eppendorf tubes kept under agitation for 30 minutes (200 rpm at 37°c) with pegs solution (10μl of a solution with 1mg/ml and ph 7.5) or without pegs solution (when used as control). 2.3.2 procedure for adherence assay after incubation, the plasma was allocated into a standard neubauer hemocytometer counting chamber with a plain, non-metallic glass surface and left to stand for 2 hours at 37 °c in the humidified atmosphere of the covered water bath to allow leukocytes to adhere to the glass. next, we counted the leukocytes, removed the coverslip, and washed the chamber by immersion in a beaker with pbs at 37°c. then, we added a drop of pbs to the hemocytometer’s chamber and allocated a clean coverslip over it. the remaining cells were counted in the same squares as previously examined. 2.3.3 procedure for calculating the adherence inhibition the percentage of leukocyte adherence (la) of each assay was estimated as: (the number of leukocytes observed on the hemocytometry chamber after washing divided by the number of leukocytes observed on the hemocytometry chamber before washing) and multiplied by 100 (%). the leukocyte adherence ratio (lar) was estimated based on the ratio between the la from the antigen-specific challenged plasma and the la from the unchallenged control plasma: olivier et al.; asian j. immunol., vol. 7, no. 1, pp. 100-111, 2024; article no.aji.119914 104 lar = la of the challenged sample divided by la of unchallenged control plasma multiplied by 100 (%). to further calculate the leukocyte adherence inhibition (lai), we subtracted the lar from 100 (%). we employed the lai results for the cascade distribution chart and the statistics calculations, both performed with the help of the microsoft excel® statistical package. 2.4 in vitro investigation: tube titration of precipitins (ttp) as previously reported, the semi-quantitative ttp against the pegs solution was performed in a transparent vitreous tube [52]. shortly, the patient’s blood was collected in a clot-activator collecting tube. after separation, the serum was centrifugated at 2,000 rpm for 10 minutes. the allergen extracts were allocated in sets of eleven glass tubes at progressive duplicated serum dilutions. the progressive dilutions were combined with the 15 μl of the antigen (pegs solution 1 mg/ml) with 250 μl of the patient’s serum, progressively diluted into physiological saline solution (nacl 0,9%) in the dilution ratios of 1:1; 1:2; 1:4; 1:8; 1:16; 1:32; 1:64; 1:128; 1:256; and 1:512. one tube was a blank control done with the water and serum to observe occasional spontaneous precipitation (sia test). after 24 hours, one of us examined the tubes, and the titers (the highest dilution factor that yields a positive visual reading) were recorded [53]. 3. results as a retrospective survey, there was no research protocol; therefore, we report the incidental immune investigation as registered in the digital medical charts. the ttp showed a wide distribution range of results. there were two negative results. most positive results concentrated on the higher dilutions (fig. 1). the mean was estimated at 1:246; the median was 1:192; the sd was estimated at 1:188; the mode was 1:512 (appeared 30 times). all sia tests were negative. the lait showed a wide distribution range of results. the lai ranged from 0% to 98%. the mean was 41.9%; the median was 45%; the sd was 28.5%; the mode was 0% (appeared seven times). the cascade distribution demonstrates a wide range of distribution of lai results (fig. 2). six patients ignored the presence of the allergen on the plasma and presented no inhibition of leukocyte adherence (lai = 0%) after contact with the pegs extract (6% of the tests). some patients showed any or low immunoreactivity during the ex vivo challenge test, while most displayed moderate or strong immunoreactivity, which could reflect the participation of pegs antibodies in a non–ige-mediated hypersensitivity condition. fig. 1. cascade distribution chart of the tube titration of precipitins (ttp on the x-axis %) resulting from the pegs solution against the serially diluted serum of a cohort of 100 tests/subjects (y-axis) olivier et al.; asian j. immunol., vol. 7, no. 1, pp. 100-111, 2024; article no.aji.119914 105 fig. 2. cascade distribution chart of the range groups of leukocyte adherence inhibition (lai) results (x-axis %) of ex vivo pegs solution monitored by the leukocyte adherence inhibition test (lait), according to the respective number of outcomes over a cohort with 100 tests/subjects (y-axis) 4. discussion more and more patients are being diagnosed with peg allergy as an increasing number of processed foods, cosmetics, household products, vaccines, and medicines add peg to their composition [54]. the development of new pegylated compounds is advancing, especially among biological immune therapeutics [55]. pegs are the polymer of choice for the delivery of small drugs, proteins, oligonucleotides, and liposomes due to their biocompatibility and ability to improve pharmacokinetics; however, pegs are a mixture of different polymers that can stimulate unwanted immunogenic reactions, leading to efforts to develop synthetic (monodisperse) more uniform pegs [56]. peg hypersensitivity is associated with medicine-induced anaphylaxis, and pegylateddrug reactions should prompt pegs hypersensitivity investigations, especially by use of skin allergic tests with high molecular weight pegs [57]. diagnosing peg allergy is challenging since the traditional allergy testing approaches are insufficient to demonstrate all kinds of hypersensitivity reactions [58]. besides the ige-mediated hypersensitivity reaction, the production of specific igm and igg antibodies and complement activation were also described [59]. since a methodology for specific-ige research has not yet been described, the feasibility of in vitro challenge tests, such as the basophil activation test, is being studied [60]. brand new allergic diagnostic techniques (such as performing tests with the help of pegylated liposomes) are currently being designed to endotype pegs allergies [61]. the precise diagnosis of the phenotypes and endotypes of hypersensitivities to pegylated medications is paramount for prescribing a successful pegs desensitization [62]. this retrospective compilation of our data showed a large distribution of results when we ascertained the results of ttp and tial to explore humoral and cellular immunoreactivity against pegs. these immunoassays do not identify the exact mechanisms responsible for the clinical condition. instead, they provide clues about sensitization and immunoreactivity distributed into an extensive spectral range between immune tolerance and symptomatic hypersensitivity. the semi-quantitative study of the serological reactions through the titration of precipitins was the most basic laboratory exam upon which the fundamentals of immunology were constructed [63]. precipitating antibodies are classically olivier et al.; asian j. immunol., vol. 7, no. 1, pp. 100-111, 2024; article no.aji.119914 106 associated with a robust immune humoral response against an antigen or allergen [64]. the lait is an ex vivo challenge test performed with a viable leukocyte buffy coat that can theoretically explore most known immune pathways as it allows the interaction of all immune-circulating participants with the allergen [65]. similarly, with the ttp, the lait did not indicate which pathways are involved in producing the immune cellular response demonstrated by the leukocyte adherence inhibition [66-69]. ttp and lait must be interpreted as humoral and cellular markers of the immune response after contact with a specific antigen, configuring themselves as techniques to endotype the exposome phenomenon, as proposed by the exposome-wide association study [70]. ttp and lait are complementary triage tests used at our facilities to select worthwhile antigens to proceed with more laborious in vivo provocation tests when the specific ige is undetectable. none of our patients presented an exclusive reaction to pegs. every patient was simultaneously tested with several chemical and biological allergens, demonstrating positive results for some of them. our clinical experience suggests that reactive allergic patients may impair their symptoms by an additional immunoreactivity against pegs. this preliminary retrospective survey demonstrated an extensive range of results from the ttp and the ex vivo challenge test monitored by lait against pegs in two cohorts of patients with various allergic symptoms. the preliminary results support that the ttp and lait performed with 1 mg/ml pegs 4000 solution may discriminate diverse degrees of in vitro and ex vivo immunoreactivity in patients suffering from diversified allergic phenotypes. it is worth carrying out more in-depth studies to evaluate the usefulness of ttp and lait in endotyping non–ige-mediated hypersensitivity to pegs. 5. conclusion our society is now flooded with chemical derivatives through industrialized foods, cosmetics, cleansing products, and medicines producing unknown immunoreactivity [71]. our preliminary results show that the lait and ttp may differentiate diverse degrees of immunoreactivity against pegs in patients clinically diagnosed with non–ige-mediated allergies. this methodology can provide a socioeconomic impact since the technology to perform tial and ttp is inexpensive and can be achieved in a single lab room attached to the clinical facility with minimum laboratory equipment. however, the propaedeutic meaning of these results and the possibility of interferents must be better established [72]. studies designed to establish diagnostic cutoffs focused on the quality-by-design approach with prospective larger double-blind cohorts need to evaluate the potential contribution of lait and ttp for endotyping immunoreactivity in patients suspected of symptomatic hypersensitivity against pegs [73,74]. 6. limitations this study is a retrospective analysis of data collected over six years. there was no protocol research, and the subject’s data were limited to the essentials available on our electronic sheets. therefore, we could not establish a crosscomparison between positive and negative controls to validate the results. the number of subjects is appropriate for a preliminary study; however, future studies must be more comprehensive and include patients’ follow-up information. the lack of a research protocol implies the possibility of a bias produced by the physician’s point of view, which indicated the exam (ceo) based on a clinical suspicion led by the anamnesis, physical examination, and in vivo provocation tests. the study lost many of these patients to follow-up, so assuring the relationship between the immunoassays’ results and the patient’s clinical outcome is impossible. disclaimer (artificial intelligence) authors hereby declare that no generative ai technologies such as large language models (chatgpt, copilot, etc) and text-to-image generators have been used during the writing or editing of manuscripts. consent it is not applicable. ethical approval the authors have collected and preserved written ethical approval per international standards. olivier et al.; asian j. immunol., vol. 7, no. 1, pp. 100-111, 2024; article no.aji.119914 107 acknowledgements the instituto alergoimuno de americana funded this work. we want to thank our new laboratory technician, alessandra vieira de oliveira, for participating in the exams. competing interests authors have declared that no competing interests exist. references 1. ravve a. principles of polymer chemistry third edition. springer, new york. 2012;1. 2. jang hj, shin cy, kim kb. safety evaluation of polyethylene glycol (peg) compounds for cosmetic use. toxicol res. 2015;31(2):105-36. 3. expert committee on food additives. evaluation of certain food additives. twenty-third report of the joint fao/who in world health organization technical report series. geneve. 1980; 648:17-18. 4. younes m, aggett p, aguilar f, crebelli r, dusemund b, filipič m, et al. refined exposure assessment of polyethylene glycol (e 1521) from its use as a food additive. efsa journal. 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to people or property resulting from any ideas, methods, instructions or products referred to in the content. _________________________________________________________________________________ © copyright (2024): author(s). the licensee is the journal publisher. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: https://www.sdiarticle5.com/review-history/119914 https://www.sdiarticle5.com/review-history/119914 _____________________________________________________________________________________________________ *corresponding author: e-mail: harunaisamohammed@nsuk.edu.ng; asian journal of immunology 4(1): 135-145, 2021; article no.aji.77674 molecular detection and sequencing of rotavirus vp4 among children aged 0-5 years with gastroenteritis in 2 selected healthcare centres in keffi, nigeria i. ibrahim1, r. u. usman2, h. i. mohammed3*, d. ishaleku3 and a. b. shuaibu4 1 medical microbiology unit, medical laboratory department, federal medical centre, p.m.b. 004, keffi, nasarawa state, nigeria. 2 science department, brightway international academy, keffi, nasarawa state, nigeria. 3 department of microbiology, nasarawa state university, p.m.b. 1022, keffi, nasarawa state, nigeria. 4 department of veterinary medicine, usman danfodio university, p.m.b. 2346, sokoto, nigeria. authors’ contributions this work was carried out in collaboration among all authors. authors ii and him designed the study, collected samples, performed laboratory and statistical analyses and wrote the first draft of the manuscript. authors ruu, di and abs designed the study, managed literature searches, wrote the protocols and managed the analyses of the study. all authors read and approved the final manuscript. article information editor(s): (1) dr. wagner loyola, brazilian agricultural research corporation, brazil. reviewers: (1) nirmal kumar mohakud, kiit university, india. (2) ahmed samy el-shafey, tanta university, egypt. complete peer review history, details of the editor(s), reviewers and additional reviewers are available here: https://www.sdiarticle5.com/review-history/77674 received 20 october 2021 accepted 27 december 2021 published 29 december 2021 abstract aims: this study was conducted to detect and sequence rotavirus vp4 among children aged 0-5 years with gastroenteritis in 2 selected healthcare centres in keffi, nigeria. study design: the study was a cross sectional study. place and duration of study: keffi, nasarawa state, between march and june 2019. methodology: stool samples were collected from 303 (203 from fmc keffi and 100 from phc angwan waje, keffi) children with gastroenteritis and information about them were obtained by original research article ibrahim et al.; aji, 4(1): 135-145, 2021; article no.aji.77674 136 structured questionnaires. all collected samples were screened for the presence of rotavirus antigen using aria rotavirus antigen detection test kit (ctk biotech, inc, san diego, usa). vp4 was detected from rotavirus positive samples by reverse transcriptase polymerase chain reaction (rt-pcr) using specific primers. the sequences of the amplified vp4 genes were verified using mega software version 7 and rotavirus strains were determined by pasting the fasta (text based format for representing nucleotide sequence) format into the basic local alignment search tool (blast) from the national center for biotechnology information (ncbi). data collected were analysed using smith’s statistical package (version 2.8, california, usa) and p value of ≤ 0.05 was considered statistically significant. results: of the 303 children screened, 54(17.8%) were positive for rotavirus infection. highest prevalence of the viral infection was recorded among males (19.7%) aged 0-12 months (24.8%). gender was found to be associated with rate of rotavirus infection in this study (p<0.05). however, age was not significantly associated with the viral infection (p>0.05). furthermore, based on the rt-pcr carried out, 3(5.6%) out of the 54 rotavirus positive samples were positive for the vp4 gene and sequences of this gene were all found to be of type p [11] strain n115. conclusion: this study reveals the presence of infection with type p [11] strain n115 (5.6%) of rotavirus in the study population. the detection of this rare rotavirus strain in this study is a cause for concern and hence there is an urgent need for the nigerian health authorities to implement a nationwide surveillance system for monitoring rotavirus molecular epidemiology. keywords: vp4; rotavirus; gastroenteritis; children; keffi; nigeria. 1. introduction rotavirus is the leading cause of severe gastroenteritis particularly among infants and young children worldwide with over 70% of cases occurring in nigeria and other endemic subsaharan african countries [1-4]. it is believed that almost every child in the world is infected with rotavirus at least once by the age of five [5]. however, immunity develops with each infection, so subsequent infections are less severe and hence adults are rarely affected [6]. rotavirus belongs to the virus family reoviridae, it is a non-enveloped and has an icosahedral nueleocapsid structure, enclosing a double stranded (ds) rna genome segmented into 11 compartments. the genome codes for six structural proteins, (vp1 to vp4, vp6 and vp7) and five nonstructural proteins (nsp1 to nsp5) [7]. vp4 is on the surface of the virion that protrudes as a spike [8]. it binds to molecules on the surface of cells called receptors and drives the entry of the virus into the cell [9]. it has to be modified by the protease enzyme trypsin, which is found in the gut, into vp5 and vp8 before the virus is infectious [10]. it also determines how virulent the virus is and determines the ptype of the virus [11]. there are at least 10 distinct species/groups of rotavirus (ai, j), differentiated by their vp6 antigenic properties [12]. groups a, b and c are found to cause infection in both humans and animals, whereas groups d, e, f, g and i have been found only in animals [13]. there are 32 g (vp7) genotypes and 47 p (vp4) genotypes identified through molecular epidemiology [2]. rotaviruses are transmitted by the fæcal-oral route, via contact with contaminated hands, surfaces and objects, and possibly by the respiratory route [3,4,14]. the viral infection is usually associated with fever, nausea and vomiting, followed by abdominal cramps, frequent watery diarrhea, which may last for 3-8 days, cough and runny nose occurring mostly among infants [3,15]. rotaviruses are stable in the environment and can survive for long periods of time on toys and ordinary surfaces found in most homes. they also are relatively resistant to most soap and commonly used disinfectants, hence preventing a child from exposure can be difficult [3]. therefore, vaccination still remains the best hope for preventing infection with rotavirus especially in resource constraint nations such as nigerian [1,16]. additionally, most hospitals and diagnostic centres in such nations do not routinely diagnosed rotavirus infection probably due to it high cost and may be because it share similar signs and symptoms with other infantile gastroenteritis [14,15,17].this consequently usually leads to misdiagnosis and mistreatment of the viral infection. ibrahim et al.; aji, 4(1): 135-145, 2021; article no.aji.77674 137 thus the aim of this study was to detect and sequence rotavirus vp4 among children aged 05 years with gastroenteritis in 2 selected healthcare centres in keffi, nigeria. 2. materials and methods 2.1 study area this study was conducted at federal medical centre (fmc) and primary healthcare centre (phc), angwan waje in keffi local government area, nasarawa state, nigeria. keffi town, where the 2 centres are located is approximately 68 km from abuja, nigeria’s federal capital territory and 128 km from lafia, the capital of nasarawa state. it is located geographically between latitude 8°3’n of the equator and longitude 7°50’e and situated on an altitude of 850 m above sea level [18]. 2.2 study population the study participants were male and female children with gastroenteritis aged 0-5 years accessing health care in the 2 selected healthcare centres in keffi, nasarawa state, nigeria. their socio-demographic and other required information were obtained from their parents/guardians by the use of a designed questionnaire. 2.3 sample size determination to determine the sample size, the formula by naing et al. [19] for sample size calculation at 0.05 level of precision was used; where: n = required sample size z = standard normal deviation at the required confidence interval (1.96) which corresponds to 95% confidence interval. p = prevalence of rotavirus infection from previous study (25.0%) (0.2) [20]. q = 1 – p = 0.9 d = degree of precision expected (0.05) to ensure minimum error however, this was rounded up to 303 samples. 2.4 sample collection and storage a total of 303 stool samples (203 from fmc keffi and 100 from phc angwan waje, keffi) were collected from children with gastroenteritis aged 0-5 years. the samples were collected into sterile wide mouth universal containers, labeled accordingly and stored at -20 o c until ready for use [21]. 2.5 laboratory analysis 2.5.1 detection of rotavirus antigen all collected stool samples were screened for the presence of rotavirus antigen using aria rotavirus antigen detection test kit (ctk biotech, inc, san diego, usa). the tests procedure and results interpretation were done according to the instructions of the manufacturer. 2.5.2 test procedure the specimen was brought to room temperature and the test device was removed from the pouch and placed on a clean flat surface and labeled accordingly. two drops of the watery stool was added into the labeled sample dilution vial followed by vigorous shaking to mix. thereafter, 2 drops of the mixture was dispensed into the sample well of the test device and a timer was set up for 15 minutes after which the results were read and interpreted as positive, negative or invalid according to the manufacturer’s instructions. 2.5.3 molecular detection of rotavirus vp4 the vp4 was detected by a reverse transcriptase polymerase chain reaction (rtpcr) system previously described by gentsch et al. [22] using specific primers (consensus primers vp4f/vp4r) adopted from the work of simmonds et al. [23]. 2.5.4 rotavirus rna extraction viral rna was extracted and purified from 10% faecal suspensions in phosphate-buffered saline using the trizol method (gibco brl, invitrogen, burlington, canada). 2.5.5 rotavirus vp4 rt-pcr the vp4 gene was reversely transcribed and amplified using the following primers as adopted from the work of simmonds et al. [23]: ibrahim et al.; aji, 4(1): 135-145, 2021; article no.aji.77674 138 con2 forward (5′-atttcggaccatttataacc3′) con3 reverse (5′tggcttcgctcatttatagaca-3′) the complementary dna was generated by reverse transcription at 45 o c for 30 minutes with initial denaturation at 95 o c for 5 minutes followed by 35 cycles of denaturation at 95 o c for 1 minute. 2.5.6 agarose gel electrophoresis the pcr products were analyzed by running a 1% agarose gel stained with ethidium bromide. the sizes of pcr products were estimated in relation to the migration pattern of a 100bp to 1000bp increments plus dna molecular marker (bioneer daejeon, north korea). 2.5.7 rotavirus vp4 gene sequencing the amplified vp4 genes obtained were sent to inqaba biotec, south africa for sequencing. the method described by sanger et al. [24] with modification was used. briefly: 10µl of the amplified vp4 genes were cleaned by mixing with 2.5µl exo/sap master mix and incubated at 37 o c for 15 minutes. the reaction was stopped by heating the mixture at 80 o c for 15 minutes. sequencing was then done on the mixture using nimagen; brilliant dye™ terminator cycle sequencing kit version 3.1 brd. the labelled products were then cleaned using zr-96 dna sequencing clean-up kit and analysed using applied biosystems abi3500xl genetic analyser yielding sequence chromatogram. sequences were verified using mega software version 7. the strains were determined by pasting the fasta (text based format for representing nucleotide sequence) format into the basic local alignment search tool (blast) from the national center for biotechnology information (ncbi). 2.6 data analysis the data obtained were analyzed using smith’s statistical package (version 2.8, california, usa). chi-square test was conducted at 95% confidence interval and p values ≤ 0.05 were considered statistically significant. 3. results and discussion this present study was conducted to detect and sequence rotavirus vp4 among children aged 05 years with gastroenteritis in 2 selected healthcare centres in keffi, nigeria. a total of 303 children (203 from fmc keffi and 100 from phc angwan waje, keffi) majority of which were males (157/303) aged 0-12 months (153/303) were recruited and screened for rotavirus antigen. overall, 54 (17.8%) children tested positive for rotavirus infection giving a total prevalence of 17.8% (table 1). the 17.8% prevalence of rotavirus infection recorded in this study was higher than the 6.0% reported among children in kwara state [25], 7.4% among children with diarrhoea in kano state [suleiman et al., 2020] and 12.5% among infants in calabar [26]. it was however lower than the 24.8% reported among children with gastroenteritis in akure [27], 25.0% among children less than five years of age in abuja satellite towns [28] and 56.0% among children with diarrhea in enugu state [29]. interestingly, researchers from other parts of africa and the world also reported varying rates of the viral infection. for instance, it was 14.5% in kenya [30], 16% in sudan [31], 18% in india [32], 39.2% in benin republic [33], 44.8% in indonesia [34] and 56% in portugal [35]. the differences observed in the prevalence rates from different studies were possibly due to differences in testing methods used, location of the studies, time and season of sampling and study population types with different associated risk factors [2]. this study did not record significant association between rotavirus infection and age of the participants (p>0.05). however, most of the children infected were between the age group 012 months (24.8%), followed by 13-24 months (19.7%), 37-48 months (4.8%) and 25-36 months 1(2.9%) while none was detected in age group 49-60 months (table 1). this observation agrees with the report of theophilus et al. [20], okebugwu et al. [27] and dhital et al. [36] among children with gastroenteritis in abuja, akure and eastern india respectively but disagrees with that of mohammed et al. [37] and aliyu et al. [38] who reported higher prevalence of the viral infection among nigerian children within the age group 2550 months. the higher prevalence of the infection recorded among younger children in this present study may be attributed to the absence of well-developed immune system among them as older children are expected to acquire protective immunity during repeated exposures to the virus and therefore, subsequent infections are mild or asymptomatic [39]. ibrahim et al.; aji, 4(1): 135-145, 2021; article no.aji.77674 139 table 1. prevalence and distribution of rotavirus infection in relation to age and gender among children between ages 0-5 years with gastroenteritis in 2 selected healthcare centres in keffi, nigeria parameter no. examined no. positive prevalence (%) p-value (n=303) (n=54) (overall=17.8) age (months) 0–12 153 38 24.8 0.7792 13–24 71 14 19.7 25–36 37 1 2.9 37–48 21 1 4.8 49–60 21 0 0.0 gender male 157 31 19.7 0.0004* female 146 23 15.8 *statistically significant fig. 1. agarose gel electrophoretogram of rotavirus vp4 amplified gene. samples 1, 6 and 9 were positive for rotavirus vp4 gene while samples 2, 3, 4, 5, 7, 8, 10, 11, 12, 13, 14, 15 and 16 were negative. *l represents the molecular ladder, ‘ −ʼis the negative control while ‘ + ’ is the positive control there was significant association between gender and prevalence of rotavirus infection in this study (p<0.05) as males were more infected (19.7%) compared to their female counterparts (15.8%) (table 1). this is consistent with the reports of most other previous studies conducted in nigeria [3, 15, 18, 26, 28, 29, 37] and other parts of the world [30, 31, 34, 40]. the higher prevalence of the infection among male subjects in most previous studies may be connected to the fact that females mount stronger humoral and cellular immune responses to infection or antigenic stimulation than the males [41]. furthermore, based on the rt-pcr carried out in this study, 3(5.6%) out of the 54 samples that tested positive with the lateral flow immunoassay kit were positive for the vp4 gene (fig. 1). the low rate of rotavirus vp4 gene detected by rtpcr in this present study could possibly be as a result of pcr inhibitors in the faeces that were carried over into the rna extracts [42]. it could also be attributed to prolonged storage of the stool samples, method of rna extraction and effects of freezing and thawing [43]. the 3 amplified vp4 genes were sequenced and were found to be of type p [11] strain n115 (figs 2a and 2b). this strain showed 88.08% nucleotide identity to human rotavirus a strain g12 p [11] with accession number mh559158.1 ibrahim et al.; aji, 4(1): 135-145, 2021; article no.aji.77674 140 in the genbank. the nucleotide sequence of the n155 strain detected in this study has been deposited in the genebank under the accession number: eu200796.1 (figs 3 and 4). fig. 2a. chromatograph of the sequenced rotavirusvp4 genes (forward primer) fig. 2b. chromatograph of the sequenced rotavirus vp4 gene (reverse primer) ibrahim et al.; aji, 4(1): 135-145, 2021; article no.aji.77674 141 fig. 3. image of blast of sample showing sequence with significant alignment with that in the gen bank fig. 4. phylogeny of rotavirus vp4 gene this strain of rotavirus detected in this present study has not been reported in nigeria. in contrast however, other genotype combinations have been reported in nigeria and other african countries. for instance, genotype g12 was detected in asaba, delta state [44], ibadan, oyo state, nigeria [45], nairobi, kenya [46], dakar, senegal [47], democratic republic of congo [48] and in tunisia [49]. furthermore, in india, giri et al. [50] identified genotype g12 p [11] while ray et al. [51] and ramani et al. [52] reported the detection of strain n155 with a difference in the g-type (g10) ibrahim et al.; aji, 4(1): 135-145, 2021; article no.aji.77674 142 among neonates with gastroenteritis in india. additionally, libonati et al. [53] also reported the detection of g10 p [11] type in india. these strains of group a rotavirus are believed to be from bovine origin as a result of bovine-human gene re-assortment [50]. 4. conclusion this study recorded 17.8% prevalence of infection with type p [11] strain n115 (5.6%) of rotavirus among children aged 0-5 years with gastroenteritis in 2 selected healthcare centres in keffi, nigeria. the detection of this rare rotavirus strain in this study is a cause for concern and hence there is an urgent need for the nigerian health authorities to implement a nationwide surveillance system for monitoring rotavirus molecular epidemiology, before considering introduction of rotavirus vaccination into the expanded program on immunization (epi). this will help to give necessary information on current genotypes and novel introductions as well as evolution of mutant strains to help augment current rotavirus prevention and control. consent all parents/guardians of the children included in this study completed and signed an informed consent form. ethical approval formal ethical approval to conduct this study was obtained from the research ethics committee of federal medical centre, keffi (fmc/kf/hrec/207/17). permission was also obtained from the management of primary healthcare center angwan waje, keffi. acknowledgement the study team would like to thank the management of federal medical centre and primary healthcare centre, angwan waje, keffi, nigeria for their kind permission to conduct the research work. we are also grateful to all parents/guardians of the children who voluntarily participated in the study. however, this research did not receive any form of grant from governmental or non-governmental organizations. competing interests authors have declared that no competing interests exist. references 1. troeger c, khalil ia, rao pc, cao s, blacker bf, ahmed t, et al. rotavirus vaccination and the global burden of rotavirus diarrhea among children younger than 5 years. jama pediatrics. 2018; 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≡ director; *corresponding author: e-mail: bawazir56@gmail.com; asian journal of immunology 4(1): 146-153, 2021; article no.aji.83681 prevalence and classes of sars-cov-2 antibodies among covid-19 suspected patients who attended a health care setting in sana’a, yemen talal a. sallam a#, mokhtar al-youssefi b≡ and amen a bawazir c*# a department of medical microbiology, faculty of medicine and health sciences, sana`a university, yemen. b alyoussefi specialized laboratories, sana’a, yemen. c department of public health and epidemiology, faculty of medicine and health sciences, aden university, yemen. authors’ contributions this work was carried out in collaboration among all authors. authors tas and may designed the study and author may carried out the data collection. authors tas and aab analysed and interpreted the data. all authors read and approved the final manuscript. article information editor(s): (1) prof. cynthia aracely alvizo báez, autonomous university of nuevo leon, mexico. (2) dr. wagner loyola, brazilian agricultural research corporation, brazil. reviewers: (1) sadeeq sheshe, kano university of science & technology, nigeria. (2) ji dejun, yangzhou university, china. complete peer review history, details of the editor(s), reviewers and additional reviewers are available here: https://www.sdiarticle5.com/review-history/83681 received 19 november 2021 accepted 28 december 2021 published 29 december 2021 abstract aims: this study investigates sarscov-2 antibody prevalence and classes among covid-19 suspected patients in sana’a, yemen. antibody response to sars-cov-2 infection remains to be fully elucidated. currently, no reports on sars-cov-2 antibody response from yemen are available. study design: this cross-sectional study investigates sars-cov-2 antibody prevalence and classes among covid-19 suspected patients. place and duration of study: this study was conducted in sana’a the capital of the republic of yemen from june 2020 through january 2021. methodology: serological investigation for anti-sars-cov-2 antibody tests was conducted for 259 original research article sallam et al.; aji, 4(1): 146-153, 2021; article no.aji.83681 147 suspected covid-19 patients who attended a health care facility for antibody testing to confirm the diagnosis on c. results: the mean age was 40.8 ±16.6 years. of all subjects, 180 (69.5%) were males and 79 (30.5%) were females, 73% were < 50 years of age. a total of 133 (51.4%) had at least one antisars-cov-2 antibody class, 6 (2.3%) had isolated igm, 80 (30.9%) had concomitant igm and igg and 49 (18.9%) had isolated igg. only the seropositivity of isolated anti-sars-cov-2 igg significantly (p=0.002) differs among various age groups. there was a significantly higher (p=0.017) igm seropositivity among females than among males. conclusions: among subjects with suspected covid-19, > 30% had concomitant igm and igg with a minority having isolated igm or igg suggesting concurrent or close seroconversion time of both antibody classes. in addition, around 50% of subjects were sars-cov-2 seropositive suggestion low sars-cov-2 seroconversion and consequently low community seroprevalence. an antibody dynamic study based on will characteristics of covid-19 patients is required. also, a community-based seroprevalence study based on the detection of a combination of igm, igg, and iga remains essential to determine the prevalence of sars-cov-2 infection in yemen. keywords: covid-19; igg; igm; sarscov-2; seroprevalence; yemen. 1. introduction immune response to sars-cov-2 infection remains to be fully elucidated. however, the available data, although inconsistent, have shown that sars-cov-2 infection induces antibody response 4 to 15 days post infection[16] with rising levels in severe disease [7,8] and increasing seroconversion rates with progress of time [2,9,10]. although anti-sars-cov-2 antibodies response apparently follows conventical serological dynamic with sequential igm and igg, the concomitant appearance of both antibody isotypes has been reported [11,12] immunoglobulin m first appears 5-10 days post onset of symptoms in most patients, rising about 2 to 3 weeks rapidly to reach a level maintained for 1 to 4 weeks and begin to decline thereafter [8,13]. however, igg antibody has been reported to appear 10 days after infection [13], and remains detectable up to 6 months after infection [14]. reports on decline time frame of antisars-cov-2 antibody levels are inconsistent as they vary from 20 [11,12] to 63 days [15-17] for igm and 35 to > 49 days.[8,11,18,19] and even to as long as 199 days [18] in the case of igg. this variation in seroconversion time and duration of antibodies reflects variations in different studies, in patients’ populations, in age and gender of the population enrolled, in disease severity, in clinical course, and in the serological assays used. these together seem to negatively impact the attempt to reliably establish the dynamic of the antibody response and hinder a genuine estimate of seroprevalence and the herd immunity level. so far epidemiological studies have shown conflicting low seroprevalence rates of sarscov-2 antibodies, in different parts of the world. various reports have shown seroprevalence rates of 4 to 7.3% in several part of europe, 4.2% in northern america, 22% in central and southern asia [17], 0.07% in south korea [16], 3.8% in wuhan geographic regions [19] and 7.8% in malaysia [20]. this variation suggests varying time of testing through the pandemic, demographic and geographic variations and variation of serological assays used. the low seroprevalence rates is attributable to the fact that majority of the studies were mainly based on detection of igg and igm whereas iga was found in covid-19 patients in isolation of igm and igg [20,21]. thus inclusion of iga detection in seroprevalence studies detects more covid-19 infections. yemen is one of the countries where confinements were inadequately implemented during spread of covid-19 because of the current political turmoil and the collapse of the health care system. based on latest estimate of the ministry of public health and population in yemen, covid-19 might expand and potentially infect 90% of the population [2,22]. despite this gloomy scenario the other side of the story is vowing as the absence of containment measures will promote the development of natural herd immunity. despite the frequent reports of sarscov-2 antibody prevalence and insights on antibody response from a number of countries around the globe but not from sana’a, yemen. yemen as one of the countries where confinement of covid-19 was ineffectively implemented since the emergence of the disease because of the current political turmoil and the collapse of the health care system. this is one of the very few studies if not the first on antibody response and sallam et al.; aji, 4(1): 146-153, 2021; article no.aji.83681 148 seroprevalence of sars-cov-2 in the major city of sana`a, northern part of yemen. moreover, this study will provide insights on the antibody response to sars-cov-2 in yemen where the study was conducted relatively early during sars-cov-2 pandemic as well as it could work as a baseline to further studies that measure the level of herd immunity to covid-19 in the country. therefore, the aim of this study was to investigate the antibody response and to estimate the seroprevalence of sars-cov-2 among covid-19suspected patients who attended a health care setting for covid-19 testing in sana`a, yemen. 2. materials and methods 2.1 study design and settings this crosssectional study was conducted retrospectively in the sana`a region in yemen during the second week of june 2020 through january 2021. on an informed consent the antisars-cov-2 antibody tests results and the demographic data that included age and gender of 259 covid-19 suspected patients who were referred to a health care facility for antibody testing to confirm the diagnosis were anonymously enrolled in this study. 2.2 antibody testing the sera were tested for anti-sars-cov-2 igm and igg antibodies using iflash –sars-cov-2 igm and igg paramagnetic particle chemiluminescent (clia) (shenzhen yhlo biotech co., ltd. china) for qualitative determination of igm and igg according to the manufacture instructions. in summary prediluted serum samples were incubated with a recombinant sars-cov-2 antigen that was coated to paramagnetic microparticles to allow the formation of antigenantibody complex. then complex was washed under magnetic field during which magnetic particles were absorbed into the inner wall of the reaction tubes while the unbound materials were washed away from the solid phase of the magnetic field. then the complex was incubated with acridinium-labeled anti-human antibody conjugate. the newly formed complex that consisted of sars-cov-2 antigenantibody and acridinium-labeled antihuman antibody was washed. then a pre-trigger and a trigger solution were added to trigger the signal and the resulting chemiluminescent reaction was measured as relative light units (rlus) the detection of which by the iflash optical system was proportional to the antibody concentration present in the sample. the results are determined through a calibration curve by 2point calibration. antibody level ≥ 10 absorbance units per milliliter (au/ml) was considered positive. 2.3 statistical analysis data entered in a statistical package (spss version 22) for analysis. categorical variables were used for the description of the demographic characteristics of the participants (age and sex). association between the seroprevalence findings of the igm and igg with the characteristics of the participants were undertaken using a chi-square test, where p value findings of < 0.05 was considered significant. 3. results the mean age of the 259 subjects was 40.8 ±16.6 years, ranging between 3-85 years. most of the participants 180 (69.5%) were males and 79 (30.5%) were females, young adults (≤18 -49 years), who were mostly infected in week 2 of june 2020 (fig. 1). a total of 133 (51.4%) were positive for at least one anti-sars-cov-2 antibody class, and of these 6 (2.3%) had isolated igm, 80 (30.9%) had concomitant igm and igg, 49 (18.9%) had isolated igg, and 128 (49.4%) had igg with or without igm (table 1). table 1. seropositivity rate of various anti-sars-cov-2 antibody classes among covid-19 suspected subjects who attended a health care facility in in sana`a, yemen variable category n % antibody response at least one antibodies 133 51.4 absence of antibodies 126 48.6 isolated igm 6 2.3 concomitant igg/igm 80 30.9 isolated igg 49 18.9 igg with/without igm 128 49.4 sallam et al.; aji, 4(1): 146-153, 2021; article no.aji.83681 149 table 2. seropositivity rate of anti-sars-cov-2 antibody classes among males and females and different age groups of covid-19suspected subjects who attended a health care facility in in sana`a, yemen(n=259) variable at least 1 antibody class n= 133 isolated igm (n= 6) isolated igg (n= 49) igg with/without igm (n=128) concomitant igm & igg (n= 80) n (%) p n (%) p n (%) p n (%) p n (%) p sex male 88 (69.8) 0.907 1 (17.0) 0.017 144 (68.6) 0.503 89 (67.9) 0.581 125 (69.8) 0.861 female 38 (30.2) 5 (83.0) 66 (31.4) 42 (32.1) 54 (30.2) age (years) 3-18 8 (6.3) 0.069 0 (0.0) 0.721 16 (7.6) 0.002 8 (6.1) 0.112 8 (4.5) 0.074 19-49 85 (67.5) 5 (83.3) 147 (70.0) 89 (67.9) 117 (65.4) 50-64 13 (10.3) 1 (16.7) 23 (11.0) 14 (10.7) 29 (16.2) ≥ 65 20 (15.9) 0 (0.0) 24 (11.4) 20 (15.3) 25 (14.0) fig. 1. demographic characteristics of subjects covid-19 -suspected who attended a health care facility in in sana`a, yemen (n=259) 69.5 30.5 6.2 67.2 14.7 11.9 0 10 20 30 40 50 60 70 80 male female 18-mar 19-49 50-64 ≥ 65 sex age (years) p er ce n ta g es sallam et al.; aji, 4(1): 146-153, 2021; article no.aji.83681 150 3.1 seropositivity rate according to antibody classes by age and sex although, the seropositivity rate of the isolated anti-sars-cov-2 igg significantly (p=0.002) differs among various age groups, the seropositivity of the igg and the igm in combination or in isolation did not differ significantly (p>0.05) between males and females or between various age groups with the highest seropositivity rate detected being among the 19-49 years age group and the lowest detected was among ≥65 years age group (table 2). 4. discussion to the best of our knowledge, this is one of very few studies if not the first on seroprevalence of sars-cov-2 in the major city of sana`a, northern part of yemen. of all patients who were suspected of having covid-19 around half of them had at least one antibody marker, either igg or igm or both, suggesting exposure to sars-cov-2 infection. this is obviously because these patients were not clinically well characterized as cases of covid-19 but were referred for testing on suspension of the disease having presented with suggestive signs and symptoms. this explain the lower seroprevalence than has been reported among well characterized patients who were clinically diagnosed with covid-19 early in epidemic where almost 100% became antibody positive 2 to 3 weeks post disease onset [7,12]. however, owing to the attendance of the majority of the cases in a single health care facility for testing in only two weeks’ time indicating the peak of epidemic, the contagious nature of covid-19 and the lack of adequate confinement measures, a higher seropositive can be expected. one reason for the low seroprevalence rate is that the subjects may have produced transient systemic antibodies because of less severe or mild infection. it has been reported that systemic antibody production against sars-cov-2 develops mainly in severe covid-19, whereas mild disease may be associated with transient serum antisars-cov-2specific antibodies [9]. additionally, our subjects may have responded with more localized respiratory antibody response. it has been reported that some sarscov-2-exposed cohorts were negative for sarscov-2-specific serum iga and igg but showed detectable sars-cov-2-specific iga in nasal fluids and tears without serum antibody response [9]. additionally symptomatic patients who may not seroconvert despite developing persisting t cell responses have also been reported [7]. several reports have shown underestimated seroprevalence when investigation was based on igm and igg alone [20]. over 70% of our seropositive subjects were below the age of 50 years, which reflects the main age stratum of the yemeni population (< 50 years) constituting 90% of the population [23], rather than suggests that this age groups were particularly susceptible. the low seroprevalence among those 3-18 years may reflect the mild or asymptomatic infection that usually occur in this age group and thus do not seek health care. various reports have shown that less children (<18 years) than adults presented with mild to less severe and lower death rate [24-26]. the attendance of small proportion of our subjects of this age for testing further supports this inference. the low level of sars-cov-2 infection among children has been explained by the less expressed angiotensin-converting enzyme 2 (ace2) receptor of the virus, better containment of the virus in the upper tract or to the less adverse immune mechanisms arising from infection in <18 years than in adults [22] . among our subjects less females than males presented for testing which could suggests low exposure among females. however this possibly because of stay-at-home custom of yemeni women rather than due to low susceptibility to infection among females. supporting this is the seroprevalence rate among females which did not significantly differ from that among males indicating equal risk of infection. small minority of seropositive subjects (2.3%) had an isolated igm and 18.9% had isolated igg while over 30% had concurrent igm and igg. this could suggest the narrow window period of the antibody class switching from igm to igg making it difficult to detect patients with isolated igm and to less extent isolated igg. the possibility of simultaneous occurrence of both antibody classes cannot be excluded as simultaneous appearance of both antibody classes in some of sars-cov-2 infected subjects has been reported elsewhere [7,9,10,12-22,25-31] furthermore, 3 patterns of sars-cov-2 seroconversion have been proposed so far. these include sequential seroconversion of igm followed by igg, concurrent seroconversion of both igg and igm, and an unusual pattern of igg preceding igm [30]. this probably reflects the variation in different serological assays. therefore, validation and standardization of sallam et al.; aji, 4(1): 146-153, 2021; article no.aji.83681 151 sars-cov-2 serologic assays in large clinical cohort is required before coming to a final inference of serological dynamic of sars-cov-2. majority of our subjects with isolated igm were females (83%). the significance of this is difficult to point out due to the small sample size (6 subjects). however, this merit further investigation. 5. conclusions among subjects with suspected covid-19 over 30% had concomitant igm and igg with minority having isolated igm or igg suggesting concomitant or close seroconversion time point of both antibody classes. in addition, around 50% of subjects were sars-cov-2 seropositive suggestion low sars-cov-2 seroconversion and consequently low community sars-cov-2 seroprevalence in yemen. as the systemic immune response to sars-cov-2 currently is not well established, the seroprevalence of sars-cov-2 cannot be reliably determined hence herd immunity seems to be hard to establish at this stage. a large-scale community based seroprevalence study based on detection of combination of igm, igg, and iga seems essential to determine the magnitude of sarscov-2 infection in yemen. antibody response study based on will characterized covid-19 patients is also required. consent data presented in this study were obtained in an informed consent with a clear declaration on the confidentiality of the obtained data and will not be used out of the research dimension. ethical approval this study attested and approved (# rec-772020) by relevant academic institution. acknowledgments the authors are grateful to mr. mohammad bamashmoos at the alyoussefi specialized laboratories for his efforts in the data collection. competing interests authors have declared that no competing interests exist. references 1. xiang f, wang x, he x, et al. antibody detection and dynamic characteristics in patients with covid-19. clinical infectious diseases; 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cite as: olivier, celso eduardo, daiana guedes pinto, ana paula monezzi teixeira, cibele silva miguel, raquel acácia pereira gonçalves santos, jhéssica letícia santos santana, and regiane patussi santos lima. 2024. “endotyping cellular and humoral immunoreactivity against allium spices and sulfites preservatives in allergic patients: a retrospective study”. asian journal of immunology 7 (1):185-200. https://journalaji.com/index.php/aji/article/view/143. asian journal of immunology volume 7, issue 1, page 185-200, 2024; article no.aji.125235 endotyping cellular and humoral immunoreactivity against allium spices and sulfites preservatives in allergic patients: a retrospective study celso eduardo olivier a*, daiana guedes pinto a, ana paula monezzi teixeira a, cibele silva miguel a, raquel acácia pereira gonçalves santos a, jhéssica letícia santos santana b and regiane patussi santos lima c a instituto alergoimuno de americana, brazil. b instituto de ensino e pesquisa do hospital de amor de barretos, brazil. c lavoisier laboratórios, são paulo, brazil. authors’ contributions this work was carried out in collaboration among all authors. the author ceo is responsible for the conceptualization, data curation, formal analysis, literature review, and writing the original draft. authors dgp, apmt, csm, jlss, and rpsl performed laboratory procedures. author rapgs performed cutaneous tests. all authors read and approved the final manuscript. article information doi: https://doi.org/10.9734/aji/2024/v7i1143 open peer review history: this journal follows the advanced open peer review policy. identity of the reviewers, editor(s) and additional reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here: https://www.sdiarticle5.com/review-history/125235 received: 21/08/2024 accepted: 23/10/2024 published: 28/10/2024 original research article https://doi.org/10.9734/aji/2024/v7i1143 https://www.sdiarticle5.com/review-history/125235 olivier et al.; asian j. immunol., vol. 7, no. 1, pp. 185-200, 2024; article no.aji.125235 186 abstract background: spices from the allium genus season food worldwide, and their widespread use may produce allergic reactions by contact or ingestion. however, the non–ige-mediated immunoreactivity against these allergens has not yet been adequately investigated. aim: to evaluate the potential of the tube titration of precipitins (ttp) and the leukocyte adherence inhibition test (lait) to discriminate cellular and humoral immunoreactivity against two allium spices (garlic and onion) and a sulfite derivative in patients with non–ige-mediated allergic phenotypes. methodology: two cohorts of allergic patients suspected of non–ige-mediated hypersensitivity against allium spices and sulfite derivatives were investigated with the help of ttp or lait, simultaneously against extracts of garlic, onion, and sodium bisulfite. the results of the semiquantitative serum ttp against 1 mg/ml garlic extract, 1 mg/ml onion extract, and 1 mg/ml sodium bisulfite were distributed in ranges through cascade distribution charts. the results of the leukocyte adherence inhibition (lai) percentage promoted by the ex vivo challenges against 1 mg/ml garlic extract, 1 mg/ml onion extract, and 1 mg/ml sodium bisulfite were distributed in ranges through cascade distribution charts. the statistical differences inside these cohorts were calculated. results: paired t-test indicated a non-significant difference between garlic ttp and onion ttp (pvalue = 0.761); a non-significant difference between sulfites ttp and garlic ttp (p-value = 0.112); a non-significant difference between sulfites ttp and onion ttp (p-value = 0.058); a nonsignificant difference between garlic tial and onion tial (p-value = 0.942); a significant difference between sulfites tial and garlic tial (p-value < 0.001) and a significant difference between sulfites tial and onion tial (p-value < 0.001). conclusion: results support that the ttp and lait performed with sodium bisulfite, garlic, and onion extracts may discriminate diverse degrees of humoral and cellular immunoreactivity in patients suffering from diversified allergic phenotypes. keywords: allium sativum, allium cepa; endotype; garlic; hypersensitivity; leukocyte adherence inhibition test; non–ige-mediated immunoreactivity; onion; precipitins; sulfite derivatives. abbreviations lai : leukocyte adherence inhibition lait : leukocyte adherence inhibition test ttp : tube titration of precipitins 1. introduction allium is a genus of monocotyledonous plants, characterized by their odorous volatile sulfurcontaining compounds (most derived from cysteine sulfoxides) with more than eight hundred species, among which quite a few are edible and cultivated as lore for culinary purposes or therapeutic indications, such as several kinds of garlic (e.g., allium sativum l.), onions (e.g., allium cepa l.), chives (e.g., allium schoenoprasum) and leeks (e.g., allium porrum l.), among others [1]. allium spices are reported to have been used as food and as medicines in the ancient histories of egypt, china, japan, india, greece, and rome [2]. the first reported clinical case of allergy to an allium species was described in 1950 by edelstein, who treated a food-pack worker presenting occupational dermatitis after skin contact with garlic and "severe cardiospasm" immediately after ingestion [3]. shortly after that (1952), the canadian medical association published a report of a cook who was also diagnosed with an occupational allergy to onion and garlic [4]. finally (1954), the "classic aspects of onion and garlic dermatitis in housewives" was published by the american college of allergists [5]. hypersensitivities to allium spices have been reported to produce contact dermatitis, gingivitis, stomatitis, allergic rhinitis, allergic conjunctivitis, and asthma [6,7]. in spain, the reported prevalence of documented hypersensitivity against garlic and/or onions in a cohort of allergic patients submitted to routine skin allergic tests was almost 3% [8]. in saudi arabia, from a cohort of 108 patients with clinical suspicion of food allergy, 15 (13.8%) had garlic and onionspecific ige antibodies in their sera [9]. olivier et al.; asian j. immunol., vol. 7, no. 1, pp. 185-200, 2024; article no.aji.125235 187 skin tests with spices gradually became routine to diagnose cooks with hand dermatitis, and the early attempts to identify the sensitizer revealed that it was soluble in water, ethanol, and acetone [10]. in the last decade of the 20th century, doctors became progressively aware of other clinical manifestations that could be caused by hypersensitivity to components of the allium genus: several cases of bronchial asthma, rhinitis, conjunctivitis, and dermatitis were attributed to onion allergy, demonstrated by bronchial provocation tests, nasal provocation tests, and skin tests with onion extracts that cross-reacted with garlic and leek extracts; and in some cases, detection of specific ige against onion and garlic was possible by phadezyme® and cap system rast feia® [11]. allergic rhinitis elicited by allium allergens was also demonstrated by anterior rhinomanometry [12]. susceptible patients may also develop urticaria, angioedema, and anaphylactic reactions, especially to young garlic (an unripe and underdeveloped bulb) and raw onions [13,14]. an experience done with volunteers and patients with gastroesophageal reflux submitted to an esophageal ph probe who had ingested onions did not increase any reflux variables measured in asymptomatic volunteers. however, the ingestion of onions significantly increased reflux episodes in symptomatic subjects compared with asymptomatic subjects after onion ingestion and when compared with meals with no-onion [15]. even before the reports of allium allergic reactions, the sulfur compounds of allium species were already studied, particularly the diallyl disulfide, an organic molecule with formula c6h10s2 [16]. diallyl disulfide is one of the principal components of the distilled oil of garlic and other allium genus plants, produced during the decomposition of allicin released after crushing [17,18]. identified as an active component due to its larvicidal activity, the diallyl disulfide was also suspected of producing allergic reactions and was employed to perform diagnostic allergic skin tests (with the help of a 1% solution) [19-21]. non-ige-mediated allergic contact dermatitis is one of the most common symptoms of allium allergy, mainly in cooks who handle the spices, despite the use of gloves permeable to diallyl disulfide [22]. occasionally, when used as a naturopathic remedy by topical application, garlic has also been linked to allergic contact dermatitis [23]. with the help of immunoblotting, in 2001, asero et al. identified ige-binding to 15-kda and 43-kda onion proteins; after preabsorption of the patient's serum with peach lipid transfer protein (ltp), the ige-binding to the 15-kda protein disappeared [24]. later (2007), enrico et al., by immunoblotting an onion extract with the serum of a patient with onion allergy and anti-ige, revealed a 12-kda ige-binding protein band that was inhibited by onion extract and the peach allergen rpru p 3 (a marker allergen for ltp) [25,26]. with the help of mass spectrometry, a taiwan group marked the ige-binding of a 56kda protein as a major allergen for patients with garlic allergy, identified as the alliin lyase (e.c.4.4.1.4), the enzyme responsible for the lysis of alliin into the biologically active allicin molecule upon crushing of a garlic clove [27]. italian investigators also reported cellular responses, and they identified specific b cell proliferation in onion-allergic patients' plasma incubated with onion extracts (but not in healthy controls) [28]. cellular techniques using cell sorting, elispot, flow cytometry, and confocal microscopy have recently boosted a crescent interest in studying the interaction of ltp allergens with immune cells, particularly with group 2 innate lymphoid antigen-presenting cells, promoting th2 cell responses involved in allergy pathogenesis [29]. besides diallyl disulfide, allium species also produce other organic sulfides such as diallyl trisulfide, diallyl sulfide, dipropyl disulfide, dipropyl trisulfide, 1-propenyl propyl disulfide, allyl methyl disulfide, and dimethyl disulfide [30]. in organic chemistry, "sulfide" or "thioether" refers to the linkage "c–s–c". in inorganic chemistry, "sulfides" are corrosive ionic salts composed of the anion s2−, while "sulfites" (or chiefly british "sulphites") are salts composed of the sulfur dioxide (so2 − ) which is found in aqueous solution as free so2, hso3 − and/or so3 2−. in biological fluids, the sulfite ions are usually combined with carbonyl compounds such as acetaldehydes, proteins, sugars, and others [31]. the solution's free sulfite ions associate and dissociate in equilibria with carbonyl compounds according to the solution's ph at diverse equilibrium constants (pks) [32]. allium species' characteristic flavor and pungency depend on the content of "sulfates" (the sulfur tetraoxide so4 -2) in the soil the plant grows [33]. in naples, it was reported a particular association of pizza makers' hand dermatitis with positive skin allergy tests done with diallyl olivier et al.; asian j. immunol., vol. 7, no. 1, pp. 185-200, 2024; article no.aji.125235 188 disulfide and ammonium persulfate, a flourstrengthening salt with formula (nh4)2+(s2o8)2 [34]. there seems to be some hypersensitivity crossreactivity among the organic sulfides in allium spices and the inorganic sulfites used as antioxidants and preservatives in industrialized foods, cosmetics, and medicines [35]. sulfites are the group of inorganic compounds elected by the american contact dermatitis society as the "allergen of the year for 2024" [36]. sulfites were anciently aggregated into the human diet employing saccharomyces cerevisiae fermentation, which reduces sulfate into sulfites and sulfides [37,38]. nowadays, sulfites are almost universally added to industrialized foods and beverages to control non-enzymic browning, enzymic browning, oxidation, and microorganisms growing [39]. several sulfite salts are qualified to be added to industrial food, such as sulfur dioxide (e 220; so2), sodium sulfite (e 221; na2so3), sodium bisulfite (e 222; nahso3), sodium metabisulfite (e 223; na2s2o5), potassium metabisulfite (e 224; k2s2o5), calcium sulfite (e 226; caso3), calcium bisulfite [e 227; ca(hso3)2] and potassium bisulfite (e 228; khso3) [40]. since the seventies, sulfur dioxide has been known as an atmospheric pollutant associated with the rapid development of laryngeal and bronchial spasms, excessive bronchial secretions, and pulmonary edema in sensitive patients, either inhaled or ingested [41]. anaphylaxis after ingestion of sodium bisulfite was also reported [42]. besides respiratory symptoms and anaphylaxis, sulfite hypersensitivities are associated with nausea, stomach cramps, diarrhea, urticaria, angioedema, dermatitis, diaphoresis, tingling sensations, flushing, and loss of consciousness [43]. sulfites are associated with headaches in sensitive individuals [44]. sulfites are among the main suspects of allergies associated with alcoholic beverages [45]. several prescription medicines incorporate sulfites as antioxidants, such as topical ophthalmic medications; inhaled bronchodilators (salbutamol, racepinephrine); beta-adrenoceptor agonists (isoprenaline, epinephrine); local anesthetics; injectable corticosteroids; injectable antibiotics; injectable antiarrhythmics; injectable analgesics; antishock agents, including aramine®, intropin® and levophed®; and solutions for parenteral nutrition and dialysis [46-52]. the resources to diagnose sulfite hypersensitivity are in vivo and ex vivo provocation tests [53]. we routinely employ the leukocyte adherence inhibition test (lait) and the tube titration of precipitins (ttp) in our facilities as a triage to evaluate non-ige-mediated immunoreactivity against suspected allergens before the performance of more exhaustive in vivo provocation tests [54-60]. to evaluate the potential of the lait and ttp to endotyping non–ige-mediated cellular and humoral immunoreactivity against garlic, onion, and sodium bisulfite, we retrospectively compiled the electronic medical charts of patients with non– ige-mediated allergic rhinitis, allergic pharyngitis, allergic laryngitis, allergic bronchitis, allergic sinusitis, allergic migraine, atopic dermatitis, and/or urticaria who were investigated simultaneously for immunoreactivity against these three allergens by one of these assays. the present study is a proof of concept that hypothesizes that the lait and the ttp may differentiate diverse endotypes and degrees of immunoreactivity against allium species and sodium bisulfite among patients suffering from common allergic phenotypes. as the tests were performed simultaneously with the same venous sample with the three allergens, it was possible to calculate a paired t-test to discriminate crossreactivity between them. 2. materials and methods 2.1 subjects after receiving institutional review board approval from the instituto alergoimuno de americana (brazil; 07/2024), we reviewed the electronic chart of 9,450 outpatients who attended our facility from january 2018 to september 2024. a cohort of 100 consecutive outside patients (ttp cohort) had been simultaneously submitted to ttp with garlic extract, onion extract, and sodium bisulfite for presenting non–igemediated allergic rhinitis, allergic pharyngitis, allergic laryngitis, allergic bronchitis, allergic sinusitis, allergic migraine, atopic dermatitis, and/or urticaria. this cohort counted 26 males; mean age 39.7 years; sd 16.9 years; range 5 to 79 years; median 39.5 years; modes = 43 olivier et al.; asian j. immunol., vol. 7, no. 1, pp. 185-200, 2024; article no.aji.125235 189 (appeared seven times); geometric mean = 35.2 years. a cohort of 100 consecutive outside patients (lait cohort) had been simultaneously submitted to tial with garlic extract, onion extract, and sodium bisulfite for presenting non–igemediated allergic rhinitis, allergic pharyngitis, allergic laryngitis, allergic bronchitis, allergic sinusitis, allergic migraine, atopic dermatitis, and/or urticaria. this cohort counted 33 males; mean age 42.9 years; sd 21.3 years; range 2 to 91 years; median 43 years; modes = 43 and 48 years (each appeared four times); geometric mean = 35.1 years. this study did not include patients under biological and/or systemic anti-inflammatory therapy. these procedures were offered to patients with clinical suspicion of allium spices hypersensitivity who demonstrated a nonreactive or inconclusive skin test against sodium bisulfite, garlic, and onion extracts [61]. 2.2 garlic extract the peeled garlic was crushed, homogenized, and then left for 48 hours in coca's solution at 4°c for protein extraction before centrifugation and separation of the water-soluble fraction from solid particles and oily fraction [62]. the protein quantification of the allergen extracts was done according to bradford's protein-dye binding methodology [63]. the solution was diluted in antigen dilution solution (nacl 10g; kh2po4 0.72g; na3po4 2.86g; methylparaben 1g; propylparaben 0.5g; glycerin 400ml; h2o 600ml) to an estimated protein concentration of 1 mg/ml and stored at 4°c into amber opaque glass vials. the garlic extract solution was used to perform the allergic skin tests, ttp, and lait. all relevant and mandatory laboratory health and safety measures have been complied with during the experiments. 2.3 onion extract the onion extract solution was prepared using a similar technique employed for the garlic extract. 2.4 sodium bisulfite solution the sodium bisulfite powder (iupac id: sodium hydrogen sulfite; formula nahso3) was acquired from bianquimica™ and diluted with distilled water at 1 mg/ml to perform the allergic skin tests, ttp, and lait. 2.5 lait: ex vivo investigation: leukocyte adherence inhibition test 2.5.1 lait: procedure for allergen ex vivo challenging we performed the lait as previously described [64-73]. shortly, each donor's fresh plasma was divided into two parts and used in paralleled ex vivo challenging tests with the allium or onion extracts and the unchallenged plasma (added with antigen dilution solution as a control). we collected the plasma with high leukocyte content (buffy coat) from the heparinized tube after one hour of sedimentation at 37 °c. then, we distributed aliquots of 100 μl into eppendorf tubes with (or without) the challenging extract and kept them under agitation for 30 minutes (200 rpm at 37°c). 2.5.2 lait: procedure for adherence assay after incubation, the plasma was allocated into a standard neubauer hemocytometer counting chamber with a plain, non-metallic glass surface and left to stand for 2 hours at 37 °c in the humidified atmosphere of the covered water bath to allow leukocytes to adhere to the glass. next, we counted the leukocytes, removed the coverslip, and washed the chamber by immersion in a beaker with pbs at 37 °c. then, we added a drop of pbs to the hemocytometer's chamber and allocated a clean coverslip over it. the remaining cells were counted in the same squares as previously examined. 2.5.3 lait: procedure for calculation the percentage of leukocyte adherence (la) of each assay was estimated as: (the number of leukocytes observed on the hemocytometry chamber after washing divided by the number of leukocytes observed on the hemocytometry chamber before washing) and multiplied by 100 (%). the leukocyte adherence ratio (lar) was estimated based on the ratio between the la from the antigen-specific challenged plasma and the la from the unchallenged control plasma: lar = la of the challenged sample divided by la of unchallenged control plasma multiplied by 100 (%). to further calculate the leukocyte adherence inhibition (lai), we subtracted the lar from 100 (%). we employed the lai results for the cascade distribution chart and the statistics calculations, both performed with the help of the microsoft excel® statistical package. olivier et al.; asian j. immunol., vol. 7, no. 1, pp. 185-200, 2024; article no.aji.125235 190 2.6 ttp: in vitro investigation: tube titration of precipitins as previously reported, the semi-quantitative ttp against the aluminum solution was performed in a transparent vitreous tube array [74-76]. shortly, the patient's blood was collected in a clot-activator collecting tube. after separation, the serum was centrifugated at 2,000 rpm for 10 minutes. each allergen extract was allocated in sets of eleven glass tubes at progressive duplicated serum dilutions. the progressive dilutions were combined with the 15 μl of the antigen (1 mg/ml) with 250 μl of the patient's serum, progressively diluted into physiological saline solution (nacl 0,9%) in the dilution ratios of 1:1; 1:2; 1:4; 1:8; 1:16; 1:32; 1:64; 1:128; 1:256; and 1:512. one tube was a blank control done with the water and serum to observe occasional spontaneous precipitation (sia test). after 24 hours, the tubes were examined, and the titers (the highest dilution factor that yields a positive reading) were recorded [77]. 3. results as a retrospective survey, there was no research protocol; therefore, we report the incidental immune investigation as registered in the digital medical charts. the ttp for the garlic extract showed a distribution concentrated on the higher dilutions (fig. 1). there was no negative result. the mean was estimated at 1:247; the median was 1:128; the standard deviation was estimated at 1:180; the mode was 1:512 (appeared nine times). the ttp for the onion extract showed a distribution concentrated on the higher dilutions (fig. 2). there was no negative result. the mean was estimated at 1:254; the median was 1:256; the standard deviation was estimated at 1:178; the mode was 1:512 (appeared 29 times). the ttp for the sodium bisulfite solution showed a distribution concentrated on the higher dilutions (fig. 3). there was no negative result. the mean was estimated at 1:207; the median was 1:158; the standard deviation was estimated at 1:176; the mode was 1:128 (appeared 29 times). the lait for the garlic extract showed a wide distribution range of results. most results were concentrated in the more immunoreactive groups. there were five negative results. the lai ranged from 0% to 89%. the mean was 51.3%; the median was 57.5%; the standard deviation was 24.0%; the mode was 0% (appeared five times). the cascade distribution demonstrates a wide range of lai results (fig. 4). some patients showed low or moderate immunoreactivity during the ex vivo challenge test. in contrast, others displayed strong immunoreactivity, which could reflect the participation of garlic allergens in a non–ige-mediated hypersensitivity condition in these patients. fig. 1. cascade distribution chart of the tube titration of precipitins (x-axis %) resulting from the garlic extract against the serum of the ttp cohort of 100 tests/subjects (y-axis) olivier et al.; asian j. immunol., vol. 7, no. 1, pp. 185-200, 2024; article no.aji.125235 191 fig. 2. cascade distribution chart of the tube titration of precipitins (x-axis %) resulting from the onion extract against the serum of the ttp cohort of 100 tests/subjects (y-axis) fig. 3. cascade distribution chart of the tube titration of precipitins (x-axis %) resulting from the sodium bisulfite extract against the serum of the ttp cohort of 100 tests/subjects (y-axis) the lait for onion extract showed a wide distribution range of results. most results were concentrated in the more immunoreactive groups. there were three negative results. the lai ranged from 0% to 89%. the mean was 52%; the median was 51.2%; the standard deviation was 24.9%; the mode was 62% (appeared seven times). the cascade distribution demonstrates a wide range of lai results (fig. 5). some patients showed low or moderate immunoreactivity during the ex vivo challenge test. in contrast, others displayed strong immunoreactivity, which could reflect the participation of onion allergens in a non–igemediated hypersensitivity condition. the lait for sodium bisulfite showed a wide distribution range of results. most results were concentrated in the less immunoreactive groups. there were 27 negative results. the lai ranged from 0% to 88%. the mean was 27.5%; the median was 25.5%; the standard deviation was 25.1%; the mode was 0% (appeared 27 times). the cascade distribution demonstrates a wide range of lai results (fig. 6). most patients showed low or moderate immunoreactivity during the ex vivo challenge test. at the same time, a few displayed strong immunoreactivity, which could reflect the participation of sulfites in a non– ige-mediated hypersensitivity condition in these patients. olivier et al.; asian j. immunol., vol. 7, no. 1, pp. 185-200, 2024; article no.aji.125235 192 the paired t-test indicated a non-significant, minimal difference between garlic ttp and onion ttp; p-value = 0.761. the paired t-test indicated a non-significant, minimal difference between sulfites ttp and garlic ttp; p-value = 0.112. the paired t-test indicated a non-significant, minimal difference between sulfites ttp and onion ttp; p-value = 0.058. the paired t-test results indicated a nonsignificant minimal difference between garlic tial and onion tial; p-value = 0.942. the paired t-test indicated a significantly large difference between sulfites tial and garlic tial; p-value < 0.001. the paired t-test results indicated a significantly large difference between sulfites tial and onion tial; p-value < 0.001. fig. 4. cascade distribution chart of the range groups of leukocyte adherence inhibition (lai) results (x-axis %) of the ex vivo challenge against garlic extract monitored by the leukocyte adherence inhibition test (lait), according to the respective number of outcomes over the lait cohort with 100 tests/subjects (y-axis) fig. 5. cascade distribution chart of the range groups of leukocyte adherence inhibition (lai) results (x-axis %) of ex vivo challenge against onion extract monitored by the leukocyte adherence inhibition test (lait), according to the respective number of outcomes over the lait cohort with 100 tests/subjects (y-axis) olivier et al.; asian j. immunol., vol. 7, no. 1, pp. 185-200, 2024; article no.aji.125235 193 fig. 6. cascade distribution chart of the range groups of leukocyte adherence inhibition (lai) results (x-axis %) of ex vivo challenge with sodium bisulfite solution monitored by the leukocyte adherence inhibition test (lait), according to the respective number of outcomes over the lait cohort with 100 tests/subjects (y-axis) 4. discussion soon after the discovery of penicillin (along with the antibiogram) in 1928, the empirical bactericidal properties of allium spices were demonstrated to be "penicillin-like" and proved effective against gram-positive bacteria, first in animals (1931) and later in humans (1945) [78,79]. allium extracts also proved effective against fungi, especially their organosulfur derivatives [80,81]. the early biochemical studies performed with the allium species were motivated by the alliinase enzymatic ability to hydrolyze s-alkyl-substituted cysteine sulfoxide derivatives to the corresponding alkyl alkane thiosulfinates, ammonia, and pyruvic acid [82]. allium species are a natural source of bioactive compounds, including more than 180 active secondary metabolites [83]. several allium bioactive compounds have been studied for their supposed anticancer, anti-diabetic, antiinflammatory, antimicrobial, immunomodulatory, and cardioprotective properties [84]. allium spices were studied in several murine models of inflammatory diseases with promisor results [85]. murine experiments demonstrated that onion bulb extract inhibited the house dust miteinduced phosphorylation of egfr, erk1/2, and akt pathways, inhibiting airway cellular influx, perivascular and peribronchial inflammation, goblet cell hyper/metaplasia, and ex vivo eosinophil chemotaxis [86,87]. consequently, it is natural to hypothesize that such intense biological activity is probably associated with a great potential to promote immunoreactivity and/or hypersensitization, interfering with metabolic pathways, being proposed even for treating allergic diseases [88]. therefore, when treating a patient with the precision medicine approach, diagnosing or endotyping an eventual allergy, hypersensitivity, or immunoreactivity against any naturopathic therapeutics or nutraceutical food, elective for long-term treatments is remarkably advisable for the prescription of eviction measures and tolerogenic strategies aimed to mitigate diseases symptoms phenotypes [89]. the four classical hypersensitivity mechanisms described by gell and coombs have been amplified to seven types with several subtypes, increasing the complexity of the required investigation of the hypersensitivity mechanisms responsible for the disease phenotypes [90,91]. endotyping the hypersensitivity mechanisms against sulfites may also help distinguish superimposable phenotypes produced by common pathways such as allergic migraines and sinusitis [92]. the retrospective compilation of our data showed a large distribution of results when we ascertained the results of ttp and tial to olivier et al.; asian j. immunol., vol. 7, no. 1, pp. 185-200, 2024; article no.aji.125235 194 explore humoral and cellular immunoreactivity against two allium spices and a sulfite derivative. these immunoassays do not identify the exact mechanisms responsible for the clinical condition. instead, they provide clues about sensitization and immunoreactivity distributed into an extensive spectral range between immune tolerance and symptomatic hypersensitivity. the semi-quantitative research and titration of precipitins is an essential laboratory exam upon which the fundamentals of immunology were constructed [93]. precipitating antibodies suggest a remarkable immune humoral response against the tested antigens [94]. the lait is an ex vivo challenge test performed with a viable leukocyte buffy coat that can theoretically explore most known immune pathways as it allows the interaction of all immune-circulating participants with the allergen [95]. the lait did not indicate which pathways produce the resulting final phenomenon (leukocyte adherence inhibition) [96-99]. as indicators of a previous immune response, lait and ttp are suitable techniques to quantify an exposome measurement and their correspondent immune response, as proposed by the exposome-wide association study [100]. the comparative results obtained from the tial cohort demonstrated a more significative immunoreactivity from the tests performed with garlic extract and onion extract than the results obtained with the sodium bisulfite. this finding states that sulfites are not the only allergen responsible for cellular immunoreactivity against the allium species. on the contrary, several protein allergens in allium spices play a critical role in immunoreactivity against these species. this means that if a patient presents cellular immunoreactivity against allium species, he/she will not necessarily present immunoreactivity against sulfites; however, reciprocation is not probable. this preliminary retrospective survey demonstrated extensive results from the ttp and the ex vivo challenge test monitored by lait against two allium species in two cohorts of patients with various allergic symptoms. ttp and lait are complementary triage tests used at our facilities to select worthwhile antigens to proceed with more laborious in vivo provocation tests when the specific ige is undetectable. none of our patients presented an exclusive reaction to these allergens. every patient was simultaneously tested with several chemical and biological allergens, demonstrating positive results for some of them. our results suggest that reactive allergic patients may impair their symptoms by an additional immunoreactivity against allium spices and sulfite derivatives. 5. limitations this study is a retrospective analysis of data collected over six years and nine months. there was no protocol research, and the subject's data were limited to the essentials available on our electronic sheets. therefore, we could not establish a cross-comparison between positive and negative controls to validate the results. the number of subjects is appropriate for a preliminary study; however, future studies must be more comprehensive. the lack of a research protocol implies the possibility of a bias produced by the point of view of the physician who indicated the exam (ceo) based on a clinical suspicion led purely by the anamnesis and physical examination. the study lost many of these patients to follow-up, so assuring the relationship between the immunoassays' results and the patient's clinical outcome is not possible yet. unfortunately, it was impossible to compare the two procedures because they were taken from different groups of patients. 6. conclusion our preliminary results show that the lait and ttp may differentiate diverse degrees of immunoreactivity against allium spices in patients clinically diagnosed with non–igemediated allergies. this methodology may be easily incorporated into specialized centers since the technologies to perform tial and ttp are inexpensive and can be performed with minimum laboratory equipment. however, the technique depends on trained biomedical personnel performing artisanal laboratory procedures. as preliminary results, the propaedeutic meaning of these results and the possibility of interferents must be yet established [101]. more studies focused on the quality-by-design approach with prospective larger double-blind cohorts need to evaluate the potential contribution of lait and ttp for endotyping immunoreactivity of patients olivier et al.; asian j. immunol., vol. 7, no. 1, pp. 185-200, 2024; article no.aji.125235 195 suspected of symptomatic hypersensitivity against allium spices and sulfite derivatives [102]. consent as a retrospective survey of results recorded in cognito, consent was given collectively by the institution's ethics committee following the principles of the declaration of helsinki [103]. ethical approvals the authors have collected and preserved written ethical approval as per international standards. disclaimer (artificial intelligence) authors hereby declare that no generative ai technologies such as large language models (such as chatgpt, copilot, and similar) and text-to-image generators have been used during the writing or editing of manuscripts. acknowledgements the instituto alergoimuno de americana funded this work. we want to thank our new laboratory technician, alessandra vieira de oliveira, for participating in the exams. competing interests authors have declared that no competing interests exist. references 1. block e. garlic and other alliums: the lore and the science. the royal society of chemistry; 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asian journal of immunology 4(1): 175-179, 2021; article no.aji.85117 antimicrobial efficacy of four different extracts of plantago major: an in vitro study g. akkuş a and m. m. hiz-çi̇çekli̇yurt b* a department of biology, faculty of arts & science, çanakkale onsekiz mart university, turkey. b department of medical biology, faculty of medicine, çanakkale onsekiz mart university, turkey. authors’ contributions this work was carried out in collaboration between both authors. author ga is a student responsible for all antimicrobial assays and performed the statistical analysis and wrote the first draft of the manuscript. author mmç wrote the protocol, managed the analyses of the study and the literature searches. both authors read and approved the final manuscript. article information open peer review history: this journal follows the advanced open peer review policy. identity of the reviewers, editor(s) and additional reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here: https://www.sdiarticle5.com/review-history/85117 received 27 october 2021 accepted 29 december 2021 published 30 december 2021 abstract aims: plantago major is frequently used in traditional treatment for upper respiratory tract diseases such as pneumonia, cough, pharyngitis, and skin, eye, and urinary tract infections. this study aims to evaluate the antimicrobial activity and minimum effective dose of hexane, methanol, ethanol, and water extract of plantago major leaves. study design: in vitro experimental study. methodology: p. major leaves was crushed into a fine powder and dissolved in different solvents (hexane, methanol, ethanol, or water) using a soxhlet device, then the extracts were purified by evaporation of the solvent. all extracts were analyzed for antibacterial and antifungal properties using broth dilution method depending on mic value determined according to the solventmicroorganism-time trio in ddm. results: the in vivo test showed that all methods to extract of plantago major leaves have activity against all test microorganisms. both hexane and water extract showed the same activity on staphylococcus aureus, bacillus subtilis, and pseudomonas aeruginosa, c. albicans and candida tropicalis at 4 mg/ml. the lowest activity of plantago major’s (pm's) hexane and water extract was on escherichia coli and pseudomonas vulgaris bacteria as 8 mg/ml. methanol and ethanol extracts of pm showed higher activity than hexane and water extract. pm ethanol extract showed antimicrobial activity against staphylococcus aureus, enterococcus faecalis, pseudomonas aerouginosa at 2ml/ml, and bacillus subtilis escherichia coli and proteus vulgaris at 4 ml/ml. original research article akkuş and hiz-çi̇çekli̇yurt; aji, 4(1): 175-179, 2021; article no.aji.85117 176 conclusion: the results nominate the plantago major extract has potential antimicrobials and antifungals. however, the development of antimicrobial agents requires purifying the active bio components by high throughput techniques to achieve effective activity as positive controls. keywords: plantago major; antimicrobial activity; mic. abbreviations here is the definitions section. this is an optional section. tb : boiling point pm : plantago major 1. introduction plantago major (p. major) is a widely used medicinal plant in folk medicine [1]. the plant contains bioactive components such as flavonoids, polysaccharides, terpenoids, lipids, iridoid glycosides, and caffeic acid derivatives [2,3]. due to its rich components, the plant was used to treat various medical conditions such as coughs, infection, fever, bleeding, and inflammation [3,4]. currently, animal studies focus on using the plantago major (pm) on different medicinal conditions. parhizgar and colleagues have demonstrated the protective effect of pm extract in the presence of kidney damage. parhizgar has shown glomerular filtration rate (gfr), urine osmolarity, and urinary excretion rate of potassium were increased via the treatment of pm extract of kidney tissuedamaged rats [5]. in another study, hydroalcoholic extract of plantago major also had a protective role in doxorubicin-induced nephropathy. these two animal studies have been demonstrated the protective role of pm extract on the renal system. boskabadi et al. [6] have been shown potent relaxant effects of pm extract on tracheal smooth muscles of rats. the randomized, double-blind placebo-controlled clinical trial done by jazayeri and colleagues has achieved elevated liver enzymes and a better prognosis for nonalcoholic fatty liver disease by using 2 gr of pm supplementation twice daily for 12 weeks [7]. depending on the therapeutic properties shown in both animals and clinical studies, pm extract protects the kidneys and liver against toxicity. in another way of view, pm extract is not harmful to animals and humans by its protective role, thus potent bioactive compound for drug development. pm extracts were widely used for antibiotics, antioxidants, analgesics, and wound healer purposes from ancient times. the antibacterial activity of pm extract was evaluated by sharma et al. they found no activity for periodontal pathogens by the kirby-bauer disc diffusion technique [8]. however, none of the studies were performed to evaluate pm extract's antimicrobial activity by mic method against standard test microorganisms. thus, this study investigated the bioactivity of a plantago major against pathogens: staphylococcus aureus, enterococcus faecalis, bacillus subtilis, pseudomonas aeruginosa, proteus vulgaris as antimicrobial, and candida albicans and candida tropicalis as antifungal. 2. materials and methods 2.1 chemicals the medium used for this study was nutrient broth for bacterial, and rpmi-1640 medium with l-glutamine for fungi were purchased from sigma-aldrich (hamburg, germany). the microbial lines were purchased from american type culture collection (atcc®, manassas, va, usa). the antibiotics and antifungal pharmaceuticals were used as a positive control as follows: ampicillin (mustafa nevzat pharmaceuticals, turkey); gentamicin (bilim pharmaceuticals, turkey); fluconazole (pfizer pharmaceuticals, roerig division (new york, n.y.). all other analytical grade chemicals used without further purification were purchased from sigma-aldrich (hamburg, germany). 2.2 preparation of plant extract the plants leaves were washed with distilled water, dried in the shade with continuous airflow, and then grounded into powder as 1-3 mm pieces. the powder of pm was randomized aliquoted into 20 mg using filter paper (whatmann no.1) and then was macerated with 200 ml solution for 8 hours in soxhlet (wisd, wise therm) for each solution. the extraction step was done with four different solvents as follows: hexane (c₆h₁₄, tb:69ºc), methanol (ch3oh, tb:64,7 ºc), ethanol (c2h5oh, tb: akkuş and hiz-çi̇çekli̇yurt; aji, 4(1): 175-179, 2021; article no.aji.85117 177 78.4ºc and water (h2o, tb:100ºc). after this step, all extracts were evaporated until dried at 50 ºc to obtain a solvent-free extract, which will be stored in the refrigerator (0-4 ºc) until experiments. each extract was redissolved in dimethyl sulfoxide (dmso) to yield a final concentration of 32 mg/ml. all of them were sterilized by a membrane filter (0.2 µm) before use [9]. 2.3 antimicrobial and antifungal activity assay antimicrobial activity of extracts was applied to gram-positive bacteria cultures: staphylococcus aureus (atcc 6538), enterococcus faecalis (atcc 292112), bacillus subtilis (atcc 6633), whereas gram-negative bacterial cultures escherichia coli (atcc 25922), pseudomonas aeruginosa (27853), proteus vulgaris (atcc 13315). antifungal activity has been studied against candida albicans (atcc 60193) and candida tropicalis (atcc 13803). antimicrobial activity analyses of four different extracts of plantago major were performed using broth microdilution methods as recommended by clinical & laboratory standards institu 1.5 × 10 8 cfu/ml. the inoculums were pipetted in each well. the plant extracts were dissolved in dimethyl sulfoxide (dmso), and after two times concentrated extracts at 32-0,156 mg/ml concentration were pipetted in each well of microtiter plates. in brief, the wells were filled with 100 µl culture suspension as nutrient broth (sigma-aldrich, germany) for antimicrobial activity and rpmi-1640 medium with l-glutamin (sigma-aldrich, germany) for antifungal activity as a growth medium. as per protocol, all extracts have been applied to wells 32 mg/ml and diluted 1/2 respectively. from 1-12 wells, it was from 32 to 0.156 mg/ml. the standard antibiotics and fungicide were used as ampicillin, gentamicin, and fluconazole 2 mg/ml, respectively as control. the aerobic incubation condition was applied at 37 °c 18-24 h for bacteria and 48 h for fungi. the minimum inhibitory concentration (mic) was calculated by visible inhibition of the microbial growth at the lowest concentration. 3. results and discussion table 1 and table 2 are summarized the antibacterial and antifungal effects of different extracts of plantago major. the antimicrobial activity of hexane and aqueous extracts of pm against all test microorganisms except enterococcus faecalis was the same. the results revealed that the efficiency of both ethanol and methanol extracts of pm was similar to all test microorganisms. methanol and ethanol showed the highest activity (2 mg/ml) in s. aureus, e. faecialis and p. aeruginosa bacteria and c. albicans yeast however had low activity on b.subtilis, e. coli p. vulgaris bacteria, and c. tropicalis yeast. table 1. antimicrobial activity of different extracts of plantago major mic (mg/ml) test materials gram (+) gram (-) s ta p h y lo c o c c u s a u re u s a t c c 6 5 3 8 e n te ro c o c c u s fa e c a li s a t c c 2 9 2 1 1 2 b a c il lu s s u b ti li s a t c c 6 6 3 3 e s c h e ri c h ia c o li a t c c 2 5 9 2 2 p s e u d o m o n a s a e ro u g in o s a a t c c 2 7 8 5 3 p ro te u s v u lg a ri s a t c c 1 3 3 1 5 pm-nhexoh 4 2 4 8 4 8 pm-meoh 2 2 4 4 2 4 pm-etoh 2 2 4 4 2 4 pm-h2o 4 4 4 8 4 8 gentamicin 10 -3 10 -3 8.10 -3 16.10 -3 8.10 -3 16.10 -3 ampicilin 1610 -3 16.10 -3 16.10 -3 32.10 -3 32.10 -3 16.10 -3 dmso akkuş and hiz-çi̇çekli̇yurt; aji, 4(1): 175-179, 2021; article no.aji.85117 178 table 2. antifungal activity of different extracts of plantago major mic (mg/ml) test materials c a n d id a a lb ic a n s a t c c 6 0 1 9 3 c a n d id a t ro p ic a lis a t c c 1 3 8 0 3 pm-nhexoh 4 4 pm-meoh 2 4 pm-etoh 2 4 pm-h2o 4 4 fluconasole 625. 10 -3 25. 10 -1 dmso in addition, the results (tables 1-2) show that methanol and ethanol extracts showed higher activity than hexane and water extract. it is known that hexane has polar solubility, methanol and ethanol have semi-polar (methanol is more polar than ethanol), and water has apolar solubility. these differences explain that ethanol and methanol would be better solvents for pm, and bioactive compounds were more easily resolved in ethanol and methanol. the four types of pm extracts exhibited inhibitory effects against test microorganisms; however, these antimicrobial activities were below the mic value of positive controls (ampicillin gentamicin and fluconazole). thus, we can conclude that pm extracts show antimicrobial activity but are not as effective as standard therapeutics. this study provides further documentation of the applicability of the plantago major extract against microorganisms. previous studies have shown that pm has no antimicrobial activity against primary plaque colonizers or periodontal pathogens [8]. however, we clearly showed that pm ethanol extract showed antimicrobial activity against staphylococcus aureus, enterococcus faecalis, pseudomonas aerouginosa at 2ml/ml, and bacillus subtilis escherichia coli and proteus vulgaris at 4 ml/ml. in addition to ethanol extract, hexane, methanol, and water extract showed antimicrobial activity. together with this, all extracts showed antifungal activity against candida albicans and candida tropicalis. in a previous study, the kirby-bauer disc diffusion technique was used to investigate the antimicrobial activity, which is not recommended as a reference method. in contrast, we clearly showed antimicrobial activity by the mic method, which is a gold standard used in clinics to calculate the antimicrobial activity at routine practice. in addition, sharma and colleagues prepared their text extract with maceration; thus, the preparation procedure should affect the concentration of bioactive compounds in total. similar to our study, ferrazzano and colleagues [12] demonstrated a significant antimicrobial effect of plantago lanceolata and evaluated the plant extract as a natural anti-cariogenic agent. although ferrazzano et al. studied different species, both plants have the same genus and have similar phytochemicals. the broth dilution mics for plantago major extracts tested against c. albicans and c. tropicalis showed significant inhibition, which means pm extracted by different solvents might be used with its antifungal activity. in summary, we have provided documentation of pm extracts with antimicrobial and antifungal activity; therefore, pm extracts should be a good candidate for drug development. 4. conclusion this study demonstrated the antimicrobial activity of plantago major against all test microorganisms. in addition, n-etoh and nmeoh extract of pm showed better antimicrobial and antifungal activity when compared to hexane and aqueous extracts. the difference in antimicrobial efficiency at different extracts clearly showed that the extraction method is important to obtain bioactive molecules. despite significant improvements in synthetic molecules in the pharmaceutical industry, the growing antibiotic resistance problem still requires finding akkuş and hiz-çi̇çekli̇yurt; aji, 4(1): 175-179, 2021; article no.aji.85117 179 alternative antimicrobial agents. in this frame, the discovery of plant-based antibiotics is also essential to reduce the side effects of synthetic antimicrobials. thus, we recommended testing different extract types and using gold standards as antimicrobial activity determination. the results of our study show that pm extracts are effective as an antimicrobial agent. however, the active component of the plant extracts should be enhanced, or active components should be purified by high-throughput techniques to develop bio-based pharmaceuticals. competing interests authors have declared that no competing interests exist. references 1. wang h, zhao c, huang y, wang f, li y, chung hy. chemical constituents and bioactivities of plantaginis herba. hong kong med j. 2015;22:29–35. 2. samuelsen ab. the traditional uses, chemical constituents and biological activities of plantago major l. a review. j ethnopharmacol. 2000;71:1–21. 3. najafian y, hamedi ss, farshchi mk, feyzabadi z. plantago major in traditional persian medicine and modern phytotherapy: a narrative review. electron physician. 2018;10(2):6390-9. 4. ghanadian m, soltani r, homayouni a, khorvash f, jouabadi sm, abdollahzadeh m. the effect of plantago major hydroalcoholic extract on the healing of diabetic foot and pressure ulcers. a randomized open-label controlled clinical trial. int j low extrem wounds 2022; (in press). 5. parhizgar s, hosseinian s, soukhtanloo m, bideskan ae, hadjzadeh ma, shahraki s et al. plantago major protects against cisplatin-induced renal dysfunction and tissue damage in rats. saudi j kidney dis transpl. 2018;29(5):1057-64. 6. boskabadi j, saadat s, boskabady mh. the relaxant effect of plantago major on rat tracheal smooth muscles and its possible mechanisms. iran j allergy asthma immunol. 2020;19(4):386-96. 7. jazayeri sf, ghods r, hashem dabaghian f, shojaii a, moravej saa, khadem e et al. a randomized doubleblind clinical trial. evid based complement. alternat med. 2021;6693887. 8. sharma h, yunus gy, mohapatra ak, kulshrestha r, agrawal r, kalra m. antimicrobial efficacy of three medicinal plants glycyrrhiza glabra, ficus religiosa, and plantago major on inhibiting primary plaque colonizers and periodontal pathogens: an in vitro study. indian j dent res. 2016;27(2):200-4. 9. kaur k, arora s, kumar s, nagpal a.. antimutagenic activities of acetone and methanol fractions of terminalia arjuna. food and chemical toxicology. 2002;40: 1475-82. 10. clsi, method for antifungal disk diffusion susceptibility testing of yeasts, approved guideline. clsi document m44-a. clsi, 940 west valley road, suite 1400, wayne, pennsylvania 19087-1898, usa; 2004. 11. clsi, methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard-ninth edition. clsi document m07-a9. wayne, pennsylvania; 2012b. 12. ferrazzano gf, cantile t, roberto l, ingenito a, catania mr, roscetto e et al. determination of the in vitro and in vivo antimicrobial activity on salivary streptococci and lactobacilli and chemical characterisation of the phenolic content of a plantago lanceolata infusion. biomed res int. 2015;286817. _________________________________________________________________________________ © 2021 akkuş and hiz-çi̇çekli̇yurt; this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: https://www.sdiarticle5.com/review-history/85117 http://creativecommons.org/licenses/by/4.0 _____________________________________________________________________________________________________ *corresponding author: e-mail: adeshadrach1@gmail.com; asian journal of immunology 5(1): 52-64, 2022; article no.aji.88927 herd immunity, the best way out of sars cov-2 pandemic: preaching the good news about covid-19 vaccine and vaccine efficacy opeyemi shadrach ademola a,b*, emono dankano nehemiah a, gideon kehinde oyelowo c and florence oluwakemi oladeji d,e a department of chemical pathology, university college hospital, university of ibadan, ibadan, nigeria. b department of pharmaceutical technology, adventist college of health technology, km 2 igbopeigbeti road, igbope, oyo state, nigeria. c department of chemistry, usman danfodio university, p.m.b. 2346, sokoto, sokoto state, nigeria. d department of nursing, ladoke akintola university of technology, p.m.b. 4000, ogbomoso, oyo state, nigeria. e neonatal ward, bowen university teaching hospital, ilorin-ogbomoso road, p.o.box 15, ogbomoso, oyo state, nigeria. authors’ contributions this work was carried out in collaboration among all authors. author osa designed the study and wrote the protocol. author gko wrote the first draft of the manuscript. author edn performed the statistical analysis. author foo managed the literature searches. all authors read and approved the final manuscript. article information open peer review history: this journal follows the advanced open peer review policy. identity of the reviewers, editor(s) and additional reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here: https://www.sdiarticle5.com/review-history/88927 received 10 june 2022 accepted 12 july 2022 published 18 august 2022 abstract aims: to investigate vaccine efficacy amongst the vaccinated population and also to investigate the willingness to be vaccinated among the unvaccinated population of oke-ogun region of oyo state, nigeria. study design: a snowball sampling approach was used. place and duration of study: samples were collected randomly from the general populace of oke-ogun, oyo state, between 20 th of january to 20 th of april, 2022. analysis of samples was carried out at the medical laboratory unit of adeniyi memorial hospital, igbope. original research article ademola et al.; aji, 5(1): 52-64, 2022; article no.aji.88927 53 methodology: 500 consenting adults were recruited to participate in the study (250 health workers and 250 non health workers, aged 18 years and above) vaccinated or unvaccinated with sarscov-2 vaccine. a standard questionnaire was administered to the participants and consenting adults were sampled and tested using a sars-cov-2 rapid diagnostic antigen test kit. a 2 x 2 contingency table (chi-square method, 2-tailed with 1 degree of freedom test) was used to determine the efficacy of sars-cov-2 vaccine. results: among the 500 participants, 328 (65.6%) participants have been vaccinated, although 214 (42.8%) participants accounted for full vaccination. higher vaccination rate 220 (88%) occurred among the health workers compared to 153 (61.2%) among the other participants. from the nonimmunized participants, only 32 (18.6%) are willing to get vaccinated immediately, 95 (55.23%) were not willing to get the vaccination unless they know more about the virus and the vaccines. infection rate was seen to be higher among the unvaccinated population (10.24%) compared to the vaccinated population (2.6%). furthermore, infection among the unvaccinated health worker (13.3%) was seen to be higher than the unvaccinated non-health workers participants (9.2%). conclusion: covid-19 vaccine has proved to be efficient with high degree of immunity conferred on fully-immunized individuals. this will help in the reduction of the severity, mortality of the disease and reduction of the impact of the pandemic on the national health system and economy of countries. keywords: sars-cov-2; vaccination; covid-19; infection; vaccine efficacy; vaccine acceptability; rapid diagnostic test kit. 1. introduction coronavirus (sars-cov-2) is a pandemic that has destabilized human life, health, family and country’s economy. the pandemic was declared in march 2020 [1] after it was first discovered in wuhan, china. the first confirmed case in nigeria was announced on 27 february 2020, when an italian national in lagos tested positive for the virus [2,3]. on 9 march 2020, a second case of the virus was reported in ewekoro, ogun state, a nigerian citizen who came into contact with the italian national [4,5]. the number of confirmed cases as at 13 th of june, 2022 is 256,246. however, there has been far less testing for the virus in nigeria than other countries [6]. in oyo state, 10,219 cases have been reported [7]. on 17 march, 2020, oyo state recorded her first case of covid-19 through a returnee from the united kingdom [8]. several measures have been taken against the disease in order to control its transmission these include hand hygiene, physical distancing, avoidance of large gathering and the use of face mask; all these are targeted at reducing the rapid spread of the disease. in response to the pandemic, the global efforts to develop multiple vaccines to protect against covid-19 disease have been unrivalled in the history of public health. the greatest success will rely on successful vaccination plan against the sarscov-2 virus to bring an end to the epidemic. a herd immunity of the population must be achieved as this can prove greatly successful in the campaign against sars-cov-2 outbreak and to further prevent the emergence of new variants of the virus. until we reach herd immunity, covid-19 will remain a public threat and the next mutation could be more lethal [9]. in a study, vaccine acceptance rate is 74.5% in nigeria [10]. but this high rate is accounted for mostly by the health practitioners. covid-19 vaccine has proved to be efficient with high degree of immunity conferred on fullyimmunized individuals [11]. the vaccine also helps in the reduction of the severity, mortality of the disease and reduction of the impact of the pandemic on the national health system and economy [12]. however, vaccine hesitancy had been a major dilemma in the achievement of sars-cov-2 vaccination plan. to date various surveys have been developed to assess individual attitudes as well as concerns around the risks and benefits of immunization [13,14]. the population’s perception of sars-cov-2 vaccine has most likely influenced by their knowledge about the health consequences of sars-cov-2 infection and the importance of the vaccine to prevent these consequences [15] has really determined the vaccine acceptability by the population. there has been little covid-19 testing and vaccination carried out in the okeogun axis of oyo state, major covid-19 testing and vaccination has been done in the capital city, ibadan and this necessitated the reason for this https://en.wikipedia.org/wiki/nigeria https://en.wikipedia.org/wiki/italian_people https://en.wikipedia.org/wiki/lagos https://en.wikipedia.org/wiki/ewekoro https://en.wikipedia.org/wiki/ogun_state https://en.wikipedia.org/wiki/ogun_state https://en.wikipedia.org/wiki/citizenship https://en.wikipedia.org/wiki/covid-19_testing ademola et al.; aji, 5(1): 52-64, 2022; article no.aji.88927 54 study. the vaccine type used in oyo state, nigeria as at when this study was conducted was astrazeneca and pfizer though no specification of vaccine type was collected for this study. this survey is conducted to investigate vaccine efficacy amongst the vaccinated population and also to investigate the willingness to be vaccinated among the unvaccinated population of oke-ogun region of oyo state, nigeria as this is important for planning the covid-19 vaccination campaign and programme. it is also aimed to determine vaccine efficacy among the participants which can serve as a tool to preach the good news about the vaccine. 2. materials and methods 2.1 study setting and design a cross-sectional study was conducted from 20 th of january to 20 th of april, 2022 among the general population of oke-ogun, oyo state. oyo state is situated in the south-western part of nigeria. the state has 33 local government areas (lgas) while 10 0f these are located in the oke-ogun axis of the state. these lgas has varying incidence of covid-19. as at 11 june 2020, a total of 34 confirmed cases were identified all within the capital city of oyo state [8]. oyo state has a projected population of 8,929,410 with annual growth rate of 3.2% [16]. oyo state is predominantly populated by the yoruba ethnic group with a sparse presence of other ethnic nationalities. the inhabitants are majorly engaged in farming, trading and some of them are artisans and civil servants. study type: a snowball sampling approach. 500 consenting adults were recruited (250 health workers and 250 non health workers) were tested using a sars-cov-2 rapid diagnostic test kit after appropriately filling a questionnaire. 2.2 data collection individuals aged 18 years and above who gave consent participated in the survey. we collected general information and vital information about the research participants was collected using a well-structured questionnaire. the data include; demographic data, anthropometric data, medical histories, acceptability of the covid-19 vaccine and a consent on sars-cov-2 antigen test. vaccinated participants who were tested are participants with 3-month old first vaccination history. other information collected includes major reasons for vaccine hesitancy. all responses were collected and collated anonymously both manually and, on a passwordprotected laptop until the day for analysis. the specimen collected was nasopharyngeal swab. a sterile swab was inserted into the nostrils of participants so that it reaches the depth equal to the distance from nostrils to the outer opening of the ear. it was rotated 3-4 times against the surface of the nasopharyngeal and then withdrawn. the swab was then inserted into an extraction buffer tube, the buffer tube was squeezed and the swab was stirred for more than 5 times. the specimen was then mix with extraction buffer. the extracted specimen was dropped on the specimen well of the test device through the nozzle cap and result was read in 15-30 minutes. 3. interpretation of test results 1. to show that the test is working properly, a purple colored band appears on the band of the control line (c). 2. a purple colored band either faint or nonuniform or clear line appearing on the sars-cov-2 antigen (t) band shows a positive test. 3. invalid test are results that show no colored line on the band of the control line (c). 3.1 data analysis data collected from this study was analysed using the statistical software statistical package for social sciences (spss 16). age, being a continuous variable was categorized into age groups. for all categorical variables, descriptive statistics were presented as percentages (%). age and gender were included in the study to show the population’s demographics. 2 x 2 contingency table (chi-square method, 2-tailed test) was used to determine the efficacy of sars-cov-2 vaccine [17] and values was considered as statistically significant at p˂0.05 at 95% confidence level. 4. results and discussion 4.1 results 4.1.1 socio-demographic characteristics of the participants the responses of 500 participants were included in the analysis. most respondents were male ademola et al.; aji, 5(1): 52-64, 2022; article no.aji.88927 55 (65%), the majority were in the 30-50 age group (58.3%), also major respondents are into agriculture (18.2%) other than health workers which was planned to include 50% of respondents and also most respondents have tertiary education (60%). table 1. socio-demographic characteristics of the participants socio demographic characteristics number of sample (%) n=500 age group 18-30 104 (20.8%) 31-50 298 (59.6%) 51-65 84 (16.8%) >65 14 (2.8%) gender male 323 (64.6%) female 177 (35.4%) educational status primary education 25 (5%) secondary education 93 (18.6%) tertiary education 300 (60%) no education 82 (16.4%) employment industry agriculture 91 (18.2%) construction 9 (1.8%) manufacturing 27 (5.4%) trading 67 (13.4%) health care 250 (50%) art and entertainment 22 (4.4%) finance 6 (1.2%) education 10 (2%) not working 18 (3.6%) 4.1.2 participants’ health status of the 500 participants, 163 (32.6%) participants reported to have a chronic disease. amongst the health worker participants, 65 (26%) reported to have a chronic disease compared to 98 (39.2%) in the other participants. the number of positive sars-cov-2 test was 21 (4.2%) overall. in the immunized health workers, 5 (2.27%) results were positive while 4 (13.3%) was positive from the non-immunized health workers. in other participants, 3 (1.96%) results were positive from the immunized while 9 (9.2%) results were positive from the non-immunized participants. co-morbidity, with p = .50, is not significant to covid-19 test results of participants. 4.1.3 sars-cov-2 vaccine acceptability overall, 373 (74.6%) participants have been vaccinated, although 214 (42.8%) participants accounted for full vaccination. there is higher vaccination rate 220 (88%) among the health workers compared to 153 (61.2%) among the other participants. from the non-immunized participants, only 32 (18.6%) are willing to get vaccinated immediately if made readily available while others, 95 (55.23%) were not willing to get the vaccination unless they know more about the virus and the vaccines. the reason for vaccine hesitancy include: the vaccine is not safe and was made to depopulate human, religious belief, past experiences with other vaccines and some believed there is nothing true about sars-cov-2 outbreak. another vital reason for vaccine hesitancy is ‘older people and those with underlying medical conditions like cardiovascular disease, diabetes, chronic respiratory disease, or cancer are more likely to develop serious illness’ [18] if given the vaccine. it was observed that some participants from the group who wouldn’t get vaccinated even if vaccine is made available would reconsider getting vaccinated if they can be ascertained of getting maximal protection from the vaccine and wouldn’t be liable to contracting other diseases from the vaccine. 4.1.4 sars-cov-2 vaccine efficacy covid-19 infection prevention was seen to be high among the vaccinated population as only 2.14% are positive. statistical analysis has a p value > .000 and hence association of vaccine and covid-19 status is considered to be extremely statistically significant. also, among the health workers, 5 (2.27%) was seen to test positive among the vaccinated population compared to 4 (13.3%) in the unvaccinated population. statistically, p = .002 and hence, the association between vaccinated population among the health workers and their covid-19 status is also considered to be statistically significant. 3(1.96%) non-health workers were positive. statistically, p = .000 and association between vaccine status and covid-19 status was considered very statistically significant. the results also support several reports such as covid-19 tests carried out in the middle-east [17] and further helps to boast population trust in sars-cov-2 vaccine. ademola et al.; aji, 5(1): 52-64, 2022; article no.aji.88927 56 table 2. health history of participants; covid-19 vaccination status and covid-19 infection status participants’ health characteristics health worker immunized positive 5 (2.27%) negative 215 (97.73%) non-immunized positive 4 (13.33%) negative 26 (86.67%) co-morbidity cancer 4 (1.6%) obesity 8 (3.2%) diabetes 10 (4%) hypertension 20 (8%) others 23 (9.2%) other participants immunized positive 3 (1.96%) negative 150 (98.04%) non-immunized positive 9 (9.2%) negative 88 (90.72%) co-morbidity cancer 5 (2%) obesity 14 (5.6%) diabetes 18 (7.2%) hypertension 23 (9.2%) others 38 (15.2%) fig. 1. test results of participants with different chronic disease ademola et al.; aji, 5(1): 52-64, 2022; article no.aji.88927 57 fig. 2. covid-19 vaccination rate based on occupation of the participants fig. 3. covid-19 infection rate among the vaccinated and unvaccinated participants 4.2 discussion “herd immunity” is essential as this would help to prevent the spread covid-19 virus. as seen from the results above, infection rate among the vaccinated population was reduced as compared to unvaccinated population. 50% efficacy threshold was set for covid-19 vaccines because covid-19 was deemed such a severe disease, that if a vaccine is only 50% effective, it’s still worth using [18]. the emerging data on covid-19 vaccines suggests that the vaccines are very safe with high efficacy, at least against some of the variants. acceptance of the vaccine ademola et al.; aji, 5(1): 52-64, 2022; article no.aji.88927 58 was relatively high among healthcare workers (88%) but lower in the other participant group (61.2%). this high vaccine acceptance among healthcare workers has also been reported in somalia [19], mozambique [15] and south africa [20], but a contrasting report was obtained from democratic republic of congo [20]. from our study, it was shown that more participants would reconsider getting vaccinated if they can be ascertained of getting maximal protection from the vaccine and wouldn’t be liable to contracting other diseases from the vaccine. also, if vaccine efficacy is high and proven statistically, most would get vaccinated. this suggests that the more people are educated about the importance and efficacy of vaccination against covid-19, their willingness to be vaccinated will increase. this was also reported in a study in mozambique [15]. from our study, age is also a major factor affecting the population decision to accept covid-19 vaccine. the actively working group who considered their day-to-day activities necessary especially with physical meetings is the highest vaccinated group, aged 31-50 (59.6%) who are majorly traders (13.4%) and farmers (18.2%), the elderly, >65 years of age (2.8%) are the least vaccinated group. this is because of their believe that they are less prone to contacting the disease as they are majorly indoor and also because of the statement ‘older people and those with underlying medical conditions like cardiovascular disease, diabetes, chronic respiratory disease, or cancer are more likely to develop serious illness’ [15] if given the vaccine. however, in a study in ghana, populations with 36–45 years of age were less likely to accept being vaccinated compared to those aged 18–25 years [21]. in a study in mozambique [15], it was shown that the lower the age, the lower the vaccine acceptability. this was also found in saudi arabia [22] and south africa [23]. these results show that acceptability varies based on country, region, culture, and believe of different age-group bracket as to how important is covid-19 vaccine. education also plays a major role in vaccination. from our report, participants who have foreknowledge and considered vaccination important for their own health or for the health of the community were more willing to accept vaccination. this was proved as the highest population of those vaccinated has tertiary education, 300 (60%). statistically, p = .000, hence, the association between education level of participants and covid-19 vaccination is extremely statistically significant. this suggests that if more people are educated about the importance of vaccination against covid-19, their willingness to be vaccinated will increase. fig. 4. covid-19 vaccination rate based on age-group ademola et al.; aji, 5(1): 52-64, 2022; article no.aji.88927 59 fig. 5. covid-19 vaccination rate based on the level of education of participants to reach the greater population of nigeria at large, a cultural and believe barrier would have to be broken as this is another major dilemma against covid-19 vaccination plan. some participants believe that covid-19 pandemic isn’t real while some believed that the vaccine was made to reduce human population. 5. conclusion the results from our study show a strong relationship between sars-cov-2 vaccine and immunity against covid-19. this survey shows that there is a high vaccination rate among the participants with tertiary education, the actively working group (age 31-50) and health professionals and covid-19 among these groups was greatly reduced compared to the other groups in their respective categories. this implies that sars-cov-2 vaccine can confer great immunity on vaccinated individuals especially the fully vaccinated. with more education about vaccine safety and more proven statistical analysis, vaccination rate is expected to increase and governmental and societal efforts are to be directed towards sensitizing the community on covid-19 vaccination. once herd immunity is attained in a population, this will help to minimize the rapid spread of the disease, stabilize human life, health, family, country’s economy and also prevent the occurrence of other variants of the virus. consent all authors declare that ‘written informed consent was obtained from the patient (or other approved parties) for publication of this case report and accompanying images. ethical approval all authors hereby declare that all experiments have been examined and approved by the appropriate ethics committee and have therefore been performed in accordance with the ethical standards laid down in the 1964 declaration of helsinki. acknowledgements we want to acknowledge “hon. s.a. aborisade” an administrative officer at adeniyi memorial hospital for the funding to purchase the sarscov-2 rapid diagnostic test kit used for this study. other than the funding, the study sponsor had no role in the study design; in the collection, analysis, and interpretation of data; in the writing ademola et al.; aji, 5(1): 52-64, 2022; article no.aji.88927 60 of the manuscript; or in the decision to submit the paper for publication. competing interests authors have declared that no competing interests exist. references 1. who director-general’s opening remarks at the media briefing on covid-19; 2020. available:https://www.who.int/directorgeneral/speeches/detail/who-directorgeneral-s-opening-remarks-at-the-mediabriefing-on-covid-19---11-march-2020 2. first case of corona virus disease confirmed in nigeria. nigeria centre for disease control. 28 february 2020. retrieved 10 march 2020. 3. world health organization. 33rd who regulatory update on covid-19; world health organization: geneva, switzerland. 2021;1–14. available:https://www.who.int/publications/ m/item/33rd-who-regulatory-update-oncovid-19. 4. updated: coronavirus: second case confirmed in nigeria; 2020. retrieved 22 april 2022. 5. maclean ruth, dahi, abdi latif. 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"ncdc covid-19 page". nigeria centre for disease control. retrieved 13 june, 2022. 10. joao m, maroco, pero pinheiro. analise estatistica com spss, 5th ed. portugal; 2011. available: https://www.wook.pt/livro/analiseestatistica-com-o-spss-statistics-joaomaroco/24699154 11. who. coronavirus available:https://www.who.int/healthtopics/coronavirus#tab=tab_1 12. world health organization. 33rd who regulatory update on covid-19; world health organization: geneva, switzerland. 2021;1–14. available:https://www.who.int/publications/ m/item/33rd-who-regulatory-update-oncovid-19. 13. wolff er, madlonikay dj. childhood vaccine beliefs reported by somali and nonsomali parents. j am board am med. 2014;27(4):458i64. 14. adebisi ya, alaran aj, bolarinwa oa, akande-sholabi w, lucero-prisno de(iii). when it is available, will we take it? social media users’ perception of hypothetical covid-19 vaccine in nigeria. pan afr. med. j. 2021;38:230. 15. dula j, mulhanga a, nhanombe a, cumbi l, júnior a, gwatsvaira j. et al. covid-19 vaccine acceptability and its determinants in mozambique: an online survey. vaccines. 2021;9:828. available:https://doi.org/10.3390/vaccines90 80828 16. national population commission (npc). nigeria demographic and health survey; 2018. available:https://www.citypopulation.de/php/ nigeria-admin.php?adm1id=nga031 17. who. 2019-ncov-vaccine effectiveness measurement. evaluation of covid-19 vaccine effectiveness. interim guidance. 17 march 2021. 18. who. what is covid-19 vaccine efficacy? 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expert rev. vaccines 2021;0:1–13. ademola et al.; aji, 5(1): 52-64, 2022; article no.aji.88927 62 questionnaire dear respondent, i am ademola, opeyemi shadrach, a tutor at the adventist college of health technology, igbope and a master’s student of chemical pathology, university college hospital, university of ibadan. i am leading a group of researchers in carrying out a research on herd immunity, the best way out of sars cov-2 pandemic. preaching the good news about covid-19 vaccine and vaccine efficacy in oke-ogun region of oyo state, nigeria. your participation is strictly voluntary. your kind consideration and honest answers would be very much appreciated. thanks. serial number ___________________________ section a: socio-demographic characteristics 1. age _______ 2. gender a) male [ ] b) female [ ] 3. marital status a) single [ ] b) married [ ] c) divorced/separated [ ] d) widowed [ ] 4. educational status a) primary education [ ] b) secondary education [ ] c) tertiary education [ ] d) postgraduate education [ ] e) none [ ] 5. employment status a) working in person [ ] b) working remotely [ ] c) not working-temporarily laid off [ ] d) not working-student [ ] e) not working-others 6. industry (a) agriculture [ ] b) construction [ ] c) manufacturing [ ] d) trading [ ] e) health care [ ] f) art and entertainment [ ] g) finance [ ] h) education [ ] i) others [ ]. if others, please specify: _________________________________ 7. how would you define your family income? a) lower than average [ ] b) on average [ ] c) higher than average [ ] section b: co-morbidity 8. do you have any of the following disease? a) cancer [ ] b) obesity [ ] c) diabetes [ ] d) hypertension[ ] e) others if others, please specify: _______________________________ ademola et al.; aji, 5(1): 52-64, 2022; article no.aji.88927 63 section c: covid-19 prevention precautions 9. do you wear a mask? a) yes [ ] b) no [ ] c) not always [ ] d) i don’t need to [ ] 10. do you practise social distancing? a) yes [ ] b) no [ ] c) not always [ ] d) i don’t need to [ ] 11. do you often wash your hands and use a sanitizer? a) yes [ ] b) no [ ] c) not always [ ] d) i don’t need to [ ] section d: domain; covid-19 vaccination 12. have you tested for covid-19 previously? a) yes [ ] b) no [ ] c) not sure [ ] if yes, what was the test result? a) positive [ ] b) negative [ ] 13. have you received covid-19 vaccine? a) yes [ ] b) no [ ] c) not sure [ ] if yes, at what dose of vaccination are you? a) first dose [ ] b) second dose [ ] date of vaccination: 1 st dose……………… 2 nd dose…………………. 14. how likely are you to recommend getting the covid-19 vaccine to others? a) not at all likely [ ] b) somewhat likely c) extremely likely 15. has all your immediate family members been vaccinated? a) yes [ ] b) no [ ] c) i don’t know [ ] section e: for those who are unvaccinated 16. what makes it difficult for you to get a covid-19 vaccine? a) i’m not eligible [ ] b) i can’t go alone [ ] c) i don’t know where to get vaccinated [ ] d) the waiting time is too long [ ] e) always busy at work [ ] f) others. if others, please specify: _______________________________ 17. how concerned are you about getting covid-19 vaccine? a) not at all concerned [ ] b) a little concerned [ ] c) moderately concerned [ ] d) very concerned [ ] 18. how safe do you think a covid-19 vaccine would be for you? a) not at all safe [ ] b) a little safe [ ] c) moderately safe [ ] d) very safe [ ] 19. if a covid-19 vaccine were available to you, would you get it? a) yes [ ] b) no [ ] c) not sure [ ] ademola et al.; aji, 5(1): 52-64, 2022; article no.aji.88927 64 20. what would be important for you to know about to become confident in the covid-19 vaccine? a)my risk of getting sick with covid‐19 is bigger than the risk of side effects from the vaccine [ ] b) the vaccine cannot cause any immediate or long term injury [ ] c) it is impossible to get covid-19 or any other disease from the vaccine itself or its components [ ] d) the vaccine works in protecting me from covid‐19 [ ] e) the vaccine works in stopping the transmission of covid-19 from one person to another [ ] your kind participation and honest answers are very much appreciated. thanks. _________________________________________________________________________________ © 2022 ademola et al.; this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: https://www.sdiarticle5.com/review-history/88927 http://creativecommons.org/licenses/by/4.0 _____________________________________________________________________________________________________ ++ assistant professor; *corresponding author: e-mail: pranithaavunoori1310@gmail.com; cite as: gupta, n., begum, s., farooqui, v., afreen, s., & avunoori, p. (2024). a review on iguratimod: bridging hope for arthritis patients through the dual power of immunomodulation and anti-inflammation. asian journal of immunology, 7(1), 79– 89. retrieved from https://journalaji.com/index.php/aji/article/view/132 asian journal of immunology volume 7, issue 1, page 79-89, 2024; article no.aji.115805 a review on iguratimod: bridging hope for arthritis patients through the dual power of immunomodulation and anti-inflammation nikita gupta a++, sana begum a++, varda farooqui a++, summaiya afreen a++ and pranitha avunoori a++* a department of pharmacy practice, st. pauls college of pharmacy, hyderabad, telangana-510. india. authors’ contributions this work was carried out in collaboration among all authors. all authors read and approved the final manuscript. article information doi: https://doi.org/10.9734/aji/2024/v7i1132 open peer review history: this journal follows the advanced open peer review policy. identity of the reviewers, editor(s) and additional reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here: https://www.sdiarticle5.com/review-history/115805 received: 13/02/2024 accepted: 16/04/2024 published: 18/05/2024 abstract iguratimod is a small novel compound considered as a disease-modifying anti-rheumatic drug (dmard), which exhibits anti-inflammatory and immunomodulatory effects. iguratimod acts directly on b cells by inhibiting the production of inflammatory cytokines (tumor necrosis factor-α, interleukin (il)-1β, il-6, il-8, il-17), thereby suppressing the production of immunoglobulin and inhibiting the activity of nuclear factor kappa-light chain enhancer of activated b cells. preclinical studies demonstrate its positive impact on arthritis models in animals by reducing immunoglobulin production and various inflammatory cytokines. iguratimod demonstrates its efficacy as well as tolerance when used as an additional therapy for rheumatoid arthritis patients who exhibit an insufficient response to both methotrexate and biological disease-modifying anti-rheumatic drugs. review article https://doi.org/10.9734/aji/2024/v7i1132 https://www.sdiarticle5.com/review-history/115805 gupta et al.; asian j. immunol., vol. 7, no. 1, pp. 79-89, 2024; article no.aji.115805 80 additionally, iguratimod was found to possess an anabolic effect on bone metabolism, through both stimulations of osteoblastic differentiation and inhibition of osteoclastogenesis. further, the mechanism also involves suppressing nuclear factor kappa b (nf-κb) activation without blocking nf-κb inhibitor α (iκbα) degradation. although the true target molecules of iguratimod have been unclear, it would be necessary to suppose the multiple mechanisms including suppression of nf-κb. clinical trials of rheumatoid arthritis patients have shown more effectiveness and tolerability when compared to salazosulfapyridine, making iguratimod a promising dmard with unique novel properties and positive clinical outcomes. further research will determine its suitability as an alternative for patients unable to use biologics. keywords: anti-inflammation; iguratimod; arthritis; immunomodulation. 1. introduction 1.1 rheumatoid arthritis “rheumatoid arthritis (ra) is a persistent autoimmune disorder with an unknown cause, marked by synovial inflammation in the joints, gradual bone damage, and a decline in joint functionality. in the absence of treatment, deteriorating joints can result in pain and stiffness, which limit physical function and lead to long-term disability” [1]. “the development of ra is associated with genetic, environmental, and immune factors. the objective of treating individuals with ra is to achieve clinical remission or maintain low disease activity, ultimately preventing damage to joint function, current treatments for ra include non-steroidal anti-inflammatory drugs (nsaids), conventional synthetic disease-modifying anti-rheumatic drugs (csdmards), and biological disease-modifying anti-rheumatic drugs (bdmards). the use of biological dmards to treat ra is not suitable for all patients for various reasons, including complications, side effects, uncertain efficacy, and high costs that prevent their use” [2]. 1.2 iguratimod “iguratimod, is a small novel compound considered as a disease-modifying antirheumatic drug (dmard), which exhibits antiinflammatory and immunomodulatory effects.igu suppresses the production of inflammatory cytokines in cultured monocyte thp-1 cells and synovial cells derived from ra patients by inhibiting the activation of nuclear factor kappa-b (nf-κb)” [3]. “additionally, it hinders immunoglobulin production through a direct impact on b cells without inducing cytostatic effects” [4]. “igu exhibits anti-inflammatory effects and improves abnormal immunological conditions in various animal models of inflammation and autoimmune diseases, including ra” [5,6]. “recently, it has been noted that igu inhibits receptor activator of nf-κb ligand (rankl)-induced osteoclast differentiation and migration in raw264 cells by modulating the nf-κb and mitogen-activated protein kinase (mapk) pathways. this finding is especially intriguing in relation to its stimulatory effect on osteoblastic differentiation” [7]. 2. mechanism of iguratimod 2.1 anti-inflammatory “iguratimod possesses anti-inflammatory properties, as demonstrated by its ability to restrain the release of bradykinin in a mouse model of kaolin-induced arthritis. initially created as an innovative nonsteroidal anti-inflammatory drug (nsaid)” [8]. research has extensively documented the anti-inflammatory, analgesic, and antipyretic properties of iguratimod in diverse animal models. its mechanism involves suppressing the metabolism of arachidonic acid metabolite prostaglandin e2 [9], inhibiting the release of bradykinin, decreasing the production of interleukin (il)-1 and interleukin (il)-6 [10,11], and selectively inhibiting the activity of cyclooxygenase-2 [12]. “in different models of autoimmune disorders, iguratimod demonstrated notable inhibitory effects. these effects were observed in experimental autoimmune encephalitis, chronic contractile injury linked with neuropathic pain, and dextran sodium sulfateinduced colitis” [4,12-20]. “significantly, iguratimod's ability to selectively inhibit cox-2 is crucial, leading to a reduction in prostaglandin levels and accomplishing its anti-inflammatory effects. importantly, iguratimod is less prone to causing gastrointestinal ulcers because of its selective inhibition of cox-2, distinguishing it from nonsteroidal anti-inflammatory drugs (nsaids) that inhibit both cox-1 and cox-2” gupta et al.; asian j. immunol., vol. 7, no. 1, pp. 79-89, 2024; article no.aji.115805 81 [12]. “iguratimod also hampers the secretion of inflammatory cytokines induced by tnf-α and curtails nf-κb activation in human synovial cells. this interference includes impeding the translocation of nf-κb p65 into the nucleus. through the inhibition of nf-κb activity, it modulates transcriptional regulation to suppress the production of cytokines and chemokines, thereby exerting anti-inflammatory effects” [16,17]. “iguratimod has been observed to disrupt the tnf-α-induced translocation of nf-κb from the cytoplasm to the nucleus. this interference leads to the inhibition of tnf-α-induced production of il-6, il-8, and monocyte chemoattractant protein 1. hence, iguratiomud has the potential to modulate the expression of inflammatory factors by regulating the nf-κb signaling pathway” [18]. “in the regulation of neutrophils, it has been discovered that iguratimod suppressed the expression of cps by downregulating pad in neutrophils from ra patients. notably, this effect was comparable to the impact of mtx and dxm at suitable concentrations. these results offer valuable insights for refining the treatment of rheumatoid arthritis (ra). iguratimod, at an optimal concentration comparable to mtx and dexamethasone, effectively hinders the expression of citrulline proteins in neutrophils from ra patients. this inhibitory effect is attributed to the downregulation of peptidyl arginine deiminase, shedding light on the mechanism of iguratimod in ra treatment. the findings from this study can provide valuable guidance for the treatment of rheumatoid arthritis and facilitate the identification of additional therapeutic targets” [19]. 2.2 immune response 2.2.1 regulating humoral immunity “in 2003, researchers initially observed that igu had a direct inhibitory impact on b lymphocytes in both mouse and human subjects, leading to a decrease in the synthesis of immunoglobulins. notably, this inhibition did not influence the proliferation or apoptosis of b cells [4]. in mrl/lpr mice, igu demonstrated the ability to decrease circulating plasma cells through a mechanism that does not involve antiproliferative effects” [21]. “in a recent study, it was shown that igu did not influence the activation and proliferation of b cells within the established in vitro human antibody-secreting cell differentiation system. however, its inhibitory effects were observed in the differentiation of human antibody-secreting cells, achieved by targeting the protein kinase c (pkc) and early growth response 1 (egr1) axis” [22]. fig. 1. depicts the anti-inflammatory mechanism of igu (iguratimod) igu effectively suppresses inflammation through several pathways. firstly, it inhibits the activity of mif (macrophage migration inhibitory factor) and counteracts mif-induced proinflammatory effects. additionally, igu disrupts il-17-mediated signaling by interfering with the interaction between act1 and traf5, as well as ikki, thereby reducing the expression of various inflammatory factors induced by il-17. moreover, igu interferes with the tnf-α-induced translocation of nf-κb from the cytoplasm to the nucleus, consequently suppressing the production of il-6 and il-8 induced by tnf-α gupta et al.; asian j. immunol., vol. 7, no. 1, pp. 79-89, 2024; article no.aji.115805 82 2.2.2 suppression of immunoglobulin synthesis “in subsequent clinical trials, it was noted that iguratimod exhibited favorable therapeutic outcomes concerning both clinical symptoms and biological markers, including rheumatoid factor (rf) levels and immunoglobulin concentrations in plasma. consequently, our investigations were directed toward assessing its impact on b-cell functions, particularly about immunoglobulin production and proliferation. in cultures of murine b-cells, iguratimod exhibited a notable reduction in igm production and the isotype-switch to igg1 class induced by lipopolysaccharide (lps) and/or il-4. additionally, it inhibits spontaneous igg production without affecting cell proliferation in a human plasmacytoma cell line (arh-77). moreover, when human peripheral b cells were stimulated with autologous t cells and anti-cd3 antibody, iguratimod demonstrated a concentration-dependent inhibition of both igm and igg production” [4]. in contrast, iguratimod did not exhibit any influence on the mitogen-induced proliferation response [23,24] and thymus and activationregulated chemokine (tarc) production in human b cells stimulated with anti-cd40 antibody and il-4 [25]. hence, it seems that this compound hinders the production of immunoglobulins by b cells without causing a cytostatic effect.subsequently, to elucidate the hyper-immunoglobulinemia observed in rheumatoid arthritis (ra) patients and the inhibitory effects of iguratimod, we examined the secretion of immunoglobulins from ra synovial tissues. the experimentation was conducted utilizing severe combined immune deficiency (scid) mice that were implanted with human rheumatoid arthritis (ra) tissue.consequently, the sera of the mice exhibited elevated concentrations of polyclonal human igg. furthermore, the iguratimod-treated group demonstrated a noteworthy reduction in igg levels compared to the group treated with the vehicle. in chronic arthritis models such as adjuvant-induced arthritis (aia) rats and mrl/lpr mice [11], the alleviation of arthritic lesions by iguratimod was concomitant with the improvement of hyperimmunoglobulinemia [3,26]. about the clinical effectiveness of b-celltargeted anti-cd20 antibody in rheumatoid arthritis (ra) patients, [27] these findings hold significant importance in understanding the mechanisms behind the anti-rheumatic effects exhibited by small molecule disease-modifying antirheumatic drugs (dmards). 2.2.3 suppression of cytokine synthesis “another clear difference between iguratimod and classical nsaids is the inhibitory effect on cytokine production, as mentioned above. when tested on cultured monocytes/macrophages, iguratimod demonstrated the ability to inhibit the production of il-1β, tnfα, il-6, il-8, and monocyte chemoattractant protein-1 (mcp-1) with ic50 values ranging from 1 to 20 µg/ml” [28-31]. “in synovial cells obtained from rheumatoid arthritis (ra) patients, iguratimod markedly decreased the synthesis of il-6, il-8, and colony-stimulating factors (csfs) within concentration ranges of 0.3 to 30 µg/ml” [28,32,33]. “the suppression of mrna expression coincided with iguratimod's inhibitory effect on the production of these cytokines”(29,31,33). therefore, it is reasonable to suggest that iguratimod suppresses the expression of inflammatory cytokines at the gene level. additionally, the research findings demonstrate [32] that iguratimod also impedes the increased expression of co-stimulatory molecules like cd54, cd58, and cd106 in synovial cells upon stimulation with ifn-γ. “this mechanism of action seems to involve the prevention of nuclear factor-kappa b (nf-κb) activation. the inhibitory effect on cytokine production was similarly observed in animal models. in a mouse air-pouch inflammation model, oral administration of iguratimod at doses of 30 and 100 mg/kg significantly decreased mcp-1 production induced by tnfα injection” [29]. “moreover, at doses of 10 and 30 mg/kg, iguratimod reduced the rise in serum tnfα and ifn-γ levels in the concanavalin a-induced hepatitis model in mice, along with serum transaminase levels” [29]. “iguratimod was also found to suppress the onset of active experimental autoimmune encephalomyelitis (eae) in rats” [33]. “in this model, it was observed to inhibit tnfα and ifn-γ production by antigen-specific t cells and the infiltration of cells into the spinal cord of rats. notably, iguratimod exhibited an anti-cachectic effect on adenocarcinoma colon-induced cachexia in mice by inhibiting il-6 gene expression” [34]. “furthermore, recent reports indicate that cia rats treated with iguratimod showed reductions in mrna expression of il-17 in peripheral lymphocytes and circulating il-17, suggesting that the compound exerts its immunoregulatory and bone-preserving effects by shifting responses away from il-17-producing t cells (th17 cells)” [35]. these findings suggest that iguratimod's inhibition of cytokine production may gupta et al.; asian j. immunol., vol. 7, no. 1, pp. 79-89, 2024; article no.aji.115805 83 contribute to its clinical efficacy in treating rheumatic conditions, making it a characteristic feature of this drug. 2.3 osteoprotective mechanism “osteoporosis frequently arises as a secondary consequence of rheumatoid arthritis (ra), potentially resulting in joint stiffness, malformation, and significant impairment in functionality. employing early and efficient measures to safeguard bone health and enhance bone metabolism can be advantageous in thwarting joint deterioration.several signaling pathways are crucial for osteoblast proliferation, differentiation, and the regeneration of damaged bone and cartilage. these include the bone morphogenetic protein (bmp)2–smads pathway, the p38 mitogen-activated protein kinase (mapk) pathway, and the tnf-α/nf-κb pathway.research has demonstrated that igu enhances the expression of osterix (osx), a key factor in osteoblast differentiation. bmp2 has the ability to stimulate osx expression by inducing the upstream transcription factor dlx5. additionally, the p38–mapk pathway collaborates with the bmp2–smads pathway to enhance osx phosphorylation” [36]. “igu has been found to boost osteoblast differentiation by upregulating the expression of osx and dlx5” [37]. “the p38 mapk belongs to the mapk superfamily and plays a role in the initial phases of osteoblast lineage proliferation by phosphorylating dlx5, runt-related transcription factor (runx)2, and osx (36). igu has the potential to enhance the activation of p38, thereby promoting osteoblastic differentiation. additionally, the tnf-α/nf-κb signaling pathway is implicated in osteoblast proliferation, apoptosis, and differentiation” [38]. nf-κb facilitates the deterioration of osteocytes by diminishing the expression and phosphorylation of the bmp-smad1 signaling pathway. igu has the capacity to sustain decreased nf-κb activation, thus mitigating the inhibitory effects of tnf-α on osteoblasts. fig. 2. illustrates the inhibitory effects of igu on the immune response, particularly in the context of rheumatoid arthritis (ra) in ra, the initial interaction between th1 cells and antigen-presenting cells (apcs), such as dendritic cells, involves t-cell receptors (tcrs) and major histocompatibility complex (mhc). various environmental factors influence the production of autoantibodies like anti-citrullinated protein antibodies (acpa) and rheumatoid factor (rf). these immune complexes activate synovial fibroblasts (sfs) and macrophages, leading to the production of pro-inflammatory cytokines such as tumor necrosis factor-alpha (tnf-α) and il-6. additionally, they affect th17 cells, which produce il-17, further contributing to joint destruction. igu intervenes in this process by impacting the production of pro-inflammatory cytokines in both th1 and th17 cells, as well as affecting the production of immunoglobulins and antibodies in b cells. it also influences bone metabolism by inhibiting osteoclast activation and promoting osteoblast differentiation. nf-κb, a central player in the pathogenesis of ra, perpetuates the chronic cycle of inflammation underlying its pathology. inflammatory mediators like tnf-α activate cells in the synovium, particularly macrophages and sfs, largely through nf-κb activation. sfs, in response to tnf-α or il-1, synthesize many nf-κb-induced genes, including chemokines and matrix metalloproteinases (mmps), which further promote inflammation and joint destruction gupta et al.; asian j. immunol., vol. 7, no. 1, pp. 79-89, 2024; article no.aji.115805 84 “additionally, igu fosters the formation of calcium nodules in vitro” [37]. “osteoclast differentiation is primarily regulated by two essential cytokines: macrophage colonystimulating factor (m-csf) and receptor activator of nuclear factor kappa-b ligand (rankl)” [39]. “the rankl/osteoprotegerin (opg) system is crucial in bone erosion associated with rheumatoid arthritis (ra). an increase in the rankl/opg ratio contributes to bone erosion. igu has the ability to reduce the production of rankl, leading to a significant decrease in the rankl/opg ratio both in serum and in ra fibroblast-like synoviocytes (flss) induced by il1β after treatment” [40]. “rankl and its receptor rank interact with osteoclast precursor cells, initiating downstream pathways such as peroxisome proliferator-activated receptor (ppar)-γ, c-fos, and nuclear factor of activated t cells (nfat)c1” [41]. “igu has the ability to inhibit osteoclast formation and bone resorption stimulated by rankl through the pparγ/c-fos signaling pathway. furthermore, it decreases the expression of nfatc1 and subsequent osteoclast marker genes” [42]. gan et al. found that “igu effectively inhibited rankl-induced osteoclast differentiation, migration, and bone resorption in raw264.7 cells, with the effect varying depending on the dosage administered”. “this effect was attributed to the activation of the mapk and nf-κb pathways” [43]. “it indicates that igu directly suppresses the formation and activity of osteoclasts. additionally, the tnf-α/nf-κb signaling pathway inhibit the production of matrix metalloproteinases (mmps)” [38]. “mmps are pivotal in the degradation of cartilage in rheumatoid arthritis (ra), predominantly synthesized by fibroblast-like synoviocytes (flss). du et al. conducted in vitro experiments treating flss with varying concentrations of igu, followed by stimulation with tnf-α, il-1β, or il17a. they observed a significant inhibition of mmp-3 at 5 μg/ml igu, while mmp-1 inhibition occurred at 50 μg/ml. clinical trials revealed a significantly reduction in mmp-1 and mmp-3 levels following 24 weeks of igu treatment (25 mg, twice daily)” [44]. these findings indicate that igu effectively prevents mmp-1 and mmp-3, thus preserving cartilage integrity. moreover, opg serves as a natural antagonist to rankl, preventing its binding to the osteoclast receptor. the equilibrium between rankl and opg is critical for sustaining osteoclast homeostasis [45]. igu can suppress the expression of mmp-3 and the rankl/opg ratio by inhibiting the phosphorylation of erk1/2, consequently thwarting the degradation of bone in rheumatoid arthritis [46]. igu not only enhances osteoblast differentiation but also suppresses osteoclast formation and the production of matrix proteins through its interaction with various signaling pathways. consequently, igu plays a crucial role in safeguarding bone health. fig. 3 illustrates the intricate adjustments made by igu within the signaling pathways. fig. 3. illustrates the osteoprotective mechanism of igu, highlighting its impact on various signaling pathways crucial for osteoblast proliferation, differentiation, and bone/cartilage repair these pathways include the bmp2-smads signaling pathway, the p38-mapk pathway, and the nf-κb pathway. igu, or iguratimod, facilitates osteoblast differentiation by upregulating the expression of osx and its upstream regulator dlx5, while also enhancing p38 activation. furthermore, igu inhibits osteoclastogenesis and bone resorption induced by rankl via the pparγ/c-fos signaling pathway. additionally, igu decreases the expression of nuclear factor in activated t cell c1 and downstream osteoclast marker genes gupta et al.; asian j. immunol., vol. 7, no. 1, pp. 79-89, 2024; article no.aji.115805 85 3. progress in clinical research numerous clinical trials have validated igu as a promising novel treatment for rheumatoid arthritis (ra). phase i, ii, and iii trials have demonstrated the effectiveness and safety of igu monotherapy over a span of 24 weeks. typically, igu exhibits its therapeutic effects within 8 weeks, demonstrating a faster onset compared to methotrexate (mtx). igu treatment exhibits early and sustained efficacy. furthermore, the efficacy of a 50 mg dosage of igu (25 mg twice daily) is comparable to that of a 15 mg dose of mtx (15 mg per week). a multicenter, prospective observational study was conducted to assess the long-term safety and efficacy of igu in rheumatoid arthritis (ra) patients. the study enrolled all eligible ra patients who had been treated with igu since its introduction to the market in 2012. efficacy was evaluated using disease activity score 28 (das28), while adverse drug reactions such as liver and renal dysfunction, interstitial lung disease, gastrointestinal and blood disorders, and infections were monitored up to week 52. the findings indicated that extended use of igu demonstrated a manageable safety profile and led to enhanced control of rheumatoid arthritis (ra) activity. additionally, another study assessing the 3-year efficacy of igu in ra patients confirmed its effectiveness and safety. these outcomes suggest that igu can be utilized for prolonged treatment durations. methotrexate (mtx) serves as a cornerstone in the management of rheumatoid arthritis (ra), often forming the foundation of treatment. typically, ra management entails a combination of several anti-rheumatic medications. consequently, the combination of igu with mtx has become a prevalent and novel therapeutic approach for ra. ren et al. conducted a randomized study involving 82 patients, dividing them into two groups. the control group received mtx at a dose of 10 mg once weekly, which was escalated to 15 mg once weekly after 2 weeks. the observation group received igu at a dose of 25 mg twice daily in addition to the regimen followed by the control group, over a period of 6 months. the total effective rate in the observation group was 90.24%, markedly surpassing that of the control group, which stood at 78.05% [41]. the findings indicated that igu enhances the therapeutic efficacy of mtx. when used in conjunction with leflunomide (lef) for rheumatoid arthritis (ra) treatment, methotrexate (mtx) frequently leads to an increased incidence of adverse reactions. in the tranmod study, 66 patients with refractory rheumatoid arthritis (ra) were randomly assigned to two groups. the observation group received a combination of methotrexate (mtx) at a dose of 10 mg once weekly and iguratimod (igu) at a dose of 25 mg twice daily, while the control group received mtx (10 mg once weekly) combined with leflunomide (lef) at a dose of 10 mg once daily for a duration of 16 weeks. results showed that both groups experienced reductions in disease activity score 28 (das28) scores compared to before treatment. at 8 weeks, there was a significant difference between the two groups in terms of achieving american college of rheumatology (acr)20 and acr50 response criteria. however, by 16 weeks, there was no significant difference in acr20, acr50, and acr70 response rates between the two groups. compared to the combination of mtx and lef, the combination of mtx and igu demonstrated superior short-term clinical efficacy and fewer adverse reactions in ra patients. furthermore, igu is frequently employed in conjunction with biological agents like tocilizumab and etanercept. this underscores the versatility of igu, as it can be combined with various anti-rheumatic medications for the treatment of rheumatoid arthritis (ra). in real-world clinical scenarios, certain rheumatoid arthritis (ra) patients exhibit insufficient response to multiple diseasemodifying antirheumatic drugs (dmards). a study verified that the effectiveness and tolerability of igu in combination with mtx therapy persisted for a duration of up to 52 weeks in active ra patients who had shown inadequate response to mtx alone. in another study, 131 patients previously treated with traditional disease-modifying antirheumatic drugs (dmards) were divided into three groups. group 1 consisted of 44 patients treated with a combination of igu (25 mg, twice daily, orally) and mtx (10 mg weekly, orally). group 2 included 38 patients who received igu alone (25 mg twice daily, orally), while group 3 comprised 49 patients who received mtx alone (10 mg weekly, orally). therapeutic effects with igu were observed between 4 and 10 weeks after treatment initiation, effectively addressing patients with inadequate responses to prior dmard treatments. the combination of igu with mtx exhibited superior efficacy compared to igu gupta et al.; asian j. immunol., vol. 7, no. 1, pp. 79-89, 2024; article no.aji.115805 86 or mtx monotherapy. igu has demonstrated supplementary effectiveness in rheumatoid arthritis (ra) patients who exhibit inadequate responses to methotrexate (mtx). consequently, combining igu with mtx presents a promising emerging approach for treating active ra in individuals who have either inadequate responses to or intolerance to traditional diseasemodifying antirheumatic drugs (dmards). moreover, igu has shown additional effectiveness and safety in ra patients who demonstrate inadequate responses to biological dmards, such as tocilizumab. wang et al. conducted a self-controlled study involving 20 rheumatoid arthritis (ra) patients with leukopenia to address the challenge of clinical drug selection due to the myelosuppressive adverse effects of many disease-modifying antirheumatic drugs (dmards). they administered igu (25 mg twice daily) in combination with methylprednisolone (medrol; 8 mg once daily) during the early stage of the disease. as white blood cell counts returned to normal with the help of hormones during the mid-stage, mtx was added to the regimen (7.5 mg once weekly), and methylprednisolone was gradually discontinued. subsequently, igu (25 mg twice daily) and mtx (7.5 mg once weekly) were employed as longterm maintenance therapy for a duration of 12 weeks. results indicated improvements in erythrocyte sedimentation rate (esr), c-reactive protein (crp), disease activity score 28 (das28), and white blood cell counts compared to pre-treatment levels. the acr20 and acr50 response rates were 85% and 45%, respectively, and the overall rate of leukocyte elevation was 95%. only one case experienced mild liver damage during the treatment period, which resolved with liver protection treatment without discontinuing anti-rheumatic therapy. this underscores the favorable therapeutic efficacy and high safety profile of phased treatment with igu, methylprednisolone, and mtx in ra patients with leukopenia. the aforementioned clinical trials have established that igu is a valuable choice as an initial treatment for rheumatoid arthritis (ra), demonstrating notable effectiveness and safety whether used independently or in combination with other medications. 4. conclusion ra is a chronic autoimmune disorder characterized by synovial inflammation, joint damage, and decreased joint function. treatment goals include achieving remission or low disease activity to prevent further joint damage. iguratimod exerts its anti-inflammatory effects by inhibiting nf-κb activation, cytokine production, and cox-2 activity. it regulates humoral immunity by inhibiting immunoglobulin synthesis and b cell differentiation. iguratimod's osteoprotective mechanism involves enhancing osteoblast differentiation and inhibiting osteoclast formation and activity.clinical trials have demonstrated the efficacy and safety of iguratimod in ra treatment, both as monotherapy and in combination with other dmards. combining iguratimod with methotrexate or other dmards has shown superior efficacy compared to monotherapy. iguratimod has shown promise in patients with inadequate responses to traditional dmards or biological agents.phased treatment with iguratimod, methylprednisolone, and methotrexate has been effective in ra patients with leukopenia. iguratimod is a valuable treatment option for ra, offering early and sustained efficacy with a manageable safety profile. its mechanism of action, including antiinflammatory, immunomodulatory, and osteoprotective effects, makes it a promising candidate for ra therapy. competing interests authors have declared that no competing interests exist. 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(2) augustine ikhueoya airaodion, federal university of technology owerri, nigeria. complete peer review history: http://www.sdiarticle4.com/review-history/65262 received 28 november 2020 accepted 02 february 2021 published 23 february 2021 abstract background: type 1 diabetes mellitus (tid), is a disease that has long been connoted to as insulin-dependent, childhood-onset, young adult-onset or juvenile onset diabetes with essential insulin deficiency that requires daily insulin administration with peak onset during puberty (10-15) years of age. aim: this study was aimed at assessing the burden of tid among diagnosed autoimmune disease in saint vincent and the grenadines. methods: from 2014 to 2018, individuals with autoimmune, immunological and rare disease were identified from the hospital records of milton cato memorial hospital, which records information on all patient coming in for healthcare services. a structured data extraction tool was employed to extract the data from the hospital record using the open data kit (odk). data was analysed using statistical package for social sciences (spss) version 23 and r studio statistical software for analysis. the chi-square test was used to test for association. all statistical tests were two-tailed and level of confidence was set at 95%, and p values < 0.05 was considered to be statistically significant. original research article adedeji et al.; aji, 4(1): 1-14, 2021; article no.aji.65262 2 results: the mean age of patient with type 1 diabetes was 26.58 ± 11.73 yrs. old and the median age was 31 years. old, almost two-third 81(64.8%) were females. yearly, women showed a significantly higher incidence of t1d than men, there was an annual decrease in the incidence from 2014 to 2018, with a peak incidence in 2014 (0.49/1000 person-years). there was an annual decrease in the incidence from 2014 to 2018, with a peak incidence in 2017 for male (0.40/1000 person-years) and in 2014 for females (0.69/1000 person-years). the lowest incidence was noted in 2018 (0.00/1000 person-years) and (0.08/1000 person-years) for both male and female respectively. there was increased mortality in people with t1d with a baseline of 3% in 2014 to 33% in 2018. conclusions: sequel to the findings of this study, the incidence of autoimmune disease, type 1 diabetes mellitus have decreased in the last decade, whereas the mortality rates have increased. this finding of increased mortality of t1d suggests that this disease is no longer rare and will have implications for future healthcare planning. keywords: type 1 diabetes mellitus; incidence; saint vincent and the grenadines. 1. introduction type 1 diabetes mellitus (tid), is a disease that has long been connoted to as insulin-dependent, childhood-onset, young adult-onset or juvenile onset diabetes with essential insulin deficiency that requires daily insulin administration with peak onset during puberty (10-15) years of age [1]. the pathophysiology of type 1 diabetes is an insulin-requiring chronic disorder which stems from an autoimmune demolition of the pancreatic β cells through cell mediated immunity as well as a humoral immune response, culminating in elevated concentrations of blood sugar and gradual functional excoriation and degeneration of various organs and tissues. elevated blood glucose concentrations activate oxidative stress with concomitant degeneration of dna, lipid and protein macromolecules by free radicals with accelerated diabetes-linked non enzymatic glycosylation or glycation of proteins and tissues damage in non-healthy persons, but not so in healthy persons. the etiology of type 1 diabetes is unknown; t1d often starts suddenly and may include symptoms such as the most important polydipsia, polyuria, polyphagia, lack of energy, excessive fatigue, sudden weight loss, slow healing of wounds, frequent infections and blurred vision and diabetic ketoacidosis following severe dehydration especially in children and adolescents. the symptoms are more severe in children than adults. economically disadvantaged families, access to self-care tools including self-management education as well as access to insulin is limited. this pitfall leads to severe disability and reducing the quality-of-life years of the individual. even though, type 1 diabetes permeates all age groups, numerous epidemiological investigations emphasize disease features with clinical disease in childhood and adolescence, and sometimes present difficulties of differentiation from certain forms of type 2 diabetes or latent autoimmune diabetes in adults, lada [2]. type 1 diabetes is an important and excruciating chronic disease of children globally, with resultant 8to 10-fold excess risk of death in developed regions, whereas most cases die within a short period in developing countries, or where not adequately followed-up or registered may elude healthcare personnel in developed countries [3]. a 60-fold internal gradient in the incidence of type 1 diabetes and epidemic periods have ostensibly been identified and reported. in 1995, the global prevalence of diabetes mellitus (dm) in adults was estimated to be 4.0% and projected to rise to 5.4% by the year 2025 [4]. however, by 2011, the international diabetes federation (idf) estimated the global prevalence of diabetes mellitus to be 8.3% and projected a rise to 9.9% by 2030. in absolute numbers, this translates to 366 million persons with diabetes mellitus in 2011 which will rise to 552 million people by 2030. eighty percent of those with diabetes live in lowand middle-income countries [5]. in the caribbean, the overall prevalence of diabetes mellitus is estimated to be approximately 9% and is responsible for 13.8% of all deaths among adults in the region [5, 6]. diabetes mellitus is therefore one of the major public health challenges for the caribbean in the twenty-first century. living with type 1 diabetes remains a challenge for the child and the whole family even in countries with access to multiple daily injections adedeji et al.; aji, 4(1): 1-14, 2021; article no.aji.65262 3 or an insulin pump, glucose monitoring, diabetes education and expert medical care. poor metabolic control may result in the acute complications of hypoglycemia and ketoacidosis, chronic microvascular and macro vascular complications and death [79]. children are more sensitive to a lack of insulin than adults and are at higher risk of a rapid and dramatic development of diabetic ketoacidosis. episodes of severe hypoglycemia or ketoacidosis, especially in young children, are risk factors for structural brain abnormalities and impaired cognitive function, which may cause schooling difficulties and limit future career choices [10, 11]. many children and adolescents find it difficult to cope emotionally with their condition. diabetes causes them embarrassment, results in discrimination and limits social relationships. it may impact on school performance and family functioning. many schools and nurseries are reluctant to receive children with diabetes [12]. like many developing nations, caribbean countries are undergoing significant demographic changes. as such, these countries have a double burden of infectious/communicable diseases (e.g., hiv/aids) and chronic, non-communicable diseases (especially diabetes), and these diseases are assuming epidemic proportions. few reviews of diabetes in this population have been conducted; however, this article summarizes the available information on the epidemiology of diabetes, the types of diabetes, the etiologic factors and complications of diabetes, and the public health burden associated with diabetes in the caribbean. due to the paucity of information on the incidence and mortality of diabetes mellitus type 1 within the population of saint vincent and the grenadines. this study was aimed at assessing the burden of tid among diagnosed autoimmune disease in saint vincent and the grenadines. this would provide empirical information in characterizing the incidence and mortality of type 1 diabetes in order to effectively and efficiently collate and evaluate healthcare and economics of diabetes, promote and establish domestic programs in the epidemiology of diabetes to prevent and curb the debilitating disorder and its concomitant complications. 2. methodology 2.1 study area this study was carried out in kingstown, saint vincent and the grenadines. st. vincent and the grenadines comprises of 32 islands and cays, of which 9 are inhabited. the largest is st. vincent, where the nation’s capital, kingstown, is located. st vincent and the grenadines has a population of 111,000, which has remained fairly flat since 1990. in 2019, st vincent and the grenadines has an estimated population of 110,589. the country is densely populated with 307 people per square kilometer (792/sq. mi). the capital and largest city is kingstown, with a population estimated at 35,000. most vincentians are the descendants of african slaves brought to the region to work plantations, as well as portuguese and east indians, who were brought to the island after slavery was abolished by the british living in the region. the largest ethnic group was african (66%), followed by those of mixed descent (19%), east indian (6%), europeans (mostly portuguese (4%), and carib amerindian (2%). there is a growing community of chinese people in the country. in 2012, the male population (55,551) outnumbered the female (53,637). the population is young, with almost 25 per cent under the age of 15 and 41.7 per cent under the age of 35. although this under-35 age group has decreased since the 2001 census by 6.1 per cent, it remains the largest proportion of the total population. the 2012 census determined the population aged under 5 years to be 8,645. a little over 9.1 per cent of the population is over the age of 65.5. st vincent has both public hospitals and private clinics in the area around kingstown. being a small developing nation, the level of care is well below that of the us or europe and the public facilities are often stretched way beyond capacity. there are public hospitals and clinics throughout the islands, with each place (with the exception of mayreau) having some form of medical center. any serious problems, however, will require a trip to out of st vincent. public spending on health in st vincent and the grenadines was four per cent of gdp in 2011, equivalent to us$310 per capita. in the most recent survey, conducted between 1997 and 2010, there were 75 doctors, and 379 nurses and midwives per 100,000 people. additionally, in the period 2007-12 virtually all births (99 per cent) were attended by qualified health staff and in 2012, 94 per cent of one-year-olds were immunized with one dose of measles. adedeji et al.; aji, 4(1): 1-14, 2021; article no.aji.65262 4 the milton cato memorial hospital (mcmh) is a 215-bed hospital serving the 110,000 inhabitants of st. vincent and the grenadines. the hospital was originally called the colonial hospital, built in the early 19 th century by the british government under the colonial system, and later renamed kingstown general hospital. in the late 1800s, the imperial parliament granted permission for construction of a new wing, which was opened in 1889, and contained a total of seven beds. in 1914, the princess mary louise wing was completed, and used mainly as nurses’ quarters. as a public hospital, all of milton cato memorial hospital’s services operate under the auspices of the ministry of health, wellness and the environment. patients are required to pay user fees for medical services, which don’t often recover the services’ true costs. that being said, patients who cannot afford to pay are not prevented from accessing healthcare. 2.2 study design the study utilized a retrospective populationbased study which consists of secondary data derived from milton cato memorial hospital of patients with autoimmune disease. 2.3 inclusion criteria  data on immunological disease and rare disease 2.4 exclusion  incomplete data on immunological disease and rare disease 2.5 data collection procedure a semi-structured interviewer-administered questionnaire with close and open ended questions will be used to collect relevant information with the aid of an android mobile device using the open data kit (odk). the interview schedule consisted of 15 sections: a structured data extraction tool was employed to extract the data from the hospital record with the aid of an android mobile device using the open data kit (odk). the data extraction tool was developed and modified with reference to existing tools used in similar studies. the data extraction tool comprises of information on sociodemographic (age, sex), year of diagnosis and diagnosis. 2.6 outcome measures and data analysis for annual incidence, the year-specific numerator included subjects with incident cases of autoimmune disease in the specific calendar year, and the denominator included the mid-year population from the population and demographic health survey (dhs) from 2013-2019 which are cross-sectional surveys conducted every year, compiled by the statistical office ministry of finance, economic planning, sustainable development and information technology of the government of saint vincent and the grenadines population. this nationally representative survey involved a multi-stage sampling design up to the household level with enumeration areas distributed by region and type of residence using the most recent national census as its sampling frame. crude rates, sexand age-specific rates, standardized rates adjusted for sex and age using the 2014-2018 mid-year population, and their 95% confidence intervals (cis) were calculated. incident cases of t1d disease were defined as those with disease in a particular year (e.g., 2014) and the preceding year (e.g., 2013 to 2014) that met the algorithm in that year (e.g., 2015) and the following year (e.g., 2016). subgroup analyses were performed according to age and sex, the mortality rate in cases of diabetes mellitus type 1 was estimated by dividing the number of incident diabetes mellitus type 1 who died during the study period by the number of person-years for incident of diabetes mellitus type 1. mortality rates were also stratified by time since diagnosis. data was being edited, collated and entered into the 2019 microsoft excel data sheet, after which it was exported into the international business machine (ibm) statistical package for social sciences (spss) version 23.0 and r studio statistical software for analysis. the analysis involved the calculation of descriptive statistics (such as frequency distributions, percentages and means) and inferential statistics. continuous variables were expressed as means ± standard deviation while categorical variables were expressed as absolute frequencies. parametric analysis was used after tests for normality adedeji et al.; aji, 4(1): 1-14, 2021; article no.aji.65262 5 confirmed that continuous variables were normally distributed. the chi-square test was used to test for association. all statistical tests were two-tailed and level of confidence was set at 95%, and p values < 0.05 was considered to be statistically significant. test of normality was done to check for normal distribution of data using the shapiro-wilk test and kolmogorov-smirnov test with significance level set at 0.05. assumptions were set that if the sig. value of both test (p>0.05), the data is normal. if it is below 0.05, the data significantly deviate from a normal distribution and nonparametric testing will employed such as the median will be used instead of the mean to represent summative statistics due to the median is no affected by outliers or extreme values. the information provided by the probability value (p-value) does not provide an estimate for the magnitude of the effect of interest and the precision of this magnitude. as a result of this, most of the inferential statistics reported in this report, did not only provide information on the pvalue but also on the magnitude of the effect (effect size statistics) in the form of correlation coefficient, regression coefficient and also their confidence intervals (cis). confidence intervals (cis) were interpreted as the value that encompasses the population or ‘true’ value. this style of reporting both the effect sizes and their cis gave a clear understanding of the relationships between the variables. 3. results 3.1 socio-demographics characteristics of patients with diabetes mellitus type 1 table 1 shows the socio-demographics distribution of patient with diabetes mellitus type 1 in respect to age, sex. from 2014 to 2018, the total number of cases of diabetes mellitus type 1 in milton cato general hospital was 125, with almost one-third 38(30.4%) occurring in the year 2014. among the cases of diabetes mellitus type 1 the mean age was 26.58 ± 11.73yrs old and the median age= 31 years old, almost twothird 81(64.8%) were females. fig. 2 shows the trend in incidence by year. every year, women showed a significantly higher incidence of diabetes mellitus type 1 than men, there was an annual decrease in the incidence from 2014 to 2018, with a peak incidence in 2014 (0.49/1000 person-years). the lowest incidence was noted in 2018 (0.04/1000 person-years). among sex, there was an annual decrease in the incidence from 2014 to 2018, with a peak incidence in 2017 for male (0.40/1000 personyears) and in 2014 for females (0.69/1000 person-years). the lowest incidence was noted in 2018 (0.00/1000 person-years) and (0.08/1000 person-years) for both male and female respectively. fig. 1. saint vincent and the grenadines map adedeji et al.; aji, 4(1): 1-14, 2021; article no.aji.65262 6 fig. 3 shows that the overall peak age of incidence was 31 to 35 years in 2014. in 2014, the peak age incidence among men was different 36-40 years. however, the peak age of prevalence among women was similar to the overall incidence graph 31 to 35 years of age. fig. 4 shows that the overall peak age of incidence was 36 to 40 years in 2015. in 2015, the peak age incidence among men and women similar to the overall incidence graph 36 to 40 years of age. fig. 5 shows that the overall peak age of incidence was 36 to 40 years in 2016. in 2016, the peak age incidence among men was different 26-30 years. however, the peak age of prevalence among women was similar to the overall incidence graph 36-40 years of age. fig. 6 shows that the overall peak age of incidence was 36 to 40 years in 2017. in 2017, the peak age incidence among men and women was similar to the overall incidence graph 36-40 years of age. fig. 7 shows that the overall peak age of incidence was 6-10 years in 2018. in 2018, the peak age incidence among men and women was similar to the overall incidence graph 6-10 years of age. 3.2 association between social demographic characteristics in the table 2, among sex, the females had higher proportions across the years (2014-2018) compared to that of the male we hereby fail to reject the null hypothesis which postulates that, there is no significant higher proportion of females to males who have diabetes mellitus type 1 from 2014 -2018 in saint vincent and the grenadines due to there was no statistically significant association observed (p>0.05). among the age group, those within the age group of 31-40 years had significantly higher proportions across the years (2014-2018) compared to that of other age group, this difference was not statistically significant (p>0.05). we hereby fail to reject the null hypothesis which postulates that there is no significant higher proportion of individuals ≤ 20 years of age compared to other age groups who have diabetes mellitus type 1 from 2014 -2018 in saint vincent and the grenadines. in the table 3, those within the age group of 3640 years had higher proportions of both male and female compared to that of other age group to having diabetics mellitus type 1, however, there was no statistically significant association observed between age and sex (p>0.05). table 1. socio-demographics characteristics of patients with diabetes mellitus type 1 variable frequency (n=125) percentage (%) age ≤5 9 7.2 6-10 7 5.6 11-15 12 9.6 16-20 10 8.0 21-25 14 11.2 26-30 9 7.2 31-35 28 22.4 36-40 34 27.2 >40 2 1.6 mean ± s.d (26.58 ± 11.73) yrs. old, 95% c.i for mean (24.50-28.65), median age= 31 yrs. old sex male 44 35.2 female 81 64.8 year 2014 38 30.4 2015 25 20.0 2016 23 18.4 2017 36 28.8 2018 3 2.4 s.d = standard deviation, c.i = confidence interval adedeji et al.; aji, 4(1): 1-14, 2021; article no.aji.65262 7 fig. 2. incidence of diabetes mellitus type 1 from 2014 -2018 fig. 3. peak age of incidence of diabetes mellitus type 1 in 2014 fig. 8 shows the case fatality from diabetics mellitus type 1 of the total, 2018 had the highest case fatality of 33% compared to the other years with 2016 having no case fatality at all. 4. discussion in this study we observed a decreasing trend in the incidence of type 1 diabetes mellitus (t1d) with a baseline of 0.49 per1000 person-years 2014 to 0.04 per 1000 person-years in 2018. the decrease in trend reported in the present study might be explained due to the use of the hospital database could stem from the lack of accurate diagnosis. another explanation of the findings is that data analyzed in this study were obtained when subjects visited healthcare institutions. therefore, no information was available for t1d patients who did not visit a healthcare institution, which could underestimate the t1d burden. the incidence rate of t1d reported was lower compared to findings from a similar study who found an age-adjusted type 1 diabetes incidence difference from 0.1/100,000 per year in china and venezuela to 36.8/100,000 per year in sardinia and 36.5/100,000 in finland. findings from the study also showed that lowest incidence (<1/100, 000 per year) was realized from china and south america populations. similar study carried out by the diamond project group (2006), also showed that with age-adjusted incidence of type 1 diabetes varied from 0.1 per 100,000/year in china and venezuela to 40.9 per 100,000/year in finland [13]. this difference between the incidence rate of both studies can be overestimated or underestimated due to various population size of both countries and the difference in multiplier rate (1000 vs 100000) by both studies which impairs the basis of comparison. the population size in the caribbean was approximately 120,000 people as at the time of the study. 4.1 social demographic characteristics of individuals who have t1d the incidence of t1d increased continuously with age until it reached a peak, after which, it 2014 2015 2016 2017 2018 total population 0.49 0.32 0.30 0.47 0.04 male 0.30 0.25 0.15 0.40 0.00 female 0.69 0.40 0.45 0.53 0.08 0.00 0.20 0.40 0.60 0.80 in c id en c e r a te ( p er 1 0 0 0 p er so n s) incidence of diabetes mellitus type 1 from 2014 -2018 adedeji et al.; aji, 4(1): 1-14, 2021; article no.aji.65262 8 fig. 4. peak age of incidence of diabetes mellitus type 1 in 2015 fig. 5. peak age of incidence of diabetes mellitus type 1 in 2016 <5 6_10 11_20 16_20 21-25 26_30 31_35 36_40 41-45 total population 0.34 0.12 0.30 0.20 0.35 0.00 0.88 0.79 0.00 male 0.00 0.23 0.00 0.19 0.23 0.00 1.01 0.77 0.00 female 0.69 0.00 0.64 0.21 0.47 0.00 0.75 0.81 0.00 0.00 0.20 0.40 0.60 0.80 1.00 1.20 in c id en c e r a te ( p er 1 0 0 0 p er so n s) peak age of incidence of diabetes mellitus type 1 in 2015 <5 6_10 11_20 16_20 21-25 26_30 31_35 36_40 41-45 total population 0.00 0.35 0.10 0.20 0.23 0.48 0.38 1.05 0.00 male 0.00 0.23 0.00 0.19 0.00 0.46 0.25 0.26 0.00 female 0.00 0.47 0.21 0.21 0.00 0.49 0.50 1.88 0.00 0.00 0.50 1.00 1.50 2.00 in c id en c e r a te ( p er 1 0 0 0 p er so n s) peak age of incidence of diabetes mellitus type 1 in 2016 adedeji et al.; aji, 4(1): 1-14, 2021; article no.aji.65262 9 fig. 6. peak age of incidence of diabetes mellitus type 1 in 2017 fig. 7. peak age of incidence of diabetes mellitus type 1 in 2018 declined slowly. the incidence of t1d among female in this study peaks occurring at 31 to 35years. this pattern might be related to a type of diabetes termed late onset type 1 diabetes mellitus which dissimilar with other studies which showed that t1d is the major type of diabetes in youth, accounting for ≥85% of all diabetes cases in youth < 20 years of age worldwide [14, 15]. in general, the incidence rate increases from birth and peaks between the ages of 10–14 years during puberty [16] which is in contrast with this study. the increasing incidence of t1d throughout the world is especially marked in young children. registries in europe suggest that recent incident rates of t1d were highest in the youngest age-group (0– 4 years) [16]. incidence rates decline after puberty and appear to stabilize in young adulthood (15–29 years). however, in a similar study which reported that the incidence of t1d in adults is lower than in children, although approximately one fourth of persons with t1d are diagnosed as adults. clinical presentation occurs at all ages and as late as the 9th decade of life. up to 10% of adults initially thought to have type 2 diabetes are found to have antibodies associated with t1d and beta cell destruction in adults appears to occur at a much slower rate than in young t1d cases, often <5 6_10 11_20 16_20 21-25 26_30 31_35 36_40 41-45 total population 0.23 0.00 0.30 0.30 0.57 0.48 0.38 2.09 0.00 male 0.23 0.00 0.19 0.39 0.68 0.00 0.25 2.04 0.00 female 0.23 0.00 0.42 0.21 0.47 0.98 0.50 2.15 0.00 0.00 0.50 1.00 1.50 2.00 2.50 in c id en c e r a te ( p er 1 0 0 0 p er so n s peak age of incidence of diabetes mellitus type 1 in 2017 <5 6_10 11_20 16_20 21-25 26_30 31_35 36_40 41-45 total population 0.11 0.23 0.10 0.00 0.00 0.00 0.00 0.00 0.00 male 0.00 0.23 0.00 0.00 0.00 0.00 0.00 0.00 0.00 female 0.23 0.24 0.21 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.05 0.10 0.15 0.20 0.25 in c id en c e r a te (p er 1 0 0 0 p er so n s) p ea k a g e o f i n c i d en c e o f d i a b et es m el l i t u s t y p e 1 i n 2 0 1 8 adedeji et al.; aji, 4(1): 1-14, 2021; article no.aji.65262 10 table 2. trend analysis by age group and sex variable df χ2 (p-value) 95% confidence interval (pvalue) sex 2014 2015 2016 2017 2018 freq (%) freq (%) freq (%) freq (%) freq (%) total (%) lower limit upper limit male 12(31.6) 10(22.7) 6(26.1) 16(44.4) 0(0.) 44(35.2) female 26(68.4) 15(60.0) 17(73.9) 20(55.6) 3(100.0) 81(64.8) 4 3.783 (0.392) f 0.308 0.476 total 38(100) 25(100) 23(100) 36(100) 3(2.4) 125(100) age ≤5 3(7.9) 3(12.0) 0(0.0) 2(5.6) 1(33.3) 9(7.2) 32 0.088 (0.708) f 0.308 0.476 6-10 2(5.3) 1(4.0) 3(13.0) 0(0.0) 1(33.3) 7(5.6) 11-15 4(10.5) 3(12.0) 1(4.3) 3(8.3) 1(33.3) 12(9.6) 16-20 3(7.9) 2(8.0) 2(8.7) 3(8.3) 0(0.0) 10(8.0) 21-25 4(10.5) 3(12.0) 2(8.7) 5(13.9) 0(0.0) 14(11.2) 26-30 1(2.6) 0(0.0) 4(17.4) 4(11.1) 0(0.0) 9(7.2) 31-35 15(39.5) 7(28.0) 3(13.0) 3(8.3) 0(0.0) 28(22.4) 36-40 4(11.8) 6(24.0) 8(34.8) 16(44.4) 0(0.0) 24(27.2) >40 2(5.3) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 2(1.6) total 38(100) 25(100) 23(100) 36(100) 3(2.4) 125(100) *statistically significant (p<0.05). f (fisher’s exact test) ci = confidence interval) χ2= chi-square test statistics, df= degree of freedom adedeji et al.; aji, 4(1): 1-14, 2021; article no.aji.65262 11 table 3. association between social demographic characteristics variable sex df χ2 (p-value) 95% confidence interval (p-value) sex male female freq (%) freq (%) total (%) lower limit upper limit age ≤5 1(2.3) 8(9.9) 9(7.2) 8 10.632 (0.231) f* 0.158 0.303 6-10 2(4.5) 5(6.2) 7(5.6) 11-15 2(4.5) 10(12.3) 12(9.6) 16-20 6(13.6) 4(4.9) 10(8.0) 21-25 7(15.9) 7(8.6) 14(11.2) 26-30 2(4.5) 7(8.6) 9(7.2) 31-35 8(18.2) 20(24.7) 28(22.4) 36-40 15(34.1) 19(23.5) 34(27.2) >40 1(2.3) 1(1.2) 9(1.6) total 44(100) 81(100) 125(100) *statistically significant (p<0.05). f (fisher’s exact test) ci = confidence interval) χ2= chi-square test statistics, df= degree of freedom fig. 8. case fatality from type 1 diabetes mellitus delaying the need for insulin therapy after diagnosis. individuals diagnosed with autoimmune diabetes when they are adults have been referred to as having latent autoimmune diabetes of adults [17-19]. also, findings from the study confirmed female predominance in the incidence rate of t1d, with ap-proximately 2-fold higher incidence in women than in men. this finding is similar to some studies who highlighted a distinctive pattern with an observation that regions with a high incidence of t1d (populations of european origin) have a male excess, whereas regions with a low incidence (populations of noneuropean origin) report a female excess [20, 13, 21, 22] which is resonate with this study with a baseline of 0.49 per1000 person-years 2014 to 0.04 per 1000 person-years in 2018. 4.2 case-mortality and morbidity from t1d the study findings found increased mortality in people with t1d compared with the general population with a baseline of 3% in 2014 to 33% in 2018. this which that one-third of person that have t1d dies from it. this finding has serious 3% 8% 0% 28% 33% 0% 5% 10% 15% 20% 25% 30% 35% 2014 2015 2016 2017 2018 c as e fa rt al it y r at e % diabetes mellitus type 1 case fatality rate % adedeji et al.; aji, 4(1): 1-14, 2021; article no.aji.65262 12 implication on the healthcare of the country, this finding reveals the gap in the management and treatment of tid. this rate was higher than that found by a norwegian cohort of 1,906 t1d patients diagnosed at <15 years of age between 1973–1982 (46,147 person-years) reported an smr for all-cause mortality of 4.0 with an smr of 20 for ischemic heart disease. acute metabolic complications of t1d were the most common cause of death <30 years of age [23]. however, this difference could be attributed to both rates were unadjusted. the smr is more informative than the crude mortality rate because this compares mortality rates to people without sle of the same age and gender and therefore assesses the excess mortality due to sle. alternatively, it may be due to our cases having milder disease or to the different study methods used. however, our study didn’t compute age specific mortality rate and sex-specific mortality rate due lack of availability of data and poor management health information system in the country as at the time of the study. 5. conclusion sequel to the findings of this study, this study showed that the incidence of autoimmune disease, type 1 diabetes mellitus have decreased in the last decade, whereas the mortality rates of type 1 diabetes mellitus have increased. this finding of increased mortality of t1d suggests that this disease is no longer rare and will have implications for future healthcare planning. 6. recommendations the study highlights the increase in the mortality due to tid in saint vincent and the grenadines. primary prevention of diabetes by lifestyle modification, screening for individuals at high risk and prevention of disease complications in established disease through nonpharmacological and therapeutic interventions are all needed to reduce the high burden of diabetes and associated mortality in saint vincent and the grenadines and similar population. type 1 diabetes treatment is a challenging issue in developing regions, but not so in developed countries where those living with the disease have easier access insulin, glucometer strips and other materials from government subvention or personal savings. non-industrialized countries are faced with inadequate resources, limitation in diagnosis, insulin initiation and storage, family, marital and emotional issues and challenges. since type 1 diabetes affects a few people compared to the general population, it is palpably ignored by governments and policy maker. the socio-economic status in developing regions does not provide the latitude for the required insulin therapy and inextricably-linked monitoring of blood glucose. it is pertinent to spread awareness regarding the metabolic disorder and its sequelae and to undergird government health and healthcare ambient regarding the consequences, pros and cons, in the administration of medicinal drugs for the treatment of diabetes. from key findings from study, it reinforces the need for improved access to insulin and blood glucose meters and test strips in lower income countries and the training of healthcare workers in such countries to recognize and treat this condition. three tiers of care (minimal, intermediate and comprehensive) have been defined by availability of insulin and blood glucose monitoring regimens, requirements for hba1c testing, complications screening, diabetes education, and multidisciplinary care, and it is to be hoped that policy-makers will aspire to attain the highest levels of care possible given the resources available. provide increased training opportunities for health care professionals by establishing collaborative training programs between professional and non-profit health organizations and clinical programs for research in t1d. thus, it is imperative to focus on expansive clinical trials which compare the appropriateness of diverse diabetes medications to provide the guidelines for healthcare providers on which patients to prescribe certain drugs. there tends to be decrease in diabetes complications in certain parts of the world, and the survival and quality of life have improved tremendously, but financial constraints and awareness have restricted ample access to type 1 diabetes prevention, control and treatment, as well as meeting the informed inventiveness and creativity of gadgets, such as the closed-loop systems. the essential management and access to medicinal drugs are more imperative than high-tech systems in developing countries or elsewhere. there is extant optimism with opportunities for the future in unravelling the metabolic and cellular processes in convergence adedeji et al.; aji, 4(1): 1-14, 2021; article no.aji.65262 13 for researchers, clinicians, healthcare providers and policy makers to undertake intensive measures regarding the issues, challenges and presenting opportunities underlying type 1 diabetes and its sequelae which are solvable. consent it is not applicable ethical approval approval was gotten to access medical information of patients from ministry of health and wellness and hospital administrator at milton cato memorial hospital in saint vincent and the grenadines. competing interests authors have declared that no competing interests exist. references 1. world health organization. diabetes fact sheets;2020. available:https://www.who.int/newsroom/fact-sheets/detail/diabetes 2. diaz-valencia pa, bougneres p, vallenon a-j. global epidemiology of type 1 diabetes in young adults and adults: a systemic review. bmc public health. 2015;15:255. available: https://doi.org/10.1186/s12889015-1591-y 3. who. diamond project group. who multinational project for childhood diabetes diabetes care. 1990;13:10621068. available:https://doi.org/10.2337/diacare.1 3.10.1062 4. nelson a. unequal treatment: confronting racial and ethnic disparities in health care. j natl med assoc. 2002;94(8): 666–8. 5. international_diabetes_federation. idf diabetes atlas. in: international diabetes federation. 5th ed; 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[pubmed: 1348306] 22. gale ea, gillespie km. diabetes and gender. diabetol: 2001;44:3–15. 23. skrivarhaug t, bangstad hj, stene lc, sandvik l, hanssen kf, joner g. long-term mortality in a nationwide cohort of childhood-onset type 1 diabetic patients in norway. diabetol. 2006;49:298– 305. _________________________________________________________________________________ © 2021 adedeji et al.; this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: http://www.sdiarticle4.com/review-history/65262 http://creativecommons.org/licenses/by/4.0 _____________________________________________________________________________________________________ *corresponding author: e-mail: meshackkugama@gmail.com; cite as: y.a., onaolapo, mazadu, r.m., baraya, k.y., fasuyi, f.h., irhue, a.e., ahmed, b., hassan, a., shehu, a.a., bala, s.b., kasim, s.n., and kugama, m.a. 2024. “effect of alstonia boonei and morinda lucida on renal histology of wistar rats infected with trypanosoma brucei brucei”. asian journal of immunology 7 (1):292-301. https://doi.org/10.9734/aji/2024/v7i1152. asian journal of immunology volume 7, issue 1, page 292-301, 2024; article no.aji.127386 effect of alstonia boonei and morinda lucida on renal histology of wistar rats infected with trypanosoma brucei brucei onaolapo, y.a. a, mazadu, r.m. a, baraya, k.y. a, fasuyi, f.h. b, irhue, a.e. a, ahmed, b. a, hassan, a. a, shehu, a.a. a, bala, s.b. a, kasim, s.n. c and kugama, m.a. a* a nigerian institute for trypanosomiasis research, federal ministry of science, technology and innovation, nigeria. b national biotechnology development agency, bioresource development center, onipanu, ogbomoso, oyo state, nigeria. c department of applied biology, college of science and technology, kaduna polytechnich, nigeria. authors’ contributions this work was carried out in collaboration among all authors. all authors read and approved the final manuscript. article information doi: https://doi.org/10.9734/aji/2024/v7i1152 open peer review history: this journal follows the advanced open peer review policy. identity of the reviewers, editor(s) and additional reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here: https://www.sdiarticle5.com/review-history/127386 received: 02/10/2024 accepted: 05/12/2024 published: 20/12/2024 original research article https://doi.org/10.9734/aji/2024/v7i1152 https://www.sdiarticle5.com/review-history/127386 onaolapo et al.; asian j. immunol., vol. 7, no. 1, pp. 292-301, 2024; article no.aji.127386 293 abstract animal african trypanosomiasis is a protozoan disease caused by trypanosomes and transmitted through the bite of an infected tsetse fly. the disease has an adverse effect to the economy of the affected areas and drugs used for the treatment of the disease are faced with several challenges ranging from resistance to the parasite to high level of toxicity. our study aimed at evaluating the effect of alstonia boonei and morinda lucida plant extracts on the renal histology of wistar rats infected with trypanosoma brucei brucei. phytochemical screening of the methanol extracts indicated the presence of alkaloids, saponins, tannins, flavonoids, carbohydrates, phenols and steroids/terpenes, while glycosides were not detected. only four of the eight constituents tested for were detected in the chloroform leaf extract of a. boonei, namely, alkaloids, tannins, flavonoids, and carbohydrates. alkaloids, tannins falvonoids, and carbohydratses were detected in both the chloroform and methanol extract of morinda lucida. histological lesions of the infected-untreated rats revealed severe cellular degeneration in the renal cortex of t. brucei infected rats. furthermore, the glomerular tufts were shrunken leaving a large bowman’s. the renal photomicrographs of rat treated with 500mg/kgbwt of alstonia boonei revealed that the renal cortex presented apparently normal histological features of malpighian renal corpuscle containing glomerulus and bowman’s space with slightly shrunken glomerulus. the proximal convoluted tubules had narrow lumina and were lined with cuboidal cells with rounded vesicular basal nuclei. groups treated with 1000mg/kgbwt revealed erythrocytic remains of degenerated renal cells with scanty inflammatory cells in the cortical region of kidney of the wistar rats. however, the glomerulus, the proximal and distal convoluted tubules presented normal histological features. the results indicated that treatment with graded doses of methanol extract of a. boonei elicited varying effects on the visceral organs of the infected animals. it was noted that alstonia boonei ameliorated the effect of the infection on the kidney of the infected animals with the histology sections being comparable with those treated with the reference anti-trypanosomal drug (diminazine aceturate) and the uninfected rats. however, morinda lucida had little or no ameliorative effect on the kidney of wistar rats infected. therefore, we recommend that extracts of astonia boonei should be characterized and active components responsible for the ameliorative effect be detected and elucidated structurally. keywords: trypanosomiasis; parasitaemia; phytochemicals; toxicity. 1. introduction animal trypanosomosis has a serious effect of ravaging both lower and higher ruminants in subsaharan africa and is caused by a protozoan parasite of the genus trypanosoma [1]. the species responsible for this disease known as nagana in africa are trypanosoma vivax, trypanosoma congolense and to a lesser extent trypanosoma brucei brucei. the disease is transmitted through the bite of an infected tsetse fly of the glossina specie. it is implicative in causing severe anaemia, weight loss, reduced productivity, infertility and abortion, with death occurring in some animals during the acute and chronic phase of the infection [2]. in nigeria, studies of ethno medicinal plants used in the traditional management of trypanosomiasis indicated both significant in vitro and in vivo antitrypanosomal activity [3]. the country is richly endowed with indigenous plants, which are used in herbal medicine to cure diseases and heal injuries, some of these plants are used as food or medicine. these indigenous plants exhibit a wide range of biological and pharmacological activities such as anticancer, anti-inflammatory, diuretic, laxative, antispasmodic, antihypertensive, antidiabetic and antimicrobial functions. it is generally assumed that the active medicinal constituents contributing to these protective effects are the phytochemicals, vitamins and minerals. some of these secondary metabolite can act singly or in synergy to bring about healing [4]. currently, homidium chloride, isometamidium and diminazene aceturate are used for the treatment of this disease. each of these drugs has one or more of these challenges: expensive, highly toxic, need parenteral administration and parasites increasing resistance. also, attempt to produce vaccine for the prevention of trypanosomiasis proves abortive because of the parasite changing its antigenic identity. therefore, the need for alternative new molecules that are safe, effective and affordable is urgent [5]. onaolapo et al.; asian j. immunol., vol. 7, no. 1, pp. 292-301, 2024; article no.aji.127386 294 scientific studies revealed that natural products derived from plants offer novel possibilities to obtain new drugs that are active against trypanosomes and investigation of antitrypanosomal activity of traditionally used plants has been a major area of concern [5,6]. there is need for the development of new agents to complement the existing drugs for the treatment of african trypanosomiasis. alstonia boonei and morinda lucida are used as traditional remedy for several infectious and noninfectious diseases including antihelminthic, antiinflammatory, analgesic/pain-killing, antimalaria/antipyretic, antidiabetic (mild hypoglycaemic), antiprotozoan and antibiotic properties [7]. these justify the need to explore these plants as potent drugs for trypanosomiasis. 2. materials and methods 2.1 plants materials the plant materials were harvested in the morning in, zaria local government area of kaduna state. it was authenticated in the herbarium, department of biological science, kaduna state university and given the voucher number (a765 and q567) for alstonia boonei and morinda locida respectively. it was air dried at room temperature, pulverized in a mortal using pestle to obtained fine powder. 2.2 experimental animals sixty (60) wistar rats were used for this study. the animals were purchased from the animal house of nigerian institute for trypanomiasis research, kaduna. they were kept in a standard rat cage, fed with standard pellet diet with water ad libitum for thirty (30) days. 2.3 infection of animals with trypanosomes the parasite trypanosoma brucei brucei was obtained from nigerian institute for trypanosomiasis research, kaduna. the animals were inoculated with 1000 parasite per meal of blood intraperitoneally. the number of parasite was compared with harbert and lumsden rapid matching method [8]. 2.4 extraction from plant materials extraction was done using soxhlet apparatus. one thousand grams (1000g) of the powdered plant was divided into five hundred (500g) each. 2,000ml of each of methanol and chloroform were used for the extraction of each of the powdered plant. the solvent was recovered and concentrated in vacuoe to obtain the extract. 2.5 phytochemical analysis of the plant parts the phytochemical analysis was carried out according to the method of sofowora. the presence of tannins, saponins, flavonoids, glycosides, anthraquinones, carbohydrate, phenols, terpens and alkaloids was tested qualitatively [9]. 2.6 histopathological examination of harvested organs the kidney specimens from each rat was stored immediately in 10%v/v formalin in normal saline after gross histological examination and dehydrated using increasing concentrations of isopropyl alcohol (80e100%). paraffin sections at 5mm thickness was made from the paraffin embedded organs using a leica rotary microtome (bright b5143 huntington, england). this was followed by routine staining with hematoxylin and eosin which involved the process of deparaffinization, hydration, staining, rinsing and clearing in xylene. slides was viewed under light microscope with photomicrographs taken with a leica dm750 camera microscope (400 x) [10]. histopathological lesions were scored using semi-quantitative approach as follows: 0 for normal, 1 (1%e30%) for mild, 2 (31%e70%) for moderate, and 3 (>70%) for severe. 2.7 statistical analysis of data values of the data obtained from the study was summarized and expressed data analysis was performed using statistical package for social science (spss) 2018 version 23.0. 3. results 3.1 phytochemical constituents screening of extracts of alstonia boonei and morinda lucida using methanol and chloroform as solvents the results of the phytochemical constituents and screening of the methanolic leaf extract of alstonia boonei indicated the presence of seven onaolapo et al.; asian j. immunol., vol. 7, no. 1, pp. 292-301, 2024; article no.aji.127386 295 constituents, namely, alkaloids, saponins, tannins, flavonoids, carbohydrates, phenols and steroids/terpenes, while glycosides were not detected. only four of the eight constituents tested for were detected in the chloroform leaf extract of a. boonei, namely, alkaloids, tannins, flavonoids, and carbohydrates. four phytoconstituents namely, alkaloids, tannins, flavonoids, and carbohydrates were detected in the methanol extract of morinda lucida. in the chloroform extract of m. lucida, only three of the eight phytoconstituents were detected namely, alkaloids, tannins, and flavonoids; saponins, glycosides, carbohydrates, phenols, and steroids were not detected. all three constituents that were present in the chloroform extract of m. lucida were detected in relatively high quantities. glycosides were not detected in all the solvents leaf extracts of the plants, while carbohydrates were detected, albeit in relatively minute quantities, in the methanol and chloroform extracts of a. boonei and in the methanol extract of m. lucida. phenols as well as terpenes were detected in the methanol extract of a. boonei; none was detected in the chloroform extract of the plant nor in either the methanol or chloroform extracts of m. lucida (table 1). table 1. phytochemical screening of the methanol and chloroform leaf extracts of alstonia boonei and morinda lucida phytochemical constituents abme abce mlme mlce alkaloids +++ +++ +++ +++ +++ +++ +++ +++ saponins +++ ------ tannins +++ +++ +++ +++ flavonoids +++ +++ +++ +++ glycosides -------- carbohydrates + + + -- phenolic compounds +++ ------ terpenes ++ ------ abme: alstonia boonei methanol extract; abce: alstonia boonei chloroform extract; mlme: morinda lucida methanol extract; mlce: morinda lucida chloroform extract +++: highly present; ++: moderately present; +: present; ---: undetected plate 1. photomicrographs of renal section from t. brucei infected rat administered 500 mg/kgb.wt of methanol extract of a. boonei (group a). the renal cortex presents apparently normal histological features of malpighian renal corpuscle containing glomerulus (g) and bowman’s space (arrowhead) with slight shrunken glomerulus. the proximal convoluted tubules (p) have narrow lumina and are lined with cuboidal cells with rounded vesicular basal nuclei. the distal convoluted tubules (d) have wider lumina and are lined with cuboidal cells. h&e x25 onaolapo et al.; asian j. immunol., vol. 7, no. 1, pp. 292-301, 2024; article no.aji.127386 296 plate 2. photomicrographs of renal section of t. brucei brucei infected rats administered 1000 mg/kgb.wt of methanol extract of a. boonei (group b). the renal cortex shows what appeared to be erythrocytic (circle) remains of degenerated renal cells with scanty inflammatory cells (arrowhead). the glomerulus (g), proximal (p) and the distal convoluted tubules (d) present normal histological features. h&e x250 plate 3. photomicrographs of renal section t. brucei brucei infected rats administered 500 mg/kgb.wt of chloroform extract of m. lucida (group c). the renal cortex shows severe atrophy of the glomerular tuft (g), with nearly all the tubular cells showing degeneration (arrow head). note the focal aggregation of mononuclear cells around one of the glomerulus (circle). h&e x250 3.2 histopathological studies of kidney organs of trypanosoma brucei brucei infected wistar rats treated with leaf extracts of alstonia boonei boonei and morinda lucida effect of 500 mg/kgb.wt of the methanol extract of alstonia boonei on kidney histology of trypanosoma brucei brucei infected rats: the renal photomicrographs of wistar rat in group a revealed that the renal cortex presented apparently normal histological features of malpighian renal corpuscle containing glomerulus and bowman’s space with slightly shrunken glomerulus. the proximal convoluted tubules had narrow lumina and were lined with cuboidal cells with rounded vesicular basal nuclei. the distal convoluted tubules had wider lumina and were also lined with cuboidal cells (plate 1). effect of 1000 mg/kgb.wt of the methanol leaf extract of alstonia boonei on kidney histology of trypanosoma brucei brucei infected wistar rats: erythrocytic remains of onaolapo et al.; asian j. immunol., vol. 7, no. 1, pp. 292-301, 2024; article no.aji.127386 297 degenerated renal cells with scanty inflammatory cells were seen in the cortical region of kidney in the rats in group b. however, the glomerulus, and the proximal and distal convoluted tubules presented normal histological features (plate 2). effect of 500 mg/kgb.wt of the chloroform extract of m. lucida on kidney histology of t. brucei brucei infected rats: the renal cortex of the rat in group c showed severe atrophy of the glomerular tuft; in addition, almost all the tubular cells showed degeneration. there was also focal aggregation of mononuclear cells around one of the glomeruli (plate 3). effect of 1000 mg/kgb.wt of the chloroform extract of morinda lucida on kidney histology of trypanosoma brucei brucei infected wistar rats: renal section from t. brucei brucei infected rats treated with 1000 mg/kgb.wt of chloroform extract of m. lucida (group d) showed features of cellular degeneration and shrunken glomerular tuft, as well as hemorrhage (plate 4). plate 4. photomicrographs of renal sections t. brucei brucei infected rats administered 1000 mg/kgb.wt of chloroform extract of m. lucida (group d). the section is hemorrhagic (circle), showing features of cellular degeneration and shrunken glomerular tuft (g). h&e x250 plate 5. photomicrographs of renal section t. brucei brucei infected rats administered diminazine 3.5 mg/kgb.wt (group e). the renal cortex shows apparently normal renal corpuscle of glomerulus (g), proximal and distal convoluted tubules (p and d) with few focal aggregated mononuclear infiltrated cells (circle). h&e x250 onaolapo et al.; asian j. immunol., vol. 7, no. 1, pp. 292-301, 2024; article no.aji.127386 298 effect of diminazine aceturate (3.5 mg/kgb.wt) on kidney histology of t. brucei brucei infected rats: the renal cortex of rats in group e showed apparently normal renal corpuscle of glomerulus, proximal and distal convoluted tubules, with few focal aggregated mononuclear infiltrated cells (plate 5). kidney histology of untreated trypanosoma brucei brucei infected rats: severe cellular degeneration was seen in the renal cortex of t. brucei infected rats that received no treatment (group f). furthermore, the glomerular tufts were shrunken leaving a large bowman’s space (plate 6). kidney histology of uninfected rat: the renal cortex of uninfected rat (group g) revealed apparently normal glomerulus, proximal and distal tubules, with few infiltrated inflammatory cells (plate 7). plate 6. photomicrographs of renal section of untreated t. brucei brucei infected rats (group f) the renal cortex is showing severe cellular degeneration and shrunken glomerular tufts (g) leaving a large bowman’s space (bs). h&e x250 effect on liver histology: plate 7. photomicrographs of renal sections of uninfected-untreated (group g). the renal cortex shows renal corpuscle with apparently normal glomerulus (g), proximal and distal tubules (p and d) with few infiltrated inflammatory cells (circles). h&e x250 onaolapo et al.; asian j. immunol., vol. 7, no. 1, pp. 292-301, 2024; article no.aji.127386 299 4. discussion the result from the phytochemical screening obtained in this study revealed the presence of bioactive components in the aqueous and chloroform extracts of the leaves of a. boonei which is in agreement with the reports of [11] who noted the presence of phytochemicals in the water and methanol leaf extracts of a. boonei. [12] had also reported the presence of some bioactive agents in the methanol stem bark and leaf extracts of the plant extracts. these reports indicated that a. boonei is an excellent source of chemical moieties that could find important medicinal and pharmacological applications. similarly, extracts of morinda lucida showed an array of phytoconstituents including alkaloids, tannins, flavonoids, and carbohydrates. this also agreed with the reports of [13,14] who had previously reported the presence of bioactive agents in solvent extracts of the stem bark and root of m. lucida (saponins, tannins, anthracenes, flavonoids, alkaloids, carbohydrate and protein). the present results indicated that the class of phytochemicals varied between the two plants (a. boonei and m. lucida) and between the solvent used for extracting a particular plant. according to [15], the metabolic profiles of medicinal plants are mainly controlled by genetic factors, adding that variations in the levels of phytochemicals may represent long-term ecological and evolutionary interactions. thus, the genetic differences between the two plants. the present results indicated that t. brucei brucei infection in rats is characterized by several histopathological derangements of visceral organs (liver) which may impair their functionality and result in death of untreated animals. the histopathological abnormalities observed in the kidney of untreated t. brucei brucei infected rats were consistent with the reports of other authors who had reported the effect of trypanosoma infections in animals. also, [16] had noted alterations in kidney, liver, lungs and spleen of mice infected with t. brucei. in the study, the kidneys were characterized by congested intertubular spaces, and the lungs were seen with congestions of the blood vessels, interstitial expansion and emphysema in infected mice. again, [17] who assessed the histological changes associated with infection with t. evansi, a phylogenetic close relative of t. brucei, noted marked changes in the visceral organs including cellular infiltration of the liver, myocardial necrosis, tubular necrosis of the nephrons and destruction of the bowman’s capsule. the changes in the visceral organs seen in trypanosome have been attributed to any one or a combination of the following factors: mechanical damage, increased vascular permeability, deranged immunological mechanisms, and/or the activities of trypanosome toxins [17]. the results of the histology tissue indicated that treatment with graded doses of either the methanol extract of a. boonei or the chloroform extract of m. lucida elicited varying effects on the kidneys of the infected animals. it was noted that alstonia boonei ameliorated the effect of the infection on the kidney of the infected animals with the histology sections being comparable with those treated with the reference antitrypanosomal drug (diminazine aceturate) and the uninfected rats [18]. the m. lucida extract was shown to exhibit lower tissue protective effect against t. brucei brucei infection when compared with a. boonei: in the morinda lucida treated animals, the kidneys revealed gross pathologies that were also seen in the untreated rats. from the aforementioned postulations by [17] on the probable mechanisms of tissue damage seen with trypanosome infection, it may be inferred that a. boonei act to prevent or ameliorate the tissue damages by inhibiting several of these mechanisms. the results of the phytochemical screening of the methanol extract of a. boonei and the chloroform extract of m. lucida indicated that a. boonei contained phenols, saponins, steroids, and terpenes, in addition to alkaloids, tannins, and flavonoids which were detected in m. lucida [19,20]. these differences might lend credence to the renoprotective effects observed in the a. boonei treated rats. 5. conclusion our findings revealed that crude extracts of alstonia boonei and morinda lucida contain variety of phytochemicals which have documented antimicrobial effects. the crude extract of alstonia boonei demonstrated ameliorative in vivo effects on some histological lessions of wistar rats infected with trypanosoma brucei brueci. consent it is not applicable. ethical approval ethical clearance for the use of laboratory animals was obtained from kaduna state ministry of agriculture and forestry, kaduna. onaolapo et al.; asian j. immunol., vol. 7, no. 1, pp. 292-301, 2024; article no.aji.127386 300 disclaimer (artificial intelligence) author(s) hereby declare that no generative ai technologies such as large language models (chatgpt, copilot, etc.) and text-to-image generators have been used during the writing or editing of this manuscript. competing interests authors have declared that no competing interests exist. references 1. abd el-ghani mm. traditional medicinal plants of nigeria: an overview. agriculture and biology journal of north america. 2016;7(5):220-247. 2. adeyemi na, wada ya, oniye sj, hussaini m, muhammad ad. effects of trypanosoma brucei brucei infection on haematological profile, testosterone level, and oxidative stress status in wistar rat (rattus norvegicus). bima journal of science and technology. 2024;8(1):30-38. 3. adotey jpk, adukpo ge, opoku boahen y, armah fa. a review of the ethnobotany and pharmacological importance of alstonia boonei de wild (apocynaceae). international scholarly research notices. 2012;(1):58-71. 4. ahmadi h, fatahi r, zamani z, shokrpour m, sheikh-assadi m, poczai p. rna-seq analysis reveals narrow differential gene expression in mep and mva pathways responsible for phytochemical divergence in extreme genotypes of thymus daenensis celak. bmc genomics. 2024;25(1):237. 5. akinseye or, morayo ae, olawumi as. qualitative and quantitative evaluation of the phytochemicals in dry, wet and oil extracts of the leaf of morinda lucida. j. biol. agric. healthcare. 2017;7(7):22-25. 6. bashir l, shittu ok, sani s, busari mb, adeniyi ka. african natural products with potential anti-trypanosomal properties: a review. international journal of biochemistry research and review. 2015;7(2):45-79. 7. boakye yd, agana ta, oteng-amankwah ea, boamah ve, agyare c. evidencebased review of medicinal plants for the management of onchocerciasis. natural products in vector-borne disease management. 2023;27-49. 8. buguet a, mpanzou g, bentivoglio m. human african trypanosomiasis: a highly neglected neurological disease. neglected tropical diseases and conditions of the nervous system. 2014;165-181. 9. chukwudi ic, omemgboji oc, anene bm. efficacy of isometamidiumin combination with verapamil, chlorpromazine or sodiumethylenediaminetetra-acetic acid in treatment of experimental diminazene aceturate-resistant strain of trypanosoma brucei brucei infection in rats. sahel journal of veterinary sciences. 2020;17(4):37-45. 10. dhawan d, gupta j. research article comparison of different solvents for phytochemical extraction potential from datura metel plant leaves. int j biol chem. 2017;11(1):17-22. 11. feldman at, wolfe d. tissue processing and hematoxylin and eosin staining. histopathology: methods and protocols. 2014;31-43. 12. ferreira da vinha a, barreira s, castro a, machado m. comparison between the phytochemical and antioxidant properties of plants used in plant infusions for medicinal purposes. journal of agricultural science. 2013;5(11):11-19. 13. herbert wj, lumsden whr. rapid estimation of trypanosome parasitaemias by matching. transactions of the royal society of tropical medicine and hygiene. 1976;70(4):282-282. 14. hussain s, sultana t, sultana s, hussain b, mahboob s, al-ghanim ka, riaz mn. seasonal monitoring of river through heavy metal bioaccumulation and histopathological alterations in selected fish organs. journal of king saud university-science. 2021;33(8):101626. 15. maichomo mw, orenge co, gamba do. introduction of african animal trypanosomosis (aat)/nagana. in combating and controlling nagana and tick-borne diseases in livestock. igi global. 2021;1-23 16. ogbuanu cc, nwagu ln, ezeh cn, achara ni, onwuatuegwu jtc. proximate, nutraceutical composition and antimicrobial activities of uvaria chamea (udagu) seeds and oil. newport international journal of scientific and experimental sciences; 2023. 17. omoya f, oyebola tf. antiplasmodial activity of stem bark and leaves of alstonia onaolapo et al.; asian j. immunol., vol. 7, no. 1, pp. 292-301, 2024; article no.aji.127386 301 boonei (de wild). journal of microbiology and experimentation. 2019;7(5):241-245. 18. shuaibu za, lawal ai, samuel fu, ereke so, sani na, ibrahim ma, fanaiye go. histopathological changes associated with trypanosoma congolense infection in albino rats; 2018. 19. sofowora a. research on medicinal plants and traditional medicine in africa. the journal of alternative and complementary medicine. 1996;2(3):365-372. 20. ungogo ma, ebiloma gu, ichoron n, igoli jo, de koning hp, balogun eo. a review of the antimalarial, antitrypanosomal, and antileishmanial activities of natural compounds isolated from nigerian flora. frontiers in chemistry. 2020;8:617-648. disclaimer/publisher’s note: the statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of the publisher and/or the editor(s). this publisher and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. _________________________________________________________________________________ © copyright (2024): author(s). the licensee is the journal publisher. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: https://www.sdiarticle5.com/review-history/127386 https://www.sdiarticle5.com/review-history/127386 _____________________________________________________________________________________________________ ++ professor; *corresponding author: e-mail: docteurlakrafiyasser@gmail.com, docteuryasser273@hotmail.com; cite as: yassir, lakrafi, benhayoun basma, cherfouf rihabe, bouroyene el mehdi, barakat leila, benzakour merieme, khadija echchilali, mina moudatir, and hassan el kabli. 2024. “a 6-year statistical study of systemic sclerosis in the region of casablanca-settat, morocco”. asian journal of immunology 7 (1):159-74. https://journalaji.com/index.php/aji/article/view/140. asian journal of immunology volume 7, issue 1, page 159-174, 2024; article no.aji.122682 a 6-year statistical study of systemic sclerosis in the region of casablancasettat, morocco lakrafi yassir a*, benhayoun basma b, cherfouf rihabe b, bouroyene el mehdi a, barakat leila b++, benzakour merieme b++, khadija echchilali b++, mina moudatir b++ and hassan el kabli b++ a internal medicine department and geriatrics unit of the ibn rochd university hospital center in casablanca, morocco. b internal medicine department and geriatrics unit p38 of the ibn rochd university hospital center in casablanca, morocco. authors’ contributions this work was carried out in collaboration among all authors. all authors read and approved the final manuscript. article information doi: https://doi.org/10.9734/aji/2024/v7i1140 open peer review history: this journal follows the advanced open peer review policy. identity of the reviewers, editor(s) and additional reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here: https://www.sdiarticle5.com/review-history/122682 received: 03/07/2024 accepted: 06/09/2024 published: 16/09/2024 abstract systemic sclerosis is a non-organ-specific autoimmune disease of the connective tissue, characterized by excessive production of collagen leading to fibrosis of the skin as well as organs such as the lung, it is responsible for aesthetic discomfort, an alteration of the functional prognosis and potentially life-threatening due to damage to the heart, lungs and kidneys. we, therefore, original research article mailto:docteurlakrafiyasser@gmail.com https://doi.org/10.9734/aji/2024/v7i1140 https://www.sdiarticle5.com/review-history/122682 yassir et al.; asian j. immunol., vol. 7, no. 1, pp. 159-174, 2024; article no.aji.122682 160 decided to carry out a retrospective study extending over 6 years at the internal medicine department and geriatrics unit of the ibn rochd university hospital center in casablanca that concerned the medical files of 2164, in order to see if systemic scleroderma in our region presented any particularities. we noted that scleroderma was relatively rare in our context with only 42 patients in whom the diagnosis was made in 06 years with a female predominance (the sex ratio in our studies was 4.25 women for 1 man), we noticed that the provinces the most concerned are casablanca, el jadida and settat with a predominance in urban areas, that the family history of scleroderma or autoimmune disease is weakly present in our context, the main profession at risk found in our context is automobile sheet metal work and it concerned 3 male patients (7.14%), exposure to tobacco was however present in 11 patients or 26.9%. we also noted that the first symptoms were diverse (arthralgia, myalgia, raynaud's phenomenon) and often appeared between 1 and 5 years (in 59.6% of patients) before the diagnosis to be made. the clinical picture is classic with varied skin involvement in 100% of our patients with abnormalities on capillaroscopy in 95.2% of our patients, orofacial involvement in 54.8% of patients, muscular involvement in 57.1%, joint damage in 64.3%, bone damage in 23.8%, lung damage in 61.9%, heart damage in 23.8%, kidney damage in 26.2%, digestive damage in 54.8%, and urogenital involvement in 33.3% of patients. 7 patients died (16.66%), 3 patients (7.14%) had progression of the disease leading to complications, and 20 patients (47.61%) reported improvement while the disease remained stationary in 11 patients (26.19%). keywords: systemic scleroderma; autoimmune disease; connective tissue; microcirculation. 1. introduction systemic scleroderma is a non-organ-specific autoimmune disease of the connective tissue characterized by damage to the microcirculation leading to raynaud’s phenomenon and excessive collagen production resulting in fibrosis of various organs. it is a rare condition with a poorly known prevalence and predominantly affecting women, with a femalemale ratio ranging from 3:1 to 8:1. this condition not only impacts aesthetic and functional aspects but also vital due to the potential involvement of the lungs, heart and kidneys [1]. our work consists of a retrospective study reporting the different particularities of scleroderma in the casablanca-settat region. 2. materials and methods our work consists of a retrospective study over 6 years extending from january 1 of 2018, to december 31 of 2023, carried out in the internal medicine and geriatrics unit of the ibn rochd university hospital center in casablanca and which drains patients from the region of casablanca-settat and which concerned the medical files of 2164 patient with 42 patients in whom scleroderma was newly diagnosed. the objective of our study is to determine the different epidemiological particularities of scleroderma, the different visceral damage, the response to different treatments and the evolution of this condition, as well as the impact of this condition on the fertility of patients. for this we used the following operating sheet created using google forms. 3. results during the period of 06 years from january 1, 2018 to december 31, 2023 we welcomed 2164 patients to the internal medicine and geriatrics unit of the ibn rochd university hospital center in casablanca for different reasons and the diagnosis of systemic scleroderma was retained according to the acr/eular 2013 criteria in 42 patients, which represents 1.94% of all admissions to the service during this period, of which 8 were men and 34 were women. the skin manifestations observed in patients included raynaud's phenomenon in 95.2%, pulp ulceration in 35.7%, pulp scarring in 61.9%, sausage-like fingers in 54.8%, skin sclerosis not extending beyond the mcp joints in 45.2% and extending beyond the mcp joints in 45.2%, thinning of the lips and nose in 40.5%, loss of forehead wrinkles in 23.8%, exaggerated perioral folds in 14.3%, skin hypopigmentation in 11.9%, telangiectasia in 28.6%, and calcinosis in 4.8%. 4. discussion scleroderma is a non-specific autoimmune disease of connective tissue, which makes it a systemic disease. it is characterized by damage yassir et al.; asian j. immunol., vol. 7, no. 1, pp. 159-174, 2024; article no.aji.122682 161 to the microcirculation, the most classic of which is raynaud's phenomenon, as well as excessive production of collagen leading to organ fibrosis, the most common of which is pulmonary fibrosis. it has an aesthetic and functional prognosis and can be life-threatening due to damage to the kidneys, lungs and heart. the pathophysiological mechanism is partially understood, and the etiology is unknown. [1] fig. 1. distribution of patients according to gender fig. 2. distribution of patients according to rural/urban origin 71.4% (n=30) were of urban origin, while 28.6% (n=12) were of rural origin. fig. 3. distribution of patients according to province of origin 35.7% (n=15) were from casablanca, 28.6% (n=12) from el jadida, 19% (n=8) from settat, 4.8% (n=2) from each of mohammedia, médiouna, and sidi bennour, and 2.4% (n=1) from berrechid 35.70% 28.60% 19% 4.80% 4.80% 4.80% 2.40% distribution of patients according to province of origin casablanca el jadida settat mohammedia mediouna sidi bennour berrechid yassir et al.; asian j. immunol., vol. 7, no. 1, pp. 159-174, 2024; article no.aji.122682 162 fig. 4. percentage of patients with a family history of systemic sclerosis 85.7% (n=36) had no family history of systemic scleroderma, 9.5% (n=4) were unaware if they had any, and 4.8% (n=2) reported a family history of scleroderma fig. 5. percentage of patients with a family history of autoimmune disease other than scleroderma 76.2% (n=32) had no family history of autoimmune disease, 14.3% (n=6) had at least one family member with an autoimmune disease, and 9.5% (n=4) were unaware if they had a family history of autoimmune disease fig. 6. distribution of patients according to age of onset of first symptoms the age of onset of the first symptoms was below 15 years in 7.1% of patients, between 16 and 35 years in 28.5% of cases, between 36 and 55 years in 40.5% of cases, between 56 and 65 years in 14.3%, and over 65 years in 9.5% of cases 4.80% 85.70% 9.50% percentage of patients with a family history of systemic sclerosis yes no unknown yassir et al.; asian j. immunol., vol. 7, no. 1, pp. 159-174, 2024; article no.aji.122682 163 fig. 7. summary diagram of the initial symptoms of systemic scleroderma in our patients the initial symptoms included raynaud's phenomenon in 83.3% of cases, skin sclerosis in 42.9%, dyspnea in 23.8%, myalgia in 9.5%, and arthralgia in 35.7% fig. 8. distribution of patients according to age of diagnosis the age at diagnosis was between 16 and 35 years in 19.1% of cases, between 36 and 55 years in 52.3%, between 56 and 65 years in 16.7%, and over 65 years in 11.9% table 1. results of the immunological assessment in our patients immunological assessment number of patients percentage immunological assessment not done 1 2.4% negative immunological test 3 7.1% positive anti-nuclear antibodies 38 90.5% anti-topo-isomerase/scl 70 antibodies positive 12 31.57% anti-rna polymerase iii antibodies positive 7 18.42% positive anti-centromere antibodies 11 28.94% anti-centromere antibodies + positive anti-scl 70 antibodies 3 7.89% anti-centromere antibodies + positive anti-rna-olymerase iii antibodies 3 7.89% anti scl 70 antibodies + anti rna-polymerase iii antibodies positive 2 5.26% 19.10% 52.30% 16.70% 11.9% distribution of patients according to age of diagnosis 16-35 years 36-55 years 56-65 years over 65 years yassir et al.; asian j. immunol., vol. 7, no. 1, pp. 159-174, 2024; article no.aji.122682 164 fig. 9. summary diagram of the different skin manifestations found in our patients fig. 10. summary diagram of the presence or absence of abnormalities on capillaroscopy 95.2% of patients had abnormalities in periungual capillaroscopy, 2.4% had a normal capillaroscopy, and 2.4% did not undergo the procedure fig. 11.summary diagram of rodnan score at diagnosis 57.1% of patients experienced myalgia, and 4.8% had actual myositis 95.20% 2.40% 2.40% abnormalities on capillaroscopy yes no not done yassir et al.; asian j. immunol., vol. 7, no. 1, pp. 159-174, 2024; article no.aji.122682 165 fig. 12. summary diagram of muscle damage 61.9% of patients experienced inflammatory arthralgia, and 4.8% had true arthritis fig. 13. summary diagram of joint damage 19% of patients had osteoporosis, and 4.8% had acro-osteolysis fig. 14. summary diagram of bone damage yassir et al.; asian j. immunol., vol. 7, no. 1, pp. 159-174, 2024; article no.aji.122682 166 fig. 15. summary diagram of dyspnea present fig. 16. 6-minute walk test result . fig. 17. summary diagram of thoracic scanner results 42.9% of patients had diffuse interstitial lung disease on chest ct, 33.3% had a normal scan, 19% had pulmonary fibrosis, 2.4% had chronic obstructive pulmonary disease, and 2.4% had a posterior-basal parenchymal infiltrate 9.50% 38.10% 7.10% 1.2 38.10% summary diagram of dyspnea stage i stage ii stage iii stage iv no dyspnea 33.30% 42.90% 19% 2.40% summary diagram of thoracic scanner results normal scan diffuse interstitial lung disease pulmonary fibrosis chronic obstructive pulminory disease posterrior basal parenchymal infiltrate yassir et al.; asian j. immunol., vol. 7, no. 1, pp. 159-174, 2024; article no.aji.122682 167 fig. 18. summary diagram of the results of the lung functional exploration 66.6% of patients underwent pulmonary function tests. among them, 26.2% had inconclusive results, 19% had normal results, 19% had a restrictive syndrome, and 2.4% had a mixed ventilatory disorder fig. 19. summary diagram of cardiac damage 7.1% of patients had conduction disturbances on ekg, 7.1% had left heart failure, 19% had pulmonary hypertension on echocardiography, 2.4% had mitral insufficiency with tricuspid insufficiency and dilation of the inferior vena cava, and 7.1% had pulmonary arterial hypertension detected by catheterization fig. 20. summary diagram of renal damage 23.8% of patients had hypertension, 11.9% had renal insufficiency, and 2.4% had proteinuria 26.20% 19% 19% 1.2 results of the lung functional exploration inconclusive results normal results restrictive syndrome mixed ventilatory disorder yassir et al.; asian j. immunol., vol. 7, no. 1, pp. 159-174, 2024; article no.aji.122682 168 fig. 21. summary diagram of digestive damage 50% of patients had gastroesophageal reflux, 21.4% had dyspepsia, 19% had constipation, 11.9% had malabsorption, 2.4% had dysphagia with solids, 2.4% had dysphagia with liquid diarrhea, and 2.4% had abnormalities on esogastroduodenoscopy. fig. 22. summary diagram of the different treatments adopted fig. 23. summary diagram of the different modes of evolution of the pathology in our patients 47.6% of patients reported progression of the disease, 26.2% had stable disease, 16.7% died, 7.1% reported complications, and 2.4% were lost to follow-up 47.60% 26.20% 7.10% 16.70% evolution of the disease progression of the disease stable disease complication death lost to follow up yassir et al.; asian j. immunol., vol. 7, no. 1, pp. 159-174, 2024; article no.aji.122682 169 fig. 24. summary diagram of the different causes of death 4 patients died from cardiorespiratory failure, 1 patient died from cardiac arrest due to septic shock, and 2 patients died from cardiac decompensation we therefore carried out a retrospective study spanning 06 years from january 1, 2018 to december 31, 2023 in the internal medicine department and geriatrics unit of the ibn rochd university hospital center in casablanca during which we highlighted the following particularities: • on the epidemiological level: this is a rare condition in our context, of 2164 patients hospitalized in our department, the diagnosis of systemic sclerosis was only retained in 42 patients or 1.94%. we noticed a strong female predominance with 34 female patients for 8 male patients, i.e. a sex ratio of 4.25 women for 1 man, which is consistent with data from the french literature [1]. 71.4% of our patients were of urban origin compared to 28.6% who were of rural origin, dominated by the province of casablanca from which 35.7% originated, followed by the province of el jadida from with 28.6% than the province of settat in 3rd position from which 19% of patients came, totaling 83.3% of patients. several studies have highlighted the physiopathological role of certain organic solvents such as aliphatic hydrocarbons, halogenated aliphatic hydrocarbons, white spirits or naphtha solvents; toluene, xylenes, trichloroethylene and aromatic hydrocarbons, which made it possible to identify so-called risky professions such as car painting, sheet metal work and textiles [2]. during our study only 3 patients, all male, i.e. 7.1% of patients had occupations known to be at risk and worked in automobile sheet metal, while 71.4% of our patients were without a profession and finally 21.4% had occupations not known to be at risk. the genetic component has been suggested in the bibliography about the pathophysiology of systemic scleroderma, evoking some familial cases of scleroderma or even an area of familial autoimmunity although specific genomic studies attempt to precisely identify the genomic actors. incriminated are still ongoing. [3,4] we only found a family history of systemic sclerosis in 4.8% of our patients while a family history of autoimmune diseases other than systemic sclerosis was found in 14.3% of type patients including systemic lupus erythematosus, rheumatoid arthritis and immunological thrombocytopenia. it has been noted in the french literature that the peak frequency of scleroderma is between the age of 45 and 64 years [1], but in our study the peak frequency was between 36 and 55 years in 52.3% of our patients, however we noted that the onset of symptoms was way before with a delay between the appearance of the first symptoms and the diagnosis varying between 2 to 5 years in 42.9% of patients, greater than 5 years in 21.4% of patients while it was equal or less than to 1 year in 35.9% of patients. • on the clinical level: the first clinical symptoms related to the disease were essentially cutaneous (made of raynaud's yassir et al.; asian j. immunol., vol. 7, no. 1, pp. 159-174, 2024; article no.aji.122682 170 phenomenon and cutaneous sclerosis found respectively in 83.3% and 42.9% of patients), joint (type of arthralgia found in 33.7% of patients), respiratory (type of dyspnea found in 23.8% of patients) and muscular (type of myalgia found in 9.5% of patients). it should be noted that patients in whom the pathology first appeared in the respiratory system were able to be diagnosed quickly in less than a year, while patients with joint, muscular and skin involvement could’nt be diagnosed quickly with a delay between the appearance of symptoms and the making of the diagnosis which can exceed 5 years, which can be explained by the severity of the condition with a rapid manifestation of sufficient classification criteria when the pulmonary involvement is inaugural, while the diagnostic delay in patients showing isolated cutaneous, muscular or joint symptoms does not allow the diagnosis to be made quickly, especially since patients in the moroccan context only consult late after prolonged and often random selfmedication. at the time of diagnosis of systemic sclerosis in our patients, the clinical picture was dominated by skin involvement with raynaud's phenomenon found in 95.2% of patients, followed by pulp scars found in 61.9% of patients, then curled fingers found in 54.8%, of cutaneous sclerosis not exceeding the mcp in 45.2% and exceeding the mcp in 45.2%, then refinement of the lips and nose found in 40.5% of patients, then pulpal ulcerations found in 35.7%, telangiectasias in 28.6%. the rodnan scores at the time of diagnosis varied between 0 for scleroderma sin scleroderma and 40 in patients with extensive sclerosis, with a majority rodnan score at 13. orofacial involvement was present in 54.8% of patients with limitation of the mouth opening in these and calcinosis of the facial in only 2 of them. muscular damage mainly due to myalgia was found in 57.1% of patients, only 2 of whom presented true myositis with a clinical muscle deficit, biological rhabdomyolysis and a myogenic trace on the electro-neuro-myogram. joint damage was found in 61.9%, mainly suffering from arthralgia, of which only 2 patients presented true arthritis. bone damage was found in 23.8% of patients with 2 patients having presented acro-osteolysis and 8 having presented osteoporosis (a frequent manifestation of scleroderma [1]); in a study conducted jointly by the rheumatology department of pasteur hospital in nice and the nephrology department of chu nîmes on 102 patients, the incidence of osteoporosis was 35%. in contrast, another study conducted at the internal medicine department of chu tunis in tunis found an incidence of acro-osteolysis at 37.03% [5,6]. • for visceral damage dyspnea was found in 61.9% of patients at different stages, with a 6-minute walk test returning pathological in 47.6%. parenchymal pulmonary involvement was found in 64.3% of patients, respiratory functional exploration found a restrictive syndrome in 19% of patients, and evaluation of the release of carbon monoxide found a decrease in it in 21.4% of patients, none of our patients benefited from bronchoscopy with bronchoalveolar washing, a controversial practice but which has demonstrated its effectiveness in early detection [7], other schools suggest a histological sampling in order to confirm the diagnosis, classify the lung involvement and make a prognostic assessment, however this practice is still not part of the recommendations [8]. renal damage was found in 26.2% of patients, 23.8% presented with arterial hypertension, while 11.9% had renal failure and 2.4% of patients presented positive proteinuria. cardiac involvement was found in 23.8% of patients with 7.1% of patients having presented abnormalities on the ekg, 7.1% had right heart failure, 4.8% had left heart failure, 19% of patients had pulmonary hypertension on echocardiography and pulmonary arterial hypertension was found by catheterization in only 3 patients (7.1%). it has been reported in the literature that severe microcirculation abnormalities demonstrated by periungual capillaroscopy were strongly associated with pulmonary vascular damage [9], but in our study capillaroscopy was performed in 41 patients (97.6%), and revealed various abnormalities ranging from mega capillaries to vascular desertification in 95.2% of patients while ph was only found in 19% of patients. digestive involvement was found in 54.8% of patients with gastroesophageal reflux disease (gerd) in 50% of patients, dyspepsia in 21.4%, constipation in 19%, fluid diarrhea with malabsorption in 2.4% of patients and solid dysphagia with endo-brachyesophagus on esogastroduodenal fibroscopy. urogenital involvement was found in 33.3% of patients, 21.4% of patients presented with urinary incontinence, 4.8% with dyspareunia and 11.9% with erectile dysfunction. we also observed a reduction in fertility in these patients, 100% of patients who had children had them before the diagnosis of scleroderma, and yassir et al.; asian j. immunol., vol. 7, no. 1, pp. 159-174, 2024; article no.aji.122682 171 34.3% of patients attempted unsuccessfully to have children after the diagnosis of scleroderma all by natural means, we have not found any study corroborating this result and we cannot exclude the effect of different treatments which leaves the question open. • therapeutically for raynaud's phenomenon, 40 patients were placed on calcium channel blockers, 24 of whom required the addition of 5 phosphodiestherase inhibitors and 8 of whom required the addition of fluoxetine. for extensive skin involvement without visceral involvement, 17 patients were placed on methotrexate, 3 of whom also benefited from local treatment with cannabis oil, when this was associated with pulmonary involvement. 18 patients were placed on mycophenolate mofetil, due to lack of average 6 patients with pulmonary involvement benefited from cyclophosphamide and the 3 patients with pah confirmed by catheterization benefited from a prostacyclin analog, and only 2 patients benefited from nintedanib-type antifibrosis for pulmonary fibrosis. despite the therapeutic means made available to patients, we remain behind what is done in renowned specialized centers and in research centers, for skin damage we have demonstrated innovation by attempting to add a local treatment with cannabis oil which has demonstrated a certain effectiveness in blocking cb2 receptors suspected in the pathophysiology of systemic sclerosis [10], the use of hyaluronidase by local injection has also demonstrated its effectiveness without proof of toxicity and the only negative point of which remains the need for multiple injections [11] as well as non-pharmacological treatments such as sporting and physical activity which makes it possible to modulate immunity [12] or even ultrasound treatment which makes it possible to soften the skin thus making it possible to reduce functional limitation [13], with regard to the management of raynaud's phenomenon our approach is not far from the latest recommendations developed by the rheumatology team of portugal in this year and which revolves around 3 main axes: patient awareness against potential triggering factors, pharmacological treatment allowing the microcirculation to dilate and finally promoting the healing of ulcerations before reaching a possible amputation [14], a chinese team carried out a literature review in 2021 showing the effectiveness of botulinum toxin injection in the management of raynaud's phenomenon [15]. regarding the management of pulmonary involvement, the combination of nintedanib, mycophenolate mofetil and a low dose of corticosteroid therapy has demonstrated their superiority to other conventional treatments [16], however in our context we remain limited by the means and the difficulty of insurance procedures, moreover only 2 patients were able to benefit from nintedanib. other studies are underway to improve the management of systemic sclerosis, such as trials of janus kinase inhibitors, tgf pathway inhibitors, anti-interleukins 6 and 8 and more recently transplantation especially of adipose and mesenchymal stem cells and which remains the most promising therapy [17,18, 19,20]. • evolution 20 patients reported an improvement (47.6%), 11 patients (26.3%) remained stable, 3 patients (7.1%) reported various complications, 7 patients (16.7%) died while only 1 patient was lost to follow-up (2.4%). the improvement reported by patients concerned cutaneous sclerosis in 6 patients, raynaud's phenomenon in 9 patients, lung damage in 8 patients, digestive damage in 3 patients and joint and muscle damage respectively in 4 patients. regarding the complications, 13 patients presented progression of the pathology, 4 patients manifested a new autoimmune disease, and only 1 patient presented a secondary respiratory infection. for the deceased patients, 3 were aged between 56 and 62 years at their death, while the other 4 were over 65 years old at their death. the causes of death were dominated by cardiac decompensation secondary to pulmonary failure in 4 patients, followed by cardiac failure in 2 patients and 1 patient who had septic shock. 5. conclusion systemic sclerosis is an autoimmune disease that is certainly rare but bothersome on an aesthetic and functional level, and can be lifethreatening due to its various visceral damage affecting the lung, heart and kidney. it has an unknown cause and a complex and poorly understood physiopathological mechanism but is increasingly better understood thanks to progress made in genetics. the diagnosis is yassir et al.; asian j. immunol., vol. 7, no. 1, pp. 159-174, 2024; article no.aji.122682 172 based on the acr/eular classification criteria based on clinical, biological and radiological criteria and the management of which, although codified with clear recommendations, is no less complicated and costly and always makes the object of research with the need for global and multidisciplinary care. in the context of the casablanca university hospital, although the frequency of the disease is relatively low, we find no difficulty in making the diagnosis and detecting the various visceral disorders, the real challenge remains on the therapeutic level which requires a simplification of administrative procedures. and easier access to different therapeutic molecules. annexe annexe is available in the following link: https://journalaji.com/index.php/aji/libraryfiles/d ownloadpublic/8 disclaimer (artificial intelligence) author(s) hereby declare that generative ai technologies such as large language models, etc have been used during writing or editing of manuscripts. this explanation will include the name, version, model, and source of the generative ai technology and as well as all input prompts provided to the generative ai technology details of the ai usage are given below: 1. chatgpt 4 o of open ai 2. used for translating the text from french to english consent it is not applicable. ethical approval it is not applicable. competing interests authors have declared that no competing interests exist. references 1. eric hachulla, christian agard, yannick allanore, jérôme avouac, brigitte badermeunier, alexandre belot, alice berezne, anne-sophie bouthors, géraldine condette-wojtasik, joël constans, pascal de groote, elisabeth diot, florence dumas, alain lescoat, patrick jego, francisca joly, david launay, véronique le guern, janine-sophie le quintrec, géraldine lescaille, christophe meune, bruno moulin, christelle nguyen, nadine omeish, frédéric pene, marie-aleth richard, juliette rochefort, alexandra roren, olivier sitbon, vincent sobanski, marie-elise truchetet, luc mouthon and collaborators national protocol for diagnosis and care of systemic scleroderma ; 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pmcid: pmc8935249. disclaimer/publisher’s note: the statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of the publisher and/or the editor(s). this publisher and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. _________________________________________________________________________________ © copyright (2024): author(s). the licensee is the journal publisher. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: https://www.sdiarticle5.com/review-history/122682 https://www.sdiarticle5.com/review-history/122682 _____________________________________________________________________________________________________ *corresponding author: e-mail: drmichaeloku@gmail.com, michaeloku@unical.edu.ng; cite as: m.e, oku, akpaso, m.i, odey, p.a, eru, e.m, anani, s.e., and umoh, n.m. 2024. “stereological studies on ameliorative role of ethanolic extracts of vernonia amygdalina and gongronema latifolium against streptozocininduced diabetic splenic tissue damage in wistar rats”. asian journal of immunology 7 (1):131-48. https://journalaji.com/index.php/aji/article/view/138. asian journal of immunology volume 7, issue 1, page 131-148, 2024; article no.aji.122015 stereological studies on ameliorative role of ethanolic extracts of vernonia amygdalina and gongronema latifolium against streptozocininduced diabetic splenic tissue damage in wistar rats oku, m.e a*, akpaso, m.i a, odey, p.a a, eru, e.m a, anani, s.e. a and umoh, n.m a a department of anatomy, university of calabar, nigeria. authors’ contributions this work was carried out in collaboration among all authors. all authors read and approved the final manuscript. article information doi: https://doi.org/10.9734/aji/2024/v7i1138 open peer review history: this journal follows the advanced open peer review policy. identity of the reviewers, editor(s) and additional reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here: https://www.sdiarticle5.com/review-history/122015 received: 18/06/2024 accepted: 20/08/2024 published: 29/08/2024 abstract background: diabetes mellitus is a chronic metabolic dysfunction marked with prolonged excessive glucose level, with disruptions in the metabolism of starch, lipids, and amino acids due to excessive production and/or inadequate utilization of glucose. objective: to assess the protective impact of vernonia amygdalina (va) and gongronema latifolium (gl) on splenic cytoarchitechure in stz induced glycemic disorder rats using quantitative and histological methods. original research article https://doi.org/10.9734/aji/2024/v7i1138 https://www.sdiarticle5.com/review-history/122015 oku et al.; asian j. immunol., vol. 7, no. 1, pp. 131-148, 2024; article no.aji.122015 132 materials and methods: a total of thirty wistar rats were randomly divided into six groups of five rats each, weighing between 150-200g. the groups were marked as a, b, c, d, e and f. group a [normal control] received distilled water, b [diabetic control] received 45mg/kg body weight of streptozocin, c received 100mg/kg of the extract va only; d received 100mg/kg of gl extract only. e received 5mg/kg of metformin and f received combined 100mg/kg of va and gl for fourteen days. twenty four hours after the last administration, the animals were sacrificed and the spleen was harvested, fixed, processed, stained with h&e and the spleen volume was estimate using cavalieri method and data were analyzed using anova at p<0.05. results: the diabetic group treated with stz only revealed reduced white pulp and the marginal zone and an apparent decrease in the cellular density of the lymphocyte component of the pulp, with positive perl’s stain. the stereological analysis of the spleen showed a significant decrease of the splenic pulp volume density and a significant increase in the connective tissue volume density, which significantly improved after treatment with the combined extract of va+gl-treated rats. the reduction of splenic pulp was mainly due to the decrease in the volume density of all structural components of the white pulp. conclusion: it can be suggested that combined ethanolic extracts of va+gl when used in combination can be used in management of diabetes. keywords: diabetes; spleen; vernonia amygdalina; gongronema latifolium; stz. 1. introduction diabetes is a chronic disorder associated with many debilitating multi-systemic complications. the spectrum of the diabetic pathology is complex and thus would require beyond single therapy for management of such complications. the more acceptable approach should embrace alternatives to orthodox care. diabetes remains a public health concern with an estimated 463 million cases worldwide [8.8% of the adult population] [1]. “diabetic patients have significant defects of antioxidant protection which may increase their vulnerability to oxidative damage and the development of diabetic complications” [2]. “reactive oxygen species [ros] are chemically reactive molecules containing oxygen, such as, h2o2, hocl, and free radicals such as superoxide anion, hydroxyl radicals” [3]. “antioxidants are molecules involved in scavenging of free radicals. this defense mechanism involves both enzymatic and nonenzymatic strategies” [3]. “in 2017, diabetes resulted in approximately 4.2 million deaths. it is the seventh leading cause of death globally” [4]. the pooled prevalence of 5.77% in nigeria suggests that about 11.2 million people [1 out of every 17 adults] are living with the disease. recent research has also shown a connection between diabetes and dementia, hearing loss, and some forms of cancer [5]. diabetes increases the risk of early death, and diabetesrelated complications can lower quality of life [6]. the high cost of anti-diabetic drugs, its side effects and numerous complications associated with poor prognosis arising from orthodox drugs has led many to embrace plant-based therapy [7]. as several studies revealed amelioration of alzheimer’s disease post-administration of telfairia occidentalis and talinium triangulare on rats induced with scopolamine [8,9,10,11,12], musa paradisiaca stem juice limit the extent of status epilepticus, increased neuronal protein synthesis, reduced cytoarchitectural damage and astriogliosis as well as enhancing long term recognition memory in rats [13,14], star fruit ameliorates neurotoxicity in rats [15], other researchers also reported the antihyperglycemic and hypoglycemic action of vernonia amygdalina [va] and gongronema latifolium [gl] in diabetic and non-diabetic rats [16]. the aqueous leaf extract has been shown to possess antihyperlipidemic and hypolipidemic effects on diabetic and non-diabetic rats [17]. according to [18], va has continued to receive a lot of attention due to the numerous curative potentials that it has demonstrated; it is purported to possess antioxidant activity from radical scavengers [19]. many people also believe in traditional ways of living, and this has influenced their health-seeking behavior [20], hence, the rationale to investigate the potentials of va and gl on the cytoarchitecture of the spleen in streptozocin-induced diabetic wistar rats in the present study. 2. materials and methods a total of 30 rats [150-200g] with ethical approval [farec/pa/[uc/049] were acquired from the oku et al.; asian j. immunol., vol. 7, no. 1, pp. 131-148, 2024; article no.aji.122015 133 animal house, college of medical sciences, university of calabar. the rats were kept under standard conditions of temperature [27oc-30oc] and fed with rat chow purchased from agro feed mill nigeria limited, calabar and provided with distilled water for drinking. they acclimatized for a week prior to commencement of the experiment. the animals were housed in properly ventilated plastic cages. 2.1 plant extracts preparation the fresh leaves of va and gl were purchased from marian market, calabar, cross river statenigeria. these plants were identified by a botanist with reference number bot/ herb/ucc/0188 and bot/herb/ucc/0718 respectively. the plants were cleaned and air dried, after which they were grounded into powered form. a measured amount of 120 grams of powdered leaves was extracted using 2.5 liters of absolute ethanol for 48hours with intermittent gyration of sample holder. after 48hours of soaking, the extract solution was first double filtered with chess cloth, then with filter paper [whatman/filter paper]. the filtrate [extract] was thereafter concentrated under reduced pressure at 45oc in rotary evaporator to 10% volume and then to complete dryness using regulated temperature water bath, yielding 22 grams [about 19.5g] of crude extract. the extract obtained was stored in a refrigerator until required. 2.2 induction of diabetes to the experimental rats diabetes was induced in overnight fasted experimental animals by a single dose of intraperitoneal injection of freshly prepared streptozocin [stz] at 45mg/kg body weight reconstituted in 0.1m sodium citrate buffer at ph 4.5-5.0 as solvent using lorke’s method. diabetes was confirmed by use of glucometer to test for the fasting blood glucose concentration of experimental animals 48hrs after stz administration, values above 180mg/dl were considered diabetic and found suitable for the study. 2.3 determination of ld50 ld50 of ethanol leaf extracts of va and gl was established to be >2000mg/kg according to lorke’s method [1983]. the dosage was determined using 5% [100mg/kg] of 2000mg/kg body weight of ethanol extract of va and gl leaves. 2.4 plant extract and metformin administration group a, the normal control rats were given only distil water and feed, group b was the diabetic control rats, induced with diabetes [received 45mg/kg of stz], group c animals were induced with diabetes [received 45mg/kg of stz] and were given vernonia amygdalina [100mg/kg] only. group d were induced for diabetes [received 45mg/kg of stz] and were given gongronema latifolium [100mg/kg] only. group e were induced with diabetes [received 45mg/kg of stz] and were given metformin [100mg/kg] and group f was induced with diabetes [received 45mg/kg of stz] and were given both extracts of vernonia amygdalina [100mg/kg] and gongronema latifolium [100mg/kg]. pre and post induction of stz fasting blood sugar [fbs] and weight of animals were assessed. these procedures were carried out daily during the course of extracts administration. 2.5 tissue processing procedure twenty four hours after the last administration, the experimental animals were sacrificed and the spleen was fixed with 10% formol saline. the tissues were dehydrated using ascending grades of alcohol followed by clearing with xylene, infiltrated, embedded with paraffin wax and sectioned [5µm] with rotary microtome. the ribbons of the sections were collected using clean slides and first straighten with 10% ethanol before being gently lowered into the surface of a warm water bath so as to straighten wrinkles on sectioned tissues. the floated sections were mounted on an albumenized slides and it was dried in the oven at 60 degrees celsius, for one hour before staining. 2.6 hematoxylin and eosin staining procedure [cole, 1943] tissue sections were dewaxed in xylene for 5-10 minutes, transferred through descending grades of alcohol [100%, 95% and 70%] 5mins respectively. sections were rinsed in running tap for 10mins and stained in hematoxylin for 10 minutes. they were rinsed in water and blueing in tap water for 5 minutes. sections were differentiated in 1% acid alcohol so as to help nucleus absorbs the stain`. they were counter oku et al.; asian j. immunol., vol. 7, no. 1, pp. 131-148, 2024; article no.aji.122015 134 stained in 1% eosin for 30 seconds. various sections were blued under a running tap water for 2-3mins. finally, sections were passed through ascending grades of alcohol, cleared in xylene and mounted in distyrene, plasticizer and xylene [dpx]. 2.7 stereology the absolute volume of the spleen was estimated using the cavalieri estimator of volume according to the method of west et al. [1999]. to achieve this, spleen of each wistar rat per group was isolated, processed and sectioned serially using a microtome, as shown by [21] to provide a number of sections after pilot study on how many slices could be derived and how many slices will give the lowest coefficient of error, each section had a section of the spleen. tissue sections of the spleen were selected using a systematic uniform random sampling method. the sections derived were stained using h and e stains. a transparent counting grid was placed randomly over the cut surface of every spleen slice. the number of points hitting the spleen was counted. the volume was then estimated using the cavalieri’s principle [22] as follows: v total [mm3]: =𝑇 × 𝑎 𝑝⁄ × ∑𝑝𝑖 where ‘‘: =’’ indicates that the result is the estimated value rather than the true value, “v” is the total volume of spleen, “t” = 100. slice thickness =5 µm x100 = 500 µm. convert 500 µm to mm, 500/1000 = 0.5 mm is the average slice thickness, “a/p” ¼ is the area per point associated with each point in the counting grid [4mm²], and “ʃp” is the total number of points hitting the spleen. 2.8 data analysis results obtained at the end of the experiment were analyzed using the statistical software, statistical package for social science [ibm spss version 23.0] and microsoft office excel 2019 were used for charts. results were expressed as mean ± s.e.m. one-way analysis of variance [anova] was used to compare mean difference between groups followed by least significant difference [lsd] post hoc test. paired t-test was employed for the comparisons of means as appropriate. values were considered significant when p<0.05. 3. results h&e stained sections from the control group showed normal histology of the spleen with normal white pulp [wp] and numerous white blood cells, red pulp [rp], the marginal zone and the central vessels [bv] following h&e staining [plate 1]. group b revealed shrunken white pulp [wp] and enlarged red pulp [rp] with reduced germinal center using, the spleen also showed numerous lymphocytes in the shrunken white pulp area indicating the presence of inflammation and immune response and plate 1. photomicrograph of spleen of wistar rats in normal control group treated with h20 showing normal white pulp and red pulps with germinal center, h&e stain, [mag x 100] oku et al.; asian j. immunol., vol. 7, no. 1, pp. 131-148, 2024; article no.aji.122015 135 distortion of the splenic cytoarchitecture, [plate 2]. group c revealed signs of regenerating white pulp [wp] compared to the diabetic group, with lymphocytes infiltrating into the red pulp [rp] area [plate 3]. group d showed prominent white pulp (wp) with numerous white blood cells and reduced red pulp [rp] [plate 4]. group e showed normal appearing white pulp [wp] with white blood cells and reduced red pulp [rp] [plate 5]. group f revealed signs of regenerating normal white pulp, red pulp, increased lymphocytes in the white pulp with normal central vessel [plate 6]. stereological estimation of spleen volume of diabetic control group b [970±250.98] [ce = 0.035] was significantly [p<0.05] lower when compared to normal control group a [2398.7±352.70] [ce = 0.018]. spleen volume of all treated groups: group c [1630±321.23] [ce = 0.028], group d [2538±284.32] [ce = 0.015], group e [3458±515.54] [ce = 0.004] and group f [2548±871.34] were significantly [p<0.05] higher when compared to the volume of the diabetic control group b [970±250.98] [ce = 0.023]. plate 2. photomicrograph of spleen of wistar rats in diabetic control group treated with 45 mg/kg streptozocin only showing shrunken and distorted white pulp and reduced germinal center with blood vessel, h&e stain, [mag x100] plate 3. photomicrograph of spleen of wistar rats in group c treated with 45 mg/kg streptozocin and 100 mg/kg of va only showing regenerating white pulp [rwp] with lymphocytes [lym] and reduced red pulp [rp] area, h&e stain, [mag x 100] oku et al.; asian j. immunol., vol. 7, no. 1, pp. 131-148, 2024; article no.aji.122015 136 plate 4. photomicrograph of spleen of wistar rats in group d inducedwith 45 mg/kg streptozocin and treated with 100 mg/kg gl only showing regenerating white pulp [wp] filled with increased white blood cells [wbc] and reduced red pulp [rp] area, h&e stain, [mag x100] plate 5. photomicrograph of group e [metformin solution 5mg/kg] showing regenerating splenic white pulp with white blood cells infiltrating the reduced red pulp [rp], h&e stain, [mag x 100] oku et al.; asian j. immunol., vol. 7, no. 1, pp. 131-148, 2024; article no.aji.122015 137 plate 6. photomicrograph of the spleen of wistar rats in group f treated with 45 mg/kg streptozocin and combined extracts of 200 mg/kg va + gl showing regenerating white pulp, with reduced red pulp with presence of numerous normal lymphocytes [lym] in the white pulp and central vessel. h&e stain [mag x100)] plate 7. photomicrograph of spleen of wistar rats in normal control group treated with distilled water showing weakly positive perl’s stain with sparse hemosiderin deposits. perl’s prussian stain, [mag x100] oku et al.; asian j. immunol., vol. 7, no. 1, pp. 131-148, 2024; article no.aji.122015 138 plate 8. photomicrograph of spleen of wistar rats in group b treated with 45 mg/kg streptozocin only showing intense perl’s staining of hemosiderin deposits [arrows] within the red pulp. perl’s prussian stain [mag x 100] plate 9. photomicrograph of group c splenic tissue treated with 45 mg/kg streptozocin and 100 mg/kg of va only showing moderate staining of hemosiderin deposits [bluish colouration] within the red pulp [perl’s stain x 100] oku et al.; asian j. immunol., vol. 7, no. 1, pp. 131-148, 2024; article no.aji.122015 139 plate 10. photomicrograph of spleen of wistar rats ingroup d treated with 45 mg/kg streptozocin and 100 mg/kg of gl only showing moderate perl’s stain with moderate hemosiderin deposits [arrow] within the red pulp. perl’s prussian stain, [mag x 100] plate 11. photomicrograph of the spleen of wistar rats in group e treated with 45 mg/kg streptozocin and 5mg/kg metformin showing moderate perl’s stain of hemosiderin deposits [arrow] within the red pulp. perl’s prussian stain, [mag x 100] oku et al.; asian j. immunol., vol. 7, no. 1, pp. 131-148, 2024; article no.aji.122015 140 plate 12. photomicrograh of spleen of wistar rats in group f treated with 45 mg/kg streptozocin and combined extract of 200 mg/kg va and gl showing weak perl’s stain with moderate hemosiderin deposits [bluish coloration] within the red pulp. perl’s prussian stain, [mag x100] plate 13. stereological image of the spleen in normal control group of wistar rat showing normal morphology and splenic volume oku et al.; asian j. immunol., vol. 7, no. 1, pp. 131-148, 2024; article no.aji.122015 141 plate 14. stereological image of splenic tissue of diabetic group that received 45mg/kg of stz only, showing splenic tissue atrophy with distorted morphology and reduced spleen volume plate 15. stereological image of splenic tissue of animals that received 45mg/kg of stz and treated with 100mg/kg of va only showing increased splenic volume compared to the diabetic control group oku et al.; asian j. immunol., vol. 7, no. 1, pp. 131-148, 2024; article no.aji.122015 142 plate 16. stereological image of splenic tissue of animals that received 45mg/kg of stz and treated with 100mg/kg of gl only showed increased volume of the spleen when compared to the diabetic group plate 17. stereological image of splenic tissue of animals that received 45mg/kg of stz and treated with metformin at 5mg/kg daily, showed significantly increased volume of the spleen when compared to the diabetic group oku et al.; asian j. immunol., vol. 7, no. 1, pp. 131-148, 2024; article no.aji.122015 143 plate 18. stereological image of splenic tissue of animals that received 45mg/kg of stz and treated with 200mg/kg of combined extract of va+gl showed significantly increased volume of the spleen compared to the diabetic control group 4. discussion diabetes decreases immune response capacity, including the suppression of immune cell function, atrophy of immune organs [22,23]. studies have been done to ascertain the potential utility of natural products as immunomodulatory agents to enhance hosts responses to diseases [24]. this study focuses on the effect of diabetes on one of the important immunologic organsthe spleen and the possible ameliorative effect of combined extract of vernonia amygdalina and gongronema latifolium. diabetes-related spleen damage increases immune dysfunction, which often results in the high risks of infection, morbidity and mortality in diabetic patients. “herbs have been used in many capacities to improve human health and also serve as a source of natural templates for pharmaceutical synthesis of drugs. many local communities across afro-asian regions of the world rely heavily on their ethno-botanical heritage to meet most of their primary healthcare needs” [25]. this study demonstrated that anti-diabetic effect of va+gl in rats is mediated through complex multiple mechanisms. “immunodeficiency is one of the major causes of diabetic complications. the spleen, as a secondary lymphoid organ, acts as a site of initiation of most of the immune responses. spleen harbors stem cells that act as precursors to insulin producing β cells of the pancreas. stz selectively accumulates in the pancreatic β cells and induces hyperglycemic conditions through mitochondrial complication mediated glucotoxicity of the β cells” [26]. in this study, administration of the combined extracts post diabetes induction was found to reduce the increased blood glucose level and restore the normal level of serum insulin, spleen size and the spleen weight in the treated rats compared to the diabetic rats. in the diabetic control [group b] induced with 45mg/kg of streptozocin, the spleen showed signs of depleted white pulp compared to the enlarged size in the normal control group which is in line with the work of [27] who reported that the lymphocyte number were dramatically declined in both peripheral blood and the spleen following a distortion of the white pulp, dilation in the blood vessels, an increase of collagen deposition and a depletion in the iron particles detected in the red pulp. this indicates that the oku et al.; asian j. immunol., vol. 7, no. 1, pp. 131-148, 2024; article no.aji.122015 144 lymphocytes are stressed by diabetic toxicity with high levels of free radicals which, increases the levels of pro-inflammatory cytokines leading to programmed cell death. the white pulp of the spleen is responsible for both adaptive and humoral immunity involving the b and tlymphocytes. shrunken white pulp leads to a reduction in immune response, thereby making the body susceptible to diabetic complications. in groups c and d, which received 100mg/kg of vernonia amygdalina [bitter leaf] and gongronema latifolium [utazi] respectively showed significant reversible changes in the histological integrity of the spleen compared with the results in the diabetic group. this result suggests the extracts individually has effect on the diabetic alterations that occurred in the spleen after stz induction to cause reversible changes in the splenic cytoarchitecture, with regenerating white pulp and presence of numerous normal lymphocytes in the spleen. in group e, treated with 5mg/kg of metformin, changes in the histology of the spleen were observed. the shrunken white pulp seen in the diabetic group b was seen regenerating with increased size in this group. this shows that metformin has anti-oxidant properties in line with [28]. there was a significant increase in the number of white blood cells which help to strengthen the body's immune response. in group f that received 100mg/kg of both v. amygdalina and g. latifolium showed similar histological profile to that of the normal control. this suggests a possible regeneration of the splenic cell functions and a significant reversal of diabetic insults on the splenic cytoarchitechure by the combined extract. this observation is in line with previous studies on the potentials of vernonia amygdalina, gongronema latifolium and azadirachta indica to cause a regeneration of pancreatic beta cells of stz induced diabetic rats. [29,16,30]. the effects of the plant extracts were more pronounced when used in combination. this study observed that the improvement in the histology of the spleen was more remarkable in the group that received the combined extract [va+gl] than in the groups that received individual extracts, [va and gl]. in another study by [27], the administration of 100mg/kg of whey proteins restored the histological integrity of the spleen following stz induced diabetic degenerative changes were seen. plant extracts can be useful in reversing diabetic insults on the histology of the spleen as was discovered from this study. the findings of this research are consistent with previous research on the regenerative potential of vernonia amygdalina, gongronema latifolium, and azadirachta indica in restoring pancreatic beta cells in stz-induced diabetic rats [29]. moreover, the synergistic impact of the mixed extract [va+gl] was more pronounced in improving the histology of the spleen compared to the individual extracts [va and gl] in this study. these results align with research by [27], which illustrated that the administration of whey proteins at a dosage of 100mg/kg restored the histological integrity of the spleen in the presence of stz-induced diabetic degenerative changes. therefore, the use of plant extracts shows promise in reversing diabetic-induced damage to the spleen's histology, as evidenced by the findings of this study. staining intensity of the white pulp by perl’s prussian blue in the spleen sections of the normal control and the treated groups [c, d, e and f] groups were weakly positive, by contrast to the diabetic group which showed higher intensity staining. the immunosuppressive states induced by hyperglycemia is largely responsible for the decline in lymphocyte population and subsequent increased hemosiderin deposition in the red pulp area as a consequence of impaired phagocytic activity [27]. the findings of [31] further support the current study's results, as they reported that the administration of whey protein to diabetic rats improved the lymphocyte population in the white pulp of the spleen. this improvement may be attributed to the inhibition cascade of the programmed cell death pathway, indicating a potential mechanism by which whey protein restores lymphocytic activity. additionally, the restoration of iron deposition to approximately normal levels observed in the study suggests a further improvement in lymphocytic function. therefore, the use of whey protein as a therapeutic agent holds promise for enhancing lymphocyte activity and restoring the normal functioning of the spleen in diabetic conditions. changes observed in the cytoarchitecture of the spleen suggested that whey proteins could enhance immune response. this suggests that the ethanolic extract in single or combined doses administered reversed splenic cell destruction from stz induced diabetes. the immunosuppressive states induced by hyperglycemia is largely responsible for the decline in lymphocyte population and subsequent increased hemosiderin deposition in oku et al.; asian j. immunol., vol. 7, no. 1, pp. 131-148, 2024; article no.aji.122015 145 the red pulp area as a consequence of impaired phagocytic activity [27]. this agrees with the findings in another study by [31] who reported that the improvement of the lymphocyte population in the white pulp by whey protein treated diabetic rats may be due to the inhibition cascade of the programmed cell death pathway. whey proteins was found to restore the iron deposition to the approximately the normal level, indicating improvement of lymphocytic activity. changes observed in the cytoarchitecture of the spleen suggested that whey proteins could enhance immune response. this suggests that the ethanolic extract in single or combined doses administered reversed splenic cell destruction from stz induced diabetes. the stereological analysis of the spleen showed significant decrease of the splenic pulp volume density in the diabetic group when compared to the control group and the treated group of wistar rats. reducing the presence of splenic pulp was mainly due to the decrease in the volume density of all structural components of the white pulp. this observation aligns with another study by [32], following administration of dexamethasone at 150mg/kg body weight, which showed significant decrease of the splenic pulp volume density and significant increase of the connective tissue volume density. the cytoarchitecture of the spleen was restored following treatment with the combined extracts of va+gl. stereological studies also revealed that combined extracts of va+gl treatment remarkably improved the volume of the splenic white pulp depleted by stz induced diabetes. the point-counting method was utilized to study the volume densities of the following tissue compartments: red pulp; white pulp [this compartment was divided in two sub compartments: follicles and per arteriolar lymphocyte sheath]; marginal zone; and connective tissue. stereology is a number of mathematical and statistical methods that permit the evaluation of 3dimensional structural information from 2dimensional sections [or histological slices]. thus, researchers obtain important quantitative structural information, such as the volume, surface area or numbers of cells within described regional lines. the need for such quantitative information biological studies is of importance when evaluating the effect of various experimental treatments on any specific organs, tissues and cells in the body. spleen volumes were significantly increased in animals treated with va+gl and individual extracts of va and gl. stereological studies also revealed that va+gl treatment remarkably improved the volume of the splenic tissues and the numerical density depleted by stz diabetes. possible antidiabetic mechanism of va+gl in stz induced diabetes is through the regeneration of atrophic splenic structural integrity and its strong antioxidant potential. diabetes is known to involve oxidative stress and changes in lipid metabolism. gongronema latifolium has been shown to possess antihyperglycemic, anti-lipidemic, antioxidant and anti-inflammatory properties. [33], showed that ethanolic extracts of gl leaves have antihyperglycemic effects in streptozocin induced diabetic rats, thought to be mediated through the activation of hexose and glucose -6-phosphate dehydrogenase, g6pdh. va strengthens the immune system through its effect on cytokine regulation [34]. a study by [35], showed ferulic acid provides protection against hyperglycemia induced, oxidative stress mediated splenotoxicity by regulating the plasma insulin level, blood glucose level, intracellular inflammation and mitochondria dependent intrinsic pathway of apoptosis in the spleen. this study highlighted that the spleen volume of diabetic control group [970±250.98] [ce = 0.035] was significantly [p<0.05] lower when compared to normal control group [2398.7±352.70] [ce = 0.018] and group f [2548±871.34] were significantly [p<0.05] higher when compared to the volume of the diabetic control group [970±250.98] [ce = 0.023] [36-41]. 5. conclusion from this study, it could be inferred that combined and individual ethanolic extracts of va+gl possesses anti-diabetic and antioxidant properties that can mitigate the impact of stz induced diabetes on the spleen of wistar rats. disclaimer (artificial intelligence) author(s) hereby declare that no generative ai technologies such as large language models (chatgpt, copilot, etc) and text-to-image generators have been used during writing or editing of manuscripts. oku et al.; 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cite as: olivier, celso eduardo, daiana guedes pinto, ana paula monezzi teixeira, cibele silva miguel, raquel acácia pereira gonçalves santos, jhéssica letícia santos santana, and regiane patussi santos lima. 2024. “assessing cellular and humoral immunoreactivity to lanolin: a retrospective cohort study in atopic and contact dermatitis patients”. asian journal of immunology 7 (1):316-25. https://doi.org/10.9734/aji/2024/v7i1154. asian journal of immunology volume 7, issue 1, page 316-325, 2024; article no.aji.128121 assessing cellular and humoral immunoreactivity to lanolin: a retrospective cohort study in atopic and contact dermatitis patients celso eduardo olivier a*, daiana guedes pinto a, ana paula monezzi teixeira a, cibele silva miguel a, raquel acácia pereira gonçalves santos a, jhéssica letícia santos santana b and regiane patussi santos lima c a instituto alergoimuno de americana, brazil. b instituto de ensino e pesquisa do hospital de amor de barretos, brazil. c lavoisier laboratórios, são paulo, brazil. authors’ contributions this work was carried out in collaboration among all authors. the author ceo is responsible for the conceptualization, data curation, formal analysis, literature review, and writing the original draft. authors dgp, apmt, csm, jlss and rpsl performed laboratory procedures. author rapgs performed cutaneous tests. all authors read and approved the final manuscript. article information doi: https://doi.org/10.9734/aji/2024/v7i1154 open peer review history: this journal follows the advanced open peer review policy. identity of the reviewers, editor(s) and additional reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here: https://www.sdiarticle5.com/review-history/128121 received: 24/10/2024 accepted: 26/12/2024 published: 28/12/2024 original research article https://doi.org/10.9734/aji/2024/v7i1154 https://www.sdiarticle5.com/review-history/128121 olivier et al.; asian j. immunol., vol. 7, no. 1, pp. 316-325, 2024; article no.aji.128121 317 abstract background: several publications report lanolin as responsible for hypersensitivity reactions in patients with atopic dermatitis and/or contact dermatitis, as diagnosed by in vivo provocation tests. there is no standardized lab exam that can endotype the mechanisms responsible for these phenotypes. aim: to evaluate the potential of the tube titration of precipitins (ttp) and the leukocyte adherence inhibition test (lait) to discriminate and endotype immunoreactivity against lanolin in patients with atopic dermatitis and/or contact dermatitis. methods: we retrospectively examined the medical charts of two cohorts of patients diagnosed with atopic dermatitis and/or contact dermatitis with clinical suspicion of lanolin hypersensitivity, who were investigated with the help of ttp or ex vivo challenge tests monitored by lait against lanolin. the registered results were distributed in ranges through cascade distribution charts. the statistical characteristics of these cohorts were calculated. results: the ttp results spread a distribution concentrated over the more diluted titrations (fig. 1). there was no negative result. the mean was estimated at 1:343; the median was 1:512; the standard deviation was estimated at 1:187; the mode was 1:512 (appeared 53 times). the lai ranged from 0% to 95%. the mean was 41.5%; the median was 41.5%; the standard deviation was 27.3%; the mode was 0% (appeared sixteen times). the cascade distribution demonstrates a widespread distribution of lai results. conclusion: ttp and lait performed with lanolin solution were able to discriminate diverse degrees of humoral and cellular immunoreactivity in patients suffering from atopic and/or contact dermatitis. it is worthwhile conducting more in-depth studies to evaluate the usefulness of ttp and lait in endotyping non–ige-mediated hypersensitivity to lanolin. keywords: atopic dermatitis; contact dermatitis; endotype; hypersensitivity; lanolin; leukocyte adherence inhibition test; precipitins; precision medicine. abbreviations lai : leukocyte adherence inhibition lait : leukocyte adherence inhibition test ttp : tube titration of precipitins 1. introduction in the past, when synthetic garments were not yet produced, natural wools were dominant allergens in cold months, associated with atopic dermatitis, eczema, or textile contact dermatitis, especially in infants [1]. wool allergy has been traditionally diagnosed by scratch, patch, and intracutaneous tests with wool extracts [2]. wool allergy has been successfully treated by desensitization since the 1930s [3]. concomitantly, the cause of wool allergy was attributed to the wool fat: lanolin [4]. lanolin is a complex mixture of free fatty acids, fatty acid esters, cholesterols, and other hydrocarbons extracted from sheep's wool [5]. lanolin is produced from the secretion of the sebaceous glands of sheep to serve as a wool's protective coating, which is a composition that varies according to the breed of sheep, geographic location, method of extraction, and level of purification [6]. the first extraction of this "wool wax" is attributed to the ancient greeks, who documented, in 700 bc, boiling wool in water produced a top layer of greasy froth that could be skimmed [7]. in 1882, the germans otto braun and oscar liebreice applied a patent describing a sequential centrifugation of the wool froth along with water to incorporate both elements, resulting in a suspension which, after refrigeration, boiling, and alkalinization became what they coined "lanolin" (us letters patent no. 271,192 dated january 23, 1883) [8]. nowadays, several more sophisticated modified techniques are employed to produce several varieties of lanolin-based products and derivatives through physical and chemical modifications (hydrolysis. hydrogenation, acetylation, ethoxylation, transesterification, and so forth) [9]. very soon, the industrial production of lanolin was widespread. it began to be incorporated into skin-cream products as an emulsifier, stabilizer, emollient, and skin moisturizer, such as toilet articles, shaving creams, toilet soaps, hair lotions, shampoos, hair conditioners (to prevent hair drying, scaling and brittleness), cosmetic creams, lipsticks, nail polish removers, eye make-up (for more uniformly dispersion of the pigment), hair sprays (as a plasticizer), hair bleaching agents, and used by pharmacists as olivier et al.; asian j. immunol., vol. 7, no. 1, pp. 316-325, 2024; article no.aji.128121 318 an excipient for topical medications [10]. studies done by freeze-fracture and transmission electron microscopy of stratum corneum found that most applied lanolin occupied intercellular spaces. some penetrated corneocytes and had a special affinity for the cell junction [11]. allergic contact dermatitis to lanolin was first described by ramirez and eller in 1929 [12]. very soon, several case reports of hypersensitivity to lanolin were published worldwide [13-15]. the 1950s saw the beginning of studies on the allergenic components of lanolin, as well as on the differences in immunoreactivity between the different versions, demonstrating, through patch tests, that the main allergenic components could be into the hydroalcoholic or the fatty lanolin components [16,17]. allergy to lanolin is more common in children (4.5%) than in adults (3.2%) and patients with a previous history of eczema and hay fever [18]. although lanolin is considered a weak sensitizer over undamaged skin, there is a higher risk of sensitization when applied over skin-damaged conditions such as contact dermatitis, atopic dermatitis, psoriasis, dermatitis herpetiformis, seborrheic dermatitis, stasis dermatitis, leg ulcers, perineal dermatitis, and so forth [19]. in the eighties, the widespread use of this moisturizer exposed a lanolin allergic "crisis", which was also described as a "myth", and a "comedy", when several pharmaceutical ointments began to declare on their packages to be "lanolin free" [20, 21]. the presence of lanolin is unanimous among the diverse batteries historically recommended for making diagnostic contact test kits (patch tests) [22]. the biggest obstacle to diagnosing hypersensitivity to lanolin is its multiple varieties, components, allergens, versions, contaminants, and additives, such as preservatives and antioxidants [14]. the standard patch test agent for diagnosing lanolin contact allergy is lanolin alcohol 30% in petrolatum [23, 24]. however, this method alone has a very low sensitivity to diagnosis of lanolin hypersensitivity, a fact that had been contributing to the "lanolin paradox", leading the physicians to use for tests the suspected materials brought in by the patients [25]. several papers report discrepancies among cutaneous tests performed with lanolin derivatives such as lanolin alcohols and amerchol l101 (a mixture of 10% lanolin alcohols and mineral oil) [26, 27]. atopic dermatitis and contact dermatitis are interrelated phenotypes with several associated endotypes [28]. while the atopic dermatitis phenotypes usually develop in patients presenting hypersensitivity to food allergens or house dust mites, the typical contact dermatitis phenotype develops in patients presenting hypersensitivity to organic and inorganic contactants [29]. some patients presenting predominantly the atopic dermatitis phenotypes may also present less evident contact dermatitis phenotypes and vice versa [30]. there is not yet a satisfactory laboratory exam to diagnose the non-ige-mediated atopic dermatitis or contact dermatitis endotypes. several trial evaluations were conducted on the relationship between leukocytes and allergy tests. however, the results did not significantly yield definitive conclusions [31]. the better way to diagnose lanolin hypersensitivity is the exclusion/provocation test when the patient excludes the use of the suspected allergen(s) until the symptoms disappear, and then the allergen is re-introduced to observe reactions. however, this is particularly difficult when polysensitization dominates the clinical picture. in order to shorter the list of suspected allergens, we performed in our facilities the leukocyte adherence inhibition test (lait) and the tube titration of precipitins (ttp) as triage tests to elect the allergens that will be emphasized in the exhaustive in vivo exclusion/provocation tests [32-37]. the present study hypothesizes that the lait and the ttp may help differentiate diverse endotypes and degrees of immunoreactivity against lanolin among patients suffering from atopic dermatitis and/or contact dermatitis. to evaluate the potential of the lait and the ttp to endotyping non–ige-mediated immunoreactivity against lanolin, we retrospectively compiled the electronic medical charts of patients with these conditions who were investigated with these procedures. 2. materials and methods 2.1 subjects after receiving institutional review board approval from the instituto alergoimuno de olivier et al.; asian j. immunol., vol. 7, no. 1, pp. 316-325, 2024; article no.aji.128121 319 americana (brazil; 10/2024), we proceeded with the electronic chart review of 9,700 outpatients who attended our facility from january 2018 to november 2024. a cohort of 100 outside patients had been submitted to ttp with lanolin solution for presenting non–ige-mediated atopic dermatitis and/or contact dermatitis. this cohort counted 23 males; mean age 38.9 years; sd 19.9 years; range 7 to 91 years; median 37.5 years; modes: 16; 23; 37; and 70 years (each appeared 4 times); geometric mean = 32.8 years. a cohort of 100 outside patients had been submitted to an ex vivo allergen challenge test with lanolin solution monitored with lait for presenting non– ige-mediated atopic and/or contact dermatitis. this cohort counted 25 males; mean age 40.9 years; sd 18,5 years; range 3 to 86 years; median 40.5 years; mode = 50 (appeared five times); geometric mean = 35.0 years. this study did not include patients under biological and/or systemic anti-inflammatory therapy. these procedures were offered to patients with clinical suspicion of lanolin hypersensitivity who demonstrated a nonreactive or inconclusive skin test against lanolin solution [38]. 2.2 lanolin solution in a becker flask, 10 ml of lanolin (farma norte™) and 10 ml of glycerin [glycerin p.a. – acs, c3h5(oh)3, dinâmica™] were homogenized and kept under refrigeration (4 °c) to perform the allergic skin tests, ttp, and lait. 2.3 ex vivo investigation: leukocyte adherence inhibition test 2.3.1 procedure for allergen ex vivo challenging we performed the lait as previously described [39-49]. shortly, each donor's fresh plasma was divided into two parts and used in paralleled ex vivo challenging tests with lanolin solution and the unchallenged plasma assay. we collected plasma with high leukocyte content (buffy coat) from the heparinized tube after one hour of sedimentation at 37 °c. then, we distributed aliquots of 100 μl into eppendorf tubes kept under agitation for 30 minutes (200 rpm at 37 °c) with lanolin solution (10μl) or without lanolin solution (when used as control). 2.3.2 procedure for adherence assay after incubation, the plasma was allocated into a standard neubauer hemocytometer counting chamber with a plain, non-metallic glass surface and left to stand for 2 hours at 37 °c in the humidified atmosphere of the covered water bath to allow leukocytes to adhere to the glass. next, we counted the leukocytes, removed the coverslip, and washed the chamber by immersion in a beaker with pbs (phosphatebuffered saline) at 37 °c. then, we added a drop of pbs to the hemocytometer's chamber and allocated a clean coverslip over it. the remaining cells were counted in the same squares as previously examined. 2.3.3 procedure for calculation the percentage of leukocyte adherence (la) of each assay was estimated as: (the number of leukocytes observed on the hemocytometry chamber after washing divided by the number of leukocytes observed on the hemocytometry chamber before washing) and multiplied by 100 (%). the leukocyte adherence ratio (lar) was estimated based on the ratio between the la from the antigen-specific challenged plasma and the la from the unchallenged control plasma: lar = la of the challenged sample divided by la of unchallenged control plasma multiplied by 100 (%). to further calculate the leukocyte adherence inhibition (lai), we subtracted the lar from 100 (%). we employed the lai results for the cascade distribution chart and the statistics calculations, both performed with the help of the microsoft excel® statistical package. 2.4 in vitro investigation: tube titration of precipitins (ttp) as previously reported, a transparent vitreous tube array performed the semi-quantitative ttp against the lanolin solution [50-54]. shortly, the patient's blood was collected in a clot-activator collecting tube. after separation, the serum was centrifugated at 2,000 rpm for 10 minutes. the allergen extracts were allocated in sets of eleven glass tubes at progressive duplicated serum dilutions. the progressive dilutions were combined with the 15 μl of the antigen solution with 250 μl of the patient's serum, progressively diluted into physiological saline solution (nacl 0,9%) in the dilution ratios of 1:1; 1:2; 1:4; 1:8; olivier et al.; asian j. immunol., vol. 7, no. 1, pp. 316-325, 2024; article no.aji.128121 320 1:16; 1:32; 1:64; 1:128; 1:256; and 1:512. one tube was a blank control done with the water and serum to observe occasional spontaneous precipitation (sia test). after 24 hours, the tubes were examined, and the titers (the highest dilution factor that yields a positive reading) were recorded [55]. 3. results as a retrospective survey, there was no research protocol; therefore, we report the incidental immune investigation as registered in the digital medical charts. fig. 1. cascade distribution chart of the tube titration of precipitins (x-axis %) resulting from the lanolin solution against the serum of a cohort of 100 tests/subjects (y-axis) fig. 2. cascade distribution chart of the range groups of leukocyte adherence inhibition (lai) results (x-axis %) of ex vivo lanolin solution monitored by the leukocyte adherence inhibition test (lait), according to the respective number of outcomes over a cohort with 100 tests/subjects (y-axis) olivier et al.; asian j. immunol., vol. 7, no. 1, pp. 316-325, 2024; article no.aji.128121 321 the ttp showed a distribution concentrated over the more diluted titrations (fig. 1). there was no negative result. the mean was estimated at 1:343; the median was 1:512; the standard deviation was estimated at 1:187; the mode was 1:512 (appeared 53 times). all sia tests were negative. the lait showed a wide distribution range of results. the lai ranged from 0% to 95%. the mean was 41.5%; the median was 41.5%; the standard deviation was 27.3%; the mode was 0% (appeared sixteen times). the cascade distribution demonstrates a wide range spread of lai results (fig. 2). half of the patients presented moderate immunoreactivity during the ex vivo challenge test (lai = 22 to 60%). at the same time, one quarter displayed strong immunoreactivity, while the other quarter displayed low or no immunoreactivity. 4. discussion lanolin-based creams are frequently prescribed for patients with atopic and/or contact dermatitis due to their emollient properties [54]. the american contact dermatitis society elected lanolin as the "allergen of the year 2023" [55]. despite the emphasis borrowed by this title, there is a total lack of scientific studies about the physiopathology of the hypersensitivity (or hypersensitivities) against lanolin. the only tools to verify hypersensitivity against lanolin are the cutaneous tests. there is no record in the literature of any standardized lab examination (such as specific ige, associated cytokines, or any serologic technique) to diagnose lanolin hypersensitivity or suggest lanolin immunoreactivity. endotyping the mechanisms responsible for allergic phenotypes is crucial, not only for diagnosis but also for supervising treatments under the perspective of personalized medicine and recognizing differential diagnosis among phenotypes [56]. as a proof of concept, we submitted lanolin to an ex vivo challenge monitored by the lait to testify to cellular immunoreactivity. in the same proofof-concept mentality, we research precipitins against lanolin to testify to humoral immunoreactivity through the ttp. despite nonreactive or inconclusive skin tests, we propose these procedures to patients with atopic dermatitis and/or contact dermatitis with a strong clinical suspicion of hypersensitivity against lanolin. the clinical reasoning to indicate these tests is in the assumption that they may function as triage tests to reinforce the need for a more exhaustive exclusion-provocative test (when the patients exclude the suspected allergen until the symptoms disappear and then re-introduces the allergen to observe the reactions). there was no prospective plan. we spreadsheeted the results of a retrospective compilation of data produced by ttp and tial, exploring humoral and cellular immunoreactivity against lanolin. these assays provide clues about humoral and cellular immunoreactivity, and the results distribute themselves in an extensive spectral range between immune tolerance and symptomatic hypersensitivity. results provided by lait and ttp were interpreted as markers of the immune response after contact with the specific antigen, configuring themselves as techniques to identify exposition to the antigen, as proposed by the exposome-wide association study [57]. this retrospective survey demonstrated significative cellular and humoral immunoreactivity demonstrated by the ttp and the ex vivo challenge test monitored by lait against lanolin in two cohorts of patients with atopic and/or contact dermatitis. all patients evaluated with ttp demonstrated some degree of humoral immunoreactivity (there was no negative result), and most presented positivity by the more diluted titrations, demonstrating that lanolin allergens are widespread among patients with dermatitis and are highly immunogenic. sixteen patients did not present cellular immunoreactivity against lanolin (lai = zero%). in contrast, others presented an extensive range of inhibition of the leukocyte adherence after the ex vivo provocation test, suggesting that cellular immunoreactivity must be a better parameter to differentiate hypersensitivity reactions among patients. none of our patients presented an exclusive reaction to lanolin. we assessed every patient simultaneously with several chemical and biological allergens, demonstrating positive results for some of them, according to the clinical suspicions. the most vital suggestion driven by the results is that allergic patients may impair their symptoms by using creams or cosmetics produced with lanolin. in our practice, we have observed immediate cutaneous reactions (obtained by the skin scrape olivier et al.; asian j. immunol., vol. 7, no. 1, pp. 316-325, 2024; article no.aji.128121 322 test) and delayed reactions obtained by a fortyeight-hour contact test or a photosensitized ninety-six-hour contact test (patch test). based on this clinical experience, we can hypothesize that at least three endotypes are associated with lanolin hypersensitivity. the results presented by this work demonstrated that there is yet a lot unknown about the endotypes responsible for lanolin hypersensitivity, which may be produced by at least three mechanisms: a predominantly humoral, a predominantly cellular, and a compound of both. 5. limitations this study is a retrospective analysis of data collected over six years. there was no protocol research, and the subject's data was limited to the essentials available on our electronic sheets. therefore, we could not establish a crosscomparison between positive and negative controls to validate the results. the number of subjects is appropriate for a preliminary study; however, future studies must be more comprehensive. the lack of a research protocol implies the possibility of a bias produced by the physician's point of view, who suggested the exam barely on clinical suspicion led purely by the anamnesis and physical examination. the study lost many of these patients to follow-up, so assuring the relationship between the immunoassays' results and the patient's clinical outcome is impossible. 6. conclusion our preliminary results show that the lait and ttp may differentiate diverse cellular and humoral immunoreactivity degrees against lanolin in patients clinically diagnosed with non– ige-mediated allergies. this methodology can provide a socioeconomic impact since the technologies to perform tial and ttp are inexpensive and can be performed in a single lab room attached to the facilities with minimum laboratory equipment. however, the propaedeutic meaning of these results and the possibility of interferents must be better established [58]. more studies focused on the quality-by-design approach with prospective larger double-blind cohorts need to evaluate the potential contribution of lait and ttp for endotyping immunoreactivity of patients suspected of symptomatic hypersensitivity against lanolin and other similar food processing additives [59]. we are planning future work with control groups to draw more substantiated conclusions. 7. future directions and recommendations for clinical practice the primary intended use of in vitro or ex vivo allergen challenge tests is to spare the patients from being submitted to unnecessary, exhaustive, and dangerous in vivo challenge tests. exploring the humoral and the cellular arms of immune systems, the ttp and tial alone or combined may represent, in the near future, a tool for allergists to construct an etiologic diagnosis from their patients, as well as determine the endotypes (mechanisms) of hypersensitivity, in order to choose more convenient and personalized therapies for them. disclaimer (artificial intelligence) author(s) hereby declare that no generative ai technologies such as large language models (chatgpt, copilot, etc.) and text-to-image generators have been used during the writing or editing of this manuscript. ethical approval as a retrospective survey of results recorded in cognito, consent was given collectively by the institution's ethics committee following the principles of the declaration of helsinki [60]. acknowledgements the instituto alergoimuno de americana funded this work. we want to thank the laboratory technicians alessandra vieira de oliveira and luciana sacilotto carvalho for their help with the exams. competing interests authors have declared that no competing interests exist. references 1. osborne ed, murray pf. atopic dermatitis; 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cite as: olivier, celso eduardo, daiana guedes pinto, ana paula monezzi teixeira, cibele silva miguel, raquel acácia pereira gonçalves santos, jhéssica letícia santos santana, and regiane patussi santos lima. 2024. “endotyping cellular and humoral immunoreactivity against tartrazine in allergic patients: a retrospective study”. asian journal of immunology 7 (1):217-27. https://journalaji.com/index.php/aji/article/view/146. asian journal of immunology volume 7, issue 1, page 217-227, 2024; article no.aji.126461 endotyping cellular and humoral immunoreactivity against tartrazine in allergic patients: a retrospective study celso eduardo olivier a*, daiana guedes pinto a, ana paula monezzi teixeira a, cibele silva miguel a, raquel acácia pereira gonçalves santos a, jhéssica letícia santos santana b and regiane patussi santos lima c a instituto alergoimuno de americana, brazil. b instituto de ensino e pesquisa do hospital de amor de barretos, brazil. c lavoisier laboratórios, são paulo, brazil. authors’ contributions this work was carried out in collaboration among all authors. author ceo conceptualized the study, did data curation, formal analysis, literature review and wrote the original draft. authors dgp, apmt, csm, jlss and rpsl performed laboratory procedures. author rapgs performed cutaneous tests. all authors read and approved the final manuscript. article information doi: https://doi.org/10.9734/aji/2024/v7i1146 open peer review history: this journal follows the advanced open peer review policy. identity of the reviewers, editor(s) and additional reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here: https://www.sdiarticle5.com/review-history/126461 received: 22/10/2024 accepted: 06/11/2024 published: 11/11/2024 original research article https://doi.org/10.9734/aji/2024/v7i1146 https://www.sdiarticle5.com/review-history/126461 olivier et al.; asian j. immunol., vol. 7, no. 1, pp. 217-227, 2024; article no.aji.126461 218 abstract background: several publications report that tartrazine is responsible for ige-mediated and non– ige-mediated hypersensitivity reactions. there is no standardized lab exam to endotype non–igemediated immunoreactivity against tartrazine besides in vivo provocation tests. aim: to evaluate the potential of the tube titration of precipitins (ttp) and the leukocyte adherence inhibition test (lait) to endotype humoral and cellular immunoreactivity against tartrazine in patients clinically diagnosed with non–ige-mediated allergic phenotypes associated with various non-ige-mediated allergic conditions. study design: we retrospectively examined the medical charts of two cohorts of patients diagnosed with the aforementioned allergic phenotypes with clinical suspicion of tartrazine hypersensitivity, who were investigated with the help of ttp (first cohort) or ex vivo challenge tests monitored by lait (second cohort) against tartrazine. methodology: the registered results of the semi-quantitative serum ttp against 1 mg/ml tartrazine solution were distributed in ranges through a cascade distribution chart to outline the variability of the results inside the first cohort. the registered results of the leukocyte adherence inhibition (lai) percentage promoted by the ex vivo challenges with 1 mg/ml tartrazine solution were distributed in ranges through a cascade distribution chart to outline the variability of results inside the second cohort. the statistical characteristics of these cohorts were calculated. results: most positive ttp results concentrated on the higher dilutions. the mean was estimated at 1:290; the standard deviation was estimated at 1:200. the lai ranged from 0% to 88%. the mean was 35.4%; the standard deviation was 24,7%. the cascade distribution graph demonstrates that the lait distribution is mainly over the negative and weaker results. conclusion: our preliminary results support that the ttp and lait performed with 1 mg/ml tartrazine solution may discriminate diverse humoral and cellular immunoreactivity degrees in patients suffering from several non–ige-mediated allergic conditions, encouraging further prospective studies and validation. keywords: asthma; rhinitis; endotype; hypersensitivity; leukocyte adherence inhibition test; precipitins; tartrazine; urticaria. abbreviations lai : leukocyte adherence inhibition lait : leukocyte adherence inhibition test ttp : tube titration of precipitins 1. introduction tartrazine is a tasteless synthetic yellow-lemon water-soluble aromatic mono-azo dye used as its sodium salt, potassium salt, calcium salt, and aluminum lake [1]. azo dyes (organic compounds bearing the functional group r−n=n−r′ where r and r′ are aryl groups) such as tartrazine, sunset yellow, and carmoisine are additives aggregated to provide color to medicines and processed foods in products such as canned vegetables, chewing gum, frankfurters, macaroni, soft drinks, spaghetti, bread, butter, cheese, concentrated fruit juices, ice cream, jellies, tomato ketchup, jam, candies, pickles, and others, regardless any intrinsic nutrition value, preservative activity or health benefit [2,3]. tartrazine may be used alone or associated with aluminum lakes or blue colorants to produce green shades [1,4]. tartrazine is also known by the codes: e102, ins 102, ci 19140, fd&c yellow 5, yellow 5 lake, acid yellow 23, food yellow 4, and trisodium 1-(4-sulfonatophenyl)-4(4-sulfonatophenylazo)-5-pyrazolone-3carboxylate [5]. the european food safety authority panel calculated a theoretical maximum daily exposure to tartrazine of 8.1 mg/kg/day for adults and 13.1 mg/kg/day for children, establishing an acceptable daily intake (adi) of 7.5 mg/kg/day [6]. however, these limits do not represent enough environmental safety since they can damage aquatic fauna such as the freshwater zebrafish (danio rerio) [7]. tartrazine has been known to cause allergic reactions since 1958 [8]. the extensive clinical spectrum of tartrazine hypersensitivity includes urticaria, angioedema, anaphylactic shock, asthmatic bronchitis, rhinitis, throat tickle, cough, vasculitis (purpura), and contact dermatitis [9-14]. a patient with textile contact dermatitis after using a yellow cloth was diagnosed with the help of a skin contact test with tartrazine [15]. olivier et al.; asian j. immunol., vol. 7, no. 1, pp. 217-227, 2024; article no.aji.126461 219 the fact that tartrazine is a drug additive with no proper function besides coloring pills and solutions makes it one of the first aims to diagnose when facing allergic conditions elicited by colored medications such as antihistamines and steroids used to treat allergies and asthma [16]. tartrazine belongs to a select group of aromatic substances (aspirin, salicylates, sulfites, benzoates, and azo dyes) known to produce non–ige-mediated “allergic-like” dose-dependent symptoms such as urticaria and bronchospasm at remarkably similar molar doses in allergic patients submitted to progressive oral challenges [17-19]. these reactions appear to have a familiar incidence, presenting cross-reactivity among these substances [20]. tartrazine hypersensitivity has been associated with aspirin sensitivity; however, the mechanism is obscure since aspirin inhibits the cyclooxygenases responsible for producing prostaglandins, while tartrazine does not have this effect [21,22]. a multicenter study performed with patients with aspirin-induced asthma found a frequency of 2.6% of patients with tartrazine hypersensitivity [23]. due to tartrazine hypersensitivity, there is a worldwide concern to substitute tartrazine from medicines and industrialized food for natural colorants such as curcumin (e100), riboflavin (e101), beta-carotene (e160a), annatto (e160b), or other chemical dyes such as quinoline yellow (e104), or yellow iron oxide (e172) [24,25]. azo dyes are relatively small compounds that cannot interact alone with antibodies, so in hypersensitivity reactions, they function as haptens, usually linked to complex proteins, such as albumins [26]. commercial anti-tartrazine polyclonal antibodies are usually produced for research purposes by conjugating tartrazine with ovalbumin or allophycocyanin [27]. although one can find commercial lab kits to detect specific ige against conjugated tartrazine, antibodies of the ige class are unlikely to mediate tartrazine hypersensitivity [28]. the primary laboratory marker of patients reacting to dyes is eosinophilia [29]. a radioimmunoassay inhibition assay was designed to detect anti-tartrazine igd and ige antibodies. however, patients diagnosed with tartrazine hypersensitivity could be distinguished from controls only by their specific igd, not by their specific ige antibodies against tartrazine [30]. there is not yet any reliable routine lab exam to quantify immunoreactivity against tartrazine, and the clinical diagnosis of hypersensitivity to tartrazine is founded chiefly on in vivo tests based on oral or cutaneous provocations. cellular immunoreactivity against tartrazine (and other food additives) has already been demonstrated by ex vivo challenging tests monitored by the granulocytic myeloperoxidase release reaction [31]. ex vivo challenges employing leukocytes to detect immunoreactivity against tartrazine were also already monitored by sulfidoleukotriene production in allergic patients [32]. the direct effect of tartrazine on lymphocytes is suggested by cytotoxic experiments employing ex vivo experiments employing human lymphocytes [33]. ex vivo studies in cultured human leukocytes demonstrated that tartrazine at 70 μg/ml induces dna damage, suggesting a genotoxic potential [34]. the leukocyte adherence inhibition test (lait) and the tube titration of precipitins (ttp) are performed in our facilities as triage tests to identify immunoreactivity against suspected allergens executed before the performance of more exhaustive in vivo provocation tests [3541]. the present study hypothesizes that lait and ttp may differentiate endotypes and degrees of immunoreactivity against tartrazine among patients suffering from common allergic phenotypes. to evaluate the potential of the lait and the ttp to discriminate humoral and cellular immunoreactivity against tartrazine, we retrospectively compiled the electronic medical charts of patients clinically diagnosed with non– ige-mediated allergic phenotypes associated with chronic and/or recurrent conditions such as rhinitis, sinusitis, conjunctivitis, bronchitis, allergic contact dermatitis, intrinsic atopic dermatitis, urticaria systemic anaphylactic reactions and/or gastrointestinal disorders who were investigated with these procedures. 2. materials and methods 2.1 subjects after receiving institutional review board approval from the instituto alergoimuno de olivier et al.; asian j. immunol., vol. 7, no. 1, pp. 217-227, 2024; article no.aji.126461 220 americana (brazil; 08/2024), we reviewed the electronic chart of 9,500 outpatients who attended our facility from january 2018 to october 2024. the first cohort (ttp cohort) consisted of 100 outside patients clinically diagnosed with non–ige-mediated allergic phenotypes associated with chronic and/or recurrent conditions such as rhinitis, sinusitis, conjunctivitis, bronchitis, allergic contact dermatitis, intrinsic atopic dermatitis, urticaria systemic anaphylactic reactions and/or gastrointestinal disorders. these patients had been submitted to ttp with 1 mg/ml of tartrazine solution. the ttp cohort counted 23 males and 77 females; mean age 35,5 years; sd 21.8 years; range 1 to 79 years; median 35 years; modes = 9, 27, 42, 43, and 69 (each appeared four times); geometric mean = 25.3 years. the second cohort (lait cohort) consisted of 100 outside patients clinically diagnosed with non–ige-mediated allergic phenotypes associated with chronic and/or recurrent conditions such as rhinitis, sinusitis, conjunctivitis, bronchitis, allergic contact dermatitis, intrinsic atopic dermatitis, urticaria systemic anaphylactic reactions and/or gastrointestinal disorders. these patients had been submitted to an ex vivo allergen challenge test with tartrazine solution 1mg/ml monitored with lait. the lait cohort counted 36 males and 64 females; mean age 40 years; sd 21.2 years; range 4 to 90 years; median 38 years; mode = 22 (appeared four times); geometric mean = 33.2 years. this study did not include patients under biological and/or systemic anti-inflammatory therapy. these procedures were offered to patients with clinical suspicion of tartrazine hypersensitivity who demonstrated a nondetectable specific ige against tartrazine and a non-reactive or inconclusive skin test done with tartrazine 1 mg/ml solution [42]. 2.2 tartrazine solution the tartrazine solution was prepared with powdered tartrazine diluted with distilled water at 1 mg/ml to perform the allergic skin tests, ttp, and lait. 2.3 ex vivo investigation: leukocyte adherence inhibition test 2.3.1 procedure for allergen ex vivo challenging we performed the lait as previously described [43-52]. shortly, each donor's fresh plasma was divided into two parts and used in parallel ex vivo challenging tests with tartrazine acetate solution 1 mg/ml and the unchallenged plasma assay. we collected plasma with high leukocyte content (buffy coat) from the heparinized tube after one hour of sedimentation at 37°c. then, we distributed aliquots of 100 μl into eppendorf tubes kept under agitation for 30 minutes (200 rpm at 37°c) with tartrazine solution (10μl of a solution with 1mg/ml) or without tartrazine solution (when used as control). 2.3.2 procedure for adherence assay after incubation, the plasma was allocated into a standard neubauer hemocytometer counting chamber with a plain, non-metallic glass surface and left to stand for 2 hours at 37 °c in the humidified atmosphere of the covered water bath to allow leukocytes to adhere to the glass. next, we counted the leukocytes, removed the coverslip, and washed the chamber by immersion in a beaker with pbs (phosphatebuffered saline) at 37°c. then, we added a drop of pbs to the hemocytometer's chamber and allocated a clean coverslip over it. the remaining cells were counted in the same squares as previously examined. 2.3.3 procedure for calculation the percentage of leukocyte adherence (la) of each assay was estimated as: (the number of leukocytes observed on the hemocytometry chamber after washing divided by the number of leukocytes observed on the hemocytometry chamber before washing) and multiplied by 100 (%). the leukocyte adherence ratio (lar) was estimated based on the ratio between the la from the antigen-specific challenged plasma and the la from the unchallenged control plasma: lar = la of the challenged sample divided by la of unchallenged control plasma multiplied by 100 (%). to further calculate the leukocyte adherence inhibition (lai), we subtracted the lar from 100 (%). we employed the lai results for the cascade distribution chart and the statistics calculations, both performed with the help of the microsoft excel® statistical package. olivier et al.; asian j. immunol., vol. 7, no. 1, pp. 217-227, 2024; article no.aji.126461 221 2.4 in vitro investigation: tube titration of precipitins (ttp) as previously reported, the semi-quantitative ttp against the tartrazine solution was performed in a transparent vitreous tube array [53-55]. shortly, the patient’s blood was collected in a clot-activator collecting tube. after separation, the serum was centrifugated at 2,000 rpm for 10 minutes. the allergen extracts were allocated in sets of eleven glass tubes at progressive duplicated serum dilutions. the progressive dilutions were combined with the 15 μl of the antigen (1 mg/ml) with 250 μl of the patient’s serum, progressively diluted into physiological saline solution (nacl 0,9%) in the dilution ratios of 1:1; 1:2; 1:4; 1:8; 1:16; 1:32; 1:64; 1:128; 1:256; and 1:512. one tube was a blank control done with the water and serum to observe occasional spontaneous precipitation (sia test). after 24 hours, the tubes were examined, and the titers (the highest dilution factor that yields a positive reading) were recorded [56]. 3. results as a retrospective survey, there was no research protocol; therefore, we report the incidental immune investigation as registered in the digital medical charts. the cascade distribution graph showed a distribution range of ttp results. there was one negative result, while more positive results concentrated on the higher dilutions (fig. 1). the mean was estimated at 1:290; the median was 1:256; the standard deviation was estimated at 1:200; the mode was 1:512 (appeared 42 times). all sia tests were negative. the lai ranged from 0% to 88%. the mean was 35.4%; the median was 36.5%; the standard deviation was 24,7%; the mode was 0% (appeared nineteen times). the cascade distribution graph demonstrates distribution mostly over the negative and weaker results (fig. 2). nineteen patients (19%) ignored the allergen, presenting no inhibition of leukocyte adherence (lai = 0%) after contact of the plasma with the tartrazine solution. some patients showed low or moderate immunoreactivity during the ex vivo challenge test, while most displayed strong immunoreactivity, suggesting tartrazine's participation in the hypersensitivity condition. 4. discussion to detect humoral and cellular immunoreactivities against tartrazine, we retrospectively compiled the data registered in an excell® spreadsheet resulting from ttp and tial against tartrazine at our facilities. these triage tests are performed before the in vivo provocation tests when skin tests are unfeasible due to the patient’s skin conditions. the humoral and the cellular immunoreactivity profiles present divergent results. the humoral profile, represented by the ttp, presented the most results in the more diluted titrations, suggesting that most patients produced prominent levels of anti-tartrazine antibodies. however, the cellular profile, represented by the tial, presented most results in the negative or the weaker lai results, suggesting a less determinant participation of the cellular immunoreactivity. the fact that the results were obtained from two cohorts and measured by different methodologies (quantitative and semiquantitative) did not allow us to perform a paired t-test analysis. the primary strategy of personalized medicine is to diagnose the endotypes responsible for the disease’s phenotypes [57]. the former limited capacity to diagnose hypersensitivity had created in physicians the common idea that diagnosing a single hypersensitivity would be enough to treat their patients, conferring them a “monosensitization label”. as medical knowledge and resources advance, more diagnoses are being performed, making physicians aware that polisensitization is more a rule than an exception. panallergens responsible for cross-reactivity between allergens from diverse sources, such as tropomyosins or profilins, contribute to this awareness, allowing a more extensive comprehension of the allergic conditions [58,59]. cross-reactivity among azo dyes and similar preservatives, such as the sulfides, a group of inorganic salts added to processed foods and also naturally found in allium spices and fermented beverages, is a large field for studies that must be explored to understand hypersensitivity conditions better, endotyping their mechanisms, and tailor the treatment of allergic patients [60,61]. the great insight gell and coombs gave in the sixties resisted the test of time and now is amplified as a compass to understand the wide variety of hypersensitivity reactions that are still olivier et al.; asian j. immunol., vol. 7, no. 1, pp. 217-227, 2024; article no.aji.126461 222 far from being completely elucidated [62,63]. endotyping underlying hypersensitivity mechanisms may help distinguish superimposable phenotypes presenting similar symptoms that may hamper establishing a precise diagnosis [64]. ttp and lait assays do not identify the exact immune mechanisms responsible for clinical hypersensitivity; instead, they are general immune markers of the humoral and cellular responses against allergens, quantifying an exposome measurement [65]. this retrospective proof of concept analysis demonstrated several degrees of humoral and cellular immunoreactivity, as demonstrated by ttp and lait against tartrazine in two cohorts of patients with several allergic conditions. none of the patients presented an exclusive reaction to tartrazine. every patient was simultaneously evaluated for other suspected allergens, demonstrating positive and negative results. our results suggest that tartrazine can elicit humoral and cellular immunoreactivity in allergic patients, theoretically able to impair their allergic symptoms. fig. 1. cascade distribution chart of the tube titration of precipitins (ttp on the x-axis) resulting from the tartrazine solution against the serum of a cohort of 100 tests/subjects (y-axis) fig. 2. cascade distribution chart of the range groups of leukocyte adherence inhibition (lai) results (x-axis %) of ex vivo tartrazine solution monitored by the leukocyte adherence inhibition test (lait), according to the respective number of outcomes over a cohort with 100 tests/subjects (y-axis) olivier et al.; asian j. immunol., vol. 7, no. 1, pp. 217-227, 2024; article no.aji.126461 223 5. conclusion in vitro humoral assays and ex vivo cellular challenges can detect immunoreactivity against potentially lethal allergens without posing any risk for the patient and are occasionally employed in daily routine when conventional allergy diagnostic procedures are not elucidative or contra-indicated [66]. our preliminary results show that the ttp and lait may differentiate diverse degrees of immunoreactivity against tartrazine in patients clinically diagnosed with non–ige-mediated cutaneous allergies. this methodology can provide a socioeconomic impact since the methodologies to perform ttp and lait are inexpensive and can be performed in a single lab attached to the facilities with minimum laboratory equipment. however, the propaedeutic meaning of these results and the possibility of interferents must be better established [67]. more studies focused on the quality-by-design approach with prospective larger double-blind cohorts need to evaluate the potential contribution of ttp and lait for endotyping immunoreactivity of patients suspected of symptomatic hypersensitivity against tartrazine and other similar food processing additives [68]. 6. future directions and recommendations for clinical practice the primary intended use of in vitro or ex vivo allergen challenges is to spare the patients from being submitted to exhaustive and dangerous in vivo challenge tests. exploring the humoral and the cellular arms of immune systems, the ttp and tial alone or combined may represent, in the near future, a tool for allergists to construct an etiologic diagnosis from their patients, as well as determine the endotypes (mechanisms) of hypersensitivity, in order to choose more convenient and personalized therapies for them. 7. limitations this study is a proof-of-concept retrospective analysis of data collected over six years. there was no protocol research, and the subject's data was limited to the essentials available on our electronic sheets. therefore, we could not establish a cross-comparison between positive and negative controls to validate the results. the number of subjects is appropriate for a preliminary study; 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brian.churchill@iqvia.com; asian journal of immunology 3(1): 208-222, 2020; article no.aji.57464 tnf-α, tnf receptors and their complex implications in therapy brian mark churchill1*, pallavi patri2,3, rashna cama4 and jula k. inrig5,6 1 department of medical science and strategy (asia), iqvia, etamin block, prestige tech park, kadubeesanahalli, bengaluru, karnataka, 560103, india. 2 department of nephrology, columbia asia hospital – sarjapur road, ambalipura, bengaluru, karnataka, india. 3 weill cornell medical college, new york, usa. 4 department of medical sciences and strategy (asia), iqvia, natraj by rustomjee, 702 western express highway, sir mathuradas vasanji road junction, mumbai, maharashtra, 400069, india. 5 global head renal center of excellence, therapeutic science and strategy unit, iqvia, 10188 telesis ct suite 400, san diego, ca 92121, usa. 6 department of medicine, uc irvine and duke university, usa. authors’ contributions this work was carried out in collaboration among all authors. author bmc conceptualized and designed the review paper, performed the literature search, wrote the paper and wrote the first draft of the manuscript, collected feedback, incorporated the changes, prepared subsequent drafts and then the final manuscript. authors pp, rc and jki reviewed the manuscript and helped in shaping it. all authors read and approved the final manuscript. article information editor(s): (1) dr. cynthia aracely alvizo báez, autonomous university of nuevo leon, mexico. reviewers: (1) joseli lannes vieira, brazil. (2) juei-tang cheng, christian university, taiwan. complete peer review history: http://www.sdiarticle4.com/review-history/57464 received 10 may 2020 accepted 02 june 2020 published 10 june 2020 abstract tumor necrosis factor alpha (tnf-α), also called cachectin or cachexin, is a naturally occurring cytokine produced by activated macrophages and monocytes, and to a smaller scale by tlymphocytes, b-lymphocytes, fibroblasts and astrocytes. it has both inflammatory and immunomodulatory roles effectuated through its action on tumor necrosis factor receptor-1 (tnfr1) and tumor necrosis factor receptor-2 (tnfr2). tnf-α overexpression (especially of tnfr1 mediated actions) or under expression (especially of tnfr2 mediated actions) may review article churchill et al.; aji, 3(1): 208-222, 2020; article no.aji.57464 209 contribute to several diseases including rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, crohn’s disease, ulcerative colitis, and juvenile inflammatory arthritis. several anti-tnf therapies are proven to be beneficial in these diseases, including etanercept, infliximab, adalimumab, certolizumab and golimumab. biological responses of tnf-α include inflammation, apoptosis, immunomodulation, tissue repair, antiviral and antitumor activity and mediation of endotoxin-induced septic shock, recruitment of inflammatory cells including neutrophils, monocytes and lymphocytes to sites of infection; and cell activation through action on different receptors (tnfr1 and tnfr2). low grade chronic inflammation occurs in several other diseases including atherosclerosis, hypertension, chronic kidney disease, alzheimer’s disease, parkinson’s disease and multiple sclerosis. researchers have explored whether anti-tnf therapies can be helpful in these diseases too. both positive and negative effects of anti-tnf therapies have been observed in these diseases. these observations suggest that tnf-α and tnf receptors system have an incredibly complex pathophysiologic role, and hence blockade of this pathway results in complex effects on different organ systems. in this review we have explored the myriad effects on various organ systems due to tnf blockade, including covid-19. keywords: tumor necrosis factor alpha (tnf-α); rheumatoid arthritis; covid-19; tnfr1; tnfr2. 1. introduction tumor necrosis factor (tnf) is a naturally occurring inflammatory cytokine the tnf superfamily that consists of 19 members (ligands) [1]. they are type ii transmembrane proteins that can bind to 29 receptors of the tnf receptor superfamily including fas, cd40, cd27 and rank [2]. tnf-α, also called cachectin or cachexin, is produced by activated macrophages and monocytes, and to a smaller scale by tlymphocytes, b-lymphocytes, fibroblasts, and astrocytes [3]. it can bind to tnfr1 (tumor necrosis factor receptor-1) and tnfr2 (tumor necrosis factor receptor-2), and has both inflammatory and immunomodulatory roles [4]. it has been implicated in pathogenesis of several diseases including rheumatoid arthritis, systemic lupus erythematosus, ulcerative colitis and crohn’s disease [1,3]. tnf-α and tnf receptors system is complex and has complicated implications in therapy [5]. anti-tnf agents including etanercept, infliximab and adalimumab are helpful in treatment of many diseases where chronic inflammation is involved for example, rheumatoid arthritis, psoriatic arthritis, ulcerative colitis, and crohn’s disease [5-8]. these therapies may predispose patients to increased risk of infections (including reactivation of tuberculosis), cardiovascular events, autoimmune diseases, malignancy, hepatitis, nephritis and tubulointerstitial diseases, as has been discussed later in this paper. several other diseases including atherosclerosis, hypertension, chronic kidney disease, alzheimer’s disease, parkinson’s disease and multiple sclerosis also show evidence of lowgrade chronic inflammation. researchers have explored whether anti-tnf therapies can be helpful in these diseases too. both positive and negative effects of anti-tnf therapies have been observed in these diseases. these observations suggest that tnf-α and tnf receptors system have a complex pathophysiologic role, and hence blockade of this pathway results in complex effects on different organ systems. in this review we have explored the effects of tnf blockade on various organ systems. 2. biological properties of tnf-α and tnf receptors 2.1 tumor necrosis factor-α tumor necrosis factor-α (tnf-α) was discovered by lloyd j old together with elizabeth carswell in 1975 [6,9]. they discovered that tnf-α is produced by lymphocytes and macrophages that cause lysis of specific type of cells like tumor cells [6]. this discovery led to the discovery of a number of other cytokines including lymphotoxin alpha (tnf-β), cd40l, cd27l, and fasl [6,9]. biological responses of tnf-α include inflammation, apoptosis, immunomodulation, tissue repair, antiviral and antitumor activity and mediation of endotoxin-induced septic shock. it is involved in recruitment of inflammatory cells including neutrophils, monocytes and churchill et al.; aji, 3(1): 208-222, 2020; article no.aji.57464 210 lymphocytes to sites of infection; and cell activation through its action on different receptors (tnfr1 and tnfr2) [3,4,10]. tnf-α exhibits both pro-inflammatory and immunoregulatory properties. in addition to its function as an endogenous pyrogen, tnf-α has been shown to initiate a powerful inflammatory response by stimulating the generation of other inflammatory cytokines such as interleukin-6 (il6), interleukin-8 (il-8), interleukin-10, corticosteroids, and metalloproteinases [3,10]. once the triggering factor for the immune system activation is eliminated, tnf-α steps in again to control and resolve the inflammatory process [3]. the gene that codes for human tnf-α is located on short arm of chromosome 6, linked to human leucocyte antigen (hla) b [11-13]. there are two forms of tnf-α: transmembrane tnf-α or membrane-associated tnf-α (mtnf-α) and soluble tnf-α (stnf-α). membrane-associated tnf-α (mtnf-α) is cleaved by metalloproteinases adam-17[tnf-α converting enzyme (tace)] to soluble tnf-α [stnf-α] that can subsequently be detected in blood plasma [4,6,11,14]. please refer to table 1 for an overview [4,6,11-14]. 2.2 tnf receptors tnf-α acts through two cell-surface receptors tumor necrosis factor receptor 1 (tnfr1), also known as p55, tnfrsf1a, cd120a; and tumor necrosis factor receptor 2 (tnfr2) also known as p75, tnfrsf1b, cd120b [15-17]. although both tnfr1 and tnfr2 can bind with the soluble and transmembrane forms of tnf-α, tnfr2 has high affinity for the transmembrane form of tnf-α [16]. jonathan holbrook et al have described tnf-α and tnf receptors in detail [14]. tnfr1 is expressed on almost all cells [14]. it has ‘death domain or dd’ in its intracellular region. when tnf-α binds with tnfr1, it leads to recruitment of tnfr1-associated dd (tradd) [14]. this triggers an inflammatory response or cell survival response through activation of nf-kb (nuclear factor kappa-light-chain-enhancer of activated b cells), or cell death response depending on the physiological state of the cell [3,14,17]. tnfr2 is expressed in immune cells [predominantly in t regulatory cells (tregs)], neurons and endothelial cells [3,14]. besidesleading to direct actions after tnf-α binding occurs, tnfr2 may have a ‘ligandpassing’ role as well: soluble tnf-α may dissociate from tnfr2 rapidly and then bind with tnfr1 [16,17]. there is no death domain (dd) in tnfr2. after interacting with tnf-α, tnfr2 binds with the proteins tnfr-associated factors 1 and 2 (traf1 and traf2) activating nf-kb. nf-kb then triggers a cell survival response including immune modulation, inflammation and tissue regeneration depending on the conditions [3,14,17]. tnfr2 has an important role in pancreatic regeneration, protection of cardiomyocytes, remyelination, survival of some neuronal cells and in stem cell proliferation too [3]. since tnfr2 does not bind with tradd, it does not lead to cell death response. please refer to fig. 1. please note that the figure is symbolic for easy understanding. it is not representative of true molecular structure of tnf or its receptors. 3. tnf-α polymorphism and diseases tnf-α polymorphisms may have an influence on susceptibility of diseases. a few examples are given below. 3.1 rheumatoid arthritis role of tnf-α -308g/a polymorphism and differential expression of tnf-α in pathogenesis table 1. the two forms of tnf-α soluble tnf-α transmembrane tnf-α amino acids 157 amino acid residues 233 amino acid residues molecular weight 17 kda 26 kda receptor interaction attaches to type 1 and type 2 tnf receptors (tnf-r1 and tnf-r2); cellular actions mainly mediated through tnf-r1. attaches to tnf-r1 and tnf-r2; actions mainly mediated through tnf-r2. sites of action acts at sites distant from tnf-α producing cells. acts locally churchill et al.; aji, 3(1): 208-222, 2020; article no.aji.57464 211 fig. 1. an overview of tnf-α signaling pathway of rheumatoid arthritis was evaluated by somdatta das, et al. [18]. they conducted a case-control study, including 126 rheumatoid arthritis patients and160 community matched age and sex controls. the study duration was 3 years. they found that presence of tnf-α -308a allele and differential tnf-α expression are associated with increased risk susceptibility for rheumatoid arthritis [18]. this study suggests that polymorphism of tnf-α has a role in pathogenesis of rheumatoid arthritis. 3.2 systemic lupus erythematosus y-j lin, et al investigated the association of tnfalpha genetic polymorphisms (-1031t/c, 863c/a, -857t/c, -308a/g and +489a/g) in sle patients and controls in taiwanese population [19]. their observations included [19]:  the frequency of the polymorphisms at 1031 and -857 were significantly different in patients with antinuclear antibodies and hematological disorders respectively.  the frequency of a allele of the polymorphisms at -308 was significantly increased in patients with malar rash, discoid rash, photosensitivity, oral ulcers and serositis.  the frequency of the polymorphisms at +489 was significantly different in patients with discoid rash and photosensitivity. churchill et al.; aji, 3(1): 208-222, 2020; article no.aji.57464 212 julian ramírez-bello, et al investigated whether the tnf -238g/a, -308g/a, -376g/a (rs1800750), and -1031t/c (rs1799964) polymorphisms are associated with sle or lupus nephritis susceptibility in a mexican population [20]. the study included 442 patients with sle and 495 controls. the investigators found that tnf -238g/a and -1031t/c polymorphisms were associated with sle susceptibility [20]. these observations of different investigators suggest that tnf-alpha genetic polymorphisms contribute to increased sle susceptibility. 3.3 crohn’s disease genoile santana, et al conducted a case-control and cross-sectional study to analyze whether tnf-α -308 polymorphism is associated with crohn's disease and its clinical features [21]. they enrolled 91 patients with crohn's disease and 91 controls (either with gastroesophageal reflux disease or functional dyspepsia) in salvador [21]. the investigators observed that high tnf-α producing predicted phenotype was associated with the penetrating form of crohn’s disease and colectomy. they found that the tnf-α -308 polymorphism was not associated with the perianal disease [21]. 4. tnfr1 gene polymorphism and diseases 4.1 tumor necrosis factor receptorassociated periodic syndrome (traps) missense mutations of tnfr1 gene results in development of tumor necrosis factor receptorassociated periodic syndrome (traps), an autosomal dominant disease [22]. the gene mutation results in structural alteration of tnfr1 receptor rendering it nonfunctional and its retention in endoplasmic reticulum [23]. the disease is associated with unprovoked prolonged periodic or episodic fever (fever lasting from a few days to months, average duration 21 days), severe abdominal pain, localized inflammation affecting multiple organs, and migrating rash on the limbs [23]. since these inflammatory episodes are unprovoked, they are described as ‘autoinflammation’ [14]. there is an elevation of cytokines il-6 and tnf-α in this disease [23]. there is a paradox in the pathogenesis of traps: as tnfr1 is associated with inflammation, nonfunctional tnfr1 should not lead to inflammation, contrary to the observation of inflammation affecting multiple organs in this disease [23]. this suggests that mechanism of action of the tnf-α and tnf receptors is complex, and further studies are needed to elucidate these mechanisms. treatment modalities include high doses of prednisolone, high dose non-steroidal antiinflammatory drugs, colchicine, etanercept, anakinra (il-1 receptor antagonist), canakinumab (anti-il-1β), and tocilizumab (anti-il-6) but each have variable success [14,23]. presently, antiinterleukin1β therapy is the standard to treat more severe cases of traps [14]. 4.2 susceptibility to develop invasive pulmonary aspergillosis investigators have found that polymorphism of both tnfr1 and tnfr2 is associated with increased susceptibility to invasive pulmonary aspergillosis. sainz et al found that tnfr1 gene polymorphism results in greater susceptibility to invasive pulmonary aspergillosis [24]. the group had found in an earlier study that tnf-α gene polymorphism or tnf-α levels had no correlation with susceptibility to invasive pulmonary aspergillosis, while polymorphism in tnfr2 gene was strongly associated with susceptibility to the infection [25]. 5. tnfr2 gene polymorphism and diseases tnfr2 gene polymorphism may affect susceptibility and/or severity of some diseases. a few examples are given below. in all these examples, the sample size is small. more studies are needed to evaluate this area. 5.1 schizophrenia renata suchanek-raif, et al conducted a case control study involving 401 patients and 657 controls to study whether the three single nucleotide polymorphisms (rs3397, rs1061622, and rs1061624) in tnfr2 gene were associated with a predisposition to paranoid schizophrenia in caucasian population [26]. they observed that tnfr2 gene polymorphism of rs3397, rs1061622, and rs1061624 are associated with a higher risk of developing schizophrenia and a more severe course of progression in males [26]. in this study, the rs3397 single nucleotide polymorphisms were found to be protective for women [26]. these observations suggest that tnfr2 gene polymorphisms churchill et al.; aji, 3(1): 208-222, 2020; article no.aji.57464 213 table 2. anti-tnf therapeutic agents tnf inhibitor mechanism of action specificity dosage schedule approved indications etanercept fully human soluble tnf receptor inhibitor sol tnf, tmtnf, subcutaneous injection weekly ra, pa, jia, as lymphotoxin a infliximab mouse chimeric monoclonal antibody to tnf-α sol tnf, tmtnf intravenous injection every 6-8 weeks ra, pa, jia, as, cd, uc adalimumab fully human monoclonal antibody against tnf-α sol tnf, tmtnf subcutaneous injection every 2 weeks ra, pa, jia, as, uc, cd certolizumab human fab fragment against tnf attached to 2 polyethylene glycol molecules sol tnf, tmtnf subcutaneous injection every 4 weeks ra, pa, as, cd golimumab fully human monoclonal antibody against tnf-α sol tnf, tmtnf subcutaneous injection every month ra, as, pa, uc ra-rheumatoid arthritis; pa-psoriatic arthritis; asankylosing spondylitis; jia-juvenile inflammatory arthritis; uc ulcerative colitis; cdcrohn’s disease; sol tnfsoluble tumor necrosis factor; tm tnftransmembrane tumor necrosis factor have a role in determining severity of schizophrenia. 5.2 coronary artery disease v.h sankar, et al conducted a study to evaluate whether polymorphism at position 196 in exon 6 of tumor necrosis factor 2 (tnfr2) gene is associated with coronary artery disease [27]. the investigators found that the incidence of coronary artery disease in patients with mm genotype was 65% and in those with rm genotype was 42% [27]. the investigators concluded that mm genotype of tnfr2 increases the risk of developing coronary artery disease, and the rm genotype has a protective role [27]. 5.3 chagas disease libeth criado, et al included 313 chagas disease patients from colombia who were serologically positive for trypanosoma cruzi [28]. out of these 313 patients, 159 had cardiomyopathy, and 154 were asymptomatic [28]. the investigators found that distribution of the tnfa -1031c and -308a alleles between cardiomyopathic and asymptomatic subjects was significantly different [28]. the investigators suggested that tnfa 1031c and -308a gene polymorphisms may influence susceptibility to develop cardiomyopathy in chagas disease [28]. 6. anti-tumor necrosis factor therapeutic agents tnf-α is involved in pathogenesis of several diseases including rheumatoid arthritis, systemic lupus erythematosus, ulcerative colitis and crohn’s disease [1,3]. anti-tnf therapeutic agents are used for several therapeutic indications including rheumatoid arthritis (ra), ankylosing spondylitis (as), psoriatic arthritis (pa), juvenile inflammatory arthritis (jia), crohn’s disease (cd) and ulcerative colitis (uc) [5-8]. table 2 depicts a brief overview of these agents [5-8,29]. 7. impact of tnf blockade on different organ systems 7.1 immune system and infections the pro-inflammatory role of tnf-α is a vital component of host defense against infections [10]. tnf-α blockers increase the risk of infections (bacterial, viral, and mycotic), including tuberculosis, histoplasmosis, candidiasis, aspergillosis, listeriosis, infection with atypical mycobacterium, pneumocystis carinii [30,31]. lymphoma, neuropathy and bowel obstruction have also been noted with the use of anti tnf-α therapeutic agents [31]. tnf-α together with other cytokines including interferon-delta (ifn-δ), generates a cellmediated immune response to intracellular pathogens including mycobacterium tuberculosis, listeria and histoplasma species leading to granuloma formation [10]. granuloma formation is required to contain the growth of and for the resolution of these infections [10]. use of anttnf-α therapeutic agents inhibits this cell churchill et al.; aji, 3(1): 208-222, 2020; article no.aji.57464 214 mediated immune response and granuloma formation, hence increasing the risk of granulomatous diseases including tuberculosis. reactivation of infections may also occur, including reactivation of latent tuberculosis and hepatitis b virus [30,31]. use of live vaccines during anti-tnf therapy is not recommended, as this may cause vaccine associated disease, including disseminated infection [32]. the risk of serious infections in highest in the first few months. the incidence of infection declines over time. galloway et al evaluated and compared data of 11,798 anti-tnf treated patients and 3,598 non biologic diseasemodifying antirheumatic drugs (nbdmard) treated rheumatoid arthritis patients. they observed that the incidence rate of serious infections was 42 per 1000 patient-years in antitnf treated patients, and 32 per 1000 patientyears in nbdmard treated patients indicating a significant increase in serious infection risk with the use of anti-tnf therapy [33]. the adjusted serious infection rate was 20% higher in anti-tnf treated individuals than nbdmard treated subjects [33]. this risk was highest in the first six months of therapy, and then started to decline [33]. liao et al conducted a meta-analysis and found that the risk of serious infections defined as those that were life‐threatening, requiring hospitalization and/or intravenous antibiotic therapy, or leading to significant disability/death, and general infections (infections regardless of severity and microorganisms) were lower in etanercept (soluble receptor) treated individuals, as compared with the monoclonal antibodies (adalimumab and infliximab) [34]. the risk of tuberculosis was also lower in etanercept treated individuals as compared to the individuals treated with adalimumab or infliximab [34]. 7.1.1 leprosy tnf plays an important role in pathogenesis of nerve lesions in leprosy. high levels of tnf have been detected in reactional skin lesions in leprosy [35]. tnf may be involved in early stages of schwann cell infection by mycobacterium leprae [35]. tnf induces il-6 and il-8 production that contribute to neuroinflammation [35]. mycobacterium leprae also induces il-23 production in schwann cells that contributes to demyelination [35]. more studies are required to evaluate the effect of antitnf therapy in patients with leprosy. 7.1.2 chagas disease chagas disease is caused by protozoan parasite trypanosoma cruzi [36]. chronic chagas disease cardiomyopathy (chronic chagasic cardiomyopathy), the main manifestation of chagas disease, is associated with heart failure [36,37]. elevated levels of tnf-α occur in chronic chagas disease cardiomyopathy and are related to the severity of left ventricular dysfunction [36,37]. low grade inflammation occurs in cardiac tissue in presence of elevated tnf and interferon-γ levels. pereira i.r found in their experimental studies in mice that short term anti-tnf therapy together with trypanocidal treatment may have beneficial effect in chronic chagasic cardiomyopathy [36]. however other investigators have observed conflicting results in animal studies, and have noticed that anti-tnf therapy may actually aggravate the cardiomyopathy [37]. more studies are required to evaluate the effect of anti-tnf therapy in chronic chagasic cardiomyopathy. 7.1.3 covid-19 high mortality associated with coronavirus disease 2019 (covid-19) has prompted researchers worldwide to accelerate the research activities to find possible safe and effective treatment options and preventive measures including vaccines [38]. severe acute respiratory syndrome coronavirus 2 (sars-cov-2) that causes covid-19, enters the host cells through angiotensin converting enzyme-2 expressed by type ii surfactant-secreting alveolar cells in the lungs [38]. the most important mediators that drive inflammatory response to covid-19 are not completely known. upregulation of different cytokines including interleukin-1 (il-1), interleukin-6 (il-6), tnf, and interferon-γ occurs in covid-19 [38]. ‘cytokine storm’, in which too many cytokines are suddenly released into circulation, can cause severe inflammatory response that causes more harm than good. marc feldmann, et al have discussed this topic in detail and have proposed that clinical trials are needed to evaluate safety and effectiveness of anti-tnf therapies, particularly infliximab and adalimumab [38]. they speculate that these antitnf agents may be beneficial in decreasing the intensity of the ‘cytokine storm’, reduce inflammation, and help in severe covid-19 [38]. churchill et al.; aji, 3(1): 208-222, 2020; article no.aji.57464 215 7.2 autoimmune disorders the mechanisms leading to disease through tnf-α and tnf receptors pathway are complex showing both positive and negative effects on autoimmune disorders. a few cases have been reported of etanercept and adalimumab inducing systemic lupus erythematosus and lupus serositis [39-43]. but there are other reports favoring anti-tnf therapeutic agents having beneficial effect on these autoimmune diseases. uppal, et al investigated the effect of infliximab in active systemic lupus erythematosus subjects and found that the drug significantly decreases the systemic lupus erythematosus disease activity index (sledai). they concluded that anti-tnf therapeutic agents are interesting candidates that can be explored further for treatment of active sle [44]. there are conflicting experiences with other anti-tnf therapies and autoimmune diseases too. it is known that there is deficiency of tnfr2 in several autoimmune diseases that could result in uncontrolled inflammatory activity through soluble tnf-α, tnfr1 pathway. instead of using non-specific anti-tnf-α therapeutic agents that cause varying impact on both tnfr1 and tnfr2 pathways resulting in unpredictable consequences, using specific agents to target tnfr1 or tnfr2 pathways may yield better and predictable results. yang et al have proposed that either using agents that specifically block tnf-α or tnfr1 and preserve the available tnfr2; or using tnfr2 agonists to increase the number of tnfr2 and promote tissue regeneration may prove to be good therapeutic strategies in autoimmune diseases [3]. further studies are required to evaluate whether these strategies are indeed beneficial and could be safely implemented. 7.3 cardiovascular system tnf-α levels have an important relationship with cardiovascular diseases and events including congestive heart failure [30,31]. the level of tnf-α has been shown to be predictive of recurrent myocardial infarction [43]. also, tnf-α level may play a role in hypertension, dyslipidemia, and atherosclerosis [45-48]. investigators have proposed that tnf-α induces atherogenesis by promoting induction of vascular cell adhesion molecule-1 (vcam-1) and intercellular adhesion molecule-1 (icam-1) expression by endothelial cells and vascular smooth muscle cells, endothelial cell apoptosis, and smooth muscle cell migration and proliferation [45]. interestingly, the cellular location of tnfr1 may have effects on atherogenesis. as an example, tnfr1 in macrophages may decrease atherogenesis, whereas the tnfr1 in the arterial walls may promote atherogenesis [45,48]. tnf-α might have a role in arterial inflammation and intimal hyperplasia [15]. kitagaki et al showed that tnf-α is an important contributor to arterial inflammation in mice and that blockage of tnfr1 inhibits intimal hyperplasia following experimentally induced arterial inflammation (post injury) [15]. tnfr2 signaling mediates inhibition of neointimal formation by decreasing adherent cells and apoptosis of endothelial cells as well as the activation, proliferation and migration of endothelial cells [15]. 7.4 malignancy tnf-α is an important cytokine that is involved in tumor surveillance and hence anti-tnf therapy theoretically poses a risk for developing malignancies. this has been substantiated in several studies indicating that anti-tnf therapeutic agents may be associated with an increased risk of malignancies including lymphoma, breast cancer, colorectal cancer, and melanoma [30,31,49]. berghen et al observed incidence of malignancy in 365 rheumatoid arthritis patients receiving antitnf therapy at a single center from january 2000 until january 2012 that received anti-tnf therapy [49]. they observed occurrence of 34 malignancies in 30 patients after the start of antitnf treatment. among these, 20 patients developed a solid malignancy, 6 a hematologic, 2 a solid and a hematologic malignancy, and 2 patients developed multiple (2 solid) malignancies. they also observed that some immune modulation-related lymphoproliferative disorders regressed spontaneously after stopping anti-tnf therapy. they concluded that the malignancy risk in rheumatoid arthritis patients treated with anti-tnf therapy was slightly higher than in the normal population [49]. however, there are other studies that have shown conflicting results. mercer et al compared the rates of solid tumors in 11,767 anti-tnf treated patients with 3,249 non-biologic or synthetic disease modifying anti-rheumatic drugs (s-dmardsincluding methotrexate, azathioprine and cyclophosphamide) in 5-year data was obtained from british society for churchill et al.; aji, 3(1): 208-222, 2020; article no.aji.57464 216 rheumatology biologics register [50]. none of these patients had prior history of cancer. they observed that solid tumors developed in 81 per 10,000 patient-years in anti-tnf treated patients, and 117 per 10,000 patient-years in s-dmards treated patients. there was no statistically significant difference in risk of solid cancer for anti-tnf treated versus sdmard treated patients [50]. another study by min jung et al also showed similar results [51]. the investigators collected information of 45,423 seropositive rheumatoid arthritis patients from korean nationwide health insurance claims data. they compared incidence of tumor in patients receiving anti-tnf therapy (2,337 patients), and those receiving conventional synthetic disease-modifying anti-rheumatic drugs (csdmardsincluding methotrexate, leflunomide, hydroxychloroquine, sulfasalazine, cyclosporine, tacrolimus, azathioprine, mizoribine, and bucillamine) (43,086 patients) from the 5 years of data. they found that 1732 patients in csdmard group and 49 patients in anti-tnf group developed malignancy. on statistical analysis, the investigators found that incidence of cancer was similar in ra patients treated with anti-tnf and csdmards [51]. 7.5 nervous system increased production of tnf-α has been noticed in several central nervous system disorders including multiple sclerosis, alzheimer’s disease, parkinson’s disease, hiv encephalopathy, meningitis, stroke, demyelinating disorders, neuropathy, and myelosuppression [30,31,5256]. high levels of tnf in nervous system disorders promote demyelination, axonal degeneration and increased permeability of blood brain barrier [35]. tnfr2 has a neuroprotective role and serves an important role in oligodendrocyte regeneration and remyelination [3,53]. fischer et al synthesized a soluble, human tnfr2 agonist (tnc-sctnfr2) by genetic fusion of tenascin c to a tnfr2-selective single-chain tnf molecule and showed that tnc-sctnfr2 rescues human differentiated neurons from oxidative stress induced cell death [57]. they speculated that tnfr2 agonists may be good therapeutic options in several diseases, including neurodegenerative, cardiac and autoimmune and diseases [57]. alzheimer’s disease: tnf-α and tnfr1 levels are increased, and tnfr2 levels are decreased in human alzheimer’s disease brain tissues [30,53]. though theoretically it appears that antitnf therapy should be helpful in alzheimer’s disease, investigators have observed conflicting results. as an example, experimental studies in animals have shown that infliximab that binds tnf-α (anti-tnf-α therapy) reduced amyloid plaques and tau phosphorylation in alzheimer’s disease in mice as early as 3 days after the intracerebroventricular injection; and improves object recognition memory impairment [58,59]. shi et al reported that intrathecal injection of infliximab improved cognition significantly in a female suffering from alzheimer’s disease [60]. peri-spinal injection of etanercept has been shown to improve cognitive impairment within minutes of administering the injection [61]. it is generally accepted that total inhibition of tnf (especially inhibition of the neuroprotective tnfr2) may nullify the positive effects of antitnf therapies [53]. though there are case reports and experimental data supporting a beneficial role [58-61], more studies (large scale, randomized controlled trials) are needed to explore the safety and efficacy of anti-tnf therapies in alzheimer’s disease. multiple sclerosis: experimental studies in animals with multiple sclerosis has shown beneficial effect of anti-tnf therapies. however, investigators have also reported conflicting evidence: several cases of development of multiple sclerosis after initiating anti-tnf therapy [54-56]. titelbaum et al. reported new onset multiple sclerosis in a rheumatoid arthritis patient who was on etanercept therapy for two years. on stopping etanercept, there was radiological resolution of lesions. however, after 6 months, new lesions developed [62]. these conflicting evidences suggest that more studies are required in this therapeutic area to get a clearer picture regarding whether anti-tnf therapy is beneficial in multiple sclerosis. 7.6 kidneys tnf-α blockers used in rheumatoid arthritis and crohn’s disease occasionally result in the development of autoantibodies, lupus-like syndrome, and glomerulonephritis [63]. this has raised a concern regarding the use of tnf-α blocking agents in patients who are at risk of developing renal disease or who have underlying renal disease [63]. churchill et al.; aji, 3(1): 208-222, 2020; article no.aji.57464 217 tnf-α may be produced in inflamed kidneys from infiltrating monocytes and macrophages, and intrinsically by renal tissue including podocytes, mesangial cells, proximal tubules, thick ascending limb of loop of henle, and the collecting ducts [16,64]. tnf-α is cytotoxic to renal cells and is involved in renal scarring [6567]. tnf-α induces the production of reactive oxygen species including superoxide. superoxide increases glomerular permeability to albumin [68]. animal studies have shown that nitric oxide is also produced in several glomerular diseases. nitric oxide has a protective effect and protects the kidneys from the injurious effect of superoxide, and even reverses the damage [68]. in kidneys, tnfr1 is primarily present in glomeruli and peritubular endothelial cells [16]. tnfr2 is usually absent in the kidneys in normal state [16]. in several renal diseases, tnfr2 levels increase in the kidneys especially at the site of injury [16]. tnf-α has been implicated in acute kidney injury (aki) and chronic kidney disease (ckd) [69]. studies in animal models have shown that inflammatory cytokines like tnf-α play a role in development of aki and its inhibition may protect kidneys from the damage [70,71]. however, antitnf therapy may also result in acute kidney injury. michael b stokes et al reported 5 cases of rheumatoid arthritis who developed acute kidney injury possibly due to anti-tnf therapy [72]. tnf-α and tnfr1 and tnfr2 are higher in chronic kidney disease [65,73]. richard et al found that higher levels of tnf-α are associated with more rapid worsening of renal functions over time in patients with chronic kidney disease [65]. it has also been observed that high levels of tnfr 1 and tnfr2 in diabetes are associated with progression of diabetic kidney disease, and higher mortality [74]. tnf-α may play a role in renal ischemiareperfusion injury post kidney transplantation [69]. in the pathogenesis of renal ischemiareperfusion injury, tnf-α induces fibrin deposition, cellular infiltration, vasoconstriction and apoptosis [69]. investigators have found that inhibition of tnfr2 pathway may be helpful in decreasing incidence of glomerulonephritis. the possible underlying mechanisms that are involved in this are not completely understood. tnfr2 (but not tnfr1) is found in high concentration in renal tubular epithelial cells in renal tubulointerstitial nephritis and interacts with ‘sef’ or il-17rd, causing activation of nf-kb pathway and inflammation [16,17]. tnf-α may enhance the interaction of tnfr2 and il-17rd, but the presence of tnf-α is not absolutely necessary for the interaction to occur (upregulated tnfr2 may trigger interaction with il-17rd independent of tnf-α) [17]. this may be one of the pathogenic mechanisms explaining the role of upregulated tnfr2 contributing to nephritis and tubulointerstitial renal diseases [17]. the evidence is helpful, but more studies are needed to understand the role of tnf-α and tnf receptors in renal diseases. kidney transplant recipients are on immunosuppressive medications and are already at an increased risk of infections [75]. anti-tnf therapy may increase this risk of infections and malignancy in kidney transplant recipients and should be used with caution [76]. 8. conclusion tnf-α, tnfr1 and tnfr2 systems are complex. increased concentration of tnf-α is a prominent feature of many diseases including rheumatoid arthritis, ankylosing spondylitis, juvenile inflammatory arthritis, crohn’s disease, ulcerative colitis and psoriatic arthritis. the beneficial effects of anti-tnf therapies in these diseases led to the speculation that these therapeutic agents may be helpful in other ailments that have abnormalities in concentration of tnf-α and tnf receptorsfor example, in alzheimer’s disease, parkinson’s disease, systemic lupus erythematosus, tubulointerstitial nephritis, diabetic nephropathy, chronic kidney disease, and cardiovascular diseases including hypertension and atherosclerosis. however, investigators have found that this is not entirely true. while some studies have shown beneficial effects of anti-tnf therapy, other studies have shown conflicting results that point towards the complex nature of tnf-α and tnf receptors pathway. more extensive research is needed to understand this system, so that better and more selective anti-tnf therapeutic agents may be developed to help in diseases including alzheimer’s disease, multiple sclerosis, parkinson’s disease, renal and cardiovascular diseases. declaration we thank iqvia (communications, branding, and legal units) for giving us permission, and ‘no churchill et al.; aji, 3(1): 208-222, 2020; article no.aji.57464 218 objection’ note for publication of this paper. we thank iqvia for providing us the support and infrastructure needed to complete this paper, including giving us the opportunity to form this great team that was instrumental in bringing this paper to conclusion. consent it is not applicable. ethical approval it is not applicable. competing interests authors have declared that no competing interests exist. references 1. chen x, li p, yang x, miao x, luo h. tumor necrosis factor receptor ii (tnfr2) promotes the growth of mouse ct26 colon cancer. j immunol. 2018;200(1 supplement):178.7. 2. idriss ht, naismith jh. tnf alpha and the tnf receptor superfamily: structurefunction relationship(s). microsc res tech. 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this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: http://www.sdiarticle4.com/review-history/57464 https://doi.org/10.1038/ki.2009.142 https://doi.org/10.1038/ki.2009.142 https://doi.org/10.2215/cjn.13121215 https://doi.org/10.2215/cjn.13121215 https://doi.org/10.1155/2015/948417 https://doi.org/10.1155/2015/948417 https://doi.org/10.1681/asn.2007091048 https://doi.org/10.1681/asn.2007091048 https://doi.org/10.1111/j.1523-1755.2005.00744.x https://doi.org/10.1111/j.1523-1755.2005.00744.x https://doi.org/10.1097/00005392-199907000-00068 https://doi.org/10.1097/00005392-199907000-00068 https://doi.org/10.1155/2009/137072 https://doi.org/10.1155/2009/137072 https://doi.org/10.1152/ajprenal.00533.2012 https://doi.org/10.1152/ajprenal.00533.2012 https://doi.org/10.1093/ndt/gfh832 https://doi.org/10.1093/ndt/gfh832 https://doi.org/10.2215/cjn.03500412 https://doi.org/10.2215/cjn.03500412 https://doi.org/10.1038/srep43520 https://doi.org/10.1038/srep43520 https://doi.org/10.1097/md.0000000000005108 https://doi.org/10.1097/md.0000000000005108 http://creativecommons.org/licenses/by/4.0 _____________________________________________________________________________________________________ *corresponding author: e-mail: shayanthebest911@gmail.com; cite as: ashfaq, muhammad shayan, and muhammad hasan. 2025. “anti-n antibody reacting at 37°c: an unusual reaction in antibody screening in a 20-year-old male liver donor”. asian journal of immunology 8 (1):82-85. https://doi.org/10.9734/aji/2025/v8i1162. asian journal of immunology volume 8, issue 1, page 82-85, 2025; article no.aji.135018 anti-n antibody reacting at 37°c: an unusual reaction in antibody screening in a 20-year-old male liver donor muhammad shayan ashfaq a* and muhammad hasan a a department of pathology and laboratory medicine, section of hematology & transfusion medicine, aga khan university hospital, karachi, pakistan. authors’ contributions this work was carried out in collaboration between both authors. authors msa and mh contributed to the design, drafting and critical revision of the manuscript. both authors read and approved the final manuscript. article information doi: https://doi.org/10.9734/aji/2025/v8i1162 open peer review history: this journal follows the advanced open peer review policy. identity of the reviewers, editor(s) and additional reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here: https://pr.sdiarticle5.com/review-history/135018 received: 23/02/2025 published: 29/04/2025 abstract anti-n antibodies usually possess cold-reactive properties which make them clinically insignificant and are mostly classified as naturally occurring igm antibodies. these antibodies sometimes trigger reactions at body temperature (37°c) or in the anti-human globulin phase resulting in serious medical outcomes such as delayed hemolytic transfusion reactions or hemolytic disease of the newborn. this case shows an extraordinary naturally occurring anti-n antibody which was detected in a 20-year-old male liver donor during standard antibody testing because of its unexpected reactivity at 37°c. the absence of any previous blood transfusions or medication use in the patient made this case stand out as particularly intriguing. the antibody identification was successful case report https://doi.org/10.9734/aji/2025/v8i1162 https://pr.sdiarticle5.com/review-history/135018 ashfaq and hasan; asian j. immunol., vol. 8, no. 1, pp. 82-85, 2025; article no.aji.135018 83 following the 'pre-warm' technique implementation while antigen phenotyping validated it as an iggtype anti-n antibody. this medical case demonstrates the critical importance of recognizing unusual antibody responses during blood transfusions and organ transplants because uncommon reactions may lead to serious consequences. keywords: anti-n antibody; mns blood group system; transfusion medicine; serological testing; delayed hemolytic transfusion reactions. 1. introduction the mns blood group system, discovered by landsteiner and levine in 1927, was the second to be identified after the abo system. among the antibodies in the mns system, anti-m is a common "naturally occurring" antibody (perrault, 1973). the s antigen was identified in 1947 by walsh and montgomery after the development of the antiglobulin test (harmening, 2018). most anti-m antibodies are cold-reactive and do not activate complement or react with enzymetreated rbcs (thakral et al., 2010). they are rarely associated with hemolytic transfusion reactions (sancho et al., 1998). anti-n antibodies are less common than anti-m and are also typically naturally occurring, coldreactive igm or igg agglutinins that do not activate complement or react with enzymetreated rbcs (perrault, 1973; taj et al., 2025). they are clinically insignificant unless reactive at 37°c and have been linked to rare cases of mild hemolytic disease of the fetus and newborn (hdfn) (ballas et al., 1985; wiebe et al., 2024). potent anti-n antibodies are more frequently found in individuals of african descent with a specific rbc phenotype (m+ nss-) due to the absence of the n antigen (harmening, 2018; katagiri et al., 2024). immune anti-n antibodies are extremely rare (klein & anstee, 2013). we report a case of naturally occurring anti-n that reacts at 37°c, identified during routine antibody identification testing (kumawat et al., 2015). 2. case report a 20-year-old guy from karachi, who’s healthy and doesn't have any other medical issues, decided to donate a liver to his dad. his father had been struggling with chronic liver disease because of a hepatitis b infection, so they set up a liver transplant at dow university of health sciences. this donor had never had a blood transfusion or taken any medication. for the usual pre-transplant checks, they sent a test request to aga khan university hospital to identify any antibodies. the first blood tests came back showing a positive auto-control, which means his red blood cells reacted with his own serum at room temperature. but when they did a direct antiglobulin test (dat) with anti-igg + c3d, it came back negative, ruling out any autoantibodies. they then checked three different antibody screening panels (id-diacell iii-iii, biorad) and found positive reactions in all of them (2+, 3+, and 3+). they also noticed that the auto-control was positive right from the spin phase. so, they decided to run the antibody screening again using a method where they warmed everything up, and surprise! they got positive results across all three panels. to dig deeper, they used the papain treatment method (id-diacell papain kit), which surprisingly turned up a negative result for the red cell antibody screening. but when they went ahead with the antibody identification using the warm technique, they found out he had an anti-n antibody using this 11-cell identification panel (id-diacell, biorad). the reaction was pretty strong, 3+, with homozygous n+ n+ cells (panels 4, 10, and 11) and negative with heterozygous m+ n+ cells (panels 1, 3, 7, and 8), plus also negative with nnegative cells (panels 2, 5, 6, and 9). they also checked other antigens for n, s, s, and m and found that he was m-, n-, s-, and s-. they treated his plasma with dithiothreitol, which confirmed there was an igg-type anti-n antibody present. the antibody titer was 1:2. this report emphasizes a naturally occurring anti-n antibody that reacts at 37°c, which could actually be pretty important even though the donor hadn’t had any blood transfusions before. it really brings home the need to check for these naturally happening antibodies during routine blood tests because their reactions at body temperature can affect transfusion practices. 3. discussion anti-n antibodies belong to the mns blood group system and are usually naturally occurring and mainly cold-reactive igm antibodies. these antibodies are often clinically insignificant unless they decide to react at 37°c or during the antihuman globulin (ahg) phase of testing. in this ashfaq and hasan; asian j. immunol., vol. 8, no. 1, pp. 82-85, 2025; article no.aji.135018 84 case, we found the anti-n antibody in a 20-yearold male liver donor who had a serological profile showing an uncommon blood group discrepancy. while anti-n antibodies are generally more of a cold-reactive type and don’t usually bind complement, if they do react at 37°c, that’s when we start to worry about their clinical significance. normally, anti-n antibodies don’t cause major issues, as they are linked to non-pathological clinical outcomes. they typically don't lead to hemolytic transfusion reactions (htrs) or hemolytic disease of the fetus and newborn (hdfn) unless we see them react at body temperature (37°c), like in this instance (perrault, 1973). a transfusion reaction is more likely if these antibodies show strong reactivity at 37°c, but that's pretty rare. when it happens, it can cause delayed hemolytic reactions, resulting in some transfusion-associated complications. besides, while hdfn can be a concern in cases of maternal-fetal blood group incompatibility, instances of hdfn linked to anti-n are super rare (harmening, 2018). interestingly, the igm class of anti-n antibodies usually reacts in colder conditions and doesn’t typically bind to complement or react with enzyme-treated red blood cells (rbcs). this is kind of similar to anti-m antibodies, which show the same cold-reactive behavior and limited clinical relevance, unless they react at body temperature (thakral et al., 2010). however, in our case, the antibody was behaving unusually by reacting at 37°c, reminding us to stay on our toes about atypical antibody behaviors in blood donors, especially when we’re talking about organ transplant recipients who might need careful crossmatching and serologic evaluations. the phenotyping results were pretty noteworthy since the donor's red cell antigen profile showed the absence of the m, n, s, and s antigens, which are usually part of testing in the mns blood group system. plus, after treating with dithiothreitol (dtt), we confirmed that the anti-n antibody was of the igg nature, hinting that it could have some clinical significance in certain transfusion scenarios (sancho et al., 1998). finding an anti-n antibody in a healthy individual with no prior blood transfusions is pretty unusual, given that naturally occurring antibodies in the mns system are typically igm types and don’t usually trigger immune responses. the titer of 1:2 here suggests the anti-n antibody could definitely be clinically important at 37°c (kumawat et al., 2015). this just goes to show how important it is to do a thorough antibody screening for blood donors; even naturally occurring antibodies might need our attention in a clinical setting. 4. conclusion in conclusion, this case really emphasizes the importance of rare blood group antibodies in organ transplantation and transfusion medicine. it’s a reminder for clinicians and lab staff to keep an eye out for unusual antibody profiles, especially when there are discrepancies in routine serological testing. we need more studies and awareness about these antibodies to avoid adverse reactions in transfusions and organ transplant procedures. disclaimer (artificial intelligence) author(s) hereby declare that no generative ai technologies such as large language models (chatgpt, copilot, etc.) and text-to-image generators have been used during the writing or editing of this manuscript. consent as per international standards or university standards, patient(s) written consent has been collected and preserved by the author(s). ethical approval it is not applicable. competing interests authors have declared that no competing interests exist. references ballas, s. k., dignam, c., harris, m., & marcolina, m. j. (1985). a clinically significant anti-n in a patient whose red cells were negative for n and u antigens. transfusion, 25(4), 377– 380. harmening, d. m. (2018). modern blood banking & transfusion practices. fa davis. katagiri, t., iwasaki, h., fujieda, a., kasashima, s., ozaki, s., uemori, m., ... & nakao, s. ashfaq and hasan; asian j. immunol., vol. 8, no. 1, pp. 82-85, 2025; article no.aji.135018 85 (2024). a case of hepatitis‐associated aplastic anaemia following living‐donor liver transplantation for fulminant hepatitis showing loss of heterozygosity in the 6p chromosome in the affected liver. british journal of haematology, 204(2), 623– 627. klein, h. g., & anstee, d. j. (2013). mollison's blood transfusion in clinical medicine. john wiley & sons. kumawat, v., jain, a., marwaha, n., & sharma, r. r. (2015). anti-n antibody reacting at 37°c: an unusual occurrence interfering with routine testing: two interesting cases. asian journal of transfusion science, 9(1), 92–93. perrault, r. (1973). naturally-occurring anti-m and anti-n with special case: igm anti-n in a nn donor. vox sanguinis, 24(2), 134– 149. sancho, j. m., pujol, m., fernández, f., soler, m., manzano, p., & feliu, e. (1998). delayed haemolytic transfusion reaction due to anti-m antibody. british journal of haematology, 103(1), 268–269. taj, r., ng, k., cuddapah, s. r., rand, e. b., bleicher, m., amaral, s., ... & abt, p. l. (2025). liver transplant from a deceased donor with cystinosis: a case report. jimd reports, 66(1), e12467. thakral, b., saluja, k., sharma, r. r., & marwaha, n. (2010). phenotype frequencies of blood group systems (rh, kell, kidd, duffy, mns, p, lewis, and lutheran) in north indian blood donors. transfusion and apheresis science: official journal of the world apheresis association: official journal of the european society for haemapheresis, 43(1), 17–22. wiebe, n., stueck, a., & mcleod, m. (2024). steatotic liver disease arising in an asymptomatic 20-year-old man with panhypopituitarism and elevated transaminases. canadian liver journal, 7(4), 511–516. disclaimer/publisher’s note: the statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of the publisher and/or the editor(s). this publisher and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. _________________________________________________________________________________ © copyright (2025): author(s). the licensee is the journal publisher. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: https://pr.sdiarticle5.com/review-history/135018 https://pr.sdiarticle5.com/review-history/135018 _____________________________________________________________________________________________________ *corresponding author: e-mail: wwalana@uds.edu.gh; cite as: walana, williams, george billak, jennifer suurbaar, and hawa malechi. 2025. “t cell exhaustion and immune dysfunction in spontaneous abortion: a targeted review”. asian journal of immunology 8 (1):179-89. https://doi.org/10.9734/aji/2025/v8i1170. asian journal of immunology volume 8, issue 1, page 179-189, 2025; article no.aji.139815 t cell exhaustion and immune dysfunction in spontaneous abortion: a targeted review williams walana a*, george billak b, jennifer suurbaar b and hawa malechi c,d a department of clinical microbiology, school of medicine, university for development studies, tamale, ghana. b department of biochemistry and molecular medicine, school of medicine, university for development studies, tamale, ghana. c department of obstetrics and gynaecology, school of medicine, university for development studies, tamale, ghana. d department of obstetrics and gynaecology, tamale teaching hospital, northern region, tamale, ghana. authors’ contributions this work was carried out in collaboration among all authors. authors ww and gb conceived the idea. authors ww, gb, js and hm performed a literature search. all authors reviewed and made significant contributions to finalise the manuscript and agreed to the submission of this manuscript for consideration. all authors read and approved the final manuscript. article information doi: https://doi.org/10.9734/aji/2025/v8i1170 open peer review history: this journal follows the advanced open peer review policy. identity of the reviewers, editor(s) and additional reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc, are available here: https://pr.sdiarticle5.com/review-history/139815 received: 10/05/2025 published: 15/07/2025 abstract background: immune tolerance is established between the foetus and the mother in normal pregnancies. however, this tolerance may be breached, resulting in undesired pregnancy outcomes such as spontaneous abortion, foetal anomalies, and maternal complications. reported review article https://doi.org/10.9734/aji/2025/v8i1170 https://pr.sdiarticle5.com/review-history/139815 walana et al.; asian j. immunol., vol. 8, no. 1, pp. 179-189, 2025; article no.aji.139815 180 studies suggest a potential link between immune response and unplanned pregnancy termination. a comprehensive understanding of immune responses in pregnancy loss harbours the potential to control the associated mechanisms. objective: this review synthesised existing studies on t cell exhaustion (tce) in spontaneous abortion and pregnancy complications. methodology: a comprehensive database search from scopus, google scholar, pubmed, and web of science identified 21 eligible studies that reported original data on spontaneous abortion and pregnancy complications and tce. results: the review has shown that tce is common in spontaneous abortion, characterised by the upregulation of inhibitory receptors and a suboptimal or lack of t-cells to perform their normal functions, such as proliferation, cytokine production, and cytotoxicity. decreased expression of pd1, lag-3 and tim-3 may contribute to an enhanced inflammatory response. increased proportions of th1 and th17 cells are associated with pro-inflammatory responses and decreased immune regulatory function, resulting from altered pd-1/pd-l1 expression, which may contribute to pregnancy loss. decreased anti-inflammatory cytokines (tgf-β1, il-10, and il-4) promote an imbalanced homeostasis, which can lead to pregnancy loss. these anomalies are partly anchored on chronic antigen exposure, oxidative stress, and hormonal changes, phenomena frequently associated with recurrent spontaneous abortion or poor pregnancy outcomes. conclusion: optimal modulation of immune response to control tce may prevent or minimise the occurrence of spontaneous abortion and promote successful pregnancies. keywords: t cell exhaustion; immune exhaustion; spontaneous abortion; pregnancy; immune regulation; reproductive immunology; systematic review. 1. introduction pregnancy is a complex immune state where tolerance and immune activation coexist (hu et al., 2016). despite its prevalence, the mechanisms governing maternal-foetal immune tolerance remain unclear. pregnancy failure affects approximately 15% of known pregnancies, with more than 50% attributed to immune tolerance defects (kuon et al., 2015). research has shown that natural killer cells expressing tim-3 play a defensive role in the early stages of pregnancy, promoting immunosuppressive activities and the induction of regulatory t cells (kuon et al., 2015). spontaneous abortion, also known as miscarriage, is the unplanned termination of a pregnancy before the foetus can survive outside the womb, typically before the 20th week of gestation (miller et al., 2020). spontaneous abortion can occur due to various reasons, including chromosomal abnormalities, hormonal imbalance, infections, immune system disorders, uterine or cervical abnormalities, and advanced maternal age, among others (miller et al., 2020). spontaneous abortion, affecting close to 30% of pregnancies, is a common complication, with recurrent pregnancy loss affecting 1 in 100 women (miller et al., 2020). while chromosomal abnormalities and thrombotic complications are established causes, immunologic factors, such as t-cell exhaustion, have also been implicated (miller et al., 2020). programmed cell death protein 1 (pd-1), t cell immunoglobulin and mucin-domain containing-3 (tim-3), and cytotoxic t-lymphocyte-associated protein 4 (ctla-4) are critical immune checkpoint receptors (catakovic et al., 2017). their dysregulation can contribute to spontaneous abortion by disrupting immune tolerance during pregnancy. these markers maintain a balance of t helper cells (th1/th2) and regulatory t cells (tregs), along with their respective cytokines, which are essential for pregnancy success (li et al., 2017). altered expression of these markers can lead to t-cell exhaustion and recurrent miscarriages, underscoring their importance in maintaining the delicate balance of the maternal-foetal immune system (li et al., 2020). this systematic review synthesised existing literature on tce in spontaneous abortion and pregnancy, exploring its prevalence, mechanisms, and clinical implications to identify knowledge gaps and areas for future research. 2. methodology 2.1 study design this systematic review spanned from january 2014 to march 2024, adhering to the prisma walana et al.; asian j. immunol., vol. 8, no. 1, pp. 179-189, 2025; article no.aji.139815 181 2020 guidelines for a comprehensive and transparent approach. the research process involved a structured identification, screening, eligibility, and inclusion framework conducted independently to minimise errors and bias. studies were selected based on their relevance to understanding t cell exhaustion in pregnancy, with a focus on peer-reviewed journals. data extraction encompassed t cell functionality, inhibitory receptor expression, cytokine profiles, and outcomes related to spontaneous abortion. furthermore, a comprehensive review of animal models and clinical studies was conducted to gain a deeper insight into the role of t cell exhaustion in spontaneous abortion and pregnancy complications, providing a more complete understanding of this complex phenomenon. 2.2 search strategy a systematic literature search was conducted, encompassing a range of databases, including scopus, google scholar, pubmed, and web of science, to identify and synthesise the findings of previous studies. applicable keywords, utilising medical subject headings (mesh) for pubmed, were combined using logical operators to develop a comprehensive search strategy. a combination of keywords from the title was used to construct boolean search strings (“t cell exhaustion”) or (“immune exhaustion”) and (“spontaneous abortion”) or (“miscarriage”) and (“pregnancy”) in order to search and identify relevant studies. this methodology ensured a rigorous and systematic approach to understanding t cell exhaustion in the context of spontaneous abortion and pregnancy. 2.3 inclusion criteria available studies and data were included based on the following predefined criteria: (1) original research papers published between january 2014 and march 2024, (2) experimental and observational studies, (3) availability of full text, and (4) articles written and published in english. fig. 1. schematic flow diagram of the study retrieval process walana et al.; asian j. immunol., vol. 8, no. 1, pp. 179-189, 2025; article no.aji.139815 182 table 1. summary of included articles. rsa: recurrent spontaneous abortion: rsa is defined as three or more consecutive spontaneous miscarriages before the 22nd week of gestation; wrsa: women with rsa; ipl: induced pregnancy loss; hc-p: healthy control pregnant; hc-np: healthy control non-pregnant; decidual macrophagesmφs; nk-cells: natural killer cells authors; year population characteristics t-cells & exhaustion markers practical implications observational (zych et al., 2024) n = 50; 20 = wrsa, 20 = hc-p, and 10 = hc-np rsa women exhibit decreased expression of pd-1 and lag-3 on various t cells and increased expression of tigit on nkt cells. • the decreased expression of pd-1 and lag-3 may contribute to an enhanced inflammatory response. • in women with rsa, elevated tigit expression on nkt cells compensates for reduced pd-1 and lag-3 expression. (zargar et al.,2024) n = 57; 22 = wrsa, 18 = recurrent implantation failure, and 17 = hc-p the mean numbers of nk cells (cd16+ and cd56+) and ifn-γ levels were significantly higher in the rsa • a significant correlation was found between the levels of ifn-γ and nk cells in the rpl group, indicating that immune dysfunction and ifn-related cytotoxicity may contribute to pregnancy loss. (zhang et al., 2022) n=98, 17 = wrsa, 81 hc-p decreased levels of tim-3+ peripheral nk-cells and pd-1 in women with miscarriage • lower tim-3 levels in wrsa cases may indicate poor immune adaptation to pregnancy, suggesting tim-3 could be a marker for predicting miscarriage risk. (talukdar et al., 2018) n = 43; 26 women with idiopathic recurrent pregnancy loss (rpl) and 17 healthy fertile controls significantly elevated frequencies of γδ t cells correspond to elevated ifn-γ and tnf-α, but decreased il-10, in rpls. • these findings suggest a shift in γδ t cell cytokine profile toward a pro-inflammatory phenotype, which may contribute to immune dysregulation and pregnancy failure in women with idiopathic recurrent pregnancy loss. (zych et al., 2021) n = 36, 24 = wrsa, 12 = hc-p elevated pd-1 expression was observed on t helper (cd4+) cells in wrsa, while tim-3 expression was decreased on t cytotoxic (cd8+) cells. • the dysregulation of the maternal immune system in rsa is characterised by a shift towards a th1 cytokine profile and increased cytotoxicity, which may contribute to pregnancy loss. (liu et al., 2021) n = 150; 50 = wrsa, 50 = hc-p and 50 = hc-np wrsa had increased naïve cd4+ t cells, central memory cd4+ t cells, and mature nk cells, while terminally differentiated cd4+ t cells and effective memory cd4+ t cells were decreased. • higher naïve cd4+ t cells may weaken the immune system, while increased central memory cd4+ t cells suggest altered immune responses. elevated mature nk cells can cause cytotoxic effects, potentially leading to pregnancy loss. decreased terminally differentiated and effective memory, cd4+ t cells, indicate reduced walana et al.; asian j. immunol., vol. 8, no. 1, pp. 179-189, 2025; article no.aji.139815 183 authors; year population characteristics t-cells & exhaustion markers practical implications immune protection and a weaker response to pregnancy-related antigens. (wang et al., 2020) n=65; 45 =wrsa; 20 = hc-p increased th1 and th17 cells, and decreased regulatory treg cells, were associated with a significant decrease in the expression of pd-1 on th1 and th17 cells in wrsa. • increased proportions of th1 and th17 cells are associated with pro-inflammatory responses and decreased immune regulatory function due to altered pd-1/pd-l1 expression, which may have led to pregnancy loss. (zhu et al., 2019) n=68; 33 = wrsa; 35 = hcp wrsa exhibits an activated immune system, characterised by high levels of cytotoxic nk cells and low levels of immunoregulatory il-10+ cd56bright nk cells. nk cells also spontaneously produce a high amount of tgfß1. • this indicates a persistent inflammatory response that cannot be efficiently counter-regulated. this suggests that down-regulating the cytotoxic immune response and enhancing immunoregulatory mechanisms could be beneficial for wrsa (abdolmohammadi vahid et al., 2019) n= 100; 50 = wrsa and 50 = hc-p treg and exhausted t cells were decreased, while th17 and exhausted treg cells were increased in wrsa. • the decrease in treg cells and exhausted t-cells suggests a failure in the regulatory mechanisms that prevent the maternal immune system from attacking the foetus. • increase in th17 and exhausted treg cells indicates a shift towards a pro-inflammatory immune environment. (qian et al., 2018) n = 75; 37 = wrsa, 38 = hc-p wrsa has a higher th17/treg cell ratio at the maternal-foetal interface compared to women with normal pregnancies • wrsa exhibited elevated pro-inflammatory cytokine levels due to the skewed th17/treg cell ratio at the maternal-foetal interface, contributing to immune tolerance failure in wrsa. (kuon et al., 2015) n = 123; 97 = wrsa; 26 = hc-np wrsa showed higher levels of activated t-cells (cd3+dr+, cd4+dr+, and cd8+dr+) that were less responsive to mitogens than the controls. a significant increase in neopterin levels and lower levels of cd16+cd56+ nk cells and cd19+ b lymphocytes • the chronic activation of t-cells and the deficiency in nk and b-cells in wrsa may contribute to the inability of the maternal immune system to tolerate the developing foetus, leading to miscarriage. walana et al.; asian j. immunol., vol. 8, no. 1, pp. 179-189, 2025; article no.aji.139815 184 authors; year population characteristics t-cells & exhaustion markers practical implications were observed in wrsa. experimental (li et al., 2022) n = 135, 40 = wrsa, 86 hcp, 9 hc-np tim-3 expression is significantly decreased in wrsa, while it is increased in normal pregnancies. • decreased tim-3 results in the dysfunction of dmφs, which leads to an increase in pro-inflammatory cytokines and a decrease in anti-inflammatory cytokines, subsequently increasing foetal loss. (huang et al., 2020) n = 43; 8 = wrsa, 35 = hcp the proportion of cd69+cd103+ resident and memory t-cells is significantly higher in women with rsa than in hc-p. • the resident memory t-cells expressed high levels of chemokine receptors such as cxcr3 and cxcr6. they had an enhanced capacity to produce both effector cytokines, tnf-α and ifn-γ, which may have led to pregnancy loss. (liu et al., 2020) 200 specific pathogen-free female cba/j mice decreased pd-1 and pd-l1 expression in the placenta and spleen • thyroiditis in mice led to reduced pd-1 and pd-l1 expression in the placenta and spleen, resulting in a decrease in treg cells and an increase in th17 cells at the maternal-foetal interface, resulting in miscarriage. (luo et al., 2020) n = 12; 60 wrsa and 60 hc-p decreased expression of foxp3 both at the protein and mrna levels and reduced frequency of cd4+cd25highcd127low/− regulatory t cells in patients with rsa • in wrsa, there is a reduction in the number and function of tregs, which may lead to the immune system attacking the foetus, resulting in miscarriage. (yu et al., 2021) n = 42; 10 = wrsa-p; 11 = rsa-up; 11 = hc-p; 10 = hc up rsa patients exhibit increased cd107a expression on gdt cells, a higher frequency of il-17asecreting vδ2+ gdt cells, upregulated ccl8 expression, and enhanced infiltration of cd8+ t cells and m2 macrophages. additionally, pd1 expressions on vd2+ gdt cells are elevated in these patients. • activated gdt cells, particularly those expressing cd107a and il-17a, may contribute to inflammation at the maternal-foetal interface; these cells have an enhanced ability to kill other cells, potentially leading to rsa. (wang et al., 2019) n = 37 18 normal pregnancies 19 abortion-prone model decreased frequency of splenic t cells co-expressing ctla-4 and tim-3 in miscarriage models • in the abortion prone model, there is a decrease in splenic t cells co-expressing ctla-4 and tim-3, which is linked to changes in cytokine; there is an increase in walana et al.; asian j. immunol., vol. 8, no. 1, pp. 179-189, 2025; article no.aji.139815 185 authors; year population characteristics t-cells & exhaustion markers practical implications pro-inflammatory cytokines (e.g., tnf-α) and a decrease in anti-inflammatory cytokines (e.g., il-4, il10). (li et al., 2017) n = 18; 10 = wrsa, hc-p = 8 tim-3+ nk cells from wrsa produce fewer anti-inflammatory cytokines and have increased cytotoxicity compared to those from normal pregnancies • tim-3+ nk produces more anti-inflammatory cytokines (tgf-β1, il-10, il-4) and fewer pro-inflammatory cytokines (tnf-α), fostering immune tolerance, and wrsa produces fewer anti-inflammatory cytokines, resulting in pregnancy loss (xu et al., 2017) n=18; 9 mice were normal, and nine were abortion-prone matings in the study. tim-3 and pd-1 pathways were blocked in pregnant mice. • tim-3 and pd-1 regulate cd8+ t cell function to maintain early pregnancy in mice • cd8+ t cells that co-express tim-3 and pd-1 were reduced in the decidua of mice with abortion-prone matings • blocking tim-3 and pd-1 in normal pregnant mice led to increased foetal resorption (wang et al., 2016) 36 normal pregnant mice 45 abortion-prone mice decreased pd-1 and tim-3 expression on cd4+ t cells in abortion prone mice. • blocking pd-1 and tim-3 pathways in experimental models led to decreased th2 cytokine production and increased foetal resorption. (sun et al., 2016) n = 80; 20 = wrsa, 30 = hc-np and 30 hc-p tim-3 levels are lower in nk cells from wrsa than in normal pregnancies. tim-3 is more abundant in dnk cells during early pregnancy than pnk cells. blocking tim-3 reduces cytokine production in dnk cells. • tim-3 influences the production of cytokines like ifn-γ and tnf-α in dnk cells, essential for immune tolerance and vascular remodelling. • tim-3's interaction with galectin-9 inhibits nk cell cytotoxicity towards trophoblasts, preventing placental damage and supporting foetal development. walana et al.; asian j. immunol., vol. 8, no. 1, pp. 179-189, 2025; article no.aji.139815 186 3. results and discussion 3.1 study identification and retrieval fig. 1 illustrates the step-by-step processes of identifying, retrieving, and selecting studies for this review. our literature search yielded 23,000 articles from google scholar, 12,959 from pubmed, and 367 from web of science. after filtering to remove duplicates and irrelevant studies, 50 articles qualified for a thorough fulltext evaluation. upon further assessment, 21 of these studies met the predetermined inclusion criteria and were included in the review. 4. discussion recurrent spontaneous abortion (rsa) is a multifaceted condition where maternal immune dysregulation is a pivotal factor. evidence consistently highlights an imbalance between pro-inflammatory and regulatory immune responses central to rsa pathophysiology. for example, reduced expression of immune checkpoints, such as pd-1 and tim-3, on t cells —crucial for suppressing inflammation —has been observed in rsa cases, suggesting impaired immune tolerance at the maternal-foetal interface (zych et al., 2021; wang et al., 2020). additionally, elevated levels of th17 cells and a reduction in regulatory t cells (tregs) further underscore a shift towards a pro-inflammatory immune environment that may lead to pregnancy loss (vahid et al., 2018; qian et al., 2018). natural killer (nk) cells also exhibit substantial dysregulation in rsa. studies have documented increased cytotoxic nk cell activity alongside reduced levels of immunoregulatory subsets, a combination that disrupts the immune balance essential for successful pregnancy (zhu et al., 2019; li et al., 2017). gamma-delta t (γδt) cells, which produce high levels of il-17a and express cytotoxic markers, contribute to this inflammatory milieu, further compromising pregnancy outcomes (yu et al., 2021; talukdar et al., 2018). dysregulation of decidual macrophages (dmφs) and t cells is critical at the maternal-foetal interface. reduced tim-3 expression in these cells correlates with increased production of proinflammatory cytokines and decreased production of anti-inflammatory cytokines, ultimately contributing to foetal loss (li et al., 2022; xu et al., 2017). experimental models underscore the significance of immune checkpoints, showing that blocking the tim-3 or pd-1 pathways exacerbates pregnancy loss by inducing inflammatory and cytotoxic responses (wang et al., 2016; xu et al., 2017). the immunological environment in rsa is characterised by heightened inflammation and impaired regulatory mechanisms. targeting immune checkpoints such as pd-1 and tim-3 offers a promising therapeutic approach to restore immune balance and improve pregnancy outcomes in rsa patients. 5. future perspective potential therapeutic strategies for preventing spontaneous abortion include targeting t-cell exhaustion. this can be achieved by enhancing treg function, blocking inhibitory receptors on t cells, and modulating cytokine environments to restore immune balance in pregnancy. diagnostic markers for t cell exhaustion, such as the expression of inhibitory receptors and cytokine profiles, could help predict and prevent spontaneous abortion. non-invasive diagnostic tools, such as blood tests or imaging techniques, could be developed to monitor immune status during pregnancy. future research should focus on elucidating the detailed mechanisms of t cell exhaustion in pregnancy, identifying additional biomarkers, and developing targeted therapies. collaborative efforts between researchers, clinicians, and pharmaceutical companies will be crucial for translating experimental findings into clinical practice. 6. conclusion studies have identified t-cell exhaustion as a significant contributor to spontaneous abortion. this phenomenon is characterised by reduced t cell functionality and proliferation in women who have experienced spontaneous abortion. studies have shown that t cell exhaustion is characterised by a decrease in the expression of stimulatory receptors and an increase in the expression of suppressive receptors, making it challenging for t cells to respond to infections. this highlights the critical role of t-cell exhaustion in spontaneous abortion. regulatory t cells (tregs) have been seen to play a vital role in maintaining immune homeostasis and preventing foetal rejection. however, women with a history of repeated unexplained pregnancy loss have been found to walana et al.; asian j. immunol., vol. 8, no. 1, pp. 179-189, 2025; article no.aji.139815 187 have reduced treg presence and functionality, which may contribute to spontaneous abortion. furthermore, t-cell exhaustion and an overactive immune response have been linked to spontaneous abortion. modulating the immune response and preventing t-cell exhaustion may be potential therapeutic strategies to prevent spontaneous abortion and promote successful pregnancy outcomes. decidual natural killer cells (dnk) have been found to play a crucial role in maintaining pregnancy and preventing spontaneous abortion. however, an imbalance in dnk cells, particularly an overactivation of dnk1 cells, may contribute to spontaneous abortion. in conclusion, t cell exhaustion and immune modulation play a critical role in spontaneous abortion and recurrent pregnancy loss. targeting these cell populations or their associated pathways may be a promising therapeutic strategy for preventing or treating pregnancy complications. further research is needed to fully understand the mechanisms underlying t cell exhaustion and spontaneous abortion and to develop practical therapeutic approaches. disclaimer (artificial intelligence) author(s) hereby declare that no generative ai technologies such as large language models (chatgpt, copilot, etc.) and text-to-image generators have been used during the writing or editing of this manuscript. consent it is not applicable. ethical approval this systematic review did not necessitate ethical clearance, as it relied on previously published studies and research works freely accessible in the public domain, eliminating the need for additional ethical oversight. competing interests authors have declared that no competing interests exist. references abdolmohammadi, v. s., ghaebi, m., ahmadi, m., nouri, m., danaei, s., aghebati-maleki, l., mousavi ardehaie, r., yousefi, b., hakimi, p., hojjat-farsangi, m., rikhtegar, r., & yousefi, m. 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(2021). differences in immune checkpoints expression (tim-3 and pd-1) on t cells in women with recurrent miscarriages—preliminary studies. journal of clinical medicine, 10(18). https://doi.org/10.3390/jcm10184182 disclaimer/publisher’s note: the statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of the publisher and/or the editor(s). this publisher and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. _________________________________________________________________________________ © copyright (2025): author(s). the licensee is the journal publisher. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: https://pr.sdiarticle5.com/review-history/139815 https://doi.org/10.1111/aji.13524 https://doi.org/10.1186/s12865-019-0290-3 https://doi.org/10.3390/ijms25179378 https://doi.org/10.3390/jcm10184182 https://pr.sdiarticle5.com/review-history/139815 _____________________________________________________________________________________________________ *corresponding author: e-mail: prince@std.uestc.edu.cn; asian journal of immunology 4(1): 96-105, 2021; article no.aji.73786 covid-19 and actor well-being: a serial mediated moderation of mask usage and personal health engagement addo prince clement1*, gumah bernard2, ato kwamena sagoe1, ohemeng asare andy3, kulbo nora bakabbey2, takyi nyankom lydia1 and kulbo bassamar dora4 1 akenten appiah-menka university of skills training and entrepreneurial development, ghana. 2 school of management and economic, chengdu, university of electronic science and technology of china, p.r. china. 3 centre for business, george brown college, canada. 4 school of business, kwame nkrumah university of science and technology, zambia. authors’ contributions this work was carried out in collaboration among all authors. all authors read and approved the final manuscript. article information editor(s): (1) dr. wagner loyola, brazilian agricultural research corporation (embrapa), brazil. (2) dr. cynthia aracely alvizo báez, autonomous university of nuevo leon, mexico. reviewers: (1) ekta damani (malvania), amc dental college and hospital, india. (2) mohammad salem hareedy, assiut university, egypt. (3) sudhir kumar atri, pandit bhagwat dayal sharma post graduate institute of medical sciences, india. complete peer review history: https://www.sdiarticle4.com/review-history/73786 received 30 august 2021 accepted 08 september 2021 published 14 september 2021 abstract wearing of face mask has become the new norm and a requirement for accessing public spaces. the current study explored the drives of self-regulation towards the purchase and use of face masks for actor wellbeing and public safety. the study adopted a cross-sectional survey approach. data from 1859 participants sampled specifically from lockdown areas are the backbone of this study. we drew inspiration from the self-regulation theory and the trending slogan "any mask is better than no mask" to propose a model based on the fear of covid-19 and actor wellbeing. we adopted hayes' process macro in analyzing the proposed model. the findings confirmed that the fear of covid-19 (β=.78, p<.001) invokes actors' self-regulation and alters attitudes (β=.521 original research article clement et al.; aji, 4(1): 96-105, 2021; article no.aji.73786 97 p<.001) to drives mask purchase intentions and use significantly. we also re-echoed the role of self-efficacy in the behavioral change decision-making under threatening conditions. the models explained a total variance of 80% in explaining how the fear of covid-1d invokes an individual's behavioral change towards public safety and actor wellbeing. policymakers, wellbeing psychologists, and healthcare practitioners can leverage the finding in this work to understand the antecedents that promote people's behavioral change towards psychological and physical wellbeing, such as that which come with covid-19 and mask use. in particular, face mask advocates can leverage this paper's fear and wellbeing understanding in their promotional and educational exercises. we recommend reconsidering mask use protocols to support the slogan "any mask is better than no mask." keywords: covid-19; fear of covid-19; face mask; self-regulation; actor wellbeing. 1. introduction the world has practically come to a standstill in recent times because of covid-19 and the strategies to fight it. individuals, scientists, nations, and governments have channelled resources into saving lives and the economy from the grips of the 2019-novel sars-2 virus known as the covid-19. the outbreak of covid-19 has affected the wellbeing and health of individuals and societies all over the world. this has imposed physical, cognitive, psychological, and social challenges with pressure on the service industry as much as on other sectors. when engaging with service providers, customers are crowned with fear for their health [1]. logistics service quality and other sectors such as education, transportation, tourism, and hospitality were greatly affected while new markets, including online shopping, door-to-door services delivery, and live streaming businesses, spike to record heights [2]. many of these are due to the fear of covid-19, change in consumer behavior and lifestyle, and individuals' self-efficacy to succeed in handling and adapting to new and complex behavioral risky situations, and the self-regulation in the avoidance of disease has become vital to survival [3]. people will voluntarily or otherwise develop a behavioral immune system that drives change in response to survival [4]. this behavior change is crucial to managers, service providers, and public institutions responsible for controlling and managing infectious diseases. research has established that humans are rooted in self-organizing structures that, during a critical event like a pandemic, are triggered to adjust, endure, and develop to manage the circumstances. with covid-19, social and physical distancing and mask use have been one of such structures triggered towards surviving and consumer safety, which require agility and efficiency [5]. several papers in the service industry have drawn the need for consumer and actor safety first from the hierarchy of needs theory [6, 7]. this study examined how fear of covid-19 alters the behavior, attitude, and intention in using and purchasing face masks towards consumer wellbeing. this research will be among the first to bridge the existing theoretical, conceptual, and practical gap by examining influential factors of individuals' behavioral intention (fear of covid-19) and the practicalities of behavior change (mask use) in examining the intentions of face mask purchase towards the actor and public safety and wellbeing. the researchers anticipate that this study's results will correspondingly provide a practical analysis for governments, and policymakers, to understand individuals' behavioral intentions to the use of face masks before any investment in face masks or applying face masks policies and measures, especially now that many countries are experiencing a second wave of covid-19. 1.1 self-regulation self-regulation has multiple dimensions (conscious or automated process) with several definitions [8,9,10,11]. self-regulation in this study is, however, operationalized according to the definition by [11], stating that self-regulation is a conscious process in which the individual dedicates the needed effort to override a natural response or behavior and substitute it with a more effortful behavior or response that is more consistent with a targeted goal(s). refraining from unsafe sex and over-eating, resisting urges to drinking and smoke might all be considered examples of self-regulation. from this definition, self-regulation may be simplified as the ability to change oneself and control an inner process for a better result. self-regulation is, therefore, a key indicator to success when viewed from a cultural, clement et al.; aji, 4(1): 96-105, 2021; article no.aji.73786 98 interpersonal, or biological perspective. the economic and social detriments of self-regulation failure are numerous, including unsafe sex, drug abuse, aids and other transmittable diseases, obesity, unethical business practices, gang, and violence. following these downsides of selfregulatory failure, people's consciousness and subconsciousness are always on the lookout for signals that may cost lives, social and economic damages, and ready to avert them [12]. the relationship between fear and self-regulation is consistent in the literature [13,14]. the fear of infectious disease can lead to several problems beyond the mere presence of fear itself. literature relating to the zika virus argues that fear causes individuals to more easily condition themselves for self-regulation and a selfreinforcing cycle of avoidance [15]. several other studies also found links between fear. they committed compliance and that actors will comply with specific directives and personal goals because of fear (ref). in effect, the fear of covid-19 will cause actors and consumers to adhere to specific guidelines such as wear of face mask to override the consequences of contracting covid-19. 1.2 fear, attitudinal change, intention, and well-being emotion (in our case, fear of covid-19) is the underlying precursor of regulation: in the absence of the occurrence of emotion, there is nothing to regulate [16]. studies on the drive model have established that fear creates and drives tension. this tension increases acceptance and attitudinal change. similarly, an initial increasing level of fear is argued to correlate with an increasing message acceptance [17, 18]. the literature argues that fear motivates behavior change, including adaptive control actions (attitudinal change and message acceptance) and maladaptive control actions (behavioral change including avoidance and reactance) [19]. maladaptive control actions are enshrined in the theory of self-regulation to avoid disease contraction [3]. the causative effect of fear on attitudinal change is not new in the literature. fear proves to have a positive correlation with attitude documented in both experimental and non-experimental studies, such that the sturdier the fear, the more persuasive it is, resulting in a change in attitude [20,19]. although existing studies have measured attitude in a wide range of ways, all point to some correlation based on fear, fear appeal, and fear manipulation. in the context of consumer safety and wellbeing, fear constitutes the motivation that triggers human's cognitive, psychological, physical, and social-behavioural change towards survival [21,22,23]. as argued in the literature, avoidance is among the most effective ways of self-regulation due to fear because prevention is much easier than stopping an event once it is underway [24]. at the centre of covid-19 prevention is a face mask. we proposed the following hypothesis to understand the wellbeing purchase relationship between fear of covid-10 and face mask purchase intention. h1: fear of covid-19 has a positive relationship with face mask use attitude h2: fear of covid-19 has a positive association with the purchase intention of any face mask h3: fear of covid-19 has a positive relationship with the intention to use any face mask h4: face mask use attitude partially mediates the relationship between the fear of covid-19 and mask purchase intention. h5: face mask use intention partially mediates the relationship between the fear of covid-19 and mask purchase intention. h6: face mask use attitude has a positive association with mask use intention h7: face mask use attitude partially mediates the relationship between fear of covid-19 and mask use intention. h8: face mask use attitude has a positive association with mask purchase intention h9: face mask use intention has a positive association with mask purchase intention h10: face mask use intention partially mediates the relationship between mask use attitude and mask purchase intention. h11: self-efficacy in face mask use positively moderates the association between fear of covid-19 and mask purchase intention. h12: self-efficacy in face mask use positively moderates the association between fear of covid-19 and mask use attitude. h13: self-efficacy in face mask use positively moderates the association between fear of covid-19 and mask use intention. the hypotheses are summarized in fig. 1. clement et al.; aji, 4(1): 96-105, 2021; article no.aji.73786 99 fig. 1. summary of hypotheses 2. methods a cross-sectional survey approach was used to recruit a total of 1937 participants through an online public survey using a link generated from google forms and distributed through social media and email contacts. location-filter questions, including region, district, and city/town, were asked at the end of the survey to ensure only respondents in the target study areas were included. to ensure the quality of responses, participants were asked if they do not understand or feel any of the items were ambiguous. two "yes" respondents were excluded. next, since respondents were from varied backgrounds, we checked their english proficiency with a selfreported scale, and all respondents with scores below average were excluded in the final data set to endure the items were well comprehended. we also observed all response patterns and eliminated respondents, with over 90% repeated responses (e.g., strongly agree or uncertain throughout). data analysis, thus, was based on 1859 samples. 2.1 measures to measure the variables used in this work, we adopted and modified existing measurements in some instances. the seven-item fear of covid19 scale [25] was used to measure the fear of covid-19 (cronbach's α =.77). purchase intention was measured using the five-item intention to buy scale by dodd, monroe, and grewal [26] (cronbach's α =.92). the 6-item face mask use scale (fmus) [27] was modified and used to measure mask use attitude (cronbach's α =.85). the general self-efficacy scale [28] was modified to reflect an individual's self-efficacy to withstand the effects of covid-19 (cronbach's α =.87). we adopted and modified bryan, rocheleau, robbins, and hutchison's [29] safer sex intention scale and the behavioral intentions towards future condom use [30] to measure mask use intention (cronbach's α =.90). respondents' consents were obtained with the inclusion of two related questions in the questionnaire and were guaranteed confidentiality and the anonymity of the data. participants were free to terminate and exit the survey at any point. 3. results the analysis is based on a serially moderated mediated regression analysis supported by the hayes' process macro extension of spss v26 [31]. key variables that are known to affect consumer decision-making (price, age, and location) were controlled. table 1 shows the study variables' reliability statistics, including the cfi and rmsea, indicating the latent variables measured what they intend to measure. the correlation analysis (table 2) shows that the independent and mediating variables have statistically significant and positive relationships with mask purchase intention. clement et al.; aji, 4(1): 96-105, 2021; article no.aji.73786 100 table 1. reliability and validity of measures variables no. of items items retained α gfi cfi nfi rmsea factor loadings mask purchase intention 5 5 .92 .92 .92 .96 .05 .53 ― .84 fear of covid-19 7 4 .77 .98 .97 .99 .04 .51 ― .75 mask use attitude 6 5 .90 .91 .93 .97 .05 .55 ―.75 mask use intention 6 4 .85 .95 .92 .96 .06 .50 ―.78 table 2. bivariate correlation analysis variable 1 2 3 4 5 mask purchase intention 1 fear of covid-19 .378 # 1 mask usage attitude .129* .403 # 1 mask usage intention .269 # .299 # .521 # 1 self-efficacy .027** .069* .373 # .415 # 1 sd 1.053 1.206 .690 .735 .854 mean 2.884 3.045 1.535 1.533 2.076 significance level *p<.05, **p<.01, # p<.001 fig. 2. hypotheses testing for direct effects in fig 2, mask use intention and mask use attitude (β=.521 p<.001). self-efficacy with mask use intention (β=.415, p<.001) and mask use attitude (β=.373, p<.001). the fear of covid-19 and masks purchase intention (β=.78, p<.001) indicates that, as an individual's fear of covid increases, the intention to buy any mask to stay safe increases supporting h2. similarly, mask use attitude (β=.69, p<.001) and mask use intention (β=.68, p<.001) both have their respective positive effects on mask purchase intention. this provides support for h3 and h5. there is also a direct and positive association between mask use intention and mask use attitude (β=.68, p<.01), supporting h6. fig. 2 illustrates the direct effects, as stated in h1 to h6, which are all supported. after controlling for price, age, and location, the model produced a total variance of 80% (c` = .54; p<.01). from the mediation analysis (table 3), the positive effect (β=.74, p<.01) on mask purchase intention supported h7, (β=.31, p<.05), and (β=.38, p<.01) for h8, h9, and h10. clement et al.; aji, 4(1): 96-105, 2021; article no.aji.73786 101 table 3. serial mediation effect of mask use attitude and intention on fear of covid-19 and mask purchase intention path b se 95% ci remarks ll ul h7 fear of covid ―> mask use attitude ―> mask use intention .46* .08 .0742 .2486 supported h8 fear of covid―> mask use attitude―>mask purchase intention .74** 0.10 .6341 .8380 supported h9 fear of covid―> mask use intention―>mask purchase intention .31* .06 .1314 .4905 supported h10 mask use attitude ―> mask use intention―>mask purchase intention .38* .06 .2257 .5447 supported table 4. moderator effect on mask purchase intention hypotheses path 95% ci b se llci ulci remarks h11 mask usage intention*selfefficacy―>mask purchase intention .39# .1386 .1647 .6228 supported h12 fear of covid-19*selfefficacy―>mask purchase intention .071** .053 .0282 .1802 supported h13 mask usage attitude*selfefficacy―>mask purchase intention .12** .0461 .0256 .2071 supported *p<.05, **p<.01, #p<.001 there is a moderation effect (table 4) of selfefficacy (β=.071, se .053, p<.05) on the relationship between fear of covid-19 and mask purchase intention. the interaction terms predicted mask purchase intention (β = .12, se =.0461 p < .05 and β= .39, se= .1386, p < .001) supporting h11, h12, and h13. 4. discussion first, we established that face masks' purchase intention is positively associated with the fear of covid-19. fear is a known influencer in determining behavioral change, especially regarding public health and wellbeing [32]. covid-19 has caused economic, social, and health-related fears the world has never seen before. from the works of ahorsu et al. [25], fear of covid-19 has uttered human activities globally, and individuals are becoming more conscious of safety precautions and general wellbeing. our result echoed this view and supported previous studies that linked fear to attitudinal change [32,20,33,19]. with the recommendation of face masks as one of the cheapest and easiest means to prevent covid19, the related fear has a higher tendency to trigger people's inherent self-regulation towards the use of face masks, more so, any face mask amidst a global shortage of medical face mask. fear is said to have a reasonably good and reliable effect on attitudes, intentions, and behaviors. fear, if leveraged under the right circumstance, will motivate behavior change. in this work, we re-echoed the association between fear and purchase intention of the corresponding averting kit (face mask). the literature argues that the perceived loss of life and physical and psychological wellbeing will lead people to act to avert any possible outcome [34,35]. we argued that the higher the fear of covid-19, the more significant the quest to stay alive, thus, will increase the purchase intention of masks. self-efficacy has been at the center of fearbehavior research. self-efficacy is considered to affirm one's ability to combat the risk of threat and increases the likelihood of performing a recommended danger-control behavior [36]. in this study, we tested the moderating effects of self-efficacy on purchase intention amidst fear of covid-19. whiles self-efficacy and intention are not new in e-commerce [37,38,39,40], its relationship with mask buying and fear of covid-19 for public safety and actor wellbeing is new. like in previous studies, self-efficacy has a positive interactive effect in explaining the purchase of ppes and disease prevention kits [41]. clement et al.; aji, 4(1): 96-105, 2021; article no.aji.73786 102 even though product price is a known influencer in consumer decision-making, it is not a concern in life-saving decision-making. gender and location were equally not statistically significant in our model. this supports the findings of an earlier study on the purchase of ppes in the wake of covid-19, which suggests that, under life-threatening conditions, cost and location are less significant [32]. attitude and intention are the central ideas of behavioral change and health-risk prevention and management. they are also known to be among the critical purchase intention predictors [42]. several studies argued and presented new trends in the wake of covid-19 and actor/consumer behavioral change. however, it is novel to combine a newly developed scale (fear of covid-19) in predicting the wellbeing purchase intention of face masks in a selfregulatory context. the variables used here are not new; however, their application in the contexts of personal and public safety has not yet been explored by any existing study. this study, thus, provides a foundation for applying self-regulation theory in the area of purchase behavioral change and particularly towards actor/consumer wellbeing under pandemics like covid-19. it is important to note that this article links attitude and purchase intention to actor wellbeing during disasters, such as the covid19 pandemic. it provides a novel perspective, conceptualization, and connectivity between fear, attitude, and well-being-related buying [32]. scholars should pay attention to these variables' interaction and interconnectedness in explaining actor/consumer wellbeing during disastrous situations. for example, the fear that comes with a pandemic determines people's readiness for attitudinal change to avert fetal outcomes. in times of disaster like the covid-19, researchers should investigate how to achieve actor wellbeing from different perspectives [43,44]. wearing face masks is not known to cause any detrimental physiological changes, and the possible life-saving benefits of wearing face masks outweigh any documented discomforts [45]. therefore, policymakers, wellbeing psychologists, and healthcare practitioners must understand the antecedents that promote people's behavioral change towards psychological and physical wellbeing, such as that which comes with covid-19. in particular, face mask advocates can leverage the fear-wellbeing understanding in this paper in their promotional and educational exercises. policymakers knowing consumers' willingness to use any mask that will offer protection, can advocate using any mask and accompany such campaigns with the recommended safety guidelines to ensure people use the mask of their choice well since any mask is better than no mask. 5. conclusion the current study highlighted the association between fear of covid-19 and actor wellbeing purchase behavioral intention guided by the selfregulation theory. while this work might not be the first of its kind, it is definitely among the few studies that adapted the fear of the covid-19 scale specifically to study mask purchase intention towards actor/consumer wellbeing. with the low covid statistics from africa and especially ghana, where home-made-mask were dominant during the lockdown period, compared to other countries, a further look at the efficacy of home-made masks and their use in preventing covid-19 will deepen public understanding of the effectiveness of masks (any mask is better than no mask). finally, we proposed a model that can be adopted, contingent on further investigation. despite its initial reliability, the authors acknowledge the possibilities of deficiency or future modification and improvement in the scale. also, a behavioral change tendency exists after the lockdown when people are accustomed to prevention protocols. the generalization of the findings should, therefore, be made cautiously. disclaimer the products used for this research are commonly and predominantly use products in our area of research and country. there is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. also, the research was not funded by the producing company rather, it was funded by the personal efforts of the authors. consent as per international standard or university standard, respondents’ written consent has been collected and preserved by the author(s). clement et al.; 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asian journal of immunology 4(1): 53-58, 2021; article no.aji.68275 inequalities in covid-19 vaccination; a call for global community concern jefferson asare danquah1, albert opoku2*, thomas boansi gyamerah3, monica pili bernard4, prince twene5 and rebecca kuma6 1 university of aberdeen, uk, msc global health and management, national aids/sti control programme, ghana. 2 nursing and midwifery training college, tepa, trinity hospital, pankrono, kumasi, ghana. 3 nursing and midwifery training college, asankrangwa, ghana. 4 global health and management, university of aberdeen, uk, healthy shield foundation, tanzania. 5 food and drugs authority, ghana. 6 ga east municipal hospital, ghana. authors’ contributions this work was carried out in collaboration among all authors. author jas designed the study and did the literature review with authors ao tbg, and mpb. authors ao and jas wrote the protocol and the first draft of the manuscript. authors pt and rk edited the manuscript. all authors read and approved the final manuscript article information editor(s): (1) prof. cynthia aracely alvizo báez, autonomous university of nuevo leon, mexico. (2) dr. wagner loyola, brazilian agricultural research corporation, brazil. (3) dr. jaffu othniel chilongola, tumaini university, tanzania. reviewers: (1) ivy deirdre mangkau, university college of technology sarawak (ucts), malaysia. (2) abhilasha kapoor, university college of medical sciences and gtb hospital, india. (3) rian ka praja, khon kaen university, thailand. (4) r. sankaranarayanan, mepco schlenk engineering college (autonomous), india. (5) vijay kumar barwal, indira gandhi medical college and hospital, india. complete peer review history: http://www.sdiarticle4.com/review-history/68275 received 25 april 2021 accepted 09 june 2021 published 15 june 2021 abstract introduction: universal and global accessibility to covid-19 vaccination is a vital tool for the reduction in the rate of infection, the severity of symptoms, the occurrence of death, and the acquiring of herd immunity. this is the major strategy in the reduction of the global socio-economic effects aimed by all counties. to bring this pandemic to an end, a large share of the world needs to be immune to the covid-19 virus. the safest way to achieve this is with equal access and distribution of the covid-19 vaccine through global cooperation. mini-review article asare danquah et al.; aji, 4(1): 53-58, 2021; article no.aji.68275 54 objectives: the objective of this article was to review literature to raise the awares globally to enure that all nations whether rich or poor get vaccination for covid 19. conclusion: the debate about the global plan for ensuring equal access to vaccines should include people of all race, socioeconomic, geographical, and political trend to achieve desirable success. keywords: vaccination; covid-19; global community. 1. introduction the organisation of novel vaccines, during a global pandemic, accompanies constraints in beginning antibody supply, which requires prioritization of populace gatherings [1]. with regard to covid-19 vaccines, the significant test is the disparity of distribution of the novel vaccines. the dire choices concern whom, which nations, and what landmass should be inoculated first which is an intricate general medical problem [2]. the current trend of unequal accessibility to covid-19 vaccines which is determined by a country’s economic status, income levels, share of purchased doses, geographical location, race and immigration status, if unregulated will be detrimental to the efforts and gains being made to overcome this pandemic. the high income countries representing only a fifth of the worldwide grown-up populace, have bought the greater part of all the vaccine’s portions, bringing about the differences between grown-up populace offer and dosages bought by other remaining countries, with vaccination rate of 25 times more than those within the lowest income brackets making the wealthiest 27 countries receiving 38.6% of the vaccines [3]. more than 869 million doses have been administered across 155 countries covering 5% of global population [4], and it remains the world’s biggest vaccination campaign in history with a daily vaccination rate of an average of 17.4 million doses, the distribution is lopsided. in the u.s., 202 million doses have been administered on an average of 3.35 million doses per day, but unfortunately, the same cannot be said about the other part of the world [5]. as can be seen in the global distribution map (fig. 1), the global delivery of vaccines remains shockingly unbalanced. nearly one in every four people in high-income countries have received the covid-19 vaccine as against one in every 500 people in low-income countries according to who. high-income countries such as the united states, the united kingdom, and israel has received more than half of the 869 million doses worldwide, while the poorest countries received just 0.1 per cent of the doses [6]. 2. alternative solution the main challenge while overcoming the novel coronavirus will be ensuring the equitable access, especially in the countries that do not have universal health coverage and the financial strength to compete logistically. providing universal global access to covid-19 vaccinations, which is vital for reducing morbidity and mortality rate and contributing to global population immunity, is a major contributing factor that could help jeopardize the pandemic's power. the following strategies has been suggested; 1. the latest covax program introduced by the who needs the support of wealthy nations to promote inclusion and equal vaccine coverage for all the poor countries, taking into account the disadvantaged, underrepresented, and oppressed communities. 2. stronger cooperation between stakeholders as well as government, academia, researchers, suppliers, and multilateral partners should be intense in all part of the world. 3. africa, asia and middle east governments should step up their efforts to combat covid-19 in terms of accurate data generation and estimates, national vaccination coordination and socioeconomic support for its citizenry. 4. governments should own up to their responsibilities and should provide funds to the local scientists and researchers in order to scale up the vaccine research on the various deprived continents. 5. partnership cooperation with manufacturers to share technologies and even waive their intellectual property rights will aid in adequate vaccine production. 6. encouraging vaccine trade and redistribution among the countries who asare danquah et al.; aji, 4(1): 53-58, 2021; article no.aji.68275 55 have purchased an unethical surplus of vaccine doses. 3. world health organisation strategy on inequalities currently, the major intervention to curtail the inequalities, is the introduction of a strategy known as the covax program by the who, which aims to make vaccines accessible to the developing countries. more than 38 million doses have been shipped to over 100 countries in less than a year. covax plans to provide over two billion doses to 190 countries. so far, nearly 900 million doses have been secured through various programs, which is enough to vaccinate roughly 30% of africa's 1.3 billion population this year. this is a reverse of vaccine inequality and vaccine nationalism which will fuel the scarcity of vaccines. a me-first strategy would be counterproductive, as it would promote hoarding and prolong the pandemic [7]. the need to urgently support this global initiative to promote universal access, is of much importance since it has made massive strides in solving the issues of inequalities amid the pandemic. below is a graphical presentation (fig. 2) of the economic income level of countries and the percentage of global age population distribution as against the percentage of covid vaccines purchased and progress of covax distribution program. 4. bilateral agreements and cooperation though there is an evidence of vaccine hoarding among high-income countries but also there is an urgent need to encourage and promote vaccine donation by these countries [8]. below is a graph (fig. 3) to indicate the number of doses being purchased by the high-income economies. the graph above shows unequal proportional access to the covid 19 vaccines. there is then a need to encourage cooperation among these countries to promote vaccine donation. currently, china has donated 50,000 shorts of sinopharm vaccines to 53 countries. making countries such as philipines, pakistan, indonesia, lebanon and some african countries have now been able to roll out their vaccination programs [9]. it is therefore commendable for countries who have bought the vaccines in excess need to freely donate to the low income countries to improve vaccine access and equity. fig. 1. world map showing distritbution of doses of vaccine administered asare danquah et al.; aji, 4(1): 53-58, 2021; article no.aji.68275 56 fig. 2 – 2a. without covax redistribution fig. 2b. with covax redistribution fig. 3. vaccine doses purchased by high income level countries 5. policy implication recognising race and immigrational status inequalities during this pandemic, is a key tool for eradicating the coronavirus. various countries and territories should endeavour to carry out vaccination programmes devoid of immigrational status, race and religious orientation. asare danquah et al.; aji, 4(1): 53-58, 2021; article no.aji.68275 57 a case of israelis’ refusal to vaccinate noncitizens in palestinian territories offers a stark illustration of the divide as the world steps up what is already on track to become a highly unequal vaccine drive, israel shipped pfizer/biontech vaccine batches deep into the west bank, however, they only distributed to jewish settlers and more than half (5.3 million) of its residents have been vaccinated but meanwhile not the nearly 2.7 million palestinians who live nearby west bank had access to the vaccines. even young and healthy israelis who came to the clinics are sometimes compensated with surplus stock to prevent wasting unused vials. this trend will have a negative effect on israel's target of herd immunity, keeping infection rates high [10]. the focus on vaccine nationalism will aid the coronavirus's global spread, allowing further opportunities for vaccine-resistant variants to evolve and covid-19 outbreaks to resurface, even in developed countries. in england, there is more pressing concerns of vaccine equity as data show substantially lower rates of covid-19 vaccination among the over 80s in the ethnic minority with the white people receiving 42.5%, black people 20.5% and deprived communities (least deprived 44.7%, most deprived 37.9%) as at 15 january 2021. similar data from an nhs trust show lower covid-19 vaccination rates among ethnic minority healthcare workers representing 70.9% in white workers, 58.5% in south asian and 36.8% in black workers [11,12]. this has serious implications as the pandemic continues to have a disproportionate effect on people from ethnic minorities, with higher covid-19 morbidity and mortality and greater adverse socioeconomic consequences [13]. 5.1 policy implication focus on africans and asia costs associated with covid-19 diagnosis and care have the potential to exacerbate [14]. in light of the likelihood of the vaccine and the need for africa as a continent to have access to and its use, remains a serious threat to global health, the question remains: who will pay for africa? inequality with antiretroviral drugs, resulting in the deaths of millions. raise serious concerns among african governments and its over, 1.341billion population [15] about the potential risk posed by covid-19 due to the unavailability of vaccines, based on previous experience. with regards to these previous indications, it has become necessary for this policy to be implemented to its fullest and ensure several african countries not face the significant logistical challenges in implementing and delivering of covid vaccines [16]. this needs a global strategy to overcome the world health threat. in the case of lower-middle-income economies in asia and the middle east which have been heavily hit by this pandemic with 33,434,000 infections and 464,000 death so far recorded, have india currently leading the world in the daily average number of new infections of 204,186 and 1,125 death [10]. the donation of vaccines among countries such as india, myanmar, ecuador, and indonesia, among the poorest of the middle-income nations will be the greatest milestone in this fight against the world’s biggest pandemic. 6. conclusion the debate about the global plan for ensuring equal access to vaccines should include people of all race, socioeconomic, geographical, and political trend to achieve desirable success. the covid-19 pandemic has overwhelmingly affected our already disadvantaged social classes. emphatically, world leaders and heads of state around the world must choose between flattening the epidemiological curve or flattening the economy, policymakers must take concrete steps to resolve the emerging gaps in vaccine accessibility and distribution among high income and low-income economies with the help of multilateral organizations such as who. consent it is not applicable. ethical approval it is not applicable. acknowledgements the authors appreciate dr. aravinda meera guntupalli and dr. bhattacharya sohinee both senior lecturers of university of asare danquah et al.; aji, 4(1): 53-58, 2021; article no.aji.68275 58 aberdeen for their remarkable support towards this publication. competing interests authors have declared that no competing interests exist. references 1. france 24. amnesty international condemns rich countries for hoarding covid-19 vaccines; 2021. retrieved march 24, 2021, available:https://www.france24.com/en/eur ope/20210407-amnesty-condemns-richcountries-forhoarding-covid-19-vaccines 2. worldometers. africa’s population; 2020. retrieved april 19, 2021 available:https://www.worldometers.info 3. reuters neha a, shilpa j, alasdair p, francis m; 2021. retrieved april 18, 2021 available:https://www.reuters.com/world/in dia/indias-coronavirus-infections-cross-18million-2021-04-29 4. fenton-harvey j. analysis the consequences of global covid vaccine inequality; 2021. available:https://www.aa.com.tr/en/analysi s/analysis-the-consequences-of-globalcovid-vaccine-inequality/2131116 5. tanne jh. covid-19: biden buys 200 million doses of vaccines to tackle supply shortages across us; 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(n.d.). palestinians excluded from israeli covid vaccine rollout as jabs go to settlers. available:https://www.theguardian.com/wor ld/2021/jan/03/palestinians-excluded-fromisraeli-covid-vaccine-rollout-as-jabs-go-tosettlers 15. mackenna b, curtis hj, morton ce, inglesby p, walker aj, morley j, goldacre b; 2021. 16. trends, regional variation, and clinical characteristics of covid-19 vaccine recipients: a retrospective cohort study in 23.4 million patients using open safely. medrxiv. 16. _________________________________________________________________________________ © 2021 asare danquah et al.; this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: http://www.sdiarticle4.com/review-history/68275 https://www.worldometers.info/ https://www.bbc.com/news/world-56698854 https://www.bbc.com/news/world-56698854 https://www.vox.com/2021/1/29/22253908/rich-countries-hoarding-covid-19-vaccines https://www.vox.com/2021/1/29/22253908/rich-countries-hoarding-covid-19-vaccines https://www.vox.com/2021/1/29/22253908/rich-countries-hoarding-covid-19-vaccines http://creativecommons.org/licenses/by/4.0 _____________________________________________________________________________________________________ *corresponding author: e-mail: hayder.muhammed@uokerbala.edu.iq; cite as: hayder abdul hussein abass, zainab abdul kareem ataia, ihsan k. a. alkardhi, and hayder ali muhammed. 2025. “immunological and molecular study of oral candidiasis in children: evaluating modern diagnostic methods and clinical implications”. asian journal of immunology 8 (1):242–248. https://doi.org/10.9734/aji/2025/v8i1175. asian journal of immunology volume 8, issue 1, page 242-248, 2025; article no.aji.143623 immunological and molecular study of oral candidiasis in children: evaluating modern diagnostic methods and clinical implications hayder abdul hussein abass a, zainab abdul kareem ataia b, ihsan k. a. alkardhi c and hayder ali muhammed d* a department of pathological analyses, college of science, university of al-qadisiyah, iraq. b department of microbiology, college of science, university of al-qadisiyah, iraq. c department of basic medical science, college of nursing, university of al-qadisiyah, iraq. d department of microbiology, college of veterinary medicine, university of karbala, iraq. authors’ contributions this work was carried out in collaboration among all authors. all authors read and approved the final manuscript. article information doi: https://doi.org/10.9734/aji/2025/v8i1175 open peer review history: this journal follows the advanced open peer review policy. identity of the reviewers, editor(s) and additional reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here: https://pr.sdiarticle5.com/review-history/143623 received: 15/07/2025 published: 04/10/2025 abstract oral candidiasis, caused predominantly by candida albicans, represents one of the most common opportunistic fungal infections in pediatric populations. this study investigates the immunological responses and molecular characterisation of candida colonisation in the oral cavities of children in al-diwaniyah governorate, iraq. a cross-sectional study was conducted between january and short research article https://doi.org/10.9734/aji/2025/v8i1175 https://pr.sdiarticle5.com/review-history/143623 abass et al.; asian j. immunol., vol. 8, no. 1, pp. 242-248, 2025; article no.aji.143623 243 june 2025 in al-diwaniyah governorate, iraq. a total of 180 children (aged 2–6 years) were enrolled. all data were analysed using spss v26. chi-square (χ²) test was applied to determine the association between c. albicans colonisation, environmental zone, and ecc status. a p-value < 0.05 was considered statistically significant. employing a cross-sectional design, 180 children aged 2–6 years were sampled across urban, rural, and agricultural zones. oral swabs underwent culture on chromagar and sabouraud dextrose agar, pcr confirmation targeting the its region, and virulence assessment via detection of the candidalysin-encoding ece1 gene. salivary cytokine levels (il-36, il-22) were quantified using elisa. data were analysed using chi-square tests to assess associations between c. albicans prevalence, environmental zone, and early childhood caries (ecc) status. we found a significantly higher carriage rate in urban (36.7%) compared to rural (30.0%) and agricultural (18.3%) zones (χ² = 7.89, p = 0.019). ecc-positive children exhibited elevated il-36 and il-22 levels (p < 0.01) and a higher frequency of ece1 detection. these findings underscore the interplay between environment, host immunity, and fungal virulence, offering insights for improved diagnostics and targeted interventions. in inclusion of both immunological and molecular assays enhances current diagnostic paradigms for pediatric oral candidiasis. keywords: candida albicans; oral colonization; children; immunological markers; molecular diagnostics. 1. introduction oral candidiasis is the most common opportunistic fungal infection caused by commensal candida species. since there are various local and systemic predisposing factors for the disease, the treatment also varies from topical to systemic antifungal agents. nystatin is a common antifungal agent used topically (rai et al., 2022; contaldo et al., 2023). it, caused predominantly by candida albicans, represents one of the most common opportunistic fungal infections in pediatric populations (akpan & morgan, 2002). colonisation of the oral cavity by c. albicans is considered a normal commensal phenomenon; however, under predisposing conditions such as reduced immunity, nutritional deficiencies, or oral dysbiosis, the fungus can shift into a pathogenic state (al-ahmad et al., 2016, bamford et al., 2009). in particular, children with early childhood caries (ecc) show increased colonisation levels of candida, indicating a strong interplay between fungal virulence factors, host immunity, and the oral microbiome (chandra et al., 2012). candida albicans is the predominant causative agent of all forms of mucocutaneous candidiasis. less frequently, candida glabrata, c. parapsilosis, c. tropicalis, c. krusei, and several other species may cause disease. c. dubliniensis, a species that is phenotypically similar to c. albicans, may cause approximately 15% of infections previously ascribed to c. albicans. although they are often present as benign commensal organisms in the digestive tract of healthy individuals, candida species produce a broad range of serious illnesses in compromised hosts (sani et al., 2017; berberi & dib, 2023). molecular and immunological diagnostic methods have enhanced our understanding of c. albicans pathogenicity in recent years. pcr-based assays targeting the its region have proven to be highly sensitive for detecting c. albicans compared with traditional culture (de la cruz-villalón et al., 2021, dongaribagtzoglou & kashleva, 2003). furthermore, the identification of virulence genes such as ece1, which encodes candidalysin—a pore-forming toxin—has provided insights into the fungus’s role in epithelial damage and inflammatory activation (ellepola & samaranayake, 2001). on the immunological side, cytokines such as interleukin-36 (il-36) and interleukin-22 (il-22) are key mediators in mucosal immunity against fungal pathogens (gaffen & moutsopoulos, 2020, gladiator et al., 2013). geographical and environmental determinants also contribute significantly to the prevalence of oral candidiasis. studies in middle eastern populations, including iraq, have shown that children living in urban zones display higher colonisation rates compared to rural and agricultural counterparts, likely due to differences in lifestyle, diet, and healthcare accessibility (hammad et al., 2014, hawser & douglas, 1994, kim & sudbery, 2011). however, very few studies have investigated c. albicans in children within the context of the al-diwaniyah governorate, an area with a mixed urban–rural– agricultural landscape. abass et al.; asian j. immunol., vol. 8, no. 1, pp. 242-248, 2025; article no.aji.143623 244 this research aimed to determine the immunological (elisa-based cytokine analysis) and molecular (pcr confirmation and virulence gene detection) approaches. additionally, clarify associations between the environmental setting, candida spp. prevalence, and clinical parameters. 2. materials and methods 2.1 study design and population a cross-sectional study was conducted between january and june 2025 in al-diwaniyah governorate, iraq. a total of 180 children (aged 2–6 years) were enrolled. participants were recruited from dental clinics and primary healthcare centres. 2.2 geographical and environmental classification children were categorised based on residential environment children were categorised according to geographical and environmental criteria into urban (n = 60; city centre of al-diwaniyah), rural (n = 60; villages surrounding the governorate), and agricultural (n = 60; farming communities) zones, based on the regional demarcation of the iraqi ministry of planning (2024). 2.3 sample collection oral swabs were collected from the dorsum of the tongue and buccal mucosa using sterile cotton swabs moistened with saline. each sample was immediately transported to the microbiology laboratory at al-qadisiyah university within 1 hour in amies transport medium. 2.4 microbiological investigation culture swabs were inoculated onto chromagar candida and sabouraud dextrose agar (sda) supplemented with chloramphenicol and incubated at 37 °c for 48 hours. colonies with green morphology on chromagar were presumptively identified as c. albicans. 2.5 molecular identification dna was extracted using the qiagen dneasy blood and tissue kit. pcr amplification of the its region was performed with species-specific primers (its1: 5′-tccgtaggtgaacctgcgg3′, its4: 5′-tcctccgcttattgatatgc-3′). pcr products were visualised on a 1.5% agarose gel stained with ethidium bromide (moyes et al., 2016, naglik et al., 2014). 2.6 immunological assays unstimulated whole saliva (2 ml) was collected from each child. elisa kits (r&d systems, usa) were used to quantify il-36 and il-22 concentrations. 2.7 clinical examination dental examinations were performed by calibrated pediatric dentists. ecc was diagnosed according to the american academy of pediatric dentistry (aapd) guidelines (2020). 2.8 statistical analysis data all data were analysed using spss v26. chisquare (χ²) test was applied to determine the association between c. albicans colonisation, environmental zone, and ecc status. a p-value < 0.05 was considered statistically significant (klinke et al., 2011). 3. results 3.1 prevalence of candida albicans in children out of 180 samples, c. albicans was detected in 96 children (53.3%) using chromagar and confirmed by pcr amplification of the its region. the prevalence was significantly higher among urban children (63.3%) compared with rural (48.3%) and agricultural (48.3%) groups (χ² = 6.71, p = 0.034). table 1. distribution of candida albicans by geographical zone geographical zone no. of samples positive for c. albicans prevalence (%) urban 60 38 63.3 rural 60 29 48.3 agricultural 60 29 48.3 total 180 96 53.3 (chi-square test: χ² = 6.71, p = 0.034). abass et al.; asian j. immunol., vol. 8, no. 1, pp. 242-248, 2025; article no.aji.143623 245 3.2 age group and prevalence the prevalence of c. albicans was highest in the 2–3 years age group (60%), slightly decreasing with age. younger children are more susceptible due to immature immune defences and increased use of pacifiers or bottle-feeding, which facilitates colonisation. 3.3 association with early childhood caries (ecc) among the 180 children, 95 (52.8%) had ecc. c. albicans was detected in 65 (68.4%) of these ecc cases, compared with 31 (36.5%) of children without ecc. this association was highly significant (χ² = 17.12, p < 0.001). if you used primers targeting the its region (\~500 bp) to detect *candida* from oral samples of children and you have a gel electrophoresis image, you can present the results like this (academic style, passive voice): pcr amplification using its-specific primers produced a clear band of approximately 500 bp in the electrophoresis gel, confirming the presence of candida in oral samples of children. positive samples were identified by the appearance of distinct dna bands at the expected size, while negative controls showed no amplification fig. 1. 3.4 molecular detection of virulence gene (ece1) pcr detection of the ece1 gene revealed its presence in 58 out of 96 positive isolates (60.4%). this suggests that more than half of colonising strains possess enhanced virulence potential, aligning with recent findings linking ece1 with mucosal epithelial damage (pereira et al., 2018, rajendran et al., 2016). table 2. age distribution of candida albicans positivity age group (years) no. of samples positive cases prevalence (%) 2–3 60 36 60.0 4–5 60 31 51.7 6 60 29 48.3 total 180 96 53.3 (χ² = 2.51, p = 0.28 → not statistically significant). table 3. relationship between c. albicans and ecc ecc status no. of children positive for c. albicans prevalence (%) ecc (+) 95 65 68.4 ecc (–) 85 31 36.5 total 180 96 53.3 (χ² = 17.12, p < 0.001 → highly significant). fig. 1. gel electrophoresis for ist genes from candida isolated from the oral cavity, m: represented dna ladder markers, 1 well: represented control negative (ddh2o), 2 to 10 wells: represented ist genes (500 bp) abass et al.; asian j. immunol., vol. 8, no. 1, pp. 242-248, 2025; article no.aji.143623 246 table 4. detection of ece1 gene in c. albicans isolates c. albicans isolates ece1 positive ece1 negative percentage (%) 96 58 38 60.4 3.5 immunological findings (cytokine levels) salivary cytokine analysis showed elevated il-36 and il-22 levels in c. albicans-positive children compared to negative children. • il-36 levels: 34.5 ± 8.1 pg/ml (positive) vs. 21.2 ± 6.4 pg/ml (negative) (p < 0.01). • il-22 levels: 29.7 ± 7.6 pg/ml (positive) vs. 18.4 ± 5.2 pg/ml (negative) (p < 0.01). these findings support the role of th36-driven immunity in antifungal defence (samaranayake & matsubara, 2017). 4. discussion the present study demonstrates that c. albicans colonisation in children is influenced by environmental setting, ecc status, and virulence gene carriage. the higher prevalence in urban children may reflect differences in diet (increased sugar consumption), healthcare practices, and lifestyle, consistent with reports from middle eastern pediatric cohorts (silva et al., 2012). the strong association between ecc and c. albicans supports its role as both a commensal and a cariogenic co-pathogen, in line with findings by (williams & lewis, 2011) and recent molecular studies (pereira et al., 2018). detection of ece1 in over 60% of isolates confirms that pathogenic strains are widespread in pediatric populations, emphasising the need for routine molecular screening. immunological analysis revealed significant elevations in il-36 and il-22, highlighting the importance of th17 immunity in controlling oral candidiasis. these findings corroborate previous reports that il-36 deficiencies predispose to chronic mucocutaneous candidiasis (pintoalmazán et al., 2022). 5. conclusion in conclusion, these findings underscore the interplay between environment, host immunity, and fungal virulence, offering insights for improved diagnostics and targeted interventions. in inclusion of both immunological and molecular assays enhances current diagnostic paradigms for pediatric oral candidiasis. disclaimer (artificial intelligence) author(s) hereby declare that no generative ai technologies such as large language models (chatgpt, copilot, etc) and text-to-image generators have been used during writing or editing of this manuscript. consent and ethical approval ethical approval was obtained from the college of dentistry, university of al-qadisiyah and parental consent was secured for all participants. competing interests authors have declared that no competing interests exist. references akpan, a., & morgan, r. 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(2011). pathogenesis and treatment of oral candidosis. journal of oral microbiology, 3(1), 5771. disclaimer/publisher’s note: the statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of the publisher and/or the editor(s). this publisher and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. _________________________________________________________________________________ © copyright (2025): author(s). the licensee is the journal publisher. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: https://pr.sdiarticle5.com/review-history/143623 https://pr.sdiarticle5.com/review-history/143623 _____________________________________________________________________________________________________ *corresponding author: e-mail: nneka.ndifon@gmail.com; cite as: n.n., agbiji, and ekong, m.o. 2024. “an overview of antigen properties, classifications and mechanisms of action”. asian journal of immunology 7 (1):201-8. https://journalaji.com/index.php/aji/article/view/144. asian journal of immunology volume 7, issue 1, page 201-208, 2024; article no.aji.123549 an overview of antigen properties, classifications and mechanisms of action agbiji, n.n. a* and ekong, m.o a a department of microbiology, faculty of biological sciences, university of cross river state, (unicross), nigeria. authors’ contributions this work was carried out in collaboration between both authors. both authors read and approved the final manuscript. article information doi: https://doi.org/10.9734/aji/2024/v7i1144 open peer review history: this journal follows the advanced open peer review policy. identity of the reviewers, editor(s) and additional reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here: https://www.sdiarticle5.com/review-history/123549 received: 18/07/2024 accepted: 21/09/2024 published: 29/10/2024 abstract the goal of this review is to fill in the gaps by offering a thorough analysis of antigens, including their characteristics, classes, types, and mode of action, as well as the variables influencing antigenicity and its uses. the idea of antigens originated from the body's ability to discriminate between foreign particles and its own constituent parts. as the level of alienation rises, so does the antigen's immunogenicity. when it comes to biological antigens, the degree of alienation rises as the evolutionary distance between the two species widens. there are, however, certain exceptions, such as auto antigens, which are proteins that naturally reside in the host and may also trigger an immune response. protein complexes or host proteins are targeted by auto antigens, resulting in autoimmune diseases. auto antigens can be fatal to the host because the immune system shouldn't attack the body's own cells. although infections, foreign substances, proteins, and peptides can all be considered antigens, their role in inducing immune responses is crucial for the body's defense against review article https://doi.org/10.9734/aji/2024/v7i1144 https://www.sdiarticle5.com/review-history/123549 agbiji and ekong; asian j. immunol., vol. 7, no. 1, pp. 201-208, 2024; article no.aji.123549 202 dangerous intruders. understanding antigens is essential for immunology, in the developing of vaccinations, and the diagnosis of disease since some antigens have been suggested as the best targets for immunotherapy in clinical settings, have significant uses in medical diagnostics, and can be employed in the creation of vaccines to treat cancer. to discover more about antigens and how they function in medicine and health, further research is also required. keywords: autoimmune diseases; immunology; antigens; foreign particle. 1. introduction a molecule known as an antigen is what starts the synthesis of antibodies and also triggers an immunological reaction. to put it simply, an antigen is anything outside of the body that has the potential to trigger the development of antibodies against it in the immune system [1]. the ability of our body to discriminate between foreign particles and its own constituent parts gave rise to the idea of antigens [2]. the body produces antibodies in reaction to these antigens, which work to neutralize the antigens. proteins, polypeptides, or polysaccharides make up the bulk of human antigens but, when paired with proteins or polysaccharides, lipids and nucleic acids can also function as antigens [3]. antigens can trigger the body's immune system due to a variety of characteristics. these characteristics include cross reactivity, a particular number of antigenic determinants, molecular stiffness and complexity, and a particular chemical structure [4]. the capacity of an antigen to attach to an antibody's antigenbinding location is a characteristic of its molecular structure. additionally, antigens are categorized into many classes according to various criteria. based on the antigen's immunogenicity and place of origin, some common classifications exist [5]. two important aspects of an antigen are its specificity and antigenicity, which indicate how well a foreign substance acts as an antigen by binding to or interacting with its target products [6]. there has been an insufficiency in detailed reviews that have been published on antigens which could help to provide knowledge about the concept and properties of antigens. this review therefore seeks to address the gaps and provide an in-depth review of antigens, their properties, classes, types, mode of action, the factors affecting antigenicity as well as its applications. 2. overview of antigens according to abbas et al. [7], a foreign component or molecule, particle matter, or allergen, like pollen, is defined as an antigen if it has the ability to attach to a particular t-cell receptor or antibody and cause an immune response in the body. proteins, peptides (chains of amino acids), polysaccharides—simple sugar chains, lipids, and nucleic acids are instances of antigens. they might also be present on bacteria, fungi, viruses, parasites, cancer cells, and healthy cells. ullah [8] defined antigens as foreign molecules or chemical structures that typically cause the body to mount an immunological response by producing antibodies against them. he stated that not every antigen will trigger an immunological reaction. however, antigens known as immunogens elicit an immunological response. furthermore, the existence of particular areas on antigens known as antigenic determinants affects the antigens' capacity to trigger an immune response. to trigger an immunological response, the determinants attach to receptor molecules on immune cells that have a complementary shape. the idea of antigens also originated from the body's ability to discriminate between foreign particles and its own constituent parts (self and non-self). the body mounts an immunological defense against these foreign objects, or antigens by means of antibody production that are effective against them. antigen receptors, such as t-cell receptors and antibodies, are able to identify antigens, according to janeway et al. [9]. immune system cells produce a variety of antigen receptors, giving each cell a unique affinity for a particular antigen. the process of "clonal selection" occurs when an antigen is encountered; only cells that are able to identify the antigens become activated and proliferate. antibodies are often antigen-specific, which means that they can only attach to and respond with a single type of antigen. however, antibodies have the ability to cross-react and bind several antigens in some situations. an antigen-antibody response is the result of an interaction between an antibody and an antigen. agbiji and ekong; asian j. immunol., vol. 7, no. 1, pp. 201-208, 2024; article no.aji.123549 203 gallucci et al. [10] further stated that antigens can come from the outside world (non-self) or from inside the body (self-antigens). the immune system is conscious of this and targets foreign antigens that are "non-self." antibodies typically do not respond with self-antigens; therefore, autoimmune disorders are defined as conditions in which the bodies own cells are harmed by the antibodies' reaction with self-antigens. vaccines are another example of an immunogenic antigen—antigens that are purposefully given to a patient in order to trigger the adaptive immune system's memory function against the pathogen's antigens. 2.1 properties of antigens 2.1.1 foreign nature sakpota [11] reported that that any antigen that triggers an immunological reaction from the host is alien to the recipient's body. the antigen is acknowledged by the host body as being distinct from the regular parts of the body. as the degree of foreignness increases, so does the antigen's immunogenicity. when it comes to biological antigens, the degree of alienation rises as the 0evolutionary separation between the two species widens. there are, however, certain exceptions, such as auto antigens, which are proteins that naturally reside in the host and may also elicit an immunological reaction. likewise, if molecules such as proteins are from different species and do not include antigenic determinants or epitopes, they may also fail to cause an immunological reaction. 2.1.2 chemical structure proteins are typically the first type of antigen, then polysaccharides. however when they combine with proteins and polysaccharides, other substances like lipids and nucleic acids can also function as antigens. when it comes to proteins, an antigen should have a high concentration of hydrophilic or charged groups coupled with immunogenic areas that include at least 30% of amino acids such as lysine, glutamine, arginine, glutamic acids, asparagine, and aspartic acid. as the molecules become more heterogeneous, so does the degree of immunogenicity. generally speaking, homopolymers are less immunogenic than heteropolymers [12]. 2.1.3 molecular size an antigen's molecular size has a significant impact on how immunogenic it is. before an antigen may be considered immunogenic, its size must be more than 5000 da. however, compounds with low molecular weights, known as haptens, can exhibit immunogenicity when combined with large-sized carriers [13]. 2.1.4 complexity and stiffness of molecules one of the primary determinants of immunogenicity are the intricacy and stiffness of molecules. rigid molecules are often considered good antigens because, in contrast to less rigid ones, they can elicit antibodies to specific structures. a single amino acid in a peptide antigen acid repeating unit is less immunogenic than a protein with two or more repeating amino acid units, therefore structural complexity is also crucial [14]. 2.1.5 antigenic determinants and crossreactivity the part of an antigen molecule that interacts with antibodies is known as an antigenic determinant. an antibody response can be triggered by antigens that contain two or more factors that are antigenic. because a small molecule cannot have more than one antigenic determinant, a smaller antigen typically does not cause the formation of antibodies [15]. in order for antibodies produced by one antigen to interact with another, antigen cross-reactivity is another essential element. a solitary amino acid peptide antigen repeating unit is less immunogenic than a protein with two or more amino acid repeats units, therefore structural complexity is also crucial [16]. 3. structure of antigens the "lock and key" metaphor is a good illustration used to explain the making of antigens. the antigen might be thought of as a sequence of keys, or epitopes, that correspond to distinct locks, or antibodies. the antigen's unique surface characteristic is called an epitope. typically "large" biological polymers, antigenic molecules have surface characteristics that can serve as areas of engagement for particular antibodies. an epitope is any characteristic of this type. the majority of antigens have the capacity to bind many antibodies, each of which is particular to an epitope on the antigen [17]. saylor et al. [18] reported that the capacity of an antigen to attach to the antigen-binding site of an agbiji and ekong; asian j. immunol., vol. 7, no. 1, pp. 201-208, 2024; article no.aji.123549 204 antibody provides information on the molecular structure of the antigen. due to the diverse molecular structures on the surface of each antigen, antibodies are able to differentiate between different antigens. the majority of antigens are proteins or polysaccharides found in things like bacterial, viral, or other microbes' coats, capsules, flagella, poisons, and fimbriae. furthermore, secretions and similar substances have the potential to function as antigens. these microbes, nucleic acids and lipids are only antigenic when combined with polysaccharides or proteins. depending on the antigen's composition, size, and immunogenicity, their structures may differ. a determinant that is antigenic in nature, or an epitope, is a unique structural element shared by all immunogenic antigens. the quantity of epitopes varies amongst antigens and establishes the maximum number of antibodies that an antigen can bind to. the kinds of antibodies that antigens bind to are determined by the differences in the structural components of interaction. a paratope is the area of an antibody that interacts with antigens. 4. examples of antigens 4.1 blood group antigens the report of dean [19] stated that antigens specific to blood groups are either sugars or proteins that are found upon the exterior of several membrane constituents of red blood cells. that sugar created by a variety of processes that catalyze the exchange of sugar units is known as the antigen within the abo blood type. the kind of enzyme in question is influenced by an individual's dna, determines the kind of sugar found in red blood cells. proteins known as blood group antigens rh are also influenced by the dna of the host. the d antigen, a big protein found on red blood cells, is encoded by the rhd gene. antigen-antibody responses that help identify various human blood types can be utilized to distinguish these antigens. 4.2 bacterial capsule according to nossal & ada [20], a coating of polysaccharides called a bacterial capsule is what develops outside of the cell envelope and causes the host to become immunogenic. the capsule is thought to be a potential source of bacterial pathogenicity since it is a properly organized layer that is difficult to remove. since phagocytosis requires the presence of a capsulespecific antibody, the capsule may possibly participate in some bacteria's ability to evade phagocytosis. in vaccinations, capsules of bacteria are usually employed as antigens since their polysaccharide component has been coupled with protein carriers. varied bacterial species have different precise capsule compositions, roles, and activities. 5. classes of antigens according to ullah [8], antigens can be categorized into several kinds according to certain criteria. in light of the antigen's immunogenicity and place of origin, some common classifications exist. 5.1 types of antigen according to origin antigens can be divided into two categories according to their origin 5.1.1 exogenous antigen exogenous antigens are antigens which are foreign to their host and can enter the body by ingestion, inhalation, or injection; they can then move throughout the body through bodily fluids; the absorption of foreign antigen is principally carried out by antigen processing cells (apcs) via phagocytosis, such as dendritic cells and macrophages. many antigens, such as intracellular viruses, can begin as exogenous antigens and change into endogenous antigens later on [21]. 5.1.2 endogenous antigen endogenous antigens those who are produced by the host's own body as a byproduct of metabolism or as a result of an intracellular bacterial or viral infection. the body's cells, as well as any broken down substances or antigenic byproducts of metabolism, are considered endogenous antigens. usually, they are broken down by macrophages and then identified by immune system cytotoxic t-cells. antigens classified as endogenous include those that are autologous, idiotype or allogenic, and xenogeneic or heterologous. they may also result in autoimmune illnesses since the host immune system recognizes its own cells and particles as immunogenic [22]. 5.1.3 auto antigens auto antigens are proteins in the host or protein complexes that the immune system targets, agbiji and ekong; asian j. immunol., vol. 7, no. 1, pp. 201-208, 2024; article no.aji.123549 205 leading to autoimmune illnesses. since the immune system shouldn't assault the cells throughout the body, auto antigens can be fatal to the host. genetic and environmental factors lead to the loss of immunological tolerance to certain antigens [23]. 5.1.4 tumor antigens according to schumacher & schreiber's findings [24], major histocompatibility complex (mhc) 1 and 11 on the surface of tumor cells present tumor antigens, also referred to as neoantigens. when normal cells undergo the malignant transformation, a mutation unique to the tumor causes the production of antigens. since the tumor cells learn how to avoid immune defense and antigen presentation, these antigens typically do not trigger an immunological response. 5.1.5 native antigens since no antigen-presenting cells (apc) process native antigens, immune cells like t-cells are unable to attach to them. however, even in the lack of processing, these antigens can activate b-cells [25]. 5.2 types of antigens on the basis of immune response based on the immunological response, antigens can be divided into two categories [26]. 5.2.1 complete antigens/ immunogens antigens that trigger a certain type of immune response are known as complete antigens or immunogens. even in the absence of any carrier particles, these antigens are capable of evoking an immunological response. these are often high molecular weight proteins, peptides, or polysaccharides (more than 10,000 da). 5.2.2 incomplete antigens/haptens haptens, also known as incomplete antigens, are substances that, by themselves, are incapable of triggering an immune response. usually nonprotein molecules, these need a carrier molecule in order to create a whole antigen. less antigenic determinant sites and a reduced molecular weight—typically less than 10,000 da—are characteristics of haptens. a protein or a polysaccharide molecule forms the carrier molecule that is bound to the hapten and is regarded as a non-antigenic component. 5.3 antigenicity and specificity the capacity of a chemical structure (either an antigen or hapten) to bind selectively with a subset of products that have adaptive immunity, such as t cell receptors or antibodies also referred to as b cell receptors, is known as antigenicity, according to the report of dowds et al. [27]. the term "antigenicity" was formerly more frequently used to refer to "immunogenicity," and the two terms are still frequently used synonymously. on the other hand, immunogenicity technically refers to an antigen's capacity to evoke a flexible reaction immune response. therefore, an antigen may attach to a t or b cell receptor selectively without triggering an adaptive immune response. an antigen is considered immunogenic if it elicits an immunological reaction; this type of antigen is known as an immunogen. according to abbas & lichtman [7], specificity is the host cell's capacity to identify an antigen as a distinct molecular entity and discriminate it from another with extreme precision. the main cause of antigen specificity is the antigen's side-chain conformations. it is quantifiable and doesn't have to follow a linear or rate-limited step formula. adaptive immunity comprises b and t cells as its biological constituents. 5.4 factors affecting antigenicity the report of kuriakose et al. [28-29] mentioned the variables influencing antigenicity. among them are: 1. foreignness: considering that the immune system typically distinguishes between self and non-self and is unable to mount an immunological defense against selfantigens, an antigen needs to be foreign. only alien molecules are hence antigenic. 2. size: a material is not necessarily allergenic over a certain size. but generally speaking, a molecule's likelihood of being antigenic increases with its size. 3. chemical composition: a substance's antigenicity increases with its chemical complexity. the polymer's primary sequence residues and/or the molecule's secondary, tertiary, or quaternary structure combine to form the antigenic determinants. 4. physical form: particulate antigens tend to elicit stronger immune responses than soluble ones, while denatured antigens agbiji and ekong; asian j. immunol., vol. 7, no. 1, pp. 201-208, 2024; article no.aji.123549 206 elicit stronger responses than their original form. 5. degradability: generally, immunogenic antigens those who are readily phagocytosed. this is due to the fact that the majority of antigens must be broken down and phagocytosed, and given to helper t cells by an antigen-presenting cell in order for an immune response to emerge. 5.5 applications of antigens gayed [30] reported the applications of antigens. they include: • antigens are used for the differentiation of bacterial species as due to the specificity of antigens. • antigens have further uses as a diagnostic tool to find out whether a sample contains any antibodies. • antigens are necessary building blocks of complexes of antigen and antibody, which are used in forensic analyses to identify human blood as well as additional materials. • these are also utilized in immunoassays to quantify different biological and chemical substances. • autoimmune disorders, some of which are fatal, are brought on by auto antigens. • vaccines employ inactivated enzymes as a passive immunity technique to treat and prevent a variety of illnesses. • it has recently been suggested that peptides known as neoantigens, which are found on the surfaces of cancer cells, are the best targets for immunotherapy in clinical settings [31]. • antigens play a significant role in medical diagnostics through the process of antigenantibody responses. antigens' existence in patient samples like blood, urine, or saliva can be determined using antibodies. antigen-antibody reactions, for instance, are employed in the widely used diagnostic test known as elisa which means enzyme-linked immunosorbent assay (elisa) [32]. 6. conclusion an essential part of the body's immune system is played by antigens. they have a crucial role in promoting the synthesis of antibodies. though antigens could be pathogens, foreign substances, proteins and peptides, they help to trigger immune responses which is essential for the body’s defense against harmful invaders. the knowledge of antigens is fundamental in immunology, vaccine development and disease diagnosis. further research is also required to discover more about antigens and their roles in medicine and health. disclaimer (artificial intelligence) author(s) hereby declare that generative ai technologies such as quillbot have been used during editing of manuscripts. this explanation will include the name, version, model, and source of the generative ai technology and as well as all input prompts provided to the generative ai technology. details of the ai usage are given below: 1. name: quillbot for chrome 2. version: v15.407.0 3. the input prompts provided to the generative ai technology is the work written. it assisted in paraphrasing the work to reduce plagiarism. competing interests authors have declared that no competing interests exist. references 1. national human genome research institute, us national institutes of health; 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2023. doi: 10.35248/2169-0138.23.12.231. disclaimer/publisher’s note: the statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of the publisher and/or the editor(s). this publisher and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. _________________________________________________________________________________ © copyright (2024): author(s). the licensee is the journal publisher. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: https://www.sdiarticle5.com/review-history/123549 https://www.sdiarticle5.com/review-history/123549 _____________________________________________________________________________________________________ *corresponding author: e-mail: xjg71@163.com; asian journal of immunology 4(1): 85-95, 2021; article no.aji.71615 fermented apple juice by two commercial lactobacillus species: changes of physicochemical composition and antioxidant activity wen-sheng yan1, yu-ru guo1, huan-yang li1 and jian-guo xu1* 1 school of food science, shanxi normal university, linfen, china. authors’ contributions this work was carried out in collaboration among all authors. all authors read and approved the final manuscript. article information editor(s): (1) prof. cynthia aracely alvizo báez, autonomous university of nuevo leon, mexico. reviewers: (1) abdul manab, , brawijaya university, indonesia. (2) valcineide oliveira de andrade tanobe, federal university of paraná, brazil. complete peer review history: https://www.sdiarticle4.com/review-history/71615 received 01 june 2021 accepted 04 august 2021 published 10 august 2021 abstract the present study was aimed to compare the changes of physiochemical composition, antioxidant activities of fermented apple juice with non-fermented apple juice by two commercial lactic acid bacteria (lab), lactobacillus casei cicc 20975 and lactobacillus bulgaricus cicc 21101. the antioxidant activity was evaluated by three systems including dpph, abts free radical scavenging methods and fe 3+ reducing power. the results showed that fermentation significantly increased the content of total phenols in apple juice (p<0.05). after fermentation, all malic acid was converted into lactic acid during fermentation with the lactic acid content up to 381.78 mg/kg. free proline 21.55 mg/kg and lysine 21.99 mg/kg were also significantly increased. similarly, fermented apple juice showed significantly higher antioxidant activities when compared to non-fermented apple juice. the scavenging activity of dpph, abts free radical and the reducing power of fe 3+ in fermented apple juice increased by 22.4%, 35.0%, 9.7%, respectively. in conclusion, fermented apple juice by two commercial lactic acid bacteria (l. casei cicc 20975 and l. bulgaricus cicc 21101) exhibited a more satisfied property and possessed great application potentials. keywords: fermented apple juice; lactic acid bacteria; physicochemical composition; antioxidant activity. original research article yan et al.; aji, 4(1): 85-95, 2021; article no.aji.71615 86 1. introduction lactic acid bacteria (lab) was a group of probiotics that can utilize carbohydrate of matrix to produce a large amount of lactic acid, including lactococcus, lactobacillus, leuconostoc, pediococcus, streptococcus and oenococcus species [1]. with an extremely wide carbohydrate utilizing profiles ranging from common monosaccharides to complex plant polysaccharides, labs were especially suitable to ferment various vegetable and fruits juice. fermented vegetable and fruit juice combined the beneficial role of probiotics with bioactive phytochemical compounds in plants such as flavonoids and polyphenols, becoming a kind of unique functional food. nowadays, fermentation by different lab was an effective technology that had been used not only to improve the flavor and stability of food but also to improve the economic and nutritional value of the original product [2,3]. apple was one of the special local fruit products in china. according to the latest statistics, china had become the world’s largest producer of apples, apple planting area and output accounted for more than 50% of the world. however, most of the apple products sold were fresh food with low processing output, and mainly concentrated apple juice, apple vinegar, which had a single processing type, single taste and low additional value. among various choices, fermented apple juice was a good choice to add economical value for apples. in many literature, lactic acid bacteria such as l. acidophilus, l. rhamnosus, l. casei, l. plantarum in fermented apple juice had been reported [4,5]. previous studies in our laboratory optimized various lab to ferment apple juice and compared their consumer preferences and the number of viable bacteria (data not published). the result showed that apple juice fermented with l. casei cicc 20975 and l. bulgaricus cicc 21101 had typical apple flavor, soft taste, delicious with slight sweetness and sour. on the basis of the above test, in the present study, we compared the physicochemical compounds, antioxidative abilities as well as aroma profile of the fermented apple juice with non-fermented apple juice to indicate the nutritional and functional changes before and after fermentation. 2. materials and methods 2.1 microorganisms and culture both l. casei cicc 20975 and l. delbrueckii subsp. bulgaricus (termed l. bulgaricus hereafter) cicc 21101 were purchased from china’s industrial microbial preservation management center (cicc). all bacterial cultures were stored frozen at -20°c in mrs medium (aoboxing biotech co. ltd, beijing, china) containing 20% glycerol. the strains were reactivated by means of double passage on mrs when needed. 2.2 raw materials and reagents in this work, fuji apples were purchased from orchard worker in jixian, shanxi, china. the fuji apples were cleaned with filtered water and chopped into small pieces. then they were soaked in color protector liquid (mixed solution of ascorbic acid concentration of 3.5 mg/l and citric acid concentration of 7.5 mg/l) for 30 minutes. the apple juice was obtained through mechanical process by pressing the pulp in juice extractor, and then apple juice was heated by microwave heating for 2 min. the heated fuji apple juice was stored frozen (-20°c) prior to use. no additive was added to the juice. methanol (hplc grade) and acetonitrile (hplc grade) were purchased from merck (germany); mrs broth medium, agar and nutrient broth medium (nb) were purchased from beijing aoboxing biotech co. ltd. (beijing, china); fructose, maltose, sucrose and free amino acid were purchased from sigma chemical co. sigma (usa); amino acid standards were purchased from shanghai anpel laboratory technology co., ltd. all other chemicals and reagents used in the experiments were of analytical grade. 2.3 preparation of fermented apple juice l. casei cicc 20975 and l. bulgaricus cicc 21101 were cultivated on mrs broth at 37°c for 24 h. cells at the late exponential phase was obtained, harvested by centrifugation (10,000×g, 10 min, 4°c), washed twice with 50 mm sterile phosphate buffer solution (pbs, ph 7.0), and then re-suspended in sterile distilled water to the final optical density. after that, l. casei cicc 20975 (3.0×10 6 cfu/ml) and l. bulgaricus cicc 21101 (3×10 6 cfu/ml) were inoculated into 200 ml freshly prepared apple juice. mixtures were statically cultured at 38°c for 60 h. and the final live bacterial counting was performed in mrs and the results showed that the live lab numbers in fermented apple juice were up to 1.53.0×10 9 cfu/ml. in the present study, an uninoculated apple juice was used as an experimental control. yan et al.; aji, 4(1): 85-95, 2021; article no.aji.71615 87 2.4 determination of free sugars the concentration of free sugars was determined by hplc (1200 series, agilent, usa) equipped with a refractive index detector (rid) according to a modified version of the method described by mousavi with some modifications [6]. the mobile phase was acetonitrile and water at a volume ratio of 7:3. chromatographic analysis was achieved using a column (agilent amino column 4.6 mm×250mm, 5 μm) maintained at 35°c. the injection volumes of 10 μl were chromatographically separated at a flow rate of 1.0 ml/min for both samples and standards. sugar content was calculated using external standards. the following chromatographic grade free sugars were used as standards. 2.5 analysis of organic acids the chromatographic system was used to quantify the organic acids consisted of a diode array detector (dad) (1200 series, agilent, usa) according to a modified version of the method described by belguesmia et al. with some modifications [7]. the mobile phase was k2hpo4 (10 mm, ph 2.55). one milliliter of sample was added 5 ml of mobile phase, ultrasonic extracted for 30 min and left for 1 h at 60 °c water bath. after being centrifuged for 10 min at 12000×g at 4 °c, the supernatant was kept for 1 h at 4 °c before filter-sterilization (0.45 μm pore size). the samples were then kept at –80 °c until analysis. chromatographic analysis was achieved using a column (agilent aq 4.6 mm×250 mm, 5 μm) maintained at 30 °c. the injection volumes of 10 μl were chromatographically separated at a flow rate of 0.5 ml·min -1 for both samples and standards. the wavelength was set at 210 nm to detect the organic acids. the following chromatographic grade organic acids were used as standards. to ensure accuracy, the working standards were prepared daily. the hplc results were qualitatively analyzed by peak retention time and quantified by peak area using the external standard method. extractions and injections were conducted in triplicate for each fermentation replicate. 2.6 determination of free amino acids faas analysis was carried out using acid hydrolysed (0.2 mm hcl) samples by reversephase high-performance liquid chromatography (hplc) after precolumn derivatization by phenylisothiocyanate (pitc), by a modified method adapted from zhao [8]. extraction of faas from apple juice: one gram of sample was added with 8 ml of 0.2 mm hydrochloric acid, shook in a vortex for 5 min, then extracted by ultrasound for 10 min. after standing in the dark for 2 h, it was centrifuged at 2057×g for 10 min, taking the supernatant for later use. instrument method: the faas contained in the samples were separated using an amino acid shiseido c18 column, 5 μm (250×4.6 mm) attached to agilent 1260 chromatography system equipped with uv-vis detector monitoring at 254 nm. the injection volume was set at 10 μl and the column was kept at 40°c. the gradient mobile phase, consisting of eluent a (prepared by mixing the 0.1 mol/l sodium acetate with acetonitrile at a ratio of 97: 3) mix well and adjust ph to 6.5 (31.815 g sodium acetate plus 3880 ml water plus 120 ml acetonitrile) and eluent b (80% acetonitrile, 20% milli-q water) was injected at a flow rate of 1 ml / min throughout the experiment. the gradient program was defined as follows: 100 % a at start, 85 % a and 15 % b at 14 min, 66 % a and 34 % b at 29 min, 0 % a and 100 % b at 30 min and for 7 min, 100% a at 38 min and for 9 min, 100 % a at 45 min, allowing the column to equilibrate for 15 min until the 60th min. 2.7 determination of the total phenolic content total phenolic content was determined based on the folin-ciocalteu colorimetric method as described by xu et al. [9]. gallic acid was used as a reference standard, and the values of total phenols were expressed as milligram of gallic acid equivalent (gae) per milliliter of fruit juice. the juice was diluted with a mixture of methanol (80%)-water and centrifuged at 6650×g for 10 min. the supernatant was used as a test sample for determining the total phenolic content. briefly, an aliquot (0.5 ml) of appropriately diluted apple juice, 2.5 ml of deionized water and 0.5 ml of 1.0 m folin-ciocalteu reagent were mixed within 10 ml volumetric flasks and vortexed. after 8min, 1.5ml of 7.5% sodium carbonate solution was added and mixed thoroughly. the absorbance of the reaction mixtures was measured using a spectrophotometer at 765 nm wavelength after incubation for 2 h at room temperature. methanol was used as the blank, and gallic acid (ga) was used for calibration of the standard curve (0-500 mg·l -1 ). phenolic content was expressed as gallic acid equivalents (milligrams of gae per gram juice). yan et al.; aji, 4(1): 85-95, 2021; article no.aji.71615 88 2.8 determination of the content of total flavonoid the total flavonoid was measured by the method of by feng and xu [10]. rutin was used as a reference standard, and the total flavonoid content was calculated as the equivalent of rutin content per ml of sample. 2.8.1 determination of the content of ascorbic acid (vc), total titratable acidity (tta), ph, and total soluble solids (tss) the content of vc was determined by 2, 6dichlorophenol titration method according to the publication of aoac [11]. taa content was measured by using the method of aoac [11]. briefly, samples were 10-fold diluted, and then titrated with 0.1 n naoh with phenolphthalein as indicator. the ph of the juice was measured using a ph meter at 25℃. tss in the juice sample was determined by a benchtop digital refractometer (pal-1, atago, japan). 2.9 determination of antioxidant activity 2.9.1 dpph free radical scavenging activity the dpph free radical scavenging activity method according to the method as previously described by guo et al. [12]. briefly, each of sample solutions was serially diluted to various concentrations in methanol respectively, and then a 0.5 ml of samples was mixed with 2.5 ml of 60 μm dpph dissolved in methanol. the mixture was shaken vigorously and left to stand for 30 min in the dark, and the absorbance was measured at 517 nm against a solvent blank. the scavenging rate on dpph free radical was calculated according to the formula: scavenging rate (%) = [1(asample – ablank) /acontrol] ×100. where, a was the absorbance of the sample, blank, or control, as indicated. the dpph free radical scavenging activity of apple juice was expressed in mm of vc. 2.9.2 abts free radical scavenging activity the abts free radical scavenging activity method as described by xu et al. [13]. abts free radical cation was generated by a reaction of 7 mmol/l abts and 2.45 mmol/l potassium persulfate. the reaction mixture was allowed to stand in the dark at room temperature for 16−24 h before use and was used within 2 days. the abts solution was diluted with methanol to an absorbance of 0.700 ± 0.050 at 734 nm. one hundred microliters of the diluted samples were mixed with 2.0 ml of diluted abts solution. the mixture was allowed to stand for 6 min at room temperature, and the absorbance was immediately recorded at 734 nm. the scavenging rate were calculated using the equation described for dpph assay. the abts free radical scavenging activity of apple juice was expressed in mm of vc. 2.9.3 determination of fe 3+ reducing power the fe 3+ reducing power method as described by kwaw et al. [14]. an aliquot of the apple juice (1 ml, diluted at 1:100) was mixed with 0.05 ml of hcl (0.01 m), 0.4 ml of potassium ferricyanide (0.02 m), 0.4 ml of 0.02 m fecl3 and 0.7 ml of distilled h2o. the mixture was consequently incubated at 37 o c in the dark for 30 min and the absorbance read at 720 nm. the fe 3+ reducing power of apple juice was expressed in mm of vc. 2.10 electronic nose measurements the aroma profiles of the juice samples were analyzed using an electronic nose system (pen3, airsence, germany) [15]. briefly, each sample (15 ml) was put into a 100 ml glass jar. then the glass jar was sealed with three layers of plastic wrap and the headspace inside it was equilibrated for 30 min at 45°c under agitation at a speed of 500 rpm. the headspace volatiles were put into the electronic nose for 9 sec at a rate of 7.7 ml·min -1 . to acquire stable signals, the acquisition duration for the sensors was 90s. the analysis system was purged with processed dry and pure air before each analysis. each sample was tested 6 times to ensure the accuracy of the data, and the last measured three data were used in the subsequent analysis. the serial number, main applications, and references of the ten sensors were listed in table 1 [16]. 3. results and discussion 3.1 changes of sugars content in apple juice the content of the free sugars and total soluble solids (tss) in fermented and non-fermented apple juice were shown in table. 2. as we can see from table. 2, the levels of tss, fructose and glucose decreased significantly (p<0.05). the soluble solid (tss) of apple juice decreased by 3.4% during fermentation. the soluble solids utilization was high in fermented apple juice yan et al.; aji, 4(1): 85-95, 2021; article no.aji.71615 89 compared to non-fermented apple juice due to the higher microbial load [17]. there was literature reported that the decrease in sugar concentrations during fermentation was largely due to not only bioconversion into lactic acid, but also the utilization for growth and metabolism of lactic acid bacteria [18]. besides, 5.3% of fructose and 1.3% glucose were utilized during fermentation, respectively. similar results were reported for sohiong juice [19] and pomegranate juice [7]. it had been reported that fructose and glucose were efficient carbon and energy source for most of lactobacillus strains [20]. among the free sugars, the concentrations of sucrose and maltose showed no significant difference when compared fermented juice with non-fermented juice (p>0.05). this may be the result that sucrose and maltose were not utilized by lactic acid bacteria during fermentation generally. in addition, there were no detectable lactose in fermented and non-fermented juices. 3.2 changes of acid substances of apple juice the total acid (ta), ph and the content of nine free sugars in fermented and non-fermented apple juice were shown in tab. 3. as shown in table. 3, the total acid content of fermented apple juice increased significantly (p<0.05), and the ph content decreased significantly (p<0.05). the total titratable acid concentration decreased by 28.1% from 0.69 g·kg -1 to 0.96 g·kg -1 . at the meantime, the ph decreased by 29.4% from 5.21 to 3.68. the low ph and high acidity were in agreement with the findings reported by ibanoglu et al. [21]. 3.3 changes of free amino acid content of apple juice it had been reported that soluble substances, some fat and some vitamins in fruit juice can be metabolized by probiotics into amino acids, fatty acids and so on [22]. the varieties and contents of free amino acids in fermented and nonfermented apple juice were shown in table. 4. according to table. 4, a total of 17 amino acids were detected in fermented and non-fermented apple juice, among which 7 were essential amino acids for human body. changes in the content of nonessential amino acids (neaa) were similar to total free amino acids (tfaa), there were no significant differences in fermented and nonfermented apple juice (p>0.05). among the 17 detected amino acids, aspartic acid was the most abundant, and there was no significant change after fermentation, followed by glutamic acid and serine, which decreased significantly from 49.13 mg·l -1 to 14.58 mg·l -1 and decreased from 293.68 mg·l -1 to 255.86 mg·l -1 , respectively. similar results were reported by xu et al. [23]. a total of seven total essential amino acids were detected and represented 4.6% and 3.6% of the total free amino acids in fermented and nonfermented apple juice, respectively. meanwhile, cysteine, phenylalanine and methionine were not detected. moreover, the content of proline was up to 21.55 mg·kg -1 and 3.7 times higher than the juice without fermentation. the similar trend was also found in the content of lysine with a concentration of 21.99 mg·kg -1 in fermented juice. 3.4 changes of phytochemical contents in the apple juice the contents of phytochemical compounds, including total polyphenols, flavonoids and vc, fermented and non-fermented apple juice were shown in fig. 1. as indicated in fig. 1, except for the content of total polyphenols, the contents of the other two active compounds in the juice with and without fermentation showed no significant differences. interestingly, total polyphenols significantly increased by 14.4% and the value was up to 0.97 milliequivalent of gallic acid. previous studies showed that vegetable or fruits juice fermented by lactobacillus showed higher contents of polyphenols and flavonoids, suggesting a bio-conversion role of lab in this process [24]. as for the change of vc content, kaprasob et al. also reported that cashew apple juice fermented with l. plantarum retained a matchable level. this may be due to the protective effect of lab fermentation and they may prevent the degradation of vc. all of these results indicated that lab fermentation can biotransform some natural botanical compounds into bioactive compounds or prevent the loss of bioactive phytochemicals, and finally enhance the quality of products. 3.5 changes of antioxidant activity of apple juice the changes of antioxidant activity of in fermented and non-fermented apple juice were shown in fig. 2. it can be seen from fig. 2 that the antioxidant activity of fermented apple juice was significantly increased (p<0.05). after fermentation, the dpph free radical scavenging rate reached up to 56.9% equivalent to 7.32 mmol·l -1 vc and it was 1.3 times of that non yan et al.; aji, 4(1): 85-95, 2021; article no.aji.71615 90 table 1. sensors used and their main applications in pen 3 number in array sensor name general description reference (ppm) s1 w1c aromatic compounds toluene,10 s2 w5s very sensitive, broad range sensitivity, react on nitrogen oxides, very sensitive with negative signa no2, 1 s3 w3c ammonia, used as sensor for aromatic compounds benzene, 10 s4 w6s mainly hydrogen, selectively (breath gases) h2, 0.1 s5 w5c alkenes, aromatic compounds, less polar compounds propane, 1 s6 w1s sensitive to methane broad range ch3, 100 s7 w1w reacts on sulfur compounds, sensitive to many terpenes and sulfur organic compounds, which are important for smell, limonene, pyridine h2s, 1 s8 w2s detects alcohols, partially aromatic compounds, broad range co, 100 s9 w2w aromatics compounds, sulfur organic compounds h2s, 1 s10 w3s reacts on high concentrations, sometime very selective (methane) ch3, 100 table 2. changes of sugar content in fermented and non-fermented apple juice sample soluble solids ( o brix) free sugar fructose (g·100g -1 ) glucose (mg·kg -1 ) sucrose (g·100g -1 ) maltose (ug·g -1 ) lactose (g·100g -1 ) aj 12.8±0.5a 6.24±0.20a 4.46±0.23a 0.77±0.09a 0.12±0.04a nf faj 9.4±0.3b 5.91±0.19b 3.80±0.16b 0.79±0.21a 0.15±0.03a nf different letters within a column indicate statistically significant differences between the means (p<0.05); nf, not found; aj, apple juice; faj, fermented apple juice. table 3. changes of acid substances of fermented and non-fermented apple juice sample tta (g·kg -1 ) ph organic acid (ug·g -1 ） oxalic acid tartaric acid malic acid lactic acid acetic acid citric acid oxalic acid maleic acid fumaric acid aj 0.69±0.12b 5.21±0.64a nf 0.59±0.18a 204.30±9.21 nf nf nf nf nf nf faj 0.96±0.06a 3.68±0.43b nf 0.45±0.09b nf 381.78±7.89 nf nf nf nf nf numbers represent mean values of three independent replicates ± sd; tta, abbreviations of total titratable acid; nf, not found; aj, apple juice; faj, fermented apple juice; different letters within a column indicate statistically significant differences between the means (p < 0.05). yan et al.; aji, 4(1): 85-95, 2021; article no.aji.71615 91 table 4. changes of free amino acid content (mg·kg -1 ) of fermented and non-fermented apple juice amino acids aj faj amino acids aj faj aspartic acid 415.61±10.91a 418.13±9.88a proline 5.84±0.38b 21.55±0.52a glutamate 49.13±5.14a 14.58±2.12b tyrosine nf nf cystine nf nf valine 7.02±0.23 nf serine 293.68±9.35a 255.86±8.89b methionine nf nf glycine 4.48±0.23 nf isoleucine 6.11±0.41a 2.42±0.34b histidine nf nf leucine 1.53±0.08a 1.43±0.19a arginine nf nf phenylalanine (phe)△ nf nf threonine 2.52±0.21a 1.66±0.17a lysine 20.3±3.18b 21.99±2.91a alanine 12.04±0.75a 10.61±2.21a teaa 37.48±3.34a 28.92±2.26b neaa 780.78±18.23a 782.31±26.39a tfaa 818.26±10.45a 811.23±12.18a numbers represent mean values of three independent replicates ± sd; nf, not found; aj, apple juice; faj, fermented apple juice; abbreviations of amino acids (teaa, total essential amino acids; neaa, nonessential amino acids; tfaa, total free amino acids); different letters within a column indicate statistically significant differences between the means (p<0.05). fig. 1. changes of antioxidant substances of fermented and non-fermented apple juice different letters within a column indicate statistically significant differences between the means (p<0.05); aj, apple juice; faj, fermented apple juice. fig. 2. changes of antioxidant activity of fermented and non-fermented apple juice different letters within a column indicate statistically significant differences between the means (p < 0.05); aj, apple juice; faj, fermented apple juice yan et al.; aji, 4(1): 85-95, 2021; article no.aji.71615 92 fermented apple juice. as for abts free radical scavenging rate, the activity reached up to 58.9% equivalent to 13.45 mmol·l -1 vc and had a 50% increase. the fe 3+ reducing power of fermented apple juice was 82.3% equivalent to 9.35 mmol·l -1 vc and it was 1.1 times of that nonfermented apple juice. similar results reported that noni juice fermented with bifidobacterium longum had greater antioxidant activity than nonfermented noni juice. 3.6 analysis of aroma composition of apple juice the aroma profiles of apple juice during fermentation were examined using an electronic nose. fig. 3 displayed a typical response of ten sensors during measurement of apple juice (0h) and fermented apple juice (60h) in which each curve represented a different sensor response with time. the ordinate represented the changing ratio between g and g0. it was apparent that, the responsive values of the sensors, after an initial period of low responsive values, increased sharply and then stabilized after 60 s. in this research, the responsive values of each sensor at 60 s point were used in analysis. as shown in fig. 3, compared with fig. (a), the (b) of response values of w5s, w1s, w1w, w2s, w6s and w2w were enhanced to varying degrees. with the increase of fermentation time, the response values of w1w and w2w decreased at first (0h~12h) and then increased (12h~60h), while the response values of w5s, w1s and w2s gradually increased to stable with the increase of fermentation time, but the response values of other sensors did not change obviously. the differences in flavor of apple juice during different fermentation stages may be due to the changes in the substances represented by the sensors. therefore, the characteristic flavor of fermented apple juice may be derived from esters, nitrogen, methane, sulfur compounds, alcohols, hydrogen, alcohols, and the dynamic changes of these substances affected the overall flavor. (a). 0h; (b). 60h fig. 3. response curves of electronic nose sensors to fermented and non-fermented apple juice 0 5 10 15 20 0 20 40 60 80 100 time(s) r es po ns e v al ue s g (g 0 ) w1c w5s w3c w6s w5c w1s wiw w2s w2w w3s yan et al.; aji, 4(1): 85-95, 2021; article no.aji.71615 93 fig. 4. changes in response curves of electronic nose sensors to apple juice at different fermentation stages 4. conclusion the results showed that the lab might had utilized carbohydrates and produced large amounts of organic acid, thus lowering the ph of the samples during fermentation. lactic acid bacteria fermentation can release the content of organic acids, free amino acids and convert phenolic compounds to enhance the antioxidant activity. in addition, the characteristic flavor of fermented apple juice may be derived from nitrogen, methane, sulfur compounds, alcohols, hydrogen, alcohols. these findings highlighted the beneficial effect of probiotic fermentation on the quality of apple juice. probiotic fermentation changed the phytochemical composition of fruits juice, which thus enhanced their antioxidant activity. all of these contribute to a more satisfied quality of the final product from both stability and nutrition perspectives. based on the findings in this study, apple juice fermented by l. casei cicc 20975 and l. bulgaria cicc 21101 was satisfied leavening agents for a health beverage. however, additional research was needed on the control of the fermentation process and the identification of the key active compounds and flavor substances produced during fermentation. all these works assist in obtaining more desirable organoleptic qualities in fermented food products. disclaimer the products used for this research are commonly and predominantly use products in our area of research and country. there is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. also, the research was not funded by the producing company rather it was funded by personal efforts of the authors. consent not applicable. ethical approval not applicable. competing interests authors have declared that no competing interests exist. references 1. freire al, ramos cl, souza pndc, et al. nondairy beverage produced by controlled fermentation with potential probiotic starter yan et al.; 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# alumni professor; *corresponding author: e-mail: bli@augusta.edu, brli1@juno.com; cite as: li, shen, and biaoru li. 2024. “optimizing cancer immunotherapy through combination therapies: advances in chemoimmunotherapy, lymphodepletion, and precision medicine”. asian journal of immunology 7 (1):247-57. https://journalaji.com/index.php/aji/article/view/148. asian journal of immunology volume 7, issue 1, page 247-257, 2024; article no.aji.126682 optimizing cancer immunotherapy through combination therapies: advances in chemoimmunotherapy, lymphodepletion, and precision medicine shen li a++ and biaoru li b,c++#* a university of chicago medical center, section of general surgery, chicago, il 60637, usa. b department of pediatrics and ga cancer center, children hospital at ga, augusta, ga 30913, georgia. c school of medicine, cwru, 10900 euclid ave, cleveland, oh 44106, usa. authors’ contributions this work was carried out in collaboration between both authors. both authors read and approved the final manuscript. article information doi: https://doi.org/10.9734/aji/2024/v7i1148 open peer review history: this journal follows the advanced open peer review policy. identity of the reviewers, editor(s) and additional reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here: https://www.sdiarticle5.com/review-history/126682 received: 10/09/2024 accepted: 12/11/2024 published: 15/11/2024 abstract chemoimmunotherapy was a challenge issue in an early clinical study. cytotoxic chemotherapy was immunosuppressive while, in the clinic, chemoimmunotherapy demonstrated supporting immunotherapy. to increase act (adoptive cell transfer) immunotherapy and decrease the effects review article mailto:bli@augusta.edu https://doi.org/10.9734/aji/2024/v7i1148 https://www.sdiarticle5.com/review-history/126682 li and li; asian j. immunol., vol. 7, no. 1, pp. 247-257, 2024; article no.aji.126682 248 of chemotherapy for tumor-infiltrating lymphocytes (tils) to treat solid tumors, we began to study chemoimmunotherapy by isolating and culturing primary tumor cells and immune cells from the removed solid tumor tissues before 1994. after about 30-40 years of efforts, results of optimizing combination treatment have demonstrated that chemotherapy combined with immunotherapy (called chemoimmunotherapy) is better than monotherapy; now there are four fields to be developed for the optimizing combination, including (1) lymphodepletion (ld) supporting act immunotherapy for advanced cancer, (2) chemoimmunotherapy (ci) supporting treatment for advanced cancer, (3) immune checkpoint inhibition (ici) combined with immunotherapy, and (4) precision medicine (pm) supporting immunotherapy for advanced cancer. precision therapy can cover all three, as described above, as well as ld, chemoimmunotherapy, and ici combination. techniques for precision therapy include tumor tissue biobanks, single cell technique, clinical genomics, and artificial intelligence to support the combination treatment. overall, the latest generation of optimizing combination therapies is more specific and sensitive in treating neoplastic diseases than older versions with fewer side effects. based on 30–40 years of r&d to improve immunotherapy for patients with advanced cancer and based on increasing research, it is time to define combinations treatment and evaluate the efficacy of optimizing combination treatments for oncological diseases keywords: cancer; chemoimmunotherapy; cytotoxic chemotherapy; oncological diseases. 1. introduction in 1986, dr. rosenberg discovered that isolated and cultured tumor-infiltrating lymphocytes (tils) from tumor tissue could be applied to the adoptive cell transfer (act) to treat advanced melanoma [1]. in addition, they also used a combined ctx before til infusion therapy for the treatment of tumor patients so that ctx could eliminate lymphocytes in vivo to support act immunotherapy [2]. lymphodepletion (ld) is routinely used in act immunotherapy, such as car-t and til therapy [3-4]. after about four decades of efforts, some research on combination therapy has demonstrated that chemotherapy coordinating with immunotherapy (called chemoimmunotherapy) is better than monotherapy; however, understanding regimens of combination therapy also includes chemotherapeutic drugs, optimal drug dose, administration time, and sequence of chemoimmunotherapy is still not clear [5-6]. we have discovered the “confused phenomenon” in our early study. to address the phenomenon, we began to study both tumor cells and immune cells from the removed same solid tumor tissues, which were published in 1994 [7]. we aimed to increase til act efficacy and decrease chemotherapy toxicity to act immunotherapy at that time. our strategies included that both til and primary tumor cells culture from the solid tumor tissues so that the cultured tils from solid tumors are activated and then infused into patients for act immunotherapy. “at the same time, the primary tumor cells from the tumor tissues are utilized as in vivo and in vitro chemosensitivity tests for specific chemotherapy drugs to decrease chemotherapy toxicity to act immunotherapy. as our early report for combination therapy for patients with solid tumors by til re-infusion and chemotherapy using an in vitro chemosensitivity test, we discovered the combination was better than the monotherapy. following thirty years of research for combination treatment, we have developed precision medicine, including tumor tissue biobanks, single cell technique, clinical genomics, and artificial intelligence to support the combination treatment. overall, the latest generation of combination therapies is more specific and sensitive in treating neoplastic diseases than older versions with fewer side effects” [8-12]. based on 30–40 years of r&d to improve immunotherapy for patients with advanced cancer and based on increasing research, it is time to define combinations treatment and evaluate the efficacy of combination treatments for oncological diseases. according to the current publications, the manual will introduce four sections for optimizing combination therapy: (1) lymphodepletion (ld) supporting act immunotherapy for advanced cancer, (2) coordinated therapy (chemoimmunotherapy) supporting treatment for advanced cancer, (3) immune checkpoint inhibition (ici) combined with immunotherapy, and (4) precision medicine supporting immunotherapy for advanced cancer. li and li; asian j. immunol., vol. 7, no. 1, pp. 247-257, 2024; article no.aji.126682 249 2. lymphodepletion supporting act immunotherapy to treat advanced cancer over the past 40 years, adoptive cell transfer (act) for the treatment of malignancies has been one of the most dynamic and fruitful advances in cancer therapy [13-14]. to support act efficacy, in 1986, dr. rosenberg first used ctx as lymphodepletion (ld) to support the til reinfusion for overcoming immune tolerance in patients with metastatic melanoma [15]. other solid tumor diseases have also routinely used ld to support til reinfusion in clinics [16-17], as shown in fig. 1. moreover, car-t (chimeric antigen receptors) has become an essential antitumor therapy component [18]. however, a significant number of these patients still relapse after car-t therapy or become resistant to cart therapy [19-20], so they need to improve the efficacy of car-t therapy by ld to play an essential role in overcoming the resistance to car-t therapy [21-22]. the main goals of ld are to (1) reduce endogenous lymphocytes to inhibit the engraftment of car-t infusions and support their long-term activity; (2) reduce tumor cells to avoid rapid exhaustion of car-ts; and (3) prepare and reprogram the microenvironment and soluble factors to ensure optimal engraftment, homing, and long-term survival of car-t cells [23-25]. as in table 1, there are several lds, such as fludarabine (flu) and cyclophosphamide (ctx, cy), and their combination is selectively used for individual situations. ld regimens and doses vary depending on the target disease (such as all, nhl, mm, or solid tumors) and the source of t cells (autologous vs. allogeneic) [26-28]. because a patient receives ld with individual differences, personalized regimens still need to be considered for ld selection with their doses for the new til or car-t therapy approaches. fig. 1. strategy for ld to increase til efficacy nb. a lymphodepletion regimens adding til treatment to increase til efficacy. table 1. lymphodepletion types components common lymphodepletion common usage fludarabine purine analogue car-t cll and indolent nhl cyclophosphamide an alkylating agent til or car-t melanoma, lymphoma, and indolent nhl flu and cy both action car-t call bendamustine an alkylating agent car-t cll and other band t-cell lymphomas alemtuzumab targeting car-t lymphoproliferative diseases oxaliplatin/cy targeting car-t car-t to solid tumors clofarabine anti-leukemic agent car-t clinical remission li and li; asian j. immunol., vol. 7, no. 1, pp. 247-257, 2024; article no.aji.126682 250 3. chemoimmunotherapy combinations supporting immunotherapy chemotherapy has been a critical part of cancer treatment for more than 70 years [29]. the cytotoxic drugs kill tumor cells and inhibit their proliferation through dna damage, inhibiting dna replication and arresting mitosis. however, cytotoxic chemotherapy is widely considered to be immunosuppressive because it can cause dose-dependent myelosuppression, suggesting antagonism with immunotherapy [30]. before 1994, to increase act immunotherapy and decrease side effects from chemotherapy for til to treat solid tumors, we began to study chemoimmunotherapy with techniques for primary tumor cells and immune cell separation and culture from the removed solid tumor tissues [31-33]. that time, to increase til efficacy and decrease toxic chemotherapy, we study chemoimmunotherapy through which the cultured tils from solid tumors are activated and then infused into patients for adoptive immunotherapy. at the same time, the primary tumor cells from the tumor tissues are utilized as in vivo and in vitro chemosensitivity tests for chemoimmunotherapy [34-35], as shown in fig.2. after more than thirty years effort, now chemoimmunotherapy combination regimens may have shown advantages over monotherapy: (1) they can maximize cancer elimination within the range of tolerable toxicity; (2) target a wider range of tumor cells with different genetic and epigenetic abnormalities in a heterogeneous tumor population; (3) slow the development of drug resistance; (4) can shrink the primary tumor mass, reduce the number of act cells providing an opportunity for their combination with immunotherapy; (5) some chemotherapeutic agents may directly stimulate antitumor immunity with low infiltration of effector t cells within the tumor [36-38]. now, we all know that the rationale in a chemoimmunotherapy combination can eliminate disseminated and metastatic cancer cells, although chemotherapy can cause myelosuppression. 4. immune checkpoint inhibiting immune suppression supporting immunotherapy now, a suppression of tumor growth is emerging through target checkpoints as a new generation of antitumor therapy. there are two kinds of molecularly targeted checkpoint inhibitors. the first one is specifically killing tumors within the tumor microenvironment (tme), inhibiting the occurrence and development of tumors [39], and the second one can target and inhibit immunepathway molecules called the tumor immune microenvironment (time) [40] so that they can restore immune cell activity and improve the body's antitumor immune function. currently, molecular target checkpoints increasingly studied within time are named immune checkpoint inhibitors (icis) for pd-1, pd-l1, ctla-4, tim-3, lag-3, and siglec-15 by their corresponding molecular target inhibitors. fig. 2. clinical strategies for chemoimmunotherapy to increase til efficacy. nb. a clinical procedure combining tils with sensitive chemotherapeutic agents, which were screened chemosensitivity assay (csa) from til cytotoxicity experiment of patient’s autogenous tumor cell to increase treatment response li and li; asian j. immunol., vol. 7, no. 1, pp. 247-257, 2024; article no.aji.126682 251 table 2. ici combination types common ici type supporting item ici-ici nivolumab ipilimumab ici-act nivolumab car-t/til ici-chemotherapy nivolumab chemotherapy ici antiangiogenic nivolumab antiangiogenic therapy ici-vaccine nivolumab hpv16 specific peptide vaccine ici-radiation nivolumab ionizing radiation ici-tme nivolumab targeting tgfβ ici-cytokine nivolumab il2/ifn-gamma icis were initially approved for the treatment of melanoma. due to single-agent icis' failure, combination regimens involving icis have been investigated, such as ici-act, ici-chemotherapy, and ici antiangiogenic doublets, as shown in table 2. nivolumab and ipilimumab have shown initial success in melanoma and renal cell carcinoma (rcc). ici-ici combination therapy is a viable approach to overcome treatment resistance. novel combination strategies to overcome ici resistance rapidly evolve, with many clinical trials underway. bispecific antibodies (bsabs) allow the targeting of specific resistance mechanisms in a single molecule, and dual checkpoint inhibition of pd-l1 and lag-3 is an example that has shown promising preclinical results. other bsabs combine icis with non-ici immunotherapies, such as pd-l1 antibodies and transforming growth factor-β (tgf-β) traps, a key player in the development of an immunosuppressive tumor microenvironment (tme). other forms of ici immunotherapy are also encouraging, including immunostimulatory cytokines (e.g., recombinant interleukin-2, interferon-α), cancer vaccines, and adoptive cell therapy. combining vaccine therapies with icis may enhance antitumor effects in preclinical models. combining car-t with icis may overcome some of the resistance mechanisms encountered. in addition, a combination of icis with ionizing radiation and icis with targeted therapies for cellular processes, including dna damage repair, phosphatidylinositol 3-kinase, and histone deacetylase pathways. a logical approach to combination study design is needed to maximize patient benefit [41]. fig. 3. strategies for precision medicine for personalized t-cell therapy to increase t-cell efficacy nb. a) improving til immune-response in experiment such as discover quiescent genes for til and neo-antigen from tumor cells; and rebuilding immune-response for til or set up car-t or tcr-t cells; b) ici improving t-cell to attack tumor cells; c) targeting block to increase t-cell activity; d) tme block to increase t-cell activity; d) epigenetics treatment improving t-cell to attack tumor cells; and e) chemotherapy to increase t-cell attack tumor cells. li and li; asian j. immunol., vol. 7, no. 1, pp. 247-257, 2024; article no.aji.126682 252 fig. 4. strategies for combination therapy to support immunotherapy 5. a new generation of precision medicine supporting immunotherapy according to those discussed above, distinct ld, chemotherapy, and ici are different for individual patients. of course, all combinations should differ from one person to another. following thirty years of research, a new generation of precision medicine techniques is emerging [42-44]. “to address the issues, successful precision medicine for tumor disease has involved immune cells/primary cell culture, tumor tissue biobanks, single cell technique, clinical genomics, and artificial intelligence in our laboratory” [45-50]. “moreover, precision medicine is discovering driver genes, tumor proliferation, and tumor metastasis genes according to the updated strategy” [51]. once we find distinct biomarkers in dna level (snp and epigenetics), rna level microrna, picorna, non-coding rna), and protein level, we can develop precision medicine as fig-3 to support immunotherapy. a. specific personalized combination immunotherapy can be discovered “in the early period, we developed til culture for clinical applications by cultured til infusion in vivo for adoptive cell therapy (act) from solid tumors. after twenty-five efforts, we successfully developed our laboratory's single-cell techniques, clinical genomics, and artificial intelligence. after, we used single-cell genomics analysis to discover a set of upregulated quiescent genes such as tob, lklf, tgf-β, erf, and rest/nrsf from the t-cells” [52-55]. “moreover, after thirty years of effort, we know that til is a group of heterogeneous immune cells, so we can perform ex vivo heterogeneous til determining immune characteristics to kill autologous tumor cells and then treat the tumor patients based on immune characteristics for the tumor diseases [56]. now we can measure quiescent status in the heterogeneous immune cells such as cd3+ t-cell (cd8+ t-cell and cd4+ t-cell), cd19+ bcell (tumor-infiltrating b-cells, til-b), cd16+/cd56+ nk cell (natural killer cell), cd16+/cd56+/cd3+ nkt cell (natural killer tcell), and other immune-cells (macrophage and neutrophil). finally, the specific immune cells that have been specifically contacted to correspond with the tumor antigen of tumor cells will be cultured for precision immunotherapy. as we all know above, if we discover the higher expression of checkpoint-inhibiting molecules such as pd-1, which blocks immune cells, we can also further stimulate immune cells with pd-1 inhibitors to combine with the specific immune cells for personalized immunotherapy” [57-58]. b. molecular therapeutic targeting can be discovered molecular targeted therapies are advanced therapeutic techniques that interfere with specific molecules to block cancer growth, progression, and metastasis [59-60]. now, molecular targeted therapies approved by the food and drug administration (fda) have demonstrated remarkable clinical success in the treatment of a li and li; asian j. immunol., vol. 7, no. 1, pp. 247-257, 2024; article no.aji.126682 253 myriad of cancer types, including breast, leukemia, colorectal, lung, and ovarian cancers. we can also further develop immune cells with molecular targeted therapies to combine with specific immune cells to create personalized immunotherapy. c. tme targeting gene can be discovered “tumor microenvironment (tme) and tumor cells in tumor tissue take many strategies to evade the host immune response by creating many immune-suppressive factors [61]. thus, we can use the strategy from tme to be personalized therapy. tme consists of tissues, cells, and signaling molecules in tumor tissue, affecting the immune response to tumor cells. furthermore, tme elements of tissues, cells, and molecule factors include those during the early period of tumor tissues and those in an aggressive period in tumor tissues. identifying and regulating tme cells and regulating molecules such as extracellular matrix (ecm) and pathways such as adenosine (ado) and indole-2,3-dioxygenase (ido) may guide a new generation of precision medicine so that we can use tme targeting components to combine with the specific immune cells to personalized immunotherapy” [5]. d. epigenetics targeting can be discovered epigenetic therapy is based on methylation assay and ptm histone assay [62-65]. methylated cytosines recruit protein complexes that promote functionally inactive heterochromatin under a global decrease in dna methylation, such as 5-azacitidine (aza) and decitabine (dec), by inhibiting dnmt1. epigenetic therapy based on ptm histone assay is hdac inhabitation, which removes the acetyl groups from the lysine residues to apply for an option for tumor treatment so that we can use epigenetic therapy to combine with the specific immune cells for personalized immunotherapy. e. specific chemotherapy can be discovered customized chemotherapy has now been applied to the field of cancer [66-70]. according to genomic data, we can also use personalized chemotherapy to develop immunotherapy with specific immune cells and specific chemotherapy for personalized immunotherapy. 6. conclusion the "foe" or "friend" of chemotherapy combined with immunotherapy is a long-term puzzling issue. cytotoxic chemotherapy is considered to be immunosuppressive while it is recommended to support immunotherapy in clinical application, to increase act immunotherapy, and to reduce the side effects of chemotherapy, we have experienced several decades to study the mechanism using isolating and culturing primary tumor cells and immune cells from patient specimens such as resected solid tumor tissues. after about 30-40 years of effort, the optimizing combination has proved that chemotherapy combined with immunotherapy is superior to monotherapy. based on our current publications, including other clinical laboratory work, the manual introduced four areas of combination therapy: lymphodepletion (ld), chemoimmunotherapy (ci), ici combined with immunotherapy, and precision medicine (pm) supporting immunotherapy as fig. 4. precision medicine based on patients’ information includes patient specimen biobanks, single-cell technologies, clinical genomics, and artificial intelligence to support combination therapy. based on 30-40 years of r&d to improve patient immunotherapy, now is an excellent time to define combination therapy and evaluate its efficacy in oncological diseases. disclaimer (artificial intelligence) author(s) hereby declares that no generative ai technologies such as large language models (chatgpt, copilot, etc) and text-to-image generators have been used during writing or editing of this manuscript. details of the machine-learning (ai) usage is given below: 1. cytoscape platform integrated by topology algorithm. 2. galaxy server for next generation sequencing analysis. 3. gene chip expression console for microarray data for gene expression and brb array tool for gene expression signature (ges). acknowledgments under the support of dr. h. d. preisler, we have set up a method to analyze genomic profiles from tumor cells and til. this work was supported by li and li; 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cite as: olivier, celso eduardo, daiana guedes pinto, ana paula monezzi teixeira, cibele silva miguel, jhéssica letícia santos santana, regiane patussi santos lima, nicole sartoreto rocha, and raquel acácia pereira gonçalves santos. 2025. “immunoreactivity to cocoa and nickel in atopic and allergic contact dermatitis”. asian journal of immunology 8 (1):35-49. https://doi.org/10.9734/aji/2025/v8i1158. asian journal of immunology volume 8, issue 1, page 35-49, 2025; article no.aji.133582 immunoreactivity to cocoa and nickel in atopic and allergic contact dermatitis celso eduardo olivier a*, daiana guedes pinto a, ana paula monezzi teixeira a, cibele silva miguel a, jhéssica letícia santos santana b, regiane patussi santos lima c, nicole sartoreto rocha d and raquel acácia pereira gonçalves santos a a instituto alergoimuno de americana, brazil. b instituto de ensino e pesquisa do hospital de amor de barretos, brazil. c lavoisier laboratórios, são paulo, brazil. d faculdade de americana, são paulo, brazil. authors’ contributions this work was carried out in collaboration among all authors. the author ceo did conceptualization, data curation, formal analysis, literature review, and writing the original draft. authors dgp, apmt, csm, jlss, rpsl and nsr performed laboratory procedures. author rapgs performed cutaneous tests. all authors read and approved of the final manuscript. article information doi: https://doi.org/10.9734/aji/2025/v8i1158 open peer review history: this journal follows the advanced open peer review policy. identity of the reviewers, editor(s) and additional reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here: https://pr.sdiarticle5.com/review-history/133582 received: 01/02/2025 published: 05/04/2025 original research article https://doi.org/10.9734/aji/2025/v8i1158 https://pr.sdiarticle5.com/review-history/133582 olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 35-49, 2025; article no.aji.133582 36 abstract background: cocoa is one of the foods more consistently associated with high nickel content, a metal that functions as a hapten responsible for various non–ige-mediated symptomatic hypersensitivities. aim: the present study is a proof-of-concept that hypothesizes that lait and the ttp may differentiate diverse degrees of cellular and humoral immunoreactivity against cocoa extract and a nickel solution, as well to verify some sort of cross-reactivity between them in patients suffering from non–ige-mediated atopic and allergic contact dermatitis. methodology: we examined the medical charts of two cohorts of patients clinically diagnosed with intrinsic atopic dermatitis and allergic contact dermatitis, who were simultaneously investigated with cocoa extract and nickel solution with the help of ttp or lait. the registered results of the semi-quantitative serum ttp against cocoa extract and nickel solution, as well as the registered results of the leukocyte adherence inhibition (lai) percentage promoted by the ex vivo challenges against a cocoa extract and a nickel solution, were distributed in ranges through a cascade distribution chart to outline the variability of the results inside the cohorts. results: the mean for the ttp for the cocoa extract was estimated at 1:385; the median at 1:512; and the sd at 1:180. the mean or the ttp for the nickel was estimated at 1:309; the median was 1:256; the sd was estimated at 1:199. the mean for for the leukocyte adherence inhibition (lai) the cocoa extract was 51.9%; the median was 56%; the sd was 26.3%. the mean for the lai for nickel solution was 33%; the median was 33%; the sd was 26,6%. the pearson correlation indicated that there is a significant medium positive relationship between ttp results between cocoa (x-axis) and nickel (y-axis), r(98) = .335; p < .001. the pearson correlation indicated that there is a significant medium positive relationship between lait results between cocoa (x-axis) and nickel (y-axis), r(98) = .425; p < .001. conclusion: the results demonstrated a more significant immunoreactivity from the tests performed with cocoa extract than obtained with the nickel solution. these findings state that cocoa possesses other allergens responsible for cellular immunoreactivity besides nickel. this means that if a patient presents cellular immunoreactivity against cocoa, he/she will not necessarily present immunoreactivity against nickel; however, reciprocation is less probable. keywords: cocoa, dermatitis, endotype; hypersensitivity; leukocyte adherence inhibition test; nickel; non–ige-mediated immunoreactivity; precipitins. abbreviations lai :leukocyte adherence inhibition lait :leukocyte adherence inhibition test ttp :tube titration of precipitins 1. introduction self-reported allergy to chocolate is a common complaint in the clinical practice of allergology; however, ige-mediated cocoa hypersensitivity is not a commonly demonstrable endotype responsible for the phenotypes described as cocoa allergies (nin-valencia et al. 2024; sloan & powers 1986). when thoroughly investigated, ige-mediated reactions to chocolate are usually due to cow's milk, hen's egg, peanut, soybean, hazelnut, almond, and other ingredients and contaminants added to cocoa to make sweet chocolate (lopes et al. 2019, pilolli et al. 2024). “the allergen nomenclature sub-committee of the world health organization and international union of immunological societies (who/iuis) do not recognize any protein derived from theobroma cacao that could officially be stated as a major (or a minor) ige-mediated allergen. a candidate to be recognized as a significant igemediated cocoa allergen is the pathogenesisrelated protein pr10 (tcpr-10), a protein with homology to pollen and food allergens derived from the birch (betula verrucosa), the pear (prunus persica), the apple (malus domestica), the cherry (prunus avium) and the carrot (daucus carota)” (menezes et al. 2012). however, cocoa is one of the foods more consistently associated with high nickel content, a metal that functions as a hapten responsible for various non–ige-mediated symptomatic hypersensitivities associated with several toxic and allergic diseases (cubadda et al. 2020). nickel is the fifth most abundant metal in the earth's crust. it is found in soil, absorbed by cocoa roots, and transported upright to accumulate in cocoa beans (mostafa et al., 2024). olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 35-49, 2025; article no.aji.133582 37 besides nickel, cocoa beans may present several toxic metals absorbed from the soil, posing human health risks (frimpong et al., 2024). besides cocoa, nickel may be present in licorice, lettuce, soybeans, oatmeal, nuts, almonds, legumes, peanuts, oats, grains, whole wheat (and whole meal flours), and mainly in canned food with high nickel contents, liberated from a nickel-plated tin alloy (sharma 2013, abeck et al., 1993; veien & menné, 1990; veien et al., 1985). nickel is one of the leading causes of allergic contact dermatitis, which is diagnosed by patch tests (isufi et al., 2025). nickel is a hapten collected by dendritic cells through the toll-like receptor 4 and presented to naïve t cells in regional lymph nodes (schmidt et al., 2010, roediger & weninger, 2011). “nickel hypersensitivity may be responsible for allergic contact dermatitis, allergic contact mucositis, and systemic nickel allergy syndrome (schäfer et al., 2001; ahlström et al., 2019; greco et al., 2023; ricciardi et al., 2014). systemic nickel allergy is a term commonly used to describe clinical manifestations of the systemic provocation by haptens associated with atopic dermatitis, psoriasis, urticaria, angioedema, rhinitis, asthma, headache, chronic fatigue, postprandial dyspnea, cystitis, vulvovaginitis, acne, and iron deficiency anemia (nijhawan et al., 2009; veien, 2011; akiba et al., 2025). “sometimes, the exposition through a systemic route of immunoreactive haptens may produce localized cutaneous hypersensitivity phenotypes such as dyshidrotic eczema (pompholyx), toxicoderma-like rash, chronic pruritus, maculopapular rash, vasculitis-like lesions, flexural dermatitis, papuloerythrodermalike eruptions, and baboon syndrome” (antico & soana, 2015). nickel hypersensitivity may be successfully treated with sublingual desensitization and a lownickel diet. (filatova & cherpak, 2020; ricciardi et al., 2013; morris, 1998). nickel allergy can be diagnosed with cutaneous and oral challenge tests (veien et al., 1987). non–ige-mediated symptomatic nickel hypersensitivity may be differentiated with the help of the leukocyte adherence inhibition test (olivier et al., 2023c). “non–ige-mediated cellular immunoreactivity against several food allergens had already been reported by our group with the help of the leukocyte adherence inhibition test (lait) (olivier et al., 2021d; olivier et al., 2022a; olivier et al., 2022c; olivier et al., 2024a). the humoral immunoreactivity against several food allergens with the help of the tube titration of precipitins (ttp) was also evaluated” (olivier et al., 2024b; olivier et al., 2024c; olivier et al., 2025). “we routinely employ the lait and the ttp in our facilities as a triage to evaluate non–igemediated immunoreactivity against suspected allergens before performing more exhaustive in vivo provocation tests” (kuratsuji, 1981; olivier et al., 2023f; olivier et al., 2023h; olivier et al., 2023d). to evaluate the potential of the lait and ttp to endotyping non–ige-mediated cellular and humoral immunoreactivity against cocoa extract and nickel solution, we retrospectively compiled the electronic medical charts of patients diagnosed with non–igemediated hypersensitivity who were investigated simultaneously for immunoreactivity against these two allergens by one of these assays (olivier et al., 2023b; olivier et al., 2023e; olivier et al., 2024f). the present study is a proof-of-concept that hypothesizes that lait and the ttp may differentiate diverse degrees of cellular and humoral immunoreactivity against cocoa extract and nickel solution, as well to verify some crossreactivity between them through the calculation of a paired t-test to distinguish some order of cross-reactivity in patients suffering from non– ige-mediated clinically diagnosed allergies. “as the tests were performed simultaneously with the same venous sample for the two allergens, it is possible to calculate a paired t-test between lait results (since they refer to the same quantitative variable), as well as to present a dispersion graph between them to distinguish some order of correlation suggesting (or not) cross-reactivity” (gosset-student 1908). 2. materials and methods 2.1 subjects after receiving institutional review board approval from the instituto alergoimuno de americana (brazil; 02/2025), we reviewed the electronic chart of 10.170 outpatients who attended our facility from january 2018 to march 2025. a cohort of 100 consecutive outside patients (ttp cohort) had been simultaneously submitted to ttp with cocoa extract and nickel solution for presenting non–ige-mediated intrinsic atopic dermatitis and contact dermatitis. this cohort olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 35-49, 2025; article no.aji.133582 38 counted 27 males; mean age 38.6 years; sd 21.4 years; range 1 to 84 years; median 37.5 years; modes = 4, 7, 27, 30, 32, 33, 41, 45, 46, 50 years (each appeared 3 times); geometric mean = 29.9 years. a cohort of 100 consecutive outside patients (lait cohort) had been simultaneously submitted to tial with cocoa extract and nickel solution for presenting non–ige-mediated intrinsic atopic dermatitis and contact dermatitis. this cohort counted 27 males; mean age 43.3 years; sd 19.4 years; range 10 to 91 years; median 40 years; mode = 35 years (appeared seven times); geometric mean = 38.7 years. this study did not include patients under biological and/or systemic anti-inflammatory therapy. these procedures were offered to patients with clinical suspicion of cocoa and/or nickel hypersensitivity who demonstrated an undetectable specific ige against cocoa and nonreactive or inconclusive skin tests against cocoa extract and nickel solution (olivier et al., 2013). 2.2 extracts 2.2.1 cocoa extract the whole cocoa (pulp, peel, and seeds) was crushed, homogenized, and then left for 48 hours in a coca-based extractor solution (propylparaben 0.5g, methylparaben 1g, sorbitol 30g, nacl 5g, nahco3 2.5g, 1,000ml h2o) at 4 °c for protein extraction before centrifugation and separation of the water-soluble fraction from solid particles and oily fraction (coca, 1922). the protein quantification of the allergen extracts was done according to bradford's protein-dye binding methodology (bradford, 1976). the solution was diluted in antigen dilution solution (nacl 10g; kh2po4 0.72g; na3po4 2.86g; methylparaben 1g; propylparaben 0.5g; glycerin 400ml; h2o 600ml) to an estimated protein concentration of 1 mg/ml and stored at 4 °c into amber opaque glass vials. the cocoa extract was used to perform allergic skin tests, ttp, and lait. all relevant and mandatory laboratory health and safety measures have been complied with during the experiments. 2.2.2 nickel solution the [niso4 (h2o)6] was acquired from labcenter campinas. the powder was weighed and diluted in a buffer solution [nacl 10g; kh2po4 0,72g; na3po4 2,86g; h2o 600ml] to achieve the final concentration of 1 mg/ml to be employed in the lait, ttp, and cutaneous tests. 2.3 lait: ex vivo investigation: leukocyte adherence inhibition test 2.3.1 lait: procedure for allergen ex vivo challenging we performed the lait as previously described (olivier et al., 2012, olivier et al., 2014, olivier et al., 2021b, olivier et al., 2021c, olivier et al., 2021e). shortly, each donor's fresh plasma was divided into two parts and used in parallel ex vivo challenging tests with the cocoa extract, the nickel solution, and the unchallenged plasma (added with antigen dilution solution as a control) (olivier et al., 2022b, olivier et al., 2022a, olivier et al., 2022c, olivier et al., 2023f, olivier et al., 2023h). we collected plasma with high leukocyte content (buffy coat) from the heparinized tube after one hour of sedimentation at 37 °c. then, we distributed aliquots of 100 μl into eppendorf tubes with (or without) the challenging extract and kept them under agitation for 30 minutes (200 rpm at 37 °c) (olivier et al., 2023a, olivier et al., 2024e). 2.3.2 lait: procedure for adherence assay after incubation, the plasma was allocated into a standard neubauer hemocytometer counting chamber with a plain, non-metallic glass surface and left to stand for 2 hours at 37 °c in the humidified atmosphere of the covered water bath to allow leukocytes to adhere to the glass. next, we counted the leukocytes, removed the coverslip, and washed the chamber by immersion in a beaker with phosphate buffer saline (pbs) at 37 °c. then, we added a drop of pbs to the hemocytometer's chamber and allocated a clean coverslip over it. the remaining cells were counted in the same squares as previously examined. 2.3.3 lait: procedure for calculation the percentage of leukocyte adherence (la) of each assay was estimated as: (the number of leukocytes observed on the hemocytometry chamber after washing divided by the number of leukocytes observed on the hemocytometry chamber before washing) and multiplied by 100 (%). the leukocyte adherence ratio (lar) was estimated based on the ratio between the la from the antigen-specific challenged plasma and olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 35-49, 2025; article no.aji.133582 39 the la from the unchallenged control plasma: lar = la of the challenged sample divided by la of unchallenged control plasma multiplied by 100 (%). to further calculate the leukocyte adherence inhibition (lai), we subtracted the lar from 100 (%). we employed the lai results for the cascade distribution chart and the statistics calculations, both performed with the help of the microsoft excel® statistical package. 2.4 ttp: in vitro investigation: tube titration of precipitins as previously reported, the semi-quantitative ttp against the cocoa extract and the nickel solution was performed in a transparent vitreous tube array (olivier et al. 2021f, olivier et al. 2024g, olivier et al. 2024d, olivier et al. 2024b, olivier et al. 2024a). shortly, the patient's blood was collected in a clot-activator collecting tube. after separation, the serum was centrifugated at 2,000 rpm for 10 minutes. each allergen extract was allocated in sets of eleven glass tubes at progressive duplicated serum dilutions. the progressive dilutions were combined with separated aliquots of 15 μl of the antigen (cocoa extract or nickel solution) with 250 μl of the patient's serum, progressively diluted into physiological saline solution (nacl 0,9%) in the dilution ratios of 1:1; 1:2; 1:4; 1:8; 1:16; 1:32; 1:64; 1:128; 1:256; and 1:512. one tube was a blank control done with the distilled water and serum to observe occasional spontaneous precipitation (sia test). after 24 hours, the tubes were examined, and the titers (the highest dilution factor that yields a positive reading) were recorded (williams & chase, 1971). 3. results as a retrospective survey, there was no research protocol; therefore, we report the incidental immune investigation as registered in the digital medical charts. the ttp for the cocoa extract showed a distribution concentrated on the higher dilutions (fig. 1). there was one negative result. the mean was estimated at 1:385; the median was 1:512; the standard deviation was estimated at 1:180; the mode was 1:512 (appeared 65 times). the ttp for the nickel showed a distribution concentrated on the higher dilutions (fig. 2). there was one negative result. the mean was estimated at 1:309; the median was 1:256; the standard deviation was estimated at 1:199; the mode was 1:512 (appeared 46 times). the lait for the cocoa extract showed a wide distribution range of results. most results were concentrated in the higher immunoreactive groups. there were seven negative results. the lai ranged from 0% to 98%. the mean was 51.9%; the median was 56%; the standard deviation was 26.3%; the mode was 0% (appeared seven times). the cascade distribution demonstrates a wide range of lai results (fig. 3). some patients showed low or moderate immunoreactivity during the ex vivo challenge test. in contrast, others displayed strong immunoreactivity, which supposedly could reflect the participation of cocoa allergens in the non–ige-mediated hypersensitivity condition of these patients. fig. 1. cascade distribution chart of the tube titration of precipitins (x-axis %) resulting from the cocoa extract against the serum of the ttp cohort of 100 tests/subjects (y-axis) olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 35-49, 2025; article no.aji.133582 40 fig. 2. cascade distribution chart of the tube titration of precipitins (x-axis %) resulting from the nickel solution against the serum of the ttp cohort of 100 tests/subjects (y-axis). fig. 3. cascade distribution chart of the range groups of leukocyte adherence inhibition (lai) results (x-axis %) of the ex vivo challenge test against cocoa extract monitored by the leukocyte adherence inhibition test (lait), according to the respective number of outcomes over the lait cohort with 100 tests/subjects (y-axis) the lait for nickel solution showed a wide distribution range of results. most results were concentrated in the lower and moderate immunoreactive groups. there were eighteen negative results. the lai ranged from 0% to 89%. the mean was 33%; the median was 33%; the standard deviation was 26,6%; the mode was 0% (appeared eighteen times). the cascade distribution demonstrates a wide range of lai results (fig. 4). some patients showed low or moderate immunoreactivity during the ex vivo challenge test. in contrast, others displayed strong immunoreactivity, which could reflect the participation of nickel in the non–ige mediated hypersensitivity condition of these patients. olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 35-49, 2025; article no.aji.133582 41 the pearson correlation indicated that there is a significant medium positive relationship between ttp results between cocoa (x-axis) and nickel (y-axis), r(98) = .335; p < .001; see fig. 5. the pearson correlation indicated that there is a significant medium positive relationship between lait results between cocoa (x-axis) and nickel (y-axis), r(98) = .425; p < .001; see fig. 6. fig. 4. cascade distribution chart of the range groups of leukocyte adherence inhibition (lai) results (x-axis %) of ex vivo ex vivo challenge test against nickel solution monitored by the leukocyte adherence inhibition test (lait), according to the respective number of outcomes over the lait cohort with 100 tests/subjects (y-axis) fig. 5. dispersion chart of the leukocyte adherence inhibition (lai) results of the tube titration of precipitins against cocoa extract (x-axis %), plotted against the lai results of the tube titration of precipitins against nickel solution (y-axis %). olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 35-49, 2025; article no.aji.133582 42 fig. 6. dispersion chart of the leukocyte adherence inhibition (lai) results of the ex vivo challenge test against cocoa extract (x-axis %), plotted against the lai results of the ex vivo challenge test against nickel solution (y-axis %). 4. discussion cocoa polyphenols have several effects on innate inflammatory response and systemic and intestinal adaptive immune systems (pérez-cano et al., 2013). animal studies have observed that polyphenols in cocoa extract prevent ige synthesis and can suppress the development of atopic dermatitis by downregulating inflammatory markers, inflammatory chemokines, and cell infiltration into lesion areas, turning this food a potential therapy for patients with skin diseases; however, these preliminary data does not justify yet its empirical prescription for the treatment of allergic patients (kang et al., 2017; abril-gil et al. ,2012). despite the results demonstrating a positive correlation between cocoa and nickel immunoreactivity, the cocoa extract demonstrated significantly higher immune responses, suggesting that nickel is not the only active allergen in the cocoa extract. this infers that evaluating one allergen does not eliminate the need to evaluate the other. this observation also demonstrates that cocoa polyphenols do not directly suppress the immunoreactivity evaluated by the assays, leading to more studies about their alleged downregulation effects over inflammatory chemokines and immune cells, as reported in animal studies. endotyping cellular and humoral immunoreactivity biomarkers against specific allergens, haptens, and their respective crossreactivities are tools to build effective strategies to personalize exclusion diets and desensitization treatments for allergic patients (agache & akdis, 2020; khan, 2016). due to the facility's ability to diagnose hypersensitivities associated with the ige-mediated endotype, there is a tendentious bias among physicians to just "ignore" the non-ige-mediated mechanisms (zhang et al., 2024). to demonstrate a non–igemediated allergic endotype, sometimes it is necessary to employ a multi-omics approach to differentiate the variety of clinical phenotypes and immune endotypes (macowan et al., 2025; yoon & bunyavanich, 2025). the substantial number of sources for nickel is a confounding factor when investigating clinical hypersensitivity against this hapten. initially, it may appear to be a polysensitization picture since the patients react to a confounder number of unrelated sources. the patient may also present unrelated symptoms, leading to the diagnosis of several allergic phenotypes. until the conclusion that all phenotypes are produced by only one endotype mechanism elicited by only one allergen, it is usually a long journey requiring complex dietary interventions (mikajiri et al., 2024). olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 35-49, 2025; article no.aji.133582 43 as a hapten, even in the presence of an igemediated hypersensitivity, it is almost impossible to develop a specific ige-antibody for a commercial immunoassay against the multitude of carrier-haptens conjugates that may be produced once nickel gains blood circulation and conjugates with albumin, nickeloplasmin, or another serum protein (barceloux, 1999). the best strategy is to challenge the patients' living plasma (ex vivo) or fresh serum (in vitro) with nickel solution and quantify the resulting immunoreactivity. the correlation and the distribution of simultaneous positive specific ige against food and inhalant allergens is usually weak. however, polysensitization is more of a rule than an exception (zhang et al., 2025; čelakovská et al., 2024). the research of precipitins is the pioneering laboratory exam upon which the bases of immunology were constructed (wells, 1911). precipitating antibodies testifies a humoral immune response against the tested antigens (gell et al.,1946). before the discovery of ige, the research of precipitins was the leading way to realize in vitro diagnosis of immunoreactivity against allergens (augustin & hayward, 1960; augustin et al.,1960a; augustin, 1953). precipitins against food proteins and circulating immune complexes are strongly associated with patients with selective iga deficiency since the secretory iga is one of the main participants of the mechanism of immune exclusion promoted into the intestinal lumen to prevent the absorption of undigested proteins to blood circulation (cunningham-rundles et al., 1978). primary selective immune deficiencies (such as iga or ige) lead to immune dysregulation, a concept that is evolving as specific endotypes associated with secondary immunodeficiencies raised by the inflammatory conditions provided by the allergic reactions (henrickson, 2025; olivier et al., 2023g; sapartini et al., 2025). the lait was designed as an ex vivo challenge test performed with a viable leukocyte buffy coat exploring several immune pathways, resulting in a final result quantified as the allergen-specific leukocyte adherence inhibition (olivier et al. 2021a; thomson, 1982; tong et al., 1979; fink et al., 1987; halliday et al.,1974). the comparative results obtained from the tial cohort demonstrated a more significative immunoreactivity from the tests performed with cocoa extract than those obtained with the nickel solution. this finding states that cocoas possess more allergens responsible for cellular immunoreactivity besides nickel. this means that if a patient presents cellular immunoreactivity against cocoa, he/she will not necessarily present immunoreactivity against nickel; however, reciprocation is less probable. this preliminary retrospective survey demonstrated extensive results from the ttp and the ex vivo challenge test monitored by lait against cocoa extract and nickel solution in two cohorts of patients with allergic contact and atopic dermatitis. ttp and lait are complementary triage tests used at our facilities to select worthwhile antigens to proceed with more laborious in vivo provocation tests when the specific ige is undetectable. none of our patients presented an exclusive reaction to these allergens. every patient was simultaneously tested for several chemical and biological allergens, demonstrating positive results for some of them. 5. limitations this study is a retrospective analysis of data collected over seven years. there was no protocol research, and the subject's data was limited to the essentials available on our electronic sheets. therefore, we could not establish a cross-comparison between positive and negative controls to validate the results. the number of subjects is appropriate for preliminary study; however, future studies must be more comprehensive. the lack of a prospective research protocol implies the possibility of a bias produced by the physician's point of view, which indicated the exam was based on clinical suspicion led purely by anamnesis and physical examination. the study lost many of these patients to follow-up, so assuring the relationship between the immunoassay results and the patient's clinical outcome is not possible yet. the two procedures were not compared with paired tests because they were taken from distinct groups of patients. 6. conclusion our preliminary results show that the lait and ttp may differentiate diverse degrees of immunoreactivity against cocoa extracts and nickel solution in patients clinically diagnosed with non–ige-mediated atopic and allergic olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 35-49, 2025; article no.aji.133582 44 contact dermatitis. tial and ttp are inexpensive, can be performed with minimum laboratory equipment, and can be incorporated into strategies to address health disparities in managing allergies (anagnostou et al., 2025). as a preliminary report, the propaedeutic meaning of the presented results and the possibility of interferents must be yet established (anouar, et al., 2024). more studies focused on the qualityby-design approach with prospective larger double-blind cohorts need to evaluate the potential contribution of lait and ttp for endotyping cellular and humoral immunoreactivity in patients suspected of hypersensitivity against nickel and nickelcontaining foods (chiarentin et al., 2023). 7. future directions and recommendations for clinical practice the primary intended use of in vitro or ex vivo allergen challenge tests is to spare the patients from being submitted to unnecessary, exhaustive, and dangerous in vivo challenge tests. exploring the humoral and the cellular arms of immune systems, the ttp and tial alone or combined may represent, in the near future, a tool for allergists to construct an etiologic diagnosis from their patients, as well as determine the endotypes (mechanisms) of hypersensitivity, in order to choose more convenient and personalized therapies for them. adding data provided by ttp and tial may also contribute to streamlining biomedical research and improving tools such as large language models, usually used by clinicians as a decision support system to enhance diagnostic accuracy (abers & mathias, 2025). consent it is not applicable. ethical approvals as a retrospective survey of results recorded in cognito, consent was given collectively by the institution's ethics committee following the principles of the declaration of helsinki (wma, 2013). disclaimer (artificial intelligence) authors hereby declare that no generative ai technologies such as large language models (chatgpt, copilot, etc.) and text-to-image generators have been used during the writing or editing of this manuscript. acknowledgements the instituto alergoimuno de americana funded this work. we want to thank the laboratory technicians alessandra vieira de oliveira and luciana sacilotto carvalho for their help with the exams. competing interests authors have declared that no competing interests exist. references abeck, d., traenckner, i., steinkraus, v., vieluf, d., & ring, j. 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(2024). causality between 91 circulating inflammatory proteins and various asthma phenotypes: a mendelian randomization study. immunotargets and therapy, 13: 617-629. disclaimer/publisher’s note: the statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of the publisher and/or the editor(s). this publisher and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. _________________________________________________________________________________ © copyright (2025): author(s). the licensee is the journal publisher. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: https://pr.sdiarticle5.com/review-history/133582 https://pr.sdiarticle5.com/review-history/133582 _____________________________________________________________________________________________________ *corresponding author: e-mail: lucyjanefrancisokolie@gmail.com; cite as: janefrancis, okolie lucy, maduka amuche stella, ojeniran taiwo paul, yahaya imran umar, lawal mustapha, oniye mariam masud, and umar muhammad muhsin. 2025. “the role of trained immunity in protection against infectious diseases: epigenetic reprogramming and long-term immune responses”. asian journal of immunology 8 (1):86-96. https://doi.org/10.9734/aji/2025/v8i1163. asian journal of immunology volume 8, issue 1, page 86-96, 2025; article no.aji.134904 the role of trained immunity in protection against infectious diseases: epigenetic reprogramming and longterm immune responses okolie lucy janefrancis a*, maduka amuche stella b, ojeniran taiwo paul c, yahaya imran umar d, lawal mustapha e, oniye mariam masud f and umar muhammad muhsin g a department of microbiology, alex ekwueme federal university ndufu-alike, nigeria. b department of science education, nnamdi azikiwe university awka, nigeria. c alex ekwueme federal university teaching hospital, abakaliki, ebonyi state, nigeria. d department of biochemistry, bayero university kano, nigeria. e department of microbiology, kebbi state university of science and technology, aleiro, nigeria. f department of environmental biology, kwara state polytechnic/science laboratory technology, nigeria. g department of microbiology, bayero university kano, nigeria. authors’ contributions this work was carried out in collaboration among all authors. all authors read and approved the final manuscript. article information doi: https://doi.org/10.9734/aji/2025/v8i1163 open peer review history: this journal follows the advanced open peer review policy. identity of the reviewers, editor(s) and additional reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here: https://pr.sdiarticle5.com/review-history/134904 received: 26/02/2025 published: 03/05/2025 minireview article https://doi.org/10.9734/aji/2025/v8i1163 https://pr.sdiarticle5.com/review-history/134904 janefrancis et al.; asian j. immunol., vol. 8, no. 1, pp. 86-96, 2025; article no.aji.134904 87 abstract trained immunity, a concept introduced more than a decade ago, defines the non-specific memory capacity of innate immunity by epigenetic and metabolic reprogramming. it involves features of epigenetic and metabolic reprogramming of innate immune cells that induce long-term functional changes in host defense. this review explores the mechanisms and duration of trained immunity, as well as the therapies related to its potential in infectious diseases. a systematic literature search was conducted across pubmed, google scholar, and web of science for studies published between 2019 and 2025. experimental and clinical studies on innate immune memory, epigenetic modifications, and metabolic pathways associated with long-term immune responses were selected as study criteria. the synthesized data was narratively summarized to assess key themes, including molecular mechanisms, sustainability, and translational applications. a total of eight studies were included in this review. the findings revealed that trained immunity is mediated by histone modifications, metabolic changes, and long-term myelopoiesis. studies also show that immune training has varying durations, with effects lasting from weeks to months. emerging therapeutic strategies will focus on vaccine enhancement, cancer immunotherapy, and sepsis management. however, challenges remain, including a lack of clinical validation, incomplete mechanistic understanding, and ethical concerns. trained immunity is a promising approach to immunomodulation between innate and adaptive immunity. future research should aim to conduct clinical trials, assess safety, and develop precision medicine approaches to efficiently utilize this concept in therapeutic applications. keywords: trained immunity; innate immune memory; infectious diseases; metabolic reprogramming; epigenetics. abbreviations bcg : bacillus calmette-guérin hand :hiv-associated neurocognitive disorders lncrna : long non-coding rna mesh : medical subject headings nk : natural killer prrs : pattern recognition receptors tiis : trained immunity-inducing agents tlr : toll-like receptors 1. introduction trained immunity, referred to as innate immune memory, is an inherent capability of innate immune cells to mount enhanced response to secondary infections after an initial exposure. in contrast to adaptive immunity where memory cells are antigen-specific, trained immunity relies on functional reprogramming of the innate immune cells, mainly monocytes, macrophages, and natural killer cells by epigenetic and metabolic modifications (dulfer & domínguezandrés, 2024). this is a phenomenon shown previously in invertebrates without an adaptive immune system suggesting that innate immune memory has provided a fundamental evolutionary advantage (netea et al., 2020). trained immunity has its historical basis as early as its prolific documentation in the nonspecific protective effects of vaccines. for example, the bacillus calmette-guérin (bcg) vaccine used for tuberculosis has been shown to boost resistance to unrelated infections and is contrary to the prevailing separation of innate and adaptive immunity (berendsen et al., 2021; ciarlo et al., 2020). further studies have demonstrated that long-lasting changes in innate immune function can be induced by microbial components like β glucans and lipopolysaccharides, mediated by epigenetic modifications e.g. histone methylation and acetylation in particular to inflammatory response gene (bomans et al., 2018; sánchezramón et al., 2018). trained immunity is mediated through changes in metabolism including the reprogramming of immune progenitor cells from the bone marrow to convert to aerobic glycolysis. however, it has been shown that histone modifications, such as h3k4me3 and h3k27ac, are associated with a heightened inflammatory response on reinfection (katzmarski et al., 2021). in addition, evidence shows that trained immunity is inherited transgenerationally by germ cell epigenetic modifications and shapes immune responses in the offspring (kaufmann et al., 2022). two differing mechanisms of immunological memory, namely trained immunity and adaptive immune memory, have a distinct cellular basis, specificity, duration, and underlying molecular janefrancis et al.; asian j. immunol., vol. 8, no. 1, pp. 86-96, 2025; article no.aji.134904 88 fig. 1. conceptual framework of the study mechanisms. training immunity involves the innate immune cells such as monocytes, macrophages, and nk cells, while adaptation immune memory is dependent on antigenspecific t and b lymphocytes which get clonally expanded and persist for a long period (netea et al., 2020). in contrast to adaptive immunity, which works via highly specific somatically revisers in antigen receptors to encounter antigens previously exposed, trained immunity based on broad pattern recognition receptors (prrs), like toll-like receptors (tlrs), to detect conserved microbial components (mulder et al. 2019). the length of immune memory is otherwise very different in these two systems. adaptive immune memory does persist for years or even a lifetime, as the presence of long-lived memory t and b cells (bekkering et al., 2021). these epigenetic and metabolic reprogramming programs that train immunity tend to be at the molecular level and include histone modifications and shifts in glycolytic metabolism to enhance the responsiveness of innate immune cells to reinfection (saeed et al., 2014). adaptive immune memory, however, is sustained through clonal expansion, long-lived plasma cells and memory t cells upon receipt of antigens with specialized effector functions (sallusto et al., 2010). these immune strategies also form an important distinction in terms of their evolutionary origin. conserved across the wide spectrum of diverse species ranging from plants and invertebrates, this ancient type of immunity definitely plays a selective role in the host defense (duggan et al., 2023). in contrast to adaptive immunity, this is unique to the vertebrates and gives highly refined and longlasting protection against pathogens (sun & lanier, 2021). the aim of this review was to investigate the complete analysis of protective mechanisms in infectious diseases through the synthesis of contemporary evidence on molecular processes and duration of effect and therapeutic possibilities of trained immunity. fig. 1 illustrates the conceptual framework of the study. 2. methodology 2.1 literature search strategy a comprehensive and systematic literature search was conducted to identify relevant studies on trained immunity, epigenetic reprogramming, and long-term immune responses in the context of infectious diseases. the search was performed across multiple databases, including pubmed, web of science, and google scholar, with a timeframe of 2019 to 2025, ensuring broad coverage of biomedical and immunological research. the search strategy combined medical subject headings (mesh) terms and keywords related to trained immunity, innate immune memory, epigenetics, histone modifications, metabolic reprogramming, and infectious disease protection. boolean operators (and, or) were used to refine searches (table 1), and databasespecific filters like full-text availability, and publication date restrictions. this review was not registered in prospero. janefrancis et al.; asian j. immunol., vol. 8, no. 1, pp. 86-96, 2025; article no.aji.134904 89 table 1. search string combination of keywords (“trained immunity” or “innate immune memory”) and (“epigenetic reprogramming” or “histone modification” or “dna methylation”) and (“infectious disease” or “pathogen defense”) and (“metabolic shift” or “glycolysis”) table 2. methodology theme method literature search pubmed, web of science, google scholar keywords “trained immunity,” “epigenetic reprogramming,” “infectious disease,” “metabolic reprogramming” timeframe 2019 to 2025 selection criteria peer-reviewed articles of original research published in english. studies that investigated trained immunity and its mechanisms, including epigenetic and metabolic changes. research focusing on infectious disease protection through innate immune memory. experimental and clinical studies involving humans and relevant animal models. data extraction & synthesis data were extracted using a standardized data extraction form. narrative synthesis approach was used to summarize key findings and highlight common themes. 2.2 selection criteria this review was conducted following the prisma guidelines, and a corresponding prisma flowchart is included in the results to illustrate the study selection process. to ensure relevance and quality, the following selection criteria were applied: 2.2.1 inclusion criteria • peer-reviewed articles of original research published in english. • studies that investigated trained immunity and its mechanisms, including epigenetic and metabolic changes. • research focusing on infectious disease protection through innate immune memory. • experimental and clinical studies involving humans and relevant animal models. 2.2.2 exclusion criteria • studies focusing exclusively on adaptive immunity without reference to trained immunity. • non-english publications. • opinion pieces, editorials, reviews, and conference abstracts without full data. 2.3 data extraction and synthesis search results were screened based on the predefined inclusion criteria and data from the selected studies were extracted independently by two reviewers using a standardized data extraction form, capturing the study design, population involved, interventions, outcomes, and key findings. when there were discrepancies, it was resolved through consensus. a narrative synthesis was used to summarize key findings and highlight common themes. 3. results and discussion 3.1 included studies fig. 2 illustrates the included studies selection processes. eight studies were included in this review, after a broad selection and screening of 205 articles. the studies included were conducted between 2019 and 2025, with a majority of them employing experimental study design. three of the studies had histone modifications as their mechanism of trained immunity while two studies identified epigenetic modulation as their trained immunity mechanism. bacterial & viral infections were observed to be the most common pathogen context across the studies and the duration of immune training was long-term. janefrancis et al.; asian j. immunol., vol. 8, no. 1, pp. 86-96, 2025; article no.aji.134904 90 fig. 2. prisma flow diagram 3.2 mechanisms underlying trained immunity trained immunity is primarily driven by epigenetic and metabolic reprogramming, enabling innate immune cells to mount enhanced responses upon secondary stimulation. key epigenetic mechanisms such as histone modifications, dna methylation and their regulation have all been the focus of several studies. as an example, jentho et al. (2021) established that heme-trained immunity results in heme long-lasting epigenetic remodelling that alters myelopoiesis and enhances monocyte function. in addition, the set7 lysine methyltransferase regulates oxidative phosphorylation plasticity for trained immunity in response to β glucan stimulation which was previously shown by keating et al. (2020). additionally, these findings are consistent with previous work that demonstrated the importance of histone methylation and acetylation of immune gene promoters to promote hypercytokinemia on secondary t-cell stimulation (fok et al., 2019). other important drivers of trained immunity are metabolic reprogramming; glycolysis, oxidative phosphorylation, and lipid metabolism are shifted. hao et al. (2025) stated that metabolic adaptations, such as enhanced glycolysis and glutaminolysis, have a crucial role in innate immune memory. this is in alignment with rusek et al. (2018) who showed that even bacterial and viral stimuli, elicit metabolic shifts that sustain immune memory. in addition, salauddin et al. (2024) rethink that both shifts from oxidative phosphorylation and lipid metabolism are crucial for the immune response, so that metabolic pathways may be targeted to amplify the response. participation of non-coding rnas in trained immunity has also been recently highlighted. in the case of fok et al. (2019), they observed that lncrnas mediate the crosstalk between cellular metabolism and epigenetic modifications to modulate innate immune reprogramming. this coincides with abhimanyu et al. (2021), who showed that targeting rnabased regulatory pathways can reverse the postinfectious epigenetic-mediated immune suppression. overall, the convergence of epigenetic and metabolic reprogramming in the form of trained immunity indicates the possibility of its therapeutic manipulation. while the reliance on antigen-specific lymphocytes for classical immunological memory, trained immunity might be a broader, non-specific protective mechanism that can be used for vaccine development and immunotherapy (hajishengallis et al., 2025). 3.3 duration and sustainability of trained immunity trained immunity varies with different persistence depending on the stimulus, the immune cell type, as well as metabolic and janefrancis et al.; asian j. immunol., vol. 8, no. 1, pp. 86-96, 2025; article no.aji.134904 91 table 3. findings from included studies author & year study objective study design mechanisms of trained immunity disease/pathogen context duration of immune training clinical/translational relevance key findings abhimanyu et al., 2021 investigate the reversal of postinfectious immune suppression via epigenetic modifications experimental study (in vitro & in vivo) epigenetic modulation through histone acetylation bacterial & viral infections long-term effects assessed potential for therapeutic reprogramming of immune responses identified key epigenetic markers that can reverse immune suppression post-infection hao et al., 2025 explore metabolic adaptations driving innate immune memory review of metabolic pathways glycolysis and oxidative phosphorylation shifts broad infectious diseases variable depending on stimulus identifying metabolic targets for immunotherapies highlighted the role of metabolic rewiring in sustaining trained immunity jentho et al., 2021 study epigenetic modulation in trained immunity induced by heme experimental study (animal models) histone methylation and dna modifications heme-related immune disorders longlasting (several months) potential for improving anemia and inflammatory conditions demonstrated sustained myelopoiesis and immune adaptation via heme exposure xing et al., 2020 examine trained immunity in tissueresident macrophages experimental & review epigenetic and metabolic modifications in macrophages respiratory infections months to years implications for vaccine strategies proposed a new vaccination model leveraging trained macrophage responses fok et al., 2019 investigate lncrna involvement in metabolic-epigenetic immunity links experimental study long non-coding rnas (lncrnas) in immune reprogramming bacterial & viral infections persistent effects possible rna-based immunotherapies found lncrnamediated regulatory pathways crucial for trained immunity table 3 cont’d author & year study objective study design mechanisms of trained immunity disease/pathogen context duration of immune training clinical/translational relevance key findings wang et al., 2024 explore trained immunity’s impact on sepsis experimental study (animal models) histone modifications, cytokine reprogramming sepsis weeks to months new therapeutic avenues for sepsis management found that trained immunity can enhance sepsis resistance but also janefrancis et al.; asian j. immunol., vol. 8, no. 1, pp. 86-96, 2025; article no.aji.134904 92 author & year study objective study design mechanisms of trained immunity disease/pathogen context duration of immune training clinical/translational relevance key findings risk hyperinflammation lajqi et al., 2024 investigate trained immunity in pediatric infections experimental study (pediatric models) histone modifications, cytokine memory pediatric bacterial & viral infections months to years potential for improving pediatric vaccine efficacy found that early-life infections induce long-lasting immune adaptations keating et al., 2020 examine set7 methyltransferase’s role in trained immunity experimental study set7-driven oxidative phosphorylation plasticity fungal infections (βglucan exposure) long-term metabolic imprinting targeting set7 for trained immunity-based therapies identified set7 as a key regulator of trained immunity in response to β-glucan janefrancis et al.; asian j. immunol., vol. 8, no. 1, pp. 86-96, 2025; article no.aji.134904 93 epigenetic adaptations. several studies show that trained immunity can persist from weeks to months and for years in some cases. according to jentho et al. (2021), when myeloid progenitors are exposed to heme, these epigenetic modifications lead to long-term immune reprogramming beyond the life span of a single immune cell through altered hematopoiesis. as in keating et al (2020), β glucan-induced trained immunity can persist for up to several months through the sustained effect of changes in oxidative phosphorylation and mitochondrial function. recovery of trained immunity also depends on repeated exposure to stimuli. by repeatedly encountering the same microbes, recurrent encounters furthered the training of the immune system, keeping it lasting much further by continuously reprogramming metabolic and epigenetic pathways (rusek et al., 2018). on the other hand, abhimanyu et al. (2021) reported that the longevity of trained immunity is contextdependent and reversible under immunosuppressive conditions. this is further corroborated by capriotti and klase (2024), who demonstrate that trained immunity mechanisms break down in chronic hiv infection and are lost more quickly than would have been otherwise the case. in addition, it has been noted in studies that trained immunity persistence is disease model-dependent. in sepsis-induced trained immunity, metabolic adaptations shown by hao et al. (2025) are transient and come back to baseline in a couple of weeks. hajishengallis et al. (2025) demonstrated that chronic inflammatory disease would maintain trained immunity for extended periods, and they suggest a possible role in disease progression. taken as a whole these findings suggest that trained immunity can be maintained on a sustained basis but its persistence depends on external stimuli disease states and the cellular environment. consequently, this opens up the potential for vaccine development, as prolonging trained immunity from a trained immunity-inducing vaccine, could provide wide-spectrum protection against infectious diseases (salauddin et al., 2024). 3.4 emerging therapeutic strategies leveraging recent advancement has highlighted trained immunity as a promising avenue for the development of new therapeutic strategies for the promotion of host defense, modulation of chronic inflammatory conditions, and other desirable effects by vaccine. using trained immunity inducing agents (tiis), a class of agents including the β-glucan and the cell wall component vaccine bacillus calmette guérin (bcg) is one of the most actively explored approaches to providing general protection against infections. bcg vaccination induces long-term metabolic and epigenetic reprogramming in monocytes and enhanced resistance to viral and bacterial pathogens beyond tuberculosis (xing et al., 2020). wang et al. (2024) also suggested that trained immunityinducing compounds could be used to strengthen innate immune responses and thus help host resilience to sepsis. trained immunity is also a target beyond infectious diseases towards autoimmune and chronic inflammatory disorders. based on their results, hajishengallis et al. (2025) proposed that the trained monocytes and macrophages seem to participate in the continuous inflammatory cascade in chronic diseases, which makes them potential target of immunomodulatory therapies. lajqi et al. (2024) explored pediatric infectious diseases and showed that manipulating trained immunity could improve immune response in immunocompromised children. an additional creative aspect is using epigenetic and metabolic modulators to control trained immunity in therapeutic areas. this led fok et al. (2019) to find that immune training is driven by long noncoding rnas (lncrnas) as critical regulators, that provide new targets of epigenetic-based therapies. metabolic intervention strategy targeting lysine methyltransferase set7 was also found by keating et al. (2020) to boost trained immunity via optimization of mitochondrial function. trained immunity is currently being studied as a potential tool for the treatment of neuro-inflammatory and chronic infection conditions, including hiv-associated neurocognitive disorders (hand). according to capriotti and klase (2024), trained immunitymodulating agents might reverse innate immune dysfunction in hiv resulting in the restoration of immune function. 4. challenges and future perspectives despite all of this interest in trained immunity as a therapeutic target, many challenges and knowledge gaps remain. the lack of an understanding of the long-term consequences of trained immunity induction is a major limitation. although many studies have uncovered the links between epigenetic and metabolic hallmarks of innate immune memory (keating et al., 2020; janefrancis et al.; asian j. immunol., vol. 8, no. 1, pp. 86-96, 2025; article no.aji.134904 94 fok et al., 2019), how long and whether these changes have sustained adverse effects remains unknown. it is of particular concern, however, how excessive immune activation can lead to chronic inflammation or autoimmunity (hajishengallis et al., 2025). in addition, most recent research relies on in-vitro models or preclinical animal studies without necessary translation to human immune responses (xing et al., 2020). with these, rigorous clinical trials for validation of the efficacy and safety of trained immunity-based interventions are required to advance the field. some of the studies have demonstrated that vaccination with bacillus calmetteguérin (bcg) and β-glucans may promote host defense against heterologous infections (xing et al., 2020; wang et al., 2024), but the results should be confirmed in rcts for various populations. in addition, clinical studies should determine what the optimal dosing is and when and with what population it does and does not respond, as it appears that the effects of trained immunity might be affected by age, genetics, and metabolic status (salauddin et al., 2024). large-scale trials of trained immunity for sepsis, cancer immunotherapy, and chronic infections would provide crucial insights into its therapeutic potential (xia et al., 2021; capriotti & klase (2024). from the perspectives of ethics and safety, there are multiple concerns when manipulating innate immune memory. trained immunity is antigen non-specific, as opposed to adaptive immunity, and therefore carries the risk of off-target effects, including inappropriate inflammation or immune exhaustion (hajishengallis et al., 2025). intergenerational epigenetic inheritance is also a cause for concern since the immune changes could be passed on to a generation, with uncertain consequences (fok et al., 2019). regulatory bodies must construct a catalog of clear guidelines for the clinical medicines involving stimulated immunity, as the use of the agents must be both safe and ethically justified, and available. to make these possible, tailored interdisciplinary research and robust clinical trial and oversight will need to be guided toward the translation of trained immunity-based strategies to practical and sustainable clinical interventions. 5. conclusion trained immunity has the potential to fundamentally change the field of immunology by providing novel information regarding the process by which innate immune cells learn a memory-like response in the context of epigenetic and metabolic reprogramming. it is reviewed for its mechanisms, duration, and potential for therapy, including how these might impact infectious diseases, cancer, as well as inflammatory disorders. emerging strategies in βglucan-based immunomodulation and vaccine adjuvants are promising, but remain challenging due to gaps in mechanistic understanding, the need for rigorous clinical validation, and ethical dilemmas of immune manipulation. future studies should focus on large size clinical trials, individualization of immunomodulatory approaches, and policies that can make implementation safe and effective. with trained immunity, disease prevention and treatment of many diseases could make this revolution, bridging innate and adaptive immune strategies for long-term health benefits. data availability: no new datasets were generated or analyzed in the current study. concent and ethical approval it is not applicable disclaimer (artificial intelligence) author(s) hereby declare that no generative ai technologies such as large language models (chatgpt, copilot, etc.) and text-to-image generators have been used during the writing or editing of this manuscript. competing interests authors have declared that no competing interests exist. references abhimanyu, ontiveros, c. o., guerra-resendez, r. s., nishiguchi, t., ladki, m., hilton, i. b., ... & dinardo, a. r. 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(2020). innate immune memory of tissue-resident macrophages and trained innate immunity: re-vamping vaccine concept and strategies. journal of leukocyte biology, 108(3), 825–834. disclaimer/publisher’s note: the statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of the publisher and/or the editor(s). this publisher and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. _________________________________________________________________________________ © copyright (2025): author(s). the licensee is the journal publisher. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: https://pr.sdiarticle5.com/review-history/134904 https://pr.sdiarticle5.com/review-history/134904 _____________________________________________________________________________________________________ *corresponding author: e-mail: ahmedkhalaf214@gmail.com; cite as: khalaf, ahmed a. m. 2025. “human immunodeficiency virus epidemic among people who inject drugs and female sex workers in north africa: a systematic review”. asian journal of immunology 8 (1):50-59. https://doi.org/10.9734/aji/2025/v8i1159. asian journal of immunology volume 8, issue 1, page 50-59, 2025; article no.aji.125999 human immunodeficiency virus epidemic among people who inject drugs and female sex workers in north africa: a systematic review ahmed a. m. khalaf a* a ministry of health, tripoli, libya. author’s contribution the sole author designed, analysed, interpreted and prepared the manuscript. article information doi: https://doi.org/10.9734/aji/2025/v8i1159 open peer review history: this journal follows the advanced open peer review policy. identity of the reviewers, editor(s) and additional reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here: https://pr.sdiarticle5.com/review-history/125999 received: 19/10/2024 published: 18/04/2025 abstract background: human immunodeficiency virus (hiv) and acquired immunodeficiency syndrome (aids) remain major health problems worldwide, with high mortality and morbidity rates, particularly in developing countries. the united nations programme on hiv/aids (unaids) reported that there were 38.4 million individuals globally who had hiv. around 1.5 million new hiv infections were recorded in 2021, while approximately 650,000 individuals worldwide lost their lives due to aidsrelated illnesses. the prevalence of hiv in people who inject drugs (pwid) and female sex workers (fsws) has not been widely investigated. information on the epidemiology of hiv infection among pwid and fsws in north africa is limited. the aim of the present study was to review the status of the hiv epidemic among pwid and fsws in north african countries by explaining hiv prevalence. methods: a comprehensive literature search was performed on studies related to the prevalence of hiv infection in pwid and fsws published between 1990 and 2024 using search engines such as pubmed, science-direct, google scholar. reports that are entirely on north africa were considered, systematic review article https://doi.org/10.9734/aji/2025/v8i1159 https://pr.sdiarticle5.com/review-history/125999 khalaf; asian j. immunol., vol. 8, no. 1, pp. 50-59, 2025; article no.aji.125999 51 including studies reporting hiv infection in high-risk groups, particularly pwid and fsws, with the data organized into a customized database. results: a total of 3605 records were retrieved in the initial search, 10 relevant records hiv among pwid and fsws were recognized and included in the study. the overall prevalence of hiv in pwid ranged from 0.15% to 87.1% and 0% to 15.7% in fsws. injection drug abuse and women engaged in sex work were commonly associated with hiv infection. conclusion: the findings of this study showed that the prevalence of hiv among pwid and fsws in north africa is high. pwid and fsws has been identified as the dominant method of transmission in various north african countries like libya, egypt, morocco and tunisia. the execution of initiatives designed to improve sanitation infrastructure, elevate educational standards, and enhance socioeconomic situations is crucial for decreasing the prevalence of hiv infections among pwid and fsws. keywords: human immunodeficiency virus; people who inject drugs; female sex workers; north africa. 1. introduction human immunodeficiency virus (hiv) is a member of the lentivirus genus belonging to the retroviridae family. in 1981, it was identified as the initial cause of the illness now referred to as acquired immunodeficiency syndromes (aids) (gruters et al., 1987; fauci, 1999). hiv is spread mainly from the body fluids of an infected person, including blood, breast milk, semen and vaginal fluids (world health organization [who], 2024a). hiv infection is characterized by mild immune system alterations that occur before any symptoms or negative emotions appear. before seroconversion, which occurs when an individual has just been exposed to hiv, this stage lasts for up to three months after infection. although the course of an infection and the amount of time it takes for clinical signs to appear might vary widely from person to person, the disease typically advances rather slowly. the onset of progressive hiv illnesses and immunosuppressive symptoms takes several years after the first infection (naif, 2013). hiv targets cells of the human immune system, such as cd4+ t cells, macrophages, and dendritic cells. cd4+ cells play an important role in maintaining the immune system. following infection, hiv utilizes cd4+ cells as a host to replicate and infect additional cells. this results in the decrease of cd4+ cells in the body, causing a complete breakdown of the immune system. the progression from hiv to aids is monitored by the sharp decrease in cd4+ cells (yousaf et al., 2011). chronic hiv infection is causing a deficiency in cellular immunity. the unexpected emergence of opportunistic infections characterizes this final stage of hiv infection. the main contributors to hiv-related morbidity an death are these last ones. the use of cotrimoxazole and antiretroviruses can significantly lower the frequency of opportunistic infections and increase the life expectancy of hiv patients. the initiation of antiretroviral therapy for an individual with hiv is determined by their level of immune system weakness (comlan et al., 2017). the sensitive enzyme immunoassays available today can identify antibodies as soon as one to two weeks following infection. other tests are necessary to support antibody investigation (p24 antigen, pcr), corroborate positive antibody screens (western blot, pcr), and give clinicians treating hivpositive patients additional information (qualitative and quantitative pcr, genotyping) (fearon, 2005). two types of hiv have been described which are hiv-1 and hiv-2. globally, hiv-1 infection is the leading cause of the aids pandemic, while the emergence of hiv-2 is concentrated in west african countries (campbell & gandhi, 2011). at present, hiv infection continues to be one of the most serious global health issues facing humans. according to statistics from the united nations (un), the global number of individuals living with hiv in 2021 tallied at approximately 38.4 million. furthermore, an estimated 1.5 million new cases of hiv infections emerged. additionally, in 2021, around 650,000 deaths were attributed to aids related illnesses. the un program on hiv/aids estimated that at the end of 2021 there were 20.6 million persons living with hiv infection in sub saharan africa, which is the highest globally. moreover, the lowest infection was in north africa and the middle east; roughly 180,000 individuals were estimated to be living with hiv. infection with hiv remains a major cause of illness and death in developing nations, including north african countries (unaids, 2022). people who inject drugs (pwid) are at increased risk of hiv; worldwide, about 11 million people inject khalaf; asian j. immunol., vol. 8, no. 1, pp. 50-59, 2025; article no.aji.125999 52 drugs, and around 1 in 8 (or 1.4 million) of these people are living with hiv. in addition, 10% of new hiv infections worldwide are due to the use of injection drugs (who, 2024b). globally, sex workers are facing a greater impact from the hiv pandemic (who, 2012). treatment abandonment among patients with hiv infection is a public health issue worldwide. at the end of december 2021, 75% of all people living with hiv were accessing treatment (unaids, 2022). adherence to antiretroviral therapy (art) and loss of follow-up impact the prognosis of individuals living with hiv/aids in various regions globally (lima et al., 2018). however, by the end of 2013, the art coverage level in the north africa and middle east region remained the lowest in the world at 11%. hiv-positive individuals who inject drugs and sex workers who have discontinued treatment and follow-up contribute to increased morbidity, mortality risks, and hiv transmission, especially in north african countries (gökengin et al., 2016). although hiv/aids is a silent and fatal disease, little is known about its infection among pwid and fsw in north african countries. however, having updated literature on hiv prevalence is crucial for tracking the spread of infection and describing the burden on global public health. hence, the aim of this study was to assess the status of the hiv prevalence among pwid and fsws in north africa to help understand the hiv epidemiology in this part of the african continent. 2. methodology present systematic review summarizes main results of the hiv epidemiology among pwid and fsws in north africa. a comprehensive literature search was performed using pub med, science-direct and google scholar, databases for articles published in english from 1990 until 2024. the data included in the present review were primarily conducted in the countries of north africa. these data sources were recognized through a comprehensive search of pertinent studies and databases. the keywords used to question the databases included hiv in combination with prevalence, “pwid”, “fsws”, as well as north africa and country names. all articles were evaluated for pertinence before inclusion. the review involved all countries contained in the north africa descriptions of the world bank and who emro. these include algeria, libya, egypt, morocco, and tunisia. in this article, the north africa high-risk groups were including people who inject drugs (pwid) as well as female sex workers (fsws). eligibility and evaluation of the studies: each study was examined and thoroughly assessed for its inclusion in the studies: all the studies were examined and thoroughly evaluated for inclusion in the study. all descriptive/cross-sectional, case control, and epidemiological studies were included. letters to the editor, editorials, case reports, hypotheses, studies on animals or cell lines, and unpublished reports were excluded. studies were eligible for review if they reported hiv epidemiology in pwid and fsw. therefore, studies reporting the prevalence of hiv in prisoners, man who have se with man, hemodialysis, hiv in patients with tb or cancer were excluded (fig. 1). data extraction: relevant information from articles reporting the prevalence of hiv and its epidemiology in north african countries were extracted. data on the number of participants in each eligible study, study country, prevalence of hiv infection population age group, and year of study were collected and classified in divide tables. 3. results a total of 3605 records were identified from three databases. duplicates studies were identified and removed, leaving 166 potential records, after review of titles and abstracts records were excluded based on selection criteria, and a total of 10 reports on the prevalence of hiv among pwid and fsws covering 4 countries were included. the review process is presented in fig. 1. seven studies recorded hiv positive among pwid, 5 recorded the hiv positive among fsws sample sizes ranged from 113 to 12,981 pwid, and 69 to1447 fsws. pwid and fsws data were obtainable for 4 of the 5 na countries. no studies were available for algeria. egypt contributed the largest number of data points of hiv prevalence measures where, there were 4 studies from egypt, 2 from tunisia, morocco, and libya, respectively. the prevalence of hiv across the key populations such as pwid and fsws reported by the 10 studies was range between 0 and 87.1%. when divided according to population categories, injecting drugs is a primary way hiv is transmitted globally (who, 2024b). study population hiv prevalence ranged from 0.15% in a study from egypt to 87.1% in a study from libya (table 1). the median hiv prevalence among pwid in all studies was 0.91%. sexual khalaf; asian j. immunol., vol. 8, no. 1, pp. 50-59, 2025; article no.aji.125999 53 hazard behavior associated with hiv infection among fsws, the country-specific estimate fsws ranged from 0% in tunisia to 15.7% in libya. egypt and morocco had a prevalence estimate < 9% (table 2). the median hiv prevalence among fsws in all studies was 1.25%. fig. 1. flowchart of study selection two studies were found in this review that recorded hiv/hcv and hiv/hbv co-infection, prevalence of hiv/hcv and hiv/hbv co infections among pwid was 83.2% and 3.7%, respectively. beside, the prevalence of hiv/hcv and hiv/hbv co-infections among fsws was 3.7% and 0%, respectively. 4. discussion hiv infection among pwid and fsws continues to be a major public health issue worldwide (mirzoyan et al., 2013). understanding the epidemiological characteristics of hiv infection are crucial and beneficial in defining the implications and problems of the infection. injecting drugs is a primary way hiv is transmitted globally (who, 2024b). the findings revealed that the hiv prevalence among north african pwid population groups ranged from 0% to 87.1%, this results is higher than the previous estimates of global prevalence (mathers et al., 2008; rashti et al., 2020). it is also higher than the findings of studies conducted in europe (wenz et al., 2016; handanagic et al., 2016; hatzakis et al., 2015), china (zhang et al., 2007), bangladesh (azim et al., 2008) and brazil (oliveira-filho et al., 2020). by contrast, it is relatively low compared with other studies reported from the usa (peters et al., 2016), and estonia (platt et al., 2006). the differences that were noticed in hiv prevalence among studies, nations, and areas could arise from diverse factors like compliance with prevention methods and variations in community spread. in this study, hiv prevalence among pwid is high in the northern countries of africa, particularly libya (mirzoyan et al., 2013). this occurrence can be ascribed to its geographical positioning, which borders three sub-saharan nations, along with socio-political challenges that have significantly facilitated the spread of hiv and hindered the adherence to hiv control measures. these results highlight a need for continuing prevention of hiv transmission among pwid. the hiv epidemic, similar to other epidemics, arises within an intricate social setting. social norms impacting transmission include sexual behaviors. between 75 and 85 percent of the approximately 28 million hiv infections that have happened thus far are due to transmission through sexual contact (royce et al., 1997). concerning the investigation of hiv and related sexual risk like sex work, this systematic review shows that the hiv prevalence amongst north african fsws population groups ranged from 0% to 15.7%. these outcomes are similar to numerous other systematic reviews conducted (paz-bailey et al., 2016; leili et al., 2021). however, it is low compared to what is observed in india (wayal et al., 2011), kenya (luchters et al., 2008). additionally, it is higher than the results of studies conducted in china (wang et al., 2009), central america (soto et al., 2007), and the democratic republic of the congo (vandepitte et al., 2007). women engaging in sex work networks appear to be the main drivers of considerable hiv transmission in libya, morocco, and egypt, which may have been due to the extensive scale of commercial sex networks in these environments, coupled with the elevated levels of hazard manners exhibited within these networks. fsws and their male clients are at high hazardous for hiv and have been significant in starting the epidemic in many african countries (hunter, 1993). the hiv epidemic is very fluid and has expanded quickly; nearly every country worldwide is impacted. however, prevalence investigations have been conducted on a global level to gain understanding of hiv epidemiology. monitoring prevalence changes is crucial for predicting pandemic evolution and developing a successful public health strategy (bokazhanova & rutherford, 2006). khalaf; asian j. immunol., vol. 8, no. 1, pp. 50-59, 2025; article no.aji.125999 54 table 1. studies reporting hiv prevalence among people who inject drugs (pwid) across the north africa study/references sampling location year of study study population age group hiv prevalence among pwid anan a et al. (2024) egypt 2019-2022 146 11-50 31.5% ghrabi a et al. (2018) tunisia 2017-2018 113 32-47 10% mirzoyan l et al. (2013) libya 2010 328 15-≥50 87.1% wahdan i et al. (2013) egypt 2012 338 <25->45 12.4% soliman c et al. (2010) egypt 2006 413 18-65 1% elmir e et al. (2002) morocco 1991-1999 12981 30-≥40 11.1% watts dm et al. (1993) egypt 1986-1990 1961 any age 0.15% table 2. studies reporting hiv prevalence among female sex workers (fsws) across the north africa study/references sampling location year of study study population age group hiv prevalence among fsws johnston l et al. (2013) morocco 2010-211 1447 18 ≥ 8.3% valadez jj, et al. (2013) libya 2010 69 15-≥50 15.7% wahdan i et al. (2013) egypt 2012 338 <25-45+ 5.9% znazen a et al. (2010) tunisia 2007 188 14-≥34 0% watts dm et al. (1993) egypt 1986-1990 349 any age 0% khalaf; asian j. immunol., vol. 8, no. 1, pp. 50-59, 2025; article no.aji.125999 55 situation in north africa and the middle east regarding hiv/aids: according to statistics from the united nations (un), around 180,000 people were estimated to be living with hiv, approximately 14000 people became newly infected with hivv. furthermore, roughly 5100 fatalities were linked to diseases related to aids in 2021 (unaids, 2022). sub-saharan africa, this region is the most heavily affected by hiv worldwide, accounting for 52.6 million people living with hiv and 42000 of aids deaths in 2021 (unaids, 2022). this region borders north africa, and many illegal immigrants come from it to north african countries. therefore, individuals in communities with hiv may not know they are infected or carry the virus, leading to potential transmission to family members or other people. on the basis of the geographical countries of north africa, the results indicated that libya (mirzoyan et al., 2013), egypt (anan et al., 2024), morocco (elmir et al., 2002) and tunisia (ghrabi et al., 2018), respectively had higher prevalence hiv among pwid. moreover, prevalence estimates at the country level indicate a high burden of infection in libya (valadez et al., 2013), morocco (johnston et al., 2013) and egypt (wahdan et al., 2013), respectively, among fsws. the observed variation in the estimates of hiv prevalence among fsws across various countries may be partially attributed to the diverse risk factors and spread pathways present in each nation. these results emphasize a continuous need for ongoing prevention of hiv spread among fsws between north african countries. in africa, despite the high hiv prevalence, the reported occurrence of hiv infection among pwid and fsws in north africa was considerably lower compared with south africa (jones et al., 2023; asher et al., 2013). the north africa region is still lacks sufficient hiv epidemiological information, leading to debates regarding the epidemic's prevalence in this part of the globe. it's crucial to have current hiv prevalence data for north african countries to understand the virus's spread. hence, additional monitoring of hiv prevalence is necessary to evaluate and track the escalating hiv impact (abu-raddad et al., 2010). the environments where the hiv pandemic is happening are becoming more varied. the epidemics are primarily influenced by social, structural, and population-level risks and protections, which in turn affect the individual risks of hiv infection (beyrer, 2007). the field of hiv prevention is constantly changing, and there is now discussion about the potential for worldwide virtual eradication of hiv (mutevedzi & newell, 2014). mitigation of hiv disease strategies will expected be required to decrease hiv prevalence in north africa, where there might be restrictions on getting health care and accessing resources. the suitable approach can vary from one country to another and additionally have to be justifiably focused on prevention of hiv transmission. while countries must focus on addressing the underlying causes of hiv exposure risk, the main emphasis should be on addressing the immediate factors that increase individuals' risk of hiv exposure, as dealing with structural factors is time-consuming and outside the purview of the public health sector. hence, there is an opportunity for prevention that should not be missed to control the epidemic in this region (abu-raddad et al., 2010). this systematic review offers the latest extensive assessment of hiv prevalence by carefully evaluating existing literature. when interpreting results, it's vital to consider and navigate through various constraints in order to gain a comprehensive and robust understanding of the outcomes. the primary limitations come from the data that is currently available, with a scarcity of studies on prevalence. despite these limitations, the present systematic review demonstrated that there is a significant burden of hiv infection in pwid and fsws in most of the north african countries. however, additional investigation is required to fill the knowledge gaps. 5. conclusion the findings of this report provide a comprehensive overview of the prevalence of hiv among pwid and fsws. variation in prevalence of hiv observed in different regions in north africa. pwid and fsws has been identified as the dominant method of transmission in various north african countries like libya, egypt, morocco and tunisia. as the hiv pandemic is still evolving, more studies need to be conducted in this part of the african continent to comprehend the right burden of the illness. studies in this region need to take into consideration societal and institutional factors. the extensive programs of monitoring of hiv prevalence are necessary to prevent these high khalaf; 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(2010). sexually transmitted infections among female sex workers in tunisia: high prevalence of chlamydia trachomatis. sexually transmitted infections, 86(7), 500–505. disclaimer/publisher’s note: the statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of the publisher and/or the editor(s). this publisher and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. _________________________________________________________________________________ © copyright (2025): author(s). the licensee is the journal publisher. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: https://pr.sdiarticle5.com/review-history/125999 https://pr.sdiarticle5.com/review-history/125999 _____________________________________________________________________________________________________ *corresponding author: e-mail: ayodeleadeli101@gmail.com; cite as: fagbemi oluwaseyi ajibola, and adedoyin elizabeth ayodele. 2025. “epigenetic dysregulation and its role in immune-mediated diseases”. asian journal of immunology 8 (1):230–241. https://doi.org/10.9734/aji/2025/v8i1174. asian journal of immunology volume 8, issue 1, page 230-241, 2025; article no.aji.144830 epigenetic dysregulation and its role in immune-mediated diseases fagbemi oluwaseyi ajibola a and adedoyin elizabeth ayodele b* a department of human anatomy, college of medicine and surgery, federal university lokoja, kogi state, nigeria. b department of microbiology, federal university oye ekiti, ekiti state, nigeria. authors’ contributions this work was carried out in collaboration between both authors. both authors read and approved the final manuscript. article information doi: https://doi.org/10.9734/aji/2025/v8i1174 open peer review history: this journal follows the advanced open peer review policy. identity of the reviewers, editor(s) and additional reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here: https://pr.sdiarticle5.com/review-history/144830 received: 18/07/2025 published: 27/09/2025 abstract immune-mediated diseases, including rheumatoid arthritis (ra), systemic lupus erythematosus (sle), multiple sclerosis (ms) and inflammatory bowel disease (ibd: crohn's disease & ulcerative colitis) represent a significant global health burden characterized by a loss of immune tolerance and chronic inflammation. while genetic predisposition plays a crucial role, it does not fully account for disease pathogenesis, onset, or flare-ups. the emerging field of epigenetics provides a critical mechanistic link between genetic susceptibility and environmental triggers. this review synthesizes current evidence on how dysregulation of key epigenetic mechanisms, dna methylation, histone modifications, and non-coding rna expression, contributes to the breakdown of immune homeostasis. we detail the aberrant epigenetic landscapes in specific immune cell subsets (e.g., t cells, b cells, macrophages) across major autoimmune disorders. furthermore, we explore the potential of epigenetic modifications as novel biomarkers for diagnosis, prognosis, and disease activity monitoring. finally, we discuss the promising therapeutic avenue of "epigenetic therapy," review article https://doi.org/10.9734/aji/2025/v8i1174 https://pr.sdiarticle5.com/review-history/144830 ajibola and ayodele; asian j. immunol., vol. 8, no. 1, pp. 230-241, 2025; article no.aji.144830 231 repurposing existing drugs and developing new compounds to reverse pathogenic epigenetic marks and restore immune tolerance. keywords: epigenetics; autoimmunity; dna methylation; histone modification; non-coding rna; rheumatoid arthritis; systemic lupus erythematosus; multiple sclerosis; biomarkers; epigenetic therapy. 1. introduction immune-mediated inflammatory diseases (imids), encompassing a broad spectrum of conditions such as rheumatoid arthritis (ra), systemic lupus erythematosus (sle), multiple sclerosis (ms), and inflammatory bowel disease (ibd), represent a formidable challenge to global public health. these chronic, debilitating disorders are characterized by a pathological breakdown of self-tolerance, leading to uncontrolled immune activation against specific tissues or systemically, resulting in persistent inflammation and organ damage (wilkinson & shapiro, 2024). the collective prevalence of imids is estimated to affect 5-10% of the population in developed nations, posing a significant economic and social burden due to long-term disability, reduced quality of life, and the need for lifelong medical management (stroeks et al., 2025) the etiological landscape of autoimmunity has long been conceptualized through the lens of genetic predisposition interacting with environmental triggers. the advent of genomewide association studies (gwas) has been instrumental, identifying over hundreds of susceptibility loci across various imids. these studies have robustly confirmed the paramount importance of the major histocompatibility complex (mhc) region and highlighted key pathways involved in immune cell signaling, cytokine production, and lymphocyte activation (ausserwinkler et al., 2025; arneth, 2024). however, a critical paradox emerged from this genetic data: the identified risk alleles, while statistically significant, often confer only a modest increase in relative risk and exhibit low penetrance. this is most strikingly evidenced by the significant discordance rates observed in monozygotic (identical) twins, who share nearly 100% of their genetic sequence. for example, concordance rates for sle, ra, and ms typically range from only 25% to 40%, leaving a substantial portion of disease risk unexplained by genetics alone (khan et al., 2024; ates et al., 2025). this compelling "heritability gap" strongly implies that non-genetic factors play a dominant role in determining whether a genetically susceptible individual will ultimately develop clinical disease. this gap is bridged by epigenetics, a discipline that has revolutionized our understanding of gene regulation. epigenetics refers to the study of heritable changes in gene expression and cellular phenotype that occur without alterations to the underlying dna nucleotide sequence (lin et al.,2024; gibson et al., 2022). these mechanisms, primarily dna methylation, histone modifications, and non-coding rna-associated gene silencing, act as dynamic regulators of chromatin architecture, determining the accessibility of genetic information to the transcriptional machinery. crucially, the epigenetic landscape is plastic and can be actively shaped by a myriad of environmental exposures, including infectious agents (e.g., epstein-barr virus), tobacco smoke, hormonal changes, dietary components, and pollutants (kwon et al., 2024; kaszycki and kim, 2025). these factors can initiate stable epigenetic reprogramming within immune cells, potentially leading to the sustained overexpression of proinflammatory genes or the silencing of key immunoregulatory pathways. this review manuscript synthesizes current evidence positing that epigenetic dysregulation serves as the crucial mechanistic link between genetic susceptibility and environmental triggers in the pathogenesis of immune-mediated diseases. we will detail the aberrant epigenetic patterns, the "epigenome" characteristic of major imids, focusing on specific immune cell subsets. furthermore, we will explore the profound translational implications of this field, discussing the potential of epigenetic marks as novel biomarkers for diagnosis and prognostication and surveying the promising frontier of epigeneticbased therapeutics aimed at reversing pathogenic marks and restoring immune homeostasis. 2. fundamental epigenetic mechanisms epigenetic regulation provides a dynamic and heritable layer of control over gene expression, ajibola and ayodele; asian j. immunol., vol. 8, no. 1, pp. 230-241, 2025; article no.aji.144830 232 enabling cellular differentiation and plasticity in response to environmental cues without altering the primary dna sequence. in the immune system, these mechanisms are paramount for the development, activation, and termination of responses, ensuring a precise balance between effective pathogen clearance and self-tolerance. the dysregulation of these processes is a cornerstone of autoimmune pathology. the three primary, interconnected epigenetic mechanisms are dna methylation, histone modifications, and non-coding rna-associated silencing. restoring immune homeostasis. 2.1 dna methylation dna methylation is the most extensively studied epigenetic modification in mammals. it involves the covalent addition of a methyl group to the 5' position of a cytosine ring, primarily within cpg dinucleotide sequences. dense clusters of cpg sites, known as cpg islands, are often found in gene promoter regions. methylation of these islands typically leads to transcriptional repression by physically impeding the binding of transcription factors and by recruiting proteins such as methyl-cpg-binding domain proteins (mbds), which then attract histone deacetylases and other chromatin-remodeling complexes to establish a closed, silent chromatin state (harris et al, 2025; smith et al., 2025). the establishment and maintenance of dna methylation patterns are catalyzed by a family of enzymes called dna methyltransferases (dnmts). dnmt3a and dnmt3b are responsible for de novo methylation, setting up new methylation patterns during embryonic development. dnmt1, the maintenance methyltransferase, faithfully copies methylation patterns to the daughter strand during dna replication, ensuring heritability of the epigenetic mark through cell divisions (sarre et al., 2025). in the context of immunity, dna methylation is critical for processes such as x-chromosome inactivation, genomic imprinting, and the silencing of endogenous retroviral elements. crucially, it governs lymphocyte lineage commitment and function. for example, the differentiation of naïve cd4+ t cells into distinct effector subsets (th1, th2, th17) and immunosuppressive regulatory t cells (tregs) is orchestrated by precise changes in the methylation status of key cytokine and transcription factor genes (roy et al., 2025). a hallmark of many autoimmune diseases, particularly sle, is a state of global dna hypomethylation in t cells, which promotes the aberrant overexpression of autoimmuneassociated genes (e.g., itgal (cd11a) and tnfsf7 (cd70)), alongside locus-specific hypermethylation events that may silence protective genes (deng et al., 2024) 2.2 histone post-translational modifications histones are the core protein components of nucleosomes, around which dna is wrapped. their n-terminal tails are subject to over 100 different post-translational modifications (ptms), including acetylation, methylation, phosphorylation, ubiquitination, and sumoylation. these ptms alter the electrostatic charge of histones and serve as docking sites for other proteins, collectively influencing chromatin accessibility and gene expression (nie et al., 2024). the most well-characterized modifications are: acetylation/deacetylation: catalyzed by histone acetyltransferases (hats) and histone deacetylases (hdacs), respectively. acetylation neutralizes the positive charge on lysine residues, reducing the affinity between histones and the negatively charged dna backbone. this results in a more open, transcriptionally permissive chromatin structure (euchromatin). deacetylation has the opposite effect, promoting chromatin condensation and gene silencing (heterochromatin) (chen et al., 2024). methylation: catalyzed by histone methyltransferases (hmts) and removed by histone demethylases (hdms). the functional outcome of methylation is highly contextdependent, varying by the specific lysine or arginine residue modified and the degree of methylation (mono-, di-, or tri-methylation). for instance, trimethylation of histone h3 lysine 4 (h3k4me3) is a mark of active promoters, while trimethylation of h3 lysine 27 (h3k27me3) is a repressive mark associated with facultative heterochromatin (chen et al., 2024). the "histone code" hypothesis posits that these combinations of modifications act sequentially or in concert to form a complex language that dictates specific functional outcomes for a chromosomal region (ueberheide et al., 2024). in immune cells, activating marks like h3k4me3 ajibola and ayodele; asian j. immunol., vol. 8, no. 1, pp. 230-241, 2025; article no.aji.144830 233 and h3k9ac are enriched at the promoters of highly expressed cytokine genes (e.g., ifng, il4, il17), while repressive marks like h3k27me3 help silence alternative lineage genes during t-cell differentiation, ensuring stable effector cell identity (jay et al., 2025). 2.3 non-coding rnas (ncrnas) non-coding rnas represent a vast and diverse class of functional rna molecules that are transcribed from the genome but not translated into proteins. they act as crucial epigenetic regulators, primarily at the post-transcriptional level. micrornas (mirnas): these are short (~22 nucleotides), single-stranded rnas that regulate gene expression by binding to complementary sequences in the 3' untranslated regions (utrs) of target messenger rnas (mrnas). this binding typically leads to mrna degradation or translational repression. a single mirna can target hundreds of mrnas, allowing it to fine-tune entire genetic networks. in immunology, mirnas are indispensable regulators of immune cell development, proliferation, differentiation, and function. for example, mir-155 is a proinflammatory mirna that promotes th1 and th17 responses, while mir-146a acts as a critical negative feedback regulator of nf-κb signaling and is often dysregulated in autoimmunity (shaheen et al., 2024; mehta et al., 2025). long non-coding rnas (lncrnas): these are transcripts longer than 200 nucleotides with limited protein-coding potential. they exert their regulatory functions through a variety of mechanisms: as scaffolds for chromatinmodifying complexes, guides for their localization to specific genomic loci, decoys for transcription factors, or sponges that sequester mirnas. the lncrna malat1, for instance, has been implicated in regulating alternative splicing and gene expression in immune cells, while others like tmevpg1 (nest) can influence epigenetic states to control interferon-γ expression (antonazzo et al., 2024). these three mechanisms do not operate in isolation but form a highly integrated regulatory network. dna methylation can influence histone modification patterns, and both can be guided by ncrnas. conversely, histone modifications can regulate the expression of ncrnas. this complex crosstalk ensures precise spatiotemporal control of the gene expression programs that dictate immune cell identity and function, and its disruption lies at the heart of epigenetic dysregulation in disease (kong et al., 2024). 3. epigenetic dysregulation in specific immune-mediated diseases the overarching principles of epigenetic regulation become profoundly altered in immunemediated diseases. the following section details the disease-specific epigenetic landscapes that contribute to pathogenesis, moving from systemic autoimmunity to organ-specific disorders. 3.1 systemic lupus erythematosus (sle) sle is arguably the best-studied autoimmune disease from an epigenetic perspective and is characterized by a profound state of global dna hypomethylation in cd4+ t cells and other immune cells. this hypomethylation mirrors the epigenetic state of activated, proliferating t cells and leads to the overexpression of genes normally silenced in resting lymphocytes, such as perforin (prf1), the t-cell adhesion molecule itgal (cd11a), and the co-stimulatory molecule tnfsf7 (cd70) (ribeiro et al., 2024; araki & mimura, 2024). this aberrant overexpression promotes autoreactive t-cell help to b cells, driving autoantibody production. mechanism of hypomethylation: the root cause involves impaired erk signaling pathway signaling, leading to reduced expression and translocation of dnmt1. this results in passive demethylation during cell division. furthermore, oxidative stress and elevated levels of nitric oxide in sle patients can directly inhibit dnmt enzymatic activity (somers et al., 2024). histone modifications: sle t cells exhibit a shift towards a transcriptionally permissive chromatin state. this is characterized by globally decreased levels of the repressive mark h3k27me3 and increased levels of activating marks like h3k18ac and h3k4me3 at the promoters of key interferon-stimulated genes (isgs) and inflammatory cytokines, amplifying the type i interferon signature that is a hallmark of sle (zhang et al., 2024). ajibola and ayodele; asian j. immunol., vol. 8, no. 1, pp. 230-241, 2025; article no.aji.144830 234 ncrnas: a distinct mirna signature is present in sle patient blood and tissues. mir-146a, a negative regulator of the interferon pathway, is under expressed, contributing to unchecked ifn production. conversely, mir-21 and mir-148a are overexpressed; they promote autoimmunity by targeting dnmt1, creating a vicious cycle of dna hypomethylation, and by dampening negative regulatory pathways (royo et al., 2025). 3.2 rheumatoid arthritis (ra) in ra, epigenetic dysregulation is most prominently studied in the hyperplastic, invasive synovial tissue, particularly in fibroblast-like synoviocytes (fls), which develop an aggressive, "transformed-like" phenotype that drives joint destruction. dna methylation: ra-fls exhibit a unique and stable "methylation signature" that distinguishes them from osteoarthritis fls and is present even in early disease. key changes include: hypomethylation of genes encoding matrixdegrading enzymes (mmp3, mmp9), chemokines (cxcl12), and critical signaling molecules (stat3, irf5), leading to their overexpression and promoting inflammation, angiogenesis, and tissue invasion (prideaux et al., 2024). hypermethylation of genes that normally promote apoptosis or cell cycle arrest, such as tnfrsf25 (dr3) and bcl2l11 (bim), conferring resistance to cell death and contributing to synovial hyperplasia (svendsen et al., 2025). histone modifications: the balance of hat/hdac activity is skewed in ra. while some hdacs are downregulated, class i and ii hdac activity is generally increased in ra synovium, contributing to the suppression of antiinflammatory genes. conversely, hat activity is also elevated, promoting the expression of proinflammatory genes like il6 and tnf (enayati et al., 2024). ncrnas: mir-155 is highly expressed in ra synovial fluid and tissue, where it promotes macrophage activation and th17 cell differentiation. mir-146a is also upregulated but may act in a feedback inhibitory role that is ultimately insufficient to control inflammation. mir-124a is significantly downregulated, leading to increased activation of the monocyte chemoattractant ccl2 (seyedi et al., 2024). 3.3 multiple sclerosis (ms) epigenetic mechanisms in ms are critical for determining the fate of t cells, pushing differentiation towards pro-inflammatory th1 and th17 lineages and away from immunoregulatory tregs. dna methylation: genome-wide studies reveal significant differences in dna methylation patterns in cd4+ t cells, cd8+ t cells, and b cells of ms patients compared to controls. intriguingly, the strongest ms-associated genetic risk variant, hla-drb1, also displays msspecific methylation patterns, suggesting a complex gene-environment interaction where epigenetics modulates the effect of the primary genetic risk factor (elbahrawi et al., 2024). key hypomethylated loci are associated with t-cell activation and adhesion pathways. histone modifications: hdacs and sirtuins (sirt1, a class iii hdac) are key regulators of t-cell plasticity in ms. sirt1 deficiency has been linked to hyperacetylation of the transcription factor foxo1, impairing treg function and promoting a pro-inflammatory t-cell state. furthermore, inhibitors of hdacs have been shown to ameliorate disease in experimental autoimmune encephalomyelitis (eae), the mouse model of ms, by promoting treg development (xie et al., 2025). ncrnas: circulating mirnas serve as promising biomarkers. mir-326 expression correlates with disease severity and promotes th17 differentiation. mir-17-92 cluster members are dysregulated, impacting t-cell proliferation. serum levels of mir-155 and mir-301a are also elevated and associated with active disease (xie et al., 2025) 3.4 inflammatory bowel disease (ibd: crohn's disease & ulcerative colitis) the intestinal epithelium and mucosal immune system in ibd patients display widespread epigenetic alterations induced by the luminal environment (microbiome, nutrients). dna methylation: studies on intestinal mucosal biopsies reveal hundreds of differentially methylated regions. crohn's disease is often associated with hypermethylation and silencing of the socs1 gene, a negative regulator of cytokine signaling, leading to enhanced jak/stat signaling. in ulcerative colitis, the tnf gene promoter is often hypomethylated, contributing to its excessive production (mahurkar‐joshi et al., 2025). ajibola and ayodele; asian j. immunol., vol. 8, no. 1, pp. 230-241, 2025; article no.aji.144830 235 table 1. summary of key epigenetic alterations in major immune-mediated diseases disease cell type/ tissue dna methylation changes histone modifications key ncrnas involved sle cd4+ t cells global hypomethylation; hypomethylation of cd11a, cd70 increased h3/h4 acetylation ↓ mir-146a, ↑ mir-21, ↑ mir-155 ra synovial fls hypomethylation of cxcl12, il6; hypermethylation of dr3, bim increased hdac activity ↑ mir-155, ↑ mir-203, ↑ mir-124a ms cd4+ t cells, pbmcs differential methylation at hla & non-hla loci altered hdac/sirt expression ↑ mir-326, ↑ mir-155, ↓ mir-17-92 ibd intestinal mucosa hypermethylation of socs1 (crohn's); hypomethylation of tnf altered hat/hdac balance ↑ mir-21, ↑ mir-29, mir-196 dysregulation histone modifications: butyrate, a short-chain fatty acid produced by commensal gut bacteria, is a natural hdac inhibitor. a deficiency in butyrate production or sensing in ibd leads to increased hdac activity, repressing the expression of anti-inflammatory genes and genes involved in epithelial barrier integrity. this links the environmental factor (microbiome) directly to epigenetic dysregulation (li et al., 2025) ncrnas: mir-21 is upregulated in ibd mucosa and promotes intestinal inflammation by targeting pdcd4, a suppressor of il-10 expression. mir-29 is also overexpressed and targets key molecules essential for intestinal barrier function. the let-7 family mirnas are involved in regulating il-13-mediated inflammation (goodarzi et al., 2025). 4. environmental triggers and epigenetic modification the compelling evidence for epigenetic dysregulation in immune-mediated diseases raises a critical question: what initiates these changes? environmental factors are the primary architects, acting upon a genetically susceptible background to instigate pathogenic epigenetic reprogramming. these exposures can induce stable alterations to the epigenome of immune cells and tissue-resident cells, effectively "embedding" the memory of a environmental insult and lowering the threshold for autoimmune activation. the timing of exposure (e.g., in utero, during adolescence) can be as critical as the exposure itself due to periods of heightened epigenetic plasticity. 4.1 tobacco smoke cigarette smoke is one of the most wellestablished environmental risk factors for ra and other imids. it is a complex mixture of over 7,000 chemicals, many of which have demonstrable epigenetic effects. mechanism: chemicals like benzene and hydroquinone can directly inhibit the activity of dnmts by forming covalent adducts or by generating reactive oxygen species (ros) that interfere with enzymatic function. this leads to dna hypomethylation. evidence: smokers exhibit global dna hypomethylation in peripheral blood mononuclear cells (pbmcs). more specifically, smoking has been linked to hypomethylation of the ahrr (aryl hydrocarbon receptor repressor) gene and site-specific hypomethylation at the cxcl12 (involved in ra pathogenesis) and il6 promoters in immune cells, promoting a pro-inflammatory state (lim & kim, 2024; svendsen et al., 2025). this effect can persist for years after smoking cessation, illustrating the long-lasting impact of epigenetic modification. 4.2 microbial infections (e.g., epsteinbarr virus) infections, particularly with the epstein-barr virus (ebv), are strongly associated with an increased risk of sle, ms, and other autoimmune conditions. mechanism: ebv infection induces widespread epigenetic changes in host b cells to facilitate its own latency and proliferation. the viral protein ebna2 acts as a transcriptional regulator that binds to numerous autoimmune risk loci ajibola and ayodele; asian j. immunol., vol. 8, no. 1, pp. 230-241, 2025; article no.aji.144830 236 identified by gwas, altering their expression and histone modification landscape. furthermore, ebv-encoded latent membrane protein 1 (lmp1) can dysregulate dnmt expression, leading to hypomethylation and activation of endogenous retroviruses and autoimmune-related genes (viel, 2024; zhao et al., 2024)) evidence: patients with sle have been shown to have aberrant hypomethylation of ebv genes, leading to loss of viral control and a higher viral load. the cross-reactivity between ebv antigens (e.g., ebna1) and self-antigens (e.g., sm in sle) in an epigenetically permissive environment may break tolerance. 4.3 diet and the gut microbiome dietary components and the metabolites produced by the commensal gut microbiota are potent epigenetic modifiers that directly influence immune cell function and mucosal integrity. mechanism: methyl donors: nutrients involved in one-carbon metabolism, such as folate, vitamin b12, and choline are essential for generating sadenosylmethionine (sam), the universal methyl donor for dna and histone methylation. deficiencies in these nutrients can lead to global hypomethylation. short-chain fatty acids (scfas): butyrate, propionate, and acetate are produced by bacterial fermentation of dietary fiber. butyrate is a potent inhibitor of hdac (particularly class i and iia). hdac inhibition in immune cells, particularly in the gut, promotes the development and function of regulatory t cells (tregs) via hyperacetylation of the foxp3 promoter and other loci, thereby enforcing immune tolerance (xu et al., 2025) evidence: a "western diet" low in fiber reduces scfa production, leading to decreased hdac inhibition, impaired treg function, and a heightened inflammatory state. butyrate supplementation has been shown to ameliorate disease in animal models of colitis and ms (fisse et al., 2024). 4.4 ultraviolet (uv) radiation uv radiation is a known trigger for sle flares and skin manifestations. mechanism: uvb exposure can induce dna damage and oxidative stress in keratinocytes, leading to the generation of ros. ros can directly inhibit dnmt1 activity and alter the activity of enzymes responsible for histone modifications. this can cause demethylation and overexpression of genes involved in apoptosis and inflammation, such as cd70 and cd154 (barnes et al., 2024). evidence: uv irradiation of skin cells in culture induces demethylation and overexpression of autoantigens like ro/ssa, potentially making them more visible to the immune system and initiating an autoimmune response. 4.5 air pollutants and chemicals particulate matter (pm2.5), organic solvents, and other environmental chemicals have been implicated in the pathogenesis of imids. mechanism: similar to tobacco smoke, many pollutants generate oxidative stress, leading to impaired dnmt function and global hypomethylation. some chemicals can also directly bind to and activate aryl hydrocarbon receptor (ahr), a ligand-activated transcription factor that recruits a variety of epigenetic modifiers to target genes, influencing th17/treg balance (wais and agrawal, 2024 ; hahn et al., 2024). evidence: epidemiological studies link exposure to silica dust and organic solvents to an increased risk of sle, ra, and systemic sclerosis. studies show that pm2.5 exposure is associated with altered dna methylation in inflammatory genes. 5. translational implications and future directions the profound understanding of epigenetic dysregulation in immune-mediated diseases is rapidly moving from bench to bedside, offering unprecedented opportunities for improving patient care. the dynamic and reversible nature of epigenetic marks positions them as ideal targets for novel diagnostic strategies and therapeutic interventions, heralding a new era of precision medicine in autoimmunity. 5.1 epigenetic biomarkers the quest for specific, sensitive, and noninvasive biomarkers is central to improving outcomes in imids. epigenetic marks, stable in biofluids and reflective of dynamic disease activity, hold immense promise. ajibola and ayodele; asian j. immunol., vol. 8, no. 1, pp. 230-241, 2025; article no.aji.144830 237 table 2. epigenetic and mirna biomarkers for diagnosis, monitoring, prognosis, and treatment response in sle and ra application example potential utility diagnosis specific mirna panel (e.g., mir-146a, mir-155) in serum for sle earlier and more accurate diagnosis, especially in seronegative patients disease monitoring global dna methylation levels in pbmcs in sle; cxcl12 methylation in ra objective measure of disease activity; predict flare-ups prognosis methylation status of apoptosis-related genes in ra-fls predict severity of joint destruction and disease course treatment response pre-treatment mir-125b levels predict response to rituximab in ra or sle diagnosis and differential diagnosis: dna methylation signatures and circulating mirna profiles can distinguish patients with a specific imid from healthy controls and, crucially, from those with other clinically similar conditions. for instance, a specific methylation signature in synovial tissue or peripheral blood can differentiate rheumatoid arthritis from other forms of inflammatory arthritis (e.g., psoriatic arthritis) with higher accuracy than current serological tests (proaño et al., 2025) monitoring disease activity and predicting flares: unlike static genetic risk alleles, epigenetic marks change with disease state. serial analysis of circulating cell-free dna (cfdna) methylation patterns or mirna levels in serum can provide a real-time "epigenetic snapshot" of disease activity, potentially predicting impending flares before clinical symptoms manifest. this allows for pre-emptive treatment adjustments (liu et al., 2024). prognosis and treatment response: epigenetic profiles may predict disease aggressiveness and likelihood of developing extra-articular manifestations. furthermore, baseline epigenetic markers could forecast response to specific therapies (e.g., predicting non-response to tnf inhibitors), enabling a more personalized and efficient treatment approach from the outset (shaikh et al., 2024). 5.2 epigenetic therapy the concept of pharmacologically reversing aberrant epigenetic marks to restore normal gene expression is a groundbreaking therapeutic strategy. while most epigenetic drugs are currently used in oncology, their repurposing for autoimmunity is actively being explored. dnmt inhibitors (dnmti): drugs like azacitidine and decitabine are approved for myelodysplastic syndromes. their use in autoimmunity is paradoxical; while they can reverse pathological hypermethylation of silenced genes, their primary effect is global hypomethylation, which could theoretically exacerbate diseases like sle. the future lies in developing targeted delivery systems (e.g., antibody-drug conjugates) to specific cell types or employing low-dose regimens to achieve gene-specific rather than global effects (wen et al., 2025). hdac inhibitors (hdaci): this class has shown greater immediate promise. pan-hdac inhibitors like vorinostat and givinostat have demonstrated efficacy in preclinical models of ra, sle, and ms by suppressing proinflammatory cytokine production and promoting treg function. more selective hdac inhibitors (e.g., targeting hdac6, hdac11) are in development to enhance efficacy and reduce offtarget effects (. notably, the scfa butyrate is a natural hdaci, providing a strong rationale for dietary and microbiome-based interventions (yue et al., 2025). bromodomain and extra-terminal (bet) inhibitors: these compounds disrupt the reading of histone acetylation marks by bet proteins. they have potent anti-inflammatory effects by downregulating key inflammatory genes (e.g., nfkb, il6) and have shown efficacy in multiple animal models of imids (khokhar et al., 2024). mirna-based therapeutics: this approach offers exquisite specificity. mimics (to restore levels of deficient mirnas like mir-146a) and antagomirs or locked nucleic acids (lnas) (to silence overexpressed mirnas like mir-155) are in various stages of preclinical and early clinical development. the major challenge remains the efficient and targeted delivery of these oligonucleotides to relevant immune cells in vivo (bannazadeh baghi et al., 2024). ajibola and ayodele; asian j. immunol., vol. 8, no. 1, pp. 230-241, 2025; article no.aji.144830 238 6. conclusion the investigation into epigenetic dysregulation has fundamentally reshaped our understanding of immune-mediated diseases. no longer viewed as disorders governed solely by a deterministic genetic code, imids are now recognized as conditions where dynamic epigenetic mechanisms interpret genetic susceptibility through the lens of environmental exposure. this review has synthesized compelling evidence that establishes aberrant dna methylation, histone modifications, and non-coding rna expression not as mere epiphenomena, but as central drivers of pathogenesis. these mechanisms directly mediate the loss of immune tolerance, hyperactivation of inflammatory pathways, and tissue damage characteristic of diseases like sle, ra, ms, and ibd. the environmental dimension is particularly pivotal, with factors such as tobacco smoke, viral infections (notably ebv), diet, and the gut microbiome acting as powerful epigenetic modifiers. these triggers can induce stable, heritable changes in gene expression that lower the threshold for autoimmunity, effectively encoding the memory of environmental insults within the immune system and explaining the notable discordance in genetically identical twins. this profound mechanistic insight unlocks immense translational potential. the dynamic nature of the epigenome offers a unique opportunity to develop novel epigenetic biomarkers for early diagnosis, precise prognosis, and real-time monitoring of disease activity, moving beyond the limitations of current serological and clinical markers. furthermore, the reversibility of epigenetic marks paves the way for a revolutionary therapeutic strategy: epigenetic therapy. the repurposing and refinement of drugs targeting dnmts, hdacs, bet proteins, and specific mirnas hold the promise of not just suppressing inflammation but potentially restoring immunological balance and inducing long-term remission by reversing the root epigenetic dysfunction. however, the path forward is not without challenges. the field must overcome hurdles related to the cell-type specificity of interventions, the complex crosstalk within the epigenome, and the long-term safety of modulating these fundamental regulatory systems. future research must focus on large-scale integrative multi-omics studies to validate biomarkers and on developing novel targeted delivery systems to enhance the precision of epigenetic drugs. in conclusion, the study of epigenetics has provided the missing link between genes and environment in immune-mediated diseases. it offers a more complete and nuanced disease model and, most importantly, illuminates a promising path toward personalized medicine. by reading and rewriting the epigenetic code that goes awry in autoimmunity, we are poised to develop more effective strategies for prediction, prevention, and treatment, ultimately aiming to restore the delicate balance of the immune system and improve the lives of millions of patients worldwide. consent it is not applicable. ethical approval it is not applicable. disclaimer (artificial intelligence) author(s) hereby declare that no generative ai technologies such as large language models (chatgpt, copilot, etc.) and text-to-image generators have been used during the writing or editing of this manuscript. competing interests authors have declared that no competing interests exist. references antonazzo, g., gaudet, p., lovering, r. c., & attrill, h. 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(2024). epstein–barr viruses: their immune evasion strategies and implications for autoimmune diseases. international journal of molecular sciences, 25(15), 8160. disclaimer/publisher’s note: the statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of the publisher and/or the editor(s). this publisher and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. _________________________________________________________________________________ © copyright (2025): author(s). the licensee is the journal publisher. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: https://pr.sdiarticle5.com/review-history/144830 https://pr.sdiarticle5.com/review-history/144830 _____________________________________________________________________________________________________ *corresponding author: e-mail: augustineairaodion@yahoo.com; asian journal of immunology 5(1): 40-51, 2022; article no.aji.85085 effect of ethanol extract of xylopia aethiopica fruit on oxidative stress indices of wistar rats emmanuel o. ogbuagu a, uloaku ogbuagu b, augustine i. airaodion b*, chika l. uche c, edmund o. ezirim d, ifeoma n. nweke a and prince c. unekwee a department of pharmacology and therapeutics, abia state university, uturu, nigeria. b department of biochemistry, federal university of technology, owerri, imo state, nigeria. c department of haematology, abia state university, uturu, nigeria. d department of obstetrics and gynecology, abia state university, uturu, nigeria. e department of pharmacology and therapeutics, nnamdi azikiwe university, awka anambra state, nigeria. authors’ contributions this work was carried out in collaboration among all authors. all authors read and approved the final manuscript. article information open peer review history: this journal follows the advanced open peer review policy. identity of the reviewers, editor(s) and additional reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here: https://www.sdiarticle5.com/review-history/85085 received 17 january 2022 accepted 21 march 2022 published 29 march 2022 abstract background: the use of xylopia aethiopica fruit in folklore medicine is on the increase without caution of its toxicity. aim: this present study tends to assess its effect on the oxidative stress biomarkers of wistar rats. methodology: the fruits of xylopia aethiopica were air-dried and extracted by soxhlet extractor using ethanol as solvent. the median lethal dose (ld50) of the extract was assessed using standard method. thirty adult wistar rats were divided into five groups of six rats each. animals in groups 1, 2, 3, and 4 were treated with 130, 259, 389 and 518 mg/kg body weight of x. aethiopica fruit extract respectively, while those in group 5 received normal animal feeds and water only. the administration was done once daily for 28 days via oral route. oxidative stress indices were measured using standard methods. results: a significant decline was observed in the antioxidant enzymes (catalase, superoxide dismutase, and glutathione peroxidase) activities in experimental animals compared with those in the control group (p<0.05). in the same vein, a significant reduction was observed in the concentration of reduced glutathione in experimental animals compared with those in the control original research article ogbuagu et al.; aji, 5(1): 40-51, 2022; article no.aji.85085 41 group (p<0.05). lipid peroxidation was however observed to increase when experimental animals were compared with those in the control group. the elevation in lipid peroxidation was significantly different when animals treated with higher doses of 259, 389 and 518 mg/kg body weight of extract were compared with those in the control group (p<0.05). conclusion: the adverse perturbation of antioxidant indices by xylopia aethiopica fruit is suggestive that it could induce oxidative stress and thus unhinged the immune system. oxidative stress has also been implicated in several diseases thus the consumption of xylopia aethiopica fruit as well as its use in folklore medicine should be discouraged especially in high doses due to its toxic nature. keywords: immune system; oxidative stress; toxic nature; xylopia aethiopica fruit. 1. introduction induction of oxidative stress elicits free radicals such as nitric oxide (no), superoxides anions (o2 ), hydroxyl radical (oh ), hydrogen peroxide (h2o2), organic hydroperoxide (rooh) via the release of reactive oxygen species (ros) [1]. these ros are highly fickle atoms because they are known to contain a lone pair of electrons in their outermost shells. they are known to be involved in processes like aging, carcinogenesis, mutagenesis, and so on, which is caused by their in-built ability to induce cellular obstruction on dna [2]. some operations of human elicit ros both endogenously and exogenously, which result in enervating some disease conditions. these diseases could arise from the adverse correlation in the induction of oxidative stress and the tendency of the living system to alleviate the free radicals generated sequel to the induction of stress [3]. worthy of note is the point that the living system is built to alleviate the destructive nature of the radicals elicited via the natural antioxidants that are enzymatic in nature [4]. however, synthetic antioxidants also exist which are absorbed by the body to elevate the activities of the natural ones but have been observed to have health-related risk [2]. this has resulted in the renewal of vigor in the search for antioxidants from plant sources which will work well with the living system and also increase the ability of natural antioxidants with the purpose of preventing the health issues ascribed to the synthetics antioxidants [5]. xylopia aethiopica has a great patronage in both nutrition and ethnomedicine. the plant which also known as african negro pepper, is popular among traditional medicine practitioners and traditional birth attendants (tba) who utilize the fruit preparations to cause the discharge of placental after a woman has giving birth [6]. a preparation of the stem bark or fruit is helpful in the management of bronchitis, stomach aches, asthma, and dysenteric conditions [6]. the seed extract is helpful as a vermifuge for roundworms [7]. several postnatal women eat the aqueous preparation of the fruit for its perceived antiseptic properties. some of the women have been reported to sometimes come to the hospitals with characteristics which suggest complications in organ [8]. medicinal plant extracts with a therapeutic property has the tendency of wrong prescription and sometimes, overdosed. the fact that xylopia aethiopica is a natural product does not automatically confers on it safety and might be risky to its consumers. chemical ingredients of the plant are perceived to be useful in preventing and managing cancerous tumors [9]. xylopia aethiopica fruit is known to have alkaloids, terpenoids, flavonoids, and organic oils [10,11]. xylopia aethiopica is characterized with numerous chemical components with various medicinal potentials [13]. the chemical components of this plant have been investigated to include saponins, sterols, carbohydrates, glycosides, mucilage, acidic compounds, tannins, balsams, cardiac glycosides, volatile aromatic oils, phenols [8,14,15], alkaloids, rutin and fixed oils [16,17]. the plant has also be known to contain vitamins such as vitamin a, vitamin b, vitamin c, vitamin d, and vitamin e, and proteins as well as several minerals such as copper, manganese and zinc [15,17]. the impact of the fruit on body weight and glucose concentration of animals has been reported [18]. the fruit has also been reported to induce dyslipidemia [19], hepatotoxicity [20] as well as renal toxicity [21]. this present study focused on examining its impact on the oxidative stress biomarkers of wistar rats. ogbuagu et al.; aji, 5(1): 40-51, 2022; article no.aji.85085 42 fig. 1. xylopia aethiopica fruit [12] 2. materials and methods 2.1 collection and authentication of plant materials the fruits of xylopia aethiopica were sourced from a market in aba, abia state. they were identified and authenticated by prof. margaret bassey of botany and ecological studies department, university of uyo. it was assigned a voucher number of uu/ph/4e and deposited in the herbarium of the department of pharmacognosy and natural medicine, university of uyo, akwa-ibom state, nigeria. 2.2 extraction of plant materials extraction of the plant was carried out in the post-graduate laboratory of department of pharmacognosy and natural medicine, faculty of pharmacy, university of uyo, nigeria. it was extracted based on the outlined method in ogbuagu et al. [13]. the fruits were rinsed under flowing tap water to eliminate contaminants and air-dried. the plant material was milled by laboratory blender. the pulverized plant material was macerated in 250 ml of 99.8% ethanol (sigma aldrich) contained in a flask attached to a soxhlet extractor coupled with condenser and heating mantle (isomantle). it was then poured into the sample holder (thimble) and inserted in the apparatus. the side arm is lagged with glass wool. the mixture was heated using the heating mantle (isomantle) at 60 °c and as the temperature rises it starts to evaporate, going via the extractor to the condenser. the condensate dripped into the reservoir housing the thimble. as soon as the solvent gets to the siphon it emptied itself into the flask and the process repeats itself. the process goes on until it is exhaustively extracted. the process runs for a total of 13 hours. as soon as it was set up, it was allowed to run without interruption as long as water and power supply were not interrupted. the apparatus was switched on and off and overnight running was not allowed, and the time for the complete process split over some days. the extract was poured into 1000 ml beaker and concentrated to dryness in water bath (a3672 graffin student water bath) at 35 °c. the total weight of the marc (residue) and the concentrated extract were noted. several days was spent on the entire process. the evaporated extract was kept in the refrigerator until when the need for it arise. 2.3 determination of median lethal dose (ld50) the median lethal dose (ld50) of the extract was determined using albino mice according to the method described by airaodion et al. [22]. this method involves two phases: in phase one, five groups containing five mice each weighing between 20 g and 27g were fasted for 18 hours. they were respectively treated with 1000 mg/kg, 2000 mg/kg, 3000 mg/kg, 4000 mg/kg and 5000 mg/kg body weight via intraperitoneal (i.p) route and were monitored for visible signs of toxicity and mortality for 24 hours. a dosage of 1000 mg/kg recorded 0% mortality while 2000 mg/kg, 3000 mg/kg 4000 mg/kg and 5000 mg/kg recorded 100% mortality within 24 hours. based on the value of phase one, phase two was conducted. ogbuagu et al.; aji, 5(1): 40-51, 2022; article no.aji.85085 43 in phase two, twenty-five albino mice weighing between 20 and 27g were grouped into 5 of 5 mice per group and were fasted for 18 hours. each group was administered 1200 mg/kg, 1400 mg/kg 1600 mg/kg, 1800 mg/kg and 2000 mg/kg body weight intraperitoneally (i.p) and was observed for physical signs of toxicity and mortality within 24 hours. 1200 mg/kg recorded 0% mortality while 1400 mg/kg, 1600 mg/kg, 1800 mg/kg and 2000 mg/kg recorded 100% mortality within 24 hours. the ld50 was computed as geometrical means of the maximum dose yielding 0% mortality (a) and the minimum dose yielding 100% death (b). ld50 = ab 2.4 experimental design thirty adult wistar rats used in this study were purchased from the university of uyo, nigeria. they were allowed to acclimatize for seven days prior to the start of the treatment. the weights were determined and were separated into five groups of six rats each. groups a, b, c, d served as the experimental groups, while group e served as the control. animals in group a were exposed to 130 mg/kg body weight (10% of ld50) of x. aethiopica fruit extract, those in group b were treated with 259 mg/kg body weight (20% of ld50) of x. aethiopica fruit extract, those in group c were exposed to 389 mg/kg body weight (30% of ld50) of x. aethiopica fruit extract, those in group d were treated with 518 mg/kg body weight (40% of ld50) of x. aethiopica fruit extract, while those in group e (control) received normal animals feeds and water only. the treatment was done once daily for 28 days via oral route. after 28 days treatment, the animals were sacrificed under ether anaesthesia in a desiccator after an overnight fast. blood was taken from the rats through cardiac puncture. 2.5 determination of oxidative stress indices 2.5.1 determination of superoxide dismutase (sod) activity principle: this procedure involves production of superoxide radical of riboflavin and its detection by hydroxylamine hydrochloride. the nitrite reacts with sulphanilic acid to yield diazonium compound which then reacts with naphthylamine to yield red azo compound whose absorbance is measured at 543 nm. this assay was done according to the method of mohammad et al. [23] in which 1.4 ml aliquot of the reaction mixture involved 1.1 ml of 50 mm phosphate buffer (ph 7.4), 0.075 ml of 20 mml -1 methionine, 0.4 ml of 1% (v/v) triton x-100, 0.075 ml of 10 mm hydroxylamine and 0.1 ml of 50 mm edta. the aliquot (1.75 ml) was added to 0.1 ml of the sample and incubated at 30 o c for 15 min. this was followed by addition of 80 µl of 50 µm riboflavin and then the tubes were exposed for 9 minutes to 200 watts philip lamp. after exposure time, 1 ml of greiss reagent was added and the absorbance of the colour formed was measured at 543 nm. one unit of enzyme activity was measured as the amount of sod capable of inhibiting 50 % of nitrate formation under the assay condition. 2.5.2 determination of catalase (cat) activity principle: the method is based on the fact that dichromate in acetic acid is reduced to chromic acetate when heated in the presence of h2o2 with the formation of perchloric acid as unstable intermediate. the acetate produced is measured colorimetrically at 610 nm. this was assayed by the method described by sinha [24]. the reaction mixture (1.5 ml) contained 1.0 ml of 0.01 m phosphate buffer, 0.1 ml of the sample and 0.4 ml of 2 m h2o2. the reaction was stopped by the addition of 2.0 ml dichromate acetic acid reagent (5% potassium dichromate and glacial acetic acid mixed in 1:3 ratio). then, the absorbance was measured at 610 nm. cat activity was expressed as µmol of h2o2 consumed/min/mg protein. 2.5.3 determination of glutathione peroxidase (gpx) activity principle: the activity of gpx was determined by measuring the decrease in gsh concentration after incubating the sample in the presence of hydrogen peroxide and sodium azide. h2o2 + 2gsh 2h2o + gssg the activity of glutathione peroxidase was assayed by the method described by rotruck et al. [25]. the reaction mixture contained 0.2 ml of 0.4 m trisbuffer, ph 7.0, 0.2 ml of edta, and 0.1 ml of 10 mm sodium azide, 0.2 ml of 10 mm glutathione and 0.1 ml of 0.2 mm. h2o2. the content was incubated at 37 o c for 10 minutes. the reaction was terminated by the addition of 0.4 ml 10% (v/v) tca and centrifuged at 5000 ogbuagu et al.; aji, 5(1): 40-51, 2022; article no.aji.85085 44 rpm for 5 minutes. the supernatant was assayed for glutathione by ellman’s method. exactly 3.0 ml disodium hydrogen phosphate solution and 1.0 ml of dntb reagent were added to 2.0 ml of the supernatant. the standard was taken and treated in similar manner. the absorbance was read at 412 nm and expressed in terms of glutathione consumed /min/mg protein. 2.5.4 determination of lipid peroxidation principles: lipid peroxidation in the supernatant fractions was determined spectrophotometrically by assessing the concentration of thiobarbituric acid reactive substances (tbars) as described by varshney and kale [26]. the results were expressed in malondialdehyde (mda) formed relative to an extinction coefficient of 1.56 x 10 6 mol/cm. procedure: acetic acid 1.5 ml (20%; ph 3.5), 1.5 ml of 0.8% thiobarbituric acid and 0.2 ml of 8.1% sodium dodecylsulphate was added to 0.1 ml of the sample and heated at 100 o c for 60 min. after centrifugation at 1200×g for 10 min, the organic layer was separated and absorbance measured at 532nm using a spectrophotometer. malondialdehyde (mda) is an end product of lipid peroxidation, which reacts with thiobarbituric acid to form pink chromogen–thiobarbituric acid reactive substance. it was calculated using a molar extinction coefficient of 1.56×10 6 mol/cm and expressed as nanomoles of mda/tissue. the concentration of mda (nmol/ml) was calculated by using the formula: concentration of the test = 2.5.5 determination of glutathione concentration principles: glutathione (reduced) was measured according to the method of jollow et al. [27]. reduced glutathione (gsh) forms the bulk of non-protein sulfhydryl groups. this method is based on the formation of relatively stable yellow colour when ellman’s reagent is added to a sulfhydryl compound, 2-nitro-5-thiobenzoic acid, the chromophoric product resulting from the reaction of ellman’s reagent with reduced glutathione. procedure: equal quantities of the sample and 10% trichloroacetic acid were mixed and centrifuged at 4000 x g for 15 minutes to separate the proteins. to 0.5 ml of the supernatant, 4.5 ml of ellman’s reagent was added. the mixture was vortexed and the absorbance read at 412 nm within 15 min. 2.6 statistical analysis data were subjected to analysis of variance using graph pad prism. results were presented as mean ± standard error of the mean (sem). one-way analysis of variance (anova) was used to compare the mean, followed by tukey's post hoc test. differences between means were considered to be significant at p<0.05. 3. results 3.1 median lethal dose (ld50) result the visible signs of toxicity of x. aethiopica fruit extract observed in this study are excitation, decreased motor activity, paw licking, increased respiratory rate, gasping and coma which could be followed up by death. in the first phase of the median lethal dose determination, no death was observed in the group administered 1000 mg/kg body weight of x. aethiopica fruit extract. however, all the animals died in the groups exposed to 2000, 3000, 4000, and 5000 mg/kg body weight of x. aethiopica fruit extract respectively (table 1). in the same vein, in the second phase of medial lethal dose determination, no death was recorded in the group treated with 1200 mg/kg body weight of x. aethiopica fruit extract while 100% mortality was recorded in the groups treated with 1400, 1600, and 1800 mg/kg body weight of x. aethiopica fruit extract respectively as presented in table 1. the median lethal dose (ld50) was computed as geometrical average of the maximum dose yielding 0% death (a) and the minimum dose yielding 100% death (b). ld50 = where a = 1200 mg/kg b = 1400 mg/kg ld50= 1296.15 mg/kg 3.2 effect of extracts of xylopia aethiopica fruit on oxidative stress indices of animals after 28 days of treatment the impact of extract of xylopia aethiopica fruit extract on oxidative stress parameters of animals ogbuagu et al.; aji, 5(1): 40-51, 2022; article no.aji.85085 45 after 28 days of treatment is presented in figs. 26. a significant reduction was seen in the activities of antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase) in experimental animals compared with those in the control group (p<0.05). in the same vein, a noticeable reduction was seen in the concentration of reduced glutathione in experimental animals compared with those in the control group (p<0.05). lipid peroxidation was however observed to increase when experimental animals were compared with those in the control group. the elevation in lipid peroxidation was significantly different when animals treated with higher doses of 259, 389 and 518 mg/kg body weight of extract were compared with those in the control group (p<0.05). table 1. the median lethal dose (ld50) of xylopia aethiopica fruit extract study phase/ (animal) dosage of extract (mg/kg) b.w no of mice per group no. of death recorded % mortality phase one i 1000 5 0 0 ii 2000 5 5 100 iii 3000 5 5 100 iv 4000 5 5 100 v 5000 5 5 100 phase two i 1200 5 0 0 ii 1400 5 5 100 iii 1600 5 5 100 iv 1800 5 5 100 v 2000 5 5 100 ld50= 1296.15 mg/kg 13 0 m g/k g x . a et hio pic a 25 9 m g/k g x . a et hio pic a 38 9 m g/k g x . a et hio pic a 51 8 m g/k g x . a et hio pic a c ontr ol 0 1 2 3 treatment groups a c ti v it y o f s o d ( u /m l ) fig. 2. effect of x. aethiopica fruit extract on the activity of superoxide dismutase (sod) in animals after 28 days of treatment each bar represent mean ± sd of treatment groups with n = 6 ogbuagu et al.; aji, 5(1): 40-51, 2022; article no.aji.85085 46 13 0 m g/k g x . a et hio pic a 25 9 m g/k g x . a et hio pic a 38 9 m g/k g x . a et hio pic a 51 8 m g/k g x . a et hio pic a c ontr ol 0 1 2 3 4 5 treatment groups a c ti v it y o f c a ta la s e ( u /m l ) fig. 3. effect of x. aethiopica fruit extract on the activity of catalase in animals after 28 days of treatment each bar represent mean ± sd of treatment groups with n = 6 13 0 m g/k g x . a et hio pic a 25 9 m g/k g x . a et hio pic a 38 9 m g/k g x . a et hio pic a 51 8 m g/k g x . a et hio pic a c ontr ol 0 1 2 3 4 treatment groups a c ti v it y o f g p x ( u /m l ) fig. 4. effect of x. aethiopica fruit extract on the activity of glutathione peroxidase (gpx) in animals after 28 days of treatment each bar represent mean ± sd of treatment groups with n = 6 ogbuagu et al.; aji, 5(1): 40-51, 2022; article no.aji.85085 47 13 0 m g/k g x . a et hio pic a 25 9 m g/k g x . a et hio pic a 38 9 m g/k g x . a et hio pic a 51 8 m g/k g x . a et hio pic a c ontr ol 0 1 2 3 4 5 treatment groups c o n c . o f g s h ( m m ) fig. 5. effect of x. aethiopica fruit extract on the concentration of reduced glutathione (gsh) in animals after 28 days of treatment each bar represent mean ± sd of treatment groups with n = 6 13 0 m g/k g x . a et hio pic a 25 9 m g/k g x . a et hio pic a 38 9 m g/k g x . a et hio pic a 51 8 m g/k g x . a et hio pic a c ontr ol 0 2 4 6 8 treatment groups c o n c . o f l p o ( u m ) fig. 6. effect of x. aethiopica fruit extract on lipid peroxidation of animals after 28 days of treatment each bar represent mean ± sd of treatment groups with n = 6 ogbuagu et al.; aji, 5(1): 40-51, 2022; article no.aji.85085 48 4. discussion the acute toxicity study of x. aethiopica fruit extracts led to 100% death at a dose of 1400 mg/kg bodyweight and above. this reveals that this fruit could be greatly toxic. the visible symptoms of toxicity seen in the animals were excitation, paw licking, increased respiratory rate, decreased motor activity, gasping and coma and death. in this study, a noticeable decrease was recorded in superoxide dismutase (sod) activity in animals exposed to xylopia aethiopica fruit extract at all tested doses when compared with control animals at p<0.05 (fig. 2). this suggested that xylopia aethiopica fruit extract might be toxic and could possibly play a role in the production of free radicals. this agreed with the results of nnodim et al. [28] who observed that xylopia aethiopica fruit induced oxidative stress. observation from this study showed that treatment with ethanol extract of xylopia aethiopica fruit was observed to greatly reduce the activity of catalase in animals treated with 259, 389 and 518 mg/kg of xylopia aethiopica fruit extract when compared to animals in the control group at p<0.05 (fig. 3). this might be an indication that xylopia aethiopica fruit extract at these doses might be toxic and possibly lead to induction of oxidative stress. this agreed with the findings of nnodim et al. [28], who reported that xylopia aethiopica fruit generated free radicals in treated animals. treatment with extract of xylopia aethiopica fruit significantly down-regulated glutathione peroxidase (gpx) activity at all tested doses (fig 4). the biological role of glutathione peroxidase is to change hydroperoxides of lipid to their respective alcohols and release hydrogen peroxide to form water [29,30]. this is in consonance with the results of nnodim et al. [28], who reported that xylopia aethiopica fruit significantly reduced the activity of glutathione peroxidase of treated animals. glutathione peroxidase is one of the enzymatic antioxidant with the capacity to defend the system and combat oxidative stress [31]. the reduction in the activity of glutathione peroxidase observed in this study might be an indication that xylopia aethiopica fruit has the propensity to induce oxidative stress. in this study, administration of ethanol extract of xylopia aethiopica fruit resulted in a decline in the concentration of reduced glutathione at all doses when compared with control group (fig. 5). the noticeable decrease observed in the reduced glutathione level in this study might be an indication that xylopia aethiopica fruit enhances the conjugation of gsh with acetaldehyde [32]. this may have resulted from the direct reactive oxygen species (ros) producing potential of xylopia aethiopica fruit and/or a decrease in gsh synthesis. in this study, administration of xylopia aethiopica fruit extract was seen to have noticeably led to a surge in the malondialdehyde (mda) levels indicating increased peroxidation and catabolism of the antioxidant defense mechanisms. malondialdehyde is a product of lipid peroxidation of polyunsaturated fatty acids [33,34]. furthermore, visible destruction of tissues in lipid peroxidation caused by free radicals could cause membrane injury followed by decrease in the membrane fluid content. this is in line with the study of nnodim et al. [28], who observed that xylopia aethiopica fruit significantly elevated lipid peroxidation (fig. 6). the depletion in antioxidant enzymes activities seen in this study agrees with the work of somnez et al. [35]. the biochemical mechanism by which xylopia aethiopica caused reduction in enzymatic antioxidants is currently unclear. it might be postulated that the consumption of this plant extract could cause oxidative stress and hence generate free radicals which could result in membrane destruction through lipid peroxidation and protein oxidation [36-38]. thus, the reduction in the activities of superoxide dismutase (sod), catalase (cat), as well as glutathione peroxidase (gpx), elevated lipid peroxidation. this became visible by the increased level of mda observed in this present investigation. 5. conclusion the adverse perturbation of antioxidant indices by xylopia aethiopica fruit is suggestive that it could induce oxidative stress and thus negatively impact the immune system. oxidative stress has also been implicated in different diseases, thus the consumption of xylopia aethiopica fruit as well as its use in folklore medicine should be discouraged especially in high doses due to its toxic nature. ogbuagu et al.; 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article no.aji.127402 a review on vadadustat: hope in treatment of anemia patients having chronic kidney disease nikita gupta a, mohd naveed khan a*, varda farooqui a, guntoju poojitha a and thipparam kavya a a department of pharmacy practice, st. pauls college of pharmacy, hyderabad, telangana-510, india. authors’ contributions this work was carried out in collaboration among all authors. all authors read and approved the final manuscript. article information doi: https://doi.org/10.9734/aji/2024/v7i1153 open peer review history: this journal follows the advanced open peer review policy. identity of the reviewers, editor(s) and additional reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here: https://www.sdiarticle5.com/review-history/127402 received: 09/10/2024 accepted: 12/12/2024 published: 21/12/2024 abstract vadadustat, a hypoxia-inducible factor prolyl hydroxylase inhibitor, may offer an oral alternative to injectable erythropoiesis-stimulating agents (esas) to treat anemia in patients undergoing peritoneal dialysis. current standard of care for anemia linked to chronic kidney disease (ckd) is the use of esas. targeting the hifs-the basic elements of rbc production-presents an oral alternative to the standard esas. due to their low oxygen environment, hif transcription factors are, by nature, constitutively activated at high altitude, which enhances iron mobilization and boosts production of endogenous erythropoietin (epo). while clinical trials have demonstrated safety and efficacy for vadadustat, whether it will promote cancer is still a topic under investigation. in fact, research has suggested that activation of hifs promotes tumor growth due to stimulation of review article https://doi.org/10.9734/aji/2024/v7i1153 https://www.sdiarticle5.com/review-history/127402 gupta et al.; asian j. immunol., vol. 7, no. 1, pp. 302-315, 2024; article no.aji.127402 303 angiogenesis through vascular endothelial growth factor (vegf). the hif system includes two subunits, α and β. under low-oxygen conditions, the hif-1α subunit accumulates and translocates to the cell nucleus, where it binds to hif-β to form the heterodimer hif-1αβ. this complex initiates the expression of sensitive hypoxic genes, including the epo gene, whose production is increased under such conditions. three isoforms of the hif-α subunit exist: hif-1α, hif-2α, and hif-3α, all of which can dimerize with hif-β and activate the transcription of other genes besides epo. in the intestine, duodenal cytochrome b (dcytb) reduces ferric iron (fe3+) to ferrous iron (fe2+), which is then taken into enterocytes by the divalent metal transporter-1 (dmt1). hif-2 controls both dcytb and dmt1. iron is exported from cells by ferroportin (fpn), and this export is repressed by hepcidin but stimulated by hif. in blood, iron is transported in a complex with transferrin (tf) to the liver, res cells, bone marrow, and other tissues. the increased erythropoietic activity in the marrow leads to the production of growth differentiation factor 15 (gdf15) and erythroferrone, which are known to inhibit hepcidin in liver cells. the inflammatory cytokine relates with increased hepcidin production in the liver, whose action is mediated by decreased ferroportin expression on the cell surface and lower blood iron levels. vadadustat has hif stabilized for the promotion of the secretion of epo but does not seem to have an effect on the production of vegf. it is also reported that increased activity of hif results in an antitumor effect. results from clinical trials support the use of vadadustat without genotoxicity, facilitating the treatment of anemia in patients with chronic kidney disease. keywords: vadadustat; anaemia; chronic kidney disease; hypoxia-inducible factor; erythropoiesis stimulating agents. 1. introduction 1.1 vadadustat vadadustat is an experimental oral inhibitor of hypoxia-inducible factor prolyl-hydroxylase, which stimulates the natural production of erythropoietin. currently, erythropoiesisstimulating agents are the first-line treatment for anemia associated with chronic kidney disease. all esas effectively treat anemia caused by ckd as they compensate for the deficit of epo produced by failing kidneys. hif-phis have been differentiated from esas by the fact that they activate the hif pathway and result in downstream effects on the epo gene transcription, expression of genes involved in erythropoiesis and iron metabolism. the conventional management of anemia in the ckd patient is intravenous esa, either as monotherapy or with iv or oral iron supplementation. although esas are highly effective for many patients with ckd, they do come with significant limitations. many ckd patients have a diminished ability to produce epo and absorb and mobilize iron. the production of hb is largely dependent on the supply of iron, which is controlled by both the liver, the kidneys, and the bone marrow through synchronized signals. normally, epo stimulates erythroblasts to produce erythroferrone, a protein that acts to inhibit the production of hepcidin in the liver [1]. “hypoxia-inducible factor is a transcription factor that controls the expression of many low oxygenregulated genes, including epo, which is an important stimulator of erythropoiesis. hif levels are controlled by oxygen-dependent degradation at the proteasome, a process mediated by a family of prolyl hydroxylases that serve as oxygen sensors. hif-prolyl hydroxylase inhibitors or hif-phis are novel drugs that stabilize hif and promote the expression of epo and may correct anemia related to ckd. on the other hand, because initiation of hif and its downstream genes results in a global response to hypoxia, there is, in theory, a potential risk for adverse events including neoplasia due to activation of the hif pathway. the oxygen dependent hydroxylation of hif-α is catalyzed by phd1, phd2, and phd3 that function as oxygen sensors within the hif pathway. phds are part of a large family of 2og-dependent dioxygenases, with more than 60 members. dioxygenases catalyze the hydroxylation reaction by using molecular oxygen, thereby linking oxygen, intermediary metabolism, and amino acid metabolism to a variety of cellular functions, including hif regulation, hypoxia responses, collagen synthesis, epigenetic gene regulation, and fatty acid metabolism. systemic activation of hif reduces hepcidin production in the liver, thereby improving iron uptake and mobilization. the anemia of ckd is caused by a relative lack of renal production of epo, together with functional or absolute iron deficiency and gupta et al.; asian j. immunol., vol. 7, no. 1, pp. 302-315, 2024; article no.aji.127402 304 resistance to epo signaling, often in the context of inflammation. activation of hif signaling by pharmacological agonists may provide a more physiologic and holistic treatment strategy for renal anemia than administration of recombinant epo alone. while initial reports suggested a direct role for hif-1 in suppressing the transcription of hepcidin, subsequent mouse models with global or liver-specific hif activation showed that suppression of hepcidin depends upon epoinduced stimulation of erythropoiesis. which would argue that hif itself does not play a role in suppressing hepcidin transcription [2]. 1.2 anemia anemia is the most common disorder diagnosed in most patients with chronic kidney disease. it could significantly affect a patient's quality of life if left unappropriately managed. there are multiple reasons for anemia in this patient population. in addition to drugs and dietary restrictions, patients may develop anemia due to iron deficiency resulting from a decrease in the renal capability. this leads to lowering the amount of iron available for the bone marrow to produce various blood elements. chronic patients with kidney diseases can hardly utilize their body's iron stores, and many of them, especially those who are hemodialysed, develop an increased need for additional iron therapy which has been primarily delivered by infusions. “the erythropoietic system maintains the balance in the supply of red blood cells, therefore, ensuring an adequate tissue oxygenation4. in order to maintain this balance, senescent erythrocytes are replaced by new cells. hypoxia is important in stimulating erythrocyte production through its interaction with the hif (hypoxia-inducible factor) system. the hif is a heterodimer, consisting of two subunits: alpha and beta. the production of hifalpha continues, but its degradation occurs in the absence of tissue hypoxia. on the opposite situation, alpha and beta subunits join and bind in the nucleus of the cell, a dna sequence called hypoxia-responsive elements. thus, the production of erythropoietin is stimulated” [3]. erythropoietin, on the other hand, is a molecule of 165 amino acids and 4 chains of carbohydrates. mainly produced in the interstitial cells of the renal cortex, the production of the liver significantly increases with reduction in glomerular filtration. the half-life of erythropoietin is 5 to 12 hours, and it acts like a true hormone that binds to the receptors of bone marrow cells to produce erythrocytes. “although reduction in erythropoietin production is one of the main causes of anemia in ckd, other causes include iron deficiency. it is assumed that iron loss varies between 1 and 3 gram per year in patients on hemodialysis. also, in the absence of dialysis, depletion in iron is observed in most patients4. this might be related to frequent phlebotomies, blood loss in the hemodialysis apparatus, and impairment in its absorption. the initiation of treatment with erythropoietin analogs demonstrated how frequent iron deficiency is among ckd patients” [4]. “iron deficiency is usually a functional deficiency and is often characterized by low tsi and normal or increased ferritin. ferritin can be raised in the presence of inflammation, infection, liver disease, and malignancy. the systemic homeostasis of iron, being regulated and managed by hepcidin 9.10, is also produced in the liver. it mediates degradation of ferroportin in duodenal enterocytes, hepatocytes and macrophages, which mainly prevents proper absorption and usage of iron. inflammatory cytokines can induce the transcription of hepcidin5.6, and its increase has been reported in patients with ckd5. anemia in ckd therefore is a multi-factorial process with three major factors being deficiency of erythropoietin, reduction in the life span of erythrocytes of poorly defined etiology, and changes in homeostasis of iron” [5]. anemia is associated with several symptoms that lead to reduced quality of life, such as fatigue, dyspnea, insomnia, and headache. it is also related to reduced cognitive capacity. in addition, as kidneys fail, patients may require erythropoietin a stimulus to bone marrow to produce more blood. this hormone is naturally produced by the kidneys but becomes relatively limited in chronic disease of the kidneys. most patients will come to require erythropoietin or equivalent injectable products. the two main treatments for anemia are erythropoiesisstimulating agents and iron replacement. however, despite these treatments, an important proportion of children remain anemic. increasing the esa dose to achieve higher hemoglobin levels has been associated with adverse outcomes in adults; this association has not gupta et al.; asian j. immunol., vol. 7, no. 1, pp. 302-315, 2024; article no.aji.127402 305 been examined in children. hemoglobin may be hard to keep within a narrow range using conservative esa dosing. careful administration of iron supplements may enhance esa’s efficacy, yet the commonly applied markers of iron storage in the clinical setting lack the sensitivity to identify which patients can benefit the most from supplemental iron. other agents that target the hif pathway, such as hypoxiainducible factor stabilizers and prolyl hydroxylase inhibitors, and iron supplements delivered via dialysis are, therefore under investigation and may provide alternative management. nonetheless, the effectiveness and safety of these treatments in children with ckd have not been reported yet [6]. for those with gfr < 30 ml/minute, it is fair to assume that anemia is secondary to renal failure. nonetheless, it is important to rule out iron deficiency among others since the disease can be easily reversed. if anemia appears disproportionate to the level of kidney dysfunction, further investigation should be made. in such cases, an example-like hemoglobin at 9 g/dl with serum creatinine of 200 μmol/l should be investigated. further. type 2 diabetes and hypertension are the predominant causes of ckd in the developed world and increase the risk of developing cardiovascular disease, hyperlipidemia, mineral and bone disorders, and anemia. anemia is characterized by a decrease in red blood cells and, consequently, hemoglobin (hb), and typical symptoms and signs include paleness, fatigue, and breathlessness. at least two factors determine the lag between ckd onset and development of anemia. first, epo, the hormone that promotes red blood cell production, is produced in smaller quantities in ckd patients than in non-ckd patients. second, hepcidin, a hormone that suppresses dietary iron absorption when levels are elevated, is elevated in greater concentrations in ckd patients. iron is one constituent of hemoglobin; it serves as a critical component in the oxygen transport process. anemia in patients with ckd is a known contributor to a lower quality of life and increased risk of clinical outcomes that are unfavorable [7]. 1.3 chronic kidney disease (ckd) chronic kidney disease is a degenerative and incurable illness with high morbidity and mortality rates and also the most common cause of morbidity in adults, especially those with diabetes and hypertension. as such, nonpharmacological interventions, for instance, dietary and lifestyle changes, and renal diseasespecific pharmacological treatments, targeting chronic kidney disease can help preserve renal function that otherwise may otherwise improve the outcomes. a plant-based diet low in protein and salt may reduce glomerular hyperfiltration and halt or slow the progression of renal deterioration and even contribute to alterations in acid-base balance and in the gut microbiome that are favorable. alteration in intrarenal hemodynamics through pharmacological therapies (e.g., reninangiotensin-aldosterone pathway regulators and sglt2 [slc5a2] inhibitors) may preserve renal function through a reduction in intraglomerular pressure that is independent of control of bp and glycemia; other pharmacological therapies are novel active moieties: nonpharmacologic steroidal mineralocorticoid receptor antagonists which may protect the kidney through antiinflammatory or anti-fibrotic mechanisms [8]. some glomerular and cystic kidney diseases may benefit from disease-specific treatment. considering the sheer number of comorbidities, the morbidity and mortality associated with them, and the role of non-traditional risk factors in ckd, managing the cardiovascular risk of ckd, minimizing infection risk, and preventing acute renal failure are some important interventions for these patients. if renal replacement therapy is unavoidable, then stepwise switching to dialysis may be considered. it has also been suggested that this may preserve residual renal function for longer. ksp and sc share similarities but are different from one another. more research is needed, along with the development of new treatment strategies in dietary and pharmacological interventions, to achieve optimal kidney-sparing treatment in these patients, increasing their life expectancy, and achieving better hrqolid anemia is a frequent complication present in ckd. both absolute and functional id are encountered in ckd patients [9]. absolute iron deficiency is defined as substantially depleted or depleted iron stores. functional iron deficiency refers to sufficient storage but insufficient availability of iron to be absorbed into erythroid precursors, primarily because of increased hepcidin levels. anemia in gupta et al.; asian j. immunol., vol. 7, no. 1, pp. 302-315, 2024; article no.aji.127402 306 patients with ckd has been tied to increased morbidity and mortality. the association of mortality with anemia may be related to the severity of anemia. all patients with ckd should be assessed for anemia when first evaluated. the concept of iron deficiency in ckd is much less similar to that in patients with normal renal function. in the case of ckd patients, absolute iron deficiency is characterized by tsat ≤20% and a serum ferritin concentration ≤100 ng/ml in predialysis and peritoneal dialysis patients and ≤200 ng/ml in hemodialysis patients [11]. iron-restricted erythropoiesis, or functional iron deficiency, is characterized by tsat ≤20% and elevated ferritin. iron supplementation is provided to all ckd patients who have anemia. furthermore, the overwhelming majority of patients do not attain optimal responses to esas. to overcome these barriers, hif-phis have been created as oral agents in the treatment of anemia in ckd.they mimic the body's exposure to moderate hypoxia, thereby stimulating endogenous erythropoietin production. some of these agents are already approved for clinical use in specific countries. clinical trial data show noninferiority compared with esas and superiority to placebo for the correction of anemia. hif prolyl hydroxylase domain inhibitors could offer patients with inflammation another advantage: enhancing iron use and mobilization as well as reducing ldl cholesterol levels. overall, non-inferiority was also established for major cardiovascular events except in one molecule in the population of non-dialysis. such a finding is rather an unexpected given that, based on their mechanism of action, these drugs have low levels of erythropoietin. more data and longer follow-up are needed to clarify safety issues and further explore the diversity of signaling pathways activated by hif that may have positive or negative effects and distinguish hif-phis from esas [12]. 2. mechanism of action of vadadustat 2.1 hif-ph inhibition “the hif system elicits an adaptive response to tissue hypoxia in order to prevent cell damage by optimizing oxygen delivery and reducing tissue oxygen utilization which causes the production of epo, primarily by interstitial perinephric cells of the kidney and by cells in the liver. epo binds to its receptor on the surface of erythroid progenitor cells in the bone marrow, thus enhancing the survival, maturation, and proliferation of red blood cells. the activity of the hif system varies according to tissue oxygenation”. fig. 1. hypoxia-inducible factor (hif) system. ph—prolyl hydroxylase, pvhl—von hippel−lindau protein, hre—hypoxia-responsive element, epo—erythropoietin, vegf— vascular endothelial growth factor gupta et al.; asian j. immunol., vol. 7, no. 1, pp. 302-315, 2024; article no.aji.127402 307 fig. 2. schematic overview of the phd/hypoxia-inducible factor (hif) pathway “the hif system consists of subunits α and β. hif-1α accumulates and translocates to the nucleus of a cell where it binds to hif-β and leads to the synthesis of the heterodimer hif1αβ. the heterodimer hif-1αβ leads to the expression of various hypoxia-inducible genes, such as the gene for epo, thereby enhancing its rate of production. there are three isoforms of the subunit hif-α: hif-1α, hif-2α, and hif-3α, which all can dimerize with hif-β and stimulate the expression of many genes other than the epo gene. thus, the dimer composed of the hif-α and hif-β subunits controls the expression of transferrin receptors, vegf, and endothelin-1 receptors” [13]. in addition, it has been postulated that the hif system is somehow involved in the control of cell metabolism and activity, including immune cells, and affects the total cholesterol and ldl fraction. the hif system is nearly ubiquitous: the transcription factor hif-1α is induced in virtually all cell types whereas hif-2α is expressed in a more tissue-restricted manner. “mrna expression of hif-2α is mainly detected in the brain, heart, lung, kidney, pancreas, and intestine. hif-3α tissue expression was observed in the heart, lungs, and kidneys. normoxic tissue oxygenation leads to the degradation of hif-1α through its hydroxylation via prolyl hydroxylase (ph) with the von hippellindau protein (pvhl) thereby preventing dimerization between hif-1α and hif-β at a lower level of gene expression that encodes epo. under hypoxia, ph is inhibited and hif-1α does not undergo degradation, and there is possibility of combination to produce the heterodimer hif-1αβ, which activates the gene in charge of epo synthesis. inhibition of ph corresponds to the site of action for a class of drugs hif-phis, used to treat anemia in patients with ckd” [14]. 2.2 the phd/hif oxygen-sensing pathway “hif-α is rapidly degraded under normoxic conditions through the hydroxylation of certain proline residues. hydroxylated hif-α is tagged for proteasomal degradation by the von hippellindau (vhl) tumor hifs are basic pleiotropic helix-loop-helix transcription factors that belong to the pas (per/aryl hydrocarbon receptor nuclear translocator [arnt]/single minded) family of transcription factors. they consist of two subunits, an oxygen-sensitive α-subunit and a constitutively expressed β-subunit, which is often referred to as the arnt. the hif-α subunit is continuously synthesized but is rapidly degraded in the presence of molecular oxygen. three hif-α-subunits have been identified, hif1α, hif-2α (also known as epas1), and hif-3α. under hypoxic conditions, hif-α no longer gets degraded, but translocates to the nucleus, forms a heterodimer with hif-β, and activates gene transcription.hif-1 and hif-2, the most intensively studied hif transcription factors, control a very wide range of hypoxia responses, such as angiogenesis, anaerobic glucose metabolism, mitochondrial biogenesis, and many others, thus ensuring oxygen delivery and adaptation of cells to hypoxia [15]. the list of genes directly hif-regulated is vast and several gupta et al.; asian j. immunol., vol. 7, no. 1, pp. 302-315, 2024; article no.aji.127402 308 hundred high stringency hif binding sites have been identified in the genome. although hif-1 and hif-2 share many transcriptional targets, some genes are not co-regulated. for example, the glucose metabolism through glycolysis is under the control of hif-1, whereas epo production and some iron genes are under the regulation of hifsuppressor protein, which acts as the substrate recognition” “under hypoxic conditions, hif-prolyl hydroxylation is decreased, hif-α is no longer degraded and translocates to the nucleus where it heterodimerizes with hif-β and initiates gene transcription. any decrease in hif-proline hydroxylation or inactivation of vhl function decreases hif degradation and leads to greater expression of hif target gene [16]. for example, patients carrying particular inactivating vhl mutations are susceptible to cns hemangioblastomas, clear cell renal cancer, and pheochromocytomas, cancers that are described by upregulated hif-regulated gene expression. in addition, particular mutations that compromise the ability of cells to faithfully hydroxylate hif-α lead to abnormal regulation of hif activity and predispose to polycythemia”. “although the oxygen-sensitive α-subunit of hif is ubiquitously expressed and continuously produced, it is degraded rapidly in normoxic conditions [17]. on the contrary, in hypoxia, the levels of cellular hif-α accumulate and hif-α moves to the nucleus, where it dimerizes with hif-β. hif-α degradation is orchestrated by the pvhl-e3-ubiquitin ligase complex, but this is only possible in the presence of oxygenand iron-dependent phd dioxygenases (phd1-3) that hydroxylate prolyl residues of hif-α. decarboxylation of 2-oxoglurate (2og produces hydroxylated hif-α, succinate and co2. inhibition of phd or von hippel-lindau leads to increased transcription of hif-regulated genes such as vegf, epo, pgk1, ldh, and other genes implicated in hypoxia response regulation, including cellular metabolism, mitochondrial function, inflammation, vascular function, oxidative stress, and other responses. here is the structure of a hif-phd inhibitor (phi), which has been found to hold promise in potently inducing endogenous epo in dialysis patients [18]. hif-α is hydroxylated in an oxygendependent reaction by phd1, phd2, and phd3” [18]. “phds represent a large family of 2ogdependent dioxygenases comprising over 60 members. since these dioxygenases catalyze the hydroxylation requiring the use of molecular oxygen, they constitute a point of intersection between oxygen, intermediary, and amino acid metabolism and a variety of cellular processes including hif regulation/hypoxia responses, collagen synthesis, epigenetic gene regulation, and fatty acid metabolism. other small molecules, including reactive oxygen species, nitric oxide, and the krebs cycle intermediates succinate and fumarate, have inhibitory effects on phd catalytic activity and stabilize and activate hifα, leading to associated transcriptional programs. the latter is clinically relevant in patients with deficiency of fumarate hydratase, who are at risk of developing the hereditary leiomyomatosis renal cell cancer syndrome associated with increased hif activity within affected tissues. structural analogues of 2og that inhibit the access of 2og and hifα to the catalytic center of the phd and thus reversibly inhibit hifα hydroxylation are now under clinical development for treatment of renal anemia and other indications” [19]. 2.3 the phd/hif axis in erythropoiesis and iron metabolism “hif-dependent regulation of iron metabolism. overview schematic overview of hif-regulated genes in iron metabolism shown in red. in the intestine, duodenal cytochrome b (dcytb) reduces ferric iron (fe3+) to fe2+, which enters via the enterocytes. dcytb and dmt1 are both bonafide hif-2-regulated genes. there is a release of iron into the circulation via ferroportin (fpn) that is hepcidin-regulated but also hifinducible. in blood, iron is transported in complex with transferrin (tf) to the liver, cells of the reticulo-endothelial system (res), bone marrow, and other organs. it has been demonstrated that increased erythropoietic activity in the bone marrow produces growth differentiation factor 15 (gdf15) and erythroferrone that suppress hepcidin in hepatocytes. inflammatory cytokines stimulate hepcidin production in the liver and lead to reduced ferroportin surface expression and hypoferremia. the small 25-amino-acid peptide produced by hepatocytes, hepcidin, targets fpn for degradation” [20]. “its production increases with iron and inflammatory cytokines, including interleukin 6. as hepcidin decreases the expression of fpn on the cell surface, high plasma hepcidin levels have led to reduced intestinal iron uptake and impaired release of iron from internal stores. gupta et al.; asian j. immunol., vol. 7, no. 1, pp. 302-315, 2024; article no.aji.127402 309 fig. 3. schematic overview of hif-dependent regulation of iron metabolism plasma hepcidin levels are generally increased in more advanced stages of ckd and enhance the pathogenesis of renal anemia by causing functional iron-deficiency, due to increased hepcidin levels. this involves systemic activation of hif, suppressing hepcidin production in the liver and thus enhancing iron uptake and mobilization [21]. the first studies implicated hif-1 directly in the suppression of hepcidin transcription; however, more detailed analysis of mouse models with either global or liver-specific hif activation suggests that suppression of hepcidin is epo-mediated stimulation of erythropoiesis. hif does not have a direct repressor role on hepcidin transcription in the liver. the interaction between hif and iron metabolism is a bi-directional one” . “the 5′ utr of the gene encoding hif-2α bears an iron response element that interacts with irps. in analogy to the inhibition of ferritin translation, irp binding to the hif2a 5′ utr will block hif-2α translation and therefore diminish the level of hif-2α protein in the cell in response to low intracellular iron [22]. from this mechanism, one would anticipate that renal epo production would be only mildly suppressed in iron deficiency anemia compared with other forms of anemia that are associated with either maintenance or even elevation of plasma iron. hypoxia and the phd/hif pathway play an important role in regulating iron metabolism; in addition, it promotes erythropoiesis through direct effects on the bone marrow, including stimulating the expression of the epo receptor and enhancing the synthesis of hemoglobin, as well as modulating the maintenance and lineage differentiation of stem cells and their maturation”. anemia associated with ckd results from relative deficiency of renal epo production, combined functional and/or absolute iron deficiency and resistance to epo signaling, a condition often seen in the context of inflammation. pharmacologic activation of hif signaling may offer a more holistic and physiologic therapeutic option for treating renal anemia than therapy with recombinant preparations of epo [23]. 3. progress in clinical research in clinical trials of a 20-week, phase 2b multicenter, randomized, double-blind, placebocontrolled study to assess the oral, once-daily vadadustat ability to correct anemia in patients with ndd-ckd. a total of 1,929 patients at nddckd stage 3a/b, 4, and 5 stages from 61 centers were stratified by esa therapy use and hb level. group 1: esa treatment naïve and hb ≤10.5 g/dl; group 2: previously treated with esa and hb ≤10.5 g/dl; group 3: actively treated with esa, hb ≥9.5 g/dl to ≤12.0 g/dl [24]. patients in each group received double blinded randomization 2:1 for vadadustat versus placebo, stratified by ckd stage and the presence or absence of diabetes mellitus. two hundred ten patients were included in the safety analyses: those patients who received the study drug were treated with either vadadustat (n = gupta et al.; asian j. immunol., vol. 7, no. 1, pp. 302-315, 2024; article no.aji.127402 310 138) or placebo (n = 72). for all efficacy analyses, the modified intent-to-treat population of 208 patients was used, those patients who had a baseline and ≥1 postbaseline measurement of hb and red blood cells [24]. the former group included 136 patients randomized to vadadustat and 72 to placebo. the per-protocol population was defined a priori as the primary population for use in analysis of the primary endpoint and consisted of all modified intent-to-treat patients who completed the study, had efficacy data through week 20, were ≥80% compliant with study medication, and did not have a major protocol deviation [25]. a total of 160 patients qualified for the perprotocol population. of the 210 patients who were treated with the study medication, 81% (n = 112) in the vadadustat group and 88% (n = 63) in the placebo group completed treatment through to week 20 of the study. in terms of demographic and disease-related characteristics, hb, hemoglobin; itt, intent-totreat population, i.e. all randomized patients who received ≥1 dose of study medication; tsat, transferrin saturation; uacr, urine albumin-tocreatinine ratio. values are n (%) or mean ± sd. if a subject had >1 reason checked for etiology of ckd, all reasons were counted. the most important endpoint of the investigation was the percentage of patients reaching or maintaining a mean hb ≥11.0 g/dl at the end of the last 2 weeks of treatment or showing an increase in hb ≥1.2 g/dl above the per dose average [26]. vadadustat treatment raised and maintained hb levels in the patients with anemia secondary to ckd. in the per-protocol population, 54.9% of patients treated with vadadustat achieved the primary endpoint compared with 10.3% of patients treated with the placebo (p < 0.0001). at an estimated odds ratio, patients receiving vadadustat were approximately 11.5 times more likely than patients receiving a placebo to attain a successful hb response (p = 0.0001) success: hb average of weeks 19 and 20 ≥11.0 g/dl or hb average of weeks 19 and 20 ≥1.2 g/dl higher than predose mean. for a missing value, a single value was used p-value for vadadustat versus placebo is from a logistic regression analysis [27]. vadadustat dose-robustly increased and maintained hb levels in patients throughout the 20-week study. by week 2, mean hb levels in the vadadustat group had increased substantially from baseline; hb levels plateaued between weeks 6 to 8 and were maintained throughout the 20 weeks of treatment hb response versus time in each of the 3 study groups based on esa treatment status is shown in hb excursions ≥13 g/dl occurred in only 4.3% (6 of 138) of patients in the vadadustat group. post hoc analysis of hb values for all weeks between 8 and 20: for vadadustat 71.2% and for placebo 42.7% were between 10 and <12 g/dl; and for vadadustat 8.9% were between 12.0 and 12.9 g/dl, 1% were ≥13.0 g/dl, while none for the placebo group were≥12.0 g/dl [28]. fewer vadadustat-treated patients required esa rescue therapy compared with placebo (4.4% vs 16.7%, p = 0.045). of the 6 vadadustat-treated patients who received esa during the study, 4 did not meet the protocol-prespecified criteria for esa rescue; of the remaining 2, 1 was in the previously treated group, and 1 in the actively treated group. out of the 12 placebo patients treated with esa rescue, 2 did not meet protocol-specified criteria; of the remaining 10, 1 was in the treatment naïve group, 3 were in the previously treated group, and 6 were in the actively treated group. no patient receiving vadadustat and 1 placebo-treated patient required transfusion rescue at any time during the trial [29]. baseline increase in mean absolute reticulocyte count occurred in the vadadustat group at week 2 (+0.022 × 10 6 /μl) compared with a slight decrease in the placebo group (–0.005 × 10 6 /μl; p = 0.0001) [30]. the mean reticulocyte count in the vadadustat group continued to decrease and reached plateau during weeks 6 to 8 at a level significantly above that of the placebo group, reflecting the new hb baseline. markedly lower hepcidin and ferritin levels and significantly higher total ironbinding capacity were noted in the vadadustat group, compared with the placebo group, at each postbaseline evaluation. serum iron and transferrin saturation did not differ between the 2 treatment groups over the course of the study [31]. at the end of 20 weeks, 45.8% of patients in the vadadustat group were also receiving iron supplementation, whereas 52.8% of those in the placebo group were receiving iron supplementation at this point. furthermore, 2.9% of the patients (4 of 136) receiving vadadustat, but none in the placebo group received i.v. iron.initial drug dosage at treatment initiation was 450 mg once daily; mean drug dosage at gupta et al.; asian j. immunol., vol. 7, no. 1, pp. 302-315, 2024; article no.aji.127402 311 week 19: 450 mg once daily [32]. for the majority of patients: 89%, 120 of 135 who received vadadustat, a stable hb level was achieved and maintained with ≤2 dose adjustments over the 20-week treatment period, and no dose adjustment was required in 24% of patients (33 of 135) [33]. patients who were ≥80% compliant with the allocated treatment had 92.8% (128/138) in the vadadustat group versus 94.4% (68/72) in the placebo group. a higher percentage of the vadadustat-treated patients experienced ≥1 adverse event (ae) compared to the placebotreated patients (74.6% vs 73.6) [34]. the incidence of ≥1 drug-related ae was reported in 25.4% of the vadadustat-treated patients (35 of 138) and in 11.1% of placebo-treated patients (8 of 72). in both treatment arms, most of the aes reported were mild or moderate in intensity [35]. commonly reported drug-related aes in the vadadustat group were diarrhea (4.3%), nausea (4.3%), whereas most commonly reported drugrelated ae in the placebo group was diarrhoea (2.8%) [36]. 10 patients treated with vadadustat (7.2%) and three patients treated with placebo (4.2%) discontinued the study due to aes.not listed in the table below is one of the 14 vadadustat-treated patients who were reported to have had a renal-related saeone of whom was subject 1490001, previously study group treated [37]. the patient was hospitalized for goodpasture syndrome worsening that occurred following 1 dose of study medication; other than hospitalization for worsening goodpasture syndrome, no other renalrelated adverse events were reported [38]. there were no trends observed (either increases or decreases) in the systolic values of blood pressure or diastolic values of blood pressure over time, and the distribution in the systolic values of blood pressure among patients receiving vadadustat was comparable to that in patients receiving placebo [39]. hypertension was an ae in 8.0% of patients treated with vadadustat (11 of 138) and in 2.8% of patients treated with placebo (2 of 72), and all patients had a pre-existing history of hypertension. one of the 11 patients on vadadustat who experienced an ae of hypertension during the study had furosemide discontinued about 1 month prior to the event. otherwise, no participant experienced withdrawal or dose reduction of antihypertensive drugs before development of the ae of hypertension; no participant stopped vadadustat due to hypertension [40]. out of these, 33 patients treated with vadadustat experienced ≥1 sae (23.9%), compared with 11 placebo-treated patients (15.3%); the higher proportion of saes was mainly due to a higher proportion of renal-related saes in the vadadustat arm (10.1%) than in the placebo arm (2.8%) [41]. the proportion of patients who received a baseline requirement for initiation of dialysis, an inorganic marker of the seriousness of renal saes, was similar between the vadadustat (11 of 138, 8.0%) and placebo (7 of 72, 9.7%) treatment groups [42]. the incidences of investigation-reported renal-related saes were not related to the study drug, according to the investigator. the clinical studies mentioned above have proven vadadustat as a worthwhile option as an induction treatment for patients with anemia who have chronic kidney disease [43]. 4. conclusion anemia is an inadequacy in the number or quality of red blood cells (rbcs) or a reduction in the amount of hemoglobin, which is the oxygen-carrying protein in rbcs in the blood [44]. it is associated with poorly oxygenated tissues and organs within the body, resulting in a variety of symptoms such as fatigue and weakness and paleness [45]. esas or new hifph inhibitors like vadadustat, which act through mimicking hypoxia by stabilizing the hypoxiainducible factor pathway and activates erythropoiesis and epo production, should be considered for achieving treatment goals [46]. vadadustat is an investigational oral hypoxiainducible factor prolyl-hydroxylase inhibitor that stimulates endogenous erythropoietin formation. at present, erythropoiesis-stimulating agents (esas) are still the treatment mainstay for anaemia of chronic kidney disease (ckd) [47]. all esas function effectively to correct anemia in ckd by countering the epo deficiency resulting from failing kidneys. in contrast, whereas esas function by antagonizing the activity of the hif pathway, hif-phis activate it, thereby stimulating the transcription of the epo gene and upregulating the expression of genes participating in erythropoiesis and in iron metabolism [48]. this unusual mechanism of action was believed to have an excellent anemic correction by promoting endogenous production of epo and concomitantly boosting enteric iron gupta et al.; asian j. immunol., vol. 7, no. 1, pp. 302-315, 2024; article no.aji.127402 312 absorption and mobilization of iron (in contrast to esa). in addition, the peak serum epo level is reportedly a few folds less in hif-phis as compared to esa treatment [49]. the vast majority of rcts with hif-phis established their non-inferiority as compared to esas. vadadustat is a potent inhibitor of the catalytic activity of all three human phd isozymes: phd1, phd2, and phd3; all three endozymes have nanomolar inhibitory constant values and are similar [50]. vadadustat inhibits phd with competition against the cellular endogenous cofactor 2oxoglutarate and is not dependent on free iron concentration. in the human hepatocellular carcinoma cell line (hep 3b) and human umbilical vein endothelial cells, phd inhibition by vadadustat leads to timeand concentrationdependent stabilization of hif-1α and hif-2α. this results in synthesis and secretion of epo in hep 3b cells, whereas vascular endothelial growth factor is not measured at detectable levels. a single oral dose of vadadustat in rats potently increases circulating levels of epo [51]. consent and ethical approval it is not applicable. disclaimer (artificial intelligence) author(s) hereby declares that no generative ai technologies such as large language models (chatgpt, copilot, etc) and text-to-image generators have been used during writing or editing of this manuscript. competing interests authors have declared that no competing interests exist. references 1. mimura, imari, tetsuhiro tanaka, and masaomi nangaku. evaluating the safety and efficacy of vadadustat for the treatment of anemia associated with chronic kidney disease. expert opinion on pharmacotherapy just-accepted; 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cite as: warmuzińska, alina, dariusz woszczyk, and sławomir tubek. 2024. “clinical picture of patients with the undetectable serum ige and low serum ige in a retrospective evaluation of patients of the opole regional hospital (20132023)”. asian journal of immunology 7 (1):112-22. https://journalaji.com/index.php/aji/article/view/136. asian journal of immunology volume 7, issue 1, page 112-122, 2024; article no.aji.120841 clinical picture of patients with the undetectable serum ige and low serum ige in a retrospective evaluation of patients of the opole regional hospital (2013-2023) alina warmuzińska a, dariusz woszczyk b and sławomir tubek b* a department of imaging diagnostics, regional hospital in opole, opole, poland. b clinical department of haematology, faculty of medicine, haematological oncology and internal medicine, regional hospital in opole, university of opole, opole, poland. authors’ contributions this work was carried out in collaboration among all authors. all authors read and approved the final manuscript. article information doi: https://doi.org/10.9734/aji/2024/v7i1136 open peer review history: this journal follows the advanced open peer review policy. identity of the reviewers, editor(s) and additional reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here: https://www.sdiarticle5.com/review-history/120841 received: 05/06/2024 accepted: 06/08/2024 published: 10/08/2024 abstract over the period 01/01/2013 until 31/08/2023, 13 907 total plasma ige determinations were performed in the analytical laboratory of the provincial hospital in opole. among these determinations, there were 377 results below 2.0 u/l (in 279 patients), with the ultraextremely low results (<0.1 u/l), i.e. virtually undetectable, in 65 (in 44 patients). original research article https://doi.org/10.9734/aji/2024/v7i1136 https://www.sdiarticle5.com/review-history/120841 warmuzińska et al.; asian j. immunol., vol. 7, no. 1, pp. 112-122, 2024; article no.aji.120841 113 the clinical picture of patients with the ultra-extremely low (undetectable) ige (defined as <0.1 u/l) group 1 and the low ige (defined as <2.0 u/l), divided into 4 ranges 0.1-0.5; 0.6 -1.0; 1.1-1.5; 1.61.9 u/l the group 2, 3, 4 and 5 respectively was compared. results: health problems, defined as a syndrome of immune dysfunction, occurred more frequently in the groups with the ultra-low (virtually undetectable) ige (<1.0) than in the group with the low ige (0.1 1.9) level, suggesting primary immune deficiency in these patients. this is also supported by the fact that the group 1 was younger than the others combined, and at the same time ‘sicker’ than the other groups. in this group 97 patients had serum ige determinations at least twice during the analysed period in 20 patients at least one of the repeated serum ige results was higher than 10 u/l, in the remaining ones in 17 patients it was in the range (2-10 u/l), in the remaining 60 patients the results of repeated serum ige determinations did not exceed 2 u/l. conclusions: for the purpose of talking about ige deficiency as an indicator of a predisposition to neoplastic diseases, it would be necessary to carry out screening tests at least 3 times, in the identified age groups, e.g. every 10 years, from the age of 5 years. this would make it possible to determine whether the ige deficiency is primary or secondary. primary, i.e. originally predisposing to the development of neoplastic diseases and being a part of primary immunodeficiency, or secondary as a symptom of ‘depletion’ of the immune system. the incidentally detected low serum ige needs to be verified. keywords: ultralow/undetectable ige plasma level (<0.1 u/l); extremely low ige plasma level (<2.0 u/l); low ige plasma level. 1. introduction interest in the significance of low plasma total ige levels has been growing in the recent years due to data demonstrating its relationship to immunodeficiency syndromes and susceptibility to neoplastic diseases [1-6]. in both cases, there is a broad concept of immune dysfunction syndrome. high levels of total ige, in turn, are thought to improve a prognosis in the selected groups of oncology patients. furthermore, there are emerging perspectives for the use of allegrooncological knowledge in the oncology treatment processes and the side effects of immunotherapy of neoplastic diseases [7-10]. awareness of differences in response to treatment depending on blood ige levels may influence the individualisation of treatment for the specific conditions [11]. the reference value for plasma ige levels is values below 100 u/l 2-100 u/l. in the literature, values below 2.0 2.5 u/l are considered low or extremely low levels of total ige. as the group of individuals with total ige values below 2.0 2.5 u/l may be clinically significantly heterogeneous due to the presence of tissue ige, it is suggested that ige values below this limit should be more accurately determined [5]. in a previous study comparing a group with the ultra-extremely low ige level (<0.1 u/l), i.e. virtually undetectable, with a group with the extremely high ige level (>10,000 u/l), a proposal was made that the ige values <0.1 u/l should be defined as the ultra-extremely low (or undetectable), values between 0.1 and 0.5 as the extremely low and values between 0.6 and 2.0 as the low ones [12]. in the retrospective study presented here, it was decided to compare the groups clinically with the ultra-extremely low ige levels (<0.1 u/l), i.e. virtually undetectable and the low ige (defined as >0.1 to <2.0 u/l). 2. materials and methodology within the period 01/01/2013 until 31/08/2023, 13 907 total plasma ige determinations were performed in the analytical laboratory of the provincial hospital in opole. assuming the data quoted above, among these determinations there were 377 results below 2.0 u/l (in 279 patients), while the ultra-extremely low results (<0.1 u/l), i.e. practically undetectable, were 65 (in 44 patients). a comparison was decided to be made between the clinical picture of patients with the ultraextremely low (undetectable) ige (defined as <0.1 u/l) the group 1 and the low ige (defined as <2.0 u/l), divided into 4 ranges 0.1-0.5; 0.6 1.0; 1.1-1.5; 1.6-1.9 u/l the groups 2, 3, 4 and 5 respectively. warmuzińska et al.; asian j. immunol., vol. 7, no. 1, pp. 112-122, 2024; article no.aji.120841 114 3. results in order to compare the study samples, statistical analyses were performed using the ibm spss statistics 29. the analyses used included student's t-test for independent samples, oneway analysis of variance and chi-square test of independence. the significance level was taken as the threshold α = 0.05, the effect size was interpreted according to cohen (cohen, j.,1992. a power primer. psychological bulletin, 112(1), 155–159. https://doi.org/10.1037/00332909.112.1.155). in the first instance, differences between all compared groups were calculated for nominal variables using the chi-square test of independence (table 1) and for the age using one-way analysis of variance (fig. 1). when differences were observed with the chi-square test, the significance of the differences was calculated using the bonferroni correction for multiple pairwise comparisons. parameters assessed were age, sex, place of hospitalisation (suggesting the most significant health problem), prevalence of bronchial asthma/chronic obstructive pulmonary disease (copd), neoplastic disease including lymphoma, immune dysfunction syndrome, atopic dermatitis (ad), bronchiectasis, sarcoidosis, psoriasis, granulomatosis with polyangiitis (formerly known as wegener's granulomatosis). analysis of the differences in terms of individual nominal variables (table 1), revealed a lack of statistically significant differences for sex and individual, and no differences were observed in the frequency of diseases such as lymphoma, sarcoidosis, psoriasis and granulomatosis with polyangiitis. bronchial asthma was found to be the least frequent in group 5 (approx. 21%) compared to the group 3 (approx. 43%), where it occurred most frequently. the groups 1, 2, 4 were intermediate in terms of the number of people experiencing asthma (approx. 31%) and did not differ from the extreme groups. similarly, neoplastic disease, irrespective of the starting point, was most common in the group 3 (approx. 44%) compared to group 5 (approx. 16%), while no differences were observed in the other groups, with the rates of neoplastic diseases ranging from 26% to 36%. immune dysfunctions were less frequent in the groups 4 (approx. 31%) and 5 (approx. 13%) compared to the groups 1, 2 and 3, where the percentage of dysfunctions was approx. 90%. analysing the results in terms of ad, it was found that the condition was most common in the group 2 (approx. 12%) compared to the group 5, where it was not present at all (0%), the other groups ranged from 1% to 6% and did not differ from the extreme groups. bronchiectasis in turn, was observed most frequently in the group 1 (approx. 23%) and least frequently in group 5 (approx. 3%), while no significant differences were observed in the other groups and, on average, this condition occurred in 10% of individuals. additional analysis also showed that there were no statistically significant differences in the age of the groups compared, f(4.318) = 1.66; p = 0.160; η2 = 0.02 (fig. 1). fig. 1. age differentiation in the groups compared (key: wiek-age, grupa-group) annotation. bars for errors represent 95% confidence intervals of mean scores grupa 1 grupa 2 grupa 3 grupa 4 grupa 5 54 56 58 60 62 64 66 68 70 w ie k warmuzińska et al.; asian j. immunol., vol. 7, no. 1, pp. 112-122, 2024; article no.aji.120841 115 table 1. clinical parameters evaluated in each group of patients: with the ultra-extremely low (undetectable) ige (defined as <0.1 u/l) the group 1 and the low ige (defined as <2.0 u/l), divided into 4 ranges 0.1-0.5; 0.6 -1.0; 1.1-1.5; 1.6-1.9 u/l the groups 2, 3, 4 and 5, respectively differentiation of the study groups in terms of the variables tested variable group 1 group 2 group 3 group 4 group 5 χ2 df p v sex m 18 (0.41) 23 (0.39) 19 (0.26) 33 (0.41) 24 (0.35) 4.53 4 0.339 0.12 k 26 (0.59) 36 (0.61) 53 (0.74) 47 (0.59) 44 (0.65) unit op 30 (0.68) 38 (0.64) 54 (0.75) 54 (0.68) 50 (0.74) 21.46 16 0.161 0.13 od 3 (0.07) 8 (0.14) 3 (0.04) 7 (0.09) 6 (0.09) oh 8 (0.18) 9 (0.15) 13 (0.18) 10 (0.13) 6 (0.09) ocw 0 (0.00) 4 (0.07) 1 (0.01) 2 (0.03) 1 (0.01) other 3 (0.07) 0 (0.00) 1 (0.01) 7 (0.09) 5 (0.07) asthma/copd no 34 (0.77) 40 (0.68) 41 (0.57) 50 (0.63) 54 (0.79) 10.92 4 0.027 0.18 yes 10 (0.23) 19 (0.32) 31 (0.43) 30 (0.38) 14 (0.21) neoplastic disease no 32 (0.73) 38 (0.64) 40 (0.56) 59 (0.74) 57 (0.84) 14.90 4 0.005 0.21 yes 12 (0.27) 21 (0.36) 32 (0.44) 21 (0.26) 11 (0.16) lymphoma no 38 (0.86) 50 (0.85) 57 (0.79) 68 (0.85) 56 (0.82) 1.48 4 0.830 0.07 yes 6 (0.14) 9 (0.15) 15 (0.21) 12 (0.15) 12 (0.18) immunological dysfunctions no 4 (0.09) 7 (0.12) 10 (0.14) 55 (0.69) 59 (0.87) 144.54 4 <0.001 0.67 yes 40 (0.91) 52 (0.88) 62 (0.86) 25 (0.31) 9 (0.13) ad no 42 (0.95) 52 (0.88) 68 (0.94) 79 (0.99) 68 (1.00) 13.25 4 0.010 0.20 yes 2 (0.05) 7 (0.12) 4 (0.06) 1 (0.01) 0 (0.00) bronchiectasis no 34 (0.77) 55 (0.93) 60 (0.83) 75 (0.94) 66 (0.97) 16.83 4 0.002 0.23 yes 10 (0.23) 4 (0.07) 12 (0.17) 5 (0.06) 2 (0.03) sarkoidosis no 42 (0.95) 54 (0.92) 67 (0.93) 72 (0.90) 64 (0.94) 1.66 4 0.799 0.07 yes 2 (0.05) 5 (0.08) 5 (0.07) 8 (0.10) 4 (0.06) psoriasis no 44 (1.00) 56 (0.95) 71 (0.99) 77 (0.96) 67 (0.99) 3.95 4 0.412 0.11 yes 0 (0.00) 3 (0.05) 1 (0.01) 3 (0.04) 1 (0.01) granulomatosis with polyangiitis no 41 (0.93) 54 (0.92) 69 (0.96) 76 (0.95) 66 (0.97) 2.35 4 0.671 0.09 yes 3 (0.07) 5 (0.08) 3 (0.04) 4 (0.05) 2 (0.03) annotation. n(%) number of observations (percentage of observations); χ² result of a chi-square test; df number of degrees of freedom; p statistical significance; v effect size. op – pulmonology department, od – dermatology department, oh – haematology department, ocw – internal diseases department warmuzińska et al.; asian j. immunol., vol. 7, no. 1, pp. 112-122, 2024; article no.aji.120841 116 table 2. clinical parameters evaluated in each patient group: with ultra-extremely low (undetected) ige (defined as <0.1 u/l) the group 1 and low ige (defined as <2.0 u/l), divided into 4 ranges 0.1-0.5; 0.6 -1.0; 1.1-1.5; 1.6-1.9 u/l the groups 2, 3, 4 and 5 respectively (this time a total of 2-5) contrast analysis of the groups studied in terms of the variables tested variable group 1 groups 2-5 χ2 df p φ/v sex men 18 (0.41) 99 (0.35) 0.48 1 0.487 0.04 women 26 (0.59) 180 (0.65) unit op 30 (0.68) 196 (0.70) 2.34 4 0.673 0.09 od 3 (0.07) 24 (0.09) oh 8 (0.18) 38 (0.14) ocw 0 (0.00) 8 (0.03) other 3 (0.07) 13 (0.05) asthma no 34 (0.77) 185 (0.66) 2.09 1 0.148 0.08 yes 10 (0.23) 94 (0.34) neoplastic disease no 32 (0.73) 194 (0.70) 0.18 1 0.668 0.02 yes 12 (0.27) 85 (0.30) lymphoma no 38 (0.86) 231 (0.83) 0.35 1 0.556 0.03 yes 6 (0.14) 48 (0.17) immunological dysfunctions no 4 (0.09) 131 (0.47) 22.40 1 <0.001 0.26 yes 40 (0.91) 148 (0.53) ad no 42 (0.95) 267 (0.96) 0.01 1 0.941 <0.01 yes 2 (0.05) 12 (0.04) bronchiectasis no 34 (0.77) 256 (0.92) 8.69 1 0.003 0.16 yes 10 (0.23) 23 (0.08) sarcoidosis no 42 (0.95) 257 (0.92) 0.62 1 0.432 0.04 yes 2 (0.05) 22 (0.08) psoriasis no 44 (1.00) 271 (0.97) 1.29 1 0.255 0.06 yes 0 (0.00) 8 (0.03) granulomatosis with polyangiitis no 41 (0.93) 265 (0.95) 0.25 1 0.619 0.03 yes 3 (0.07) 14 (0.05) annotation. n(%) – number of observations (percentage of observations); χ² result of a chi-square test; df number of degrees of freedom; p statistical significance; v effect size for the tables larger than 2x2. abbreviations – op – pulmonology department, od – dermatology department, oh – haematology department, ocw – internal diseases department warmuzińska et al.; asian j. immunol., vol. 7, no. 1, pp. 112-122, 2024; article no.aji.120841 117 table 3. characteristics of individual patients with the incidentally low serum ige <2.0 u/l who had more than one ige determination during the study period and at least one of the determinations was higher than 10 u/l sex/age at the time the ultra-low level of ige was determined health problems according to medical records number of the ige determinations and a range of values main (most frequent) place of hospitalisation / treatment 1 k 69 microcellular carcinoma of the right lung, decrease in the ige level during dissemination can be interpreted as a symptom of depletion of the immune system 3x 0.4 – 345, chronologically: 187 – 345 0.4 op 2 k 73 adenocarcinoma of the right lung subsequent progression metastasis to the left lung and adrenal glands; chronic obstructive pulmonary disease (copd), renal cysts can be interpreted as a symptom of immune depletion 2x 0.5 – 218.7, chronologically: 218.7 – 0.5 op 3 k 81 granulomatosis with polyangiitis (wegener granulomatosis), bronchial asthma, chronic sinusitis, venous insufficiency of the lower limbs can be interpreted as a symptom of depletion of the immune system 9x 0.5 – 82.6, chronologically: 82.6 – 1.1 -0.5 op 4 k 85 non-small cell cancer of the right lung, lower limb venous thrombosis, atrial fibrillation, old age can be interpreted as a symptom of immune system depletion 2x 0.7– 12.5, chronologically: 12.5 – 0.7 op 5 k 86 granulomatosis with polyangiitis (wegener granulomatosis), tuberculosis in anamnesis, osteoporosis, degenerative changes of the musculoskeletal system, pnemocystodosis can be interpreted as a symptom of depletion of the immune system 4x 0.7 – 92.1, chronologically: 92.1 – 3.7 1.1 – 0.7 op 6 k 73 tumour of the left lung, bronchial asthma / copd, emphysema, generalised atherosclerosis can be interpreted as a symptom of depletion of the immune system 2x 73.3 – 0.8 op 7 m 86 granulomatosis with polyangiitis (wegener granulomatosis), purulent arthritis, multilevel discopathy can be interpreted as a symptom of depletion of the immune system 2x 1.191.1 chronologically: 92.1 – 3.7 – 1.1 op warmuzińska et al.; asian j. immunol., vol. 7, no. 1, pp. 112-122, 2024; article no.aji.120841 118 sex/age at the time the ultra-low level of ige was determined health problems according to medical records number of the ige determinations and a range of values main (most frequent) place of hospitalisation / treatment 9 m 73 left lung tumour. bronchial asthma/copd, emphysema, generalised atherosclerosis – can be interpreted as a symptom of depletion of the immune system 2x 73.3 – 0.8 op 11 m 53 tuberculosis recurrence, copd, emphysema, fluid in left pleural cavity increase in the ige level during clinical improvement phase 2x 1.3 144 op 12 m 71 post-radiation pneumonia small cell cancer of the right lung, bronchial asthma, atrial fibrillation, circulatory failure increase in the ige level during clinical improvement phase 2x 1.4 – 107.7 op 13 k 73 copd/bronchial asthma, psoriasis last highest result during the period of requiring hospitalisation for exacerbation of bronchial asthma and psoriasis; high variability of ige levels indicates significant lability of the immune system 13x 1.4 1200 op 14 m 83 bronchiolitis, idiopathic bronchiolitis with organising pneumonia, bronchial asthma, secondary immunodeficiency, generalised atherosclerosis 7x 1.5 13 op 15 k 81 copd, emphysema, chronic respiratory failure on home oxygen therapy, graves-basedow disease – can be interpreted as a symptom of depletion of the immune system 7x 101.2 – 1.4 op 16 m 56 pulmonary sarcoidosis, chronic renal failure, chronic venous insufficiency of the lower limbs 3x 38.6-1.6-24.0 op 17 k 81 tumour of the right lung, copd, t2 dm without histopathological diagnosis of the tumour, stable clinical condition 2x 1,6 – 47,4 op 18 m 68 idiopathic pulmonary fibrosis, chronic respiratory failure on home oxygen therapy, bronchiectasis, t2 dm, secondary immunodeficiency, listeriosis in anamnesis increase in the ige level during inflammatory exacerbation of chronic 3x 0,9 – 16,0 – 1,6 op warmuzińska et al.; asian j. immunol., vol. 7, no. 1, pp. 112-122, 2024; article no.aji.120841 119 sex/age at the time the ultra-low level of ige was determined health problems according to medical records number of the ige determinations and a range of values main (most frequent) place of hospitalisation / treatment respiratory failure 19 k 71 adenocarcinoma of the right lung bronchial asthma increase in the ige level during asthma exacerbation 2x 1,8 – 485,5 op 20 m 48 granulomatosis with polyangiitis (wegener granulomatosis), granulomatous otitis media, t2 dm decrease in the ige level during the period of clinical improvement 4x 27,4 – 0,4 op op – pulmonology department, copd – chronic obstructive pulmonary disease, t2 dmtype 2 diabetes mellitus fig. 2. analysis of contrast in terms of age differences (key: wiek-age, grupa-group) annotation. bars for errors represent 95% confidence intervals of mean scores. grupy 2-5 grupa 1 56 58 60 62 64 66 68 w ie k warmuzińska et al.; asian j. immunol., vol. 7, no. 1, pp. 112-122, 2024; article no.aji.120841 120 in the next step, a contrast analysis was performed between the group 1 and the aggregated results of the group 2 5. a comparison of nominal indicators was performed using the chi-square test of independence, and the results are presented in the table 2. a comparison of ages was performed using the student's t-test for independent samples, and the results are presented in the fig. 2. the analyses performed (table 2), showed that only the presence of immune dysfunctions and bronchiectasis demonstrated statistically significant differences. immune dysfunctions were present in 91% of subjects in the group 1, while in the groups 2 5, just over 50% of subjects were observed to have immune dysfunctions. in the case of bronchiectasis, the prevalence of this condition was observed to be significantly higher for the group 1 (approx. 23%) compared to the groups 2-5 (approx. 8%). furthermore, it appeared that those in groups 25 were older by an average of six years compared to those in the group 1, t(321) = 2.17 95% ci [0.57; 11.88]; p = 0.031; d = 0.35 (fig. 2). in the material under study, serum ige determinations were not part of routine, repeated testing. hence, in some individuals, the serum ige was determined once, in spite of several hospitalisations or visits to hospital outpatient clinics, in others repeatedly (97 of 323 patients). in this group of 97 patients (with at least one ige result <2.0 u/l) in 20 patients at least one of the repeated serum ige results was higher than 10 u/l (table 3), of the remainder in 17 patients it was in the range (2-10 u/l), in the remaining 60 the repeated serum ige level determinations did not exceed 2 u/l. 4. discussion as mentioned above the analysis of differences in terms of individual nominal variables (table 1), revealed a lack of statistically significant differences in respect of sex and individual, and in addition no differences were observed in the frequency of diseases such as lymphoma, sarcoidosis, psoriasis and granulomatosis with polyangiitis. bronchial asthma appeared to occur least frequently in the group 5 (approx. 21%) compared to the group 3 (approx. 43%), where it occurred most frequently. the groups 1, 2, 4 had intermediate numbers of people experiencing asthma (approx. 31%) and did not differ from the extreme groups. similarly, neoplastic disease, irrespective of the starting point, was most common in the group 3 (approx. 44%) compared to the group 5 (approx. 16%), while no differences were observed in the other groups, where the percentage of neoplastic diseases ranged from 26% to 36%. immune dysfunctions were less frequent in the groups 4 (approx. 31%) and 5 (approx. 13%) compared to the groups 1, 2 and 3, where the percentage of dysfunctions reached approximately 90%. this supports the idea that the lower the serum ige level, the greater the probability of immune dysfunction. bronchiectasis, on the other hand, was observed most frequently in the group 1 (approx. 23%) and least frequently in the group 5 (approx. 3%), while no significant differences were observed in the other groups and, on average, this condition occurred in 10% of individuals. the distinctly more frequent occurrence of bronchiectasis in the group 1 indicates an impairment of the immune response to bacterial infections of the respiratory tract, which resulted in prolonged inflammation of the bronchi and destruction of the bronchial wall. an additional analysis also showed that there were no statistically significant differences in terms of the age of the groups under comparison, f(4.318) = 1.66; p = 0.160; η2 = 0,02 (fig. 1). the analyses comparing the group 1 with the groups 2-5 combined (table 2), showed that only the presence of immunological dysfunctions and bronchial dilatation showed statistically significant differences. it turned out that immune dysfunction was present in 91% of the group 1 subjects, while in the groups 2-5, just over 50% of subjects were observed to have immune dysfunction. in the case of bronchiectasis, the prevalence of this condition was observed to be significantly higher for the group 1 (approx. 23%) compared to the groups 2-5 (approx. 8%). moreover, it appeared that people in the groups 2-5 were older by an average of six years compared to those in the group 1, t(321) = 2.17 95% ci [0.57; 11.88]; p = 0.031; d = 0.35 (fig. 2). health issues, defined as a syndrome of immune dysfunction, were more common in the groups with the ultra-low (virtually undetectable) ige level (<1.0) than in the group with the low ige level (0.1 1.9), suggesting primary immune deficiency in these patients. this is also supported by the fact that the group 1 was younger than the others combined, but at the same time ‘sicker’ than the other groups. warmuzińska et al.; asian j. immunol., vol. 7, no. 1, pp. 112-122, 2024; article no.aji.120841 121 based on these observations, one can try to answer the question: randomly found the low serum ige ige deficiency primary or secondary? for the purpose of referring to ige deficiency as an indicator of predisposition to neoplastic diseases [1-6], screening should be carried out at least 3 times, in the specific age groups, e.g. every 10 years, beginning at 5 years of age. this would also make it possible to determine whether the ige deficiency is primary or secondary. primary, i.e. originally predisposing to the development of neoplastic diseases and forming part of the primary immunodeficiency, or secondary as a symptom of ‘depletion’ of the immune system. the low serum ige determined randomly requires a verification. in the delimited group with the low ige level, additional skin tests can be conducted to evaluate a deficiency of the tissue ig [13,14]. in the material under study, determinations of the serum ige were not part of routine, repeated testing. thus, in some patients, the serum ige was determined once, despite several hospitalisations or visits to the hospital outpatient clinics, in others it was determined repeatedly (97 of 323 patients). in this group of 97 patients in 20 patients at least one of the repeated serum ige results was higher than 10 u/l (table 3), of the others in 17 patients it was in the range (210 u/l), and in the remaining 60 patients repeated determinations of the serum ige levels did not exceed 2 u/l. this observation may suggest that the ige levels <0.1 u/l, found randomly, are more suggestive of primary immunodeficiency than levels between 0.1 and 2.0 u/l, where they are more probably suggestive of secondary immunodeficiency. 5. conclusion the low serum ige level found incidentally does not allow to conclude whether the ige deficiency is primary or secondary (as a symptom of ‘depletion’ of the immune system), and can be considered a poor prognostic factor, e.g. in chronic lymphocytic leukaemia [15]. its increase in subsequent studies argues for the secondary nature of a previous low level, but it can be considered a positive prognostic factor for regression of the underlying disease. disclaimer (artificial intelligence) authors hereby declare that no generative ai technologies such as large language models (chatgpt, copilot, etc) and text-to-image generators have been used during writing or editing of manuscripts. competing interests authors have declared that no competing interests exist. references 1. patel mb, smith jk, chi ds, krishnaswamy g, regulation and dysregulation of immunoglobulin e: a molecular and clinical perspective. clinical and molecular allergy (cma) 2010;8:3. available:http://www.clinicalmolecularallerg y.com/content/8/1/3 2. lawrence mg, palacios-kibler tv, workman lj, schuyler aj, steinke jw, payne sc, mcgowan sc, patrie j, fuleihan rl, sullivan ke, lugar pl, hernandez cl, beakes de, verbsky jw, platts-mills tae, cunningham-rundles ch, routes jm, borisch l. low serum ige is a sensitive and specific marker for common variable immunodeficiency (cvid). j clin immunol. 2018;38(3):225-33. doi: 10.1007/s10875-018-0476-0 3. matricardi pm, the very low ige producer: allergology, genetics, immunodeficiencies, and oncology. biomedicines.2023;11:1378. available:https://doi.org/10.3390/biomedici nes11051378 4. ferastraoaru dh, bax j, bergmann c, capron m, castells m, dobrowicz d, fiebiger e, gould hj, hartmann k, jappe u, jardakieva g, josephs dh, levischaffer f, mahler v, poli a, rosensterich d, roth-walter f, shamji m, stevelingklein eh, turner mc, untersmayrs e, karagiannis sn, jensen-jarolim e , allergooncology: ultralow ige, a potential novel biomarker in cancer—a position paper of the european academy of allergy and clinical immunology (eaaci). clin transl allergy. 2020; 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*corresponding author: e-mail: kaleabtesfaye35@gmail.com; asian journal of immunology 4(1): 190-214, 2021; article no.aji.78308 predictors of anemia among pregnant women attending antenatal care at public hospitals of sidama region, ethiopia, 2021 a case control study protocol kaleab tesfaye tegegne a*, abiyu ayalew assefa a, andualem zenebe a, wosenyeleh semeon bagajjo b#, berhanu bifato a, alelign tadele abebe c and sintayehu assefa d a department of public health, hawassa college of health science, hawassa, ethiopia. b hawassa college of health science, hawassa, ethiopia. c department of medical laboratory technology, hawassa college of health science, hawassa, ethiopia. d department of mid wifery , hawassa college of health science, hawassa, ethiopia. article information open peer review history: this journal follows the advanced open peer review policy. identity of the reviewers, editor(s) and additional reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here: https://www.sdiarticle5.com/review-history/78308 received 01 october 2021 accepted 04 december 2021 published 30 december 2021 abstract background: anemia is the main cause of morbidity and mortality among pregnant women in developing countries with maternal and fetal consequences, which leads to premature births, low birth weight, fetal cognitive impairment and death. objective: to determine predictors of anemia among pregnant women attending anc at public hospitals of sidama region, ethiopia, 2021. methods and materials: a facility based unmatched case-control study design will be conducted at public hospitals of sidama region. a total of 6 midwives, 6 laboratory technician and 6 supervisors will be involved in the data collection process. cases will be recruited consecutively as they present to the hospitals and immediately four controls will be allocated for each case that came after selection of cases. the data will be entered into epidata software and exported to spss software for windows version 23 for analysis. descriptive statistics will be computed and both bivariable and multivariable logistic regression will be employed to identify predictors of anemia among pregnant women. the output will be presented using adjusted odds ratio (aor) with the respective 95% confidence interval (ci). budget and work plan: a total of 184,928 ethiopian birr will be required to carry out this study. the data collection will be conducted from june 25 to july 25, 2021. study protocol tegegne et al.; aji4(1): 190-214, 2021; article no.aji.78308 191 keywords: anemia; predictors; pregnancy. 1. introduction 1.1 background anemia implies a reduction in capacity of red blood cells to transport oxygen to tissues as a result of fewer circulating erythrocytes than normal or a decrease in the concentration of hemoglobin (hgb). anemia during pregnancy is defined as a hemoglobin concentration less than 11gram per deciliter (g/dl) and classified as mild (10.0–10.9g/dl), moderate (7.0–9.9g/dl) and severe <7g/ dl. currently, world health organization (who) recognized that the hemoglobin value less than 11.0 g/dl at 1st and 3rd trimesters and less than 10.5 g/dl in the 2nd trimester is used to define anemia [1]. anemia is highly dominant among pregnant adolescents due to the dual iron requirements, for their own growth and the growth of the fetus, and is less likely to access antenatal care [2]. in the world pregnant women who are affected by anemia are 56 million, out of those 17.2 million pregnant women are from africa [3]. globally iron deficiency anemia (ida) affecting about 32 million pregnancy women and is the most common cause of anemia among pregnant women [3,4] and 50 % of all the pregnant women who develop anemia live in middle and low-income countries [5]. the magnitude of anemia among pregnant women was found to be highest in developing countries, such as subsahara africa, south-east asia were 57% and 48% respectively and lowest prevalence which is 24.1% reported among pregnant women in south america [6]. prevalence of anemia among pregnant women in africa was nearly half (46.3%) [7], 62.7% pregnant women are anemic in ethiopia [3]. pregnant women might be at risk of developing anemia due to low socioeconomic conditions. the poor nutritional intake, repeated infections, poor diet, poor antenatal care service, frequent pregnancies, low health-seeking behaviors and parasitic diseases, such as malaria and hookworm are associated with anemia [8-11]. insufficient intake and poor bioavailability of ironrich foods also have significant contribution for the onset of anemia during pregnancy [11]. despite the efforts made by the government and other stakeholders, anemia during pregnancy is still a public health problem in the ethiopia. 1.2 statement of problem in 2011, 38% (32.4 million) of pregnant women aged 15-49 years were anaemic globally [12]. africa (61.3%) and southeast asia (52.5%) are regions with the highest rate of anemia during pregnancy in the world [13]. anemia is still a public health problem in ethiopia. according to the central statistical agency of ethiopia 2016 report [14], the prevalence of anemia among pregnant women was 29% which decreases with increasing women’s education and household wealth. the most common obstetric problems of anemia during pregnancy include; less exercise tolerability, puerperal infection, thromboembolic problems, postpartum hemorrhage, pregnancyinduced hypertension, placenta previa and cardiac failure, abortion, prematurity, intrauterine fetal death, neonatal low birth weight, postnatal mortality and morbidity [15-17]. who intended to decrease anemia among reproductive-age women including pregnant women by 50% up to 2025 [18]. ethiopian ministry of health also tried to mitigate the problem of anemia and its impact through the implementation of essential nutrition action [19]. our study is important to develop strategies according to local conditions, taking into account the specific determinants of anaemia in the study area and among pregnant women. furthermore, most of the previous cross-sectional studies conducted in ethiopia recommended analytic study like case-control studies to be conducted [17,20-22]. in sidama zone, previously one case control study was conducted from february to march 2011 to identify predictors of anemia among pregnant women in hawassa and yirgalem cities [23], but our study differs in several ways from the previous study. one we will used women dietary diversity score and muac to assess nutritional status of pregnant women so this will help to identify nutritional factors associated with anemia and second we will do laboratory examination on peripheral morphology of red blood cells and this will give information on the type of anemia that a pregnant women tegegne et al.; aji4(1): 190-214, 2021; article no.aji.78308 192 developed and third we include water sources and sanitation ,this help to test the association between safe water sippy and anemia among pregnant women and fourth we include all public hospitals in sidama region. a lot has been done to minimize the risk of anaemia, but the complication of anaemia is still a problem amongst pregnant women. the true predictors of anaemia were not well addressed in the study area. therefore, this study will tried to investigate the stated information gaps among pregnant women so as to give evidence based action. 1.3 significant of the study despite the efforts made by the government and other stakeholders, anemia during pregnancy is still a public health problem in ethiopia. research findings have revealed that determinants of anemia vary from place to place. this highlights the importance of determining the problem-based factors associated with anemia using a strong study design in order to obtain local data in the regions. in sidama, to the best of current knowledge, no research exists that has used case control study design to identify predictors of anemia among pregnant woman. the finding of this study would help to guide the antenatal care service providers and other concerned stakeholders to work more towards alleviating the problem. also it might be used as a base line data for other researchers who are interested on this area. 2. literature review 2.1 factors associated with anemia anemia during pregnancy has a variety of causes and contributing factors including socioeconomic conditions, abnormal demands like multiple pregnancies, teenage pregnancies, malnutrition, maternal illiteracy, unemployment, short pregnancy intervals, age of gestation, primigravida and multigravida, loss of appetite and excessive vomiting in pregnancy [24]. a study conducted in canada revealed a strong significant association between intestinal parasitic infection and anemia in pregnant women [25]. a study conducted among pregnant women receiving antenatal care (anc) at fatima hospital in jashore, bangladesh showed that monthly family income, family size, gestational age (third trimester), birth spacing < 2 years [aor (95% ci), excessive blood loss during previous surgery (yes), food group eaten 24 hours (1 – 4 groups), breakfast regularly were predictors of anemia among pregnant women [26]. an analysis of recent national survey data to identify determinants of anemia among women and children in nepal and pakistan elucidate that anemia was significantly higher among women from the poorest households in pakistan, women lacking sanitation facilities in nepal, and among undernourished women (bmi < 18.5 kg/m2) in both countries (nepal and pakistan) [27]. furthermore, a research findings from study done in yemen cited that low family monthly income, short pregnancy spacing, never consumed liver, and presence of health problems as a risk factors associated with anemia [28]. across sectional study which was done at different gestational periods of 320 pregnant woman visiting antenatal care clinic at kakamega county (kenya) shows that, anemia was not significantly associated between age and anemia but there was significantly association between anemia and socio-economic status of the expectant mothers [29]. prior studies in ethiopia have reported significant associations between anemia in pregnancy and parasitic infections (e.g. schistosomiasis, hookworm infection), prior use of contraceptives, use of iron supplementation, birth spacing/ intervals, parity and gravidity, educational attainment, age, body weight, trimester of pregnancy and wealth status [21,30-36]. a cross sectional study which was done to assess prevalence of anemia and associated risk factors among pregnant women attending antenatal care in azezo health center gondar town, northwest ethiopia show that, anemia was significantly associated with age groups ranged from 26-34 years old and age groups greater than 34 years old. rural residence was significantly associated with reduced anemic cases [37]. institution based cross sectional study which was done to assess prevalence of anemia and its associated factors among all pregnant women tegegne et al.; aji4(1): 190-214, 2021; article no.aji.78308 193 attending antenatal care (anc) in mizan tepi university teaching hospital, south west ethiopia revealed that, anemia was significantly associated with history of malaria attack, and infections with hookworm, ascaris lumbricoids, s. mansoni, giardia intestinalis, and entamoeba histolytica/dispar. however, iron supplement was protective of anemia [38]. a study on prevalence of anemia and associated factors among pregnant women in ethiopia indicated that undernutrition as seen in mid upper arm circumference (muac) being less than 23cm and food consumption score being poor and borderline category, were factors independently associated with anemia [39]. a result of study conducted at jigjiga revealed that previous chronic diseases with, knowledge about anemia, excessive menstrual bleeding, history of malaria attack and history of abortion had significant association with anemia [40]. a community based crosssectional study was conducted to assess prevalence of anemia and associated factors among pregnant women in southern ethiopia revealed that household monthly income, level of food security, availability of latrine, frequency of meal per day, eating animal source of food at least once per week, history of malaria infection, and nutritional status; low socio economic class, trimester second and third, gravidity three to five and six and above, iron not supplemented, hookworm infection and low dietary diversity score showed statistically significant association (p<0.05) with anemia [41]. fig. 1. conceptual framework showing anemia and associated factors among pregnant women attending anc in sidama region, 2021. developed from different literatures reviewed [34-42] tegegne et al.; aji4(1): 190-214, 2021; article no.aji.78308 194 unmatched-case control study conducted at hawassa and yirgalem cities found lower educational level, prolonged menstruation period 6-8 days before the index pregnancy, intestinal parasitic infection, gastritis with duodenal ulcer bleeding and not taking meat/organ meats as major predicting risk factors for occurrence of anemia among pregnant women [23]. a study conducted to determinants of anemia among pregnant women attending antenatal clinic in public health facilities at durame town also identified were parasitic infection, not taking additional diet during pregnancy, consuming tea/coffee immediately after food, not eating meat, previous heavy menstrual blood flow, and being housewife as a major determinant factors of anemia among pregnant women [42]. 3. objective to identify predictors of anemia among pregnant women attending anc service at public hospitals of sidama region, ethiopia, 2021. 4. methodology 4.1 study area the study will be conducted in public hospitals of sidama region. it is located about 275 kilometers away from addis ababa. it has 30 districts, 1 city administration and 6 town administration with a total of 576 kebeles of which 524 of them are rural and 52 are urban. it is one of the highly populated areas in ethiopia, having a total population of about 4 million people residing on 72100 hectare of land. out of the total population 5.7% are urban and 94.3% rural residents [43]. sidama is characterized by three agro-ecological zones: the dry midlands/lowlands (20%), the midlands (48%) and the highlands (32%). in sidama region mixed agriculture (crop and livestock production) is practiced. major crops grown include: enset, coffee, maize, wheat, teff, barley, haricot bean and khat. enset is the main staple crop both in highlands and midlands while maize is so in the lowlands. there are two cropping seasons in sidama zone: belg and meher. belg rains are mainly used for land preparation and planting of long cycle crops such as maize and sorghum and seed bed preparation for meher crops. the meher rains are used for planting of cereal crops like barley, teff, wheat and vegetable crops. besides, meher rains are also responsible for the growth and development of perennial crops such as enset, coffee and khat. food security is more precarious in the lowland areas of aleta wondo, borecha, darra, bensa, loka abaya and hawassa zuria woredas mainly due to moisture stress and water logging in some pocket areas hampering agricultural production, less diversification of food sources and minimum use of improved farm inputs due to lack of cash and credit facilities to purchase the inputs. the sidama region administration has a total of 4063 health professional of different disciplines and 524 health posts, 127 health centers, 1 general and 12 district hospital owned by government and additionally there are 21 private and 3 ngo clinics, 65 private rural drug venders. the overall potential health service coverage of the zone by public health facilities are 90.3%. 4.2 study design and period a facility based unmatched case-control study will be conducted 4.3 source population all pregnant women attending anc service at public hospitals of sidama region will be the source population. 4.4 study population all pregnant women attending anc at public hospitals during study period and fulfilled the inclusion criteria will be the study population for this study. 4.5 eligibility criteria 4.5.1. inclusion criteria  pregnant woman who attending first anc visit  permanent resident pregnant woman (at least 6 months). 4.5.2 exclusion criteria  pregnant woman with severe illness and unable to speak and second and third visit. 4.6 sample size determination epinfo version 7 software will be applied to compute the sample size for cases and controls with an assumption of 95% confidence level, 80 % power of the study, 4:1 (r=4) ratio of nonanemic over anemic, the odds ratio = 2.5 from factors that has association with anemia from tegegne et al.; aji4(1): 190-214, 2021; article no.aji.78308 195 table 1. sample size calculation for the study factors aor % of controls exposed power ratio of controls to cases ci anemic non anemic final sample size source intestinal parasite(yes/no) 2.9 11.6 80 1:4 95 62 248 310 (23) previous heavy menstrual flow(yes/no) 2.62 12.2 80 1:4 95 75 300 375 (42) meat (yes/no) 2.8 18.7 80 1:4 95 51 201 252 (23) additional food(yes/no) 2.5 9 80 1:4 95 105 419 524 (42) tegegne et al.; aji4(1): 190-214, 2021; article no.aji.78308 196 fig. 2. schematic representation of sampling procedure recent study conducted in durame and proportion of controls exposed 9.0% [42], the maximum sample size after adding the potential none response rate of 10% the total sample size became 576 (115 cases and 461 controls). the computation is depicted in the following table (table 1). 4.7 sampling techniques from 18 public hospitals found in the region we randomly selected 6 hospitals. enumeration of prior three month’s anc register was conducted in order to know monthly flow of each hospital. then, based on the number of anc register, the sample size was allocated proportionally for all selected public hospitals in the region (fig. 2). cases will be recruited consecutively as they present to the hospitals and immediately four controls will be allocated for each case that came after selection of cases. 4.8 study variables dependent variable for objective one: anemia. independent variables:  sociodemographic; clinical and reproductive; nutrition and lifestyle; and knowledge and health service related factors are independent variables. 4.9 operational definitions anemia: any hemoglobin level below 11g/dl in first and third trimesters and below 10.5gm/dl in the second trimester of gestation is considered as anemia [1]. tegegne et al.; aji4(1): 190-214, 2021; article no.aji.78308 197 nutritional assessment: nutritional assessment of woman will be done using anthropometry and dietary methods.  a mid-upper arm circumference (muac) measurement of < 23cm and ≥23cm will be classified as malnutrition and normal nutritional status, respectively [43].  dietary diversity (dd) will be categorized as adequate (consumption of at least five of the ten food groups) and inadequate (consumption of less than five food groups) [44]. knowledge about anemia: we will used eight items composite score to measure the knowledge level of respondents regarding anemia which includes: general signs of anaemia, iron-rich foods, foods that increase iron absorption, foods that decrease iron absorption, causes of anaemia, consequences of anaemia for pregnant women and infants and young children, and prevention of anaemia. the cumulative mean score of knowledge of participants about anemia will be estimated using mean score. based on this, those who had scored less than the mean will be considered to have poor knowledge and those who had scored greater than or equal to the mean value will be considered as having good knowledge. 4.10 data collection procedure data will be collected using a structured and pretested questionnaire, anthropometry and laboratory analysis of stool specimens. a total of 6 midwives, 6 laboratory technician and 6 supervisors will be involved in the data collection process. 4.10.1 questionnaires the questionnaire included information on sociodemographic; clinical and reproductive; nutrition and lifestyle; and knowledge and health service related factors will be collected using a structured and pre-tested questionnaire, anthropometry and laboratory analysis of stool and blood sample. the part of the questionnaire on dietary diversity (dd) was adopted from a standard tool [44]. 4.10.2 anthropometric assessment 4.10.2.1 anthropometric assessment of nutritional status nutritional status of pregnant mother is measured using muac which is the only anthropometric measure for assessing nutritional status among pregnant women [43]. mid-upper arm circumference (muac) of the mother will be measured using flexible non-stretchable standard tape measure as measure of nutritional status. 4.10.2.2dietary methods of assessing nutritional status the dd was assessed using 24 hours recall method. respondents will be asked whether they had taken any food from predefined 10 food groups on the preceding day. accordingly, the level of dietary diversity score (dds) will be computed out of 10 [44]. 4.10.3 fecal sample collection and laboratory analysis 4.10.3.1 fecal sample collection following the completion of questionnaire, a wide screw capped containers pre-labeled with names will be distributed to each respondent. participant who will not able to provide sample on the first day were asked again on the following day. 4.10.3.2 fecal sample laboratory analysis initially, an adhesive cellophane tape with a glass slide and a fecal specimen container will be distributed to the pregnant women. approximately 2mg fecal specimen in labeled plastic vials containing 10% formaldehyde for the preservation of helminth eggs, protozoan cysts, and trophozoites in the fecal specimens will be collected. the stool specimens will be examined for the presence of parasites, helminth eggs, and larvae and protozoan trophozoites or cysts using direct wet mount. 4.10.4 blood sample collection and laboratory analysis labeled venous or heparinized blood samples giving sequential numbers of the study participants will be used. blood samples will be used for hemoglobin determination (by using hemocue) and rbcs morphology identification. hemoglobin determination venous blood sample will be taken, filled to micro cuvette, wipe of excess blood from the outside of the micro cuvette tip, and then placed in the cuvette holder of the device for measuring hemoglobin concentration [45]. this hgb determination will be done by selected hospitals as parts of routine anc service. tegegne et al.; aji4(1): 190-214, 2021; article no.aji.78308 198 4.11 data quality control three day intensive training will be given on how to perform muac measurement and on interviewing techniques using standard checklist and structured questionnaire. the checklist and questionnaires will be translated into a regional working language (sidaamu afoo). supervision will be conducted. double data eatery will be done and the questionnaire will be pretested on 5 % of total sample size at leku primary hospital. during data collection, continuous supervision will be done by the supervisors and principal investigator. 4.12 data processing and analysis the data will be entered into epidata software and exported to spss software for windows version 23 for analysis. descriptive statistics will be computed and both bivariable and multivariable logistic regression will be employed to identify predictors of anemia among pregnant women. 5. work plan and budget 5.1 work plan table 2. showing the work plan of the study in sidama region, 2021 activity apr may jun jul aug sep oct nov dec research proposal preparation final proposal submission and defense obtaining ethical clearance giving training data collection and processing result writing final paper submission defense and dissemination of result 5.2 budget table 3. showing the stationary cost for the study in sidama region, 2021 no items source of budget unit amount unit price(birr) total price(birr) 1 pen hchs pieces 30 6 180 2 pencils >> pieces 50 3 150 3 paper for duplication >> packet 30 300 9,000 4 pencil sharpener >> pieces 18 5 90 5 pencil eraser >> pieces 18 5 90 6 notebook >> pieces 22 20 440 sub total 9,950 table 4. showing the services cost for the study in sidama region, 2021 no items source of budget unit amount unit price(birr) total price(birr) 1 photo copying (including the pre-test) >> pieces 634 8 5,072 2 lab investigation for stool specimen specimen 10 10*634 6,340 sub total 11,412 tegegne et al.; aji4(1): 190-214, 2021; article no.aji.78308 199 table 5. showing the training cost for the study in sidama region, 2021 no items source of budget unit unit price(birr) total price(birr) 1 12 data collectors hchs 12 339*12*1 4,068 2 6 supervisors >> 6 6*339*1 2,034 3 trainer(pis) 4 4*339*1 1,356 sub total 7,446 table 6. showing the perdiem payment for the study in sidama region, 2021 no items source of budget unit unit price (birr) total price(birr) 1 data collector hchs per questionnaire 100*634 63,400 2 supervisor >> 339 per day 339*6*30 61,020 sub total 124,420 table 7. showing the total cost of the study in sidama region, 2021 no items/activities source of budget unit total price(birr) 1 stationary hchs birr 9,950 2 service (transportation etc) >> >> 11,412 3 training >> >> 7,446 4 perdiem for data collection and supervisors >> >> 124,420 grand total 153,228 6. conclusion the findings will be presented to the hawassa college of health science community and submitted to hawassa college of health science research and publication core process owner. the findings will also be communicated to local health planners and other relevant stake holders in the area to enable them take recommendations in to consideration during their planning process. it can also be communicated to health planners and managers at regional level through hawassa college of health science website and library. efforts will be made to publish in peer reviewed national and international journals. consent written permission will be obtained to undertake the study from the selected hospitals. participation in the study will be based on voluntary base and the participants will be informed about the right to withdraw at any time from the study. confidentiality will be assured by using anonymity. pregnant women who had anemia (hb<11g/dl) will be provided with iron-folate tablets and those who were in the third trimester and infected with intestinal parasites will be dewormed. written consent will be requested from every study participant included in the study during data collection time after explaining the objectives of the study. for this purpose, a one page consent letter was attached to the cover page of each questionnaire stating about the general objective of the study and issues of confidentiality which was discussed by the data collectors before proceeding with the interview. ethical approval prior to data collection appropriate ethical clearance and supportive letter will be obtained from the ethical review committee of hawassa college of health science. competing interests authors have declared that no competing interests exist. references 1. who. haemoglobin concentrations for the diagnosis of anaemia and assessment of severity. vitamin and mineral nutrition information system. geneva: world health organization; 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2020. doi: 10.25259/ijms_5_2020 29. mulambalah chrispinus siteti sdn, ogutu philip ariya, siteti darwin injete waw. anemia in pregnancy: prevalence and possible risk factors in kakamega county, kenya. science journal of public health. 2014;9(4):76-81. 30. melku m, addis z, alem m, enawgaw b. prevalence and predictors of maternal anemia during pregnancy in gondar, northwest ethiopia: an institutional based cross-sectional study. anemia; 2014. 31. kefyalew aa, abdulahi md. prevalence of anemia and associated factors among pregnant women in an urban area of eastern ethiopia. anemia. 2014;561–7. 32. desalegn s. prevalence of anaemia in pregnancy in jima town, southwestern ethiopia. ethiop med j. 1993;31(4):251–8. 33. abriha a, yesuf me, wassie m. prevalence and associated factors of anemia among pregnant women of mekelle town: a cross sectional study. bmc res notes. 2014;7:888. 34. bekele a, tilahun m, mekuria a. prevalence of anemia and its associated factors among pregnant women attending antenatal care in health institutions of arba minch town, gamo gofa zone, ethiopia: a crosssectional study. anemia. 2016;2016:1073192. 35. gedefaw l, ayele a, asres y, mossie a. anemia and associated factors among pregnant women attending antenatal care clinic in wolayita sodo town. southern ethiopia ethiop j health sci. 2015;25(2):155–62. 36. getachew m, yewhalaw d, tafess k, getachew y, zeynudin a. anaemia and associated risk factors among pregnant women in gilgel gibe dam area, southwest ethiopia. parasites vectors. 2012;5: 296. 37. alem m, enawgaw b, gelaw a, kena t, seid.m, olkeba y. prevalence of anemia and associated risk factors among pregnant women attending antenatal care in azezo health center gondar town, northwest ethiopia. j interdiscipl histopathol. 2013;1(3):137-144. 38. zekarias b, meleko a, hayder a, nigatu a, yetagessu t. prevalence of anemia and its associated factors among pregnant women attending antenatal care (anc) in mizan-tepi university teaching hospital, https://doi.org/10.4236/fns.2019.109076 https://doi.org/10.4236/fns.2019.109076 tegegne et al.; 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2016. 45. cheesbrough m. district laboratory practice in tropical countries 2009, part 1. 2nd ed. cambridge: cambridge university press; 2009. https://doi.org/10.1155/2018/8938307 https://doi.org/10.1155/2018/8938307 tegegne et al.; aji4(1): 190-214, 2021; article no.aji.78308 203 annex i: information sheet good morning/good afternoon. my name is ______________i am here on the behalf of research team of hawassa health science college. the team is conducting research on ‘predictors of anemia among pregnant women attending antenatal care at public hospitals of sidama region, ethiopia, 2021’. you are selected by random sampling technique to participate in this study because you are currently taking anc service at this hospital. your participation will only be based on your willingness .you have the right to choose not to take part in this study. if you choose to take part, you have the right to stop at any time. if you are willing to participate or refuse or decide to withdraw later, you will not be subjected to any ill-treatment. if you agree to participate in the study, you will be interviewed about socio-demographic characteristics, knowledge and health service related factors, clinical and reproductive factors, and nutrition and lifestyle related factors. your name will not be written on the questionnaire. no one will have access to the non-coded data except the principal investigator and the data will only be used for this study. your willingness and honest answers are very important for the success of this study. we would like to appreciate your help in responding to these questions, and it will not take more than 30 minutes. annex ii. informed written consent form i (the respondent), the undersigned, am told that the researchers are going to conduct study in sidama region governmental hospitals to assess the risk factors of anemia and its effect on birth weight and s/he acquainted with me the first time s/he meets. i also informed that both the government and the woreda health office to commence appropriate strategies to battle this problem would use the result of the study. i am, too, told that the research will benefit the community in general including me, the respondent, and that the research will not inflict any harm to me. besides, i briefed that i will be interviewed for not more than 20 to 30 minutes. in addition, i let know that the investigators selected me randomly. moreover, i am notified that my participation in the study is entirely voluntarily, and that i can quit from the study any time i want. likewise, i am enlightened that i will not be subjected to any form of punishment following my failure to participate in the study. in the same way, i am explained that the information collected from me will not by any means be disclosed to any people other than those participating in the study unless obtained permission from me. equally, i told that i could ask them questions i found difficult or any type otherwise. are you willing to participate in the study? yes _______ no____ signature _________ study area: sidama region health facility___________________ name of the interviewer______________ sig. _____ date ________ name of supervisor _____________sign _______ date_______ questionnaire code ____________________ annex iii: questionnaires i. socio-demographic characteristics of pregnant women code variables coding categories skip 100 age of respondent ___________ 101 residence 1. rural 2. urban 102 marital status 1. never married 2. married 3. separated 4. widowed tegegne et al.; aji4(1): 190-214, 2021; article no.aji.78308 204 103 religion 1. protestant 2. orthodox 3. muslim 4. other 104 mothers educational level 1. no formal education 2. primary (1-8) 3. secondary (8-12) 4. college and above 105 husband educational level 1. no formal education 2. primary (1-8) 3. secondary (8-12) 4. college and above 106 occupation status 1. government employee 2. self employed 3. housewife 4. farmer 5. other (specify)_____________ 107 what is the source of the food for the family? 1. buying from market 2. farming(crops, animals) 3. food aid/donation 4. other(specify) 108 who has the primary responsibility of providing food for the house hold? 1. father 2. mother 3. grandmother 4. relatives 5. other(specify) 109 do you have a bank saving account? 1.yes 2. no 110 average monthly income of the family in birr________ 111 family size 112 what is the main source of drinking water for members of your house hold? 1. purified water 2. tap water 3. public tab/stand pipe 4. protected well 5. unprotected well 6. other specify 113 did you take any action to make the water safer to drink? 1.yes 2. no if no, skip to q115 114 if yes, what do you usually do to make the water safer to drink? 1. boiling 2. chlorine-water 3. sand 4. guard/bishangari/aqua tab 5. other(specify) 115 what kind of toilet your family members usually used? 1. flush to piped sewer system 2. flush to septic tank 3. ventilated improved pit latrine 4. pit latrine with slab 5. pit latrine without slab 6. no facility/ bush/field 7. other( specify) ii. clinical and reproductive related factors of pregnant women code variable coding categories skip 200 when did you see your last menstrual 1. ____/_____/___ tegegne et al.; aji4(1): 190-214, 2021; article no.aji.78308 205 period? ( enter date, month & year) __e.c 2. i don’t know 201 have you ever given a birth? 1. yes 2. no 202 parity 1. para one 2. para two 3. para three or more 203 gravidity _______________ 204 gestational age 1. 1st trimester 2. 2nd trimester 3. 3rd trimester 205 what is the interval between current and previous pregnancy? 1. < 2 years 2. ≥ 2 years 206 age at first pregnancy(years) 207 age at first marriage (years) 208 menstrual cycle 1. regular 2. irregular 209 menstruation by no of pads 210 duration of menstruation in days __________________ 211 number of miscarriages (spontaneous abortion) __________________ 212 number of stillbirths __________________ 213 children <5 yrs death 215 1. 216 gastritis 1. yes 2. no 217 duodenal bleeding(presence of hematemesis) 1. yes 2. no 218 history of medication (quinine, cephalosporin, penicillin, methyldopa, nsaids(aspirin, ibuprofen)) 1. yes 2. no 219 history of induced abortion 1. yes 2. no 220 contraceptives use 1. yes 2. no if no skip to 222 221 types of contraceptive methods 1. pills 2. injectable 3. implant 4. iucd 5. other 222 intestinal parasite 1. yes 2. no if no skip to224 223 list types of i/p 3. 224 do you have any diagnosed chronic disease previously? 1. yes 2. no 224 if yes, specify the name of diagnosed chronic disease? ---------------- 225 did you infected with malaria in the last 3 months? 1. yes 2. no 226 diarrhea illness in past two week 1. yes 2. no 227 hemorrhoids 1. yes 2. no 228 gum bleeding 1. yes 2. no 229 presence of current blood loss 1. yes tegegne et al.; aji4(1): 190-214, 2021; article no.aji.78308 206 2. no stis 1.yes 2. no hiv status (by review records) 1.positive 2. negative iii. nutrition and lifestyle related factors of pregnant women code variable coding categories skip 300 muac in cm _____________ 301 what is your staple diet? 1. teff 2. enset based 3. maize based 4. roots 5. others(speci fy) 302 main meal frequency in a day 1.one times 2.two times 3. three times 303 additional meal during pregnancy 1. yes 2. no 304 eat foods made from teff 1. yes 2. no 305 average weekly consumption of animal source foods like meat, chicken, milk, egg, cheese, fish? 1. 1-2 times 2. ≥3 times 3. i have not eaten 306 eating condition during pregnancy 1. increased 2. no change 3. decreased 307 substance use (cigarate, alcohol, khat) 1. yes 2. no 308 drinking tea immediately after meal 1. yes 2. no 310 drinking coffee immediately after meal 1. yes 2. no 312 consistent use of bed net 1. yes 2. no 313 wearing shoes consistently 1. yes 2. no iv. 24 hour food frequency questionnaire code food groups coding categories consumed yes = 1 no = 0 400 grains, white roots and tubers, and plantains maize, rice, wheat, sorghum, millet or any other grains or foods made from these (e.g. bread, porridge). 401 pulses mature beans or peas (fresh or dried seed), lentils or bean/pea products. 402 nuts and seeds beans, peas, lentils, nuts, seeds or foods made from these 403 dairy milk, cheese, yoghurt or other milk products 404 meat, poultry and fish fresh or dried fish, beef, goat, chicken 405 eggs eggs from poultry 406 dark green leafy vegetables cassava leaves, kale, spinach etc. 407 other vitamin a-rich fruits and mangos, apricots tegegne et al.; aji4(1): 190-214, 2021; article no.aji.78308 207 vegetables 408 other vegetables tomato, onion etc. 409 other fruits other fruits, including wild fruits v. knowledge and health service related factors code variable coding categories skip 500 have you heard about anaemia? 1.yes 2. no 501 if yes: can you tell me how you can recognize someone who has anaemia? (at least two) less energy/weakness paleness/pallor spoon nails/bent nails (koilonychia) more likely to become sick (less immunity to infections) other don’t know 1.yes 2. no 502 what are the health risks for infants and young children of a lack of iron in the diet? (at least one) delay of mental and physical development other don’t know 1.yes 2. no 503 what are the health risks for pregnant women of a lack of iron in the diet? (at least one) risk of dying during or after pregnancy difficult delivery other don’t know 1.yes 2. no 504 what causes anaemia? (at least two) lack of iron in the diet/eat too little, not much sickness/infection (malaria, hookworm infection, other infection such as hiv/aids) heavy bleeding during menstruation other don’t know 505 how can anaemia be prevented? (at least two) eat/feed iron-rich foods/having a diet rich in iron eat/give vitamin-c-rich foods during or right after meals take/give iron supplements if prescribed treat other causes of anaemia (diseases and infections) seek health-care assistance other don’t know 506 can you list examples of foods rich in iron? (at least two) organ meat (liver, kidney, heart etc.) flesh meat (beef, pork, lamb, goat, rabbit, dog, chicken, duck, etc.) fish and seafood (fresh fish, dried fish, canned fish etc.) tegegne et al.; aji4(1): 190-214, 2021; article no.aji.78308 208 506 when taken during meals, certain foods help the body absorb and use iron. what are those foods? (at least one) vitamin-c-rich foods, such as fresh citrus fruits (orange, lemons, etc.) other don’t know 507 some drinks decrease iron absorption when taken with meals. which ones? (at least one) coffee tea other don’t know 508 nutrition education in pregnancy 1. yes 2. no 509 one-way walking distance from nearby health facility 1. 0-30 minutes 2. 0-30 minutes 510 at what gestational age did you start your first anc visit 1. weeks________ 2. i don’t know 511 did you get any iron supplement in this pregnancy? 1. yes 2. no 512 if yes, how many days did you take? 1. no. of days_______ 2. i don’t know 513 did you take any anti helminthic drugs previously? 1. yes 2. no 513 home delivery (previous) 1.yes 2. no vi. data related with laboratory analysis type of investigation result stool examination hgb determination perpheral morphology annex iv: keere galtini/keere hossini ani ______________kowe leelomohu hawaasi fayyimate koleejje xiinxallote gaamo riqiweeti.tini gaamo xiinxallo asitanni noohu 2013m.d sidaamu qoqowi giddo heedhanno hospitaalera ilate albise buuxirate daanno amuuwi mundeete xe’ne abbanore buuxateeti. ati hedeweelchote tenne xiinxallora beeqancho ikakki ati xaa yannara tenne tenne hospitaalera ilate albiidi buuxo aana nootahuraati.beeqokki umikki fajjonni calaati.tenne xiinxallora dawaro aa giwate wo’ma dandoo noohe.dawaro aate sumuu yiitarono aye yannarano agurate qoosokki agarantinote. dawaro aate sumuu yiitaro woyi giwittaro woyi agure fulate sumuu yiitarono owaante adhate ledo xaadannokkita xawinseemmo. tenne xiinxallora dawaro aate sumuu yiitaro heeshokki gari daafira,fayyimatenna xaadonorira noohe egenora,kilinikaalenna sirote daafiranna saga’latenna hee’nanni gari daafira afuu xa’mo xa’mineemohe. su’mikki xa’mote woraqati aana diboreesamanno. jawiidi hajajaancho aguranna ayeeno ikkiro uyinoonni kodde hunara dandoo dinosi qoleno dawarootta dawaro tenne xiinxallora calla horonsi’nannite. ate sumuu yaakkinna halaalancho dawarokki tenne xiiinxallo gumulate lowo horo noosete. tegegne et al.; aji4(1): 190-214, 2021; article no.aji.78308 209 tenne xa’muwa qolate asoota kaa’lora wodaninni galatoofantanni 30 daqiiqi aleenni keeshishannokitano qummi asineemmo annex v: sumuu yaa xawisate forme ani (dawaro eemati) woroonni malaatisa’yanni kuni xiinxallo assanohu sidaamu qoqowi giddo mangitete hospitaalera mundeete xe’ne abannorenna godowi qaaqi lophpho aana abanno qarrira xa’maera umi yannara xaadeenaeeti.mangitenna woradu fayimate biiro tenne xiixalote guma adhite soorote horonsidhara sumuu yitinotano xawiseenaeti.tini xiixallo aneno ikko wole dagooma horo uyitanote yee hedeemahuraati.qoleno dawaro aa’yanni ane aano daanno qarri nooikihuraati.konnira 20-30 daqiiqa sainokki yanna giddo isi/ise ledo hasaawate wo’naaleemma. qoleno xa’mituerino hedeweelcho doortinoetano roorimankanni dawareemahuno umi’ya fajjoni ikkinota xawisa hasireemma hasirumma yannara dawara agurammara woyi hasaawa uurisamara wo’ma fajjo nooeta xawisaniiti.xiinxallote xa’mo dawara hooga’yanni qorochishanoehu nooekita afa’ya lede xawisaniiti.qoleno anewiini afi’nooni dawaro ane fajjo nookiha wolehu reqecci assa dandiinanikkita yaano xiixallo asitanno bissa gobaani wolu afara dandaanokitano xawinseenaeeti.taaloteno duushitinoekki xa’mo heedhuro xa’me dawara afira dandeematano afeeti. dawarate maaye yaatta? eewa………………….dee’ni…………….malaate…………… xiinxallote darga.sidaamu qoqowo fayyimate mine…………………………. xa’amanohu su’ma………………………….malaate……………………barra……………… loosisannohu su’ma……………………….malaate……………………barra……………… xa’mote woraqati koodde……………………….. ledote hasattora aliidi xinxaalaano afirate su’ma abiyu ayaalew bilbilu kiiro;-+251912417806 e-mail su’ma alelinyi taadele bilbilu kiiro;-+251913440394 e-mail su’ma qaleaabi tesifaaye bilbilu kiiro;-+251911066932 e-mail su’ma sintaayehu aseffa bilbilu kiiro;-+251911392734 e-mail annex vi: xa’mote woraqata i godowii noo ama daafo addi addi xa’mo koodde variable koodete dana agure sa’a 100 dirose ………………………… 101 teesose 1. baadiyye 2. quchumaho 102 adhamate gara 1. adhantinoikite 2. adhantinote 3. tirtinote/babaxitinote 4. adhinohu reenosete 103 ama’nose 1. ortodokisete 2. kaatolikete 3. protestaantete 4. musiliimete 104 amase rosu deerra ……………………… 105 galtese rosu deerra …………………….. 106 loosise dana 1. umise looso 2. minu amaati 3. mangitete looso 4. wolu heeriro xawisi….. tegegne et al.; aji4(1): 190-214, 2021; article no.aji.78308 210 107 maatese sagale afidhanowa? 1. dikkote hidhite 2. gatiiidi jajinni 3. sagalete kaa’lonni 4. wolu nooro xawisi…… 108 maaatete sagale abate umiidi qeechchi noohu ayeerati? 1. annaho 2. amate 3. ahaahete 4. fiixaho 5. wolu nooro xawisi……………. 109 baankete suuqo noohe? 1. ee nooe 2. dinoe 110 mereerima maatese eo ……………………. 111 maatese kiiro? 112 waa afidhannowa 1. xalala waa 2. wolu heeriro xawisi….. 3. gosa hinkii’li.tanoha 4. qorowantino bue 5. qorowantinoikki bue 6. wolu nooro xawisi…… 113 waa xalala assate asatari nooni? 1. ee no 2. dino 114 nooha ikkiro maa assa? 1. waa gafe aga 2. kiloorine leda 3. shaafunni xinbiiwa 4. wayi agaraancho/bishangarwora 5. wolu nooro xawisi…… 115 hiito shumate mine horonsiratta? 1. waa duneena tuubotenni harannoha 2. sepric taanke eannoha 3. aleenni ayere eesanoha/vip/ 4. tuanchu noo shumate bale/pit with slab 5. tuanchu nooikki shumate bale/ pit without slab 6. xawoho/mulla bayichcho/ ofolla 7. wolu nooro xawisi…… ii godowii noo ama fayyimasenna sirote kifile daafo addi addi xa’mo koodde variable koodete dana agure sa’a 200 qaccete aganu mundee mamoote laoo? 1. ………/…………/……….. 2. di afoomma 201 ilte egenootta 1. ee 2. diegenoomma 202 iltinohu ino kiiro (me’e iltino) 1. mitto iltino 2. lame iltino 3. sasenna sau ale 203 godobino kiiro (me’e higge godobino) ………. 204 godowa noohu diro(ga) 1. 1 st trimester 2. 2 nd trimester tegegne et al.; aji4(1): 190-214, 2021; article no.aji.78308 211 3. 3 rd trimester 205 godowaminohu albidiihuwinni noo xeertinynye 1. <2 diro 2. >2 diro 206 umiseha meikki dirisenni godobino 207 meikki dirisenni adhantino 208 aganunni du’nantanno mundee 1. garunni daganno 2. garimalete 209 du’nantanno mundee batinynye (saanete bikkiniro) …………………. 210 du’nantanni keeshitanno barra ……………… 211 umose kayisinosero kiiro (abortion) …………………….. 212 godowa bae ilamohu kiiro (still birth) 213 5 diri woro reyinose qaaqi nooro 214 luuxichchu xisso 1. ee noose 2. dinose 215 mundee tufa 1. ee noose 2. dinose 216 egemitanno/horonsidhanno xagichchi quinine,pencillin,methyldopa,nsa ids(asprin,ibuprofen) 1. ee noose 2. dinose 217 mannu kaa’lonni leelitino umu kao (induced abortion) 1. ee noose 2. dinose 218 ila gargadha 1. ee gargadhitanno 2. digargadhitanno gargadhitano kkiro 220 sai 219 hiikoye gargaraancho horonsidhanno egeminanniha/pills/ marfenniha/injectable/ cigilete waamaniha/implant/ sirote kifile giddo woranniha/iucd/ wolu nooro xawisi/other/ 220 heleete da’muulchchi/parasite/ 1. ee no 2. dinose nooikkiro kiiro 222 sai 221 heleete da’muulichi nooro danasi xawisi? 3. 222 albaanni wole keeshitino xidanna/chronic disease/ 1. ee no 2. dinose 223 nooha ikkiro su’mansa xawisi …………. 224 sai sasu again giddo shekeerete xiwani nooheni? 1. ee no 2. dino 225 sau 2 lamalara godowa gobara qola/deeishsha 1. ee no 2. dinose 226 qunxushote xiwani/hemmoroids/ 1. ee no 2. dino 227 egireggu mundaa 1. ee no 2. dinose 228 xaa mundeete anje 1. ee no 2. dinose 229 siimu xaadinni sa’anno xisso 1. ee no tegegne et al.; aji4(1): 190-214, 2021; article no.aji.78308 212 2. dino 230 hiv buuxo 1. positive 2. negative iii saga’litannonna heeshose gade daafira addi addi xa’mo koodde variable koodete dana agure sa’a 300 giwoodu bikko (muac)(cm) ………………….. 301 wo’mu woti sagalese 1. gaashshete qixaabinote 2. weesete qixaabinote 3. badalate qixaabinote 4. umanni rumuddate 5. wolu heeriro xawisi………… 302 barrunni me’e higge saga’litanno 1. mitte higge 2. lame higge 3. sase higge 303 godowi yannara ledote sagale no? 1. ee no 2. dino 304 gaashshe gumi karsinni loosantino sagale ittanno 1. ee itanno 2. dittanno 305 lamalatenni saadate karso me’e hige itannoro 1. 1-2 hige 2. sauna sasu ale 3. horo ditanno 306 godowii noo yannara saga’litanno gari 1. lexayi lexay haranoho 2. soorro dinooho 3. ajayi ajay harannoho 307 wole ledonire adha (alkoole,sigaara caate 1. ee no 2. dino 308 hurbaatu gedeno mulenni shae aga 1. ee no 2. dino 309 hurbaatu gedeno mulenni buna aga 1. ee no 2. dino 310 agobere wo’ma wote horonsira 1. ee no 2. dino 311 koatte wo’ma wote wodha 1. ee 2. dee’ni iv 24 saate itanno sagale deerra xa’mo koodde sagalete dana koodete dana ittanoro=1 itaakiro=0 400 gummate sagale badala,ruuze,bashanqa,hayixe, loonsoonire 401 pulses baaqela ,atara, nuugetenni loonsonire 402 saadannire ado,buuro,burbuxxo 403 maala lukkotenna asu maala freshe meu maala 404 quuphphe lukkotenni 405 haanja muro boye ,spinachete daro 406 vit a noosere mango 407 wole muro timatime,shunkurta 408 wole gumma dubbu afi’nanni gumma v afasenna keeranchimate afanshi ledo xaadanno xa’mo koodde variabile koodete dana agure sa’a 500 mundeete xe’ne abanorira egenno 1. ee noose tegegne et al.; aji4(1): 190-214, 2021; article no.aji.78308 213 noose? 2. dinose 501 ee yituro afatare kula dandaatae  wolqate anje  ille wajira  wolu herirono  diafoomma 1. ee noose 2. dinose 502 keeranchimate qarri maati mundeete xe’ne qaaqu aana abitannori (ajanna 1)  aimirote qarra  wole  diafoomma 1. ee affino 2. diaffino 503 godowii noo ama aana abanno qarri(ajanna 1)  reyo  ilate qarama  wolere  diafoomma 1. ee affino 2. diaffino 504 mundeete xe;ne abbanori(ajanna 2)  sagalete irene hooga  xisso (shekeere,hookworm)  mundeete du;nama  wolere  diafoomma 1. ee affino 2. diaffino 505 mundeete xe;ne hiito gargadhinayi(ajanna 2)  irone noo sagale itatenni  vit c sagale itatenni  irone xagichcho adhatenni  wole xisso akamatenni  wolere  diafoomma 1. ee affino 2. diaffino 506 ironr gidose noo sagale kulatae (ajanna 1)  bisu maala(afale,mule)  duumo maala  asu maala 1. ee affino 2. diaffino 507 saga’lineemo sagale bisinke iron gudanoti hiitet (ajanna 1)  vit c noo sagale  wolere  diafoomma 1. ee affino 2. diaffino 508 bisinke ironete adho qanasanno sagale  shae  buna  wolere  diafoomma 1. ee affino 2. diaffino tegegne et al.; aji4(1): 190-214, 2021; article no.aji.78308 214 509 godowii noo ama saga’litano rosichchi 1. ee noose 2. dinose 510 keeranchimate mine minisenni noo xeertinynye 1. 30 daqiiqi ale 2. 30 daqiiqi woro 511 meikki trimesteraati umi buuxo asirittahu(anc)? 1. lamala ………………. 2. diafoomma 512 godowi yannara ironete adhite egenootta 1. ee 2. degenoomma 513 ee yituro me’e higge 1. barru kiiro…………. 2. diafoomma 514 godowi damuula garagat-rtanno xagga adha 1. ee 2. degenoomma 515 alba mine ilte egenoota 1. ee 2. degenoomma vi labratorete xaadanno data types of investigation result stool examination hgb determination peripheral morphology _________________________________________________________________________________ © 2021 tegegne et al.; this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: https://www.sdiarticle5.com/review-history/78308 http://creativecommons.org/licenses/by/4.0 _____________________________________________________________________________________________________ *corresponding author: e-mail: arvindkumarshukla@pusan.ac.kr; arvindkumarshuklapnu@gmail.com cite as: shukla, sandhya, arvind kumar shukla, navin ray, adarsha mahendra upadhyay, giovanna mirone, and raj kumar mongre. 2024. “long-lasting response of human circulating t-follicular helper cells (ctfh) to post sars-cov-2 mrna immunization”. asian journal of immunology 7 (1):228-46. https://journalaji.com/index.php/aji/article/view/147. asian journal of immunology volume 7, issue 1, page 228-246, 2024; article no.aji.125387 long-lasting response of human circulating t-follicular helper cells (ctfh) to post sars-cov-2 mrna immunization sandhya shukla a, arvind kumar shukla b*, navin ray c, adarsha mahendra upadhyay d, giovanna mirone e and raj kumar mongre f a department of pharmacology, bharati vidyapeeth deemed university medical college, pune-411043, india. b school of biomedical convergence engineering, pusan national university, yangsan-50612, south korea. c laboratory of mucosal exposome and biomodulation, department of integrative biomedical sciences, pusan national university, yangsan-50612, south korea. d department of gastrointestinal surgery, school of overseas education, guizhou medical university, guiyang, guizhou-550025, china. e department of scienze e tecnologie ambientali, biologiche e farmaceutiche, university of campania luigi vanvitelli; via vivaldi 43, caserta-81100, italy. f department of surgery, boston children's hospital, harvard medical school, harvard university, 300 longwood ave, boston, ma 02115, usa. authors’ contributions this work was carried out in collaboration among all authors. all authors read and approved the final manuscript. article information doi: https://doi.org/10.9734/aji/2024/v7i1147 open peer review history: this journal follows the advanced open peer review policy. identity of the reviewers, editor(s) and additional reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc. are available here: https://www.sdiarticle5.com/review-history/125387 received: 04/09/2024 accepted: 06/11/2024 published: 13/11/2024 review article https://doi.org/10.9734/aji/2024/v7i1147 https://www.sdiarticle5.com/review-history/125387 shukla et al.; asian j. immunol., vol. 7, no. 1, pp. 228-246, 2024; article no.aji.125387 229 abstract the ongoing covid-19 pandemic underscores the urgent need for effective vaccination strategies to mitigate disease burden. the development of neutralizing antibodies is a critical indicator of host defence mechanisms against life-threatening infectious diseases, such as those caused by sarscov-2. understanding the fundamental biological mechanisms and substantiality of these antibodies' production is essential for developing effective vaccines, particularly in the face of emerging variants. circulating t follicular helper (ctfh) cells, which have emerged as significant predictors of neutralizing antibody levels, are of utmost importance in shaping long-term immunity following vaccination. we emphasize the pivotal role of ctfh cells in shaping long-term immunity, providing reassurance about the effectiveness of vaccines. in this study, we elucidate the functions of ctfh cells and their lymphoid counterparts during immune responses to sars-cov-2, particularly in the context of spike protein vaccination. we explore the phenotypic diversity of ctfh cells and their potential as biomarkers for development of sars-cov-2 vaccine efficacy with long-lasting immunity. the identification of specific ctfh subgroups may inform strategies for enhancing vaccine responses, especially concerning new sars-cov-2 variant-specific vaccines. future research directions will focus on harnessing the predictive capabilities of ctfh cells to optimize vaccine development and improve immunological outcomes against evolving sars-cov-2 strains. keywords: t follicular helper cells; sars-cov-2; covid-19; cd4+ t cells. abbreviations pd-1 : programmed cell death protein-1 ccr7 : cc-chemokine receptor 7 cxcr3 : cxc-chemokine receptor 3 ccr6 : cxc-chemokine receptor 6 cxcr5 : cxc-chemokine receptor 5 icos : inducible t cell co-stimulator tfh : follicular helper t cell ctfh : circulating tfh cell 1. introduction the emergence of sars-cov-2, the virus responsible for the covid-19 pandemic, has prompted an unprecedented global effort to develop effective vaccines. among the most promising vaccine platforms are those based on mrna technology, which offers several advantages, including rapid development, scalability, and the potential for modification [1]. as vaccination campaigns have progressed worldwide, understanding the immune responses elicited by these vaccines has become paramount. investigating the dynamics of circulating t-follicular helper cells (ctfh), a subset of cd4+ t cells crucial for b cell activation and antibody production is essential for evaluating vaccine efficacy and durability [2]. the role of ctfh cells in orchestrating humoral immune responses has been extensively studied in the context of various infections and vaccinations. these specialized t cells live within secondary lymphoid organs, interacting with b cells to promote germinal center reactions, affinity maturation, and the generation of longlived plasma cells and memory b cells. importantly, ctfh cells play a central role in shaping the size and quality of antibody responses, which are critical for protection against viral infections [3]. in the context of sars-cov-2 mrna vaccination, understanding the kinetics and durability of ctfh cell responses is particularly interesting. mrna vaccines, such as those developed by pfizer-biontech and moderna, encode the viral spike (s) protein, which mediates viral entry into host cells. upon vaccination, antigen-presenting cells process and present s protein fragments to t cells, leading to the activation and differentiation of ctfh cells. these ctfh cells then migrate to b cell follicles within lymphoid organs, where they provide help to b cells undergoing somatic hypermutation and class-switch recombination, ultimately promoting the production of high-affinity antibodies against sars-cov-2 [4,5]. initial studies investigating ctfh responses to sars-cov-2 mrna vaccination have offered valuable insights into the kinetics and size of these responses. for instance, several reports have shown robust expansion of ctfh cells following vaccination, peaking within weeks after the first dose and declining gradually thereafter. significantly, the frequency and functionality of ctfh cells have been correlated with the size and persistence of antibody responses, suggesting their crucial role in vaccine-induced immunity. furthermore, shukla et al.; asian j. immunol., vol. 7, no. 1, pp. 228-246, 2024; article no.aji.125387 230 emerging evidence suggests that ctfh cell responses to sars-cov-2 mrna vaccination may show heterogeneity across individuals, influenced by factors such as age, sex, and immunological history. understanding the determinants of ctfh cell dynamics and their impact on vaccine efficacy and durability is essential for optimizing vaccination strategies and informing public health policies [6,7]. the immunoglobulin family consists of five classes: igm, iga, ige, igg, and igd. each class has its characteristics and function [8]. primary immunoglobulin m (igm) is expressed in the early stages of b cell maturation and is linked to primary immunological responses. iga protects mucosal surfaces from bacteria, viruses, and toxins. ige plays a role in allergic reactions, hypersensitivity reactions, and the defense against parasitic infections. igg, which is functionally divided into four subclasses (igg1, igg2, igg3, and igg4), is essential for both the effectiveness of vaccinations and antiviral defences. igd has a short serum half-life and is present at low concentrations, and its specific function is currently unknown [8,9] (fig. 1). this review aims to comprehensively evaluate the current literature on human ctfh cell responses to sars-cov-2 mrna vaccination, focusing on the kinetics, size, durability, and correlates of protection. we will discuss key findings from clinical studies and preclinical models, highlighting gaps in knowledge and areas for future research. additionally, we will explore the implications of ctfh cell responses for vaccine development, including strategies to enhance immunogenicity and durability. by elucidating the role of ctfh cells in vaccineinduced immunity against sars-cov-2, this review aims to contribute to the ongoing efforts to control the covid-19 pandemic and prepare for future outbreaks. fig. 1. shows how several tfh cell subsets contribute to the production of immunoglobulins with several functions. pre-tfh (ptfh) cells can differentiate into tfh1, tfh2, or tfh17 phenotypes in response to different microenvironmental signals from different healthy or pathological conditions. because each subgroup of tfh cells has specific functional characteristics, they can coordinate b cell development towards producing various immunoglobulin types essential for various immunological responses. reproduced/adapted with permission [9]. shukla et al.; asian j. immunol., vol. 7, no. 1, pp. 228-246, 2024; article no.aji.125387 231 2. emerging role of tfh cells for novel sars-cov-2 vaccine development according to the findings, cd4 t-cell responses to sars-cov-2 were 80–100% on antigenspecific t-cell responses in patients treated after sars-cov-2 infection, with most research mainly concentrating on spike (s) protein [10-13]. according to other investigations, t-cells have also been found to target the nucleocapsid (n) and membrane (m) proteins [14]. developing effective sars-cov-2 vaccines has been recognized as a significant step toward preventing the covid-19 pandemic. neutralizing antibodies (nab) are a reliable indicator of immunity against infection and immunization in rhesus macaques [15,16]. neutralizing antibodies have been identified as the critical factor of vaccination effectiveness in phase i/ii trials of the pfizer and moderna vaccines and many other candidate vaccines [17-20]. whereas the pfizer vaccine was showcased to induce cd4 and cd8 t-cell responses in trial participants, and vaccine-induced cd4 t-cell responses correlated with antibody levels [21], with our knowledge of both the precise immune factors that are associated with protection as well as the durability of this immune function is still lacking. furthermore, the factors that trigger the production of nab in the presence of spontaneous sars-cov-2 infection are crucial to investigate, especially in the situation of new viral variations that may be different mechanisms of pathogenic infections developing [22,23]. several studies have found a significant increase in activation and exhaustion markers on t cells in moderate and severe sars-cov-2 illnesses [2427]. whereas studying lymphoid tissues directly within patients is challenging, circulating t follicular cells (ctfh), also known as t follicular helper cells (tfh), act as a helpful alternative for investigating tfh responses in germinal centers. however, significant debate was over the optimum method to recognize these cells. it was widely agreed that they express cxcr5, a lymph node homing receptor, but several researchers also utilized pd1 expression in combination with cxcr5 to identify ctfh [28-30]. even though the number of cxcr5+ pd1+ cd4 t cells in the bloodstream is generally low, these cells are closely related to tfh in lymphoid tissue [31] and enhance adaptive immunity defences [32,33]. in the circumstances of infection and vaccination against numerous diseases, antigen-specific ctfh is associated significantly with neutralizing antibodies [29,34-38]. even if ctfh responses were not discussed in the specific situation of sars-cov or mers-cov infection, cd4 t-cell responses were demonstrated to be crucial in regulating sars-cov throughout mouse models [39], as well as tfh frequencies in draining lymph nodes associated with neutralizing antibodies throughout the latest research on the mers-cov vaccine in mice [40]. much research about sars-cov-2-specific t follicular helper cellular transformation by different signaling pathways exists. thevarajan et al. were among the first to publish sars-cov-2 ctfh frequencies, discovering that overall ctfh frequencies are increased during acute infection [41]. several studies have discovered a link between total cd4 t-cell frequencies and antibody levels [42,43]. another research report enhanced gene expression of cxcr5 and icos, two tfh markers, among sars-cov-2-specific cd4 t-cells but did not confirm ctfh effects [44]. however, kaneko et al. discovered that bcl6expression throughout germinal centre tfh has been lost within thoracic lymph nodes of deceased donors with covid-19, implying that tfh response initiation may be inhibited in serious sars-cov-2 infection [45]. the initiation of antigen-specific tfh responses, especially in asymptomatic patients with covid-19, is still a mystery. in their study, juno et al. investigated circulating tfh, recognized as cd45ra cxcr5+ cd4 t cells, in the blood of sars-cov-2 infected people. they discovered a link between s protein-specific ctfh and nab, indicating that effective tfh responses were produced in moderate sars-cov-2 infection [46]. unfortunately, these findings leave other aspects unresolved, particularly once those responses change throughout the recovery period. even though this research work provided a preliminary look at antigen specific tfh responses, which included pd1, a conventional tfh marker, within the research frame, pd1 expression needed more utilized to characterize the tfh population and was not published. furthermore, ox40 and cd25 were utilized as initiation markers to detect antigen-specific responses that have already been reported and demonstrated to contain a significant fraction of regulating t-cells [47]. a current study has the durability of s proteinspecific cd4 t-cell responses after recovery [48]. the authors have considered only the incidence of circulating tfh (icos+ cxcr5+ cd4 t cells) each month and three months after the symptoms started. while they found responses higher than backgrounds after three months, there was no change throughout the duration. shukla et al.; asian j. immunol., vol. 7, no. 1, pp. 228-246, 2024; article no.aji.125387 232 additional analysis of cd4 t-cell and ctfh responses during the recovery period might help to determine how much these responses change and evolve. several recent studies [49,50] have investigated ctfh groups up to 6 months after the onset of symptoms. (investigations used too much). unfortunately, this ongoing research failed due to the failure to examine connections among antigen-specific ctfh and sars-cov-2specific antibodies. in addition, these types of research were predominantly concerned with s protein-specific effects. for example, infrastructural assistance emerges in hiv infection during vaccination, when cd4 t-cell responses towards internal structural proteins correspond with neutralizing antibodies against the outer envelope protein [51,52], emphasizing the necessity of studying ctfh responses from across the sars-cov-2 proteome. boppana et al. report on sars-cov-2-specific cd4 t-cell activation to membrane (m), nucleocapsid (n), and spike (s) proteins in 26 recovered patients who were studied in real-time. they studied antigen-specific ctfh (cxcr5+ pd1+ cd4 t cells) and found connections among antigenspecific ctfh responses across all protein specificities with antibody neutralization during the first convalescent visit. they observed that the m protein-specific ctfh responses increase during check up 1 towards check up 2 (>5% activation of the total ctfh population). these responses did not correspond to antibody neutralization at only the second recovered visit and more than thirty days after illness onset. such findings are always the earliest to investigate the speed of ctfh responses following sars-cov-2 infection and the link between neutralizing antibodies and ctfh sensitivities to sars-cov-2 m and n proteins. this research also suggests that ctfh development may be postponed in sars-cov-2 infections [53]. 3. phenotypes and functions of follicular helper t cells (tfh) of sars-cov-2 infection follicular helper t cells (tfh) cells are used as a functional indicator; they can promote b cells by increasing antibody production, long-lived plasma cells, and memory b cells [54,55]. tfh cell markers, which typically include chemokine receptor cxcr5, transcription factor bcl-6, pd-1, cd40 ligand (cd40l), and icos in humans and mice, are essential for identifying tfh cells with their various subgroups not only in lymphoid tissue but also in circulation [56-59]. additionally, the phenotypes of tfh cells were linked to distinct phases of immunological responses [60,61]. within secondary lymphoid organs, naïve cd4+ t cells develop into tfh cells through cxcr5 overexpression and ccr7 reduced expression, which is controlled by antigen-specific conventional dendritic cells (dcs) and monocytederived dcs [58,62,63]. tfh cells migrate into cxc-chemokine ligand 13 (cxcl13)-enriched b lymphoid follicles inside the germinal centre (gc) due to enhanced cxcr5 and reduced ccr7 [58,64]. in human and mouse gcs, the unique transcription factor bcl-6 was exclusively expressed among tfh cells and was highly expressed within cxcr5hiccr7low/-tfh cells [64-67]. a cytokine, interleukin-21 (il-21), is firmly and selectively released only tfh cells, which increases tfh cell proliferation and enhances b cell differentiation and antibody production, which is typical for tfh cells [68-72]. in both mice as well as humans, icos reduction greatly lowers gc reactions but also tfh cells, indicating that icos expression in tfh cells is required during tfh cell differentiation and maintenance, gc production, and b cell differentiation, including antibody responses [7375]–66]. as an essential effector molecule, icos could also promote il-21 release within tfh cells [75-77]. tfh cell development and activation could be considerably enhanced by increased pd-1 expression [78-80]. tfh cells were usually classified as having three phenotypes: precursortfh (pre-tfh) cells, canonical gc tfh cells with pd-1++ and icos++bcl-6+ ccr7cxcr5++cd4+ t cells characterized as pd-1+ icos+ bcl6lowccr7lowcxcr5+ cd4+ t cells, and memory tfh cells similar to pre-tfh cells in lymphoid tissue [33,81,82]. tfh cells regulate b cell differentiation, becoming memory b cells as well as plasma cells in gc, and even select for highaffinity antibody production to form long-term innate immunity [83-86]. circulating tfh (ctfh) cells in the blood typically have two different phenotypes: central memory tfh cells (pd-1icos-ccr7highbcl-6cxcr5+ cd4+ t cells) and effector memory tfh cells (pd1+icos+ccr7lowbcl-6cxcr5+ cd4+ t cells) [61,87,88]. similarly, ctfh cells were categorized under three subgroups depending on the expression of either cxcr3 as well as ccr6: tfh1 (cxcr3+ ccr6-), tfh2 (cxcr3ccr6), tfh17 (cxcr3ccr6+), and tfh1/17 (cxcr3+ ccr6+) cells that contain the hallmark gene transcription factors and cytokines comprising th1 (t-bet as well as ifn-g), th2 (gata3, il-4, il-5, and il-13), with th17 (rorgt, il-17, and shukla et al.; asian j. immunol., vol. 7, no. 1, pp. 228-246, 2024; article no.aji.125387 233 il-22) cells [61,87,89]. ctfh2 with ctfh17 cells potentially stimulates b cell differentiation with antibody production and regulates (ig-antibodies) isotype changing. although ctfh1 cells are not commonly considered b cell helpers, icos+ pd1highccr7lowctfh1 cells control b cell differentiation and trigger antibody responses [89-95]. depending on the expression of icos, pd-1, and ccr7, as well as cxcr3 and ccr6, these findings show functionally different ctfh cell subgroups. furthermore, emerging unique subgroups distinguish among th1, th2, and th17 cells while sharing some fundamental properties. tfh-like cells were also recognized in non-lymphoid tissues, such as arthritis synovium and skin, but also salivary glands of patients, that also widely express low expression of cxcr5 and bcl-6 and high pd-1, icos, ox40, and il21, especially in comparison to tfh cells in secondary lymphoid organs, that also express tissue-specific chemokine receptors, such as ccr2, ccr5, cx3c-chemokine receptor 1 (cx3cr1) and cxcr4 [82,96-100]. tfh13 cells, a unique tfh cell subgroup that produces and secretes il-4 with il-13, have been newly found to be essential for ige production in both human and mouse allergies, and they exhibit the signaling molecules bcl-6 and gata3 [101-103]. according to current research, specific phenotypes for tfh cells are required during b cell differentiation and high-affinity antibody production (table 1). more importantly, follicular regulatory t (tfr) cells are a subpopulation of foxp3+ treg cells inside the gc, launched through foxp3+/-precursors and not just from tfh cells [104-107]. tfr cells express tfh cell markers such as cxcr5, bcl-6, pd-1, and icos and treg cell molecules such as cd25, foxp3, blimp-1, and ctla-4 [108-111]. tfr cells, like treg cells, play a significant role in immunosuppression that is greater than that of tfh cells. tfh cells can restrict gc responses and reduce tfh and b cell activation inside gcs via inhibitory molecules such as ctla-4, pd-1, and il-10, as well as tgf-b production. tfh/tfr cell equilibrium was required to regulate immunological homeostasis and modulate innate immunity [92,96,111-114]. 4. mechanism of sars-cov-2 mrna “presently, the emerging sars-cov-2 infectious outbreak is causing a significant challenge for global healthcare across the globe. innate immunity is required for neutralizing antibodies and is essential in vaccination reactions against pathogenic virus infections, such as sars-cov2, which have been linked to tfh cell differentiation and function” [55,115-120] (fig. 2). tfh cells have been studied for their role in regulating the eradication of sars-cov-2 infections and developing novel vaccines. much research showed a high frequency of ctfh cells with the cxcr5+icos+pd-1+ phenotype. in a patient with especially non-recovering covid19 patient, specific plasma sars-cov-2-binding igm and igg antibodies increased exponentially approximately 20 days after infection, with a combination of enhanced specific plasma sarscov-2-binding igm as well as igg antibodies [41]. single-cell investigation showed that individuals with active covid-19 infection had higher frequencies of ctfh cells and a large percentage with specific anti-sars-cov-2 antibodies, such as iga and igg [121]. “in recovered covid-19 patients, the percentages of spike (s)-specific ctfh cells (cd3+cd4+cd45ra–cxcr5+) are continuously produced after s-peptide activation and show a significant phenotypic inclination toward the accr6+cxcr3-ctfh17 cell phenotype. another study discovered that significantly enlarged cxcr3+ctfh1 cells were associated with such a solid neutralizing immunological response to influenza vaccination and live attenuated yellow fever immunization” [37,122]. the latest research found that increasing numbers of ccr7lowpd-1+ ctfh-effector memory (em), ctfh1 but instead ctfh2 cells, and also high il-1b and tnf-a, are found in cxcr5+cd45racd25-cd4+t cells, and therefore, ctfh1 cells were related to increased sars-cov-2-specific igg/igm antibodies. while ccr7highpd-1-ctfh-central memory (cm) and ctfh17 cells within cxcr5+cd45racd25cd4+t cells were reduced in recovered patients compared to healthy patients, ctfr cells within treg cells were increased. the frequency of high ctfh-em, low ctfh-cm, and ctfr cells was also associated with disease severity [43]. these findings suggest that ctfh cell morphological characteristics can produce significant neutralized antibodies toward sars-cov-2 in covid-19-recovered patients, which will help develop antibody-based treatments and vaccines for covid-19. shukla et al.; asian j. immunol., vol. 7, no. 1, pp. 228-246, 2024; article no.aji.125387 234 table 1. phenotypes of tfh cell subsets in blood and lymphoid tissue location cell subsets phenotypic markers ref. lymphoid tissues pre-tfh cells pd-1+ icos+ ccr7lowbcl-6lowblimp-1cxcr5+ [62,87,88] gc tfh cells pd-1++icos++ccr7bcl-6+ blimp-1cxcr5++ memory tfh cells pd-1+ icos+ ccr7lowbcl-6lowblimp-1cxcr5+ blood ctfh1/17 cells ifn-g + il-17a+ bcl-6blimp-1cxcr5+ (or) cxcr3+ ccr6+ bcl-6blimp-1cxcr5+ ctfh17 cells il-17a+ bcl-6blimp-1cxcr5+ (or) cxcr3ccr6+ bcl-6blimp-1cxcr5+ ctfh2 cells il-4+ bcl-6blimp-1cxcr5+ (or) cxcr3ccr6bcl-6blimp-1cxcr5+ ctfh1 cells ifn-g + bcl-6blimp-1cxcr5+ (or) pd-1+ icos+ ccr7lowcxcr3+ ccr6bcl-6blimp-1cxcr5+ effector memory tfh cells cd40l+ /pd-1+ /icos+ ccr7lowbcl-6blimp-1cxcr5+ central memory tfh cells pd-1icos-ccr7highbcl-6blimp-1cxcr5+ ctfh13 cells il-13hiil-4hiil-5hiil-21lowbcl-6+ gata3+ cxcr5+ [101-103] † pd-1, programmed cell death protein-1; ccr7, cc-chemokine receptor 7; cxcr3, cxc-chemokine receptor 3; ccr6, cxc-chemokine receptor 6; cxcr5, cxc-chemokine receptor 5; icos, inducible t cell co-stimulator shukla et al.; asian j. immunol., vol. 7, no. 1, pp. 228-246, 2024; article no.aji.125387 235 table 2. tfh cell responses in various vaccine candidates of sars-cov-2 vaccine candidates phenotypes function antibody isotypes ref. mrna vaccines bnt162b2 mrna vaccine aim+ cxcr5+ cd45ra-cd3+ ctfh cells expansion, aim cells include cd69+ ox40+ or cd69+ cd40l+ (or) cd69+ 4-1bb+ (or) ox40+ 4-1bb+ (or) cd40l+ 4-1bb+ (or) cd40l+ ox40+ positively correlate with anti-spikespecific iga and igg titers. iga, igg [124] rbd mrna (receptor binding domain, rbd) b220-cd4+ cd44hicd62l-cxcr5+ bcl-6+ tfh cells, b220 cd4+ cd44hicxcr5+ pd-1hi il-21+ tfh cells, b220cd4+ cd44hicxcr5+ bcl-6+ icos+ tfh cells, b220cd4+ cd44hicxcr5+ pd-1hi ifn-g + tfh cells notable expansion elicit potent sars-cov-2-specific gc b responses and induce robust and specific antibody responses, including neutralizing antibodies. gg1, igg2a, igg2b, full sδ furin mrna b220cd4+ cd44hicd62l-cxcr5+ bcl-6+ tfh cells, b220cd4+ cd44hicxcr5+ pd-1hi il-21+ tfh cells, b220-cd4+ cd44hicxcr5+ bcl-6+ icos+ tfh cells b220-cd4+ cd44hicxcr5+ pd-1hi elicit potent sars-cov-2-specific gc b responses and induce robust and specific antibody responses, including neutralizing antibodies. igg1, igg2a, igg2b, [130] mrna-1273 il-21+ cxcr5+ pd−1+ icos+ tfh cells expansion. induce robust and specific antibody responses, including neutralizing antibodies. iga, igg [123] protein vaccines strifk-fh002c pd-1+ cxcr5+ cd4+ tfh cells expansion induce specific antibody responses, including neutralizing antibodies. igg, igg1, igg2a, igg2b [129] spike (s) and receptor binding domain (rbd) protein subunit vaccine cxcr5++bcl-6+ cd4+ cd3+ b220-tfh cells expansion induce specific antibody responses, including neutralizing antibodies. igg [125] nvx-cov2373 cxcr5+ pd-1+ cd4+ tfh cells expansion induce specific antibody responses, including neutralizing antibodies. igg [4] rrbd-addavax b220-cd4+ cd44hicd62l-cxcr5+ bcl-6+ tfh cells, b220cd4+ cd44hicxcr5+ pd-1hi il-21+ tfh cells b220-cd4+ cd44hicxcr5+ pd-1hi il-4+ tfh cells slight expansion delay in eliciting potent sarscov-2-specific gc b responses induces robust and specific antibody responses, including neutralizing antibodies. igg1 [130] † tfh, follicular helper t cell; ctfh, circulating tfh cell shukla et al.; asian j. immunol., vol. 7, no. 1, pp. 228-246, 2024; article no.aji.125387 236 fig. 2. the involvement of signaling pathways in controlling tfh cell differentiation and function in sars-cov-2 infection with vaccines. when exposed to sars-cov-2 or viral antigens, naive cd4+ t cells were activated through apcs (dcs), which are triggered against antigen-specific pretfh cells by the association of mhc-ii molecules between dcs with cognate tcr on cd4+ t cells, and the expression of costimulatory molecules with cytokine production, pre-tfh cells associate with active b cells at the t-b boundary in the follicular zone, wherein they develop into diverse tfh cell subtypes that move to the gc, wherein tfh cells stimulate b cell differentiation and specific antibody production. reproduced/adapted with permission [131] shukla et al.; asian j. immunol., vol. 7, no. 1, pp. 228-246, 2024; article no.aji.125387 237 fig. 3. lymphoid and circulating tfh responses in covid-19. sars-cov-2 antigen in the lymph nodes activates antigen-specific b cells and tfh cells. their interaction leads to the initiation of the germinal centre reaction. this results in the development of memory b cells with increased somatic hypermutation (shm) and increased affinity, as well as long-lived plasma cells that traffic to the bone marrow and provide a long-term source of neutralizing antibodies. a population of short-lived antibody-secreting cells (ascs) appears in the circulation and provides a rare source of neutralizing antibodies. concurrently, a population of activated (cd38+, pd-1+, icos+) ctfh cells appears in the circulation. this population contains antigen-specific ctfh cells (not depicted). although memory b cells and ascs are primarily located in lymphoid tissues, they are typically measured in blood samples, which correlate with activated ctfh cells. activated ctfh cells also correlate with the development of neutralizing antibodies. these ctfh cells are a potential biomarker of tfh activity in lymphoid tissues. however, it remains to be determined if this population of ctfh cells are predictive of long-term neutralizing antibodies or the development of long-lived plasma cells and the prolonged evolution of the mbc pool. reproduced/adapted with permission [135]. rapid advances have been made in designing and developing sars-cov-2 vaccines, such as inactivated dna, mrna, and specific sarscov-2 proteins [123]. the mrna-1273 vaccine can effectively activate th1 and interleukin-21 expressing cxcr5+pd1+icos+tfh cellular responses as well as trigger powerful sarscov-2 neutralizing potential, providing quick protection against sars-cov-2 infection in the vertical and horizontal airways of rhesus macaques [4]. in contrast to the sars-cov-2 recombinant sars-cov-2 receptor-binding domain (rrbd) developed for the addavax (rrbd-addavax) protein vaccine gene, the shukla et al.; asian j. immunol., vol. 7, no. 1, pp. 228-246, 2024; article no.aji.125387 238 sars-cov-2 mrna vaccine gene encodes rbd as well as full-length spike protein efficiently to prompt sars-cov-2-specific gc b and tfh cellular responses, which also enhance specific neutralizing antibody responses in inoculated mice. surprisingly, the rrbd-addavax vaccination produced a significant proportion of il-4+ tfh cells [124]. the bnt162b2 mrna vaccine against sars-cov-2 produced significant aim+cxcr5+cd45ra-cd3+ctfh cell responses in humans. aim (activation-induced marker) cells are including cd69+ox40+ or cd69+cd40l+ or cd69+4-1bb+ or ox40+4-1bb+ or cd40l+4-1bb+ or cd40l+4-1bb+ or cd40l [4]. the above results show that sars-cov-2 mrna vaccines can help enhance antigenspecific tfh cell differentiation and b cell responses, as well as the production of protective immune responses by producing antibodies, making them especially potential for eliciting high-quality adaptive immune responses to regulate as well as eradicate sars-cov-2 infection. moreover, “target protein vaccines such as the sars-cov-2 subunit vaccine (nvx-cov2373) with both the full-length spike (s)-protein, strifkfh002c, as well as the spike (s)/receptor binding domain (rbd) protein subunit vaccine remarkably stimulate specific ctfh cell but also gc b cell responses, leading to high sarscov-2” [125-127] neutralizing levels of antibodies (table 2). several human clinical studies show that immobilized sars-cov-2 vaccinations can produce sufficient high neutralizing levels of antibodies to decrease the rate of patients developing severe covid-19 [127129]. such findings imply that sars-cov-2 vaccinations can boost host immune response, increase neutralizing antibody levels, and reduce the death rate of critically ill patients. meanwhile, in serious covid-19 patients, the absence of gc structures lowers bcl-6+ tfh cells. interestingly, sars-cov-2-specific tfh cells significantly increased in moderate and asymptomatic covid-19 patients. vaccines also can stimulate tfh cell differentiation and gc production, including defensive antibody responses. 5. human circulating t follicular helper cells (ctfh) response against covid-19 activated ctfh (pd-1+icos+) [41,43,132-134] with enhanced expression of cd38 [135] but also decreased expression of ccr7 [43] is seen in the blood during acute infection. such activated pd-1+icos+ ctfh cells appear spontaneously throughout the infection but typically decrease 14 days after symptom onset. as a result, antigenspecific t cell tests (activation-induced marker (aim) and intracellular cytokine staining) were crucial to evaluating sars-cov-2-specific ctfh responses throughout recovery. these studies have also shown that s-specific ctfh cells that arise following acute infection [136] remain in convalescent patients for at least 6 months [32], with a half-life of approximately 129 days [50]. the link between ctfh frequencies, morphological and functional polarization, and sars-cov-2 serologic responses have been investigated. most of the pd1+icos+ ctfh populations formed during the acute stage of covid-19 are cxcr3+ctfh1 cells [132,134], similar to influenza infection [137]. moreover, the investigation of s-specific ctfh showed a completely dominant population of cxcr3– ccr6+ cells [46,49,136]. remarkably, the fraction of cxcr3–ccr6+ s-specific ctfh cells during late convalescence (6 months) is more significant than that in initial convalescence (1–2 months) or even during the acute stage [49,136]. despite the presence of cxcr3ccr6+ ctfh17 cells, antigen-specific ctfh cells from covid-19 patients routinely release ifn and il-21, even when il-17 is not present, according to multiple independent studies [46,48-50,133,136,137]. the development of effective neutralizing antibody responses has been associated with a phenotypic polarization of ctfh. furthermore, high levels of serum spike binding and neutralizing antibodies were strongly associated with cxcr3+ ctfh1 cells. this was true not only for the overall icos+pd-1+ ctfh1 populations (which corresponds to asc responses and plasma cxcl13) [43,132-134], but also for sspecific ctfh1 [46]. the activation of ctfh1 cells in acute covid-19 significantly corresponds with both the confirmatory testing and antibody production of rbd-specific igm antibodies [46]. “the association between ctfh2, ctfh17, and antibody response, on the other hand, varies among cohorts and tests. the discovery of sspecific ctfh led to the conclusion that the occurrence of ctfh2 responses was positively correlated with increasing neutralizing levels of antibodies, whereas s-specific ctfh17 showed a significant negative correlation with neutralizing activity” [46]. overall, icos+pd1+ ctfh2/17 cells were significantly negatively correlated with antibody response in different populations [43,46,133,134]. therefore, the present shukla et al.; asian j. immunol., vol. 7, no. 1, pp. 228-246, 2024; article no.aji.125387 239 findings show that cxcr3+ ctfh1 cells are a significant correlate of neutralizing as well as overall antibodies towards sars-cov-2, but the roles of cxcr3–ctfh2 and ctfh17 cells, as well as the differences between total icos+pd1+ and aim+ ctfh cells, deserve additional exploration. whereas most research has concentrated on sspecific ctfh cells, n and m-specific ctfh cells were also investigated [53,136,138]. the proportion of ctfh cells specific for s, n, and m was shown to be favourably linked to increased plasma neutralization activity with n-specific igg antibodies [53]. remarkably, for ctfh cells specific towards various sars-cov-2 antigens, polarisation throughout ctfh1, ctfh2, and ctfh17 subgroups has been shown to change [138], but the relevance of this result is still unknown. a possible decrease of tfh cells has also been identified in some patients with acute covid-19. in particular, gc-b cells and tfh cells were shown to be reduced in lymphoid tissues of a subgroup of deceased covid-19 patients [45,53]. “furthermore, a population with ctfh cells expressing cytotoxicity-associated genes, including prf1 and gzmb (encoding perforin and granzyme b), was enhanced in hospitalized vs. nonhospitalized patients. it was also linked with decreased antibody levels to s” [139]. such findings contradict the more significant antibodies found in acute covid-19 [132] because decreased tfh function would be predicted to decrease antibody levels. such findings highlight the need for more research into the nature and function of tfh cells in acute covid-19 and whether they correspond to a specific population of patients. finally, total stimulation of ctfh cells and associated phenotypic polarisation throughout covid-19 are indicators of b-cell reaction neutralization (fig. 3). furthermore, the phenotypic traits of the different ctfh subgroups, including their link with gc tfh activities, must be clarified. it is crucial to investigate the potential for ctfh cells as indicators of the formation and recall of humoral immunity to sars-cov-2, particularly in developing variants of concern (vocs) with a higher potential for evasion of humoral immunity. it is essential to investigate the potential for ctfh cells as indicators of the formation and recall of humoral immunity to sars-cov-2, particularly in developing variants of concern (vocs) with a higher potential for evasion of humoral immunity. 6. human circulating t-follicular helper cells (ctfh) responses after sars-cov-2 vaccination vaccination with certified covid-19 vaccines produces antibody responses linked to infection prevention. following mrna vaccination, examination of axillary draining lymph nodes showed substantial gc responses that were maintained for a minimum of 12 weeks following booster vaccination [140]. s-specific tfh cells were significantly produced at those sites because they were associated with s-specific gc b cells [124,141]. according to an evaluation of associated lymph nodes and blood samples, s-specific ctfh cells show an activated phenotype (cd38+hla-dr+icos+) increase during the first month before reverting to a resting phenotype and then decreasing in frequency. within a minimum of 60 days, the frequency of s-specific tfh cells within lymph nodes is majorly consistent [141]. despite being confined to a limited number of donors, our results indicate that mrna vaccines trigger significant gc responses, which may highlight the vaccine's outstanding immunological characteristics. vaccination of naive (previously uninfected) humans produces s-specific ctfh cells [142-148] with such a cxcr3+ phenotype [142] as well as the potential to produce ifn, although without il-17a [4]. at a minimum, sspecific ctfh cell frequency increases approximately one month after immunization and subsequently decreases, compared with sspecific th1 cell frequency, which remains constant for a minimum of 6 months [144]. at two weeks following vaccination, the number of sspecific ctfh cells and s-specific conventional cd4+ th1 cells corresponds to neutralizing antibodies against spike and vocs and s with rbd-specific mbc responses [86]. this suggests that ctfh cells serve as indicators again for the production of neutralizing antibodies with mbcs and subsequent spike vaccination. vaccinating people who have recovered from covid-19 usually results in higher s-specific ctfh responses versus naïve people [4]. significantly, the number of s-specific ctfh cells among recovering patients before vaccination increases significantly post-vaccination, neutralizing antibody levels observed in both the ancestor's viruses and voc [4,143]. therefore, it indicates that ctfh cells play a significant function as biomarkers of innate immunity after sars-cov-2 vaccination. this will be critical to better classifying and correctly understanding lymphoid and circulating tfh cell responses after shukla et al.; asian j. immunol., vol. 7, no. 1, pp. 228-246, 2024; article no.aji.125387 240 fig. 4. circulating tfh subsets as correlates of antibody responses. flow cytometry visualization of ctfh subgroups depending on cxcr3 as well as ccr6 expression and its relationship with antibody responses in different configurations. reproduced/adapted with permission [135]. administration of various newly invented vaccine systems, as well as whether/how they differently activate such responses, particularly in the context of heterologous prime-boost vaccination. 7. future prospective of ctfhmediated with other therapies for sars-cov-2 infection the future prospects of ctfh-mediated therapies in the context of sars-cov-2 infection are promising, particularly in light of the remarkable adaptability of these cells to emerging variants. as our understanding of ctfh cell dynamics evolves, it becomes increasingly clear that their ability to modulate immune responses could be harnessed in conjunction with other therapeutic approaches. integrating ctfh-mediated strategies with innovative hormonal therapies may enhance vaccine efficacy and promote robust long-term immunity. by targeting the plasticity of ctfh cells, future interventions could be designed to optimize immune responses to current variants and potential future mutations of sars-cov-2, thereby improving clinical outcomes and overall public health strategies. 8. conclusion there are strong indications that the ctfh study can provide valuable information on the quantitative and qualitative characteristics of neutralizing antibodies against sars-cov-2 infection during vaccination (fig. 4). this evidence suggests that ctfh cells, specifically the ctfh1 subgroup, were valuable indicators for producing neutralizing antibodies and mbcs targeting both wild-type spike and vocs. however, critical issues exist regarding the involvement of cxcr3/ccr6 ctfh subgroups in generating neutralizing antibodies or maintaining spike-specific ctfh memory. to obtain specific results on tfh's ability to produce significant neutralizing antibody responses via vaccination, it will be crucial to understand why only specific ctfh subgroups correlate positively to antibody levels and how tfh quality may be modified using innovative vaccine platforms. furthermore, whereas the ctfh initiation, as well as the frequency of antigen-specific ctfh cells, are biomarkers of neutralizing antibodies in the acute as well as early phases of covid-19 infection but also vaccination, it is unknown whether ct cells indicate the development of long-lived plasma cells or long-term neutralizing antibodies. researchers should start understanding more about the duration of gc tfh responses and their relationship to ctfh frequencies and phenotype as research aims to comprehend the immunologic processes underpinning the persistent development of the mbc pool [145147]. resolving these concerns is critical to realizing their promise of developing efficient vaccination approaches. consent it is not applicable. shukla et al.; 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cite as: mamman, godiya peter, caleb ndako angulu, aminu, ado, mohammed evuti mahmud, ruqayyatu aliyu, samuel angulu, and adeoye daniel owoyale. 2025. “metabolomics role in health and disease: current status and future directions”. asian journal of immunology 8 (1):190-206. https://doi.org/10.9734/aji/2025/v8i1171. asian journal of immunology volume 8, issue 1, page 190-206, 2025; article no.aji.140476 metabolomics role in health and disease: current status and future directions godiya peter mamman a, caleb ndako angulu b*, aminu, ado b, mohammed evuti mahmud c, ruqayyatu aliyu a, samuel angulu d and adeoye daniel owoyale e a department of biology nigeria army university, biu born state, nigeria. b department of microbiology, federal university dutsin-ma katsina state, nigeria. c department of natural and applied sciences, college of nursing sciences, bida, niger state, nigeria. d department of basic and natural sciences, niger state, college of agriculture mokwa, niger state, nigeria. e school of life sciences, department of microbiology, federal university of technology, minna, niger state, nigeria. authors’ contributions this work was carried out in collaboration among all authors. all authors read and approved the final manuscript. article information doi: https://doi.org/10.9734/aji/2025/v8i1171 open peer review history: this journal follows the advanced open peer review policy. identity of the reviewers, editor(s) and additional reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here: https://pr.sdiarticle5.com/review-history/140476 received: 01/06/2025 published: 29/07/2025 review article https://doi.org/10.9734/aji/2025/v8i1171 https://pr.sdiarticle5.com/review-history/140476 mamman et al.; asian j. immunol., vol. 8, no. 1, pp. 190-206, 2025; article no.aji.140476 191 abstract the field of metabolomics involves the high-throughput identification and measurement of all endogenous and exogenous low-molecular-weight (<1kda) small molecules or metabolites in a biological system by analyzing the metabolome in cells, biofluids, tissues, or organisms. metabolome-wide association studies (mwas), metabolic phenotyping, single-cell epidemiologic population studies, precision metabolomics, and in combination with other omics fields like integrative omics, biotechnology, and bioengineering are some of the applications of metabolomics in health and disease scenarios. metabolomics and its potential to enhance human health, along with its developments and implications for pharmacometabolomics, lifespan, cancer, and the exposome, are the main focus of this review. metabolomic profiles will facilitate the development and improvement of therapeutic strategies to treat human diseases. future years will see a rise in the application of metabolomics to drug development, aging, and disease monitoring and diagnosis. its potential extends to food science, and environmental research. through clinical metabolomics studies, which may also uncover diagnostic biomarkers that predict disease risk, cardiometabolic disorders can be better understood. metabolomics is already being applied in biomedical settings to develop drugs by using acylcarnitines, phospholipids, genomes, and branchchain amino acids that are specific to monitoring the emergence of metabolic diseases such as obesity and diabetes. indicators derived from metabolomics should be evaluated for therapeutic efficacy and adaptability, and their optimal application in large clinical settings should be the focus of future research. keywords: metabolomics; health; disease; metabolites; biomarker. 1. introduction the rapidly developing field of metabolomics aims to accurately identify and measure all endogenous and exogenous low-molecularweight (<1kda) small molecules or metabolites in a biological system in a high-throughput way. the proteome, genome, lifestyle, environment, drugs, and underlying disease all have an upstream influence on the composition of these endogenous compounds (zhou and zhong, 2022). the study of metabolites, which are tiny chemical entities involved in biological systems' cellular processes, is known as metabolomics. it is used in molecular and personalized healthcare in clinical chemistry, transplant monitoring, newborn screening, pharmacology, and toxicology (zhou and zhong, 2022). the development of analytical techniques and bioinformatics has led to the emergence of metabolomics, a state-of-the-art omics technique. in order to identify and describe the metabolome, the metabolomics approach typically uses advanced analytical chemistry tools like nuclear magnetic resonance (nmr) and mass spectrometry (ms) in conjunction with different chromatographic techniques, such as gas chromatography (gc-ms) or liquid chromatography (lc-ms), with an emphasis on molecules smaller than 1500 da found in cells, organs, tissues, or biofluids (wishart, 2018; sun et al., 2019). the new field of medical genomics offers cuttingedge technological instruments for identifying genetic susceptibilities to diseases. but metabolomics allows us to go further by linking disorders and defects in gene expression to the pathological phenotype. although the concept of "clinical metabolomics" was first introduced in 2008, the term was first used in the literature in 2009 (le gouellec et al., 2023). the goal of clinical metabolomics is to identify metabolic signatures in bodily fluids (plasma, urine, saliva, cerebrospinal fluid, etc.) or tissues that are impacted by genetics, epigenetics, dietary patterns, environmental factors, and behavior in order to evaluate and forecast a subject's health and disease risk (ceglarek et al., 2009; mallu et al., 2021). a collection or combinations of impacted metabolites make up metabolic signatures (wishart, 2018). metabolomics' use in both health and disease has grown in importance. based on a particular metabolic signature, metabolomics makes it possible to identify biomarkers for disease diagnosis, prognosis, and response to treatment (cheng et al., 2012). the pathophysiology of complex diseases like cancer, neurodegenerative diseases, cardiovascular diseases, and metabolic syndrome can be largely explained by such metabolic profiles (neergaard et al., 2017; mallu et al., 2021; vo and trinh, 2024). furthermore, metabolomics is mamman et al.; asian j. immunol., vol. 8, no. 1, pp. 190-206, 2025; article no.aji.140476 192 crucial to precision medicine because it allows for the identification of individual metabolic variations that can support tailored treatment approaches (beger et al., 2016; nielsen, 2017). according to that perspective, metabolomics is now a potent addition to personalized medicine, bringing fresh insights into the mechanisms underlying illnesses. biomarkers are important in healthcare, particularly in diagnosis, prognosis, and treatment monitoring, since they are the quantifiable foundation of biological states and conditions (califf, 2018). biomarkers are crucial in diagnostics because they enable early and precise disease diagnosis, frequently before symptoms appear. this is especially true for diseases like cancer, where early intervention greatly enhances results. particular metabolite profiles, for instance, can help distinguish between disease stages or subtypes in order to improve diagnostic accuracy and support patient stratification (prat et al., 2021; deng et al., 2022). identifying markers of disease severity or recurrence can help healthcare providers predict disease trajectory and appropriately plan interventions, especially for chronic and complex diseases where metabolic biomarkers may indicate the risk for complications or rapid development of the disease process. biomarkers help monitor treatments by providing real-time responses in patients, allowing for the examination of efficacy and possible treatmentrelated side effects, allowing for modifications of therapeutic regimens for maximum efficacy with minimum side effects (vo and trinh, 2024). according to vargas and harris (2016), biomarkers play a significant role in advancing precision medicine by facilitating more individualized, proactive, and efficient approaches to healthcare delivery. new technical tools for identifying genetic susceptibilities to diseases have emerged with the recent rise of medical genomics. however, by linking the pathological phenotype to disorders and defects in gene expression, metabolomics allows us to go further. while the concept was first introduced in 2008, the term "clinical metabolomics" was first used in the literature in 2009 (damiani et al., 2020). in clinical metabolomics, metabolic signatures in bodily fluids (plasma, urine, saliva, cerebrospinal fluid, etc.) or tissues which are influenced by genetics, epigenetics, dietary patterns, environmental factors, and behavior are identified in order to evaluate and forecast a subject's health and disease risk (cheng et al., 2012). a group or combination of metabolites that are impacted make up metabolic signatures. in addition to being studied peripherally, as in biofluids like blood plasma, which contains a variety of metabolites that reflect organ metabolic activity and offer important pathophysiological information, the metabolome can also be studied intracellularly, exposing functional abnormalities at the cellular level (vo and trinh, 2024). human plasma contains both endogenous and environmental metabolites. a number of factors, including dietary patterns, gut microbiota, and lifestyle choices (such as smoking or physical activity), greatly impact the metabolome's variability, which is partially inherited. individual metabolic variability is mostly influenced by diet and microbiota, which makes each person's metabotype distinct (vo and trinh, 2024). nonetheless, a person's metabotype stays largely constant over time if there are no notable changes in their health. the analysis of metabolites is predicated on two broad methods: 1. targeted metabolomics is the study of identifying a particular group of metabolites. 2. untargeted metabolomics compares and identifies as many metabolites as possible between samples, including unknown ones, using an objective method. the latter strategy allows for the routine detection of numerous metabolite features peaks that correspond to individual ions with unique mass-to-charge (m/z) ratios and retention times (rt) using lc/ms-based techniques (vo and trinh, 2024).the particular intended application will determine which of these methods is best. the general trends of recent discoveries and applications of metabolomics-based biomarkers, as well as their revolutionary effects on personalized medicine and precision health, are summarized in this review. it is becoming more feasible to find disease biomarkers, even for complex conditions like cancer, neurodegenerative diseases, cardiovascular diseases, and metabolic syndrome, thanks to technological advancements and metabolomics analysis capabilities. although early diagnosis and classification are greatly enhanced by these biomarkers, they also enable a much more nuanced understanding of individual variability in mamman et al.; asian j. immunol., vol. 8, no. 1, pp. 190-206, 2025; article no.aji.140476 193 disease progression and response to treatment. this review's objective is to examine these new metabolomics biomarker discoveries with an emphasis on how they may help with healthcare personalization. the translational development of metabolomics for use in mainstream clinical practice is also covered, with particular emphasis on how metabolomics can transform healthcare from a one-size-fits-all model to a highly individualized one where any health risk can be promptly identified and treatment can be tailored for optimal efficacy. methods for metabolomics research: there are two main methods for metabolomics research: 1. mass spectrometry (ms)-based metabolomics. 2. nuclear magnetic resonance (nmr) spectroscopy. by combining these methods, metabolome coverage can be improved by addressing many of their individual shortcomings (wilkins and trushina, 2018). while nmr is useful for identifying core metabolites in important metabolic pathways, ms-based metabolomics offers a broad metabolite coverage that includes polar metabolites as well as non-polar lipids and is particularly good at detecting low-abundance metabolites (wilkins and trushina, 2018). additionally, nmr allows for the study of a large number of participants due to its low cost, high throughput, and analytical power (surendran et al., 2022). although the discovery of new biomarkers in metabolic pathways has contributed to the pathogenetic understanding of disease pathways, untargeted metabolomics has the potential to identify metabolites that may not be recognized due to their absence from the software library (odom and sutton, 2021). 2. biomarkers of metabolic and cardiovascular diseases using metabolomics 2.1 cardiovascular biomarkers the metabolomics of cardiovascular disease have identified lipid biomarkers and other metabolites that describe key pathophysiological characteristics, such as inflammation, oxidative stress, and disrupted lipid metabolism (upadhyay, 2015; vona et al., 2019). in addition to helping to track the progression of the disease, these biomarkers have excellent diagnostic and prognostic values for identifying individuals who are at risk of cardiovascular diseases (cvd). by offering details on alterations in lipid metabolism that result in atherosclerosis and cvd, lipid biomarkers play a crucial role in determining the risk of cvd. higher levels of ceramides have been linked to heart failure and atherosclerosis (vona et al., 2019), indicating their involvement in inflammatory pathways and cellular apoptosis. more recently, certain lipid metabolites have emerged as novel biomarkers in atherosclerosis. cvd is linked to other non-lipid metabolites, including betaine (millard et al., 2018), branchedchain amino acids (bcaa) (doestzada et al., 2022), and trimethylamine n-oxide (tmao) (park et al., 2019). as a byproduct of the gut microbiota's metabolism of carnitine and choline, tmao increases the amount of cholesterol that deposits in arterial walls, increasing the risk of atherosclerosis (fig. 1) (zhu et al., 2020). cardiovascular problems are linked to elevated bcaa levels, which are known to cause insulin resistance and metabolic syndrome (lynch and adams, 2014). betaine may help prevent heart disease because it is a metabolite that is involved in the metabolism of homocysteine, which is linked to improved endothelial function and decreased inflammation (zhao et al., 2018). in addition to improving cvd risk prediction, these biomarkers offer potential targets for therapeutic intervention, which aids in the development of more individualized and preventive strategies for cardiovascular health. clinicians can better risk-stratify patients, track the course of the disease, and improve their treatment plans by characterizing these particular lipid and metabolic biomarkers. 2.2 metabolic syndrome and diabetes in diabetes and metabolic syndrome, biomarkers based on metabolomics provide information about insulin resistance, obesity, and overall metabolic dysfunction that can be linked to either of these conditions (park et al., 2015). indeed, it has been suggested that biomarkers improve metabolic health management by enabling early detection, focused intervention, and therapeutic response monitoring. insulin resistance and general metabolic dysfunction are modulated by amino acid biomarkers (gar et al., 2018). high levels of the bcaas leucine, isoleucine, and valine are frequently linked to insulin resistance and are observed in metabolic syndrome and type 2 diabetes (t2d) (andersson-hall et al., 2018). because mamman et al.; asian j. immunol., vol. 8, no. 1, pp. 190-206, 2025; article no.aji.140476 194 bcaa interferes with insulin signaling, muscle cells are unable to absorb glucose as well as lipids build up (fig. 2) (vanweert et al., 2022). fig. 1. tmao's function in the development and formation of atherosclerotic lesions. high levels of tmao in the bloodstream are essential for the development of foam cells and endothelial dysfunction; tmao can stimulate platelets and encourage the formation of thrombi, which increases the risk of rupture of the atherosclerotic plaque copyright wiley (2020) (zhu et al., 2020) fig. 2. diagrammatic representation of the processes that connect insulin resistance and bcaa catabolism. glut4 glucose transporter type 4, irs-1 insulin receptor substrate-1, pdh pyruvate dehydrogenase complex, s6k ribosomal s6 kinase, mtor mammalian target of rapamycin complex, and bcaa branch-chain amino acids. (vanweert et al., 2022). copyright nature publishing group (2022) mamman et al.; asian j. immunol., vol. 8, no. 1, pp. 190-206, 2025; article no.aji.140476 195 a higher risk of type 2 diabetes has also been linked to aromatic amino acids like phenylalanine and tyrosine, which are a sign of impaired amino acid metabolism in obesity and insulin resistance (luo et al., 2020). since impaired lipid metabolism is one of the factors contributing to insulin resistance and systemic inflammation, lipid biomarkers are essential in the development of diabetes and metabolic syndrome. high levels of obesity are associated with free fatty acids (ffas), which exacerbate inflammation and insulin resistance (marko et al., 2024). according to roszczyc-owsiejczuk et al. (2021), ceramides, a class of sphingolipids, are closely linked to lipid-induced cellular stress and apoptosis and are implicated in insulin resistance. ceramides are a potential target for therapy because they are predictive of metabolic dysfunction and the onset of type 2 diabetes (chaurasia et al., 2021). metabolites of the tricarboxylic and glycolytic acid cycles are examples of additional biomarkers. the end products of glycolysis, lactate and pyruvate, have been found to be elevated in metabolic syndrome, indicating a disruption in the metabolism of glucose (rabinowitz and enerbäck, 2020). insulin resistance-related mitochondrial dysfunction and elevated levels of tricarboxylic acid cycle intermediates, such as citrate and succinate signaling, have been linked to disruptions in energy metabolism in obese individuals (ives et al., 2020). additionally, biomarkers like these are crucial for the early detection of metabolic diseases and have bearing on individualized treatment plans. in addition to identifying individuals at high risk for diabetes and metabolic syndrome, such surveillance of particular metabolic signatures can give clinicians the ability to track the progression of the disease in real time, allowing them to adjust their intervention to maximize metabolic health (ives et al., 2020). 3. metabolomics-based biomarkers for cancer 3.1 changes in metabolism in cancer compared to normal cells, cancer cells exhibit significant metabolic reprogramming that enables them to invade, multiply, and survive quickly (schiliro and firestein, 2021). this type of metabolic change has been referred to as the "warburg effect" (liberti and locasale, 2016; deberardinis and chandel, 2020). even when oxygen is present, aerobic glycolysis is preferred over oxidative phosphorylation. due to this process, cancer cells generate massive amounts of lactate, which alters the tumor microenvironment's composition and encourages growth and immune evasion (beloribi-djefaflia et al., 2016). furthermore, in order to maintain high cell division rates, cancer cells exhibit altered lipid metabolism, amino acid dependence (lieu et al., 2020), and increased nucleotide synthesis (mullen and singh, 2023). for identifying specific biomarkers linked to these specific cancer changes in metabolism, metabolomics has proven to be very helpful. according to danzi et al. (2023), metabolomics profiling of metabolites in blood, tissue, and other biofluids reveals metabolic signatures linked to cancer that can differentiate cancerous cells from healthy cells. some oncometabolites, like fumarate, succinate, and 2-hydroxyglutarate (2-hg) in gliomas, act as genetic mutations in metabolic enzymes in addition to being biomarkers for particular types of cancer (sciacovelli and frezza, 2016; liu and yang, 2021). potential treatment targets are revealed by these tumor-specific metabolites, which provide insight into tumor metabolism. additionally, the biomarkers obtained through metabolomics enable the advancement of personalized and precision oncology by facilitating early cancer diagnosis, treatment response, and disease surveillance (mateo et al., 2022). through metabolomics, this gives physicians the ability to see how cancer cells alter their metabolic activity in real time as a treatment progresses; as a result, these facts open the door to targeted interventions and individualized therapeutic approaches. 3.2 important biomarkers in cancer in cancer diagnosis, prognosis, and treatment targeting, biomarkers particularly oncometabolites are important (kes et al., 2020). mostly caused by mutations in metabolic enzymes, oncometabolites are metabolic intermediates that, when present in abnormal amounts, cause neoplastic disease (yong et al., 2020). in general, these biomarkers are extremely valuable for predicting disease outcomes and treatment response in addition to being used to identify cancers. mutations in the isocitrate dehydrogenase 1 (idh1) and 2 (idh2) enzymes in gliomas and acute myeloid leukemia mamman et al.; asian j. immunol., vol. 8, no. 1, pp. 190-206, 2025; article no.aji.140476 196 (aml) (fig. 1) produce 2-hg, a well-known oncometabolite (rakheja et al., 2013). because 2-hg is a factor that damages cellular differentiation at high levels, which leads to tumorigenesis, high levels of 2-hg function as both a diagnostic and a prognostic biomarker (wang et al., 2013). for example, dinardo et al. 2013 emphasize the value of 2-hg in aml for both diagnosis and prognosis (dinardo et al., 2013). this study showed that idh1 and idh2 mutations were linked to significantly elevated serum 2-hg levels, and at a threshold of 700 ng/ml, the diagnostic sensitivity and specificity were high, at 86.9% and 90.7%, respectively. clinical outcomes were also linked to serum 2hg levels, with higher levels being associated with a worse prognosis and a greater tumor burden. the results suggest that 2-hg may be a promising non-invasive biomarker for detecting idh mutations and tracking the progression of the disease and response to treatment in aml patients. giving some methodological or standard information about the clinical validation and application of biomarkers would also be pertinent to the research on them. furthermore, miller et al. highlight the importance of idh mutations and their metabolite, 2-hg, in the diagnosis and management of gliomas (miller et al., 2023). a reliable biomarker for idh-mutant gliomas, elevated 2-hg levels can be found using non-invasive techniques. clinically speaking, idh mutations differ from gliomas with wild-type idh in that they offer higher survival rates than are otherwise observed. targeted treatments for these mutations also have the potential to alter the tumor ecosystem in order to enhance therapeutics. these findings demonstrate how crucial idh mutations are to improving glioma diagnosis and treatment accuracy. according to dallas pozza et al. (2020), succinate is another significant biomarker that builds up in some forms of paragangliomas and pheochromocytomas as a result of mutations in the enzyme succinate dehydrogenase (sdh). similarly, hereditary leiomyomatosis and renal cell carcinoma cause fumarate to build up as a result of mutations in fumarate hydratase (fh) (trpkov et al., 2016; wang et al., 2024). thus, these metabolites play a role in identifying hereditary cancer syndromes and assessing cancer risks (collins et al., 2017; di gregorio et al., 2021) by acting as "oncometabolites" that drive dna methylation (lanzetti, 2024) and hypoxia-like responses (fandrey et al., 2019). according to vo and trinh (2024), alterations in lipid metabolism metabolites, such as phosphocholine, are also being found to be biomarkers for prostate and breast cancers. tumor growth is indicated by elevated levels of phosphocholine and other lipid-related metabolites, which can also provide diagnostic details about different cancer subtypes. in addition to supporting early diagnosis, these important biomarkers also offer valuable insights into tumor behavior and enable personalized prognosis, which leads to targeted therapies. as science continues to advance, the identification of biomarkers and their clinical applications continue to grow. according to vo and trinh (2024), these enhance precision oncology and enable therapies that are truly customized to a patient's metabolic profile. 4. biomarkers for mental health and neurodegenerative disease 4.1 neurodegenerative diseases and metabolomics a powerful tool in the hunt for neurodegenerative diseases is metabolomics, which makes it possible to identify biomarkers connected to the course of the illness and use them for diagnosis and treatment monitoring. in the early stages of detection and differential diagnosis, diseases like alzheimer's disease (ad), parkinson's disease (pd), and amyotrophic lateral sclerosis (als) are useful because they exhibit specific metabolic alterations linked to the underlying pathophysiological mechanisms in these conditions (dubois et al., 2023). glutamate, myoinositol, and phosphatidylcholine are biomarkers of alzheimer's disease that are associated with inflammation, oxidative stress, and neuronal damage (zhang et al., 2023; reveglia et al., 2023; wang et al., 2024). low levels of acetyl-lcarnitine are linked to mitochondrial dysfunction and cognitive decline (pennisi et al., 2020), while elevated myoinositol levels have been linked to amyloid plaque deposition, one of the hallmark events of ad (voevodskaya et al., 2016). early intervention may be possible because other lipidrelated metabolites, which involve specific phospholipids, are being investigated as biomarkers of the preclinical stages of ad. dopamine, uric acid, and homovanillic acid are the main metabolomics biomarkers that have been investigated in the context of parkinson's disease (kremer et al., 2021). dopamine depletion brought on by dopaminergic neuron mamman et al.; asian j. immunol., vol. 8, no. 1, pp. 190-206, 2025; article no.aji.140476 197 degeneration is a key component of parkinson's disease. conversely, there is an increase in homovanillic acid, a dopamine metabolite that could indicate the severity of the illness (kremer et al., 2021). oxidative stress has been implicated in a higher risk of parkinson's disease (pd), as evidenced by lower levels of the antioxidant uric acid (seifar et al., 2022). in als, biomarkers like creatine, ascorbate, and different amino acids show abnormalities in energy metabolism, neuroinflammation, and oxidative stress (kori et al., 2016; lanznaster et al., 2018). while branched-chain amino acid changes reflect disruptions in the muscle metabolism of these vital nutrients in als patients, lower creatine levels signify deficiencies in cellular energy (parvanovova et al., 2024). profiling these biomarkers using metabolomics offers a better understanding of the mechanisms underlying neurodegenerative disease, which may lead to early diagnosis and help distinguish it from other conditions. additionally, because metabolite levels can be tracked to evaluate treatment effectiveness and select individualized therapeutic approaches in neurodegenerative care, these biomarkers can provide insights into the development of targeted therapies (parvanovova et al., 2024). 4.2 the biomarkers of mental health the field of mental health research has seen a lot of use of metabolomics, particularly in the hunt for biomarkers linked to a range of psychiatric conditions, such as anxiety, bipolar disorder, schizophrenia, and depression (abidargham et al., 2023). the examination of neurotransmitter pathways and metabolites associated with the gut-brain axis may yield markers for the biochemical underpinnings of mental health disorders, enabling more precise diagnosis and customized treatment plans. because they directly affect brain activity and mood regulation, neurotransmitter metabolites are significant in psychiatric disorders. for instance, serotonin and its metabolite 5hydroxyindoleacetic acid (5-hiaa) is the subject of much research in relation to mood disorders (jayamohananan et al., 2019). likewise, dopamine and homovanillic acid dysregulation is associated with schizophrenia and bipolar disorder (wada et al., 2022), while depression is said to be associated with lower levels of serotonin and its metabolites (moncrieff et al., 2023). a known inhibitory neurotransmitter, gamma-aminobutyric acid (gaba), has been linked to symptoms of anxiety and depression. variations in gaba levels reflect shifts in mood fig. 3. potential pathways through which scfas influence gut–brain communication. copyright frontiers media sa (2020) (silva et al., 2020) mamman et al.; asian j. immunol., vol. 8, no. 1, pp. 190-206, 2025; article no.aji.140476 198 regulation and stress modulation (prévot and sibille, 2021). furthermore, metabolites generated by the gut-brain axis are becoming more widely acknowledged for their role in mental health (góralczyk-bi´ nkowska et al., 2022). intestinal microbiota activity produces short-chain fatty acids (scfas), like butyrate and propionate, which impact neuroinflammation, stress reactions, and emotional regulation systems (fig. 2) (silva et al., 2020). given their role in reducing inflammation and maintaining the integrity of the blood-brain barrier, low levels of scfas have been proposed to be linked to anxiety and depression (silva et al., 2020). tryptophan and kynurenine, two other gut-derived metabolites, have been linked to mood disorders and schizophrenia and are linked to immunological responses and neurotransmitter synthesis (marx et al., 2021). not only can these metabolomic biomarkers reveal the biochemical foundations of psychiatric disorders, but they will also enable a more individualized approach to mental health treatment. continuous monitoring of these biomarkers would assist medical professionals in streamlining diagnostics, customizing treatments, and probably improving patient outcomes by providing more targeted interventions (marx et al., 2021). 4.3 potential early diagnosis biomarkers for mental health and neurodegenerative diseases in the diagnosis and treatment planning of neurological and psychiatric disorders, biomarkers based on metabolomics hold great promise (török et al., 2020; mallu et al., 2021). through the identification of particular metabolic alterations that take place even prior to the manifestation of clinical symptoms, these biomarkers are anticipated to aid in early intervention, improving the prognosis by delaying the course of the disease (sun et al., 2023). they are useful for early diagnosis because, for example, a preclinical stage of the disease has been noted in which ad patients exhibit elevated myo-inositol and alterations in phospholipid profiles (ahanger et al., 2024). if these biomarkers are discovered early, prompt interventions that postpone cognitive decline will be possible. metabolomics as a foundation for biomarker discovery for the early diagnosis of psychiatric and neurodegenerative disorders has produced very encouraging results in clinical studies. alpha-synuclein aggregates and urates have been identified as interesting early indicators of parkinson's disease (pd) due to their lower levels in csf (ganguly et al., 2021). according to averina et al. (2024), depression and schizophrenia have been linked to alterations in serotonin and dopamine metabolism. moreover, increased kynurenic acid levels are associated with oxidative stress and neuroinflammation, providing insight into the early phases of mood disorders and schizophrenia (o'farrell and harkin, 2017; mor, et al., 2021). these gut-brain axis-derived altered scfas are emerging as promising biomarkers for mental health conditions, particularly depression (o'riordan et al., 2022). furthermore, als and ms can be accurately diagnosed early thanks to neurofilament light chain levels in als and sphingomyelin reductions in ms (yang et al., 2022). according to sacchet et al. (2024), elevated cortisol levels in anxiety disorders raise the risk and help make a diagnosis even before all symptoms appear. in addition to offering tools for early detection, these biomarkers also enable the application of tailored treatment to enhance prognosis and impede the progression of disease. early detection of mental health conditions enables physicians to use treatment plans based on individual biochemical profiles that optimize an intervention's efficacy (sacchet et al., 2024). it is hoped that the use of metabolomics-based biomarkers will eventually lead to a paradigm shift in how doctors choose treatments that are specific to each patient and diagnose illnesses early. this will improve patient outcomes by more effectively managing psychiatric and neurodegenerative conditions (sacchet et al., 2024). 5. disease and health metabolomics applications of metabolomics in health and disease include single-cell, epidemiologic population studies, metabolic phenotyping, metabolome-wide association studies (mwas), precision metabolomics, and integrative metabolomics, which is the use of metabolomics in conjunction with other omics disciplines (dalamaga, 2024).live single-cell mass spectrometry (lscms), lc-ms/ms, gc-ms/ms, mamman et al.; asian j. immunol., vol. 8, no. 1, pp. 190-206, 2025; article no.aji.140476 199 and single-cell metabolomics and lipidomics technologies enable high-dimensional characterizations of individual cells, disease heterogeneity and complexity, and the identification, expression, and abundance of disease-associated metabolites and small mole cules (lempesis et al., 2022). cell-cell interactions and tumor heterogeneity were discovered through imaging ms analysis of human breast cancer samples at the single-cell level (lempesis et al., 2022). metabolomics fingerprinting and footprinting techniques, mwas, and clinical biomarkers and various metabotypes of disease severity correlated to exposures (zhang et al., 2023) and biological outcomes (sun et al., 2023) have been studied and identified in individuals and populations through metabolomics fingerprinting and footprinting techniques, which will facilitate precision medicine and public healthcare (guo et al., 2023; wang et al., 2023; xu et al., 2023; ou et al., 2024; zhou et al., 2024; xu, et, al., 2024). the transition from genome-wide association studies (gwas) to metabolome-wide association studies (mwas) was initially defined in 2008 as "an investigation of the relationships between phenotype variation and disease risk factors through environmental and genomic influences" (xu et al., 2023; ou et al., 2024). exposures to single individual phenotypes and populations, epidemiologic research, disease risk, metabolome-wide association studies, and precision medicine were proposed by rattray and colleagues (ou et al., 2024). 6. metabolomics and the exposome unique opportunities to identify food ingredients and comprehend their role in the dietary exposome and food quality are provided by metabolomics. modern metabolomics techniques were employed in a study by nikou et al. (2020) to find chemical biomarkers relevant to the production process, cultivation methods, and geographic origin of olive oil, a component of the mediterranean diet that has been described as a nutritious dietary pattern. to examine intact oil and the associated polyphenols of extra virgin olive oils, metabolomic profiling using flow injection analysis-magnetical resonance mass spectrometry (fia-mrms) and the lc-orbitrap ms platform was employed. the approach may provide a means of combating food fraud and adulteration while also identifying elements of a nutritious diet. occupational exposures and patterns, such as shift work, are a significant component of the exposome. in recent years, shift work has been linked to a number of physiological changes and may be related to a number of diseases. report by borroni et al. (2023) assessed how night shift work affected the serum metabolome in a group of italian female nurses who worked nights and female coworkers who did not. they found that there were variations in the levels of taurine, serotonin, aspartic acid, and certain lipids, all of which provide hints about the biological changes brought on by working nights. according to barupal et al. (2022) reprocessing metabolomics datasets that have already been published and made publicly available can alter the number of metabolite identifications made and possibly reveal new biomarkers that were overlooked during the initial processing, as the authors of the paper by barupal et al. (2022) showed. in contrast to the initial data preprocessing method that employed the mass spectrometer manufacturer's software, the authors used ms-dial, a publicly available program, for data pre-processing and applied less strict data processing thresholds. different software and even small adjustments to preprocessing parameters can affect the quality and quantity of data reported, as the manuscript illustrated. this presents both opportunities and challenges. keski-rahkonen et al. (2021) and barupal et al. (2022) two additional comments on the manuscript sparked a heated discussion that added to the metabolomics community's broader conversations regarding data pre-processing techniques, software and parameters, reporting standards and formats, and intercomparability among data pre-processing methods. 7. future direction of metabolomics metabolomics technology will be integrated with other cutting-edge technologies to guarantee its expansion. ms-based metabolomics will be more precise, and a number of combined techniques, including gc/ms, lc/ms, and others, will provide a strong scientific basis for tackling the challenge of metabolite analysis and metabolic pathway discovery. extended mass spectrometry holds a prominent position as a vital analytical tool in the field of metabolomics. the growth of this field has provided insight into the etiology of many diseases and assisted in the identification of numerous potential illness biomarkers (xiong et al., 2020). mamman et al.; asian j. immunol., vol. 8, no. 1, pp. 190-206, 2025; article no.aji.140476 200 some of the studies that have been assessed include research for recurrence testing, appropriate treatment, medication prognosis and prediction, and early disease diagnosis (sun et al., 2019). lc, gc, or ce is used in most ms analytical procedures because biological mixtures are very complex. rapidly growing applications using evolving separation mechanisms and protocols do present both opportunities and challenges, though. with the advent of chromatographic techniques, the capacity to separate metabolites and enhance the number of metabolites identified has surely improved (li et al., 2017; zhang et al., 2019). the inability to analyze and relate the results of the latter studies, which are carried out on comparable or identical samples gathered by multiple research institutions, is a major barrier. this is the biggest obstacle to progress in this field. other factors, such as sample preparation, sample matrix, and residual effects, can also contribute to data variability. to overcome these challenges, it is essential to switch from calculating relative metabolite concentrations to more accurate absolute concentration measurements, regardless of the analytical platform, approach, and procedure. this approach is important even though ms finds it difficult. 8. conclusion a helpful method for identifying disease-related metabolites in biofluids or tissue as well as for classifying and/or characterizing molecular patterns linked to disease or treatment that are produced from metabolites is mass spectrometry (ms) based metabolomics/lipid omics. we continue to discover new biomarkers in metabolomics that redefine health and disease diagnostic precision and provide insight into the biochemical etiology of a wide range of diseases. this makes metabolomics a potentially useful platform for prognosis, early detection, and the application of tailored treatment plans founded on a thorough comprehension of metabolic changes brought on by illness and environmental influences. the metabolic profiling biomarkers have the potential to improve diagnosis and enable targeted interventions in metabolic syndromes, cancers, cardiovascular diseases, and neurodegenerative disorders. but before the biomarkers can truly reach the clinic, a lot more work needs to be done. to ensure that the biomarkers are accurate and repeatable across a range of populations, considerable attention must be paid to inter-individual variability, environmental factors, and data complexity in metabolomics. confounding factor considerations and a strict methodology in biomarker validation with large cohorts are necessary for that. depending on the features of metabolic profiles, biomarker-focused research and translation to clinical practice are constantly pushing this field away from standard treatment and toward personalized therapies. in order to translate biomarkers from metabolomics into clinical practices for improved patient outcomes and to steer the trajectory of precision medicine, it will be necessary to overcome many of the current obstacles through ongoing research and collaboration among clinicians, researchers, and technologists. disclaimer (artificial intelligence) author(s) hereby declare that no generative ai technologies such as large language models (chatgpt, copilot, etc.) and text-to-image generators have been used during the writing or editing of this manuscript. consent it is not applicable. ethical approval it is not applicable. competing interests authors have declared that no competing interests exist. references abi-dargham, a., moeller, s. j., ali, f., delorenzo, c., domschke, k., horga, g., jutla, a., kotov, r., paulus, m. p., & rubio, j. m. 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(2020). gut microbiota in atherosclerosis: focus on trimethylamine n-oxide. apmis – all purpose medical information system, 128, 353–366. disclaimer/publisher’s note: the statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of the publisher and/or the editor(s). this publisher and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. _________________________________________________________________________________ © copyright (2025): author(s). the licensee is the journal publisher. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: https://pr.sdiarticle5.com/review-history/140476 https://pr.sdiarticle5.com/review-history/140476 _____________________________________________________________________________________________________ *corresponding author: e-mail: ibrahimshnawa3@gmail.com; cite as: shnawa, ibrahim m s. 2025. “an alum loaded macrophage driven autoimmune myopathy”. asian journal of immunology 8 (1):97-103. https://doi.org/10.9734/aji/2025/v8i1164. asian journal of immunology volume 8, issue 1, page 97-103, 2025; article no.aji.134974 an alum loaded macrophage driven autoimmune myopathy ibrahim m s shnawa a,b* a department of medical biotechnology, college of biotechnology, al-qasim green university, qasim, babylon, iraq. b college of nursing, university of hilla, babylon, iraq. author’s contribution the sole author designed, analysed, interpreted and prepared the manuscript. article information doi: https://doi.org/10.9734/aji/2025/v8i1164 open peer review history: this journal follows the advanced open peer review policy. identity of the reviewers, editor(s) and additional reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here: https://pr.sdiarticle5.com/review-history/134974 received: 25/02/2025 published: 03/05/2025 abstract immune mediated diseases and syndromes are rare and attributed at most to genetic and environmental interactions. macrophagic myofasciitis mmf is one of these syndromes sub-entities. in the present opinion the immunobiology of mmf was being reviewed. the molecular autoimmune mechanisms can be as follows; concurrent release of alum nano-molecules are taken up by macrophage persist in, combine with cellular proteins forming metalloprotein. it is now modified cellular protein in a modified macrophage which have dc marker, but still of macrophage morphology. metalloprotein, when being extracellular on cellular burst or on diffusion to extracellular space will reach immune cells, in presence of; chronic induction, pathogenic allele, the hla dr1 01 and the affected tissue microenvironment. molecular mimicry, antigen bystander and/or epitope spreading response may operate and autoimmune tissue changes happened within continuum of granulomatous lesion developed in skeletal muscle at the injection site. modified macrophage may migrate to regional lymph node and spleen the finally reach the brain. as a result, disturbance occurs in skeletal muscle functions and in brain cognition function. opinion article https://doi.org/10.9734/aji/2025/v8i1164 https://pr.sdiarticle5.com/review-history/134974 shnawa; asian j. immunol., vol. 8, no. 1, pp. 97-103, 2025; article no.aji.134974 98 keywords: alum; allele; autoimmune; environment; genetics; granuloma; hla; macrophage myopathy. 1. introduction immuno-prophylactants are vaccines and serotherpeutics. these standard biologics are helpful for prevention and therapy, both in man and animals. vaccines and adjuvants so far they are helpful but they are associated with an adverse effects. vaccine and adjuvant adverse effects can be ramified into; vaccine and adjuvant associated disease enhancement vade and vaccine failure vf (shnawa, 2017, 2023, shnawa & alkhafaji 2023). one of the known vade is shoenfeld syndrome ss (shoenfeld et al., 2011). ss grouped five disease sub-entities as; ipostvaccination with adjuvanated vaccine illness, ii-macrophagic myofasciitis illness mmf, iii – sick building illness, iv-gulf war illness and v siliconosis. this syndrome sub-entity is a molecular immunogenetic disease with presentation of an autoimmune reactions. it is an inducive chronic rare syndrome associated with specific human leukocyte antigen haplotype (calarelli et al., 2024). the objective of the present opinion paper was to tackle the immunobiology [box one] of mmf. box-one: relevant terminology (abbas et al., 2015) 1. anergy: a state of unresponsiveness to antigenic stimulation. lymphocyte anergy is failure of t and/or b cell clones to react antigen and is a mechanism of maintaining immune tolerance to self antigens. 2. antigen bystander: continued immune responses to infection modified proteins, an attendant inflammation allow exposure of autoantigens to immune responses. theoretically, this could operate through t cell recognition resulting in help of potentially, b lymphocyte. 3. clonal ignorance: a form of lymphocyte unresponsiveness in which self antigens are ignored by the immune system even though lymphocyte specific for their antigens remain viable and functional. 4. clonal deletion: a mechanism of lymphocyte tolerance in which an immature t cell in the thymus or immature b cells in bone marrow undergoes apoptotic death as a consquences of recognizing self antigens. 5. epitope spreading: in autoimmunity it is found that the development of immune responses to multiple epitopes as an autoimmune disease originally target one epitope progresses, likely caused by further breakdown in tolerance and release of additional tissue antigen due to self protein stimulated by the initial response. 6. molecular mimicry: a postulated mechanism of autoimmunity triggered by infection with a microbe that cross react with self antigen. immune response to the microbe results in reactions with self tissue antigen. 7. sequestrated antigens: there are certain tissue niches in which their specific antigens are not recognized to immune system cells during the ontogeny of the individuals. like, eye vetrus fluid, semen plasm, and synovial fluid when exposed to immune cells will be recognized as foreign. 8. tolerance: unresponsiveness of adaptive immune system to antigen, as a result of inactivation or death of the antigen specific lymphocyte induced by the exposure to antigen. 2. macrophagic myofasciitis mmf concept mmf is an uncommon immune mediated inflammatory disorder of muscle and is believed to be due to an alum in a vaccine combination exhibited persistence at the site of injection. the conditions characterized by diffuse myalgia, arthralgia and fatigue (ravindran 2024, dittmann 2000, ieraeli et al 2011). it is a rare inflammatory condition that affect skeletal muscle and connective tissue characterized by infiltration of macrophages into muscle tissue, in which the affected subject presents local or systemic manifestation. the local manifestation can be an immune active lesion of granuloma, in a rare muscle disease characterized by; microscopic lesions found in muscle biopsies that showed infiltration of the muscle tissue by pas positive macrophages in light microscope and alum crystal inclusion in electron microscopic studies and they are traced in epimysium, perimysium, prefascular endomysium with crystal lesion composed of aluminum salt (tervaert et al, 2023, gibson, 2024). hence, aluminum containing vaccines have been implicated. mmf lesions result from aluminum hydroxide adjuvant hidden within the tissue with frequent steady state release of alum causing immune reactions (watad et al., 2017, santos et al 2018, caldarelli et al., 2024). shnawa; asian j. immunol., vol. 8, no. 1, pp. 97-103, 2025; article no.aji.134974 99 3. timeline knowing the past will in light the present and pin point directions to the future. so, the mmf timeline made in table 1. table 1. macrophagic myofasciitis timeline achievement date reference mmf was initially described as an emergency entity by franch myo-pathologist. the reported in lancet 1993,1998 shivane et al., 2012 amoura et al., 2000 between 1993 and 1999 more than 50 cases of mmf have been described in france 1999 amoura et al., 2000 mmf patient presents central nervous system disease 2001 authier 2001 long term persistence of vaccine driven alum in muscle 2001 gherardi et al., 2011 mmf present local and systemic forms 2003 papo 2003 electron microscopic study of mmf lesion description 2003-2005 shmgdi et al., 2005 reporting mmf unrelated to vaccination 2005 park et al., 2005 experimental induction of mmf in rats.th1 bias response, th1/th2 balance unchanged in norma lesion size 2006 authier et al., 2006 mmf being prove of vaccine autoimmune related disease 2011 israeli et al., 2011 macrophage take up alum in tendon through fluerescent alum translocate to drainage lymph node then to blood, spleen and brain 2012 gherardi & authier 2012 mmf several reports in uk 2012 shivahe et al., 2012 a report of an atypical presentation of mmf 2020 dias et al., 2020 al (oh)3 vaccine associated with mmf pseudo-lymph node and causing hypersensitivity 2020 kim et al., 2020 4. immunobiology relatively, immune cells both naïve and active forms could be involved in immunobiology of mmf. though the main player cells is the naïve and active macrophage. autoreactive b cells are evidently involved, autoreactive t cells are of unclear role. modified macrophages of dc surface markers, modified morphology and modified function but still of macrophage morphotype are noted .th1/th2 are note changed and th1 biased responses. these are the main immune-biological features of mmf as presented in the following paragraphs. alum nano-molecules may reach distant organs of the body including brain through the migration of the an alum loaded macrophage or though out diffusion process across the semipermeable membranes. this accompanied by an active liver detoxification of this chemical insult. clearance of alum from the body have been found a species dependent process (gherardi et al., 2019). alum adjuvant induces humoral immune th2 responses via primary and secondary response events in mice and mixed humoral and cellular responses in human being whereby vaccine adjuvant supports the activation of cd8 t cells but these cells does not differentiated to cytotoxic t cells (hogen-esch 2013). in an in-vitro culture system al(oh)2 stimulate isolated macrophages that contains large and persistent intracellular crystalline inclusion, the “alum loaded macrophage ”alm.alm exhibit phenotypical and functional modifications as they showed myeloid dendritic cell surface markers[hladr high,/cd1a-/cd14-] and displayed potent ability to induce mhc restricted antigen specific memory response but kept macrophage morphology. this suggest a key role of alm in relation to the alum-vaccine and important role in this memory response (ann-ceclle et al., 2004). the vaccine alum adjuvant formulation when applied into the muscle, months to years later, the alum persists hidden in the inoculation site. then released frequently in a steady state manner leading to chronic induction of immune reaction in the application site macrophages and to lesser extent lymphocytes accumulate in the injection site took up alum nano-molecules may be through pinocytosis. local granulomatous response is initiated and developed. in continuum with this local reaction alum may combined with self cellular protein from metalloprotein. a modified self protein such as a metalloprotein of mmp initiated specific immune and autoimmune responses in presence of the pathogenic allele hladr 1 01 and the activated shnawa; asian j. immunol., vol. 8, no. 1, pp. 97-103, 2025; article no.aji.134974 100 tissue microenvironment as a pathology system. the net result of such reactions is the production of myo-specific autoantibody and cd8+t cell predominantly existed along with modified macrophages in the lesions (shnawa 2023). this alum loaded modified macrophages my migrate through out blood stream to lymph nodes, spleen and brain by this they may lead to skeletal muscle and cognitive disorders (gherardi et al 2021). mmf has been reported after contact with metals and /or vaccines.it is typically occurs in individuals with genetic predisposition like hladrb1 andptpn22.such contact may initiate over-immune reaction of the immune system that propriates to production of autoantibodies and fully cause autoimmune disorder. mmf is a subentity of asia syndrome results from interaction between genetic and environmental factors with adjuvant through modulation receptors such as tlr, nlr and clr triggering aberrant immune response prompting development of an autoimmune disorder (caldarelli et al., 2024). aluminum adjuvant is well known enhancer of th2 responses. however, it has been suggested that aluminum induces th1 response in presence of other th1 inducing compounds such as lps or rec. influenza antigen, a bystander effect through which alum adjuvant trigger autoimmunity via activation of dormant autoreactive t lymphocyte in some individuals (fan et al., 2022). 5. mmf autoimmune mechanisms in general autoimmune mechanisms are of classical and recent mosaic nature (shnawa 2023). mmf autoimmune mechanisms composed of a paradigm of three heritability features and four govering etiology roles (li et al., 2015). as mentioned in the following paragraphs; three main molecular mechanisms valid for explaining autoimmunity as; tolerance, molecular mimicry and epitope spreading. tolerance can be established through clonal deletion, anergy, clonal ignorance and regulatory t cell function (perricon et al., 2019). the heritability of mmf has been well documented, quantified and exhibit three important features; i – all genetic diseases have strong genetic components, ii relatively large numbers of risk alleles are shared between multiple autoimmune diseases and iii the product of most of the autoimmune associated genes are parts of immunological pathways in particular t cell signaling, tnf signaling and innate immunity (zherankova et al., 2009). the reaction of the immune system to an environmental stimuli, produce autoimmune disease should pass four governing roles in a stepwise manner and start with; i-foundation of predisposing genetic architecture representing autoimmunity, iichronic repeated skewed and biased responses over years yield pathological system, iiithe pathological system induces loss of immune tolerance and ivadopting nocuous potentials (li et al., 2015, zherankova et al., 2009, shnawa 2023). hence, the proposed autoimmune mechanisms of the mmf are as follows; the genome of mmf patients has strong genetic component which is the autoimmune associated genes that contains large numbers of risk alleles. the gene expression products of these risk alleles are parts of the immunological pathway. alum chronic repetitive induction over years face the genome of the patient leading to a pathological system. such pathological system induces loss of tolerance. then affected immune system adopt nocuous potentials [molecular mimicry, antigen bystander and/or epitope spreading] the autoimmune condition. 6. mmf immune features (li et al., 2015, zherankova et al., 2009, shnawa 2023) the major immune features of mmf can be pointed out in the followings; i -rare immune mediated disease ii – it is of an inducive and constitutive nature. iii – adjuvant-alum driven. iv-alum form metalloprotein a modified self protein. v-mmf macrophage appeared to uselc3 associated phagocytosis to alum vaccine, and secret pain inducing molecules (masson et al., 2024). vimodified self protein, antigen bystander and/or molecular mimicry in presence of hladr 1a 01 haplotype induce autoimmune response. vii-the nature of the autoimmune response in mice is humoral while in man is mixed humoral and cellular. shnawa; asian j. immunol., vol. 8, no. 1, pp. 97-103, 2025; article no.aji.134974 101 viii-alum modified macrophage adopt new surface marker dc cells but they still of macrophage morphology. such macrophage is believed to have a role in memory response. ix-th1/th2 balance found stable, but with bias th1 response. x-local muscle lesion nature is granulomatous. xi-the immune whole mark of ongoing reactions leads to functional problems in skeletal muscles and in cognition. 7. disease entity the mmf is a rare macrophage driven myopathy. it stands as a molecular immunogenetic condition with an autoimmune presentation linked to hladr 1a 01 susceptibility haplotype. mmf is grouped within the shoenfeld’s syndrome (shoenfeld et al., 2011). 8. laboratory immunology patient’s blood samples collect for the systemic humoral and cellular investigation. the humoral for check of myo-specific autoantibodies and for macrophage and t cell subsets. biopsy sample for detection of the local cellularity nature of the granulomatous tissue reactions. electron microscopic preparation from the lesion to elucidate the alum crystallization in cells. immune laboratory animal model can be prepared to recheck the founding in man. 9. laboratory animal immune models several number of laboratory animal models have been tempted by specialist workers in this field (ruiz et al., 2017, colafrancesco et al., 2013). mmf lesions have been reproduced in; mice, rat, rabbit, monkeys and sheep by iv and im routes. iv associated with rapid elimination. while, rabbit im injection elimination lasts four weeks post injection (gherardi et al., 2019). in an experimental animal setting, fluorescent nanotagged alum within the phagocyte have shown translocation of alum from the site of injection through blood circulation to the regional lymph nodes, spleen then to brain (gherardi & authier 2012). spurg-dully and lewis rat injected with 10ul of ai(oh)3 adjuvant vaccine and watched over one year for the appearance of the lesions and the possible reduction of their sizes per time elapse. shrinkage of the lesion size happened over time. humoral th2 and b cell immune responses mounted.th1/th2 balance. the function of cytotoxic t cell interferes with alum clearance process (authier et al., 2006). 10. animal immune model suggestion a group of 18 spurg-dully rats will be elected and grouped into three groups each of six. one sham saline control, one for alum solution a lone and one with vaccine -alum adjuvant. rats of the three groups im injected, and will follow up to score the lesions and possible reduction over time elapse. the formed lesions will be subjected to histopathological evaluations in month wise manner for six months. 11. fact sheet (park et al., 2019, israeli et al., 2011, cruz-tapias et al 2013) terminology: macrophagic myofasciitis. disease nature: rare immune mediated liked to haladr1*01 susceptibility haplotype. onset duration:3 months to eight years. inducer: vaccine-alum formulation, or unknown tissue lesion nature: local sterio-typed immunologically active lesion. histology: tissue sections from biopsy made, stained with pas searching for cellularity. immunology: determination of myo-specific autoantibodies in patients sera. lesion description: infiltration of epimysium, perimysium, and perivascular endomysium with alum loaded pas positive macrophage accumulation at the site of injection. muscle necrosis is typically absent, spars cd8 t cells and minimal myofiber damage the lesion is of granulomatous nature (gherhardi, authier 2003). chemical analysis: chemical microanalysis and atomic laser spectrophotometery reveals alum crystals within the macrophage. systemic reactions: skeletal muscle and cognitive disorders (israeli et al., 2011, park et al., 2019). age prevalence: all ages. shnawa; asian j. immunol., vol. 8, no. 1, pp. 97-103, 2025; article no.aji.134974 102 12. conclusion mmf is an alum loaded macrophage driven autoimmune myopathy a syndrome sub-entity. it is linked with specific hla haplotype susceptibility and grouped within shoenfeld syndrome. mmf immunobiology and autoimmune mechanisms, immune features and factsheet were issued. consent it is not applicable. ethical approval it is not applicable. disclaimer (artificial intelligence) author(s) hereby declare that no generative ai technologies such as large language models (chatgpt, copilot, etc) and text-to-image generators have been used during writing or editing of this manuscript. competing interests author has declared that no competing interests exist. references abbas, a. k., lichtman, a. h., & pillai, s. 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(2009). detecting shared pathogenesis from shared genetics of immune related diseases. nature reviews genetics, 10, 586– 594. disclaimer/publisher’s note: the statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of the publisher and/or the editor(s). this publisher and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. _________________________________________________________________________________ © copyright (2025): author(s). the licensee is the journal publisher. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: https://pr.sdiarticle5.com/review-history/134974 https://pr.sdiarticle5.com/review-history/134974 _____________________________________________________________________________________________________ *corresponding author: e-mail: samueladavba@gmail.com; cite as: adavba, samuel adinoyi. 2025. “the role of monocytes and macrophages in the pathogenesis of non-aids defining events: mechanisms and therapeutic implications”. asian journal of immunology 8 (1):120-33. https://doi.org/10.9734/aji/2025/v8i1166. asian journal of immunology volume 8, issue 1, page 120-133, 2025; article no.aji.136909 the role of monocytes and macrophages in the pathogenesis of non-aids defining events: mechanisms and therapeutic implications samuel adinoyi adavba a* a kaduna state university, kaduna, nigeria. author’s contribution the sole author designed, analysed, interpreted, and prepared the manuscript. article information doi: https://doi.org/10.9734/aji/2025/v8i1166 open peer review history: this journal follows the advanced open peer review policy. identity of the reviewers, editor(s) and additional reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc, are available here: https://pr.sdiarticle5.com/review-history/136909 received: 25/03/2025 published: 06/06/2025 abstract monocytes and macrophages play a pivotal role in the pathogenesis of hiv infection, contributing not only to viral persistence but also to the development of non-aids-defining events (nades) in people living with hiv (plwh). these innate immune cells act as long-lived viral reservoirs, driving chronic inflammation through persistent immune activation, oxidative stress, and tissue-specific damage. hiv-infected monocytes infiltrate tissues, including the cardiovascular system, liver, kidneys, and central nervous system, where they differentiate into macrophages and release proinflammatory cytokines (e.g., tnf-α, il-6), reactive oxygen species (ros), and matrix metalloproteinases (mmps). these mediators promote endothelial dysfunction, fibrosis, and organ damage, underpinning conditions such as atherosclerosis, neurocognitive disorders, and review article https://doi.org/10.9734/aji/2025/v8i1166 https://pr.sdiarticle5.com/review-history/136909 adavba; asian j. immunol., vol. 8, no. 1, pp. 120-133, 2025; article no.aji.136909 121 hepatorenal disorder. emerging evidence highlights the role of macrophage polarization (m1/m2 imbalance) and epigenetic modifications in sustaining inflammation despite antiretroviral therapy (art). understanding these mechanisms provides critical insights for developing targeted therapies, including immunomodulators (e.g., il-6 inhibitors), antioxidant agents, and reservoirelimination strategies. this review synthesizes current knowledge on monocyte/macrophage-driven pathogenesis in hiv-associated nades. it explores plausible novel therapeutic approaches to mitigate chronic inflammation and improve clinical outcomes in plwh. keywords: hiv; monocyte; macrophages; inflammation; immune activation; viral reservoirs; tissuespecific damage; non-aids-defining events; reservoir elimination. 1. introduction human immunodeficiency virus (hiv) infection continues to pose a significant global health challenge, despite the remarkable success of art in reducing aids-related morbidity and mortality. individuals living with hiv now face an increased risk of certain chronic illnesses collectively referred to as non-aids defining events (nades), which are disproportionately common among plwh (deeks et al., 2013; serrano-villar et al., 2014). this spectrum includes cardiovascular disease, neurocognitive disorders, liver/kidney fibrosis, metabolic syndrome, and non-aids cancers, which can arise despite effective art, contributing to morbidity and mortality in the post-art era (deeks et al., 2013). these conditions occur in plwh at a younger age than in the general population. they are driven mainly by chronic immune activation, inflammation, and immune dysregulation, in which monocytes and macrophages play a central role (campbell et al., 2014; joseph et al., 2022). monocytes and macrophages, integral members of the myeloid lineage, are hiv tropic and express hiv entry receptors, cd4 and ccr5 (ancuta et al., 2008). these cells not only serve as targets for the infection and propagation of hiv but also contribute to the virus's persistence and disease progression over the long term (campbell et al., 2014; kruize & kootstra, 2019). following their differentiation from monocytes, hiv-infected macrophages migrate into tissues where they help spread the infection to nearly every tissue in the body, including the gut, testes, lung, gut-associated lymphoid tissue, brain, liver, urethra, and lymph nodes (tso et al., 2018; rose et al., 2016; ganor et al., 2019). additionally, macrophages reside in anatomical sanctuaries with restricted art penetration, which benefits viral persistence even during therapy (clayton et al., 2018; borkham-kamphorst & weiskirchen, 2016). human monocytes were first identified in 1880 when paul ehrlich and ilya metchnikoff recognized them based on their distinct morphological features (teh et al., 2023; gramegna et al., 2011). however, the development of flow cytometry in the 1970s enabled the creation of a specific antibody panel that classifies monocytes according to the degree of expression of specific surface proteins, namely the pattern recognition receptor cd14 and the fc gamma iii receptor cd16 (kapellos et al., 2019). this parameter categorizes circulating monocytes into three key subpopulations: classical, non-classical, and intermediate. these subpopulations, which differ in abundance and primary functions, can be identified by their size, granularity, and expression of these specific surface markers (ziegler-heitbrock et al., 2010; liberale et al., 2020). classical monocytes, characterized by the expression profile cd14++cd16−, constitute the majority (80–90%) of monocytes. they are the first responders to infections or tissue damage and primarily initiate immune responses through phagocytosis, antigen presentation, and the release of pro-inflammatory cytokines (zieglerheitbrock et al., 2010; potula et al., 2008). non-classical monocytes exhibit a cd14+cd16++ expression pattern and are the least abundant (2-8%). they respond to viral infections, play a crucial role in antibodydependent cellular cytotoxicity (adcc), patrol the endothelial lining of blood vessels, and participate in tissue repair and regeneration. the intermediate subset, designated cd14++cd16+, shows characteristics that bridge classical and non-classical monocytes (tanwar et al., 2020). they possess enhanced capabilities for antigen presentation, aid in tissue repair and angiogenesis, and engage in resolving adavba; asian j. immunol., vol. 8, no. 1, pp. 120-133, 2025; article no.aji.136909 122 inflammation, making them significant in various inflammatory diseases (wong et al., 2011). macrophages are specialized, long-lived phagocytic cells of the innate immune system involved in recognizing, engulfing, and degrading cellular debris, pathogens, and other foreign substances. until recently, they were thought to differentiate solely from monocytes, but newer findings have shed light on the complexities of their differentiation and immunological profiles. macrophages can originate from two distinct sources: from classical monocytes, which migrate into tissues to differentiate into macrophages or dendritic cells (monocytederived macrophages [mdm]), and from selfrenewing tissue-resident macrophages that arise from primitive embryonic precursors (sattentau & stevenson, 2016). this article explores the mechanisms by which monocytes and macrophages contribute to the pathogenesis of hiv and the development of nades, with a focus on their roles as viral reservoirs, mediators of inflammation, and drivers of tissue damage. 2. hiv infection of monocytes and macrophages: key factors in the pathogenesis of hiv monocytes and macrophages are significant targets for hiv-1 infection and play crucial roles in viral transmission, dissemination, and persistence. while monocytes can be infected by hiv-1, their susceptibility to the virus varies depending on their differentiation status. as monocytes differentiate into macrophages, they become more receptive to infection, acting as important reservoirs for the virus. these macrophages can productively replicate hiv-1 even in the presence of antiretroviral therapy (art) (parihar et al., 2010). in contrast to cd4+ t cells, which are rapidly depleted during acute infections, hiv-infected monocytes and macrophages exhibit greater resistance to the cytopathic effects of the virus. this resilience allows them to harbor hiv for extended periods, contributing to viral persistence (miguel et al., 2023). this, in turn, hinders efforts to achieve a cure and drives chronic immune activation and inflammation, leading to various consequences (campbell et al., 2014). the causes of chronic immune activation in hiv infection are complex and not yet completely understood, encompassing both direct factors (such as the enduring presence of hiv rna) and indirect triggers (microbial translocation and coinfections) (mazzuti et al., 2022). but ultimately, the persistent antigenic stimulation leads to sustained elevations of pro-inflammatory cytokines, such as il-6 and tnf-α, along with immune activation markers like cd38 and hladr (paiardini & müller-trutwin, 2013; lederman et al., 2013). monocytes, particularly the cd16+ intermediate and non-classical subsets, play a vital role in this inflammatory environment, exhibiting an activated phenotype characterized by increased expression of adhesion molecules, chemokine receptors, and toll-like receptors (tlrs), which enhances their migration into tissues and responsiveness to microbial products (ancuta et al., 2008). the ongoing inflammation contributes to tissue damage and plays a role in the development of non-aids events (nades) (burdo et al., 2011). moreover, hiv infection alters the functional properties of monocytes and macrophages, diminishing their ability to eliminate pathogens and regulate immune responses. for instance, hiv-infected macrophages demonstrate reduced phagocytosis and impaired antigen presentation capabilities, while monocytes exhibit downregulated mhc ii expression. this downregulation inhibits the formation of mhc iiantigen complexes and diminishes the ability of monocytes to uptake antigens for processing and presentation to t cells (nabatanzi et al., 2018). such impairments contribute to immune exhaustion, increasing susceptibility to opportunistic infections (kedzierska & crowe, 2001). additionally, hiv can induce a pro-fibrotic phenotype in macrophages, potentially leading to organ fibrosis and dysfunction in conditions such as liver cirrhosis and chronic kidney disease (kaspar & sterling, 2017). in hiv infection, excessive production of reactive oxygen species (ros) and impaired antioxidant defenses in macrophages and monocytes lead to oxidative stress, which exacerbates chronic inflammation, immune dysfunction, and organspecific injury, even in individuals receiving antiretroviral therapy (art) (ivanov et al., 2016). several mechanisms contribute to this issue. one key mechanism is the upregulation of nadph oxidase, a significant ros-producing enzyme in macrophages, induced by hiv proteins such as tat and gp120. this results in the overproduction of superoxide and hydrogen adavba; asian j. immunol., vol. 8, no. 1, pp. 120-133, 2025; article no.aji.136909 123 peroxide, which can damage lipids, proteins, and dna. additionally, hiv disrupts mitochondrial electron transport, causing an increase in mitochondrial ros (harshithkumar et al., 2024). it also depletes glutathione, the primary cellular antioxidant, by reducing the expression of glutamate-cysteine ligase in macrophages (lin et al., 2023). these mechanisms vary in intensity across different organs and contribute to the organspecific damage observed in people living with hiv (plwh), forming the basis for the development of non-aids-related events (nades). 3. organ-specific effects of monocytes and macrophages in hiv disease: mechanisms of tissue damage 3.1 cardiovascular disease (cvd) hiv is the main contributing factor to cvd in plwh, accounting for approximately a two-fold increase in the relative risk of this disease (ancuta et al., 2008). cvd is the leading cause of death worldwide, and has atherosclerosis as the main underlying pathology (jebaribenslaiman et al., 2022). atherosclerosis is a condition in which plaque, made up of fat, cholesterol, and other substances, accumulates in the intima of arteries, causing them to narrow and stiffen. this process begins with damage to the endothelium, accompanied by the retention and modification of low-density lipoprotein (ldl) in the intima (mundi et al., 2017). the recruitment of monocytes into the intima triggers an inflammatory state critical to the development of atherosclerosis. monocytes recruited into the arterial wall differentiate into macrophages and foam cells, facilitating plaque formation (subramanian et al., 2012). moreover, the increased elaboration of proinflammatory cytokines by hiv-infected monocytes and macrophages maintains a systemic inflammatory state, accelerating atherosclerosis and endothelial dysfunction (deeks et al., 2013). elevated levels of markers of macrophage activation, soluble cd163 and soluble cd14, are strongly associated with subclinical atherosclerosis and cardiovascular events in plwh (serrano-villar et al., 2014). furthermore, the increased expression of adhesion molecules (icam-1, vcam-1) and chemokine receptors (ccr2, cx3cr1) by hivinfected monocytes enhances their adhesion to and infiltration into the vascular endothelium. this endothelial barrier disruption promotes vascular inflammation, exacerbates endothelial dysfunction, and vascular remodeling, thereby increasing cardiovascular risk (hsue et al., 2012). additionally, cytokines derived from macrophages (tnf-α, il-6) and hiv proteins (tat, nef) directly activate endothelial cells, resulting in increased permeability that accelerates leukocyte recruitment (ganor et al., 2019). in addition, hiv infection and art disrupt lipid metabolism, leading to elevated levels of triglycerides and ldl cholesterol. macrophages in plwh accumulate lipids and exhibit impaired cholesterol efflux due to downregulation of abca1 and abcg1 transporters (clayton et al., 2018). these proteins are crucial atp-binding cassette transporters in macrophages that facilitate cholesterol efflux and play a role in reverse cholesterol transport. deficiency or downregulation of these transporters can lead to increased cholesterol accumulation in macrophages, contributing to foam cell formation and potentially accelerating atherosclerosis (yvan-charvet et al., 2007). hiv is a major contributing factor to cardiovascular disease (cvd) in plwh, resulting in approximately a two-fold increase in the relative risk of this condition (ancuta et al., 2008). cvd is the leading cause of death worldwide, with atherosclerosis being the primary underlying pathology (jebari-benslaiman et al., 2022). atherosclerosis occurs when plaque, composed of fat, cholesterol, and other substances, accumulates in the intima of arteries, causing them to narrow and stiffen. this process begins with damage to the endothelium, leading to the retention and modification of low-density lipoprotein (ldl) in the intima (mundi et al., 2017). the recruitment of monocytes into the intima triggers a critical inflammatory response that is essential for the development of atherosclerosis. once in the arterial wall, these monocytes differentiate into macrophages and foam cells, which aid in plaque formation (subramanian et al., 2012). additionally, hiv-infected monocytes and macrophages produce increased levels of pro adavba; asian j. immunol., vol. 8, no. 1, pp. 120-133, 2025; article no.aji.136909 124 inflammatory cytokines, maintaining a systemic inflammatory state that accelerates atherosclerosis and endothelial dysfunction (deeks et al., 2013). elevated levels of markers for macrophage activation, such as soluble cd163 and soluble cd14, have a strong association with subclinical atherosclerosis and cardiovascular events in plwh (serrano-villar et al., 2014). furthermore, the increased expression of adhesion molecules (like icam-1 and vcam-1) and chemokine receptors (such as ccr2 and cx3cr1) on hiv-infected monocytes enhances their ability to adhere to and infiltrate the vascular endothelium. this disruption of the endothelial barrier promotes vascular inflammation, worsens endothelial dysfunction, and contributes to vascular remodeling, all of which increase cardiovascular risk (hsue et al., 2012). cytokines released from macrophages (like tnf-α and il6) and hiv proteins (such as tat and nef) directly activate endothelial cells, resulting in increased permeability and accelerated recruitment of leukocytes (hsue et al., 2012). lastly, hiv infection and art can disrupt lipid metabolism, leading to elevated levels of triglycerides and ldl cholesterol. in plwh, macrophages accumulate lipids and demonstrate impaired cholesterol efflux, largely due to the downregulation of critical atp-binding cassette transporters, abca1 and abcg1 (yvan-charvet et al., 2007). these transporters play a vital role in cholesterol efflux and reverse cholesterol transport. their deficiency or downregulation can lead to increased cholesterol accumulation in macrophages, promoting foam cell formation and potentially accelerating atherosclerosis (yvancharvet et al., 2007). 3.2 neurocognitive disorders hiv-associated neurocognitive disorders (hand) refer to a range of neurological and cognitive impairments linked to hiv infection and aids. these disorders remain common despite art and can affect up to 50% of plwh. hand ranges from mild cognitive deficits to severe dementia and can significantly impact daily functioning (adhikary et al., 2025). the blood-brain barrier (bbb) is compromised in hiv infection, allowing for the establishment of viral reservoirs in the brain. the hiv-1 envelope glycoprotein, gp120, plays a key role in damaging the integrity of the bbb by altering tight junction proteins in human brain microvascular endothelial cells (said & venketaraman, 2025). however, the initial trigger for hand is the movement of hiv-infected monocytes across the bbb into the central nervous system (cns), where they differentiate into perivascular macrophages (león-rivera et al., 2021). perivascular macrophages are long-lived, tissueresident cells that occupy the spaces around blood vessels. they express cd14, cd16, and cd163, and, together with microglia, contain hiv even in individuals receiving art, contributing to chronic low-level viral replication (kruize & kootstra, 2019). additionally, hiv-infected monocytes secrete matrix metalloproteinases (mmp-9), which degrade tight junction proteins (such as claudin-5 and occludin), thereby increasing bbb permeability. similar to the pathogenesis of cardiovascular disease, the increased expression of adhesion molecules and chemokine receptors (such as ccr2 and cx3cr1) in hiv-infected monocytes enables their adhesion to and crossing of the bbb. activated astrocytes and endothelial cells produce chemokines like ccl2, also known as monocyte chemoattractant protein-1 (mcp-1), which help recruit monocytes into the cns (hernandez et al., 2024). the degradation of tight junctions, the apoptosis of endothelial cells, and the consequent bbb leakage allow plasma proteins (e.g., fibrinogen) and peripheral immune cells to infiltrate the cns, exacerbating neuroinflammation and white matter damage (salimi & klein, 2019). moreover, activated microglia can engage in "synaptic stripping," phagocytizing dendritic spines and synapses, causing synaptic loss, especially in the hippocampus and prefrontal cortex areas critical for memory and executive function (schlachetzki et al., 2022). soluble viral proteins (such as gp120, tat, and nef) secreted by infected macrophages contribute to excitotoxicity, oxidative stress, and synaptic damage (marino et al., 2020). the cd16+ monocyte subset, which has a pro-inflammatory profile (ong et al., 2018), plays a critical role in hiv neuroinvasion due to its high mmp-9 secretion (which leads to bbb breakdown) (campos et al., 2014) and enhanced chemotaxis (kruize & kootstra, 2019). however, inflammation and immune activation are central mechanisms driving the development of hand, with various contributing factors. hiv adavba; asian j. immunol., vol. 8, no. 1, pp. 120-133, 2025; article no.aji.136909 125 infected macrophages release pro-inflammatory cytokines (such as tnf-α, il-1β, and il-6) and chemokines (like mcp-1/ccl2), activating astrocytes and microglia while recruiting additional monocytes into the cns, sustaining chronic neuroinflammation (saylor et al., 2016). additionally, hiv rna and proteins activate the nlrp3 inflammasome in macrophages, increasing the release of il-1β and il-18. nlrp3 is primarily found in myeloid cells (monocytes, macrophages, and neutrophils) and acts as a sensor for pathogen-associated molecular patterns (pamps) and damage-associated molecular patterns (damps), initiating an inflammatory response (swanson et al., 2019). this activation occurs in two steps: first, hiv infection primes macrophages by increasing nlrp3 expression, and then specific viral components activate the inflammasome, resulting in the maturation and release of il-1β and il-18 (chivero et al., 2017). activated macrophages and microglia produce quinolinic acid, an n-methyl-d-aspartate (nmda) receptor agonist that induces excitotoxicity by overstimulating neurons. nmda receptors, which are the primary excitatory neurotransmitter receptors in the human brain, play a crucial role in synaptic plasticity, a neuronal mechanism thought to be fundamental for memory formation. quinolinic acid also generates ros, leading to mitochondrial dysfunction and neuronal apoptosis (lugohuitrón et al., 2013). these pathological processes ultimately result in a range of cognitive deficits, including asymptomatic neurocognitive impairment (subtle deficits detectable only through neuropsychological testing), mild neurocognitive disorder (functional impairment in daily activities), and hiv-associated dementia (severe cognitive and motor decline, which is now rare in the art era) (adhikary et al., 2025). 3.3 liver and kidney disease common to the mechanism of tissue damage in other organs, chronic inflammation and immune activation driven by hiv-infected monocytes and macrophages play a central role in liver and kidney pathology among plwh. however, the dynamics underlying the damage differ between the two organs. fig. 1. mechanisms of hand. hiv-infected cd16+ monocytes migrate across the bbb enabled by ccr2/mmp-9 expression, seeding cns reservoirs. persistent viral proteins (tat/gp120) and cytokines (tnf-α/il-1β) drive neuroinflammation, oxidative stress, and synaptic damage, culminating in cognitive decline adavba; asian j. immunol., vol. 8, no. 1, pp. 120-133, 2025; article no.aji.136909 126 most of the focus on liver disease has been on individuals co-infected with hcv or hbv. however, even though the apparent net effect of hiv on liver fibrosis is most evident in the presence of a second contributor, such as hcv infection, hbv infection, excessive alcohol use, or metabolic liver disease, hiv infection itself biases the liver toward fibrosis and synergistically promotes these other processes. there is also significant evidence suggesting that even without primary liver disease, hiv itself may cause liver steatosis and fibrosis (sherman & thomas, 2022). various seemingly overlapping mechanisms have been proposed to explain how hiv may potentiate or cause liver fibrosis, but they all converge on the central role of the hepatic stellate cell (hsc). hscs are pivotal in liver fibrosis, where the liver develops scar tissue secondary to chronic injury. when hscs are activated, they can transdifferentiate from a quiescent to an activated phenotype, producing excess extracellular matrix (ecm), which contributes to the development of fibrosis, cirrhosis, and other liver diseases (hoffmann et al., 2020). hiv-infected kupffer cells (resident liver macrophages) and circulating monocytes that migrate to the liver become chronically activated due to viral persistence. they create a proinflammatory microenvironment in the liver by releasing tnf-α, il-6, il-1β, and ccl2. (mosoian et al., 2016), while hiv proteins (tat, nef), which directly enhance macrophage activation, may stimulate signaling pathways such as nf-κb, amplifying cytokine production. additionally, activated macrophages secrete tgf-β, a key fibrogenic cytokine, and plateletderived growth factor (pdgf), a potent mitogen (mosoian et al., 2016). these factors are crucial in transitioning hscs from a quiescent to an activated state. furthermore, hiv-infected macrophages generate ros, which directly activate hscs and enhance tgf-β signaling, exacerbating the process of tissue damage. in addition, hivinduced gut barrier dysfunction allows translocation of microbial components, especially lipopolysaccharides (lps), to the liver. these bind to tlr4 on kupffer cells, further amplifying cytokine production (e.g., il-1β via nlrp3 inflammasome activation) (hoffmann et al., 2020). ultimately, the resulting accumulation of ecm disrupts liver architecture, leading to cirrhosis and impaired hepatic function. hiv-infected monocytes and macrophages significantly contribute to hiv-associated kidney disease, particularly hiv-associated nephropathy (hivan) and immune complex-mediated glomerulonephritis, through direct viral infection, chronic inflammation, and podocyte injury. hivan, characterized by focal segmental glomerulosclerosis with collapsing glomerulopathy and tubulointerstitial inflammation, is underpinned by various pathological processes. hiv-infected macrophages expressing pro-inflammatory cytokines (tnf-α, il-6, and tgf-β) promote fibrosis and glomerular scarring (medapalli, he & klotman, 2011). additionally, infected macrophages release il-1β via nlrp3 inflammasomes, amplifying glomerular damage (ekabe et al., 2021). podocytes can also be infected, but through non-conventional mechanisms (hiv-receptor-independent). one such mechanism is through virological synapses and tunneling nanotubes between cd4+ t cells, macrophages, and renal cells. these facilitate hiv spread among these different cell types (hughes et al., 2021). infection of podocytes triggers apoptosis, loss of function, and dedifferentiation, all contributing to glomerular damage (hughes et al., 2021). 3.4 cancer while art has reduced the incidence of aidsdefining cancers, plwh still experience an elevated risk for certain non-aids-defining cancers due to multiple mechanisms, including improvements in life expectancy with art, immunosenescence, and the loss of control over oncogenic infections due to hiv-related immune suppression. but additionally, macrophagederived chronic inflammation and immune activation create a microenvironment conducive to carcinogenesis (grulich et al., 2007). hodgkin lymphoma and cancers of the lung, anus, liver, which are non-aids malignancies have become more prevalent in plwh (wang, silverberg & abrams, 2014). while art has significantly reduced the incidence of aids-defining cancers, plwh still face an increased risk for certain non-aidsdefining cancers. this heightened risk arises from various factors, including the prolonged life expectancy associated with art, immunosenescence, and diminished control over oncogenic infections due to hiv-related immune suppression (wang, silverberg & abrams, 2014). furthermore, macrophage-driven immune dysregulation contributes to a microenvironment adavba; asian j. immunol., vol. 8, no. 1, pp. 120-133, 2025; article no.aji.136909 127 fig. 2. mechanisms of hiv-associated nephropathy (hivan) that favors carcinogenesis (grulich et al., 2007). consequently, non-aids malignancies, such as hodgkin lymphoma and cancers of the lung, anus, and liver, have become more prevalent among plwh (wang, silverberg & abrams, 2014). the chronic inflammatory state induced by the increased secretion of proinflammatory cytokines from hiv-infected monocytes and macrophages promotes dna damage, inhibits apoptosis in malignant cells, and supports tumor cell proliferation (grivennikov, greten & karin, 2010). for instance, il-6 activates jak-stat3 signaling in epithelial cells by binding to its receptor (il6r). activating this signaling pathway leads to downstream effects on gene expression and cellular behavior, including driving the expression of genes involved in cell survival, proliferation, angiogenesis, and metastasis, ultimately facilitating oncogenic transformation in certain cells (thuya et al., 2025). secondly, hiv alters macrophage polarization, steering them toward a pro-tumor m2-like phenotype, characterized by increased pd-l1, il-10, and tgf-β expression. these factors suppress the cytotoxic t-cell responses and encourage the expansion of regulatory t-cells (tregs), which favors immune escape by tumours (li et al., 2024). furthermore, chronic hiv activation boosts the population of monocyte-derived myeloid-derived suppressor cells (mdscs), which impede the functions of nk and cd8+ t-cells. mdscs are linked to aggressive tumor growth and poorer outcomes in hiv-related cancers (tumino et al., 2022). moreover, metabolic reprogramming within the tumor microenvironment—exemplified by increased glycolysis in hiv-infected monocytes and macrophages—results in the production of lactate. the lactate acidifies the tumor environment, suppresses antitumor immunity, and promotes metastasis. additionally, the elevated production of ros from dysfunctional mitochondria in these hiv-infected cells causes dna damage in adjacent cells. this damage affects oncogenes and tumor suppressor genes, thereby increasing the risk of carcinogenesis (zhou et al., 2022). 4. therapeutic implications given the substantial evidence highlighting the crucial involvement of monocytes and macrophages in the development of nades, targeting the mechanisms by which these cells contribute to these pathologies presents a promising strategy for alleviating chronic inflammation and enhancing clinical outcomes in plwh in the era of viral suppression. nonspecific, potential therapeutic approaches include: adavba; asian j. immunol., vol. 8, no. 1, pp. 120-133, 2025; article no.aji.136909 128 fig. 3. conceptual basis for employing reservoir targeting strategies table 1. plausible mechanisms of amelioration of nades following successful reservoir reduction/elimination nade mechanism of amelioration neurocognitive (hand) ↓ hiv-infected microglia, ↓ neuroinflammation cardiovascular ↓ il-6/tnf-α → ↓ endothelial dysfunction liver/kidney fibrosis ↓ tgf-β → ↓ collagen deposition 4.1 reservoir-targeting strategies the persistence of latent or transcriptionally active viruses in myeloid cells despite art remains a major barrier to cure efforts and a major contributor to the chronic immune dysfunction underlying the development of nades. reservoir-targeting strategies aim to eliminate persistent hiv and reduce chronic inflammation, directly addressing the root cause of nades. while challenges remain (e.g., myeloid reservoir penetration (chun, moir & fauci, 2015)), combined approaches promise to improve long-term outcomes for plwh. eliminating hiv reservoirs in monocytes and macrophages is critical for achieving a cure and lowering the risk of nades. recent research provides evidence supporting strategies to target these reservoirs by employing latency-reversing agents such as tlr agonists (li et al., 2023) and gene-editing technologies using crispr to excise hiv dna from infected macrophages (das et al., 2019). the ultimate aim is to reduce reservoir load, lower immune activation and inflammation, and decrease the incidence of nades. 4.2 anti-inflammatory therapies agents that modulate the activation of monocytes and macrophages, such as statins, aspirin, and cytokine inhibitors (e.g., il-6 and tnf-α blockers), may help reduce inflammation and lower the risk of nades. specifically, statins can influence vascular atherosclerotic inflammation by directly enhancing the function of endothelial cells, vascular smooth muscle cells, platelets, and immune cells. additionally, rosiglitazone, a ppar-gamma agonist, has demonstrated potential in halting brain inflammation and inhibiting hiv replication in murine models of hiv encephalitis. 4.3 restoring immune homeostasis interventions designed to restore the functional properties of monocytes and macrophages, such as enhancing phagocytosis and promoting antiinflammatory phenotypes, may contribute to mitigating tissue damage and improving overall outcomes. in particular, modulating macrophage function by shifting polarization from proinflammatory m1 to anti-inflammatory m2 (and vice versa) could have significant implications for hiv pathogenesis and the development of nonantibody-dependent enhancement (nades) (luo et al., 2024). 5. conclusion monocytes and macrophages play crucial roles in the pathogenesis of hiv disease and the development of nades. their functions as viral reservoirs, drivers of chronic inflammation, and mediators of tissue damage underscore the need for targeted therapeutic strategies to address these mechanisms. while current challenges such as low myeloid cell penetration, reservoir heterogeneity, and limited cns penetration still need to be overcome, understanding and modulating the functions of monocytes and adavba; asian j. immunol., vol. 8, no. 1, pp. 120-133, 2025; article no.aji.136909 129 macrophages presents a promising strategy for reducing the burden of nades and enhancing the long-term health outcomes of plwh. consent and ethical approval it is not applicable. disclaimer (artificial intelligence) the author hereby declares that no generative ai technologies such as large language models (chatgpt, copilot, etc.) and text-to-image generators have been used during the writing or editing of this manuscript. competing interests author has declared that no competing interests exist. references adhikary, k., banerjee, a., sarkar, r., banerjee, r., chowdhury, s. r., ganguly, k., & karak, p. 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(2010). nomenclature of monocytes and dendritic cells in blood. blood. 116. e74-80. 10.1182/blood-2010-02-258558. disclaimer/publisher’s note: the statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of the publisher and/or the editor(s). this publisher and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. _________________________________________________________________________________ © copyright (2025): author(s). the licensee is the journal publisher. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: https://pr.sdiarticle5.com/review-history/136909 https://pr.sdiarticle5.com/review-history/136909 _____________________________________________________________________________________________________ *corresponding author: e-mail: ibrahimshnawa3@gmail.com; cite as: ims, shnawa, algerbori hsh, and thewaini qno. 2024. “immunogenicity of bacterial protoplasmic sonicate proteins”. asian journal of immunology 7 (1):175-80. https://journalaji.com/index.php/aji/article/view/141. asian journal of immunology volume 7, issue 1, page 175-180, 2024; article no.aji.123078 immunogenicity of bacterial protoplasmic sonicate proteins shnawa ims a,b*, algerbori hsh c and thewaini qno a a department of medical biotechnology, college of biotechnology, al-qasim green university, qasim, babylon province, iraq. b college of nursing technology, university of hilla, babylon province, iraq. c department of basic sciences, college of dentistry, university of babylon, babylon provine, iraq. authors’ contributions this work was carried out in collaboration among all authors. all authors read and approved the final manuscript. article information doi: https://doi.org/10.9734/aji/2024/v7i1141 open peer review history: this journal follows the advanced open peer review policy. identity of the reviewers, editor(s) and additional reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here: https://www.sdiarticle5.com/review-history/123078 received: 04/07/2024 accepted: 06/09/2024 published: 20/09/2024 abstract bacterial protoplasmic sonicate proteins bpsp were recovered from skin burn pseudomonas aeruginosa and klebsiella oxytoca infections. these proteins were considerd as immunogens in rabbits. mucosal and systemic, humoral and cellular immune immune responses were matched. the p. aeruginosa protoplasmic sonicate proteins induces an increase in; nbt % neutorphil phagocytosis, significant leukocyte migration inhibition cytokines, il6 cytokine elevation and rise up of humoral agglutinins responses. while, k. oxytoca protoplasmic sonicate proteins induces increase of; humoral agglutinins and il6 cytokine as well as non-signifcant increase in leukocyte inhibitory cytokines. both at mucosal and systemic compartments. the study highlights the potentials of these proteins as candidates for developing vaccines against multidrug resistance infections, particularly in burn patients. these findings underscore the importance of immunogenicity in advancing bacterin development in clinical use. original research article https://doi.org/10.9734/aji/2024/v7i1141 https://www.sdiarticle5.com/review-history/123078 shnawa et al.; asian j. immunol., vol. 7, no. 1, pp. 175-180, 2024; article no.aji.123078 176 keywords: antigens; bacterin; cellular; humoral; immunogen; protein; vaccine. 1. introduction bacterial antigens [bags] were being investigated to be of use in, bacterial diagnosis, bacterial infection diagnosis, vaccine development and vaccine production. immunogenicity of bags holds a backbone position in vaccine development strategies [1–7]. the objective of the present work was the investigation of immunogenicity of protoplasmic sonicate protein antigens of skin burn infections with p. aeruginosa and k. oxytoca. 2. materials and methods 2.1 protoplasmic sonicate protein antigens p spa the pspa separation was in direct way for p. aeruginosa [8], while, by in direct way as it needs removal of the capsule [9,10] before processing for obtaining psps. the method for separation, identification, purification and quantitation of these psps were as in the method described by bjorn et al. [8]. the details of the processing method was as in the followings; six mls of tris buffer 0.01 n and ph 8 was added to the surface of 24 hrs growth of p. aeruginosa and k. oxytoca (after suspending bacteria then removal of capsule [9,10]) on the nutrient agar plates. growth were vortoxed in vortox test tubes for thee minutes. suspensions were centrifuged at 5000 rpm for ten minutes. supernatant were discarded and pellets ps were kept. ps suspensions were tubbed and washed three times with tris buffer 0.01n.ps were reconstituted with 6 mls, and were tubbed in the cell disintegrator tubes. then jacketed with cooled ice. the best sonication conditions were five times for five minutes at 20 oscillation ampiltude. the sonicated cell suspensions were centrifuged at 5000 rpm for ten minutes. supernats were collected and ultrafiltred with 0.22 um millipore filter. fiterates were collected in sterile plastic tubes, then proteins were separated with peg 6000 ,6% as inshnawa and alsadi [11]. the protein concentration determination was measured by biurt test [12]. to this end the preparations can be designated as papsp and kopsp both p. aeruginosa and k.oxytoca respectively. these psps were distributed into alliqoutes of 0.5 mls. in an appendroff plastic tubes and kept at 20c,till use. 2.2 immune reagents specific immune priming of rabbits were done with the psp concentrations of 2.71 mg/l for papsp and 1.8 md/l for kopsp [12]. the somatic antigens for both bacteria were prepared as heat killed as in [13].complete freund ajuvant that from difco, co.ltd. the test proposed immunogens were made as; one volume of cfa mixed with one volume the test proteins immunization protocols 2.3 immunization protocols a mounthwise twice dosage of psp-cfa in 2 mls amounts were dosaged in the first and second months to the rabbits. the specific immune priming was that of multisite injection protocol [14]. 2.4 rabbits a local breed rabbits brought from the local market were checked for ecto and endoparasites. as well as for pyrogens and found to be free off. they were acclimatized for housing conditions two weeks befor experimentation at and libitum conditions. among which nine were elected and subdivided into two test and one control group each of three rabbits. 2.5 samplings and immune function tests at the temination of the specific immune priming protocols, blood with and with out heparin were collected from the test and control rabbits by cardiac puncture for humoral and cellular immune tests. sera were saved for serology and cytokine studies. heparinized blood were used for leukocyte inhibitory factor [15] and for nbt phagocytosis [16]. appendix for test and control rabbits were collected and open up, washed from digesta and processed for separation of mucosal globulins [17]. mucosal leukocytes were separated by dextran 2% as in [18]. il 6 determinations were made as in the recommendation of the instruction of the manufacturer. standared tube agglutination test were made as in [19]. 3. results the nbt neutrophil phagocytosis percentages in papspa (56.75 % for mucosal and36.5 for shnawa et al.; asian j. immunol., vol. 7, no. 1, pp. 175-180, 2024; article no.aji.123078 177 systemic) and kopspa (48.5% for mucosal, 41% for systemic) primed rabbits were higher than that of control rabbits (20% for mucosal,18% for the systemic). leukocyte inhibitory cytoknine lif % in papsp primed rabbits were 56.6% for mucosal and 58.45 for systemic as compared to control ,the mucosal was 90% and systemic was 86%.while for kopspa primed rabbits were; 88% for mucosal and 87.5% for systemic lif as compared to normal control were 93% for mucosal and 89% for systemic responses. the il6 concentration determinations were shownig that papspa and kopspa primed rabbits have got higher il6 concentration means than normal control rabbits. papspa primed rabbits il6 concentration means were 92.8 for mucosal and 72.7 for systemic responses. in kopspa primed rabbits the mucosal concentration means were 83.26 and for systemic were 76.79 as compared to normal control were 8.83 for mucosal and 9.25 for systemic responses. the humoral specific agglutinin titre levels for papspa were 128 for mucosal and 320 for the systemic responses and kopsp primed rabbits were 64 for mucosal and 640 for systemic responses as compared to control rabbits were 4 for mucosal and 20 for systemic responses. mucosal agglutinins were resistant to treatment with 2me, tables 1 and 2. 4. disscussion the concept, application and continuity of the immunogenicity theme for bacterial protein antigens are still in the current mode of researchers allover the world [1-7]. immunogenicity appeared to have two main facets.first that of theoritical immunologisets which advocate that immunogenicity is denoted to self-nonself recognition theme [6]. while the second facets was that for most of the proper immunologists which can be summerized as the ability of an antigen to initiate humoral and/or cellular conversion from the normal baseline immune functions to an optimized cellular immune reactions outcomes that are finalized by the optimest synthesis and production of cellular secretory proteins (antibodies, cytokines) concentrations and /or optimised depression of such secretory proteins [1-5,7]. immunogenicity appeared to be essential for diagnosis and prophylaction of human infections as well as in cancer personalized prophylactic and therpeutic medicine. hence the presnt study for pseudomonas and klesiella immunogenicity may participate in developing of a porototype bacterial protein based vaccine and/ or in development for an autovaccine for multidrug resistant skin burn infection [20]. the antigenic make up of bacteria [1-5,7] like that of p. aeruginosa are formed from; flagella, pili, exottoxin a, exoppolysaachride, lps, omp, hemolysin, elastases, proteasis, heat stable phospholipid, heat stable glycolipid, exoenzyme s and ribosome [21-23]. while that of klebsiella oxytoca are; capsule, somatic antigens and lps [24]. hence, papsp and kopsp protein table 1. the immunogenicity of papsp in primed rabbits and controls rabbits groups nbt% lif% il6 pg/ml. agglutinin titres papspa m s 46.75 36.6 56.6 58.45 2.8 72.7 128 320 control m 20 93 92.8 4 s 18 89 72.7 20 table 2. the immunogenicity of kopsp in primed and control rabbits rabbit groups nbt% lif % il6 pg/ml agglutinin titres kopspa m s 48.5 41 88 87 48 72 64 640 controls m s 20 18 93 89 8.83 9.25 4 20 shnawa et al.; asian j. immunol., vol. 7, no. 1, pp. 175-180, 2024; article no.aji.123078 178 table 3. the immune features of the study bacterial proteins features papsp [25] kopsp [25] 1-chemical nature 2-origin 3-location 4-processing method 5-non-specific immune function 6specific cellular immune function 7-induction of cytokine network 8-humoral immune responses 9-functional epitope mapping [26,27] 10-immuen system compartments 11-expected immune potentials [20] protein bacterial intracellular ultrasonication rise up of phagocytosis by nbt% significant inhibition of lif cytokine rise up of il6 cytokine rise up of specific agglutinins t cell dependent and t cell inndepedent epitopes mucosal and systemic prototype protein based vaccine and an autovaccine protein bacterial intracellular ultrasonication rise up of phagocytosis by nbt % nonsignificant inhibition of lif cytokines rise up of il6 cytokines rise up of specific agglutinins t cell independent and /or, th2 dependent b cell epitope mucosal and systemic prototype protein based vaccine and an autovaccine. antigens are novel antigens prepared in this experimental settings. paps was proved to be immunogenic in rabbits model, table 1, promoting humoral and cellular immune responses both at mucosal and systemic copartments. these findings were in contradiction with that of [24]. while, kopsp immunogenicity, table 2, were by promoting humoral immune responses both at mucosal and systemic level [21]. different burn infecting bacterial protein preparations have shown different immune potential features, table 3,[25]. the functional epitope mapping for papsp may be t cell depndent and t cell independent, while that of kopsp may be of t cell independent or th2 cell depent b cell epitopes [26,27]. since immunogenicity is an integral part for developing bacterins. such findings holds the position of novelity, based upon the need for an autogenous bacterins for multidrug resistant skin burn bacterial infections [28,29]. 5. conclusions p. aeruginosa papsp and k. oxytoca kopsp protoplasmic sonicate proteins were found as lapin immunogens. papsp mediate humoral and cellular immune responses both at mucosal and systemic compartments. while, kopsp mediate humoral immune responses both at mucosa and blood stream. these proteins may be of expected immune potentials as protein based vaccine candidate and as an autovaccine for multidrug resistant skin burn infections. disclaimer authors hereby declare that no generative ai technologies such as large language models (chatgpt, copilo, etc) and text-to-image generations have been used during writing or editing of this manuscript. consent it is not applicable. ethical approval the housing, handling and interventions on rabbits were done following the international acts for regulation of housing, handling and interventions. competing interests authors have declared that no competing interests exist. references 1. tian g, qin c, hu j et al. side chain functional groups in immunogenicity of bacterial surface glycans. molecules. 2023; 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article no.aji.123078 180 27. shnawa ims. functional epitope mapping of a vaccine.vaccines vaccin. 2016;1(1) :000101 28. jin x et al. recent adavances in vaccine development against multidrug resistant bacteria.vaccines. 2022;10(9): 1456. doi: 10.3390/vaccines.10091456 29. smith r et al.2023.new insight into the immunogenicity of bacterial antigens: implications for vaccine development. j. clin. microbiol. 2023;61(5):e1425 23. doi: 10.1128 /jcm.01423-23 disclaimer/publisher’s note: the statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of the publisher and/or the editor(s). this publisher and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. _________________________________________________________________________________ © copyright (2024): author(s). the licensee is the journal publisher. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: https://www.sdiarticle5.com/review-history/123078 https://www.sdiarticle5.com/review-history/123078 _____________________________________________________________________________________________________ *corresponding author: e-mail: esmat.shaker@stu.edu.iq; cite as: shaker, esmat esam. 2025. “frequency and patterns of cardiac involvement in systemic lupus erythematosus”. asian journal of immunology 8 (1):60-69. https://doi.org/10.9734/aji/2025/v8i1160. asian journal of immunology volume 8, issue 1, page 60-69, 2025; article no.aji.134457 frequency and patterns of cardiac involvement in systemic lupus erythematosus esmat esam shaker a* a department of medical laboratories at the technical medical institute in the southern technical university, basra, iraq. author’s contribution the sole author designed, analysed, interpreted and prepared the manuscript. article information doi: https://doi.org/10.9734/aji/2025/v8i1160 open peer review history: this journal follows the advanced open peer review policy. identity of the reviewers, editor(s) and additional reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here: https://pr.sdiarticle5.com/review-history/134457 received: 13/02/2025 published: 23/04/2025 abstract the aim of the study was to determine the prevalence of myocardial and circulatory complications among lupus patients, such as pericarditis, heart failure, endocarditis, ventricular dysfunction, and myocarditis. it was also to assess the association of lupus with increased myocardial injury and to evaluate therapeutic (choriokinin, corticosteroids, and glucocorticoids) interventions to reduce the complication of cardiovascular disease. this study focused on the immunological aspect of the effect of immunosuppressive therapy in systemic lupus erythematous (sle), specifically choriokinin, corticosteroids, and glucocorticoids (gcs), on the exposure of cardiovascular disease. the study period extended from 2022 to early 2024 and relied on the results of screening of 88 sle patients and a control group of 85 patients. the causes of heart damage in sle patients were identified to assess the relationship with disease activity, duration, and rheumatic treatment. previously, we first determined n_terminal prohormone of brain natriuretic peptide (nt_probnp) levels in sle patients not receiving specific rheumatic original research article https://doi.org/10.9734/aji/2025/v8i1160 https://pr.sdiarticle5.com/review-history/134457 shaker; asian j. immunol., vol. 8, no. 1, pp. 60-69, 2025; article no.aji.134457 61 treatment and identified a relationship between biomarker and immunological marker concentrations of sle activity (increased serum levels of anticardiolipin igg, anti_dsdna, antinuclear antibodies, and decreased complement c4) and markers reflecting impaired kidney function (nasonov, 2010, kim, et al., 2017). corticosteroids are effective in preventing the progression of sle and increasing patient survival, while reducing the exposure of atherosclerosis and thrombosis (nasonov, 2010, tan, et al., 1982, hochberg, 1997) and hypercholesterolemia (kim, et al., 2017, petri, et al., 2012), ultimately reducing a exposure of cardiovascular disease at sle patients, despite isolated reports of cardiac toxicity (hochberg, 1997). noticed a statistical difference between the two groups in nt_probnp, as it was shown that the second group had a lower nt_probnp result 0.05 than the first group, and the same applies to anti_jo1, which gives us evidence beyond doubt that immunosuppressive treatments work positively in treating heart muscle diseases resulting from lupus. keywords: immunosuppressant; sle; immunosuppressive; myocarditis; bnp; aca; anti_jo1. 1. introduction systemic lupus erythematous (sle) is a chronic, multisystem immunological disorder of unknown causality and genetic predisposition it presents with diverse clinical features and follows an unpredictable progression (nasonov, 2010). myocarditis affects more than 55% of sle patients and is a leading cause of fatal condition. all cardiac anatomical structures can be affected by sle, such as the pericardium, endocardium, coronary vessels, and myocardium, with heart failure (hf) being the final stage of cardiac and circulatory disease (kim, et al., 2017). recent studies indicate that systemic lupus erythematosus (sle) is a major risk factor for myocarditis and has a significant impact on causing actual damage to the myocardic, pulmonary valve, and ventricular, endocardial, and pericardial dysfunction, which requires continuous monitoring in the intensive care unit (icu) (tan, et al., 1982, cieślik & holecki, 2022). alarming results have been recorded for deaths due to heart failure in several countries around the world. in the middle east, saudi arabia occupied an advanced position in deaths, with 36% of deaths due to heart muscle failure due to pericarditis, followed by egypt with a rate of 21.5% of deaths due to myocardial infarction, and jordan with a rate of 12% due to myocardial infarction (mazurek, et al., 2022). while the death rate due to heart failure in latin america was recorded in 2023 alone, more than 470 deaths due to myocardial infarction resulting from lupus, in mexico there are no official statistics on these cases, but medical investigative reports suggest that the percentage is increasing due to myocardial infarction (adawi & blum, 2020, mazurek, et al., 2022). the american heart association reported that approximately 2 million patients suffer from fatal myocardial infarctions, most of whom are women of childbearing age. there are myocardial infarctions that have become chronic and life-threatening, and diseases resulting from hypertension and repeated myocardial infarction (foret, et al., 2022, mazurek, et al., 2022). the pathogenesis of cardiac damage in sle is thought to depend on the combined or independent effects of chronic immunological disorder inflammation, accelerated progression of atherosclerosis, and the use of certain ant rheumatic drugs (tan, et al., 1982). according to an epidemiological study (n = 500 people), in patients with systemic lupus erythematous m cardiac damage was more frequently recorded (gladman & ibanez, 2002): arrhythmia and electrical disturbances in 22% and 6%, coronary myocarditis in 13% and 4%, major valve insufficiency in 11% and 2%, myocardial infarction (mi) in 6% and 2%, cor pulmonale in 2.2% and 0.2%, heart failure (hf) in 1.0%. in iraq, there are no statistics that show us the death rate resulting from systemic lupus erythematosus. the last statistics for iraq reached 11% in 1989, and baghdad, the capital, topped the list. all deaths were among fertile women between the ages of 25 45, and the statistics indicated myocardial infarction. after years of war, annual statistics were absent. in this research, i focused on studying the effect of lupus on the heart muscle for the year 2022 to the beginning of 2024, in terms of the effect of lupus on heart function within the available medical data and for the same age group in 1989. shaker; asian j. immunol., vol. 8, no. 1, pp. 60-69, 2025; article no.aji.134457 62 2. methodology the study included 88 patients, 80 (92%) females and 8 (9.5%) males, with a mean age of 35 years, making it an ideal sample for the study. a control group of 85 patients was selected. we did not care about gender matching, although the majority of participants in the control group were female and had similar symptoms and conditions to the study group. the patients were monitored at almawaddah private hospital and private laboratories specializing in immunodiagnostics from 2022 to 2024. 1. the medical history of each participating patient was analyzed, including confirmation of lupus and cardiac symptoms such as impaired carbohydrate metabolism and cardiac and circulatory complications in patients with rheumatoid arthritis and systemic lupus erythematosus. 2. identifying the immune damage caused by lupus other than cardiac muscle, to help us understand the context of cardiac damage, whether direct or indirect (table 1). 3. all participants underwent an ecg and echo-cardiography to confirm cardiac involvement or detect signs of myocardial ischemia (table 2). 4. laboratory and immunological examinations of the participants were performed to provide a clear statistical picture. note / this study paid special attention to the immunological aspect of the effect of systemic lupus erythematosus on the cardical seeking to understand the role of autoimmune diseases (lupus) on the cardic muscle. table 1. key immune related manifestation of condition. main immunological manifestations of diseases sledai_2k 415 coordinates slicc_di (damage index) 01 coordinates hematological disorders 22% nephritis 31% arthritis 25% skin lesions 22% sororities 22% antinuclear factor (anf) 94%, antibodies to double-stranded dna (anti_dsdna) ant phospholipid syndrome 76% of patients (positive) sjogren's syndrome 7% (positive) fig. 1. volumetric effect of immunological disorder manifestations of the disease sororities, 18% skin lesions, 18% arthritis, 21% hematological disorders, 18.00% nephritis, 25%sororities skin lesions arthritis hematological disorders nephritis shaker; asian j. immunol., vol. 8, no. 1, pp. 60-69, 2025; article no.aji.134457 63 table 2. immunodiagnostic with systemic lupus erythematous cautilizing myocardial infection n. sle immunological disorder cardiomyopathy by systemic lupus erythematous rate values 16 pericardium 26% 18 adhesive pericarditis 30% 15 exudative pericarditis 10% 4 acute myocarditis 5% 5 ischemic myocarditis (ihd) 3% 10 heart failure (hf) 15% 2 myocardial infarction (mi) 1% 16 cardiac arrhythmias 9% 2 patients with sle respectively 1% fig. 2. values cardiomyopathy by systemic lupus erythematous exclusion criteria: this study specifically targeted patients aged 30 ± 15 years, based on the statistical system issued by the iraqi ministry of health before 1989 regarding the rate of infections and deaths from heart diseases resulting from lupus. the following categories were excluded: 1. the following categories of people under the legal age of 18 were excluded for legal considerations, which require the research body to obtain judicial approval and be under judicial supervision 2. those over 45 years of age were excluded, as most of the study participants were women and must be fertile. as is the practice in current research regarding lupus, participants must be no older than 45. 3. those with comorbidities such as tumors, malignancies, or infectious diseases the medical reports provided by cardiologists were reviewed for all patients, and they had conventional hazard factors for cardiac and circulatory disease. transthoracic echocardiography was performed. patients were evaluated for left ventricular diastolic and systolic function, ejection fraction (ef), left ventricular myocardial thickness, chamber sizes, systolic pulmonary artery and diastolic pressure, pericardial status, and valves. laboratory and statistical diagnosis: concentration of the n-amino terminal fragment of (probrainnatriuretic – peptide) (nt_probnp) in serum was determined by electrochemical analysis (ecl) on a cobas analyzer utilizing the elecsys probnp ii test system. typically, myocardial infarction (mi), 1.00% heart failure (hf), 20.00% ischemic heart disease (ihd), 4.00% acute myocarditis, 7.00% exudative pericarditis, 14% adhesive pericarditis, 41% cardiac arrhythmias, 12% patients with sle respectively, 1% myocardial infarction (mi) heart failure (hf) ischemic heart disease (ihd) acute myocarditis exudative pericarditis adhesive pericarditis cardiac arrhythmias patients with sle respectively shaker; asian j. immunol., vol. 8, no. 1, pp. 60-69, 2025; article no.aji.134457 64 nt_probnp levels are ≤125 pg/ml, according to procedural guidelines (mcmurray, et al., 2012). statistical analysis was performed utilizing parametric and nonparametric statistical methods of applied statistics nt_probnp in the two groups is equal to 12. variables were presented as medians (me), with the upper and lower quartiles indicated in parentheses (25th percentile; 75th percentile). the reliability of variations between the two groups was assessed utilizing the (spss software) criterion. qualitative indicators were compared in two unrelated groups in association table utilizing the χ2 test. the significance level was p < 0.05 (rebrova, 2002). procedures: median duration of sle was two years, with moderate disease activity (sledai_2k) 9 coordinates (hochberg, 1997), and the slicc_di damage index was 2 coordinates (kim, et al., 2017). the main clinical manifestations of the disease embraced hematological disorders (49%), nephritis (45%), arthritis (37%), skin lesions (34%), and synovitis (33%). eighty-six patients (98%) had immune disorders: elevated levels of antinuclear factor (anf) were recognition in 95%, and anti_dsdna antibodies were recognition at 78% of sle patients. associated antiphospholipid syndrome (allergic fungal sinusitis (afs) and sjögren's syndrome were identified in 9 (9%) and 10 (11%) patients, respectively (miyakis, et al., 2006). valvular regurgitation with varying degrees of regurgitation (not requiring surgical treatment) was the most frequently observed cardiac abnormality in systemic lupus erythematous (sle), identified in 79 patients (90%): 78 patients (89%) with tricuspid regurgitation, 72 patients (82%) with mitral regurgitation, 57 patients (7%) with pulmonary artery regurgitation, and 15 patients (14%) with aortic regurgitation. endocarditis was identified in 26 patients (30%), and mitral or tricuspid valve prolapse in 30 patients (34%). the rarest was mitral, tricuspid, and aortic stenosis, identified in one patient (1%). the median concentration of nt_probnp was 92 (27–332) pictograms per milliliter, an elevated level of nt_probnp (> 125 pictograms per milliliter) was identified in 29 patients (32%). the possible association of cardiac disorders with therapy used in sle, patients had divided into two groups. • group 1: embraced 43 patients (40 women 3 men) with a median age of 30 ± 15 years who, at time of examination, had not received glucocorticoids (gc), immunosuppressant • group 2: embraced 45 patients (40 women 5 men), the median age was 30 ± 15 years patients took a variety of combinations of these drugs. choroquine, corticosteroids at a dose of 150 mg/day. among them were both newly ill and longterm patients with sle, but who canceled previously prescribed therapy. ha was received by 44 (98%) of them, the median dose was 20 mg /day when calculated for prednisone. during the entire period of the disease, cyclophosphamide was used in 23 (48%) patients, azathioprine in 17 (38%), mycophenolate mofetil in 16 (36%), methotrexate in 9 (19%), choroquine, corticosteroids in 43 (96%). 3. results immunological immunology and laboratory characteristics of patients of both groups are presented in table 2. the groups were comparable in age and gender, women predominated (93% 90%). patients of the 2nd group had a longer duration of illness than participants of the 1st group p < 0.00001), less activity (sledai_2k and 12 coordinates, p < 0.001), a higher damage index (slicc_di 1 0 coordinates, p < 0.001), they exhibited a lower incidence of skin lesions (11% and 57%, p < 0.0001), joints (22% 52%, p < 0.05) and hematological disorders (24% and 73.8%, p < 0.0001) . the main immunology examination manifestations of sle in group 1 patients have hematological disorders (74%), skin lesions (57%), kidneys (55%) and joints (52%), all also revealed immunological disorders an increase the level of antinuclear factor (anf) in 100%, anti dsdna in 80% patients. • group 1: nephritis (36%), sororities (29%), hematological disorders (24%) and arthritis (22.2%). • group 2: immunological disorders have identified in 96% of patients. no relevant variations were identified in the concomitant sjogren's syndrome and allergic fungal sinusitis (afs). this, division of patients with sle into groups upon taking ant rheumatic therapy shaker; asian j. immunol., vol. 8, no. 1, pp. 60-69, 2025; article no.aji.134457 65 (immunosuppressant, gibp) allowed us to assess its relationship with the activity and duration of sle, since the groups differed relevantly in these indicators. value level of nt_probnp in untreated patients was elevated than in group 2nt (151 33 pictograms per milliliter, respectively, p < 0.01), while exceeding normal values. note / that the mean value of and the mean value of the control group is equal to 4, noting that p. value nt_probnp equal 0.05 mean value lupus value is equal 19.37. table 3. comparison immunological and laboratory characteristics of patients of both groups are presented characters sle (n = 88) control (n=85) p. value notes nt_probnp (mean) 19.37 4 0.05 based on the assumption that the overall mean for both groups is equal to 12, the probability value indicates the difference in statistical significance age (mean ± sd) 30 ± 15 30 ± 14 <0.001 there is a difference in the statistical results regarding the distribution of ages gender (male) 3 (10%) 5 (4%) <0.0001 the statistical difference in results is very high between the two genders gender (female) 40 (90%) 40 (96%) <0.0001 the statistical difference in results is very high between the two genders total sample size 88 85 n. of individuals with determined gender 43 45 anti_ena (extractable nuclear antibodies): antibody ranges intermediate to sle sufferers and manipulate organization is supplied in table 5. the evaluation outcomes reveal there haven’t extensive variations (p > 0.015) within the detection results of anti_jo1 and anti_scl70 antibody <0.001 intermediate to sle patients and manage institution. but, the sle patient institution exhibited relevantly raised of antirnp levels <0.001, anti_sm <0.001, anti ssa, and anti ssb compared with group of control, with all variations being statistically relevant (p < 0.01). these results in the bottom. table 4. comparison levels anti_ena antibody sle (n = 88) control (n = 85) p. values anti_jo1 negative 6(7%) 78(92%) <0.001 positive 82(93%) 7(8%) anti-rnp <0.001 negative 13(15%) 72(85%) positive 75(85%) 13(15%) anti_scl70 0.15 negative 77(88%) 81(95%) positive 11(12%) 4(5%) anti_sm <0.001 negative 66(75%) 83(98%) positive 22(25%) 2(2%) anti_ssa <0.001 negative 35(40%) 68(80%) positive 53(60%) 17(20%) anti_ssb 0.005 negative 72(82%) 77(90%) positive 16(18%) 8(10%) shaker; asian j. immunol., vol. 8, no. 1, pp. 60-69, 2025; article no.aji.134457 66 analysis of associate chartered accountant (aca) levels: results comparing aca levels in patients with sle to those in the control group are outlined in table 6. the analysis revealed that positive rate of aca antibodies had increased over time, aca (iga, igg, igm), are elevated in sle group compared to the control group, with these variations achieving statistically relevant (p < 0.05) as shown in table 6. • p25 represents the first quartile of the 25% percentile. • p75 represents the middle quartile of the 75% percentile. • interquartile range (iqr) measure of spread and dispersion shows the least effect of infection and the result is the first quartile the middle quartile. table 5. comparison of aca detection results between sle group and the control group aca (n (%) sle (n = 88) control (n = 85) p. values negative 59(67%) 53(62%) <0.001 positive 29 (33%) 32(38%) antibody median iqr = p75p25 p. values aca iga (p75-p25) 5 (7.67-2.32) 5.35 0.017 aca igg (p75-p25) 6 (8.75-2.63) 6.12 0.001 aca igm (p75-p25) 9 (13.51-4.82) 8.69 0.004 table 6. comparison of b2_gpi autoantibodies detection results between sle group and control group antibody sle (n = 88) control (n = 85) p. value b2_gpi autoantibodies 25(28%) 14(17%) <0.001 iga (median) 8(12.94-4.08) 6(8.36-2.77) <0.001 igg (median) 6(8.36-2.8) 5(7.84-2.53) <0.001 igm (median) 6(8.35-3) 5(7.84-2.53) <0.001 table 7. operational definition immunological test examination (manufacturer) aim sllicc_di n/n the aim of the examination is to assess the amount of accumulated damage to the organs resulting from lupus anti_dsdna elisa test (thermo fisher scientific) it aims to detect autoantibodies, which are responsible for attacking the body's tissues. nt_probnp cobas (beckman coulter) the purpose of screening is to diagnose a heart condition by detecting the level of peripheral brain peptide aca elisa test (euroimmun) the aim is to detect anticentromere antibodies responsible for attacking healthy tissues in an immunological disorder disease. anti_ena elisa test (inova) it aims to detect antibodies to nuclear antigens and is used to detect sjögren's syndrome and polymyositis. sledai_2k n/n the index is used to evaluate lupus disease and helps doctors assess the patient's response to treatment. anti_jo_1 antibody elisa test (inova) type of anti _ena test anti_rnp antibody elisa test (inova) type of anti _ena test anti_sci70 antibody topoisomerase test the aim of detecting sci70 antibodies in the blood is to diagnose systemic scleroderma. anti_sm _antibody elisa test (inova) type of anti _ena test anti_ssa antibody elisa test (inova) type of anti _ena test anti_ssb antibody elisa test (inova) type of anti _ena test b2_gpi autoantibodies elisa test (euroimmun) the aim of detecting antiphospholipid syndrome is to detect antibodies to beta-2 protein. shaker; asian j. immunol., vol. 8, no. 1, pp. 60-69, 2025; article no.aji.134457 67 b2_gpi autoantibodies levels: when detecting antibodies b2_gpi autoantibodies and comparing the results with the lupus disease and control group, it was identified that positive results for antibodies b2_gpi autoantibodies were higher in lupus patients compared to the control group as shown in table 7. 4. results and discussion the influence of immunological disorder inflammation in the endocardium, valves, aortic dissection and hypertension and ha therapy are discussed as possible causes of these changes. the use of ha can lead to rapid "healing" of inflammation of a valve especially of the heart (valvulitis) (gladman & ibanez, 2002). despite the fact that bacterial or nonbacterial thrombotic endocarditis is a characteristic classic valve lesion in sle (adawi & blum, 2020), most researchers agree that the most common change in heart valves is insufficiency with varying degrees of regurgitation (mazurek, et al., 2022). some indicate a high incidence of aortic valve insufficiency, others indicate mitral valve insufficiency (kim, et al., 2017). in my study, tricuspid and mitral valve insufficiency was more common, and aortic valve insufficiency was less common; the changes were not clinically relevant and did not require surgical correction. an increase in the level of natriuretic peptide (bnp) is not specific for myocarditis, based on the dynamics of the nt_probnp level, the effectiveness of treatment is evaluated and a prognosis is made. while maintaining its high concentration, it can be assumed that there is active inflammation, its carbonization. therefore, according to the recommendations immunologic and cardiologists, all patients with clinical suspicion of myocarditis should conduct a study of the initial level of nt_probnp and its dynamics (hochberg, 1997). they do not have any special features, and associations with sle-specific autoantibodies and therapy have also not been identified. in the present study, rhythm disturbances were recognition in 18% of patients. the number of atherosclerotic plaques in the coronary and femoral arteries is twice as high in patients with disease as in people suffering from rheumatoid arthritis and dm diseases with high cardiovascular hazard (petri, et al., 2012). in my study, i did not observe a statistical difference in the incidence of various myocarditiss and heart failure between those who received immunosuppressant and high blood pressure treatments and those who did not receive them, but the difference was clear in those who developed lupus. 98% of cases, ha was used, hypertension, an increase in total cholesterol and bmi values were more often observed, which confirms the opinion about the effect of ha on the frequency of tfr. currently, the determination of the concentration of nt_probnp is used for the screening of heart failure (hf), assessment of its severity and prognosis, as well as monitoring the effectiveness of therapy. in addition, a high level of nt_probnp is an independent hazard factor not only for heart failure (hf). there are isolated studies on the concentration of bnp nt_probnp in immunological disorder inflammatory diseases. in sle, the level of this biomarker was determined only in patients receiving pathogenesis therapy (tan, et al., 1982, gladman & ibanez, 2002). the concentration of bnp nt_probnp in patients with sle was higher than in the control group, this increase was primarily associated with myocardial dysfunction, but was not associated with vascular damage, including atherosclerotic, inflammatory markers, and sle activity. 5. conclusion despite the young age of patients, therapy (primarily with glucocorticoids, ha) and a long duration of certain sle are linked to a higher prevalence hazard factors (hypertension, hypercholesterolemia, overweight), and myocarditis a practicing rheumatologist should pay attention to the need for joint management of patients with sle with a cardiologist and immunological , assessment of markers of heart failure (hf) as a potentially fatal complication, especially in patients with high disease activity under medical control of tfr utilizing a minimum dose of ha during remission and low activity. 6. limitations 1. despite the extreme importance of the study, we were unable to include larger shaker; asian j. immunol., vol. 8, no. 1, pp. 60-69, 2025; article no.aji.134457 68 areas, at least in southern iraq, due to legal restrictions on allowing data collection and involving a larger study sample. 2. medical records were used to collect information for patients, especially the control group. 3. there is no similarity between patients in the severity and duration of the injury 4. limited laboratory materials and ct scan results for patients prompted us to communicate directly with patients for reevaluation. 7. recommendations 1. raising awareness among doctors about the risks of lupus and its direct impact on heart function, especially in its early stages. 2. it is necessary to deepen the studies and establish a longitudinal study to increase understanding of the effect of lupus on the heart. 3. encourage teamwork among cardiologists, rheumatologists, and immunologists to develop an early picture of lupus disease progression and the potential for cardiac damage. 4. requiring lupus patients to undergo heart function tests even if they do not show heart symptoms and making it a mandatory work routine consent as per international standards or university standards, patient(s) written consent has been collected and preserved by the author(s). ethical approval as per international standards or university standards written ethical approval has been collected and preserved by the author(s). disclaimer (artificial intelligence) author(s) hereby declare that no generative ai technologies such as large language models (chatgpt, copilot, etc) and text-to-image generators have been used during writing or editing of this manuscript. acknowledgement i would like to extend my sincere gratitude and appreciation to professor dr. fateh shaker abdul hussein, a specialist in cardiology and vascular diseases, for his valuable insightful guidance to completion of this research. competing interests author has declared that no competing interests exist. references adawi, m., abu-gabel, t., sabbah, f., yehuda, i., tamir, s., & blum, a. 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(1996). the development and initial validation of the systemic lupus international collaborating clinics/american college of rheumatology damage index for systemic lupus erythematous. arthritis & rheumatism, 39(3), 363–369. https://doi.org/10.1002/art.1780390303 gladman, d. d., ibanez, d., & urowitz, m. b. (2002). systemic lupus erythematous disease activity index 2000. the journal of rheumatology, 29(2), 288–291. hochberg, m. c. (1997). updating the american college of rheumatology revised criteria for the classification of systemic lupus erythematous. arthritis & rheumatism, 40(9), 1725. https://doi.org/10.1002/art.1780400928 kim, c. h., al-kindi, s. g., jandali, b., et al. (2017). incidence and hazard of heart failure in systemic lupus erythematous. heart, 103(3), 227–233. shaker; asian j. immunol., vol. 8, no. 1, pp. 60-69, 2025; article no.aji.134457 69 https://doi.org/10.1136/heartjnl-2016309561 mazurek, a., gryga, k., bugala, k., iwaniec, t., musial, j., podolec, p., et al. (2022). influence of statin therapy on antiphospholipid antibodies and endothelial dysfunction in young patients with coronary artery disease and systemic lupus erythematosus. archives of medical science, 18(1), 18–24. https://doi.org/10.5114/aoms.2019.90271 mcmurray, j. j. v., adamopoulos, s., anker, s. d., et al. (2012). esc guidelines for the diagnosis and treatment of acute and chronic heart failure 2012. journal of cardiology, 17(4s3), 1–68. https://doi.org/10.15829/1560-4071-20124s3 miyakis, s., lockshin, m. d., atsumi, t., et al. (2006). international consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (aps). journal of thrombosis and haemostasis, 4(2), 295–306. https://doi.org/10.1111/j.15387836.2006.01753.x nasonov, e. l. (ed.). (2010). rheumatology. clinical guidelines (pp. 429–481). geotar-media. petri, m., orbai, a.-m., alarcón, g. s., et al. (2012). derivation and validation of the systemic lupus international collaborating clinics classification criteria for systemic lupus erythematous. arthritis & rheumatism, 64(8), 2677–2686. https://doi.org/10.1002/art.34473 rebrova, o. yu. (2002). statistical analysis of medical data. use of the statistica software package (p. 312). mediasfera. tan, e. m., cohen, a. s., fries, j. f., et al. (1982). the 1982 revised criteria for the classification of systemic lupus erythematous. arthritis & rheumatism, 25(11), 1271–1277. https://doi.org/10.1002/art.1780251101 disclaimer/publisher’s note: the statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of the publisher and/or the editor(s). this publisher and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. _________________________________________________________________________________ © copyright (2025): author(s). the licensee is the journal publisher. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: https://pr.sdiarticle5.com/review-history/134457 https://pr.sdiarticle5.com/review-history/134457 _____________________________________________________________________________________________________ *corresponding author: e-mail: chidisteve.cn@gmail.com; cite as: ndukwu, chidi l.c., and jane ugochi chinedu-madu. 2024. “seroprevalence of hepatitis b and hepatitis c viral infections in port harcourt, nigeria”. asian journal of immunology 7 (1):209-16. https://journalaji.com/index.php/aji/article/view/145. asian journal of immunology volume 7, issue 1, page 209-216, 2024; article no.aji.125624 seroprevalence of hepatitis b and hepatitis c viral infections in port harcourt, nigeria chidi l.c. ndukwu a* and jane ugochi chinedu-madu a a faculty of medical laboratory science, federal university, otuoke, nigeria. authors’ contributions this work was carried out in collaboration between both authors. both authors read and approved the final manuscript. article information doi: https://doi.org/10.9734/aji/2024/v7i1145 open peer review history: this journal follows the advanced open peer review policy. identity of the reviewers, editor(s) and additional reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here: https://www.sdiarticle5.com/review-history/125624 received: 20/08/2024 accepted: 24/10/2024 published: 30/10/2024 abstract introduction: viral hepatitis which includes five the main strains of hepatotropic viruses, hepatitis a (hav), hepatitis b (hbv), (hba), hepatitis c (hcv), hepatitis d(hdv), and hepatitis e (hev), are leading causes of morbidity and mortality globally. much public health attention is however focused on the two blood-borne hepatitis viruses hbv and hcv, due largely to their high prevalence and pervasive rates of morbidity and mortality. this study was thus aimed at determining the prevalence of hbv and hcv. materials and methods: this retrospective study was conducted in diobu, a high-density group of urban communities situated in the heart of port harcourt metropolis in the niger delta of nigeria. the medical laboratory records 206 patients, including 123 males and 83 females of various ages, residing in diobu and closely adjoining urban communities of port harcourt metropolis were reviewed; to extract data on the hbv and hcv tests. results: the mean age in years was 37.04± 12.06 the median age: 36, mode: 33, while the minimum and maximum ages were 12 and 69 years, respectively. the seroprevalence of hepatitis original research article https://doi.org/10.9734/aji/2024/v7i1145 https://www.sdiarticle5.com/review-history/125624 ndukwu and chinedu-madu; asian j. immunol., vol. 7, no. 1, pp. 209-216, 2024; article no.aji.125624 210 b in the study area was found to be 5.8%, while that for hepatitis c was 0.5%. the males had a seroprevalence of 7.3% and females 3.6% for hepatitis b. among the age groups, the highest prevalence was found in the 31-40 years age bracket at 10.6%, followed by 11-20 years (59%), 2130 years, (5.0%) and 41-50% (3.6%) conclusion: the prevalence of 5.8% for hbv in this study, represents an intermediate level of transmission on the who criteria of endemicity, while the 0.5% is low level of transmission. there is need for concerted public health policy makers and all stakeholders to put more efforts curtail the menace of viral hepatitis. keywords: blood-borne infections; hepatitis b; hepatitis c. 1. introduction hepatitis is an inflammation of the liver which may be caused by a number of viruses and noninfectious factors including excessive use alcohol, drugs, toxins and autoimmune disorders; the commonest type of hepatitis however, are the viral hepatitis. the viral hepatitis infections are caused by five main strains of hepatitis viruses namely, hepatitis a, b, c, d, and e viruses [1,2]. though all the hepatitis viruses cause liver diseases, they vary in modes of transmission, nature and severity of the illness, geographical distribution and prevention methods. while types a, b and c are the most prevalent, types b and c attracts more public health attention because they cause chronic disease in hundreds of millions of people and together are the most common cause of liver cirrhosis, liver cancer and viral hepatitis-related deaths worldwide [1,2]. about 354 million people across the globe are living with hepatitis b or c, and most of them are ignorant of the condition and could not access treatment [2]. the global prevalence hepatitis b virus (hbv) or hepatitis c virus (hcv) infections are put at 296 million and 58 million persons, respectively [3,4,5]. while hepatitis b is estimated to cause 780,000 deaths annually, the estimate for hepatitis c is about 400,000 deaths every year [1]. in nigeria, it is estimated that about 20 million people are chronically infected with hepatitis b and c; with prevalence of 8.1% and !.1% respectively [6]. hepatitis b is spread through contact with infected body fluids including infected blood and blood products, saliva, vaginal fluids and semen. it can also be transmitted from a mother to her to child during pregnancy, childbirth or breastfeeding [7]. over 95% of immunocompetent adults infected with the virus are able to have it cleared from the system even without knowing about the infection [8]. the infection is preventable by a safe and effective vaccine. the vaccine gives almost full protection against the virus, and is usually given soon after birth with boosters within few weeks [7]. hepatitis c is a bloodborne infection, transmitted through contact with infected blood, which may be through the sharing needles or syringes, or from unsafe medical procedures such as unsafe blood transfusions and blood products [9]. the symptoms of hcv infection may include fever, fatigue, loss of appetite, nausea, vomiting, abdominal pain, dark urine and jaundice. though there is no vaccine for hepatitis c, but it can be treated with antiviral medications; early detection and treatment are important to prevent serious liver damage and wellbeing [9]. though the prevalence of hcv infection in nigeria is considerably low, some scholars opined that the prevalence is on the increase [10]. this is an indication for concerted efforts in the monitoring and control of the spread of the virus. there is currently inadequate data on the prevalence of hepatitis b and hepatis c in port harcourt, nigeria. this study was thus aimed at filling the gap by determining the prevalence of hbv and hcv in urban communities in port harcourt. 2. materials and methods 2.1 study area this retrospective study was conducted in diobu, a high-density group of urban communities situated in the heart of port harcourt metropolis in the niger delta of nigeria. the notable urban communities are mile i, mile ii and mile iii urban communities of diobu. the coordinates of the area are: 4°47'24"n, 6°59'36"e (latitude:4.772152; longitude:6.994514). the communities are bordered on the north by the port harcourt new gra, on the northeast by dline, on the northwest rivers state university, nkpolu-oroworukwu, port harcourt, on the east https://en.wikipedia.org/wiki/new_gra,_port_harcourt https://en.wikipedia.org/wiki/d-line,_port_harcourt https://en.wikipedia.org/wiki/d-line,_port_harcourt https://en.wikipedia.org/wiki/rivers_state_university ndukwu and chinedu-madu; asian j. immunol., vol. 7, no. 1, pp. 209-216, 2024; article no.aji.125624 211 by the poth harcourt old gra, on the southeast by kidney island, and on the southwest by eagle island. the are a number of public and privatelyowned hospitals and other healthcare facilities in the area, including primary health centres, maternity homes, medical laboratory facilities and community pharmacy outlets patronized by many residents. on the other end, harsh economic climates and pervasive ignorance and economic have made many residents, particularly those in overcrowded area, who lack amenities like clean water and decent housing; such as water fronts to resort to self-medication and patronizing different kinds of purveyors of unregulated healthcare products. 2.2 design of the study this study was conducted between january 2022 to december 2023 among 206 males and female of all ages, residents of diobu and closely adjoining urban communities of port harcourt metropolis; attending public and private healthcare facilities within the area. the inclusion criteria include persons who live within mile i, mile ii and mile iii and closely adjoining neighborhoods such as agip, eagle island, d-line etc.; and who within the study period, conducted serological laboratory investigations for hbv and hcv antibodies (sd bioline, korea) at diagnostix and scientifique laboratories, port harcourt. persons living far from diobu or did not perform the two tests and those with incomplete records were excluded. 2.3 data collection patients’ data were obtained by going through their medical laboratory records. the outcomes of the laboratory tests of hbv and hcv infections and relevant socio-demographic data about the ages, genders and areas of residence were extracted, coded and anonymously analyzed. specimen collection and assay: about 3 ml of venous blood was collected from each study participant, transferred to a test tube and allowed to stand until clotted the serum was separated from the clotted by centrifugation at 5000 revolutions per minute for 10 min and tested for hbsag and anti-hcv using a one-step hbsag test strip (sd bioline) and a one-step hcv test strip (sd bioline), respectively, following the manufacturer instructions. the sensitivity and specificity of rapid test kits of hbsag and onestep hcv test strips were 99.1% and 99.6%, respectively. 2.4 data analysis data were analyzed with ibm spss statistics version 25. descriptive statistics were employed in presenting the data were in counts and percentages. pearson chi-square test were employed to ascertain associations between the categorical variables. 3. results in this retrospective cross-sectional study, we reviewed the laboratory records of 206 persons; 123 (59.7%) males and 83 (40.3%) females, attending public and private healthcare facilities in port harcourt who conducted serological investigations of hepatitis b and hepatitis c viral infections at diagnostix and scientifique laboratories, port harcourt. the mean age in years was 37.04± 12.06 the median age: 36, mode: 33, while the minimum and maximum ages were 12 and 69 years, respectively (fig. 1). 3.1 seroprevalence of hepatitis b and hepatitis c viral infections in port harcourt, nigeria the seroprevalence of hepatitis b in the study area was found to be 5.8%, while that for hepatitis c was 0.5%. the males had a seroprevalence of 7.3% and females 3.6%. among the age groups, the highest prevalence was found in the 31-40 years age bracket at 10.6%, followed by 11-20 years (59%), 21-30 years, (5.0%) and 41-50% (3.6%) (table 1). in the residential areas, the highest prevalence of 7.0% was observed in mile ii diobu, followed by mile iii diobu (6.4%), mile i diobu (5.8%) and adjoining communities (4.0%) (table 1). 3.2 seroprevalence of hepatitis c viral infections the seroprevalence of hepatitis c virus infection as determined in the study area was 0.5% as only one of the 206 tests recorded positive. the prevalence for males was 0.8%, while females. was 0%. the 31-40 age group had a prevalence had a prevalence of 1.9% while the rest had zero prevalence (table 2). 3.3 statistical analysis pearson’s chi-square test of independence and fisher’s exact test were performed to evaluate https://en.wikipedia.org/wiki/old_gra,_port_harcourt ndukwu and chinedu-madu; asian j. immunol., vol. 7, no. 1, pp. 209-216, 2024; article no.aji.125624 212 the relationship between the residence, age and gender (independent variables) and results of hbv and hcv test results (dependent variable). the association between these variables were found not be significant, given that the p values were not less than 0.05, we therefore failed to reject the null hypothesis which states that the variables are independent. in other words, there was no sufficient evidence to conclude that a significant association exists between the variable and the test results obtained for the hepatitis b and hepatitis c screening. fig. 1. frequency distributions of the hbv and hcv specimens by age table 1. seroprevalence of hepatitis b viral infection characteristics number tested positive tests prevalence % age groups 11-20 16 1 5.9 21-30 38 2 5.0 31-40 59 7 10.6 41-50 53 2 3.6 51-60 17 0 0.0 61-70 11 0 0.0 total 206 12 5.8 gender males 123 9 7.3 females 83 3 3.6 females 206 12 5.8 total 206 12 5.8 residence mile i diobu 49 3 5.8 mile ii diobu 53 4 7.0 mile iii diobu 44 3 6.4 adjoining communities 48 2 4.0 total 206 12 5.8 ndukwu and chinedu-madu; asian j. immunol., vol. 7, no. 1, pp. 209-216, 2024; article no.aji.125624 213 table 2. seroprevalence of hepatitis c viral infections in port harcourt, nigeria characteristics number tested positive tests prevalence age groups 11-20 16 0 0.0 21-30 38 0 0.0 31-40 59 1 1.5 41-50 53 0 0.0 51-60 17 0 0.0 61-70 11 0 0.0 total 206 1 0.5 gender males 123 1 0.8 females 83 0 0.0 total 206 1 0.5 residence mile i diobu 49 1 1.9 mile ii diobu 53 0 0.0 mile iii diobu 44 0 0.0 adjoining communities 48 0 0.0 total 206 1 0.5 4. discussion the findings in this study indicate an intermediate prevalence for hepatitis b and low prevalence for hepatitis c. majority of the people screened were those who wanted medical tests to ascertain their health status, while a few were referred based on presumptive diagnosis for viral hepatitis. majority of the were collected from males (59.7%), who also recorded a higher prevalence of 7.3% than females (3.6%). the proportion of males to females was similar to that of a study in northern nigeria with 60.4% males and 39.4%% [11]. the prevalence of 5.8% obtained here for hepatitis b infection was higher than the 1.2% prevalence among members of a university community in port harcourt [12], it however aligned closely with the prevalence of 6.2% reported in a study among abattoir workers in port harcourt [13]. the discrepancy between this and the result from the university may be due to the nature of the population. the university community is populated by persons with good knowledge of the infections and the preventive measures, above what is obtainable in the general population. the prevalence of 0.5% observed here however aligned very closely with the 0.6% prevalence in the university community [12]. the prevalence for both hepatitis b and c were lower than the prevalences of 12.6% and 15.2% for hbv and hcv respectively in a study in northern nigeria for persons investigated due to ill-health; but aligned with 6.6% for hbv among healthcare workers in the same study, but lower than 6.5% for hcv among the healthcare workers [11]. another study in northern nigeria reported a seroprevalence of hbv infection of 14.0% and hcv infection (10.4%) [14]; these were higher than the outcomes of this study. the lower prevalence in this study may be attributed to a variety of factors such as geographical and cultural factors, level of awareness and compliance with preventive measures among others. it may also be as a result of reported decline in the prevalence of hbv infection [15]. the prevalence of 5.8% is the same as the pooled global prevalence of 5.8%, less than the prevalence of 7.8% for the who african region which bears the largest burden of global prevalence for hbv infection [16]. in the same northern nigerian study, hbv seroprevalence of 9.1% for males was higher than that for females (4.9%), which was similar to the results in this study; conversely, the hcv seroprevalence for females (6.2%) was higher than that for males (4.2%) [14]. the hbv ndukwu and chinedu-madu; asian j. immunol., vol. 7, no. 1, pp. 209-216, 2024; article no.aji.125624 214 prevalence for the young adults age group (10.2%), was the highest among age groups, while the highest prevalence for hcv was 6.2% observed with the middle-aged group [14]. these results for hbv had close semblance with the findings of this study where the males had a higher prevalence than females, and the highest prevalence was found the 30-40 age group. the results hcv were however different from the findings here, the only positive hcv result was from a male within the 30–40-year group. the incidence of hcv infection is reported by several studies to be driven by intravenous drug use, men having sex with men among other factors with the prevalence found in eastern mediterranean region, south-east asia region and european region [5,17,18,19,20]. these may therefore not the critical factors in port harcourt and the country as shown with low prevalence rates for hcv infection. the limitations of the study like in retrospective studies, is based on secondary records. the samples were collected and analyzed without consideration for the study. the sociodemographic data are limited to those required for in the laboratory records, and there is no personal interaction with the participants. some information that may enrich the study are therefore not captured. tests carried out are limited to hbsag and anti-hcv; without additional analysis like elisa and the molecular analysis. future studies may be designed to overcome these limitations. 5. conclusion the prevalence of 5.8% for hbv in this study, represents an intermediate level of transmission (2–8%) on the who criteria of endemicity, while the 0.5 % for hcv is a low level of transmission. the morbidity and mortality associated the viral hepatitis can be avoided if efforts are made to curtail the viral infections. this can be done by creating greater awareness among the all strata of the populations on preventive measures. vaccination against hbv is an important tool in the fight against the menace of the virus and should be made available to everyone. young people forty years and below are at the greatest risk of being infected with viral hepatitis. it is therefore advocated that social media and other modern means of communication should be utilized in passing the message on prevention and control of the infections. consent it is not applicable. ethical approval the study was reviewed and approved by ethical review committee of the faculty of medical laboratory science, federal university otuoke, nigeria. relevant approvals were sought and obtained from the management of diagnostix and scientific laboratories. disclaimer (artificial intelligence) author(s) hereby declare that no generative ai technologies such as large language models (chatgpt, copilot, etc.) and text-to-image generators have been used during the writing or editing of this manuscript. competing interests authors have declared that no competing interests exist. references 1. mehta p, grant lm, reddivari akr. viral hepatitis. 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asian j. immunol., vol. 7, no. 1, pp. 209-216, 2024; article no.aji.125624 216 19. mateu-gelabert p, sabounchi ns, guarino h, ciervo c, joseph k, eckhardt bj, fong c, kapadia sn, huang ttk. hepatitis c virus risk among young people who inject drugs. front public health. 2022 jul 29;10: 835836. doi: 10.3389/fpubh.2022.835836. pmid: 35968435; pmcid: pmc9372473. 20. mahoney bj, morford kl, biegacki et, tetrault jm. hepatitis c virus and integrated care for substance use disorders. clin liver dis (hoboken). 2024 jun 28;23(1):e0241. doi: 10.1097/cld.0000000000000241. pmid: 38952692; pmcid: pmc1121667 disclaimer/publisher’s note: the statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of the publisher and/or the editor(s). this publisher and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. _________________________________________________________________________________ © copyright (2024): author(s). the licensee is the journal publisher. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: https://www.sdiarticle5.com/review-history/125624 https://www.sdiarticle5.com/review-history/125624 _____________________________________________________________________________________________________ *corresponding author: e-mail: mfonakpaso@unical.edu.ng; cite as: akpaso, mfon i, elton n takim, michael e oku, anani sadeyeng, lydia b edim, and gabriel u udo-affah. 2024. “gonadal histo-morphology and enhanced fertility potential of curcuma longa in male stz-induced diabetic rats”. asian journal of immunology 7 (1):258-73. https://doi.org/10.9734/aji/2024/v7i1149. asian journal of immunology volume 7, issue 1, page 258-273, 2024; article no.aji.126504 gonadal histo-morphology and enhanced fertility potential of curcuma longa in male stz-induced diabetic rats mfon i akpaso a*, elton n takim a, michael e oku a, anani sadeyeng a, lydia b edim a and gabriel u udo-affah a a department of anatomy, faculty of basic medical sciences, university of calabar, calabar, nigeria. authors’ contributions this work was carried out in collaboration among all authors. all authors read and approved the final manuscript. article information doi: https://doi.org/10.9734/aji/2024/v7i1149 open peer review history: this journal follows the advanced open peer review policy. identity of the reviewers, editor(s) and additional reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here: https://www.sdiarticle5.com/review-history/126504 received: 14/09/2024 accepted: 18/11/2024 published: 04/12/2024 abstract diabetes mellitus is a disorder in which the body fails to produce enough or respond normally to insulin, leading to abnormally high blood sugar levels. the study was aimed at investigating the effects of ethanolic extract of curcuma longa (tumeric) on fertility profile and gonadal histomorphology of streptozotocin-induced hyperglycemic male wistar rats. semen parameters (count, motility and viability) and testicular histology of streptozotocin “stz”-induced hyperglycemic male wistar rats were investigated following the administration of curcuma longa rhizome extract. twenty-four (24) rats weighing 110-180g were grouped into four. the groups were labelled a to d and contained six rats each. group a (normal control) rats were given rat pellets and distilled water original research article https://doi.org/10.9734/aji/2024/v7i1149 https://www.sdiarticle5.com/review-history/126504 akpaso et al.; asian j. immunol., vol. 7, no. 1, pp. 258-273, 2024; article no.aji.126504 259 only. group b (diabetic control) rats were given 65mg/kg body weight “bw” of streptozotocin ip. group c served as the standard group and was given 65mg/kg of streptozotocin ip + 250mg/kg of metformin ip, while group d (test group) rats were given 65mg/kg bw of streptozotocin ip + 500mg/kg of curcuma longa extract. curcuma longa extract was administered through the oral route with the aid of an oro-gastric tube. the administration lasted for twenty-eight (28) days. the rats were then anaesthetized and the testes and semen obtained for histological and semen analysis (sperm count, sperm motility, sperm viability) respectively. the diabetic group showed seminiferous tubules with reduced germinal epithelium and distorted interstitial connective tissue with a significant (p<0.05) reduction in sperm motility, count, viability compared to the normal group that showed normal histological features and normal sperm parameters (motility, count and viability). however, the administration of c. longa showed significant (p<0.05) increase in levels of sperm parameters (motility, count and viability) and progressive restoration of histological integrity within the testes. in conclusion, ethanolic extract of c. longa has the potential to reverse the damage caused by hyperglycemia in male reproductive function. keywords: curcuma longa; semen analysis; testis; diabetes mellitus. 1. introduction diabetes mellitus “dm” is a metabolic disorder with a characteristic high level of blood sugar (hyperglycemia) over a prolonged period of time and is usually accompanied by disturbed metabolism of proteins and fats [1]. blood glucose rises because it cannot be metabolized in the cells due to lack of insulin production by the pancreas, or the inability of the cells to effectively use the insulin that is being produced [1]. insulin is produced by the beta cells of the pancreas and its function is to regulate the uptake of glucose from blood into the different cells and tissues [2]. three major symptoms of diabetes mellitus include frequent urination (polyuria), increased hunger and appetite (polyphagia) and increased thirst (polydipsia) [1]. dm is also associated with complications which include diabetic ketoacidosis, cardiovascular disease, foot ulcers, damage to nerves and eyes, encephalopathy, cardiomyopathy [1,3,4] and reproductive dysfunction [5]. the diabetic complications on different cells, tissues and organs have been linked to the reactive oxygen species (ros) that are generated due to hyperglycemia [6]. prolonged and poorly controlled diabetes has been reported to cause sexual dysfunction in males and females. sexual dysfunction can also be an early sign of diabetes [7]. in males, diabetes mellitus has effects on the endocrine control of spermatogenesis and causes erectile, as well as dysfunction and impaired ejaculation [8]. it also negatively impacts sperm parameters such as volume, count, motility and morphology [9]. due to the above-mentioned complications, it became imperative by scientists around the world to find affordable solutions. studies have shown that to achieve a good metabolic control of diabetes and energy balance, a combination of lifestyle, nutrition, exercise and pharmaceutical treatment are important [10]. they are also said to be major factors in the management of diabetes [11]. pharmaceutically, metformin is considered to be the first-choice agent for treatment of diabetes [12]. another class of drugs of choice is sulfonylureas [13]. these drugs however are seen to have undesirable side effects. on account of these side effects, there is advocacy for the use of medicinal plants [14], because they have been found to have little or no side effects and are easily affordable and affordable. the use of medicinal plants as an alternative medicine has in the last millennium, been accepted all over the world [15]. in the united states of america for instance, about 38% of the population uses herbal medicine [16] and in turkey, 48.8% of the population are said to use herbal medicines [17] to treat chronic diseases and illnesses. in africa, the world health organization states that at least 80% of the population relies on medicinal plants [18]. some of these plants and herbs possess antioxidant properties. they contain carotenoids, flavonoids, alkaloids, glycosides and some are said to have anti diabetic effects [19]. one of such plants is curcuma longa commonly called turmeric. curcuma longa, the turmeric plant is commonly used as a spice but has been recognized by the scientific community for its antioxidant property. in asia, it has been used as a natural therapeutic medicine since ancient times [20]. its main akpaso et al.; asian j. immunol., vol. 7, no. 1, pp. 258-273, 2024; article no.aji.126504 260 constituent is curcuminoid which gives it a wide range of pharmacological properties including antioxidant, anti-protozoan, anti-venom, antiinflammatory [21], antibacterial, antidiabetic, antiviral and anticancer activities [19]. it would therefore be interesting to evaluate its antidiabetic effects and its effects on the fertility profile of streptozotocin-induced hyperglycemic male wistar rats. infertility affects about 8-12% of couples worldwide and an estimated 50-80 million couples have been reported to suffer from some form of infertility [22]. previously, infertility was thought to be a problem of the females but studies have now shown that 20-30% of infertility is linked to the males [23] commonly due to poor semen quality or quantity [24]. in 2015 it was observed in a study that type 1 diabetic patients showed a lower level of spermatozoa with increased motility, altered mitochondrial function and post ejaculatory dysfunction of the epididymis [8]. diabetes has also shown an increase in prevalence over the years with an estimated 425 million persons worldwide and nearly 50% of that number is not diagnosed [25]. studies have shown that there is a decline in fertility involving males with diabetes, leaving a high percentage of them with reproductive dysfunctions including reduced libido and impotence [5]. stz is an alkylating agent that causes pancreatic islet β-cell destruction and is widely used experimentally to produce a model of type 1 diabetes mellitus (t1dm) [26]. it is a cytotoxic glucose analogue which has been used as a chemotherapeutic agent in the treatment of metastasizing pancreatic islet cell tumors and other malignancies [27] and its effect can be seen within seventy-two hours after administration depending on doses administered [28]. stz has been one of the chemical agents used for the induction of diabetes mellitus in experimental animals. stz functions as dnasynthesis inhibitor in bacterial and mammalian cells [29]. the selective pancreatic beta cell toxicity and diabetic condition, resulting from stz induction, is related to the glucose moiety in its chemical structure which enables stz to enter the beta cell via the low affinity glucose -2 transporter in the plasma membrane [30]. since diabetes mellitus is said to also cause decline of fertility in males [5], semen analysis is therefore necessary in this study as it serves as a pivotal and indispensable procedure in the assessment of male fertility status [31]. when conducted meticulously and in an in-depth manner, it possesses the ability to elucidate the underlying causes contributing to male infertility, thereby offering valuable insights and diagnostic clarity [32]. the comprehensive evaluation of semen encompasses the analysis of diverse parameters, including the measurement of ejaculate volume, quantification of sperm count, assessment of sperm motility and movement patterns, evaluation of sperm morphology and structural integrity, as well as the investigation of the composition and makeup of seminal secretions [32]. 2. materials and methods 2.1 plant collection and extract preparation rhizomes of curcuma longa (tumeric) were bought at watt market in calabar, cross river state, nigeria. it was identified and authenticated by a taxonomist in the department of botany, university of calabar, cross river state, nigeria and a voucher number (bot/herb/ucc/201) was given. the fresh turmeric rhizomes were cleaned, chopped into tiny pieces and air dried for 7days after which they were grounded into powdered form. a measured amount of 950g of powered rhizomes were extracted using 2 liters of 95% ethanol for 24 hours. the extract was first double filtered with chess cloth, then with filter paper (whatman no.1 filter paper). the filtrate (extract) was concentrated under reduced pressure at 45oc in rotary evaporator to 10% volume and then dried in a vacuum water bath, yielding 58.9g (6.2%) of crude extract. the crude extract (paste) obtained was stored in a refrigerator until it was required. 2.2 experimental animals twenty-four (24) adult male wistar rats with average weight of 160g were used for this research. the rats were kept in clean cages and divided into four groups designated a, b, c and d with six rats in each group. the rats were allowed to acclimatize for two weeks in animal house, university of calabar and allowed access to commercially available chow (livestock feed) and water. 2.3 experimental design table 1 shows the experimental design of the research. twenty-four animals were divided akpaso et al.; asian j. immunol., vol. 7, no. 1, pp. 258-273, 2024; article no.aji.126504 261 randomly into four groups containing six rats each. the groups as shown in the table includes the normal control, diabetic control, standard group and the test (c. longa treated) group. 2.4 induction of hyperglycemia stz was administered intra-peritoneally to the rats after fasting for twelve hours in order to induce hyperglycemia. the stz was reconstituted in 0.5m sodium citrate and administered at a dose of 65mg/kg bw [33]. 2.5 confirmation of diabetes mellitus diabetes was confirmed three days after administration of stz using accu-check glucometer with blood samples obtained from tails of the wistar rats. the blood glucose levels (mg/dl) was checked before and after induction and every seven (7) days during administration of the ethanolic extract of curcuma longa to ascertain hyperglycemic state. it was observed that the blood glucose of all the animals in the diabetic groups were above 170 mg/dl as compared to that of the normal control which had values less than 91 mg/dl. administration of extract: curcuma longa extract administration commenced three days after induction of hyperglycemia by oro-gastric intubation which lasted for 28 days. termination of experiment: at the end of treatment period, the experimental animals were weighed and sacrificed through chloroform inhalation. the anterior abdominal wall was incised and semen was extracted from the caudal epididymis for analysis. the testis was then obtained, weighed and preserved in 10% formal saline for tissue processing using hematoxylin and eosin staining method. determination of weight: all the animals were weighed before and after induction of hyperglycemia and every three days during the course of administration of the extract. analysis of sperm parameters: the epididymis was dissected out and placed in a physiological saline in a ratio of 1:10 weight (g) by volume (ml), and was then macerated using surgical blade to release the sperm cells. the suspension was then filtered with 80 µm stainless mesh after pipetting [34]. the following sperm parameters were estimated as follows: i) sperm motility (%): two drops of sperm suspension were placed on a clean and labelled microscope slide and covered with a coverslip. this was mounted on light microscope and the number of motile cells divided by the total number of sperm cells counted and was expressed in percentage. ii) sperm viability (%): this was done using the eosin-nigrosin staining technique. the sperm suspension was mixed with equal volume of the stain and smeared on glass slides. live sperm excluded the stain and appeared lightly colored, while dead sperm took up the stain and appeared pink in color. the counts of live sperm were divided by the total number of sperm cells and expressed as percentage. iii) sperm count (x106/ml): improved neubauer hemocytometer was used for the sperm count. a capillary tube was used to pipette the sperm suspension into the counting chamber of the hemocytometer. the hemocytometer was placed on a light microscope and the counting was estimated by multiplying the number of cells counted by the dilution factor and by the hemocytometer volume. histological study using hematoxylin and eosin stain “h&e”: the paraffin slides containing testicular tissue underwent a dewaxing process involving two rounds of exposure to xylene for a duration of 5 minutes each. subsequently, rehydration was performed by sequentially immersing the slides in decreasing concentrations of alcohol (100%, 95%, and 70%) and rinsing them under tap water. following rehydration, the sections were subjected to a 15-minute staining procedure using hematoxylin, followed by a 5-minute rinse under tap water. to enhance visualization, the sections were then differentiated in acid alcohol for 1 minute and subsequently counter-stained with eosin for another 1 minute. after a rinse in tap water, the sections underwent dehydration and clearing using xylene. following this, the sections were allowed to air dry, and a few drops of dpx were applied to the slide surface before placing a coverslip on top. the resulting tissue units were then taken using a digital camera connected to a light microscope for further examination [35]. akpaso et al.; asian j. immunol., vol. 7, no. 1, pp. 258-273, 2024; article no.aji.126504 262 table 1. experimental design groups number of rats treatment a 6 normal control (rat chow and distilled water) b 6 diabetic control 65mg/kg bw of stz c 6 65mg/kg bw of stz + 250mg/kg.bw metformin d 6 65mg/kg bw of stz + 500mg/kg.bw curcuma longa 2.6 statistical analysis data obtained from the experiment was analyzed using one-way analysis of variance and duncan post hoc test using a statistical package for social science, spss version 26.0 for windows. the results were presented as mean ± standard error of mean and considered statistically significant at p<0.05. 3. results assessment of blood glucose: the changes in fasting blood glucose “fbg” were determined in this study over the course of the 28-day experimental period and they are presented in fig. 1. at day zero, the blood glucose levels in all experimental groups were considered normal ranging from about 70 mg/dl to 107 mg/dl. from the results, it can be observed that elevated blood glucose concentration was seen in all diabetic groups following the administration of 65mg/kg body weight of stz. the elevated fbg level (fig. 1) of the diabetic control group only exhibited an increase (p<0.05) of 24.40±26.61mg/dl (gotten by subtracting the final fbg level from the initial fbg level after induction with diabetes mellitus), while that of the metformin and extract treated groups had significantly decreased fbg levels of 227.60±24.32mg/dl and -236.60±25.14mg/dl respectively (p<0.05). fig. 1. daily blood glucose levels of the different experimental groups values are expressed as mean +sem, n = 6 * = significantly different from normal control at p<0.05 akpaso et al.; asian j. immunol., vol. 7, no. 1, pp. 258-273, 2024; article no.aji.126504 263 fig. 2. initial and final body weights of the different experimental values are expressed as mean +sem, n = 6. * = significantly different from control at p<0.05 a = significantly different from diabetic control at p<0.05 fig. 3. sperm count in the different experimental groups values are expressed in mean + sem. n = 6 * = values are significantly decreased compared to normal control (p<0.05) a = values are significantly increased compared to diabetic control at p<0.05 akpaso et al.; asian j. immunol., vol. 7, no. 1, pp. 258-273, 2024; article no.aji.126504 264 fig. 4. sperm motility in the different experimental groups values are expressed in mean + sem. n = 6 * = values are significantly decreased compared to normal control (p<0.05) a = values are significantly increased compared to diabetic control (p<0.05) assessment of body weight: the results of changes in the body weight of experimental animals after 28 days’ period were assessed. the initial and final weights across the 28-day period are presented on fig. 2. observed from these results was a significant (p<0.05) reduction in body weight of the experimental rats induced with diabetes (-37.00±5.75g) after subtracting the final weight from the initial weight of the rats. the normal control group a had a significant increase of +20.00±1.14g. on treatment with the test drug (metformin), a weight increase of +0.80±1.80g was observed, while that of the curcuma longa extract was a decrease of -1.20±2.08g (p<0.05) semen analysis: sperm count: results showed a significant (p<0.05) decrease of 37.70 + 7.5 million/ml and 47.10 + 1.7 million/ml of sperm count in the diabetic control and metformin treated groups respectively compared to the normal control group that had 69.6 + 1.5 million/ml sperm count. however, 65.40 + 1.4 million/ml of sperm count was recorded in group d animals (administered with 500mg/kg.bw of c. longa). this increase in sperm concentration was statistically significant when compared with the diabetic control (p<0.05) (fig. 3). sperm motility: fig. 4 shows the result of sperm motility for the experimental animals. animals in the diabetic control group and the group treated with metformin recorded significant decrease in sperm motility values when compared with the normal control group (p<0.05). however, group d animals (placed on 500mg/kg.bw of c. longa), showed an increase in sperm motility which was statistically significant when compared with the diabetic control and the metformin treated group. sperm viability: experimental animals in the diabetic control group and the group placed on metformin recorded low sperm viability values which was significantly decreased when compared with the normal control group (p<0.05). however, group d animals (placed on 500mg/kg.bw of c. longa), showed an increase in sperm viability of which was statistically significant when compared with the diabetic control (fig. 5). akpaso et al.; asian j. immunol., vol. 7, no. 1, pp. 258-273, 2024; article no.aji.126504 265 fig. 5. sperm viability in the different experimental groups values are expressed in mean + sem. n = 6 * = values are significantly decreased when compared to normal control at p<0.05 a = values are significantly increased when compared to diabetic control at p<0.05 plate 1. testis (normal control) x400 section of testes showing normal seminiferous tubules with germinal cell layers (double-headed arrow) on the basal lamina to spermatocytes filled lumen (dotted circular lines). the interstitial connective (#) tissue was well preserved akpaso et al.; asian j. immunol., vol. 7, no. 1, pp. 258-273, 2024; article no.aji.126504 266 plate 2. h&e section of testis of diabetic control (x400) section of testes showing seminiferous tubules with reduced germinal epithelium (double-headed arrow), distorted interstitial connective tissue (#) plate 3. h&e section of testis (standard group) x400 section of testes showing normal seminiferous tubules tubules with germinal cell layers (double-headed arrow) starting from spermatogonia (arrowhead) on the basal lamina and spermatocytes filled lumen (dotted circular lines). the interstitial connective (#) tissues was distorted akpaso et al.; asian j. immunol., vol. 7, no. 1, pp. 258-273, 2024; article no.aji.126504 267 plate 4. h&e section of testis (treated with 500mg/kg c.longa) x400 section showed different stages of seminiferous tubules and the interstices. the seminiferous with germinal cell layers (double-headed arrows) showed lumen partly &completely filled (dotted circular line) with mature spermatocytes. the interstitial connective (#) tissues was distorted. although the alteration exists, there is a progressive restoration observed histological observations: histological study on sections of testes in the normal control group (x400) showed prominent seminiferous tubules with regular and consistent cellular arrangements on the germinal epithelium. the lumens of seminiferous tubules were filled with flagella of newly formed spermatozoa. their basement membranes were intact and the intervening interstitium which contained blood vessels and clusters of leydig cells showed regular pattern (plate 1). section of testes from the diabetic control group (group b) showed significant alteration in histological patterns in the testes when compared with the normal control. irregular shaped seminiferous tubules with shrunken lumen and decreased tubular diameter were observed (plate 2). section of testes in the diabetic animals treated with metformin (group c) showed normal seminiferous tubules although its germinal epithelium was distorted with differentiating cells arranged in a coarse pattern. the interstitial connective tissue was distorted (plate 3). for the test group treated with 500mg/kg.bw of c. longa, seminiferous tubules were prominent and intact with basement membrane. the interstitial connective tissue was distorted, but there is progressive restoration observed. 4. discussion complex interplay of various pathological processes, prominently characterized by oxidative stress damage, inflammation, and apoptosis are involved in diabetic testicular damage [36]. curcumin, a bioactive compound derived from curcuma longa, has emerged as a potential therapeutic agent with protective properties against diabetic testicular injury. however, the precise underlying mechanisms through which curcumin exerts its beneficial effects in this context remain a subject of ongoing investigation. the present study examined the effect of c. longa crude extract on akpaso et al.; asian j. immunol., vol. 7, no. 1, pp. 258-273, 2024; article no.aji.126504 268 the histology of the testis and semen parameters of streptozotocin-induced hyperglycemic male rats. the sustained increase in the blood glucose levels of diabetic animals observed at the end of the study was corroborated by studies by pulidomoran et al. [37] and mohammed et al. [38] which revealed hyperglycemic response in stz treated animals. they postulated that the observation was due to impaired glucose oxidation which causes nad+ depletion, ultimately culminating in the inhibition of insulin biosynthesis and secretion. the reduction in blood glucose levels in the metformin and extract-treated groups, aligns with the study by akpaso et al. [39], who reported a significant reduction in fasting blood glucose levels in animals administered curcumin. studies by shenoy [40] and kunnumakkara et al. [21] have corroborated the anti-diabetic efficacy of the extract, attributing it to its antioxidant and antiinflammatory properties. findings reported by akpaso et al. [41] also revealed the potential of c. longa to significantly reduce in blood glucose levels in experimental animals. the final bodyweight of the experimental animals in the extract-treated group showed a significant increase when compared to both the diabetic and control groups. the overall changes in bodyweight statistically indicated that both the metformin and extract-treated groups experienced significant increases in their respective bodyweights when compared to both the control and diabetic groups. these findings are consistent with studies conducted by shenoy [42] and szudelski [43] which reported a significant decrease in bodyweight in streptozotocin-injected rats. furthermore, the observed decrease in bodyweight of the diabetic rats aligns with the findings of ding et al. [44] and zhong et al. [45] who observed a similar effect on diabetic animals induced with streptozotocin. the underlying mechanisms contributing to the reduction in bodyweight within the diabetic group can be attributed to the intricate interplay of factors such as the degradation of structural proteins and muscle wasting as reported by he et al. [46]. conversely, the ameliorative effects of curcuma longa on bodyweight is supported in the work of akpaso et al. [41] where the oral administration of the plant extract demonstrated remarkable improvement in the bodyweight of experimental animals. semen analysis revealed a significant reduction in the total count of sperm cells in the diabetic control group, compared to the normal control group. this observation underscores the detrimental impact of diabetes mellitus, regardless of whether it is type 1 or type 2 diabetes, on male fertility. the findings of tsao et al. [47] demonstrated the adverse effects of experimentally induced diabetes on sperm parameters. also a study conducted by alizadeh et al. [48] showed significantly reduced sperm count in diabetic animals. while the scientific basis underlying the pathophysiological mechanisms linking diabetes to semen parameters, particularly sperm count, remains diverse in the literature, prolonged hyperglycemia triggers the body's oxidative stress response and leads to endothelial injury in blood vessels, including those within the testis and epididymis [49]. moreover, elevated blood glucose levels can disrupt the regulatory function of the hypothalamic-pituitary-gonadal axis, resulting in alterations in the number and morphology of testicular interstitial cells, degeneration of sertoli cells, reduced synthesis and secretion of testosterone, impaired sperm development and maturation, and ultimately, compromised reproductive functions. however, the administration of the extract in the treated group led to a notable improvement in the total sperm count, which was comparable to that observed in the normal control group. these findings are in line with the research conducted by chanapiwat et al. [50] who demonstrated that replacing a ketogenic diet with curcumin supplementation improved semen quality. additionally, a different study reported an increase in total sperm count among infertile men participating in a randomized clinical trial after receiving curcumin [51]. together, these studies provide further support for the positive effects of curcumin and its potential in ameliorating the adverse impacts of diabetes on semen parameters, including sperm count. the experimental findings revealed a significant decline in the percentage concentration of motile sperm cells in the diabetic group compared to the control group. however both the standard and test groups showed an increase (p<0.05) compared to the normal control group. these observed outcome in the diabetic group is consistent with the outcomes of prior investigations conducted by ricci et al. [52] and zha [53] who reported that the addition of curcumin in frozen-thawed angora goat semen improved both motility and acrosome integrity, while also enhancing the motility and functional integrity of sperm plasma membrane in frozen akpaso et al.; asian j. immunol., vol. 7, no. 1, pp. 258-273, 2024; article no.aji.126504 269 bull semen. in a report by ozbek, he stated that diabetes mellitus is capable of disrupting endocrine and metabolic pathways, leading to reduced sperm motility and altered sperm morphology [54]. the reduced sperm motility observed in the standard group agrees with studies by raghif [55] and naglaa et al. [56] which illustrated that metformin administration led to decrease in sperm motility. it was stated that vitamin b12 deficiency could be the cause of this observation as chronic metformin use is associated with lower blood levels of vitamin b12. furthermore, metformin administration was said to impair the activity of mitochondrial complex-1 which plays a vital role in maintaining the normalcy of sperm motility [56,56]. sperm viability values which decreased significantly in the diabetic control and metformin-treated groups compared with the normal control group. in the extract treated group there was an increase in sperm viability which was statistically significant when compared with the diabetic control. this finding aligns with a report by ozbek also showed that diabetes mellitus was capable of altering sperm morphology through the disruption of endocrine and metabolic pathways [54]. a study by belhan et al [57] showed that the percentage of sperm viability in stz-induced diabetic rats improved mildly in curcuma longa treated group. the reason for the increase in sperm count, motility and viability as seen in the group treated with curcuma longa extract may be due to its composite antioxidants which help reduce the oxidative stress caused by diabetes. oxidative stress is capable of damaging sperm cells, as well as impairing their function [58]. the histopathological manifestations in the tissue sections of group b (diabetic control) rats were characterized by irregularly shaped seminiferous tubules exhibiting a shrunken lumen and decreased tubular diameter, which were indicative of severe edema and pronounced hyperemia within the veins. these findings align harmoniously with the seminal research conducted by ghosh et al. [59] and barsiah et al. [60] who expounded upon the frequent occurrence of abnormal histology within the testes of diabetic animals, with discernible alterations observed in the cytoarchitecture of the seminiferous epithelium as well as disruptions in the occlusive distribution pattern. conversely, the tissue sections derived from the extract-treated group displayed notable therapeutic effects, which include prominent seminiferous tubules characterized by prominent basement membranes. studies by sudjarwo et al. [61] and singh et al. [62] documented the positive influence of curcuma longa on the cytoarchitecture of the testes. this remarkable therapeutic outcome can be attributed to the phenol compounds present in the extract as suggested by sadhwani [63] and pharm-huy et al. [64] that phenols possess the ability to counteract excessive free radicals, thereby safeguarding cells and tissues against their toxic effects. many studies have also shown the tissue-protective effects of extracts of various medicinal plants rich in antioxidants [65,66,67,68,69]. 5. conclusion the findings of the study suggest that extract of curcuma longa has the potential to improve fertility in diabetic male animals, as well as protect against the detrimental effects of hyperglycemia on the male reproductive system. disclaimer (artificial intelligence) author(s) hereby declare that no generative ai technologies such as large language models (chatgpt, copilot, etc) and text-to-image generators have been used during writing or editing of this manuscript. consent it is not applicable. ethical approval an approval for the experimental protocol of this study with registration number 220ana2423 was obtained from the faculty animal research ethics committee (farec-fbms), faculty of basic medical sciences, university of calabar, cross river state, nigeria. competing interests authors have declared that no competing interests exist. references 1. gojka r. who global report on diabetes: a summary. intl j noncom dis. 2016;1(1): 38. akpaso et al.; 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cite as: shnawa, ibrahim m s, azhar o al-thahab, and kasim n a.thewaini. 2025. “preclinical immunogenicity of capsule and outermembrane proteins of a local neurotropic isolate of hemophilus infleunzae b in a lapin model”. asian journal of immunology 8 (1):1-7. https://doi.org/10.9734/aji/2025/v8i1155. asian journal of immunology volume 8, issue 1, page 1-7, 2025; article no.aji.129951 preclinical immunogenicity of capsule and outermembrane proteins of a local neurotropic isolate of hemophilus infleunzae b in a lapin model ibrahim m s shnawa a,b*, azhar o al-thahab c and kasim n a.thewaini a a department of medical biotechnology, college of biotechnology, al-qasim green university, babylon, iraq. b college of nursing, university of hillah, babylon, iraq. c department of biology, college of science, university of babylon, babylon, iraq. authors’ contributions this work was carried out in collaboration among all authors. all authors read and approved the final manuscript. article information doi: https://doi.org/10.9734/aji/2025/v8i1155 open peer review history: this journal follows the advanced open peer review policy. identity of the reviewers, editor(s) and additional reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here: https://pr.sdiarticle5.com/review-history/129951 received: 26/11/2024 published: 06/02/2025 abstract the preclinical immunogenicity of capsule c and outer membrane protein omp of human neurotropic isolate of hemophilus influenzae b hib in a lapin model is being reported. hemophilus influenzae b is gram negative, short rods, encapsulated bacterium, microaerophilic, fastidious, need original research article https://doi.org/10.9734/aji/2025/v8i1155 https://pr.sdiarticle5.com/review-history/129951 shnawa et al.; asian j. immunol., vol. 8, no. 1, pp. 1-7, 2025; article no.aji.129951 2 x and v factor for growth with six capsular serotypes. among which serotype b was the dominant. itis associated with human pyogenic respiratory and meningeal infections. the c and omp were separated, purified, and characterized. then, quantfied to the rate of 3 mg/m l and 2.27 mg/ml accordingly. both of the preparations were used as test antigens for the invitro use and for speific immune priming of rabbits. cell free culture filtrate of test isolate was prepared and used as a skin sensitin in dth test. rabbits were were preconditioned with cfa adjuvant for two weeks then specific immune primed using multisite multi-injection protocols. capsule immunogen induce leukocyte stimulatory factor cytokines. four to six folds rise both titres and concentrations of anti capsular precipitins and anti omp hemagglutinins were noted. both c and amp are proved to be lapin immunogens stimulate humoral immune responses at both systemic and mucosal immune compartments. these findings may forms the background information and be an integral part of preclinical and clinical development and production of local h. infleunzae b systemic and/or mucosal vaccines. which could be valid on approval for local mass vaccination against childhood meningitis in this area. keywords: antigen; cellular; humoral; immunogens; vaccines. 1. introduction hemophilus influenzae b is short gram negative rods, encapsulated bacterium. microaerophilic, fastidious need x and v factor for growth with six capsular serotypes. among which serotype b was the dominant. hib is associated with human pyogenic respiratory and meningeal infections (levenison et al., 2018). meningitis is a life threatining disease affects millions of people of all ages allover the world and is considered a major global public health issue (international society of infectious diseases 2023). the nature of the immunity induced by h. infleunzae b in rabbit, rat and mice is humoral by a t cell independent epitopes (levenison et al., 2018; wisplwey et al., 1989). circulating anti capsular prp antibodies promote complement dependent bactericidal power and phagocytosis found in meningitis patients and vaccinee. there is a correlation between the presence of bactericidal antibodies and resistance to h. infleunzae b infection (international society of infectious diseases 2023; wisplwey et al., 1989). hemophilus infleunzae b omp have been proved to be immunogens in infant rat (hansen et al., 1981). the noncapsulated atypical h. infleunzae suppress and modulate cellular and humoral immune responses to atypical h. infleunzae vaccine due to interference phenomena (mawas et al., 2006; lee et al., 2010; griffiths et al., 2012; latez et al., 2004; santosham, 1993). omp of atypical h. infleunzae is affected by the function of treg on b cells specific for omp in a cell culture system (hirano et al., 2019). the objective of the present work was to map immunogenicity of capsule and outer membrane proteins of h. infleunzae b local isolate. 2. materials and methods 2.1 reviewer inquiry it is a reviva revista study that has been a part of al thahab ph. d. thesis at 2006 on hib meningitis; pathogenesis and immunology in man and laboratory animal. prof. shnawa and ass. prof. thewaini had been supervisor and coadvisor. whereby had been faculty membership at babylon university. 2.2 neuropathic h. infleunzae local h. infleunzae b isolate from a clinically proven human meningitis cases the isolate was, culturally and biochemically characterized through classical and api20e system. serotyped using difco, co, bdtm kit (gonzalez & ledeboer 2023). 2.3 preparation of immune reagents the capsule was saparated, purified, identified and quantified to the concentration of 3 mg/ml (kwapnski, 1972). omp was separated, purified, identified and quantified to the concentration of 2.27 mg/ml (catlin et al., 1972). sensitin was prepared as cell free culture filtrates as in (shnawa & thewaini 2002). 2.4 rabbits a newzeland white rabbits weighing 11.5 kgs were checked to be free of parasite, bacteria, and antibodies. these rabbits were adapted to housing conditions for one week and kept ad libitum conditions. they were grouped and assigned into three groups each of three. two test groups and one control group. shnawa et al.; asian j. immunol., vol. 8, no. 1, pp. 1-7, 2025; article no.aji.129951 3 2.5 immunization protocol the test groups were primed with complete freund adjuvant cfa in a rate of one ml for each rabbit, control group was primed with saline. test and control groups were left for two weeks. after preconditioning with cfa. one group was primed with capsule, 3 mg/l. and the other with omp 2.27 mg/ml. in a two weeks apart multisite multiinjection protocol followed by one weeek leave then test bleed (alshahery & shnawa 1989). capsule in 3 mg/l...................... three rabbits outermemberane protein 2.27 mg/l.....................three rabbits saline sham control....................three rabbits 2.6 sampling and processing blood samples were collected from test and control rabbits by cardiac punctutre with a rate of six mls from each rabbit. samples were divided into two parts each of three mls amounts. tubes without anticoaggulant and three mls., and tube with anticoaggulant. sera were saved from samples without anticoaggulants in a rate of 0.5 ml amounts in an append roff tubes and kept at 18 c. whole blood with anticoaggulant uesed for test for leukocyte inhibitory factors by capillary method (soberg, 1969). appendecies were collected from primed and control rabbits. they were opened up, freed from digesta and scrapped with sterile clean scalpel to remove mucus from the mucosal surfaces. proportional mucus-saline were mixed in a sterile petri-dish. mixtures were tubbed in centrifuge tubes and centrifuged at 5000 rpm for ten minutes. supenates of three mls amounts were mixed with equal amounts of peb 6%, 6000 and left at 4 c for one hr and centrfuged for 5000 rpm for 15 minutes. precipitates were saved and reconstituted with sterile formal normal saline (shnawa & thewaini 2002). 2.7 immune function tests the anticapsular antisera and mucosal globulin were titrated with capsule antigen using capillary tube precipitation test (turgeon, 2020). the anti omp antisera and mucosal globulin were titrated with omp coated tanned sheep red cells by micro hemagglutination test (joshi & chauhan 2022). mucus-saline mixture was mixed with dextran solutions in an equal amounts for separation of mucosal leukocytes as in metcalf et al. (1986). systemic and mucosal leukocyte inhibitory factor was done by capillary method as in soberg (1969). 3. results 3.1 baseline immune functions control rabbits reveals the normal baseline immune functions in this experimental settings. the serum globulin protein concentration was 6.75 g/l. while, the normal appendex glubulin protein concentration was 0.425 g/l. natural serum baseline titre mean was 10 both for anticapsular and anti-omp antibodies and normal mucosal globulin concentration were 0.425 and 0.4g/l. both for anti-capsular and anti-omp antibodies. while the normal leukocyte inhibitory factor cytokines were ranging between 0.95 and 0.97%. 3.2 cellular immune conversion specific omp priming to rabbits lead to nonsignificant leukocyte inhibitory factor cytokines and mild leukocyte stimulatory factor cytokines in one rabbit replicate both at mucosal and systemic responses. specific capsular primed rabbits have shown leukocyte stimulatory factor cytokines as compared to lif of normal baseline immune functions. omp and capsular immune primied rabbits indicate cellular immune conversion than that of basline cellular immune functions, table 1. 3.3 delayed allergenic immune conversion at 72 hrs post-sensitization with the skin sensitins cfcf through intradermal injection of the primed rabbits. neither classical dth nor jone-moote reaction noted in both of the primed groups. this may indicate that there were no delayed allergenic immune conversion in these immune primed test rabbits table 1. 3.4 humoral immune conversion the mean of serum anticapsular antibody titre was 533.3 compared to the baseline humoral immune function was 10. while, the mucosal anti-capsular antibody mean titre was 42.66. as compare to normal humoral baseline immune function was 2. in other word five folds increase folds increase inthe serum titer means and four folds in the mucosa mean titres. while, the mean serum anti-omp titre was 853.3 compared to normal baseline function mean titre was 10. the mucosal anti-omp mean titre was 85.33 compared to normal baseline humoral immune fuction was 2. that is to say six folds increase in shnawa et al.; asian j. immunol., vol. 8, no. 1, pp. 1-7, 2025; article no.aji.129951 4 serum and five folds im mucosal anti-omp antibodies. the folds increase in mean antibody titres in serum and in mucosa indicated humoral immune conversions, table 2. 4. discussion the omp and capsular polysaccharide of h. influenzae b are; pathogenicity determnant, virulence associated antigens and virulence factor. so both in conjugate state or in separate state they are standing as targets for vaccine candidates and human approved vaccines in more than one vaccine qualifying boards all over the world (shnawa, 2019). in the present communication tempts to investigate immunogenicity of subcellular fractions of local neurotropic neurogenic h. infleunzae b. these subfractions are; capsule and omp in a lapin model, tables 1 &2. the specific immune priming protocols tempts to precondition rabbits with freund complete adjuvant cfa for a period of two weeks that provok non-specific immune stimulation to both of innate and adaptive immune cells (alshahery & shnawa 1989). as a pre-immunization adjuvant followed by two weeks apart b capsular antigen in separate and omp antigen in separate rabbit groups. this based on the theme that immuoadjuvant can be of use either pre,mix with or post-antigen priming to an immune animal model (zhao et al., 2023). both of the prepared antigens bear forigeness characters from the organism producing them that are encoded by the genetic system of that organism (venter, 1998). parallel to this the immune system cells of test animal model are encoded by gene sets that on their expression, these cells recognize that the introduced material facing them are foreign (lagou et al., 2018). immunogenicity in theoritical immunology sense is a matter of self/non-self recogntion theme (pradeu & carssella 2006). both of capsule b and omp were found to be as lapin immunogens inducing humoral immune responses both at systemic [blood sera] and mucosal [appendex globulin] levels. the immune conversion were estimated by the folds increase of antibody titre and concentrations than the baseline titre and concentrations from the baseline immune function in normal control rabbits. both of systemic anticapsular and anti-omp were express five to six folds increase. while for mucosal antibody titres rise was four to five folds increase than normal immune function was five folds. capsular b antigen induce leukocyte stimulatory factor up to 1.08% as compared to saline control was 0.95%, which is a kind of cytokine that promote leukocyte migration in capillary tube approach (sixt & lammermann 2011; thorley et al. 2007). both of the capsule b and omp antigens does not express delayed allergenic immune conversions than the baseline control rabbits. table 1. cellular immune functions as leukocyte inhibitory factor cytokines[a] and skin dth[b] for h. infleunzae b a specific immune primed rabbit systemic leukocyte inhibitory factor mucosal leukocyte inhibitory factor capsule primed r1 r2 r3 rmean control 1 1 1 1 0.98 1 1.09 1.07 1.08 0.995 omp primed r1 r2 r3 rmean control 1 0.9 0.9 0.903 0.98 1 0.95 0.91 0.95 0.97 b/skin dth/hours erythema induration necrosis capsule primed 6 48 72 omp primed 6 48 72 shnawa et al.; asian j. immunol., vol. 8, no. 1, pp. 1-7, 2025; article no.aji.129951 5 table 2. humoral immune function for capsular [a] and omp [b] primed rabbits as precipitins a and hemagglutinins b. for the neurogenic h. infleunzae b animal groups capsule primed rabbits serum antibody titre means serum concentration means gm/l mucus titre means mucus concentration means gm/l test group a r1 r2 r3 rmean control 320 640 640 533.3 10 26.3 31.42 32.1 29.94 6.75 64 32 32 42.6 2 1 2.7 2.3 2.06 0.45 test group b omp primed r1 r2 r3 rmean control 1280 640 640 853.3 10 33.1 21.2 22.31 25.4 6.35 64 128 64 85.33 2 2.7 1.0 1.0 1.56 0.4 since they neither produce typical skin dth reaction nor jone-moote reactions (burrell, 1979), during the observation period 6 up to 72 hrs. post to sensitin intradermal injection in primed test animals groups. the possible epitope nature of capsular b antigen may be t independent epitope triggering anti-prp antibodies. while that of omp antibodies may trigger th2 cells and th2 cells in turn activate b cells to produce anti-omp antibodies. apparently, it does not contain th1 dependent epitopes but there is a possibility for shiffting from th2 to th1 dependence due to coexisting epitopes mixed with independent ones (misseneir & lary 2002; mcvernon et al., 2005; shnawa, 2006; haddadi et al., 2022; garcia-diaz et al., 2013; jackson et al., 2024). thus type b capsule and omp of h. infleunzae induced local and systemic humoral immune responses in rabbits primed with them separately. systemic humoral antibody responses were higher than that mucosal responses. this was inline with shnawa (2006), working on immunogenicity of c. fetus in rabbits. the ratio of systemic to mucosal were 10;1 in term of titre while in term of concentration for omp was 13:1 and for capsule was 14:1 (shnawa, 2006). 5. conclusions human neurotropic neurogenic local h. infleunzae b omp and capsule b are found as lapin immunogen. adult rabbits proved to be valid immune model for testing immunogenicity of these antigens. capsular b antigen induce leukocyte stimulatory factor in capillary method. capsular and omp induces humoral hemagglutinin and precipitin responses both at systemic and mucosal compartments. such findings be essential for preclinical and development of systemic and/or mucosal vaccine, on approval will be valid for local mass vaccination of childhood meningitis. disclaimer (artificial intelligence) authors herebydeclare that no generative ai technologies such as large language models(chatgtp,copilot, etc ) and text-toimage generators have been used during wrting or editing of this mnanuscript. consent and ethical approval it is not applicable. competing interests authors have declared that no competing interests exist. references alshahery, m. a. n., & shnawa, i. m. s. 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(2023). vaccine adjuvants, mechanisms, and platforms. signal transduction and targeted therapy, 8, 283. available: https://doi.org/10.1038/s41392-0157-7 disclaimer/publisher’s note: the statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of the publisher and/or the editor(s). this publisher and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. _________________________________________________________________________________ © copyright (2025): author(s). the licensee is the journal publisher. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: https://pr.sdiarticle5.com/review-history/129951 https://pr.sdiarticle5.com/review-history/129951 _____________________________________________________________________________________________________ ++ assistant professor; # pharm d iii year; *corresponding author: e-mail: keshapallysrijareddy@gmail.com; cite as: gupta, nikita, sai rama konda, k. srija, and g. pooja reddy. 2024. “the mechanism and efficacy of baricitinib as a novel therapeutic option for rheumatoid arthritis: a review”. asian journal of immunology 7 (1):123-30. https://journalaji.com/index.php/aji/article/view/137. asian journal of immunology volume 7, issue 1, page 123-130, 2024; article no.aji.120135 the mechanism and efficacy of baricitinib as a novel therapeutic option for rheumatoid arthritis: a review nikita gupta a++, sai rama konda b#, k. srija b#* and g. pooja reddy b# a department of pharmacy practice, st. pauls college of pharmacy, turkayamjal, hyderabad, telangana, india. b st. pauls college of pharmacy, turkayamjal, hyderabad, telangana, india. authors’ contributions this work was carried out in collaboration among all authors. all authors read and approved the final manuscript. article information doi: https://doi.org/10.9734/aji/2024/v7i1137 open peer review history: this journal follows the advanced open peer review policy. identity of the reviewers, editor(s) and additional reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here: https://www.sdiarticle5.com/review-history/120135 received: 03/06/2024 accepted: 05/08/2024 published: 23/08/2024 abstract rheumatoid arthritis (ra) is characterized by systemic synovitis, which destroys joints. with the discovery of biological disease-modifying anti-rheumatic medicines (bdmards) and the combination of traditional dmards, clinical remission is now seen as a reasonable and achievable aim for many patients. however, bdmards must be administered via intravenous or subcutaneous injection, and some patients may not react or lose their main response. under these conditions, targeted synthetic dmards (tsdmards), which are low. review article https://doi.org/10.9734/aji/2024/v7i1137 https://www.sdiarticle5.com/review-history/120135 gupta et al.; asian j. immunol., vol. 7, no. 1, pp. 123-130, 2024; article no.aji.120135 124 molecular-weight molecules that may be taken orally, have emerged. five phase 3 studies of baricitinib, a jak1(janus kinase1) and jak2(janus kinase 2) inhibitor, demonstrated good clinical effectiveness in patients with active ra who were naïve to sdmards or had an unsatisfactory response to sdmards, mtx(methotrexate) or bdmards. in patients with rheumatoid arthritis who had not previously been treated with biologic disease-modifying antirheumatic medications (dmards), baricitinib, an oral inhibitor of janus kinase 1 and 2, decreased disease activity. keywords: rheumatoid arthritis; baericitinib; bdmards; tsdmards; jak1 inhibitor; jak2 inhibitors; mtx. 1. introduction “rheumatoid arthritis (ra) is a systemic autoimmune illness linked to a persistent inflammatory process that can harm extraarticular organs such as the kidney, lung, heart, digestive tract, eyes, skin, and nervous system in addition to joints. to categorise arthritis into noninflammatory (osteoarthritis) and inflammatory (pseudogout, basic calcium phosphate disease, gout) types, as well as bacterial and viral infections (staphylococcus aureus, neisseria gonorrhoeae, complications of lyme disease, parvovirus, enterovirus), and autoimmune processes, a great deal of research and description has been done on arthritis” [1]. “systemic synovitis-induced joint degradation is a characteristic feature of rheumatoid arthritis (ra)” [1]. “when chondrocytes and fibroblasts are stimulated, cartilage and bone are broken down, leading to the production of osteoclasts and metalloproteinases and joint injury” [2,3]. “these processes are facilitated by the overproduction of pro-inflammatory cytokines by immune cells in the synovium, such as macrophage colony stimulating factor, interleukins-6 and-17, and tumor necrosis factorα” [2,4]. “this medication has been authorized by the fda for use in adult patients with moderately to severely active rheumatoid arthritis when other dmards, such as tumor necrosis factor antagonist therapies, have not proven to be helpful” [5]. “baricitinib is an oral selective inhibitor of jak1 and jak2, specifically formulated to treat patients with active rheumatoid arthritis” [6]. “baricitinib, a jak1 and jak2 inhibitor, has been subjected to five phase 3 trials that have demonstrated excellent clinical efficacy in patients with active ra who have not responded well to sdmards, mtx, or bdmards. adalimumab, mtx, and placebo were used as comparators in studies where there was a positive response for clinical and functional criteria. additionally, it is stated that safety was bearable within the short trial duration” [1,7,8]. “jak inhibitors provide an alternative to traditional ra treatments by targeting cytokine signaling pathways linked to the pathophysiology of ra. in clinical studies including individuals with ra 1–5, the oral, selective jak1 and jak2 inhibitor baricitinib has demonstrated clinical effectiveness and tolerability. adults with moderately-to severely active ra are eligible to receive baricitinib therapy in over 50 countries, including the us, japan, and several european nations” [9]. the main therapy goal for individuals with established ra is to achieve minimal disease activity [10,11] . “many dmard treatments have been tried by a sizable portion of patients with established ra, but they have not been successful in reaching a low disease activity state. according to clinical research, response rates to all presently prescribed medications decline as cdmard and bdmard experience increases” [12]. “studies involving patients who have not responded to prior bdmards are especially significant since this cohort is growing and has the biggest unmet demand in the ra field. the ra-beacon trial did not previously evaluate the degree to which patient variables, such as age, illness duration, serological status, or history of using certain bdmards, impact the response to baricitinib” [13]. these crucial queries are covered in the present analysis [14]. “this article aims to compile and summarize the available information about the impact of baricitinib on the development of structural joint damage as well as the underlying mechanisms of these effects. results measured by magnetic resonance imaging (mri) or radiographic progression of joint erosion and joint space narrowing in clinical studies and post analyses of patients with ra who are naive to diseasemodifying antirheumatic drugs (dmards) or who gupta et al.; asian j. immunol., vol. 7, no. 1, pp. 123-130, 2024; article no.aji.120135 125 do not respond well to conventional synthetic dmards (csdmards) are presented. these results were obtained with approved doses of baricitinib (2 mg or 4 mg once daily, except in the usa, canada, and china, where the approved dose is 2 mg once daily). furthermore, preclinical baricitinib study results are discussed” [2]. “when traditional dmards are insufficient for treating rheumatoid arthritis, baricitinib, a disease-modifying anti-rheumatic medication, may be used. adult patients with moderately to severely active rheumatoid arthritis who have not reacted well to previous dmards, including tumor necrosis factor antagonist treatments, may use this medicine, according to approval from the u.s. food and drug administration (fda). moreover, baricitinib is fda approved for the treatment of covid-19, making it a viable option for hospitalized patients when combined with other medications. this includes pediatric instances that require extracorporeal membrane oxygenation, invasive mechanical ventilation, or supplemental oxygen. reviewing all the important information on baricitinib therapy in the clinical setting, including mechanisms, side effects, and contraindications, is the aim of this exercise. this exercise is specifically designed to fulfill the requirements of an interdisciplinary medical team that treats patients with rheumatoid arthritis, alopecia areata, and covid -19” [5] concluded by et al aman ahmad. 2. mechanism of action an oral, selective, and reversible jak inhibitor is called baricitinib. the tyrosine-protein kinase family of intracellular enzymes, jak, regulates signals from growth factor receptors and cytokines that are important for immune cell activity [5,15] concluded by et al anam ahmad, amanda mogul four jak proteins—tyk2, jak1, jak2, and jak 3—form distinct pairings in different cell receptors to produce homodimers or heterodimers. these jak dimers stimulate intracellular activity, including the transcription of inflammatory mediator genes, by phosphorylating the stat proteins. this ultimately sets off an autoimmune reaction [5,16] concluded by et al anam ahmad, musumeci, francesca. there is an increased affinity of baricitinib for jak1 and jak2. the medication works by blocking these jak proteins, which stop stats from becoming phosphorylated and activated. furthermore, baricitinib modifies the signaling pathway of certain growth factors, interleukins, and interferons. additionally, baricitinib causes cell death and reduces the growth of jak1/jak2 expression in mutant cells. within one week of starting therapy, baricitinib dramatically lowered blood c-reactive protein levels in rheumatoid arthritis patients. alopecia areata is thought to be caused by a confluence of immunological dysregulation and hereditary factors. important cytokines that depend on jaks for intracellular signaling, like interleukin-15 and interferon-γ, are implicated in the illness process. baricitinib has proven to be beneficial in helping people with severe alopecia areata regrow their hair [5,17] concluded by et al anam ahmad. brett king, m.d., ph.d. 3. pharmacokinetics absorption: baricitinib has a 97% bioavailability and is quickly absorbed from the gastrointestinal tract. baricitinib takes an average of 1.5 hours to reach peak plasma concentration, although it can take anywhere from 0.5 to 3 hours. it has not been demonstrated that administering food alters the peak plasma concentration. distribution: approximately 50% of baricitinib can bind to plasma proteins. baricitinib's apparent volume of distribution is 76 l, which suggests that the medication is widely distributed throughout the body's tissues. metabolism: the hepatic metabolism of approximately 10% of the medication occurs through oxidation by the cyp3a4(cytochrome p450 3a4) enzyme and organic anion transporter-3 (oat3). the strong oat3 inhibitor probenecid causes a two-fold rise in blood levels of baricitinib. consequently, patients taking probenecid at the same time should cut their baricitinib dosage in half [5] concluded by et al anam ahmad. elimination: in clinical pharmacology trials, the gastrointestinal tract eliminates 20% of the medication, and the kidneys clear about 75% of it. in patients with rheumatoid arthritis, baricitinib has an approximately 12-hour elimination half-life and a renal clearance of about 12 l/h [5,18] concluded by et al anam ahmad. jack g. shi phd. gupta et al.; asian j. immunol., vol. 7, no. 1, pp. 123-130, 2024; article no.aji.120135 126 4. administration 4.1 available dosage forms and strengths oral administration of baricitinib is possible in two dosage forms: 2 mg and 4 mg. for rheumatoid arthritis, a dose of 2 mg taken once daily by mouth is advised. clinical trials evaluated the effects of 2 mg oral baricitinib in conjunction with 10–14 days of antiviral medication in the setting of covid-19 infection [19] concluded by et al feng huang. 4.2 adult dosage for rheumatoid arthritis, 2 mg of baricitinib taken orally once daily, with or without food, is the suggested dosage. the medication can be taken either alone or in conjunction with methotrexate or non-biologic dmards. covid-19: the national institutes of health advises against using baricitinib as a secondary immunomodulatory medication and in favor of dexamethasone in covid-19 patients who are exhibiting rapidly increasing oxygen requirements and systemic inflammation. adults should take 4 mg of baricitinib once a day for a period of 14 days, or until they are released from the hospital [20] concluded by et al s honda, m harigai. 4.3 adverse effects baricitinib is generally regarded as a safe and well-tolerated drug. however, its immunosuppressive qualities raise the risk of serious infections. the most common adverse events recorded in alopecia areata patients throughout clinical trials were upper respiratory tract infections, acne vulgaris, headaches, urinary tract infections, and folliculitis [21,22] concluded by et al egídio freitas, t bieber, n katoh. herpes zoster infections are also on the rise. baricitinib has been linked to bone marrow suppression and hematological abnormalities such as anemia, neutropenia, and lymphopenia and should be monitored regularly in the lab. another side effect commonly seen after 12 weeks of baricitinib treatment is an increase in mean cholesterol, low-density lipoprotein (ldl), and high-density lipoprotein (hdl) levels without an increase in the ldl to hdl ratio. furthermore, some individuals may have a rise in creatine phosphokinase levels [23,24] concluded by et al d van der heijde, 27 pc taylor. 4.4 baricitinib for the treatment of rheumatoid arthritis since the mid-1990s, the treatment approach to rheumatoid arthritis has undergone significant modifications in the area of rheumatology. with the increased use of traditional synthetic dmards, the advent of biologic treatments, and, most recently, the development of novel small compounds that target the jak pathway, treatment paradigms have changed. a strong and specific jak1 and jak2 inhibitor is baricitinib. it has been proposed that various medicines may exhibit differential clinical effects based on their profiles of jak inhibition [25,26] concluded by et al mark c. genovese, m.d., joel kremer, m.d., omid zamani, m.d., jj o'shea, j israel, h hakobyan. adults with mild to severe active ra are advised to take baricitinib once a day. it is usually administered as a second-choice medication after patients have tried using tnf inhibitors but did not experience meaningful relief or could not handle the medication owing to adverse effects because of several potentially dangerous side effects.both brands warn of potential significant adverse effects. both caution against administering the medicine if there is a known severe illness, such as tuberculosis (tb), and recommend tb screening prior to therapy. other adverse effects may include kidney, cardiovascular, and eye health [27,28] concluded by et al ivan urits1, jacob israel2. since the mid-1990s, the treatment approach for rheumatoid arthritis has changed dramatically. treatment paradigms have shifted with the increased use of traditional synthetic dmards, the advent of biologic medicines, and, most recently, novel small compounds targeting the jak pathway. baricitinib is a very effective and specific inhibitor of jak1 and jak2. it has been proposed that medicines with varied jak inhibition profiles may have unique clinical outcomes. the purpose of this evaluation was to evaluate the safety and effectiveness of baricitinib in patients with active rheumatoid arthritis who were using conventional synthetic dmards but had not responded adequately to earlier biologic dmard therapy. in this patient cohort, once gupta et al.; asian j. immunol., vol. 7, no. 1, pp. 123-130, 2024; article no.aji.120135 127 daily oral baricitinib resulted in substantial clinical improvements at 12 weeks when compared to placebo. the therapeutic advantages were greater with the 4 mg dosage than with the 2 mg dose. adverse events, such as non-serious infections, were more common in the baricitinib group than in the placebo group. baricitinib was associated with lower neutrophil counts, higher creatinine levels, and higher low-density lipoprotein cholesterol levels. these adjustments were primarily modest and did not result in their removal from the research [25] concluded by et al mark c. genovese, m.d., joel kremer, m.d., omid zamani, m.d. 4.5 efficacy of baricitinib in phase ii studies in the jada(journal of the american dental association) research (phase 2), 301 patients with active ra(rheumatoid arthritis) receiving steady-state mtx and mtx-ir were included. at week 12, 76% of patients in the combined baricitinib 4 and 8 mg group had an acr20(american college of rheumatology 20) [29] concluded by et al david t. felson md, mph, response, compared to 41% in the placebo group [30] concluded by et al david t. felson md, mph. the placebo and 4 mg groups showed significant differences in secondary endpoints, including minimal disease activity and remission. the jada(journal of the american dental association) research (phase 2) included 145 japanese patients with active ra and mtxir(methotrexate) receiving steady background mtx. at week 12, the combined baricitinib 4 mg and 8 mg group had a significantly greater acr20 response rate (77%) than the placebo group (31%), according to a primary efficacy study [31] concluded by et al yoshiya tanaka. at week 12, the 2, 4, and 8 mg groups improved similarly across efficacy measures. however, the 4 and 8 mg groups showed earlier onset than the 1 and 2 mg groups as early as week 2 [1] concluded by et al satoshi kubo. 4.6 efficacy of baricitinib in phase iii studies six phase 3 trials on baricitinib for ra are underway or completed to establish its efficacy, evaluate adverse effects, and compare it to other dmards or placebos. in these investigations, patients in both the baricitinib and control groups received corticosteroids at baseline. baricitinib should be compared to mtx with corticosteroids, as early ra patients typically get both medications. however, no trials have compared baricitinib to mtx with corticosteroids [1] concluded by et al satoshi kubo. the ra-beacon trial (jade(jak1 atopic dermatitis efficacy and safety) comprised 527 individuals with active ra and tnf-ir(tumor necrosis factor) . at week 12, 55% of patients who received the 4 mg dosage of baricitinib had an acr20 response, compared to 27% who received the placebo [32] concluded by et al charles ludivico, m.d. compared to the placebo, there were substantial improvements in the sdai(simple disease activity index) ≤3.3 ratio. downloaded by [weill cornell medical college] at 06:35 on july 20, 2016. week 24 for the baricitinib 4 mg group. all subgroup analyses showed a favorable treatment impact, regardless of prior bdmard usage (number or kind) [33] concluded by et al dario ummarino. 4.7 safety and tolerability of baricitinib in the ra-beacon study [32], concluded by etal charles ludivico, m.d. 527 adult patients with moderately to severely active ra who had not responded to or were intolerant to at least one biologic tnf-α inhibitor and were taking background csdmard therapy experienced more treatment-emergent adverse events (teaes) than those taking placebo. patients receiving the 2 mg and 4 mg doses of baricitinib experienced more adverse events (71%) and 77%, respectively, compared to those receiving the placebo (64%), including infections (44% and 40%, vs. 31%). mild upper respiratory tract infections were found to significantly lead to imbalances. the baricitinib group saw briefer, transitory disruptions of the study medicine. sae rates were 4%, 10%, and 7% for the three groups, respectively. the proportion of patients experiencing an sae was consistent across treatment groups. there were only two nonmelanoma skin tumors in the baricitinib 4 mg group, with no reports of solid organ, hematologic, or other malignancies. the trial found two treatment-emergent serious adverse cardiovascular events (mace): one myocardial infarction (baricitinib 4 mg) and one basilar artery thrombosis (baricitinib 4 mg), which also resulted in one death. no patients had a gastrointestinal perforation.serum creatinine and ldl cholesterol levels increased, and neutrophil counts somewhat decreased when baricitinib was administered. in the baricitinib groups, very few patients experienced a treatment-emergent laboratory abnormality that led to a permanent gupta et al.; 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43(3):504-11 31. genovese mc, kremer j, zamani o, ludivico c, krogulec m, xie l, beattie sd, koch ae, cardillo te, rooney tp, macias wl. baricitinib in patients with refractory rheumatoid arthritis. new england journal of medicine. 2016;374(13):124352. gupta et al.; asian j. immunol., vol. 7, no. 1, pp. 123-130, 2024; article no.aji.120135 130 32. ummarino d. ra-beacon illuminates baricitinib. nature reviews rheumatology. 2016;12(6):313-. 33. radu af, bungau sg. management of rheumatoid arthritis: an overview. cells. 2021;10(11):2857. disclaimer/publisher’s note: the statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of the publisher and/or the editor(s). this publisher and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. _________________________________________________________________________________ © copyright (2024): author(s). the licensee is the journal publisher. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: https://www.sdiarticle5.com/review-history/120135 https://www.sdiarticle5.com/review-history/120135 _____________________________________________________________________________________________________ *corresponding author: e-mail: angeayissi04@gmail.com, mondinde@yahoo.com; cite as: ayissi marie-ange, biwole sida ghyslaine, guiedem elise, yayah emerencia ngah, jacky bikio, emilia lyonga, martha messembe, mispa yivala mbanyamsig, and georges ikomey. 2025. “role of tumour necrosis factor-alpha and adiponectin in insulin therapy failure in type 2 diabetic patients”. asian journal of immunology 8 (1):249–256. https://doi.org/10.9734/aji/2025/v8i1176. asian journal of immunology volume 8, issue 1, page 249-256, 2025; article no.aji.146139 role of tumour necrosis factor-alpha and adiponectin in insulin therapy failure in type 2 diabetic patients ayissi marie-ange a, biwole sida ghyslaine b, guiedem elise b,c, yayah emerencia ngah d,e, jacky bikio b, emilia lyonga b,f, martha messembe b, mispa yivala mbanyamsig g and georges ikomey a,b,f* a catholic university of central africa yaounde, cameroon. b faculty of medicine and biomedical sciences, university of yaoundé 1, cameroon. c siantou higher school of health sciences, siantou university institute, cameroon. d department of public health, faculty of health science, texila american university, zambia. e regional hospital bamenda, cameroon. f cameroon centre for the study and control of communicable diseases (csccd), fmbs, cameroon. g university of skövde, sweden. authors’ contributions this work was carried out in collaboration among all authors. authors am-a and gi were responsible for development of research concept, recruitment of patients, laboratory analyses, interpretation of results and participated in the initial drafting of the manuscript. authors bsg, ge, yen were responsible for development of research concept, interpretation of results and drafting of manuscript. authors mm, el, jb, and mym contributed to the laboratory analyses and drafting of the manuscript. all authors read and approved the final manuscript. article information doi: https://doi.org/10.9734/aji/2025/v8i1176 open peer review history: this journal follows the advanced open peer review policy. identity of the reviewers, editor(s) and additional reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here: https://pr.sdiarticle5.com/review-history/146139 received: 18/08/2025 published: 03/11/2025 short research article https://doi.org/10.9734/aji/2025/v8i1176 https://pr.sdiarticle5.com/review-history/146139 marie-ange et al.; asian j. immunol., vol. 8, no. 1, pp. 249-256, 2025; article no.aji.146139 250 abstract background: tumour necrosis factor alpha (tnf-α) and adiponectin are both pro and antiinflammatory cytokines that play major roles in the pathogenesis and therapeutic management of many metabolic diseases. aim: this study aimed at evaluating the role of tnf-α and adiponectin amongst insulin-treatment failure and insulin therapy success in type 2 diabetic mellitus subjects using enzyme-linked immunosorbent assay (elisa). methodology: the study was conducted at the diabetic unit of the cité verte hospital, yaounde, cameroon, between february 2023 and march 2024. a case-control study was conducted with 80 enrolled participants (41 insulin-treated patients with poor glycemic control and 39 patients with good glycemic control on insulin. the tnf-α, adiponectin and glycated haemoglobin were measured on whole blood specimens using immunoassay techniques. the graph pad prism 5.0 and epi info 7.1. software was used for the analysis of data. results: tnf-α and glycated haemoglobin (hbaic) mean levels were higher in insulin-treated patients with poor glycemic control (tnf-α: 19.4 ± 8, hbaic: 35.26 ± 11pg/ml) compared to patients with good glycemic control (tnf-α: 17.1 ± 6 pg/ml, hbaic: 21.5 ± 5 pg/ml) with p < 0.001. the adiponectin mean levels were lower in subjects with treatment failure (5.62 ± 4 pg/ml) compared to patients with treatment success (6.33 ± 6 pg/ml), was not statistically significant, p=0.0818. a negative correlation was observed between tnf-α and adiponectin in subjects with treatment failure, with r=-0.03, which was not statistically significant, p=-0.08. conclusion: the variation of tnf-alpha and adiponectin in uncontrolled patients on insulin therapy and well-controlled patients could justify their implications in the pathogenesis of type 2 diabetes. the increased levels of tnf-α, hbaic, combined with the low levels of adiponectin, could be exploited as signalisation biomarkers for monitoring insulin treatment failure and other metabolic dysregulation and inflammation. keywords: type 2 diabetes; pro-inflammatory cytokines; insulin-treatment failure; insulin-treatment success; adiponectin. 1. introduction "diabetes mellitus (dm) is a disease characterised by the disturbance of glucose homeostasis owing to insulin malfunction in the target tissues, causing abnormalities in fat, protein, and carbohydrate metabolism. the principal hallmark of the disease is an elevated glucose level in the venous plasma, which is the gold standard for the diagnosis of dm. the elevation of plasma glucose level is caused by absolute insulin deficiency, as in type 1 diabetes (t1dm), or increased insulin resistance, as in t2dm or both" (emad-eldin et al., 2024). "the management of insulin-treatment failure (itf) in type 2 diabetes mellitus (t2dm) represents a major public health challenge worldwide. despite regular insulin injections, many patients experience persistent hyperglycemia, frequent hypoor hyperglycemic episodes, and unexplained weight fluctuations. one of the key mechanisms underlying these complications is chronic low-grade inflammation, which interferes with glucose metabolism and pancreatic β-cell function" (beutler & cerami, 1989; hotamisligil et al., 1993; hotamisligil, 2000). "cytokines play a central role in this process. pro-inflammatory cytokines such as tumour necrosis factor-alpha (tnf-α), interleukin-1β (il-1β), and interleukin-6 (il-6) are consistently elevated in t2dm patients" (chen et al., 2015; bastard et al., 2002; choi, 2016). "tnf-α, in particular, induces insulin resistance by disrupting insulin signalling pathways in peripheral tissues and promoting βcell apoptosis through oxidative stress and endoplasmic reticulum dysfunction" (lee & lee, 2014; wensveen et al., 2015; shamsi et al., 2018). "adiponectin, is referred to as adipoq, apm1, or acrp30, is composed of 244 amino acids, with a molecular weight of around 26 kilodaltons (kda) secreted by white adipose tissue" (yu et al., 2022). "these proteins are encoded by the adipoq gene and are located on the chromosome locus 3q27. they are secreted by adipose tissue, exerts protective effects by enhancing insulin sensitivity, stimulating lipid oxidation, and providing cardiovascular protection" (sajid et al., 2018; swaroop et al., 2012; jeon et al., 2018). "adiponectin exists in various isoforms— low, medium, and high marie-ange et al.; asian j. immunol., vol. 8, no. 1, pp. 249-256, 2025; article no.aji.146139 251 molecular weight—which exert their effects via receptors adipor1, adipor2, and t-cadherin, differentially expressed across tissues and immune cells. these distinct isoforms and receptor interactions underscore the complexity of adiponectin signalling and its broad spectrum of physiological effects" (gianoli et al., 2025; patel et al., 2021). "however, reduced circulating levels of adiponectin are frequently observed in patients with obesity and t2dm, which contributes to the development of insulin resistance" (liu et al., 2016; matthews et al., 1985). "they cross-talk between adipocytes and immune cells, particularly macrophages, further aggravate this inflammatory state, creating a cycle of metabolic inflammation that fuels insulin resistance" (van dielen et al., 2004; yudkin et al., 2000; sartipy & loskutoff, 2003). glycated haemoglobin (hba1c) is a measurement used for glycaemic control. the hba1c was introduced into clinical use in the 1980s and subsequently has become a major guide for clinical practice. recent studies suggest that "the balance between pro-inflammatory cytokines (e.g., tnfα) and anti-inflammatory adipokines (e.g., adiponectin) may serve as predictive markers for insulin therapy outcomes in t2dm" (morris et al., 2006; samad et al., 1997; swaroop et al., 2012). the goal of this study was to evaluate the role of tnf-α and adiponectin amongst insulintreatment failure (itf) and success (its) t2dm subjects attending the endocrinology unit of the clinical setting in a sub-saharan african population hospital, cameroon. 2. materials and methods 2.1 study design the study was a case-control study with 80 participants enrolled between february 2023 and march 2024. all participants were consecutively recruited from the diabetic unit of the cité verte hospital during daily routine consultation. 2.2 study population the study population was made up of 80 diabetic patients divided into two groups: a group of patients with insulin-treatment failure (itf) consisting of 41 subjects as the case group and a group of 39 people with insulin-treatment success (its) as the control group. the glycated haemoglobin (hbaic) levels were considered for the selection of participants in the study. specimens were aliquoted and stored at −80°c until ready to be used. demographic data and clinical information were gathered for each participant through a standard questionnaire, which covered parameters like age, gender, insulin regimen, and duration of insulin treatment. 2.3 sample collection and analysis site five millilitres (5ml) of whole blood were collected, transported and analysed at the centre for the study and control of communicable diseases (csccd) of the faculty of medicine and biomedical sciences (fmbs), university of yaoundé i, cameroon. serum specimens were obtained by centrifuging whole blood at 5000 rpm for 5 minutes and subsequently stored in cryovals at -20°c. all samples were analysed within two months of collection to prevent cytokine degradation over time. 2.4 laboratory measurements 2.4.1 measurement of tnf-α and adiponectin tnf-α and adiponectin were measured using quantitative sandwich enzyme-linked immunosorbent assay (elisa) kits from invitrogen (thermo fisher scientific, usa). a solid-phase immune enzymatic technique was employed on a microtiter plate for the quantitative determination of tnf-α and adiponectin in human plasma. samples were analysed according to the manufacturer's specifications. the absorbance was read using a spectrophotometer (biotech elx800, usa) set at a dual wavelength of 450-550 nm, with each sample run in duplicate. the concentration of both cytokines was determined by extrapolating the results from a standard curve generated by plotting the average absorbance (450-550 nm) obtained. 2.4.2 measurement of glycated haemoglobin (hba1c) using immune turbidimetry glycated haemoglobin was measured by immunoturbidimetry using antibodies specific to hba1c.the immunoturbidimetric instrument measured the changes in the sample. all the procedures as recommended by the manufacturer were respected. 2.5 statistical analyses data were analysed using the graph pad prism 5.0 software package (graph pad software inc., marie-ange et al.; asian j. immunol., vol. 8, no. 1, pp. 249-256, 2025; article no.aji.146139 252 la jolla, california, usa) and epi info 7.0 software (epi info™). comparisons between tnf-α and adiponectin within the various groups were conducted using the non-parametric student's t-test. the correlations between tnfα and adiponectin were determined using pearson’s correlation coefficient. a significance level of p < 0.05 was considered statistically significant. 3. results and discussion 3.1 patient characteristics we enrolled 80 diabetic patients, of whom 65 % (n= 52) were females and 35 % (n= 28) males. their mean age (± sd) was 56.89 ± 11.08 years. patients were classified into two groups: insulin treatment failure 51 %, n= 41, and insulin treatment success 49 %, (n= 39). 3.2 levels of tnf-α the tnf-alpha levels were higher in itf subjects compared to its subjects, with a mean ± sd of 0.1418±5 pg/ml, and -3.135±4 pg/ml, with p ≤0.001. the levels of glycated haemoglobin were higher in itf compared to its, with a mean of 9.4 ±8 % and 6.37±6 %. the tnf-alpha levels were higher in itf subjects compared to its subjects, with a mean ± sd of -0.1418±5 pg/ml, and -3.135±4 pg/ml, with p ≤0.001. the levels of adiponectin were lower in itf subjects compared to its subjects, with a mean ±sd of 5.62±4 pg/ml and 6.332±6 pg/ml, with p=0.081. 3.3 levels of adiponectin adiponectin concentrations varied between 1.60 and 8.97 pg/ml in itf subjects, and from 2.252 to 10.51 pg/ml in its subjects (fig. 2). this study revealed relatively lower levels of adiponectin in insulin-treated subjects than in our controls, but with a statistically non-significant difference (p=0.0818). the levels of glycated haemoglobin were higher in itf compared to its, with a mean of 9.4 ± 8 % and 6.37±6 % respectively. fig. 1. the dot plot illustrates the plasma levels of tnf-α in insulin treatment failure and success groups table 1. the plasma levels of tnf-α and adp in insulin treatment failure and success groups variable adp tnf itf (median) n=41 its (median) n= 39 p itf (median) n=41 its (median) n=39 p sexe female 5.94 (2.1-7.01) 6.50 (2.25-9.08) 0.79 -1.06 (-4.8 – (-5.31)) -4.83 (-6.17 3.15) 0.24 male 5.90 (1.608.48) 6.99 (2.5-10.5) 0.46 -0.73 (-4.175.60) -4.51 (-5.95 3.38) 0.32 age [20-40] 5.873 (3.5-7.49) / / -0.8406 (-3.5-5.37) / / [41-60] 5.86 (1.60-8.48) 6.36 (2.93-10.5) 0.65 -2.06 (-4.84 5.6) -5.17 (-6.18 3.38) 0.33 [61-80] 5.69 (3.7-8.97) 6.99 (2.25-9.08) 0.57 0.71(-3.51 4.49) -5.06 (-5.95 2.49) 0.27 marie-ange et al.; asian j. immunol., vol. 8, no. 1, pp. 249-256, 2025; article no.aji.146139 253 fig. 2. the dot plot illustrates the plasma levels of adp in insulin treatment failure and success groups fig. 3. association between tnf-α and adp in the general population study 3.4 correlation between tnf-alpha and adiponectin there was a negative correlation between tnfalpha and adiponectin in itf subjects, with r=0.03 but non statistically significant p=-0.08. it is understood that within the two populations, for concentrations of tnf-alpha that evolve in an increasing way, we have concentrations of adiponectin, which evolve in a decreasing our results corroborate those of chenxiao liu et al. (2016), who found after a meta-analysis that the overall risk of t2dm is strongly associated with high levels of inflammatory cytokines, including tnf-alpha, and low levels of adiponectin showing that an interaction between inflammation and the development of t2 diabetes in subjects. 4. discussion the results obtained show that the levels of tnfalpha are significantly higher in itf subjects, compared to its. among other things, our results showed a significant difference between the two populations (p≤0.001); with concentrations varying between -4.838 and 5.600 pg/ml in itf subjects and -6.170 to 3.379 pg/ml of blood in its subjects. marie-ange et al.; asian j. immunol., vol. 8, no. 1, pp. 249-256, 2025; article no.aji.146139 254 this is in line with the results of several scientific articles, which have also reported elevated levels of tnf-alpha in insulin-treated diabetics. according to the study by shamsi et al. (2018), "tnf-alpha is associated with insulin resistance in these subjects". in addition, in the study conducted by sajid et al. (2018) on "the role of tnf-alpha in the development of insulin resistance and the pathogenesis of t2dm, high levels of tnf-alpha have been shown to induce insulin resistance, which impairs insulin, contributing to the development of t2dm". swaroop et al (2012) found "a positive but also significant correlation between tnf-alpha and homa ir (homeostasis assessment method to calculate insulin resistance) in t2dm subjects, suggesting an involvement of tnf-alpha in the development of insulin resistance. it is understood that within the two populations, for concentrations of tnf-alpha that evolve in an increasing way, we have concentrations of adiponectin, which evolve in a decreasing way". our results corroborate those of chenxiao liu et al. (2016), who found after "a meta-analysis that the overall risk of t2dm is strongly associated with high levels of inflammatory cytokines, including tnf-alpha, and low levels of adiponectin, showing that an interaction between inflammation and the development of t2dm". the results of this study also support the findings of jeon et al. (2018), who evaluated "the level of adiponectin in subjects with t2dm and healthy subjects, reporting a low concentration of adiponectin in subjects with type 2 diabetes". the analyses also showed that there was an inverse (r = -0.03) and non-significant (p = 0.82) correlation between tnf-alpha and adiponectin itf subjects. similarly, there is an inverse correlation between tnf-alpha and adiponectin in non-insulin-treated subjects (r= -0.08), but this does not remain very significant (p= 0.59). 5. conclusion the increased levels of tnf-alpha, hbaic, combined with the low levels of adiponectin, could be exploited as s ignal isat ion biomarkers for monitoring insulin treatment failure and other metabolic dysregulation and inflammation. the variation of tnf-alpha and adiponectin in the serum of treatment failure and non-failure diabetic patients could justify their implications in the pathogenesis of type 2 diabetes and be a good predictive marker for its management. consent all authors declare that written informed consent was obtained from the patients (or other approved parties) for publication of this study. ethical approval all authors hereby declare that the study was conducted in accordance with ethical standards approved by the cameroon national ethics committee, n° 2023/0220409/ceirsh/ess/mim, and have therefore been performed in accordance with the ethical standards laid down in the 1964 declaration of helsinki. disclaimer (artificial intelligence) author(s) hereby declare that no generative ai technologies such as large language models (chatgpt, copilot, etc) and text-to-image generators have been used during writing or editing of this manuscript. acknowledgement the authors thank the staff of the cite vert hospital and the centre for the study and control of communicable diseases, faculty of medicine and biomedical sciences, university of yaounde, cameroon. competing interests authors have declared that they have no known competing financial interests or non-financial interests or personal relationships that could have appeared to influence the work reported in this paper. references bastard, j. p., maachi, m., van nhieu, j. t., jardel, c., bruckert, e., grimaldi, a., et al. 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(2000). the influence of improved marie-ange et al.; asian j. immunol., vol. 8, no. 1, pp. 249-256, 2025; article no.aji.146139 256 glycaemic control with insulin and sulphonylureas on acute phase and endothelial markers in type ii diabetic subjects. diabetologia, 43, 1099–1106. disclaimer/publisher’s note: the statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of the publisher and/or the editor(s). this publisher and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. _________________________________________________________________________________ © copyright (2025): author(s). the licensee is the journal publisher. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: https://pr.sdiarticle5.com/review-history/146139 https://pr.sdiarticle5.com/review-history/146139 _____________________________________________________________________________________________________ *corresponding author: e-mail: arirejay@gmail.com; cite as: islam ariremako jimoh, tobechi brendan nnanna, taiwo micheal olumuji, paul onyekachi ukegbu, michael sewanu hungbo, and kanime hussaini. 2025. “the gut–net–icb axis: microbial metabolite-mediated regulation of netosis and implications for cancer immunotherapy”. asian journal of immunology 8 (1):257–268. https://doi.org/10.9734/aji/2025/v8i1177. asian journal of immunology volume 8, issue 1, page 257-268, 2025; article no.aji.147982 the gut–net–icb axis: microbial metabolite-mediated regulation of netosis and implications for cancer immunotherapy islam ariremako jimoh a*, tobechi brendan nnanna b, taiwo micheal olumuji c, paul onyekachi ukegbu d, michael sewanu hungbo e and kanime hussaini f a department of microbiology, university of ilorin, kwara state, nigeria. b aston pharmacy school, college of life and health sciences, aston university, birmingham b4 7et, uk. c department of agricultural extension and communication technology, federal university of technology akure, nigeria. d department of pharmacology and toxicology, faculty of pharmacy, madonna university, elele campus, elele, rivers state, nigeria. e department of animal breeding and genetics, federal university of agriculture, abeokuta, nigeria. f department of veterinary medicine, university of maiduguri, nigeria. authors’ contributions this work was carried out in collaboration among all authors. all authors read and approved the final manuscript. article information doi: https://doi.org/10.9734/aji/2025/v8i1177 open peer review history: this journal follows the advanced open peer review policy. identity of the reviewers, editor(s) and additional reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here: https://pr.sdiarticle5.com/review-history/147982 received: 06/09/2025 published: 20/11/2025 review article https://doi.org/10.9734/aji/2025/v8i1177 https://pr.sdiarticle5.com/review-history/147982 jimoh et al.; asian j. immunol., vol. 8, no. 1, pp. 257-268, 2025; article no.aji.147982 258 abstract background: immune checkpoint blockade (icb) has transformed oncology, but its efficacy is limited by primary and acquired resistance. the gut microbiome is a established determinant of icb response, while neutrophil extracellular traps (nets) have recently emerged as key mediators of an immunosuppressive tumor microenvironment (tme) and icb resistance. the mechanistic link between these two phenomena remains elusive. we hypothesize that gut microbial metabolites serve as critical systemic messengers that modulate net formation (netosis), thereby influencing icb outcomes. methods & findings: through a synthesis of recent literature, we delineate a novel "gut-net-icb" axis. we provide evidence that specific microbiota-derived metabolites directly regulate netosis. short-chain fatty acids (scfas) like butyrate and polyamines such as spermidine suppress net formation by inhibiting histone deacetylases (hdacs), reactive oxygen species (ros) production, and peptidyl arginine deiminase 4 (pad4) activity. conversely, certain secondary bile acids can promote netosis. we propose a model wherein a favorable gut microbiome generates a metabolite profile that systemically suppresses pathological netosis, thereby remodeling the tme to enhance cd8+ t-cell infiltration and function, creating a state permissive to icb. conversely, dysbiosis fosters a pro-netotic environment that drives resistance. conclusion: the modulation of netosis by gut microbial metabolites represents a crucial mechanism underlying the microbiome's impact on cancer immunotherapy. this mechanistic insight positions the gut microbiome and netosis as complementary therapeutic targets. strategies to promote a netosis-suppressive metabolite profile through dietary interventions, pre/probiotics, or postbiotic supplements or to directly inhibit netosis (e.g., with pad4 inhibitors) hold significant promise for overcoming icb resistance and improving patient outcomes. keywords: gut microbiome; microbial metabolites; neutrophil extracellular traps (nets); netosis; immune checkpoint blockade; cancer immunotherapy; tumor microenvironment. 1. introduction the advent of immune checkpoint blockade (icb), which targets regulatory pathways such as pd-1/pd-l1 and ctla-4 to reinvigorate antitumor immunity, represents a paradigm shift in oncology (ribas and wolchok, 2018). despite producing remarkable and durable responses in a subset of patients, icb therapy is hampered by the fact that a significant majority of individuals experience primary or acquired resistance (mariniello et al., 2025). this stark variability in treatment outcomes has spurred intensive research into the factors that govern icb efficacy, leading to the seminal discovery that the gut microbiome is a critical determinant of therapeutic success. compelling evidence from both preclinical models and clinical studies has established a causal relationship between the composition of the gut microbiota and response to icb. fecal microbiota transplantation (fmt) from icb responders can convert non-responders into responders, demonstrating the microbiome's potent role in modulating the anti-tumor immune response (routy et al., 2018, davar et al., 2021). concurrently, the role of innate immune players in shaping the tumor microenvironment (tme) has gained prominence. neutrophils, and specifically their ability to release neutrophil extracellular traps (nets), have been implicated in cancer progression and therapy resistance. nets are web-like structures composed of decondensed chromatin decorated with histones and cytotoxic granule proteins that are extruded by neutrophils in a process called netosis (liu et al., 2025). while physiologically a host defense mechanism, aberrant net formation in the tme promotes metastasis, angiogenesis, and, crucially, immunosuppression by physically impeding t-cell infiltration and directly suppressing t-cell cytotoxic function (ayodele, 2025, teijeira et al., 2020). recent work has directly correlated elevated levels of nets with resistance to icb in both murine models and cancer patients (yang et al., 2020). a crucial, yet under-explored, link connecting these two fields is the functional output of the gut microbiome: its dynamic metabolome. gut bacteria metabolize dietary and host-derived substrates into a vast array of small molecules, including short-chain fatty acids (scfas), secondary bile acids, polyamines, and tryptophan metabolites that can enter systemic jimoh et al.; asian j. immunol., vol. 8, no. 1, pp. 257-268, 2025; article no.aji.147982 259 fig. 1. the human gut (zhang et al., 2025) circulation and directly influence the function of immune cells, both within and outside the gut (kaltenmeier et al., 2021, parada venegas et al., 2019, ajibola and ayodele, 2025, jia et al., ‘). we hypothesize that these microbial metabolites serve as systemic rheostats for neutrophil activity and netosis. this review aims to synthesize emerging evidence and provide a comprehensive mechanistic framework for the "gut-net-icb" axis. we will explore how specific gut microbial metabolites directly and indirectly modulate the molecular pathways of netosis. furthermore, we will propose an integrated model wherein a favorable microbiome, through its metabolite profile, suppresses pathological net formation, thereby remodeling the tme to be more permissive to t-cellmediated killing and enhancing icb efficacy. elucidating this axis not only deepens our fundamental understanding of immunotherapy resistance but also unveils novel therapeutic strategies to modulate the immune system for improved cancer treatment. 2. gut microbial metabolites: key mediators of host immunity the gut microbiome exerts a profound influence on host physiology not merely through its presence but through its immense metabolic activity. it functions as a virtual endocrine organ, converting dietary components and host-derived substances into a diverse array of small molecules that serve as key communicators with the host's immune system (ayodele, 2025). these metabolites can mediate their effects locally within the gastrointestinal tract or, upon absorption into the portal circulation, systemically throughout the body, thereby influencing immune responses in distant sites, including the tumor microenvironment (tme) (jia et al., 2018). jimoh et al.; asian j. immunol., vol. 8, no. 1, pp. 257-268, 2025; article no.aji.147982 260 table 1. key classes of microbiota-derived metabolites and their immunomodulatory mechanisms metabolite class key examples bacterial origin / precursor primary immunologic mechanisms impact on immunity short-chain fatty acids (scfas) acetate, propionate, butyrate fermentation of dietary fiber by genera like faecalibacterium, roseburia, lachnospiraceae (mager et al., 2020) roseburia, lachnospiraceae (mager et al., 2020). hdac inhibition (mager et al., 2020) signaling via gpcrs (gpr41, gpr43, gpr109a) (ayodele et al., 2025) energy source for colonocytes & immune cells. generally anti-inflammatory: promote treg differentiation, maintain gut barrier integrity, modulate neutrophil chemotaxis and function. secondary bile acids deoxycholic acid (dca), lithocholic acid (lca), ursodeoxycholic acid (udca) bacterial dehydroxylation of primary bile acids (cholic acid, chenodeoxycholic acid) by species like clostridium cluster xiva (parada venegas et al., 2019). signaling via nuclear receptor fxr & membrane receptor tgr5 (ma et al., 2025). context-dependent: can be proinflammatory (dca) or antiinflammatory (udca). regulate metabolic homeostasis and macrophage differentiation. polyamines spermidine, spermine, putrescine decarboxylation of amino acids (e.g., arginine, ornithine) by bacteria like bifidobacterium and lactobacillus (zhang et al., 2025). regulation of autophagy, translation (via hypusination) (qi et al., 2025) anti-oxidant effects modulation of ion channels. immunomodulatory: support t-cell differentiation and function, suppress pro-inflammatory cytokine production, can inhibit netosis. tryptophan catabolites indole, indole-3-aldehyde, indolepropionic acid (ipa) tryptophan metabolism by bacteria (e.g., lactobacillus spp.) expressing tryptophanase (tofalo et al., 2019). activation of the aryl hydrocarbon receptor (ahr) (puleston et al., 2019). barrier fortification & immune regulation: ahr activation strengthens epithelial barriers, modulates th17/treg balance, and suppresses innate immune activation. other bioactive metabolites inosine, d-lactate, trimethylamine n-oxide (tmao) purine metabolism (inosine), fermentation (d-lactate), choline/carnitine metabolism (tmao). adenosine a2a receptor signaling (inosine) (kullberg et al., 2025) precursor to pro-atherogenic tmao. variable: inosine enhances th1 differentiation and icb response; tmao is generally pro-inflammatory and linked to cardiovascular disease. jimoh et al.; asian j. immunol., vol. 8, no. 1, pp. 257-268, 2025; article no.aji.147982 261 2.1 short-chain fatty acids (scfas): microbial fermentation products with systemic reach scfas are the most extensively studied microbial metabolites. butyrate is a primary energy source for colonocytes, crucially maintaining gut barrier function and preventing systemic inflammation. beyond this local role, scfas function as potent epigenetic regulators via hdac inhibition, leading to increased histone acetylation and altered gene expression in immune cells (ajibola and ayodele, 2025, mager et al., 2020). this mechanism promotes the differentiation of regulatory t cells (tregs), which are critical for maintaining immune tolerance. scfa signaling through gpcrs like gpr43 on neutrophils can also dampen their inflammatory potential and chemotaxis, representing a direct pathway for systemic immunomodulation relevant to netosis (ayodele et al., 2025). 2.2 secondary bile acids (bas): from digestion to immunomodulation primary bile acids, synthesized in the liver, are transformed into secondary bile acids by gut bacteria. this process significantly alters their signaling properties. secondary bas act as signaling molecules through the nuclear receptor fxr and the g-protein-coupled receptor tgr5, regulating not only metabolic homeostasis but also immune cell function (ma et al., 2025). for instance, specific secondary bas can promote the differentiation of anti-inflammatory macrophages, while others, in excess, can be cytotoxic and induce inflammatory stress responses. 2.3 polyamines: crucial for cellular and immune homeostasis polyamines are essential for cell proliferation and are produced by both mammalian cells and gut bacteria. bacterial-derived polyamines can influence host immunity by modulating autophagy, a process critical for immune cell function and antigen presentation (qi et al., 2025). spermidine, in particular, has been shown to have anti-inflammatory and life-spanextending effects, partly through its ability to induce autophagy and suppress oxidative stress. 2.4 tryptophan metabolites: ahr ligands and barrier regulators gut bacteria that metabolize the essential amino acid tryptophan into ligands for the ahr play a vital role in immune education and barrier integrity. ahr activation in intestinal epithelial cells and intraepithelial lymphocytes strengthens the mucosal barrier and helps maintain a balanced immune response, preventing aberrant inflammation (puleston et al., 2019). the systemic immunologic impact of these metabolites is an area of active investigation. the gut microbial metabolome constitutes a critical interface between the external environment and the host immune system. the balance and composition of these metabolites directly influence the functional state of innate and adaptive immune cells, creating a systemic milieu that can either favor or suppress antitumor immunity. the subsequent sections will delve into how these specific metabolites interact with the process of netosis to shape this immunologic landscape (puleston et al., 2019). 3. mechanistic interplay: microbial metabolites as regulators of netosis the central hypothesis of the gut-net-icb axis posits that gut microbial metabolites systemically influence the propensity of neutrophils to undergo netosis, thereby shaping the immune landscape of the tumor microenvironment (tme). this interaction is not merely correlative; a growing body of evidence demonstrates direct and indirect mechanistic pathways through which these bacterial byproducts modulate the molecular machinery of net formation. the balance between netosis-suppressive and netosis-promoting metabolites can critically determine the outcome of anti-tumor immunity (ma et al., 2025). 3.1 suppressive metabolites: scfas and polyamines as netosis brakes short-chain fatty acids (scfas): butyrate has been demonstrated to robustly inhibit netosis induced by various stimuli, including pma and lps, in human neutrophils in vitro (zhang et al., 2025). the primary mechanism is epigenetic: as potent hdac inhibitors, scfas lead to hyperacetylation of histones. this increased negative charge makes the chromatin less accessible to pad4, thereby preventing the histone citrullination that is essential for chromatin decondensation. furthermore, scfa signaling through gpr43 on the neutrophil surface directly suppresses phorbol esterinduced ros production, dismantling a critical trigger for the netotic cascade (cai et al., 2025). jimoh et al.; asian j. immunol., vol. 8, no. 1, pp. 257-268, 2025; article no.aji.147982 262 table 2. mechanisms of gut microbial metabolites in regulating netosis metabolite class net effect on netosis direct molecular mechanisms indirect immunological mechanisms short-chain fatty acids (scfas) potently suppressive hdac inhibition: hyperacetylation of histones and other proteins interferes with pad4 activity and chromatin decondensation (zhang et al., 2025). gpcr (gpr43) signaling: downregulates nadph oxidase assembly and ros production, a key trigger for suicidal netosis (cai et al., 2025). inhibition of nf-κb pathway: reduces transcription of proinflammatory cytokines that prime neutrophils. promotes differentiation of anti-inflammatory tregs, reducing the cytokine drive for netosis. enhances gut barrier integrity, reducing systemic translocation of lps, a potent netosis inducer. polyamines potently suppressive direct inhibition of pad4: competitively binds to the enzyme's active site, preventing histone citrullination (perrone and d'angelo, 2025). suppression of ros: inhibits the assembly and function of the nadph oxidase complex. induction of autophagy: autophagic flux can clear damaged mitochondria (mitophagy), preventing mtros-induced netosis (saha and goswami, 2025). modulates macrophage polarization towards an m2-like, anti-inflammatory phenotype. supports t-cell function, creating a cytokine milieu less conducive to persistent neutrophil activation. tryptophan metabolites (ahr ligands) suppressive ahr activation: ahr signaling in neutrophils directly upregulates antiapoptotic genes and downregulates pro-inflammatory pathways, raising the activation threshold for netosis (zhao et al., 2025). ahr activation in dendritic cells and t cells promotes immune tolerance and a balanced cytokine response. strengthening of the gut barrier reduces systemic inflammatory tone. secondary bile acids context-dependent (often promotive in dysbiosis mitochondrial stress: high concentrations of dca act as detergents, disrupting mitochondrial membranes and inducing mitochondrial ros (mtros), which drives netosis (zhao et al., 2025). nlrp3 inflammasome activation: can prime and activate the inflammasome in immune cells, leading to il-1β release, a potent netosis trigger. certain bas can have anti-inflammatory effects. an imbalanced ratio of proto antiinflammatory bas creates a systemic environment that favors neutrophil activation. jimoh et al.; asian j. immunol., vol. 8, no. 1, pp. 257-268, 2025; article no.aji.147982 263 polyamines: spermidine emerges as a powerful, direct inhibitor of netosis. it exerts its effect by targeting two key nodes in the process. first, it directly inhibits pad4 enzymatic activity, preventing the initiation of chromatin decondensation (perrone and d'angelo, 2025). second, it suppresses the generation of ros by the nadph oxidase complex. additionally, its well-established role as an inducer of autophagy (saha and goswami, 2025, lu et al., 2024) helps maintain cellular homeostasis in neutrophils by clearing damaged organelles that could otherwise serve as intrinsic inducers of netosis. 3.2 inductive metabolites: the detrimental role of dysbiosis secondary bile acids: in a state of dysbiosis, an overabundance of certain secondary bile acids like deoxycholic acid (dca) can have the opposite effect. at high concentrations, dca can disrupt mitochondrial membranes in neutrophils, leading to the leakage of mitochondrial dna and the generation of mitochondrial ros (mtros). this mtros can act as a potent trigger for netosis, independent of or synergistic with the nadph oxidase pathway (saha and goswami, 2025). this highlights that the impact of microbial metabolites is not universally beneficial and is entirely dependent on the compositional and metabolic balance of the gut community. 3.3 indirect modulation via the tumor microenvironment beyond direct actions on neutrophils, microbial metabolites sculpt the broader tme, creating feedback loops that influence netosis. for instance, scfas and inosine promote the activation and tumor infiltration of cd8+ t cells and th1 cells (zhao et al., 2025). these activated t cells produce ifn-γ, which can have complex effects on neutrophils but can also contribute to an anti-tumor immune cycle that suppresses the pro-tumorigenic functions of neutrophils, including excessive net formation. conversely, a metabolite profile that favors treg expansion and m2 macrophage polarization can create an immunosuppressive environment that indirectly permits sustained netosis. gut microbial metabolites function as master regulators of netosis through a multi-pronged strategy involving direct epigenetic and enzymatic interference, receptor-mediated signaling, and systemic modulation of inflammatory tone. a favorable microbiome generates a metabolite profile that acts as a "brake" on pathological netosis, while a dysbiotic microbiome often releases the "accelerator," contributing to an icb-resistant tme (saha and goswami, 2025, korbecki et al., 2025). 4. the gut-net-icb axis: an integrated model we propose a comprehensive model wherein the gut microbiome dictates clinical response to immune checkpoint blockade (icb) through the systemic regulation of neutrophil extracellular trap (net) formation, with microbial metabolites serving as the primary signaling intermediaries. this "gut-net-icb axis" provides a mechanistic framework to explain how the intestinal microbiota can influence anti-tumor immunity in distant tissues. the model pivots on the balance between two contrasting states: a netosissuppressive, icb-permissive state and a netosis-promotive, icb-resistant state. the following table outlines the contrasting characteristics of these two states within our integrated model. 4.1 the icb-permissive cycle: a favorable microbiome suppresses nets in this virtuous cycle, a diverse and balanced gut microbiome, characterized by taxa such as akkermansia muciniphila, faecalibacterium prausnitzii, and bifidobacterium spp., generates a metabolite profile rich in scfas, polyamines, and ahr ligands. metabolite production: these microbes ferment dietary fiber to produce high levels of butyrate and other scfas. they also generate immunomodulatory polyamines like spermidine and tryptophan-derived ahr ligands. systemic signaling: these metabolites are absorbed into the portal circulation and exert systemic effects. they reach the bone marrow and the tme, where they encounter neutrophils. netosis suppression: as detailed in section 4, these metabolites directly "raise the threshold" for netosis. butyrate (via hdaci) and spermidine (via pad4 inhibition) disrupt the core biochemical pathways required for chromatin decondensation and net release. jimoh et al.; asian j. immunol., vol. 8, no. 1, pp. 257-268, 2025; article no.aji.147982 264 tme remodeling: the consequent low level of nets in the tme dismantles a major immunosuppressive barrier. cd8+ t cells can effectively infiltrate the tumor parenchyma, encounter their antigen, and execute cytotoxic functions without being suppressed by netderived components like histones and proteases. effective icb response: in this permissive environment, the administration of icb can effectively reinvigorate these functional, tumorinfiltrating t cells, leading to robust tumor control and clinical response (zhao et al., 2016, mager et al., 2020, xi et al., 2022). 4.2 the icb-resistant cycle: dysbiosis promotes net-driven immunosuppression conversely, a state of dysbiosis, potentially driven by antibiotics, a western diet, or disease, initiates a vicious cycle that culminates in icb resistance. dysbiosis and metabolite imbalance: the gut community is depleted of beneficial metabolite producers. this leads to low systemic levels of scfas and spermidine. concurrently, there may be an expansion of bacteria that produce proinflammatory metabolites or, through a weakened gut barrier, allow the translocation of microbial products like lipopolysaccharide (lps) (ajibola and ayodele, 2025). loss of netosis suppression: the absence of the "netosis brake" (scfas, spermidine) leaves neutrophils hypersensitive to activation signals. furthermore, circulating lps and certain secondary bile acids provide direct triggers for netosis. net-driven immunosuppressive tme: neutrophils in the tme readily undergo netosis, leading to high net density. these nets physically trap t cells and prevent their infiltration (deng et al., 2025). more critically, net components have been shown to directly promote t cell exhaustion by upregulating pd-1 expression and suppressing their proliferative and cytotoxic capacity (cai et al., 2025). icb failure: in this scenario, the tme is characterized by excluded and profoundly exhausted t cells. even when pd-1 is blocked by icb, the t cells are too dysfunctional or physically sequestered to mount an effective anti-tumor response, resulting in primary therapeutic resistance. table 3. the two states of the gut-net-icb axis feature netosis-suppressive, icbpermissive state netosis-promotive, icb-resistant state gut microbiome profile "favorable" / eubiotic. enriched with scfa-producers polyamine-producers, and ahrligand generators. "unfavorable" / dysbiotic. enriched with pathobionts, reduced microbial diversity, and bacteria producing pro-inflammatory metabolites. systemic metabolite profile high circulating levels of butyrate, propionate, spermidine, and indoles. balanced secondary bile acid ratio. low levels of beneficial metabolites. high levels of pro-inflammatory secondary bile acids. impact on neutrophils & netosis suppressed pathological netosis. metabolites directly inhibit pad4, reduce ros, and block hdacs. neutrophils are less prone to excessive net release. promoted pathological netosis. lack of suppression and presence of inflammatory inducers lead to rampant net formation. tumor microenvironment (tme) low net density enhanced t-cell infiltration: physical barrier is removed. functional t-cells: reduced tcell exhaustion and impairment. favorable cd8+/treg ratio. high net density impaired t-cell infiltration: nets form a physical barrier. t-cell exhaustion: net components directly suppress t-cell function. immunosuppressive milieu. final icb outcome responsive. checkpoint inhibitors effectively reinvigorate pre-existing, functional, and infiltrated t cells, leading to tumor cell killing. refractory. t cells are excluded, dysfunctional, and exhausted; icb fails to initiate an effective anti-tumor response. jimoh et al.; asian j. immunol., vol. 8, no. 1, pp. 257-268, 2025; article no.aji.147982 265 fig. 2. acquired resistance to immune checkpoint blockades (bi et al., 2025) 4.3 therapeutic implications of the axis this model directly suggests novel therapeutic strategies aimed at interrupting the resistant cycle and promoting the permissive one. these interventions can target different nodes of the axis: targeting the gut: using prebiotics, probiotics, or fmt to establish a netosis-suppressive microbiome. targeting the metabolite: using postbiotic supplements to directly deliver the inhibitory signals. targeting netosis: using pharmacological pad4 inhibitors (e.g., gsk484) or dnase i to directly prevent net formation or degrade existing nets, thereby breaking the immunosuppressive barrier and potentially synergizing with icb (bi et al., 2025, zhang et al., 2025). the gut-net-icb axis provides a powerful, mechanistic model that integrates the fields of microbiome research, neutrophil biology, and cancer immunotherapy (zhou et al., 2021). it explains how a systemic factor of the gut microbiome can locally determine treatment efficacy by controlling a specific innate immune process, netosis, that directly shapes the adaptive anti-tumor response (cai et al., 2025, pirini et al., 2025). 5. conclusion the intricate interplay between the gut microbiome, host immunity, and cancer therapy continues to redefine our understanding of oncobiology. this review has synthesized compelling evidence to establish a novel and critical pathway: the gut-net-icb axis. we have delineated a mechanistic framework wherein gut microbial metabolites serve as pivotal systemic regulators of neutrophil extracellular trap (net) formation, which in turn exerts a profound influence on the efficacy of immune checkpoint blockade (icb). the model posits two divergent states. a favorable gut microbiome, characterized by producers of short-chain fatty acids (scfas), polyamines, and ahr ligands, generates a metabolite profile that systemically suppresses pathological netosis. this creates a tumor microenvironment (tme) that is permissive to tcell infiltration and function, thereby enabling a robust response to icb. conversely, a state of dysbiosis fails to provide these suppressive signals and may even promote netosis, leading to a net-rich, immunosuppressive tme that fosters t-cell exclusion and exhaustion, ultimately resulting in icb resistance. this paradigm shift from viewing the microbiome as a simple correlate to understanding it as an active modulator of specific innate immune effector mechanisms like netosis has significant implications. it moves the field beyond taxonomic associations and towards a functional, metabolite-driven understanding of therapy response. the axis provides a unifying explanation for how a distal community of microbes can precisely tune the immune landscape of a distant tumor. looking forward, this knowledge opens up a promising frontier for clinical translation. the gut jimoh et al.; asian j. immunol., vol. 8, no. 1, pp. 257-268, 2025; article no.aji.147982 266 net-icb axis is not merely a explanatory model; it is a roadmap for therapeutic innovation. strategies targeting the gut microbiome, its metabolic output (postbiotics), or the downstream netotic process itself (pad4 inhibitors, dnase) represent tangible, combinatorial approaches to overcome immunotherapy resistance. future research must focus on validating these mechanistic links in human cohorts, identifying predictive metabolite and net-based biomarkers, and launching clinical trials that strategically manipulate this axis. by harnessing the power of the gut microbiome to disarm a key mechanism of immunosuppression, we can ultimately expand the reach and success of cancer immunotherapy for a greater number of patients. 5.1 future perspectives the elucidated gut-net-icb axis provides a powerful, mechanistic framework that connects the gut microbiome's metabolic output to a specific innate immune process, netosis, which critically determines the efficacy of adaptive immunotherapy. this model not only deepens our fundamental understanding of icb resistance but also unveils a suite of novel, actionable strategies to improve clinical outcomes. based on the evidence synthesized in this review, we put forth the following recommendations for future research and clinical translation: 1. prioritize functional metabolomics in clinical cohorts: future clinical studies correlating the microbiome with icb response must move beyond 16s rrna sequencing and integrate deep metagenomics with metabolomic profiling of patient serum and feces. the primary goal should be to define a "netosis-suppressive metabolite signature" that can serve as a superior predictive biomarker compared to taxonomic data alone. 2. establish causality in gnotobiotic models: to move from correlation to causation, welldefined gnotobiotic mouse models should be employed. colonizing germ-free mice with consortia of bacteria engineered to produce specific metabolites (e.g., scfa-producers versus non-producers) and subjecting them to icb and netosis assays will provide direct proof of the metabolite-net link. 3. explore combinatorial clinical trials: the most immediate translational path involves designing clinical trials that combine icb with interventions targeting the gut-net axis. we recommend prioritizing: icb + pre/postbiotics: testing high-fiber diets or scfa/spermidine supplements in conjunction with anti-pd-1 therapy. icb + netosis inhibition: investigating the safety and efficacy of pad4 inhibitors or dnase i in overcoming resistance in selected cancer types. 4. develop standardized net biomarkers: the field requires a concerted effort to standardize the measurement of nets in human samples. validated assays for circulating net remnants are needed to correlate systemic net load with metabolite levels and icb response in patient cohorts. 5. personalize microbiome-modulating therapies: recognizing that a "one-size-fits-all" microbiome intervention may be ineffective, we recommend developing diagnostic platforms to match patients with dysbiotic, netosis-promoting profiles to tailored interventional strategies, such as fmt from donors with a validated beneficial metabolome. disclaimer (artificial intelligence) author(s) hereby declare that no generative ai technologies such as large language models (chatgpt, copilot, etc) and text-to-image generators have been used during writing or editing of this manuscript. consent and ethical approval it is not applicable. competing interests authors have declared that no competing interests exist. references ajibola, f. o., & ayodele, a. e. 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(2021). acquired resistance to immune checkpoint blockades: the underlying mechanisms and potential strategies. frontiers in immunology, 12, 693609. https://doi.org/10.3389/fimmu.2021.693609 disclaimer/publisher’s note: the statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of the publisher and/or the editor(s). this publisher and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. _________________________________________________________________________________ © copyright (2025): author(s). the licensee is the journal publisher. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: https://pr.sdiarticle5.com/review-history/147982 https://pr.sdiarticle5.com/review-history/147982 _____________________________________________________________________________________________________ *corresponding author: e-mail: jehiaghe@biu.edu.ng; cite as: f.a., ehiaghe, nworah, c.m., ehiaghe, j.i., osakue, o.n., olisah, m.c., ize-iyamu, j., amengialue, o.o., and ogoegbunam, n.d. 2025. “serum levels of tnfα, il-10 and albumin in female patients with post-surgical wound infection in asaba delta state, nigeria”. asian journal of immunology 8 (1):134-41. https://doi.org/10.9734/aji/2025/v8i1167. asian journal of immunology volume 8, issue 1, page 134-141, 2025; article no.aji.137461 serum levels of tnfα, il-10 and albumin in female patients with postsurgical wound infection in asaba delta state, nigeria ehiaghe, f.a. a, nworah, c.m. b, ehiaghe, j.i. c*, osakue, o.n. b, olisah, m.c. d, ize-iyamu, j. e, amengialue, o.o. f and ogoegbunam, n.d. a a department of immunology and immunochemistry, nnamdi azikiwe university, anambra state, nigeria. b department of clinical chemistry, nnamdi azikiwe university, anambra state, nigeria. c department of medical laboratory science, faculty of allied health sciences, benson idahosa university, benin city, nigeria. d department of medical biochemistry, faculty of basic medical sciences, chukwuemeka odimegwu ojukwu university, uli, nigeria. e edo state health management board, benin city, edo state, nigeria. f department of medical microbiology, wellspring university, benin city, edo state, nigeria. authors’ contributions this work was carried out in collaboration among all authors. all authors read and approved the final manuscript. article information doi: https://doi.org/10.9734/aji/2025/v8i1167 open peer review history: this journal follows the advanced open peer review policy. identity of the reviewers, editor(s) and additional reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here: https://pr.sdiarticle5.com/review-history/137461 received: 03/04/2025 published: 11/06/2025 original research article https://doi.org/10.9734/aji/2025/v8i1167 https://pr.sdiarticle5.com/review-history/137461 ehiaghe et al.; asian j. immunol., vol. 8, no. 1, pp. 134-141, 2025; article no.aji.137461 135 abstract post-operative wound infections (powis), sometimes referred to as post-surgical wound infections (pswis), are a common complication following surgery. surgeries frequently elicit a variety of immunological responses subsequent to tissue damage and incision which may hinder wound healing and infection resolution leading to poor patient outcome. this cross-sectional study assessed the serum levels of tnf-α, il-10 and albumin in female patients with post-surgical wound infection in asaba delta state, nigeria. twenty-five (25) patients with pswis and 25 apparently healthy individuals (control group) were recruited by random sampling method. four millilitres (4 ml) of venous blood samples were collected for the determination of serum tnf-α, il-10 and albumin levels. tnf-α and il-10 levels were estimated with a sandwich enzyme-linked immunosorbent assay, whereas serum albumin was assessed with the bromocresol green (bcg) method. the data obtained was analyzed using an independent t-test and pearson correlation, with a p-value of <0.05 indicating statistical significance. the mean serum tnf-α (143.44 ±7.35 vs 37.32 ±8.92; p = 0.001) and il-10 (27.52 ±7.77 vs 17.12 ±6.23; p = 0.001) levels were significantly higher while serum albumin level was significantly lower (34.14 ±5.43 vs 40.19 ±3.38; p = 0.001) in the female patients with pswis than in the control group. however, no significant correlation was observed between the levels of tnf-α vs albumin and il-10 vs albumin (p>0.05). the elevated levels of proinflammatory cytokine tnf-α may lead to adverse patient outcomes by hindering wound healing and slowing infection resolution. keywords: powis; tnf-α; il-10; albumin; patients. 1. introduction post-operative wound infections (powis), sometimes referred to as postsurgical wound infections (pswis), are a common complication following surgery, characterized by complex and multifaceted pathophysiology (zabaglo et al., 2025). post-operative wound infection (powis) is still a leading cause of morbidity and death for patients having surgery, even with improvements in surgical techniques and a better understanding of the pathophysiology of wound infection (kallakuri et al., 2024). the prevalence of powis is still rising in many nations, communities, and hospitals, albeit it varies greatly (ehiaghe et al., 2025b; kallakuri et al., 2024). surgeries frequently elicit a variety of immunological responses subsequent to tissue damage and incision (weledji, 2021). this include the secretion of cytokines, stress responses, and acute phase proteins, which can be detrimental when excessive (chidambaran et al., 2024; hsing and wang, 2015). cytokines represent a viable therapeutic target, as they regulate all phases of wound healing, and abnormalities in cytokine production can lead to inflammatory non-healing wounds (wong et al., 2025). cytokines are soluble molecular entities synthesized by inflammatory cells. they can be broadly classified as either pro-inflammatory cytokines (e.g., interleukins (il)-1, 2, 8, 12; tumor necrosis factor α (tnf-α), interferon γ (ifnγ), etc.), or anti-inflammatory cytokines e.g., transforming growth factor beta (tgfβ), il-4, il10, il-13 while others, like il-6, can be both (chidambaran et al., 2024). tnf-α attracts pro-inflammatory cells including neutrophils, macrophages, and fibroblasts to the wound site, where they assist remove debris and pathogens (wong et al., 2025; jang et al., 2021), thereby facilitating wound healing or resolution under physiological state. however, exaggerated tnf-α responses can culminate in failed wound infection resolution (choy et al., 2023; jang et al., 2021). interleukin 10 (il-10) is an important antiinflammatory cytokine produced mainly by the cd4+ t cells although other leukocytes such as neutrophils, b cells, dendritic cells, natural killer cells, and macrophages also secret it as well (sun et al., 2020). its primary functions include anti-inflammatory, inhibitory, or self-regulating, as il-10 appears to be a potent negative feedback regulator that influences inflammation management and resolution (al-qahtani et al., 2024; steen et al., 2020). by suppressing the activity of macrophages and dendritic cells, il-10 directly shuts down the inflammatory immune response (cocea and stoica, 2024; saraiva et al., 2020; mittal et al., 2015). it also indirectly does so by restricting t cell activation, differentiation, and effector function and encouraging peripheral tolerance (saraiva et al., ehiaghe et al.; asian j. immunol., vol. 8, no. 1, pp. 134-141, 2025; article no.aji.137461 136 2020; mittal et al., 2015). excessive il-10 production or signaling can suppress host's effective inflammatory responses, induce tolerance and immune escape, and favor microbial persistence, which can lead to the establishment of chronic or latent infections (carlini et al., 2023; rojas et al., 2017; iyer and cheng, 2012). in contrast, during acute infections, il-10 limits the magnitude of the immune responses, preventing excessive inflammation and protecting tissues from immune-mediated damage by allowing inflammation resolution when the pathogen is cleared (carlini et al., 2023; rojas et al., 2017). nutritional deficiency has been associated with an increased risk of wound infections (tfaily et al., 2022) and serum albumin level can be used to determine an individual’s nutritional and inflammatory condition (keller, 2019). hypoalbuminemia has been identified as one of the risk factors for surgical site infections (ssis) in surgery populations (mostafa et al., 2024; ryan et al., 2018). hypoalbuminemia, on the other hand, can increase the expression of tnfɑ, c-reactive protein (crp) and several interleukins, all known to be acute-phase proteins that promote tissue damage (cooper et al., 2004; yildirim et al., 2004). albumin influences wound healing by increasing the transcription of tissue-forming proteins, which in turn triggers the nfkb pathway and speeds up the healing process (utariani et al., 2020). identifying risk factors for surgical site infections early can improve post-operative outcomes, minimize re-admission rates (he et al., 2019; fong et al., 2014), and reduce treatment costs by 23.8% (javed et al., 2019). in view of all these, it has become pertinent to evaluate the serum levels of tnf-α, il-10 and albumin circulating among female patients with post-surgical wound infection in asaba delta state, nigeria. 2. materials and methods 2.1 study site the study was conducted at the federal medical center in asaba, delta state, nigeria. 2.2 study design this cross-sectional study examined the serum levels of tumor necrosis factor-alpha, interleukin10, and albumin in female patients with postsurgical wound infection in asaba, nigeria. 2.3 study population simple random sampling was used to select 50 participants for the study. the test group consisted of 25 female patients with post-surgical wound infection, while the control group included 25 apparently healthy participants. 2.4 sample size and sample size calculation the sample size (n) was calculated using prevalence from previous studies on the prevalence of post-surgical wound infections globally which was found to be 2.5% (mengistu et al., 2023) according to the sample size calculation formula described by daniel and cross (1999). 𝑁 = 𝑍2𝑃(1 – 𝑃) 𝐷2 n = required sample size z = confidence level at 95% (standard value of 1.96) p = estimated prevalence of post-surgical wound infections globally which was found to be 2.5% (mengistu et al., 2023). d = margin of error at 5% (standard value = 0.05) 𝑁 = 1.9620.025(1 − 0.025) 0.052 3.8416 𝑋 0.025(0.975) 0.0025 n = 37 minimum sample size therefore, a minimum of 50 participants was used for this study. 2.5 inclusion criteria female patients with confirmed cases of postsurgical wound infection as well as healthy female participants who provided informed consent were included for this study. 2.6 exclusion criteria female patients with post-surgical wound infection and apparently healthy female individuals without post-surgical wound infection ehiaghe et al.; asian j. immunol., vol. 8, no. 1, pp. 134-141, 2025; article no.aji.137461 137 who did not give their informed consent were excluded from this study. 2.7 sample collection each participant provided five milliliters (5 ml) of venous blood into a plain container for cytokine and albumin analysis. blood samples were allowed to clot before being retracted and centrifuged for 10 minutes at 4000 rpm. serum samples were separated and kept frozen at 20°c until analysis. 2.8 estimation of cytokine levels the levels of tumor necrosis factor-alpha and interleukin-10 were determined using the sandwich enzyme-linked immunosorbent assay technique. 2.9 estimation of serum albumin this was determined using the bromocresol green method (bcg), as described by doumas and watson (1971) and cited by okpogba et al. (2021). 2.10 statistical analysis the data was analyzed using spss 20.0 and represented as mean ± standard deviation. the independent t-test and pearson correlation were employed to determine the statistical difference and association between variables. a p value of < 0.05 was considered statistically significant. 3. results the mean serum tnf-α level was significantly higher in the female patients with post-surgical wound infections than in the control group (143.44 ±7.35 vs 37.32 ±8.92; p = 0.001). in like manner, the mean serum il-10 level was significantly higher in the female patients with post-surgical wound infections compared to the control group (27.52 ±7.77 vs 17.12 ±6.23; p = 0.001). however, there was a statistically significant decrease in the mean serum albumin level observed in the female patients with postsurgical wound infections than in the control group (34.14 ±5.43 vs 40.19 ±3.38; p = 0.001), (see table 1). furthermore, there was no significant correlation observed between the levels tnf-α and albumin (r-value = -0.154; p = 0.470) as well as between il-10 and albumin levels (r-value = 0.271; p = 0.190) in the study participants (see table 2). 4. discussion the present study noted a significantly higher mean serum tnf-α level in the female patients with post-surgical wound infections than in the control group. elevated tnf-α levels indicate a pro-inflammatory response linked to chronic illnesses and infections (chen et al., 2017). this finding aligns with the reports of other similar studies (ehiaghe et al., 2025a; ehiaghe et al., 2025b). exaggerated tnf-α responses may culminate in failed wound infection resolution (choy et al., 2023). furthermore, the higher il-10 level observed in the female patients with post-surgical wound infections show an attempt by the body to moderate the excessive inflammatory responses, a phenomenon commonly observed in chronic inflammation, where anti-inflammatory cytokines act to avoid tissue damage. il-10 limits the magnitude of the immune responses, preventing excessive inflammation and protecting tissues from immune-mediated damage by allowing inflammation resolution when the pathogen is cleared (carlini et al., 2023). table 1. levels of serum tnf-α, il-10 and albumin circulating among female patients with post-surgical wound infection and apparently healthy individuals (mean ±sd) parameter group n mean ±sd t value p value tnf-α il-10 albumin test control test control test control 25 25 25 25 25 25 143.44 ±7.35 37.32 ±8.92 27.52 ±7.77 17.12 ±6.23 34.14 ±5.43 40.19 ±3.38 45.93 5.22 4.73 0.001 0.001 0.001 *statistically significant = p<0.05; tnf-α = tumour necrosis factoralpha, il-10 = interleukin -10. ehiaghe et al.; asian j. immunol., vol. 8, no. 1, pp. 134-141, 2025; article no.aji.137461 138 table 2. the correlation between the levels of tnf-α, il-10 and albumin in the study population parameters r-value p-value tnfα vs albumin -0.154 0.470 il-10 vs albumin 0.271 0.190 *statistically significant at p<0.05 nevertheless, elevating il-10 levels can provide some anti-inflammatory buffering, but its efficiency may be limited by pro-inflammatory cytokines like tnf-α, leading to an unsustainable immunological balance. thus, increased inflammatory response shown in our current investigation may hamper infection resolution, eventually leading to poor treatment outcome in these patients. in the present study, the mean serum albumin level was found to be significantly lower in the female patients with post-surgical wound infection compared to the control group. this result agrees with several studies that found significantly lower serum albumin levels in pswis patients (choudhary et al., 2024; sultana et al., 2024; llombart et al., 2023; singh et al., 2023; he et al., 2020). hypoalbuminemia, an indicator of malnutrition and illness, has been shown to increase the likelihood of poor postoperative outcomes, especially reduced wound healing (sultana et al., 2024). in addition to increasing albumin production, inflammatory conditions can enhance albumin breakdown, which can result in hypoalbuminemia (weaving et al., 2016). interstitial oedema results from a decrease in oncotic pressure caused by a decrease in serum albumin levels, which might impede the healing and recovery process. 5. conclusion this study found that female patients with postsurgical wound infections had significantly higher mean serum tnf-α and il-10 levels, as well as lower mean serum albumin levels. findings presented in the study suggest that higher levels of the pro-inflammatory cytokine tnf 𝛼, may adversely impact wound healing and impede resolution of infection. consent and ethical approval the ethical committee of the federal medical centre in asaba, delta state, approved the study before it commenced. the informed consent of all involved participants was sought and gained. disclaimer (artificial intelligence) author(s) hereby declare that no generative ai technologies such as large language models (chatgpt, copilot, etc.) and text-to-image generators have been used during the writing or editing of this manuscript. competing interests authors have declared that no competing interests exist. references al-qahtani, a.a., alhamlan, f.s., & al-qahtani, a.a. 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(2025). postoperative wound infections. in: statpearls treasure island (fl): statpearls publishing; 2025 jan-. available from: https://www.ncbi.nlm.nih.gov/books/nbk56 0533/ disclaimer/publisher’s note: the statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of the publisher and/or the editor(s). this publisher and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. _________________________________________________________________________________ © copyright (2025): author(s). the licensee is the journal publisher. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: https://pr.sdiarticle5.com/review-history/137461 https://doi.org/10.1038/s41598-022-21849-2 https://doi.org/10.1038/s41598-022-21849-2 https://doi.org/10.1155/2020/3254017 https://doi.org/10.37349/ei.2025.1003183 https://www.ncbi.nlm.nih.gov/books/nbk560533/ https://www.ncbi.nlm.nih.gov/books/nbk560533/ https://pr.sdiarticle5.com/review-history/137461 _____________________________________________________________________________________________________ *corresponding author: e-mail: meriche.h23@gmail.com; cite as: hacene meriche, sara zaghez, feriel sandra bouafia, boutheina abdaoui, nacima sabiha gadiri, and hichem frigaa. 2025. “symptomatic multiple myeloma in the second trimester of pregnancy: a case report”. asian journal of immunology 8 (1):222–229. https://doi.org/10.9734/aji/2025/v8i1173. asian journal of immunology volume 8, issue 1, page 222-229, 2025; article no.aji.141940 symptomatic multiple myeloma in the second trimester of pregnancy: a case report hacene meriche a*, sara zaghez a, feriel sandra bouafia a, boutheina abdaoui a, nacima sabiha gadiri a and hichem frigaa b a department of immunology, clinic sainte therese uhc, annaba, algeria. b department of nephrology, hospital ibn sina, uhc, annaba, algeria. authors’ contributions this work was carried out in collaboration among all authors. all authors read and approved the final manuscript. article information doi: https://doi.org/10.9734/aji/2025/v8i1173 open peer review history: this journal follows the advanced open peer review policy. identity of the reviewers, editor(s) and additional reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here: https://pr.sdiarticle5.com/review-history/141940 received: 10/07/2025 published: 15/09/2025 abstract multiple myeloma (mm) in pregnant women is a rare disease characterized by clonal proliferation of plasma cells producing monoclonal immunoglobulin, which suppresses the normal production of other antibodies and weakens the maternal immune response. plasma cells release inflammatory cytokines (il-6, tnf-α, il-1β) that promote their survival and bone destruction. during pregnancy, maternal immunity shifts towards complex tolerance with decreased th1 inflammatory responses, th2 predominance, and increased regulatory t cells, which often masks mm and complicates its management. treatment is generally deferred until after delivery to protect the fetus, with the vtd protocol then used to control the disease by reducing plasma cell proliferation and inflammation. case report https://doi.org/10.9734/aji/2025/v8i1173 https://pr.sdiarticle5.com/review-history/141940 meriche et al.; asian j. immunol., vol. 8, no. 1, pp. 222-229, 2025; article no.aji.141940 223 this case illustrates the importance of simple tests such as serum protein electrophoresis in diagnosing unexplained anemia during pregnancy, and highlights the need for immunological monitoring and a multidisciplinary approach to optimize maternal and fetal prognosis. keywords: multiple myeloma; pregnancy; anemia; monoclonal composant; electrophoresis. 1. introduction multiple myeloma is a malignant clonal proliferation of plasma cells characterized by excessive monoclonal production of immunoglobulins, mainly igg. the median age at diagnosis is generally around 63 to 70 years, with cases in young people, and particularly during pregnancy, remaining extremely rare, with fewer than 50 cases described in the global literature since 1965 (durand, 2024; hila et al. 2022). myeloma in pregnancy is a rare occurrence as the median age of myeloma diagnosis is around the sixth and seventh decade of life and is more commonly observed in males. the first case of myeloma in pregnancy was reported in 1965, and since then there have been fewer than fifty cases reported worldwide. although only three percent of cases are diagnosed below the age of forty, myeloma is now increasingly being diagnosed in younger ages and during pregnancy due to better screening and awareness (szydełko, 2019). gestational anemia is common, but in the presence of a monoclonal peak, further investigation is necessary to rule out an underlying blood disorder and ensure appropriate management. however, diagnosis is complicated by the overlap with the physiological symptoms of pregnancy. therefore, particular vigilance is required. 2. case presentation a 35-year-old patient from annaba (algeria), 24 weeks pregnant with her second child, with no notable family history. she complains of persistent fatigue accompanied by pale skin. clinical examination revealed marked pallor, a uterine fundus consistent with 24 weeks, a normal fetal heart rate (140 bpm), and a high body mass index of 28 kg/m². blood pressure was stable at 120/80 mmhg. 2.1 biological and hematological tests blood count reveals moderate anemia (hb 10 g/dl, hematocrit 30%, mcv 75 fl), associated with low ferritin (22 ng/ml) indicative of probable iron deficiency. blood smear shows anisocytosis, polychromatophilia, presence of erythroblasts, neutrophilic hyperleukocytosis with coarse granulations, and moderate thrombocytopenia. the sedimentation rate was accelerated (78 mm/h). serum protein electrophoresis (sebia) revealed a monoclonal igg kappa peak of 42 g/l, confirmed by immunofixation. serum free light chain assay (sebia) showed a high κ/λ ratio (3.86). bence jones proteinuria was positive. in addition, mild renal failure is observed with a slight increase in urea (9.8 mmol/l), creatinine (165 µmol/l), and proteinuria at 650 mg/24h. moderate hypercalcemia (2.85 mmol/l) and a liver profile consistent with pregnancy-related cholestasis complete the picture (figs. 1, 2, 3). 2.2 morphological and radiological examinations the bone marrow aspiration revealed significant plasma cell infiltration estimated at 65%, which was confirmed as clonal by immunophenotyping (cd38+, cd138+, cd56+) using flow cytometry (bd facs lyric). a low-dose whole-body ct scan detected a 6 mm osteolytic lesion in the pelvis, typical of bone involvement associated with multiple myeloma. abdominal and obstetric ultrasounds showed a normal fetus with no abnormalities or growth retardation, and measurements were within normal limits. these examinations, which limit fetal exposure to radiation, are essential for assessing the extent of the disease while ensuring safe, risk-free maternal-fetal monitoring. 2.3 diagnosis the diagnosis of symptomatic multiple myeloma in the patient was based on the combination of a significant monoclonal igg kappa peak associated with a high κ/λ ratio, with positive bence jones proteinuria indicating significant renal involvement, as well as high myeloid plasma cell infiltration (65%) with characteristic immunophenotyping (cd38+, cd138+, cd56+). meriche et al.; asian j. immunol., vol. 8, no. 1, pp. 222-229, 2025; article no.aji.141940 224 fig. 1. the electrophoretic profile of our patient's serum proteins, performed on agarose gel, shows a monoclonal band located in the gamma globulin zone at position 21. this observation confirms the presence of a monoclonal immunoglobulin, attesting to the clonal nature of the detected protein fig. 2. the electrophoretic pattern of the protein profile at position 21 shows a monoclonal peak in the gamma globulin zone, with an estimated concentration of 40 g/l, accompanied by a decrease (suppression) in other classes of polyclonal immunoglobulins meriche et al.; asian j. immunol., vol. 8, no. 1, pp. 222-229, 2025; article no.aji.141940 225 clinically, the presence of anemia, renal failure, hypercalcemia, and osteolytic bone lesions meets the crab criteria of the imwg (international myeloma working group), justifying a diagnosis of myeloma in the active phase. the iss (international staging system )classification of stage ii reflects a high tumor burden and a guarded prognosis. this complex diagnosis requires appropriate management, which is even more delicate in the case of pregnancy. 2.4 management and follow-up due to the high teratogenic risk, chemotherapy is postponed during pregnancy, especially during the first and second trimesters, with regular monthly clinical and biological monitoring based on blood counts, renal function tests, calcemia measurements, protein electrophoresis (fig. 4a/b), and weekly ultrasounds to ensure maternal-fetal stability and a complication-free pregnancy. after delivery, treatment with the vtd protocol (treatment using bortezomib (velcade), lenalidomide (revlimid), and dexamethasone), which is effective and well tolerated, leading to complete remission. this success highlights the importance of appropriate and deferred treatment, combined with multidisciplinary post-treatment follow-up (hematologist, gynecologist, nephrologist, immunologist) to detect any relapses or side effects and ensure optimal long-term care. fig. 3. immunofixation results from our case show that the monoclonal peak corresponds to an igg kappa immunoglobulin, with a decrease in other types of polyclonal immunoglobulins 4 a. meriche et al.; asian j. immunol., vol. 8, no. 1, pp. 222-229, 2025; article no.aji.141940 226 4b. fig. 4 a/b. regression of the monoclonal peak after postpartum treatment with the vtd protocol in our patient with gestational myeloma 3. discussion multiple myeloma (mm) is a malignant blood disorder characterized by clonal proliferation of plasma cells in the bone marrow, associated with excessive monoclonal production of immunoglobulins (kyle & rajkumar, 2004). the igg kappa form identified in our patient corresponds to the most frequently observed type in the general population, which is consistent with the epidemiological and biological data reported by palumbo and anderson (2011) and corroborated by kyle and rajkumar (2004) ; palumbo & anderson, 2011 ; johns hopkins university school of medicine, 2024). immunoparesis, resulting from the suppression of polyclonal immunoglobulins, is a mechanism well described in the literature, promoting significant immunosuppression and vulnerability to opportunistic infections (rajkumar, 1999 ; la revue du praticien, 2023). this immunosuppression was highlighted by rajkumar (2011), who emphasized its prognostic effect and therapeutic implications, in line with the clinical observations made in our patient. epidemiologically, mm primarily affects older populations, with less than 3% of cases diagnosed before the age of 40, which is linked to female fertility (steinbach, 2024 ; abdellah et al., 2020). our observation is thus consistent with the rare descriptions of mm in the context of pregnancy, as confirmed by the reviews by kumar et al. (2003) and lee et al. (2016), which emphasize the rarity and complexity of management in this specific situation (szydełko, 2019 ; pajor, 1991). the clinical presentation of mm during pregnancy is often atypical. anemia, which is very common during pregnancy, particularly due to nutritional deficiencies, is a common factor that can lead to delayed diagnosis of serious conditions such as mm. this is clearly illustrated in our observation by anemia that is refractory to iron treatment, a situation also highlighted by smith et al. (2012), who recommend thorough investigation of persistent unexplained anemia in pregnant women (mor & cardenas, 2010). serum protein electrophoresis and free light chain assay, as indicated in our diagnosis, are now considered key tests for the early detection of subtle monoclonal peaks and imbalances in the κ/λ ratio ratio imbalances (terpos & dimopoulos, 2005). these investigations are systematically recommended in the literature and their importance is unanimously recognized, particularly in the reviews by gertz (2008) and terpos & dimopoulos (2005), confirming the relevance of our diagnostic approach (faze, 2019 ; borja, meriche et al.; asian j. immunol., vol. 8, no. 1, pp. 222-229, 2025; article no.aji.141940 227 2011). pregnancy alters the immune system, inducing maternal-fetal tolerance by reducing cellular and humoral immune responses (palumbo et al., 2020). this modulation can attenuate the classic symptoms of mm (fatigue, recurrent infections, bone pain), explaining the late or incidental discovery of cases during pregnancy—a point that our observation corroborates in perfect agreement with the work of mor & cardenas (2010) and lee et al. (2016) (dispenzieri et al., 2019 ; pajor, 1991). from an immunopathological perspective, il-6 plays a major role in the proliferation of malignant plasma cells and in the development of bone lesions through the activation of osteoclasts (rajkumar, 2020). as observed in our patient, this mechanism is responsible for the common disabling bone complications of mm, suggesting the need for increased vigilance, particularly in the context of pregnancy, where osteoarticular symptoms can be confused with physiological pain associated with pregnancy (terpos & dimopoulos, 2005). this issue is discussed at length by terpos and dimopoulos (2005) and terpos et al. (2020) (rajkumar, 2020 ; elgabry et al., 2023). the therapeutic management of mm during pregnancy remains a major challenge. the literature, including the study by lee et al. (2018), recommends a cautious approach, generally deferring active treatment until after delivery to avoid the teratogenic effects of conventional chemotherapy, particularly in the first trimester (rajkumar, 2011). this strategy, with close multidisciplinary follow-up, corresponds exactly to the management protocol we applied, reflecting perfect alignment with current best practices (rajkumar, 2020; smith, 2021). the standard postpartum treatment for symptomatic multiple myeloma is the combined vtd protocol (bortezomib, thalidomide, dexamethasone), recognized for its efficacy and good tolerance, validated by several studies, including that of palumbo et al. (2020). new therapeutic options, such as monoclonal antibodies and newer proteasome inhibitors, are currently being evaluated, although data in pregnant women remain limited, as highlighted by dispenzieri et al. (2019). the protocol used in this case is consistent with current recommendations and advances in the treatment of multiple myeloma. finally, the importance of early diagnosis in cases of persistent anemia in pregnant women, as highlighted in our case, is shared by many authors (terpos,2020a, 2020b). increased awareness among clinicians to include specific immunological tests is necessary. 4. conclusion symptomatic multiple myeloma during pregnancy represents an exceptionally rare and challenging clinical condition. its diagnosis is often delayed because the presenting symptoms (fatigue, bone pain, anemia) are frequently misinterpreted as physiological changes of pregnancy.moreover, the maternal immune alterations associated with feto-maternal tolerance further complicate both the clinical assessment and the interpretation of laboratory findings. nevertheless, certain warning signs should prompt further investigation: a persistent, unexplained anemia refractory to iron supplementation, when associated with the detection of a monoclonal spike on serum protein electrophoresis, strongly suggests an underlying plasma cell dyscrasia. in such cases, a comprehensive immunological work-up (including immunofixation, quantitative immunoglobulins, and free light chain assay) is mandatory for the biological detection of multiple myeloma. from a therapeutic perspective, postponing chemotherapy until the postpartum period is generally recommended in order to minimize teratogenic risks to the fetus. during pregnancy, strict multidisciplinary monitoring involving hematologists, obstetricians, neonatologists, and anesthesiologists is required to maintain maternal and fetal stability. after delivery, treatment with the vtd protocol (bortezomib, thalidomide, dexamethasone) has proven to be both effective and well tolerated, leading to complete remission and ensuring a favorable prognosis. finally, the management of multiple myeloma during pregnancy requires a personalized and multidisciplinary approach, carefully balancing maternal safety, fetal protection, and the optimization of therapeutic outcomes in the postpartum period. consent as per international standards or university standards, patient(s) written consent has been collected and preserved by the author(s). ethical approval as per international standards or university standards written ethical approval has been collected and preserved by the author(s). meriche et al.; asian j. immunol., vol. 8, no. 1, pp. 222-229, 2025; article no.aji.141940 228 disclaimer (artificial intelligence) author(s) hereby declare that generative ai technologies such as large language models, etc. have been used during the writing or editing of manuscripts. this explanation will include the name, version, model, and source of the generative ai technology and as well as all input prompts provided to the generative ai technology details of the ai usage are given below: 1. language and style enhancement 2. aide à la traduction 3. assistance in idea generation acknowledgements we would like to thank the nephrology, immunology, and obstetrics and gynecology teams for their valuable collaboration. competing interests authors have declared that no competing interests exist. references abdellah, n., et al. (2020). implications of myc rearrangements in newly diagnosed multiple myeloma. clinical cancer research, 26(24), 6581–6588. borja, d. (2011). multiple myeloma and pregnancy: a case report and literature review. archives of gynecology and obstetrics, 284(4), 945–950. https://doi.org/10.1007/s00404-011-1985-8 dispenzieri, a., et al. (2019). emerging therapies in multiple myeloma. nature reviews clinical oncology, 16(12), 695–710. durand, r et al.(2024). combined inhibition of ctps1and atr is metabolic vulnerability in p53 deficient myeloma cells. hemasphere ,oct.8;8(10):e70016. elgabry, g., spencer, l., siddiqi, h., ojha, s., & wandroo, f. (2023). a report of a symptomatic progressive myeloma during pregnancy and postpartum period from asymptomatic state. hematology reports, 15(2), 305–311. faze, f. (2019). an approach to the diagnosis and management of multiple myeloma. samj: south african medical journal, 109(10). gertz, m. a. (2008). new targets and treatments in multiple myeloma: src family kinases as central regulators of disease progression. leukemia & lymphoma, 49(12), 22402245. hila, m. et al. (2022). diagnosis and management of multiple myeloma during pregnancy: case report, review of literature and update on current treatments.ther adv hematol. jan 21:13/20406207211066173. johns hopkins university school of medicine, msd manuals professional. (2024). myélome multiple. kumar, s. (2003) response rate, durability of response and survivalafter thalidomide therapy for relapsed multiple myeloma. mayo clinic proceedings 78(1)34-39. kyle, r. a., & rajkumar, s. v. (2004). multiple myeloma. new england journal of medicine, 350(3), 282–292. la revue du praticien. (2023). myélome multiple: aspects cliniques et thérapeutiques. lee, g. w., park, s. w., go, s. i., kim, h. g., kim, m. k., min, c. k., ... & kim, k. (2018). the derived neutrophil-to-lymphocyte ratio is an independent prognostic factor in transplantation ineligible patients with multiple myeloma. acta haematologica, 140(3), 146-156. mor, g., & cardenas, i. (2010). the immune system in pregnancy: a unique complexity. american journal of reproductive immunology, 63(6), 425–433. pajor, a. (1991). multiple myeloma in pregnancy. international journal of gynaecology and obstetrics, 35(4), 341–342. https://doi.org/10.1016/00207292(91)90669-v palumbo, a., & anderson, k. (2011). multiple myeloma. new england journal of medicine, 364(11), 1046–1060. palumbo, a., et al. (2020). treatment of newly diagnosed multiple myeloma: updated recommendations. journal of clinical oncology, 38(15), 1702–1714. rajkumar, s. v. (1999). prognostic factors in multiple myeloma. hematology/oncology clinics of north america, 13(6), 1295–131. rajkumar, s.v. (2011). multiple myeloma : 2011update on diagnosis, risk stratificationand management am j hematol . jan;86(1):57-65. rajkumar, s. v. (2020). multiple myeloma: 2020 update on diagnosis, risk-stratification and management. american journal of meriche et al.; asian j. immunol., vol. 8, no. 1, pp. 222-229, 2025; article no.aji.141940 229 hematology, 95(5), 548–567. https://doi.org/10.1002/ajh.25791 smith m. et al. (2021). early detection of myeloma.innovait volume 14 issue 7. steinbach, m. (2024). multiple myeloma in young patients: a scoping review. clinical lymphoma, myeloma & leukemia, 24(1), 15–22. https://doi.org/10.1016/j.clml.2023.08.019 szydełko, m. (2019). multiple myeloma during pregnancy as a challenge in clinical practice – a review. journal of education, health and sport, 9(8), 920– 937. terpos, e., & dimopoulos, m. a. (2005). myeloma bone disease: pathophysiology and management. annals of oncology, 16(2), 122–126. terpos, e., engelhardt, m., cook, g., gay, f., mateos, m. v., ntanasis-stathopoulos, i., ... & sonneveld, p. (2020). management of patients with multiple myeloma in the era of covid-19 pandemic: a consensus paper from the european myeloma network (emn). leukemia, 34(8), 20002011. terpos, e., & ntanasis-stathopoulos, i. (2020). clinical updates regarding multiple myeloma from the 2019 american society of hematology annual meeting. clinical lymphoma myeloma and leukemia, 20(8), 499-508. disclaimer/publisher’s note: the statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of the publisher and/or the editor(s). this publisher and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. _________________________________________________________________________________ © copyright (2025): author(s). the licensee is the journal publisher. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: https://pr.sdiarticle5.com/review-history/141940 https://pr.sdiarticle5.com/review-history/141940 _____________________________________________________________________________________________________ *corresponding author: e-mail: ibrahimshnawa3@gmail.com; cite as: shnawa, ibrahim m s. 2025. “an inter mammalian microbiome-immune-axis mia”. asian journal of immunology 8 (1):26-34. https://doi.org/10.9734/aji/2025/v8i1157. asian journal of immunology volume 8, issue 1, page 26-34, 2025; article no.aji.132768 an inter mammalian microbiomeimmune-axis mia ibrahim m s shnawa a,b* a department of medical biotechnology, college of biotechnology, al-qasim green university, qasim, babylon, iraq. b college of nursing, university of hilla, hilla-babylon, iraq. author’s contribution the sole author designed, analyzed, interpreted and prepared the manuscript. article information doi: https://doi.org/10.9734/aji/2025/v8i1157 open peer review history: this journal follows the advanced open peer review policy. identity of the reviewers, editor(s) and additional reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here: https://pr.sdiarticle5.com/review-history/132768 received: 18/01/2025 published: 25/03/2025 abstract there is a mutual bidirectional cross-talk between mammalian microbiomes mm and their respective immune systems during the host homeostatic and disease states, the microbiome immune axis. the objective of the present opinion paper was to map the current status of the microbiome-immune axis updates. human gut microbiome diversity decreases rapidly after autologous stem cell transfer. myelopoiesis in human is regulated by signals from microbiome reaching the bone marrow. some animal heat treated gut bacterial antigens induces proinflammatory cytokines in rabbits ileal and villus cultures while other animal heat treated gut bacterial antigens initiated anti-inflammatory cytokines using same test culture systems. rabbits gut microbiome modulates brain development and function with coordinative helping role of the immune system. rabbit models for rhino-sinusitis and cystic fibrosis were found associated with microbiome dysbioses in nose and gut microbiomes respectively and simulating that of man. when the development of mice microbiome became arrested the immune system undergoes stunting growth and development. obese mice have shown dysbiotic microbiome. thus, the paradigm of microbiome-immune axis mia have shown that the potentials of the monkeys, rabbit and mice microbiomes can be translated to human welfare providing some limitations. opinion article https://doi.org/10.9734/aji/2025/v8i1157 https://pr.sdiarticle5.com/review-history/132768 shnawa; asian j. immunol., vol. 8, no. 1, pp. 26-34, 2025; article no.aji.132768 27 keywords: antigen; axis; bacteria; biosis; dysbiosis; human; immune; mice; rabbit; system. 1. introduction axis in the functional sense means the presence of mutual bidirectional signaling between two rather different or inter-related biologic entities. as an example, the microbiome-immune axis and it is expressed as a crucial relationship between microbiome and the immune system. a diverse microbiome is essential for maintaining balanced and healthy immune systems (shitara, 2023). gut microbiome possess an intensive influences on gut associated lymphoid tissue galt.galt displayed an immune modulatory role on gut mucosal responses leading to maintain of tolerance to harmless bacteria and responding to pathogens. early exposure to beneficial microbiome can enhance immune maturation and protect against allergic and autoimmune diseases in early life. in the past three decades, the change in the composition and function of microbiome have shown to be associated with obesity, inflammatory bowel disease, type ii diabetes, liver cirrhosis, drug treatment responses (zeevi et al., 2016). microbiome cross-talk with t cells leading to cancer, allergic inflammations and autoimmune disease (shim et al., 2023 campbell et al., 2023). the objective of the present opinion paper was to map human and representative mammalian microbiome-immune axis. 2. an old is being revisted and recreated pioneer generation microbiologists look to normal microbe living in or no human body as commensals and can prevents establishment of pathogens (parente, 2019). in the sixteens notion of the 20th century microbiologist was the global realization of cultivable/non-cultivable normal human microflora. in 19th of the past century, molecular genomic studies had led to evolution of microbiome concept, table 1. commensal human mirco-flora have been revisited and recreated as microbiota/microbiome (grice and segre, 2011). 3. microbiome versus microbiota microbiota refers to the profile of the normal cultivable microflora. while, microbiome as a term means the profile of cultivable and genetically determined uncultivable normal microflora inhabiting both in and on various mammalian body sites. so microbiome equal to; microbiome= cultivable + in-cultivable (normal commensal microflora) table 1. microbiome timeline achievements reference/date 1. five different bacteria of human mouth leewenhook 1688 2. flora and funa within living animals leidy 1853 3. some of human associated micro-organisms prove to be pathogenic in same niche nissle 1917 4. cultivation of anaerobic microbiota in the laboratory 1940-1950 5. realization of the theme for cultivable/noncultivable microbiota 1960s 6. the theme of commensal microflora in healthy body crucishank et al.1974 7. genetic and molecular based studies on human normal microbiota woese, pace, fox and others 19962005 8. popularization of microbiome/microbiota research theme gordon group 2006 9. advisory microbiome mapping guide asm 2015 10. microbiome strategic plans asm 2016 11. national microbiome data collaboration asm2019 12. microbiome research congress asm 2021 13. inclusion of the microbiome in the usa innovation act asm 2022 shnawa; asian j. immunol., vol. 8, no. 1, pp. 26-34, 2025; article no.aji.132768 28 table 2. characteristics of human and mammalian microbiomes features human monkey rabbit mice first genome mhc mnmhc rmhc mmhc second genome microbiome genes microbiome genes microbiome genes microbiome genes regulation of neonate and ageing immune system regulate regulate regulate regulate coevolution with immune system co-evolved co-evolved coevolved coevolved association with homeostasis associated associated associated associated dysbiosis immune mediated diseases immune mediated diseases translation in applicable non-tempted till now tempted tempted human microbiome composed of trillion of microbial spectra like bacteria, viruses, fungi and archea inhabiting various body compartments such as; gut skin, genitourinary and respiratory tracts. diverse microbiome is crucial for maintaining a balanced and healthy immune system. the composition and diversity of these microbial communities vary between individuals and affected by; diet, genetics and environmental factors (crucishank et al., 1974, parente, 2019, grice and segre, 2011, whiteside et al., 2015, levinson et al., 2018) table 2. 4. microbiome-immune axis axis in the functional sense refers to the presence of mutual bidirectional cross-talk between two different or inter-related biologic entities such as microbiome and that of the human cells of the immune system cells (shitara, 2023, zeevi et al., 2016, shim et al., 2023, campbell et al., 2023). 5. first versus second genome the first genome concerned with the host/immune system genetic system, while the second genome invented to designate the collective genomes of the members forming the microbiome. microbiome signals talking to host. host genome signals to cross-talk the components of the microbiome. this sort of signaling one -another usually followed by functional events within the microbiome-immune axis (shitara, 2023, zeevi et al., 2016, shim et al., 2023, campbell et al., 2023). 6. microbiome 6.1 human microbiome human microbiota is defined as a set of microbes inhabiting and interacting with human body. these interactions took three main forms as commensalism, mutualism and parasitism (grice and segre, 2011). they form complex and solitary ecosystem that adapt to the environmental conditions of the host niche. the human microbiome is constantly evolving in response to the host factors. human microbiome hm consists of an array of; bacteria, archea, viruses and eukaryotes.hm colonize various sites on or in of the body. facultative anaerobe colonize gastro-intestinal tract while strict anaerobe colonize skin, oral cavity and respiratory tract.hm impacts human physiology and immunology both in health and disease (crucishank et al., 1974, parente, 2019, grice and segre, 2011, whiteside et al., 2015, levinson et al., 2018). 6.2 mammalian microbiome animals belongs to carnivores, omnivores and herbivores groups have shown variable compositions of microbiota and maintained complexicity and stability which explain the difficulty to induce long term change in their compositions. they are competing for the essential nutrients and they alter the conditions required for growth of bacteria through production of bacteriocins that kills the competitors and controls pathogens by shnawa; asian j. immunol., vol. 8, no. 1, pp. 26-34, 2025; article no.aji.132768 29 stimulating host immunity and mucosal barrier functions .they provide signals for optimize immune functions .generally speaking ,mammalian microbiomes are somewhat similar one to another in nature for their members but they are different in proportion quantities, composition and diversity .in healthy animals, gram negative protebacteria , bacteriodetes and gram positive firmicutes including closteridiales and lactobacillales are the major phyla inhabiting large and small intestines. all of these organisms are adapted to the intra-intestinal environment and generally formed stable and complex population (tizard, 2023). studies onto gut/ileal microbiomes of rabbit and pig have shown that dominant phyla of pig ileum were firmicutes while the dominant phyla of rabbits ileal microbiome was bacteriodatae (cui and xu, 2016). 6.3 non-human primate microbiome monkey gut microbiome offers an insight into primate nutrition, physiology and immune system function (clayton et al., 2018). geography, genetics, climate, vegetation and diet related to microbiome community structure. there was more high degree of regional specificity in the microbiome composition which was associated with; host genetics, available plant food which affect diet. genetic differences, drove differences in gut microbiome community composition, while, vegetation as a diet drove regional gut mircobiome compositional differences (mesquita et al., 2021). monkey gut microbiome adopt characteristic enterotype which was compositionally analogous to that of human. such gut microbiome enterotype have stable microbial signature over time (campbell et al., 2020, moeller et al., 2012). 6.4 rabbits microbiome rabbits are both monogastric and herbivore animal with special digestive and physiological properties. their gut microbiome be stable and diverse by this it expresses significant resistant to intestinal disease (chen et al., 2017). different rabbit maternal strains showed different microbiome compositions (biada et al., 2020). rabbits may serve medical and nonmedical uses. the non-medical use include; textile production, meat production. while medical uses includes; bioreactor to produce polyclonal antibodies and as a biomedical model for human disease. whole rabbits body microbiome has shown firmicutes 62.3 %, proteobacteria 13.44 % and bacteriodate 11.84% (montoro-dasi et al., 2022, hu et al., 2021). rabbits gut microbiome plays a key role in maintaining health and in regulation and development of the immune system (kylie, 2016). 6.5 mice microbiome mice microbiome consists of a number of bacteria, viruses, fungi and eukaryotes. members of mouse microbiome distributed to gut, skin, respiratory and genitourinary tract. microbiome composition constitutes proportional amounts of firmicuts and bacteriodaiceae. mouse microbiomes are useful tool impactful in gaining a better understanding of human microbiome. there are 80 genera of mice similar to that of man, these are constituting 15% overall similarity of mouse to human microbiome (maue and lundberg, 2017, kennedy et al., 2018, ansaldo et al., 2021). 7. microbiome-immune axis 7.1 human microbiome-immune axis human immune system co-evolved with an extensive microbiome diversity on the mucosal barrier sites. it is evident and clear that microbial antigens belong to the microbiome members engage in constant dialogue with the immune system leading to microbiota specific immune responses that occur in the absence of inflammation. this constitute a form of immunity including b cell, innate like t cell and coevolutional t cell helper and t reg cell responses. microbiome induces innate like t cell and adaptive immune responses. these microbiomes immune potentials indicate that microbiome involved with cross-talk with the immune cells during immune response development (ansaldo et al., 2021). vital period for mammals to be colonized with microbiome is in the early life. such colonization is profoundly influence the intestinal immune functions. the intestinal immune system is critical for neonates in order to resist intestinal infections (yang et al., 2022). the initial colonization of an infant gut by microbes plays a pivotal role in shaping the immune system. early exposure to probiotics can enhances the immune system maturation and protects against allergic and autoimmune disease (shitara, 2023). there are a specific microbial groups take part in the immune system development and have a role in the functional changes that occur to the immune cell population through ageing (tibbs et al., 2019). human shnawa; asian j. immunol., vol. 8, no. 1, pp. 26-34, 2025; article no.aji.132768 30 myelopoiesis in the bone marrow is regulated by environmental signals including that of microbiome. microbiota derived signals can be sensed directly or indirectly by the hemopoietic stem cells and progenitor cells in bone marrow, thereby giving rise to myeloid cell lineages at steady state and during inflammation. these microbial signals affect the myelopoiesis during inflammation and infection (kim and kamada, 2023). the human gut microbiome acts as a signaling hub that integrates exposome with genome and metabolic pathways its impacts are wide across human body systems including the immune system (institute of functional medicine, 2025). the gut and skin microbiome both act as a barrier between human body and environment through sharing similar structure and function and allowing for cross-talk between them. such cross-talk is assisted by cytokines and microbial metabolites (owlstone medical, 2024). gut microbiome diversity decreases rapidly after autologous stem cell transplantation. there are specific bacterial families and certain immune cell subsets in patients receiving autologous stem cell transplantation (becker et al., 2024). 7.2 mammalian-immune axis using rabbits ileum and villus culture to elucidate the inflammatory response induce by heat killed intestinal bacterial antigens of rabbit and pig.it has been found that rabbits intestinal bacterial antigen riba induced higher expression of tlr4,while that of pigs piba induced tlr2 and tlr3.piba and riba induced increased expression of infalpha,il6.piba stimulate inf beta and il10.high appearance of gram negative in rabbit ileum do not lead to proinflammatory cytokine but high amounts of lactobacilli in pigs ileum was more expressive to anti-inflammatory cytokines (cui and xu, 2016). 7.3 monkey microbiome-immune axis short term antibiotic treatment in rhesus monkeys induced gut microbiome dysbiosis. such dysbiosis lead to increase in cd3+ t cells, cd4+ t cells, and cd16+ nk cells. but, decreased the number of t reg cells and cd20 b cells in peripheral blood cells (li et al., 2020). 7.4 rabbit microbiome-immune axis transfer of microbiota during birth fostering regulation of the first birth reaction. the maternal gamma delta t cells shape the offspring pulmonary type2 immunity in a microbiota dependent manner .the offsprings of gamma delta t cell deficient dams displayed enhanced lung type 2 immunity .tcr delta deficient dams displayed deficient both in amp levels and microbiota composition of the skin (papotto et al., 2023). rabbits gut microbiome is important in regulating trait and played a key role in immune system development. as well as it could be related to longevity and resilience (biada et al., 2020). incorporation of probiotic with rabbits diet supplementation increased the splenic and thymic index, levels of igm, c3 and c4. hence,probiotics supplement in diet could effectively improve immune organ index and immune function (zhang et al., 2020, salvoromero et al., 2020). rabbits gut bacteria can modulate gut resident immune cells and brain resident immune cells studies on gut-brain axis have shown that gut microbiome modulate brain development and function in presence of immune system cooperation (li et al., 2020). 7.5 mice microbiome-immune axis in an experimental settings mice microbiome arrested such arrestment lead to stunting in the immune system maturation with few peripheral regulatory t cells and decreased levels of iga and increased susceptibility to salmonella infection (lubin et al., 2023). neonatal mouse gut is enriched with neuro-transimittors and with specific bacteria produce serotonin directly while down regulating mono-amin oxidase a to limit the serotonin breakdown .serotonin inhibit mtor activation to promote regulatory t cells and suppress t cell responses both in ex-vivo and in-vivo in the neonatal intestines .oral gavage of serotonin into the neonatal mice lead to long term tolerance toward diet antigens and commensal bacteria as well as gut microbiome dysbiosis (sanidad et al., 2023, sanders et al., 2023). 8. comparative view there were significant co-diversification with ten gut bacterial phyla including firmicutes, actinobacteriota. strikingly around 44% of codiversifying clades detect in african apes were absent from microbiome data of man and 54% were absent from industrialized human population (salvo-romero et al., 2020). gut microbial taxa, microbial gene family contents of the great ape and human are more related by life shnawa; asian j. immunol., vol. 8, no. 1, pp. 26-34, 2025; article no.aji.132768 31 table 3. comparative translation approach in human primate, non-human primate and small mammals model microbiome homeostasis alteration matchability to human references monkey gut dysbiosis immune impairment (li et al., 2020) rabbit/rhinosinsusitis /cystic fibrosis nasal gut dysbiosis dysbiosis matchable matchable (rowe et al., 2018) (liang et al., 2021) mice/obesity gut dysbiosis matchable (maue and lundberg, 2017) style than geography (campbell et al., 2020). human gut microbiome composition and potentials are more similar to those of old world monkeys, the baboons than african apes. though there were inter-individual variation in the functional potentials of gut microbiome with in human species. such variations suggest that human gut microbiome may exhibit more plasticity in response to environmental variation (amato et al., 2019). antibiotic treatment of rhesus monkey model induced gut microbiome dysbiosis and impair the peripheral cellular immunity (li et al., 2020). results of rabbit microbiome studies can be translated to that matching human being as in the case of rhinosinusitis accompanied by sinus microbiome dysbiosis (rowe et al., 2018). in addition to the young rabbits cystic fibrosis model that was associated with gut microbiome dysbiosis simulating that of human being cystic fibrosis (liang et al., 2021). mouse gut microbiome composition contains 85% of bacterial genera different from that of human gut microbiome. the remaining 15 % of the mouse gut bacterial flora were similar to that of man. this fraction of mouse gut microbiome includes 80 genera shared with human gut microbiome. therefore it is essential for the researchers tempting translation of mouse experimental results to human being to have a clear understanding of the benefits and limitations of their model system if they wish to translate their finding to man. mouse obesity and ibd are the most models for microbiome studies both of which associated with microbiome dysbiosis (maue and lundberg, 2017), table 3. 9. homeostasis versus dysbiosis microbiomes homeostasis, is a state of normal functional balance of the influence of microbiota on health and disease depends onto; quality, quantity, proportion of each of microbiota component. homeostasis is in line with functional balance between the component of the microbiome. which indicate normal state of each of the components. such balance plays important roles in maintenance and development of human immune system (ogunrinol et al., 2020). dysbiosis, is any alteration in human or animal microbiota is associated with a disease state. alteration in the microbiota immune axis results in immune mediated diseases such as; intestinal infection, inflammatory bowel disease, autoimmunity, hypersensitivity and cancer (campbell et al., 2023, ogunrinol et al., 2020). 10. suggestion one "human gut microbiome and the innate immunity levels in neonates, children under five years and adolescents". elect 50 male and female normal; neonates, child under five years and adolescent. collect feces and blood from each group. test the phagocytic activity, acute phase proteins, c3 and c4 levels. fecal sample will be processed for the detection of gut microbiome by the use of either dna or 16srna kits. gut microbiome results concerning diversity, composition, similarity, enterotype and differences in each group in relation to the levels of innate immune parameters. 11. suggestion two "dysbiosis of human gut microbiome in typhoid patients". a plane for gut microbiome dysbiosis in normal and typhoid patients will be planed. normal subjects, untreated and treated typhoid patients will be the test groups. fecal samples from each group will be collected and processed for gut microbiome dysbiosis using either dna or 16s rna kits. results analyzed for; composition, diversity, and enterotypes for each group. shnawa; asian j. immunol., vol. 8, no. 1, pp. 26-34, 2025; article no.aji.132768 32 12. conclusion microbiome is important for human normal immune system development from neonates till ageing. human gut microbiome acts as a signaling hub that integrate exposome with genome and metabolic pathways. dysbiosis to the microbiome is mostly in line with disease state. monkey , rabbit ,and mice microbiomes may simulate but not reach the limits of identicality to human being microbiome ,there are advantages and some limitations for each of which both in structural and functional aspects .rabbit (rhinosinusitis, cystic fibrosis) and mice(obesity, ibd) microbiota were tempted for translation to human beings .as a suggestion monkey microbiome is expected to be more similar to human than rabbit and mice but apparently no tempts for translation to human till now so far current information indicated. two suggested programs for human gut microbiome studies were made. disclaimer (artificial intelligence) author hereby declare that no generative ai technologies such as lage language models(chatgpt ,copilot ,etc…) and text to image generators have been used during writing or editing of this manuscript. consent and ethical approval it is not applicable. competing interests author has declared that no competing interests exist. references amato, k. r., mallott, e. k., mcdonald, d., et al. 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(2020). the effect of probiotic bacillus amyloliquefaciens on growth performance, immune organ index, and immune function of weaned rabbits. veteriarski arhiv, 90(3), 271-277. disclaimer/publisher’s note: the statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of the publisher and/or the editor(s). this publisher and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. _________________________________________________________________________________ © copyright (2025): author(s). the licensee is the journal publisher. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: https://pr.sdiarticle5.com/review-history/132768 https://pr.sdiarticle5.com/review-history/132768 _____________________________________________________________________________________________________ *corresponding author: e-mail: nassarasma20@gmail.com; cite as: nassar, asma, amina alliouch -kerboua, hacene meriche, nassima sabiha gadiri, and frida chettab. 2025. “clinical profile and analysis of biological markers (fecal calprotectin and crp) during relapse of inflammatory bowel diseases in a population of adults”. asian journal of immunology 8 (1):104-19. https://doi.org/10.9734/aji/2025/v8i1165. asian journal of immunology volume 8, issue 1, page 104-119, 2025; article no.aji.135189 clinical profile and analysis of biological markers (fecal calprotectin and crp) during relapse of inflammatory bowel diseases in a population of adults asma nassar a*, amina alliouch -kerboua a, hacene meriche a, nassima sabiha gadiri a and frida chettab b a department of immunology, clinique saint therese uhc, annaba, algeria. b department of gastroentero-hepatology, hospital ibn sina uhc, annaba, algeria. authors’ contributions this work was carried out in collaboration among all authors. all authors read and approved the final manuscript. article information doi: https://doi.org/10.9734/aji/2025/v8i1165 open peer review history: this journal follows the advanced open peer review policy. identity of the reviewers, editor(s) and additional reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here: https://pr.sdiarticle5.com/review-history/135189 received: 26/02/2025 published: 05/05/2025 abstract inflammatory bowel diseases (ibd) are increasingly common pathologies, affecting both adults and children. this study investigates the clinical and epidemiological characteristics of ibd among patients at the gastroentero-hepatology department of annaba university hospital. conducted from original research article https://doi.org/10.9734/aji/2025/v8i1165 https://pr.sdiarticle5.com/review-history/135189 nassar et al.; asian j. immunol., vol. 8, no. 1, pp. 104-119, 2025; article no.aji.135189 105 february 2023 to september 2024, the prospective descriptive analytical study included 77 patients diagnosed with ibd, focusing on the role of biological markers, specifically c-reactive protein (crp) and fecal calprotectin (fc), in non-invasive disease assessment. inclusion criteria were based on clinical, endoscopic and histological assessments. demographic data, clinical manifestations, crp and fc levels were analyzed. among the 77 patients, 53 patients or 68.9% had crohn's disease (cd), while 24 patients or 31.1% suffered from ulcerative colitis (uc). the most prevalent symptoms were abdominal pain and chronic diarrhea. among cd patients, ileal involvement was common in 21 cases (39.62%) with 25 cases (32,47 %) experiencing complications the study found that 34 or 46.57% of patients had elevated crp levels, with a higher prevalence in those with cd compared to uc. a statistically significant association was observed between crp levels and ibd type. additionally, the analysis revealed that among patients experiencing relapse, 20 cases or 52.63% had positive fc results, compared to 14 cases or 35.9% in remission, indicating a significant correlation between relapse status and fc levels (p=0.018). however, no significant association was found between relapse status and crp levels, although a significant difference in mean crp levels between active and remission states in uc was noted (p=0.03). a weak positive correlation (r =0.12) between crp and fc during active phases was also identified. the findings underscore the clinical and epidemiological profile of ibd in this population, highlighting the importance of fc as a valuable marker for monitoring disease activity and relapse status in ibd patients. keywords: fecal calprotectin; inflammatory bowel disease; crohn's disease; ulcerative colitis; creactive protein. 1. introduction chronic inflammatory bowel diseases (ibd), including crohn's disease (cd) and ulcerative colitis (uc), are chronic and progressive pathologies responsible for irreversible destruction of the digestive tract, leading to significant alteration in quality of life. diagnosis of ibd is often difficult in clinical practice, due to pathognomonic symptoms and considerable overlap with those of irritable bowel syndrome (ulcerative colitis pubmed, 2024; torres et al., 2017) although endoscopy has been the mainstay of ibd assessment, the feasibility of using ileo colonoscopy repeatedly for long-term follow-up is limited by its availability, invasiveness, patient tolerance and cost. there is therefore considerable interest in using non-invasive biomarkers to monitor ibd activity (ma et al., 2019). fc, a neutrophil-derived protein, is the most sensitive biomarker of intestinal inflammation. fc levels correlate well with endoscopic scores in cd and uc. today, fc is used in a variety of clinical settings, notably for the initial diagnosis of ibd (d’amico et al., 2021).although fc and crp are the most studied biomarkers in this field, there are concerns about the sensitivity and specificity of crp in ibd patients. moreover, patients often prefer blood tests to stool collection, which limits the acceptability of fc. finally, the optimal thresholds for fc to predict disease activity vary according to ibd phenotype and the tests used (swaminathan & day, 2024). our study aims to assess the clinical and epidemiological profile of ibd in adult population. it also explores the correlation between crp levels and fc in the context of ibd, to better understand their usefulness as biological markers in monitoring intestinal inflammation. 2. materials and methods the present study was conducted in the immunology laboratory at annaba university hospital, annaba/algeria, over a period of 19 months, from february 9, 2023, to september 30, 2024. this is a prospective descriptiveanalytical study involving a sample of 77 adult patients referred by the gastroenterology and hepatology department at annaba university hospital. the sample consisted of 29 males and 48 females, with an average age of 39.7 ± 17.47 years, a median age of 34 years, and a mode of 34 years. the age range was from 17 to 82 years, resulting in a male-to-female ratio of 0.6. inclusion criteria patients included in our study represent those whose diagnosis of ibd (uc/cd) was confirmed nassar et al.; asian j. immunol., vol. 8, no. 1, pp. 104-119, 2025; article no.aji.135189 106 on the basis of clinical, endoscopic, and histological criteria (montreal classifications) and referred to the immunology laboratory for immunological work-up: determination of fc and crp. exclusion criteria patients whose diagnosis of ibd has not been confirmed (still under investigation). patients with incomplete or unusable clinical records. patients diagnosed with indeterminate colitis. 77 patients met the criteria and consented to participate in the study 2.1 the included immunological analysis techniques included among the immunological analysis techniques were: – determination of the fc using the sandwich elisa technique (enzyme-linked immunosorbent assay from inova diagnostics) on stool samples – determination of crp by fai (fluorescence immunoassay) on a serum samples. 2.2 data collection and statistical analysis statistical analyses were conducted using spss software. the following statistical tests were employed to compare the different subgroups: ✓ chi-square test: used for comparing qualitative variables. ✓ student's t-test and analysis of variance (anova): utilized for comparing means across groups. ✓ spearman's rank correlation: applied to assess the correlation between fc levels and crp levels. a significance level of 0.05 was established for all analyses. 3. results 3.1 study population description the study population consisted of 77 patients referred by the gastroenterology department. among the included patients, 53 (68.9%) were diagnosed with crohn's disease (cd) and 24 (31.1%) with ulcerative colitis (uc). specifically, cd was identified in 20 men (25.97% of cases) and 33 women (42.85% of cases), resulting in a male-to-female ratio of 0.6. uc was diagnosed in 15 women (19.48% of patients) and 11 men (11.68% of patients), yielding a male-to-female ratio of 0.73. no significant association was found between gender and type of ibd (uc or cd). in our cohort of cd cases, ileal localization was the most prevalent, accounting for 21 cases (39.62%), followed by colonic localization, which involved 14 patients (23.81%). additionally, 5 patients (5.3% of the cohort) were active smokers. regarding disease activity, 38 patients (49.35%) were in the relapse phase, including 24 with cd; however, no statistically significant association was observed. in terms of treatment, 15 (19, 5 %) of patients received anti-tnf therapy, 12 (15, 6 %) were on a combination of therapies, 11 (14.3%) were treated with corticosteroids, while 8 (10.4%) received immunosuppressants and 6 (7.8%) were treated with salicylates. (table 1) complications of ibd: the results suggest that the obstructive tableau is more frequent in patients with cd, with a statistically significant difference (p = 0.04). there is a trend towards a difference for fistulas, although not significant (p = 0.07). other complications (stenosis, abdominal mass) show no significant differences between cd and uc patients (fig. 1). digestive manifestations : digestive manifestations were dominated by abdominal pain in 55 cases (71.43%), followed by chronic diarrhea in 50 cases (64.93%), rectal bleeding in 15 cases or 19.48 % (14.28 of cases with uc), purulent diarrhea and false needs in 7 cases (9.09%) and constipation in 3 cases (3.9%). rectal bleeding is significantly more frequent in uc patients (p< 0.0001), consistent with this disease. other symptoms (abdominal pain, chronic diarrhea, false needs, purulent diarrhea, and constipation) showed no significant differences between cd and uc patients. (table 2) nassar et al.; asian j. immunol., vol. 8, no. 1, pp. 104-119, 2025; article no.aji.135189 107 extra-digestive manifestations: none of the extra-intestinal manifestations evaluated (peripheral arthritis, axial rheumatism, uveitis, erythema nodosum, oral ulcerations, steatosis) showed any statistically significant difference between uc and cd patients. (table 3) table 1. description of study population sample size percentage pearson chisquare test type of ibd cd uc 53 24 68,9% 31,1% ibd by gender cd man woman uc man woman 20 33 9 15 25,97% 42,85% 11,68% 19,48% p=0,98 localization (cd) ileal ileocolic colonic 21 13 14 39,62 % 24,52 % 26,41 % smoking factor smoker 5 5,3% ibd in relapse phase cd uc 24 14 31,17% 18,18% p=0,28 treatments corticosteroids immunosuppressants anti-tnf aminosalicylates (5-asa) combination therapy 11 8 15 6 12 14,3 % 10,4 % 19,5 % 7,8 % 15,6 % fig. 1. ibd population distribution based on complications table 2. ibd cases distribution based on digestive symptoms abdominal pain chronic diarrhea rectal bleeding false needs purulent diarrhea constipation cd 40 (51, 9%) 35(45, 45%) 4(5, 19%) 6(7,79%) 5(6,49%) 1(1,3%) uc 15(19,5%) 15(19,5%) 11(14,28%) 1(1,3%) 2(2,6%) 2(2,6%) total 55 50 15 7 7 3 % 71,43 64,93 19,48 9,09 9,09 3,9 p 0,24 0,76 <0,0001 0,30 0,87 0,17 0 5 10 15 stenosis fistula abdominal mass obstructive tableau uc cd p=0.04 nassar et al.; asian j. immunol., vol. 8, no. 1, pp. 104-119, 2025; article no.aji.135189 108 table 3. population distribution based on extra-digestive symptoms peripheral arthritis axial rheumatism uveitis erythema nodosum oral ulcerations steatosis uc 3(3,9%) 2(2,6%) 2(2,6%) 0 4(5,2%) 0 cd 20(25,97%) 7(9,09%) 6(7,8%) 3(3,9%) 12(15,6%) 3(3,9%) total 23 9 8 3 16 3 % 29,87 11,69 10,39 3,9 20,78 3,9 p 0,25 0,53 0,69 0,23 0,54 0,23 3.2 fc and crp assay results 3.2.1 population distribution based on fc findings among the 77 patients, 34 (44.2%) were fc positive, 36 (46.8%) were fc negative and 7 (9.1%) were fc equivocal (table 4). 3.2.2 fc results in relapse/non-relapse status the results show a significant difference between relapsing and non-relapsing patients in terms of fc. patients in relapse had a higher proportion of positive fc (20 patients or 52.63%) compared with patients not in relapse (14 patients or 35.9%), and none of the patients in relapse had an equivocal fc. this difference is confirmed by a p-value of 0.018, indicating a significant association between relapse status and fc. (table 5) 3.2.3 fc results according to ibd the results indicate that, although there are differences in fc between the relapse and remission phases, these differences are not statistically significant, with a p-value of 0.10 for both cd and uc patients. (table 6) the comparison between uc and cd during the relapse phases yielded a p-value of 0.27, also non-significant. (table 7). table 4. population distribution based on fc findings positive fc negative fc equivocal fc total number of patients 34 36 7 77 % 44,2 46,8 9,1 100 table 5. fc results in relapse/non-relapse status positive fc negative fc equivocal fc relapse 20(52,63%) 18(47,37%) 0 nonrelapse 14(35,9%) 18(46,15%) 7(17,9%) total 34(44, 2%) 36(46, 8%) 7(9, 1%) pearson chi-square test p= 0,018 table 6. fc results according to the type of ibd and active/inactive status positive fc negative fc equivocal fc pearson chi-square test cd (relapse) 24 cases 11(45,83%) 13(54,17%) 0 p=0,10 cd (non -relapse) 29cases 11(37,93%) 13(44,83%) 5(17,24%) uc(relapse) 14 cases 9(64,29%) 5(35,71%) 0 p=0,10 uc (non -relapse) 10 cases 3(30%) 5(50%) 2(20%) total (77) 34(44,2%) 36(46,8%) 7(9,1%) nassar et al.; asian j. immunol., vol. 8, no. 1, pp. 104-119, 2025; article no.aji.135189 109 table 7. comparison of fc results between uc and cd during relapse phase positive fc negative fc equivocal fc pearson chi-square test cd (relapse) 24 cases 11(45,83%) 13(45,17%) 0 p=0,27 uc (relapse) 14 cases 9(64,29%) 5(35,71%) 0 total (38) 20(52,63%) 18(47,37%) 0 3.2.4 distribution of the population according to crp levels in our study, 34 patients (46.57%) had a crp level of ≥ 6 mg/l, while 39 patients (53.42%) had a crp level of < 6 mg/l. patients with cd had a higher proportion of positive crp compared to patients with uc. the fisher's exact test showed a statistically significant association (p=0.007) between crp levels and the type of ibd (fig. 2). 3.2.5 crp results according to active/inactive disease status the results suggest that there is no significant link between disease status (active or inactive) and crp levels (<6 and ≥6), according to fisher's exact test (p = 0.48). (table 8) 3.2.6 crp results according to the type of ibd in patients with cd, no significant difference in crp levels was observed between the active and remission phases (p = 0.80). for uc, there is a trend toward a difference, but it is not statistically significant (p = 0.08). (table 9) the comparison between patients with active cd and those with active ulcerative colitis uc also yields a non-significant result (p = 0.17). (table 10). fig. 2. distribution of the population according to crp levels table 8. crp results according to active/inactive disease status crp <6 mg/l crp≥ 6mg/l fisher's exact test relapse 37 cases 18 (48,65%) 19 (51,35%) p=0,48 non –relapse 36 cases 21 (58,33%) 15 (41,67%) total (73) 39 (53,42%) 34 (46,57%) table 9. crp results according to the type of ibd crp <6mg/l crp≥6mg/l pearson chi-square test cd (relapse) 24 cases 14(58,33%) 10(41,67%) p= 0,80 cd (non-relapse) 29 cases 16(55,17 %) 13(44,83%) uc (relapse) 13 cases 4(30,77%) 9(69,23%) p=0,08 uc (on-relapse) 7 cases 5(71,42%) 2(28,6%) total (73) 39 (53,42%) 34(46,57%) 0 10 20 30 cd uc <6 30 9 6≥ 23 11 30 9 23 11 <6 6≥ p=0,007 nassar et al.; asian j. immunol., vol. 8, no. 1, pp. 104-119, 2025; article no.aji.135189 110 table 10. comparison of crp results between cd and uc in active phase crp <6mg/l crp≥6mg/l pearson chi-square test cd (relapse) 24 cases 14(58,33%) 10(41,67%) p= 0,17 uc (relapse) 13 cases 4(30,77%) 9(69,23%) total (37) 18(48,65%) 19(51,35%) 3.2.7 crp /fc results according to different locations in crohn's disease there is no statistically significant difference between fc results and the different localization in cd (table 11). the results indicate that there is no statistically significant difference in crp levels (≥ 6 or < 6) for ileal and ileocolonic locations. however, there is a suggestive trend in the colonic location with a p-value of 0.06 (table 12). 3.3 comparison of the average rate of fc and crp 3.3.1 comparison of the average rate of fc between cd and uc during flare-up the average rate of fc is higher during uc than in cd, however these results do not establish a significant distinction between uc and cd in terms of fc (p=0.09). (table 13) 3.3.2 comparison of the mean fc levels between active and remission states in uc the mean fc levels are higher in patients in the 'active' state (531 mg/l) compared to those in 'remission' (412.6 mg/l). however, the standard deviations are very high in both groups (751.71 for patients in the active state and 879.82 for those in remission), suggesting a large variability in individual values. this could indicate a heterogeneous distribution of fc levels within each group. despite this difference in means, the comparison between the two groups does not reveal a statistically significant difference (p=0, 73) (table 14). table 11. fc results according to different locations in crohn's disease ileal localization ileocolic localization colonic localization positive fc 10(47,62%) 3(23,1) 7(50%) negative fc 10(47,62%) 8(61,54%) 6(42,86%) equivocal fc 1(4,8%) 2(15,38%) 1 (7,14%) p value 0,56 0,27 0,74 table 12. crp results according to different locations in crohn's disease ileal localization ileocolic localization colonic localization crp≥6 mg/l 8(38,1%) 5(38,46%) 9(64,29%) crp <6 mg/l 13(61,9%) 8(61,54%) 5(35,71%) p value 0,58 0,67 0,06 table 13. comparison of the average rate of fc between cd and uc during flare-up ibd n mean (mg/kg) standard deviation fc uc 14 531,11 780,1 cd 24 249,88 326,24 student's t-test p=0,09 table 14. comparison of the mean fc levels between active and remission states in uc clinical status n mean (mg/kg) standard deviation fc in a flare-up 14 531 751,71 in remission 10 412,6 879,82 student's t-test p=0,73 nassar et al.; asian j. immunol., vol. 8, no. 1, pp. 104-119, 2025; article no.aji.135189 111 3.3.3 comparison of the mean fc levels between active and remission states in cd the mean fc levels are very close between the two groups: 250 mg/l during the active phase and 258 mg/l in remission. however, the standard deviations reveal greater variability in the remission group (555.53) compared to the active group (320), indicating that the fc values are more dispersed among patients in remission. the student's t-test indicates that there is no statistically significant difference between the mean fc levels based on clinical status (p=0, 95). (table 15) 3.3.4 comparison of the mean crp levels between cd and uc during the active phase the student's t-test yielded a p-value of 0.88, indicating that the difference between the mean crp levels of the two groups is not statistically significant. (table 16) 3.3.5 comparison of the mean crp levels between active and remission states in uc the difference between the mean crp levels in the two clinical states is statistically significant (p=0.03). (table 17) 3.3.6 comparison of the mean crp levels between active and remission states in cd patients in the "active" state have a slightly higher mean crp level (19.62 mg/l) compared to those "in remission" (12.06 mg/l). however, the student's t-test (p = 0.37) indicates that this difference is not statistically significant. (table 18) 3.3.7 comparison of the mean fc levels according to disease location in cd although there are differences in the mean fecal calprotectin (fc) levels based on the location of the disease (ileal, ileocolonic, colonic), these differences are not statistically significant (p=0, 53). (table 19) table 15. comparison of the mean fc levels between active and remission states in cd clinical status n mean (mg/kg) standard deviation fc in a flare-up 24 250 320 in remission 29 258 555,53 student's t test p=0,95 table 16. comparison of the mean crp levels between cd and uc during the active phase ibd n mean (mg/l) standard deviation crp uc 14 18,12 15,76 cd 24 19,7 38,88 student's t-test p=0,88 table 17. comparison of the mean crp levels between active and remission states in uc clinical status n mean (mg/l) standard deviation crp in a flare-up 13 17,73 15,54 in remission 7 6,84 5,76 student's t-test p=0,03 table 18. comparison of the mean crp levels between active and remission states in cd clinical status n mean (mg/l) standard deviation crp in a flare-up 24 19,62 38,12 in remission 29 12.06 14,63 student's t-test p=0,37 nassar et al.; asian j. immunol., vol. 8, no. 1, pp. 104-119, 2025; article no.aji.135189 112 table 19. comparison of the mean fc levels among different locations in cd ileal localization ileocolic localization colonic localization mean fc rate (mg/kg) 269,13 127,40 403,21 standard deviation 325,9 146,84 843,10 anova test p= 0,53 table 20. comparison of the mean crp levels among different locations in cd ileal localization ileocolic localization colonic localization mean crp rate (mg/l) 7,99 13,44 20,67 standard deviation 11,1 13,15 23,82 anova test p=0,38 3.3.8 comparison of the mean crp levels according to disease location in cd although there are differences in the mean crp levels based on the location of the disease (ileal, ileocolonic, colonic), these differences are not statistically significant (p=0, 38). (table 20) 3.4 correlation test between fc and crp during the active phase the correlation between crp and fc is weak and positive (r = 0.12). this means that as the fc variable increases, crp tends to increase as well, but only very slightly. however, since p = 0.48, this correlation is not statistically significant. in practical terms, this indicates that crp and fc should be used complementarily and not interchangeably to assess inflammation in adult patients with inflammatory bowel disease (ibd). a low crp does not necessarily indicate low intestinal inflammation, highlighting the importance of measuring fc for an accurate evaluation. (fig. 3). fig. 3. scatter plot of correlation between fc and crp during the active phase spearman correlation test p= 0,48 r=0,12 nassar et al.; asian j. immunol., vol. 8, no. 1, pp. 104-119, 2025; article no.aji.135189 113 4. discussion 4.1 description of the studied population inflammatory bowel diseases (ibd) do not appear to have a gender preference (cosnes et al., 2011). however, according to delmondes lm and al., a general female predominance has been observed in ibd cases, with a male-tofemale ratio of 0.641 (lm et al., 2015). a cohort study conducted in canada in 2017 involving 45567 patients showed similar results. lima martins and al. also reported this finding in their study in 2018 (benchimol et al., 2017 ; lm et al., 2018). conversely, a study by wang zz and al. in 2017 highlighted a male predominance in ibd cases (jm et al., 2018). in a german study, the peak incidence for both crohn's disease (cd) and ulcerative colitis (uc) was observed in patients aged 30 years or younger (kostev et al., 2018). the average age in the oranian study was reported as 31.2 ± 3.4 years (masson, 2024). in a study by huguet j and al. in 2018, nearly 69% of patients were over 60 years old (jm et al., 2018). in our series, the mean age was 39.7 years ± 17.47 years. in a moroccan study, cd accounted for 46.67% of cases (ibd , n.d.). in our series, we noted a clear predominance of cd, comprising 68.9% of cases. cd can affect any part of the gastrointestinal tract but shows a preference for the ileocecal region. the montreal classification is widely used by gastroenterologists for lesion topography (satsangi et al., 2006). according to literature data, the small intestine and colon are preferred sites for cd. in our case series of cd, ileal localization was the most common, representing 39.62% of cases, followed by colonic localization at 26.41%. the observed differences may be linked to geographical, environmental, or genetic factors influencing the presentation of ibd. fistulas are among the most frequently encountered complications in cd (management of acute ano-perineal crohn’s disease, 2024). our results suggest that obstructive presentations are more common in patients with cd, with a statistically significant difference (p= 0.04). this discrepancy with existing literature may arise from geographical variations, access to care, or differences in follow-up and treatment practices. a dutch population study by smids c and al. found that 44% of patients with cd smoked, compared to 20% with crohn's disease indeterminate (cdi) and 7% with uc (smids et al., 2017). among our patients, 5.3% were smokers, all diagnosed with cd. our findings do not align with the literature and may be biased due to our small sample size; however, smoking appears more prevalent in cd cases than in uc. according to research by casellas f and al., studying disease activity among a population of 117 ibd patients, among those with cd, six subjects experienced at least one relapse compared to 58 who were in remission. for uc cases, four patients had relapses while 49 showed no disease activity (nahon et al., 2018). in our series, 38 patients (49.35%) were experiencing flare-ups, with 31.17% having cd. the increase in flare-ups within our series may be attributed to the small sample size. a moroccan study reported abdominal pain in 20 patients with cd (33.3%) and in 14 patients with uc (23.3%). false needs were reported in 32 patients (53.3%), including ten with cd (16.67%) and twenty-one with uc (35%). rectal bleeding was noted in 25% of patients with uc (ibd, n.d.). in our series, digestive manifestations were dominated by abdominal pain in 51.95% of patients with cd and in 19.48% of those with uc. chronic diarrhea was present in 45.45% of cd cases and in 19.48% of uc cases; rectal bleeding occurred in 19.48% of cases (14.28% with uc), and false needs were reported in 9.09% of cases (7.79% with cd). these results are consistent with literature data. an iranian study conducted by zobeiri m and al. highlighted that extra-digestive symptoms were marked by musculoskeletal signs (51.9%), followed by ocular signs (27.9%), and skin signs (22.7%) (gao et al., 2017). in our study, articular signs were present in 41.55% of patients; ocular manifestations occurred in 10.4%, and skin signs were seen in nassar et al.; asian j. immunol., vol. 8, no. 1, pp. 104-119, 2025; article no.aji.135189 114 3.9%. our results align well with those reported in other studies. 4.2 results of fc and crp testing in a chinese study, crp levels were found to be higher among patients with cd (wang et al., 2013). in our series, 34 patients (46.57%) had crp levels ≥6 mg/l (with 29.87% having cd). our findings are consistent with existing literature. schoepfer and al. reported a higher correlation between the endoscopic activity of uc and fc levels (r = 0.8) compared to crp (r = 0.5) (schoepfer et al., 2024). a study conducted at the gastroenterology clinic of rsud dr. soetomo hospital in surabaya from march to august 2020 included 30 participants with uc. this crosssectional analytical study aimed to evaluate colitis activity through crp and fc measurements. during flare-ups, fc was positive in 20 patients (67%), while crp was positive in 13 patients (43%). a significant relationship was observed between fc and crp, with a correlation coefficient (r) of 0.57 (p = 0.01) (anindita et al., 2023). in our study, patients in flare had a higher proportion of positive fc (52.63%) compared to patients in remission (35.9%), and none of the patients in flare had equivocal fc results. this difference was confirmed by a p-value of 0.018, indicating a significant association between flare status and fc levels. our results suggest that although there are differences in fc between flare and remission phases, these are not statistically significant with a p-value of 0.1 for patients with cd and uc. furthermore, the comparison between cd and uc during flare phases yielded a p-value of 0.27, which is also not significant. this supports the notion that fc is a good biomarker for inflammation in the context of inflammatory bowel diseases (ibd), consistent with previous studies. a study conducted between 2018 and 2019 involving 49 patients with cd and 31 healthy controls recruited from the gastroenterology and hepatology department at the university hospital in krakow, poland, found significantly higher serum crp levels in the cd group compared to the control group. additionally, in patients with cd, serum crp concentrations were markedly higher during active disease than during inactive disease (słowińska-solnica et al., 2021). a study conducted in 2006 in italy, in 76 ibd patients (29 cd and 47 uc) fecal calprotectin has been evaluated by a commercial elisa kit. results demonstrate that levels of this protein in the stool are significantly more elevated in active cd and uc patients than in normal volunteers. moreover, in cd patients levels of calprotectin are higher than in uc (amati et al., 2006). our results reveal no significant link between flare status (flare or non-flare) and crp levels (<6 and ≥6) (p = 0.48). in cd patients, no significant difference in crp was observed between flare and remission phases (p = 0.8). for uc, a trend towards a difference was noted, although it was not statistically significant (p = 0.08). differences in disease location in cd may account for some of the inter-individual variation in fc. however, fc and other stool biomarkers have been found to have low sensitivity in detecting isolated small bowel involvement seen on wireless capsule endoscopy (sands, 2015). gecse and al. demonstrated that patients with large ulcerations (>5 mm) in the ileum had significantly lower fc concentrations compared to those with ileocolonic or colonic disease (297 vs. 1,523 μg/g, p < 0.0001). a study involving 120 patients with cd examined disease activity using biological markers as well as endoscopic and radiographic methods. fc concentration was measured in stool samples. among these patients, 45% had ileocolonic cd, 36.5% had small bowel cd, and 18.5% had colonic cd. in small bowel cd patients, fc was significantly correlated with crp (p = 0.03), endoscopic activity (p < 0.0001), and radiographic assessment (p = 0.03). similarly, in colonic cd patients, fc correlated with crp (p = 0.0009) and endoscopic activity (p = 0.0002). however, among small bowel cd patients, the crohn's disease activity index (cdai) and inflammatory markers were not correlated with endoscopic and radiographic evaluations. for ileocolonic cd patients, fc showed a significant correlation with endoscopy (p=0.006), radiographic assessment (p = 0.04), cdai (p = 0.0006), as well as most biological markers. the concentration of fc reflects inflammatory activity more accurately in patients with lesions in both nassar et al.; asian j. immunol., vol. 8, no. 1, pp. 104-119, 2025; article no.aji.135189 115 the small intestine and colon compared to those with isolated small bowel disease (sands, 2015). a study involving 273 cd patients, including 41 with ileal disease and 189 with ileocolonic disease, showed that fc was significantly correlated with cdai; correlation coefficients were 0.711 for ileal disease patients and 0.687 for ileocolonic disease patients (gecse et al., 2015). in our study, although there are differences in average fc levels based on disease location (ileal, ileocolonic, colonic), these differences are not statistically significant, possibly due to sample size or individual variations. according to a study including 88 patients with an average age of 34±10.8 years, twelve (13.6%) had negative crp results. while location did not serve as a significant predictor, all patients with negative crp had ileal involvement (stawczykeder et al., 2015). in our study, results indicate no statistically significant difference between crp levels (≥6 or <6) for ileal and ileocolonic locations; however, there is a suggestive trend for colonic location with a p-value of 0.06. there is an emphasis on the similarity regarding the absence of significant predictors related to location, suggesting that despite variations across studies, there is a general trend indicating that ileal location does not appear to be strongly correlated with crp levels. 4.3 comparison between the average levels of fc / crp in a prospective study involving patients recruited from the inflammatory bowel disease unit at the university hospital of saint etienne between june 2017 and june 2018, patients were followed for 12 months or until relapse. the measurement of fc and crp showed that median fc levels progressively increased in patients who relapsed during the follow-up period, rising from 26 µg/g at baseline to 105 µg/g at three months, 177 µg/g at six months, and 292 µg/g at the time of relapse (p = 0.049 between baseline and relapse) 1. serum crp levels fluctuated during follow-up, but a significant difference in median crp levels was observed between baseline and relapse (1.5 mg/l vs. 3.3 mg/l, respectively; p = 0.008). however, median crp levels remained normal below 5 mg/l (li et al., 2023). a study involving twenty-six patients with inflammatory bowel disease, including five with crohn's disease and twenty-one with ulcerative colitis, revealed no significant correlation between fc and crp (r = 0.2) (sousa et al., 2017). in another observational prospective study of fifty-nine patients with ulcerative colitis, eighteen patients (30.5%) exhibited signs of active histological inflammation. these patients had a significantly higher median fc level (278 µg/g), while the average crp level was 16 mg/l (veyrard et al., 2022). a study conducted on 273 patients with crohn's disease at the gastroenterology division of tongji medical college in wuhan, china, found median fc levels in patients in endoscopic remission, mildly active, and moderately to severely active stages to be 26.94, 66.77, and 327.22 µg/g respectively, with significant differences observed (p < 0.001). median crp levels in clinically remission, mildly active, and moderately to severely active groups were respectively 3.30, 6.30, and 30.30 mg/l (gecse et al., 2015). in our study, the average fc level in patients with active crohn's disease was 250 µg/g, while it was 258 µg/g in remission. average crp levels were 19 mg/l during flare-ups and 12 mg/l in remission. this difference may be attributed to individual variability in inflammatory response or the presence of confounding factors such as the use of immunosuppressive medications. a separate study on fifty patients with ulcerative colitis aged between twelve and seventy-four years measured fc and crp levels based on disease severity. patients with mild activity had an average fc level of 207.46 g/kg and an average crp level of 11.37 mg/l; those with moderate to severe activity had an average fc level of 729.85 g/kg and an average crp level of 29.38 mg/l. a statistically significant difference was observed between the two groups for both fc (p = 0.001) and crp (p= 0.0001), indicating that both markers increase significantly with the severity of ulcerative colitis (kyle et al., 2021). rodriguez-moranta and al. found that fc is better correlated with the degree of inflammation than other clinical indicators and serological nassar et al.; asian j. immunol., vol. 8, no. 1, pp. 104-119, 2025; article no.aji.135189 116 markers (guardiola et al., 2014). moreover, it could also be useful for predicting mucosal healing and the risk of relapse. according to a study by mohamed and al., there was a highly significant increase in average fc values among active ulcerative colitis patients compared to inactive ones; similarly, there was a significant increase in average fc values among inactive ulcerative colitis patients compared to controls (ahmed et al., 2017). in our study, the average fc level among patients with active ulcerative colitis was found to be 531 µg/g while it was 412.6 µg/g in remission. the student's t-test yielded a non-significant pvalue of 0.73 for comparing these two groups regarding fc levels. average crp levels were found to be 17.73 mg/l during flare-ups and 6.84 mg/l in remission; the student's t-test yielded a significant p-value of 0.03 when comparing the two groups for crp. a retrospective analysis of records from inflammatory bowel disease patients followed between january 2012 and october 2014 at a gastroenterology care center in mumbai included sixty-three patients (32 with ulcerative colitis and 31 with crohn's disease). erythrocyte sedimentation rate (esr), crp, and fc were compared against endoscopic results to evaluate inflammation. patients with ulcerative colitis exhibited higher fc levels than those with ileocolic crohn's disease (median fc: 1800 mg/g vs. 619 mg/g; p = 0.04). additionally, fc levels were correlated with crp (r = 0.4; p < 0.001). among the sixtythree patients showing signs of inflammation upon endoscopic examination, a higher proportion (86.9%) had positive fc concentrations compared to those showing positive crp results (65.6%; p < 0.01) (rodríguez-moranta et al., 2013). in our study, average fc levels were higher in ulcerative colitis than in crohn's disease; however, these results did not significantly distinguish between ulcerative colitis and crohn's disease concerning fc levels. the student's ttest applied to average crp levels yielded a pvalue of 0.88, indicating no statistically significant difference between the average crp levels of both groups. finally, the correlation between crp and fc was weakly positive (r = 0.12; p = 0.48). previous studies have shown that both fc and crp significantly increase in correlation with inflammation severity; however, our analysis reveals a weak and non-significant correlation within our cohort (r = 0.12; p = 0.48). this suggests that within our cohort, crp and fc do not act together to reflect inflammation effectively (badawy et al., 2014;samant et al., 2015). 5. conclusion and perspectives our study has provided a clearer definition of the clinical and epidemiological profile of inflammatory bowel diseases (ibd) in adults, highlighting the distinctive characteristics of crohn's disease (cd) and ulcerative colitis (uc). the results indicate a higher prevalence of ibd among adult women. in terms of biomarkers, creactive protein (crp) and final concentration (fc) have proven relevant for monitoring intestinal inflammation, with significantly elevated levels of fc during flare-ups, confirming its utility as an inflammation indicator. however, the absence of a significant correlation between fc and crp during flare-ups suggests that these biomarkers reflect distinct aspects of inflammation. an integrated assessment, combining these biomarkers with clinical scores and imaging, remains essential for optimal monitoring. in conclusion, this study highlights clinical and biological differences based on the type of ibd, the location of lesions (ileal, ileocolic, or colonic), and the nature of complications, emphasizing the importance of a personalized approach to managing ibd that incorporates specific biomarkers (crp, fc) and patient profiles. larger comparative studies exploring genetic and environmental factors are necessary to refine diagnostic tools and further tailor treatments. additional research will help validate these findings and optimize clinical follow-up protocols for more precise and tailored care. disclaimer (artificial intelligence) author(s) hereby declare that no generative ai technologies such as large language models (chatgpt, copilot, etc) and text-to-image generators have been used during writing or editing of this manuscript. consent and ethical approval informed consent was obtained from all participants after they were fully informed about nassar et al.; asian j. immunol., vol. 8, no. 1, pp. 104-119, 2025; article no.aji.135189 117 the 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(2013). inflammatory bowel disease in chinese children: a multicenter analysis over a decade from shanghai. inflamm bowel dis, 19(2), 423–428. disclaimer/publisher’s note: the statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of the publisher and/or the editor(s). this publisher and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. _________________________________________________________________________________ © copyright (2025): author(s). the licensee is the journal publisher. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: https://pr.sdiarticle5.com/review-history/135189 https://pubmed.ncbi.nlm.nih.gov/27914657/ https://pubmed.ncbi.nlm.nih.gov/27914657/ https://pr.sdiarticle5.com/review-history/135189 _____________________________________________________________________________________________________ *corresponding author: e-mail: celso@alergoimuno.med.br; cite as: olivier, celso eduardo, daiana guedes pinto, ana paula monezzi teixeira, cibele silva miguel, jhéssica letícia santos santana, regiane patussi santos lima, nicole sartoreto rocha, and raquel acácia pereira gonçalves santos. 2025. “endotyping cellular and humoral cross-reactivity against chicken meat and egg yolk in non-ige-mediated food proteininduced gastrointestinal allergies”. asian journal of immunology 8 (1):207-21. https://doi.org/10.9734/aji/2025/v8i1172. asian journal of immunology volume 8, issue 1, page 207-221, 2025; article no.aji.141585 endotyping cellular and humoral cross-reactivity against chicken meat and egg yolk in non-ige-mediated food protein-induced gastrointestinal allergies celso eduardo olivier a*, daiana guedes pinto a, ana paula monezzi teixeira a, cibele silva miguel a, jhéssica letícia santos santana b, regiane patussi santos lima c, nicole sartoreto rocha d and raquel acácia pereira gonçalves santos a a instituto alergoimuno de americana, brazil. b instituto de ensino e pesquisa do hospital de amor de barretos, brazil. c biomega medicina diagnóstica, são paulo, brazil. d faculdade de americana, são paulo, brazil. authors’ contributions this work was carried out in collaboration among all authors. all authors read and approved the final manuscript. article information doi: https://doi.org/10.9734/aji/2025/v8i1172 open peer review history: this journal follows the advanced open peer review policy. identity of the reviewers, editor(s) and additional reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here: https://pr.sdiarticle5.com/review-history/141585 received: 25/07/2025 published: 09/08/2025 original research article https://doi.org/10.9734/aji/2025/v8i1172 https://pr.sdiarticle5.com/review-history/141585 olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 207-221, 2025; article no.aji.141585 208 abstract background: non-ige-mediated food protein-induced gastrointestinal allergies (fpi-gias) are poorly understood, although ige-mediated hypersensitivities have been extensively described. patients with poultry meat allergy may present several phenotypes regarding clinical presentations and a wide range of symptom severity, suggesting the existence of several endotypes underlying their diseases. aim: to evaluate the potential of the tube titration of precipitins (ttp) and the leukocyte adherence inhibition test (lait) to discriminate cellular and humoral immunoreactivity against chicken meat extract and chicken egg yolk extract in patients with non–ige-mediated gastrointestinal food protein-induced allergic phenotypes. study design: we examined retrospectively the medical charts of two cohorts of patients clinically diagnosed with non–ige-mediated gastrointestinal food protein-induced allergic phenotypes related to consumption of chicken meat and/or chicken egg yolk, who were investigated with the help of ttp or lait. methodology: the ttp and lait's registered results against chicken meat and chicken egg yolk extracts were distributed in ranges through a cascade distribution chart to outline the variability of the results within the cohorts. the pearson correlation test was used to evaluate the correlation between the results obtained simultaneously with both food allergens. results: the lait for the chicken meat extract and egg yolk extract showed a wide distribution range of results. the ttp for the chicken meat and egg yolk extracts showed a distribution concentrated on the higher dilutions. the pearson correlation test showed a non-significant positive correlation between lait for egg yolk and lait for chicken meat extract; r(98) = 0.156, p = 0.121. the pearson correlation test showed a non-significant positive correlation between ttp for the egg yolk extract and ttp for the chicken meat extract; r(98) = 0.181, p = 0.072. conclusion: our preliminary results support that the ttp and lait performed with chicken meat and egg yolk extracts may discriminate diverse humoral and cellular immunoreactivity degrees in patients suffering from food protein-induced gastrointestinal allergies. despite no statistically significant quantitative correlation between the results, these findings may improve dietary guidelines for impacted individuals and contribute to creating diagnostic biomarkers. keywords: endotype; hypersensitivity; chicken meat; food protein-induced enterocolitis syndrome; leukocyte adherence inhibition test; non–ige-mediated immunoreactivity; poultry meat; precipitins. abbreviations fa : food allergies fpi-gia : food protein-induced gastrointestinal allergies lai : leukocyte adherence inhibition lait : leukocyte adherence inhibition test lmit : leukocyte migration inhibition test ttp : tube titration of precipitins 1. introduction food protein-induced gastrointestinal allergies (fpi-gia) and food allergies (fa) are yet a conundrum for physicians, gastroenterologists, nutritionists, allergists, but mainly for patients who suffer from these conditions (olivier 2013). ige-dependent mechanisms may produce fpigia; however, most cases are produced by hypersensitivities endotypes not mediated by ige (heine 2015, khan 2016, ahmed et al. 2021). non-ige-mediated fpi-gia are associated with a large spectrum of phenotypic conditions, clinically classified by the predominant anatomic localization of the symptoms (such as food protein-induced proctocolitis, food proteininduced enterocolitis, food protein-induced enteropathy, eosinophilic esophagitis, eosinophilic gastroenteritis, food protein-induced gastro-esophageal reflux disease, celiac disease, multiple food protein intolerance of infancy, food protein-induced gastrointestinal motility disorders), as well as some “functional” olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 207-221, 2025; article no.aji.141585 209 conditions, such as infantile colic, which food proteins may also induce (groetch et al. 2025, huang & white 2025). these so-called food protein-induced allergic syndromes may manifest through exchangeable, heterogeneous, and recurrent symptoms with mild, moderate, or severe presentations, posing diagnostic and therapeutic dilemmas (heine 2004). self-reported allergy to poultry meat is not a very common complaint in clinical practice (sloan & powers 1986). however, when researched, the incidence of diagnosis of hypersensitivity to chicken meat (gallus domesticus) may be surprising. sampson reported an incidence of 16,8% of positive skin prick tests to chicken meat among children with atopic dermatitis due to fa (sampson & mccaskill, 1985). the first report of chicken meat allergy was described in 1982 as a non–ige-mediated enteropathy in a child who proved to be allergic to chicken meat and cow’s milk through provocation tests monitored by jejunal biopsies demonstrating severe villous atrophy after ingestion of chicken meat (vitoria et al. 1982). it took fourteen years for the subsequent description of a non–ige-mediated eosinophilic gastroenteritis to be documented after a chicken meat provocation test monitored by intestinal biopsies (vandenplas et al. 1994). food proteininduced enterocolitis syndrome is a non-igemediated fpi-gia that may present with symptoms such as flatulence, bloating, cramps, postprandial discomfort, vomiting, and diarrhea, due to chronic exposure to an offending food, or general symptoms, such as failure to thrive, while avoiding an offending food. (agyemang & nowak-wegrzyn 2019). initially described by pediatricians and attributed to liquid foods, such as cow’s milk and liquid infant soy formulas, it was becoming apparent that solid foods (including poultry meat) could also be responsible for food protein-induced enterocolitis syndrome (nowak-wegrzyn et al. 2003). patients with poultry meat fa may present several phenotypes regarding clinical severity and features, suggesting the existence of several endotypes underlying clinical symptoms (wanniang et al. 2022). as with any fa, the endotypes behind poultry allergy are primarily classified as ige-mediated and non–igemediated. chicken meat allergy has complex sensitization profiles with nine major established allergens and twenty-five proposed candidates (guiddir et al. 2024). the major chicken meat allergen identified by a proteomics-based approach is a myosin light chain protein designated gal d 7 (shared by several poultry species), containing the majority of ige-binding epitopes, characterized by remarkable thermal stability, refolding capacity, and resistance to salivary and gastrointestinal enzymes (klug et al. 2020). poultry species develop common allergens, making usual cross-reactivity between chicken meat and turkey meat, as well as with other species (cahen et al. 1998). allergic reactions following the administration of hen’s egg yolk-based vaccines (such as for yellow-fever and typhus) have been described since the forties (rubin 1946). allergic symptoms related to ingestion of hen’s egg yolk have been reported since the 1950s, mainly in children, including regurgitation, eczema, and respiratory symptoms (todd et al. 1957). allergy to chicken meat can also develop as a cross-sensitivity against hen egg proteins (birdegg syndrome) (hemmer et al. 2016). bird-egg syndrome is a peculiar ige-mediated crosshypersensitivity to egg-yolk alpha-livetin (chicken serum albumin or gal d 5), also associated with inhaling birds’ feathers and dander (mandallaz et al. 1988, szépfalusi et al. 1994). three chicken meat allergens: parvalbumin (gal d 8), enolase (gal d 9), and aldolase (gal d 10) are also present in fish and are responsible for a crossreactive hypersensitivity condition called the “fish–chicken syndrome” (kuehn et al. 2016). usually, patients present reactions to multiple foods, an issue reinforced by the few laboratory tests which can suggest the possibility of nonige-mediated hypersensitivities (katz & goldberg 2014). some facilities employ the lymphocyte stimulation test for diagnosing hen’s egg yolk– induced enterocolitis syndrome (kajita et al. 2023). humoral immunoreactivity against food allergens and aeroallergens had been classically evaluated by precipitins (augustin 1953, augustin et al. 1960, cunningham-rundles et al. 1978, ferguson and carswell 1972, heiner et al. 1962). we also routinely employ the tube research of precipitins (ttp) in our facilities as a triage to evaluate humoral non–ige-mediated immunoreactivity against suspected allergens before performing more exhaustive in vivo provocation tests (olivier et al. 2023b, olivier et al. 2021e, olivier et al. 2021d, olivier et al. 2024e, olivier et al. 2024c). olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 207-221, 2025; article no.aji.141585 210 the leukocyte adherence inhibition test (lait) and its similar assay, the leukocyte migration inhibition test (lmit), have classically been used to differentiate non–ige-mediated immunoreactivity against microorganisms and aeroallergens (fink et al. 1987, kallen and nilsson 1979, kuratsuji 1981, thomson 1982). the lait and the lmit have also classically been used to differentiate non-ige-mediated immunoreactivity against food allergens (allardyce & shearman 1975, george & vaughan 1962, ashkenazi et al. 1980, butler et al. 1981, papageorgiou et al. 1983). non–igemediated cellular immunoreactivity against food allergens had also been reported by our group with the help of the lait (olivier et al. 2022b, olivier et al. 2022a, olivier et al. 2022c, olivier et al. 2023a). to evaluate the potential of the lait and ttp to endotyping non–ige-mediated cellular and humoral immunoreactivity against egg yolk and chicken meat, we retrospectively compiled the electronic medical charts of patients diagnosed primarily with gastrointestinal food allergies (associated or not with other extra-intestinal allergic phenotypes) related to non–ige-mediated hypersensitivity, who were investigated for immunoreactivity by one of these two assays. the present study is a proof-of-concept that hypothesizes that lait and the ttp may demonstrate a correlation between cellular and/or humoral immunoreactivity between egg yolk and chicken meat proteins in patients suffering from non–ige-mediated fpi-gia. 2. materials and methods 2.1 subjects after receiving institutional review board approval from the instituto alergoimuno de americana (brazil; 05/2025), we reviewed the electronic chart of 10.600 outpatients who attended our facility from january 2018 to january 2025. a cohort of 100 consecutive outside patients (ttp cohort) had been submitted to ttp with chicken meat extract and egg yolk extract for presenting non–ige-mediated fpi-gia. this cohort counted 35 males; mean age 43.6 years; sd 22 years; range 5 to 94 years; median 42.5 years; modes = 46 (appeared four times); geometric mean = 36.5 years. a cohort of 100 consecutive outside patients (lait cohort) had been simultaneously submitted to lait with chicken meat extract and egg yolk extract for presenting non–ige-mediated fpigia. this cohort counted 35 males; mean age 34 years; sd 24.7 years; range 1 to 100 years; median 31 years; modes = 5 years (appeared six times); geometric mean = 22.3 years. this study did not include patients under biological and/or systemic anti-inflammatory therapy. these procedures were offered to patients with clinical suspicion of chicken meat hypersensitivity who demonstrated a nonreactive or inconclusive skin test against chicken meat (olivier et al. 2013). 2.2 extracts 2.2.1 chicken meat extract chicken meat (300g of breast) acquired from the local market (half cooked and half uncooked) was crushed, homogenized, and then left for 48 hours in a coca-based extractor solution (propylparaben 0.5g, methylparaben 1g, sorbitol 30g, nacl 5g, nahco3 2.5g, 1,000ml h2o) at 4 °c for protein extraction before centrifugation and separation of the water-soluble fraction from solid particles and oily fraction (coca, 1922). the protein quantification of the allergen extracts was done according to bradford’s protein-dye binding methodology (bradford 1976). the solution was diluted in antigen dilution solution (nacl 10g; kh2po4 0.72g; na3po4 2.86g; methylparaben 1g; propylparaben 0.5g; glycerin 400ml; h2o 600ml) to an estimated protein concentration of 1 mg/ml and stored at 4 °c in amber opaque glass vials. the chicken meat extract solution was used to perform allergic skin tests, ttp, and lait. all relevant and mandatory laboratory health and safety measures have been complied with during the experiments. 2.2.2 egg yolk extract three cooked egg yolks and three uncooked egg yolks were prepared similarly with the chicken meat extract. 2.3 lait: ex vivo investigation: leukocyte adherence inhibition test 2.3.1 lait: procedure for allergen ex vivo challenges we performed the lait as previously described (olivier et al. 2012, olivier et al. 2014, olivier et olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 207-221, 2025; article no.aji.141585 211 al. 2021a, olivier et al. 2021b, olivier et al. 2021c). shortly, each donor's fresh plasma was divided into two parts and used in parallel ex vivo challenging tests with the chicken meat extract (or the egg yolk extract) and the unchallenged plasma (added with antigen dilution solution as a control). we collected plasma with high leukocyte content (buffy coat) from the heparinized tube after one hour of sedimentation at 37 °c. then, we distributed aliquots of 100 μl into eppendorf tubes with (or without) the challenging extract and kept them under agitation for 30 minutes (200 rpm at 37 °c). 2.3.2 lait: procedure for adherence assay after incubation, the plasma was allocated into a standard neubauer hemocytometer counting chamber with a plain, non-metallic glass surface and left to stand for 2 hours at 37 °c in the humidified atmosphere of the covered water bath to allow leukocytes to adhere to the glass. next, we counted the leukocytes, removed the coverslip, and washed the chamber by immersion in a beaker with phosphate buffer saline (pbs) at 37 °c. then, we added a drop of pbs to the hemocytometer's chamber and placed a clean coverslip over it. the remaining cells were counted in the same squares as previously examined. 2.3.3 lait: procedure for calculation the percentage of leukocyte adherence (la) of each assay was estimated as: (the number of leukocytes observed on the hemocytometry chamber after washing divided by the number of leukocytes observed on the hemocytometry chamber before washing) and multiplied by 100 (%). the leukocyte adherence ratio (lar) was estimated based on the ratio between the la from the antigen-specific challenged plasma and the la from the unchallenged control plasma: lar = la of the challenged sample divided by la of unchallenged control plasma multiplied by 100 (%). to further calculate the leukocyte adherence inhibition (lai), we subtracted the lar from 100 (%). we employed the lai results for the cascade distribution chart and the statistics calculations; both performed with the help of the microsoft excel® statistical package. 2.4 ttp: in vitro investigation: tube titration of precipitins as previously reported, the semi-quantitative ttp against the chicken meat extract (or the egg yolk extract) was performed in a transparent vitreous tube array (olivier et al. 2024f, olivier et al. 2024d, olivier et al. 2024b, olivier et al. 2024a). shortly, the patient’s blood was collected in a clot-activator collecting tube. after separation, the serum was centrifuged at 2,000 rpm for 10 minutes. each allergen extract was allocated in sets of eleven glass tubes at progressively duplicated serum dilutions. the progressive dilutions were combined with separated aliquots of 15 μl of the allergen extract with 250 μl of the patient’s serum, progressively diluted into physiological saline solution (nacl 0,9%) in the dilution ratios of 1:1; 1:2; 1:4; 1:8; 1:16; 1:32; 1:64; 1:128; 1:256; and 1:512. one tube was a blank control done with water and serum to observe occasional spontaneous precipitation (sia test). after 24 hours, the tubes were examined, and the titers (the highest dilution factor that yields a positive reading) were recorded (williams & chase 1971). 3. results as a retrospective survey, there was no research protocol; therefore, we report the incidental immune investigation as registered in the digital medical charts. the ttp for the chicken meat extract showed a distribution concentrated on the higher dilutions (fig. 1). there was no negative result. the mean was estimated at 1:307; the median was 1:256; the standard deviation was estimated at 1:175; the mode was 1:512 (appeared 39 times). the ttp for the egg yolk extract showed a distribution concentrated on the higher dilutions (fig. 2). there were two negative results. the mean was estimated at 1:335; the median was 1:256; the standard deviation was estimated at 1:179; the mode was 1:512 (appeared 48 times). the pearson correlation estimates a nonsignificant, small positive relationship between ttp for the egg yolk extract and ttp for the chicken meat extract; r(98) = 0.181, p = 0.072. see fig. 3. the lait for the chicken meat extract showed a wide distribution range of results. the lai ranged from 0% to 99%. the mean was 48.5%; the median was 50.5%; the standard deviation was 29.8%; the mode was 0% (appeared eleven times). the cascade distribution demonstrates a wide range of lai results (see fig. 4). some olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 207-221, 2025; article no.aji.141585 212 patients showed low or moderate immunoreactivity during the ex vivo challenge test. in contrast, others displayed strong immunoreactivity, which could reflect the participation of chicken meat allergens in a non– ige-mediated hypersensitivity condition in these patients. the lait for the egg yolk extract showed a wide distribution range of results. most results were concentrated in the more immunoreactive groups. the lai ranged from 0% to 99%. the mean was 50.2%; the median was 51%; the standard deviation was 27%; the mode was 0% (appeared eight times). the cascade distribution demonstrates a wide range of lai results (see fig. 5). some patients showed low or moderate immunoreactivity during the ex vivo challenge test. in contrast, others displayed strong immunoreactivity, which could reflect the participation of egg yolk allergens in a non–igemediated hypersensitivity condition in these patients. the pearson correlation estimates a nonsignificant small positive relationship between lait for egg yolk and lait for chicken meat extract; r(98) = 0.156, p = 0.121. see fig. 6. fig. 1. cascade distribution chart of the tube titration of precipitins (x-axis %) resulting from the chicken meat extract against the serum of the ttp cohort of 100 tests/subjects (y-axis) fig. 2. cascade distribution chart of the tube titration of precipitins (x-axis %) resulting from the hen’s egg yolk extract against the serum of the ttp cohort of 100 tests/subjects (y-axis) olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 207-221, 2025; article no.aji.141585 213 fig. 3. dispersion chart of the tube titration of precipitins (ttp) results against hen’s egg yolk extract (x-axis), plotted against the ttp results against chicken meat extract (y-axis). the tendency line shows a non-significant, small positive relationship between the assays; r(98) = 0.181, p = 0.072 fig. 4. cascade distribution chart of the range groups of leukocyte adherence inhibition (lai) results (x-axis %) of the ex vivo challenge test against chicken meat extract monitored by the leukocyte adherence inhibition test (lait), according to the respective number of outcomes over the lait cohort with 100 tests/subjects (y-axis) olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 207-221, 2025; article no.aji.141585 214 fig. 5. cascade distribution chart of the range groups of leukocyte adherence inhibition (lai) results (x-axis %) of the ex vivo challenge test against hen’s egg yolk extract monitored by the leukocyte adherence inhibition test (lait), according to the respective number of outcomes over the lait cohort with 100 tests/subjects (y-axis) fig. 6. dispersion chart of the leukocyte adherence inhibition (lai) results of the ex vivo challenge test against hen’s egg yolk extract (x-axis %), plotted against the lai results of the ex vivo challenge test against chicken meat extract (y-axis %). the tendency line shows a nonsignificant, small positive relationship between the assays; r(98) = 0.156, p = 0.121 4. discussion the semi-quantitative titration of precipitins is a pioneering technique to evaluate humoral immunoreactivity (wells 1911, hunter 1905, olivier et al. 2025b, olivier et al. 2025a). precipitating antibodies suggest the presence of a specific humoral immune response against the tested antigens (gell et al. 1946, ishizaka et al. 1959). before the discovery of ige, the research of precipitins was the leading way to realize in vitro diagnosis of immunoreactivity against antigenic and allergenic agents (augustin & hayward 1960). the discovery of ige and its olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 207-221, 2025; article no.aji.141585 215 reaginic activity, as well as the concomitant development of the radio-allergo sorbent test (rast) to detect specific ige antibodies, focused the attention of the scientific and medical community on this particular antibody class, mainly after the incorporation of similar nonradioactive assays into routine clinical assays (wide 1967, ishizaka & ishizaka 1967). although the serum-free ige (as detected by routine immunoassays) is not held responsible for clinical symptoms (as are the tissue-bound ige, responsible for the autocoids released after the encounter with the antigen), the research on serum-specific ige constructed a paradigm in the physician’s mentality. the research of tissuebond ige, as performed by allergic skin tests, is easily performed; however, the demonstration of tissue-bond ige in mucosal sites is a complex task, substituted by the research of eosinophils and t cells in endoscopic biopsies of patients with fa. this technical hindrance did not prevent the creation of the “ige-mediated local allergic reaction” concept in food hypersensitive patients (lin et al. 2002). this concept gave further origin to the similar concept of “ige-mediated local allergic rhinitis”, which advocates the mucosal production of ige at insufficient concentrations to be reflected in the blood immunoassays or the skin tests (rondon et al. 2010). usually, the correlation and the distribution of simultaneous positive specific-ige against food allergens and inhalant allergens are weak; however, when properly investigated, polysensitization is more the rule than the exception in fpi-gia (zhang et al. 2025, čelakovská et al. 2024). the paradigm of the specific antibody to diagnose the etiology of the allergic symptoms has led to the “ige culture” and a pressing pursuit to determine the utility of the specific igg to diagnose the non–ige-mediated hypersensitivity conditions (atwah & koshak 2024). the use of specific igg against food allergens may be contentious and controversial since the igg may sometimes act as a hypersensitivity trigger and sometimes as an allergen blocker, depending on its subclass, the antigen-antibody proportion, and the participation of other immune players (alkhateeb 2020). igg antibodies can participate in type ii (antibody-dependent cell-mediated) and type iii (immune complex disease) gell and coombs hypersensitivity reactions, which may be theoretically reproduced by the lait and the ttp assays, respectively (olivier et al. 2021a). however, there is a lack of effective and practical tools to diagnose gastrointestinal inflammatory reactions due to fa in patients with no evidence of systemic circulatory ige (olivier 2022). the lait is an ex vivo challenge test performed with a viable leukocyte buffy coat that can theoretically explore most known immune pathways, as it allows the interaction of all immune-circulating participants with the allergens (olivier et al. 2021a). several immune pathways can produce the final leukocyte adherence inhibition (tong et al. 1979, halliday 1974). the present study is a proof-of-concept that hypothesizes that lait and the ttp may differentiate diverse degrees of cellular and humoral immunoreactivity against hen’s egg yolk and chicken meat allergens among patients suffering from non–ige-mediated fpi-gia. as the tests were performed simultaneously with the same venous sample with the two allergens, it was possible to calculate a correlation test to distinguish some order of cross-reactivity between them. the retrospective compilation of our data showed a large distribution of results when we ascertained the results of ttp and lait to explore humoral and cellular immunoreactivity against two chicken food allergens. these immunoassays did not precisely identify the mechanisms responsible for the clinical condition. instead, they provide evidence about cellular and humoral immunoreactivity distributed into an extensive spectral range that may suggest immune tolerance or hypersensitivity. this preliminary retrospective survey demonstrated extensive results from the ttp and the ex vivo challenge test monitored by lait against hen’s egg yolk and chicken meat allergens in two cohorts of non–ige-mediated food allergic patients. ttp and lait are complementary triage tests used at our facilities to select worthwhile antigens to proceed with more laborious in vivo provocation tests when the specific ige is undetectable. none of our patients presented an exclusive reaction to these allergens. every patient was simultaneously tested for several chemical and biological allergens, demonstrating positive results for some of them. our results suggest that patients suffering from fpi-gia due to hen’s egg yolk allergy may experience additional crossimmunoreactivity against chicken meat allergens and vice-versa. olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 207-221, 2025; article no.aji.141585 216 5. limitations this study is a retrospective analysis of data collected over seven years. there was no research protocol, and the subject's data was limited to the essentials available on our electronic sheets. therefore, we could not establish a cross-comparison between positive and negative controls to validate the results. the number of subjects is appropriate for preliminary analyses; however, future studies must be more comprehensive. the lack of a research protocol implies the possibility of a bias produced by the physician's point of view who indicated the exam (olivier, ce) based on a clinical suspicion led by anamnesis, physical examination, results of skin tests, and the research of specific ige. the study lost the follow-up of most patients, hampering the registration of the relationship between the immunoassay results and the patient's clinical outcome. unfortunately, it was also impossible to compare the two procedures with paired tests because they were taken from distinct groups of patients. 6. conclusion our preliminary results show that the lait and ttp may differentiate diverse degrees of immunoreactivity against chicken meat and egg yolk extracts in patients clinically diagnosed with non–ige-mediated gastrointestinal food allergies. lait and ttp are inexpensive, can be performed with minimum laboratory equipment, and can be incorporated into strategies to address respiratory and food allergy health disparities (anagnostou et al. 2025). as a preliminary report, the propaedeutic meaning of the presented results and the possibility of interferents must be yet established (anouar 2024). more studies focused on the quality-bydesign approach, with prospective larger doubleblind cohorts needed to evaluate the potential contribution of lait and ttp for endotyping cellular and humoral immunoreactivity in patients suspected of hypersensitivity against chicken allergens (chiarentin et al. 2023). 7. future directions and recommendations for clinical practice the primary intended use of in vitro or ex vivo allergen challenge tests is to spare the patients from being submitted to unnecessary, exhaustive, and dangerous in vivo challenge tests. exploring the humoral and the cellular arms of immune systems, the ttp and lait alone or combined may represent, in the near future, a tool for allergists to construct an etiologic diagnosis from their patients, as well as determine the endotypes (mechanisms) of hypersensitivity, in order to choose more convenient and personalized therapies for them. adding data provided by ttp and lait may also contribute to streamlining biomedical research and improving tools such as large language models, usually used by clinicians as a decision support system to enhance diagnostic accuracy (abers & mathias 2025). disclaimer (artificial intelligence) author(s) hereby declare that no generative ai technologies such as large language models (chatgpt, copilot, etc) and text-to-image generators have been used during writing or editing of this manuscript. ethical approvals as a retrospective survey of results recorded in cognito, consent was given collectively by the institution's ethics committee following the principles of the declaration of helsinki (wma 2013). acknowledgements the instituto alergoimuno de americana funded this work. we want to thank the laboratory technicians alessandra vieira de oliveira and luciana sacilotto carvalho for their help with the exams. competing interests authors have declared that no competing interests exist. references abers, m. s., & mathias, r. a. 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(2025). investigating allergen-specific ige distribution and correlations in chronic urticaria: a retrospective study in shanghai, china. european journal of medical research, 30(1), 182. disclaimer/publisher’s note: the statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of the publisher and/or the editor(s). this publisher and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. _________________________________________________________________________________ © copyright (2025): author(s). the licensee is the journal publisher. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: https://pr.sdiarticle5.com/review-history/141585 https://pr.sdiarticle5.com/review-history/141585 _____________________________________________________________________________________________________ *corresponding author: e-mail: naseemar8@gmail.com; cite as: naseema. r. 2025. “an immunocomputing approach to the development of multiepitope vaccine against mycobacterium tuberculosis”. asian journal of immunology 8 (1):269–284. https://doi.org/10.9734/aji/2025/v8i1178. asian journal of immunology volume 8, issue 1, page 269-284, 2025; article no.aji.147582 an immunocomputing approach to the development of multiepitope vaccine against mycobacterium tuberculosis naseema. r a* a department of biotechnology, mepco schlenk engineering college, sivakasi, tamil nadu, india. author’s contribution the sole author designed, analyzed, interpreted and prepared the manuscript. article information doi: https://doi.org/10.9734/aji/2025/v8i1178 open peer review history: this journal follows the advanced open peer review policy. identity of the reviewers, editor(s) and additional reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here: https://pr.sdiarticle5.com/review-history/147582 received: 17/09/2025 published: 24/11/2025 abstract tuberculosis (tb) is the second most deadly airborne infectious disease caused by mycobacterium tuberculosis, posing a major global health risk dw. nearly one-third of the world’s population is infected, with low-income countries most affected due to limited access to diagnostics and treatments. although the bacillus calmette–guérin (bcg) vaccine offers protection in infancy, its reduced efficacy beyond 20 years limits long-term tb control. this study focuses on drafting a multi-epitope peptide-based vaccine against tb using immune-informatics approaches. ten m. tuberculosis-specific antigenic proteins (ppe39, ppe68, rv0310c, pe_pgrs35, pe_pgrs31, cfp10, rv1975, lpqg, esxv, and espj) were analyzed to identify potent immunogenic regions. five cytotoxic t lymphocyte (ctl) epitopes, five helper t cell (htl) epitopes, and several b-cell epitopes are proficient of causing strong immune responses, including interferon-γ (ifn-γ) production, were selected. a total of 27 epitopes were linked using aay, gpgpg, and kk linkers to construct the vaccine, which was assessed for physicochemical properties, antigenicity, allergenicity, and toxicity. the design demonstrated stability and strong immunogenic potential. molecular docking with the tlr-4 monomer showed favorable binding affinity, indicating its original research article https://doi.org/10.9734/aji/2025/v8i1178 https://pr.sdiarticle5.com/review-history/147582 naseema; asian j. immunol., vol. 8, no. 1, pp. 269-284, 2025; article no.aji.147582 270 potential as an effective vaccine candidate. by applying immune-informatics tools for precise epitope selection and vaccine design, this research provides a cost-effective strategy to enhance tb prevention. the study offers a strong framework for developing next-generation tb vaccines and supports global efforts to eliminate tuberculosis by 2035. keywords: mycobacterium tuberculosis (m.tb); cytotoxic t lymphocyte (ctl); helper t cell (htl); interferon-γ (ifn-γ); bacillus calmette–guérin (bcg). 1. introduction “tuberculosis (tb) is an airborne disease caused by the bacterium mycobacterium tuberculosis. m. tb and its seven closely related species namely m. bovis, m. africanum, m. microti, m. caprae, m. pinnipeti, m. canetti and m. munji collectively comprises m. tuberculosis complex. but not all of these species are reported to cause disease in humans” (beresford & sadoff, 2010). “the majority cases of tb are found to be caused by m. tuberculosis (wheeler et al., 2007). tb remains one among the main global health threats which causes millions of deaths annually across the globe. majority of affected people hail from low income and middle-income countries (lmic) like countries from african subcontinent” (faust et al., 2019). “an estimated 10 million people fell ill with tb worldwide in 2019, which includes 5.6 million men, 3.2 million women, and 1.2 million children” (saha & raghava, 2006). “tb is existing in all countries and all age groups. 5 – 15 % patients infected with only tb are likely to develop active tb in their lifetime. tb is more severe in hiv patients and is one of the main reason for their mortality. bcg (bacille calmette guerin) is the only vaccine available for preventing tb in humans. although bcg is of low-cost and widely used vaccine in many of the countries, its potency has varied generally in many clinical assays spanned between 080 % (brandt et al., 2002; pablos-mendez et al., 2002). it offers poor protection in adults and adolescents against pulmonary tuberculosis” (ferraz et al., 2004). also failed to treat latent tb infection (ltbi) (weinreich olsen et al., 2000). in the presence of immune-suppression, reversion of virulence may take place and possible induction of disease may happen (sharma et al., 2021). for these various reasons, the who has framed “the end tb strategy which targets the prevention, care and control for tuberculosis. to meet all the requirements and to satisfy all the problems associated with mycobacterium tuberculosis, there is an urge to build an efficient vaccine, which offers complete safeguard against tuberculosis” (world health organization, 2018). “in the current study, we have chosen ten mycobacterial proteins from different region of difference (ppe39, ppe68, rv0310c, pe_pgrs35, pe_pgrs_31, cfp10, rv1975, lpqg, esxv, espj) and an adjuvant (rpfe) for vaccine construct. pe/ppe families are found to be associated with pathogenicity of mtb infection. as in all pathogenic mycobacteria, the members of these families are abundant and comparatively shows less presence in nonpathogenic mycobacteria. it is observed that, pe/ppe proteins play a major role in processing of macrophages” (bigi et al., 2000). adhesion of pathogenic proteins on the surface of macrophage receptors, immune response to pathogenic proteins, resistance of intracellular stress, phagocytosis, intracellular survival in the host environment and regulation of cell fate are the key events involved in the host-pathogen interactions. during various stages of infection, the cfp10/esat6 complex in mycobacterium tuberculosis could play a role in regulating macrophage death (trott & olson, 2010). “the major advantage in peptide-based vaccines is the absence of live or attenuated infectious materials. hence there is zero risk for reversion of virulence” (trunz et al., 2006). “to enhance the stability, solubility lipid, carbohydrate and phosphates groups can be readily introduced. one major advantage of epitope-based vaccines is that they rely on a small, precisely defined antigen, making it easier to understand and evaluate its immunogenicity and antigenicity (choi et al., 2015). the induction of neutralizing antibodies and a protective t helper and a ctl response by t cell and b cell epitope must be required for a good tb vaccine. to maximize coverage, the ctl and htl epitopes were tested for their ability to bind with the most prevalent human hla alleles for mhc class i and ii (martinot et al., 2016). “the adjuvant rfpe was included to the vaccine draft to enhance its immunogenicity” (bellini & horváti, 2020). several vaccine sequences were generated and analyzed for allergenicity, toxicity, antigenicity, and important physicochemical properties such naseema; asian j. immunol., vol. 8, no. 1, pp. 269-284, 2025; article no.aji.147582 271 as isoelectric point, solubility, and half-life in different hosts” (kelley et al., 2015). “homology modeling was carried out for all vaccine sequences, and the best 3d model was chosen based on the recommendations of online servers” (rapin et al., 2010). the stereochemical geometry of this model was then evaluated, both at the residue level and overall structure (sanches et al., 2021). finally, molecular docking was performed between the optimized 3d vaccine model and tlr4 to study binding interactions. 2. methods and methodology 1. htl epitopes prediction: helper t lymphocyte (htl) epitopes were forecasted using the netmhciipan 4.0 server, which is an online tool for identifying immune epitopes based on a large collection of experimentally validated data. all protein sequences of the chosen antigens were handed in simultaneously in fasta format. the default settings were used to predict epitopes across the total human hla reference set, specifying an epitope length of 9 amino acids. the predicted epitopes were then ranked and scored according to their binding affinity. 2. ctl epitopes prediction: cytotoxic t lymphocyte (ctl) epitopes were predicted using the netctlpan 1.2 server. all amino acid sequences from the selected proteins were uploaded together in fasta format. using the full human hla reference set, 9-mer epitopes were predicted with default settings, and the resulting peptides were ranked by their scores. the chosen epitopes were then examined for their allergenic, toxic, and antigenic properties. mhc i–related hla-c epitopes were identified with the netctlpan 1.1 server, with all selected protein sequences submitted at once in fasta format.this prediction is based on mhc class – i binding affinity, proteasomal cleavage efficiency and tap transport efficiency. 20 alleles of hla-c haplotype were given as input for prediction. these 20 alleles were taken as a reference set from iedb. in total five hla -c epitopes were chosen for the vaccine construct. 3. b‑cell epitopes prediction: the b cell epitopes were predicted using abcpred web server. all the amino acids sequences of the selected proteins were submitted at a time in fasta format (hougardy et al., 2007). the top 30 epitopes were selected based on the score and was further analyzed for antigenicity, toxicity and allergenicity. any epitopes flagged as allergenic, toxic, or nonantigenic by allertop v2.0, toxinpred, or vaxijen were not included in the study. altogether, fourteen b-cell epitopes were chosen from the ten selected proteins (supplementary table 1). 4. ifn-γ analysis: ifn-γ plays a central role in defending the body against intracellular pathogens, and in mycobacterium tuberculosis infection it is especially important for activating macrophages (dimitrov et al., 2014). the ifn were predicted from the ifn epitope server (http://crdd.osdd.net/raghava/ifnepitope/). here, we analyzed ifn-γ activity for all the selected epitopes. 5. vaccine construct preparation: five vaccine constructs were randomly designed using the adjuvant (rpfe), which was attached to the n-terminal end. each construct followed the same layout, placing b-cell, htl, ctl, and then hla-c epitopes in sequence. the epitopes were joined using kk, gpgpg, and aay linkers, and the adjuvant was added using an eaaak linker. 6. prediction of epitope’s antigenicity, allergenicity and toxicity: five vaccine constructs were randomly designed using the adjuvant rpfe, which was attached to the n-terminal end. in each construct, the selected epitopes were arranged in the order of b-cell, htl, ctl, and hla-c. the b-cell, htl, ctl, and hla-c epitopes were joined using kk, gpgpg, and aay linkers, respectively, while the adjuvant was connected through an eaaak linker, the relative abundance of amino acids, and β-strand forming propensity (doytchinova & flower, 2007). prediction of antigenicity was done by alignment-independent fashion based on its physicochemical properties using vaxijen online web server (gupta et al., 2013). this tool provided varied options for the organism selected for analysis was bacteria, for which the corresponding default threshold score (0.4) was applied automatically. the toxinpred server was employed to assess the toxicity of the selected sequences, as this tool generates all possible naseema; asian j. immunol., vol. 8, no. 1, pp. 269-284, 2025; article no.aji.147582 272 mutants of the input peptides for evaluation (branger et al., 2004). all parameters in both allertop v.2.0 and toxinpred were maintained at their default settings. 7. homology modelling of vaccine constructs: the five vaccine constructs were analyzed through homology modelling on the swissmodel server to identify the most structurally stable option. the generated models were saved in pdb format for later evaluation. 8. structure refinement of vaccine construct: the tertiary structure of the selected vaccine draft was refined utilizing the castp web server. castp applies structural perturbation and molecular dynamics simulations to achieve protein relaxation. the most stable and accurately refined model was selected for further evaluation, including physicochemical property assessment and molecular docking studies. 9. physiochemical properties of vaccine construct: the physicochemical characteristics of the vaccine construct were found using the protparam tool available online. this tool evaluates several properties of a protein, including molecular weight, theoretical isoelectric point (pi), instability index, protein length, estimated half life, aliphatic index, and the grand average of hydropathicity (gravy). 10. molecular docking of vaccine construct: the refined 3d structures of the vaccine constructs were utilized for molecular docking studies. the crystal structure of the human tlr4 receptor complex (pdb id: 3fxi) was retrieved from the protein data bank. docking was performed using the pydock web server with the monomeric form of the human tlr4 receptor (schorey & harding, 2016). additionally, autodock vina was employed to perform docking using the a-chain of the tlr4 crystal structure with the vaccine constructs (park et al., 2009; heo et al., 2013). using the castp server, we identified the active and passive binding pockets of the tlr4 receptor and the refined vaccine constructs. the corresponding pdb files were then uploaded to the pydock server for docking analysis. fig. 1. the schematic illustration of the final vaccine construct shows the arrangement of epitopes, represented within colored boxes and connected by linker sequences. the designed construct is made up of 623 amino acids, including a 172-amino-acid adjuvant (rpfe/rv2450c) highlighted in yellow. it incorporates 14 b-cell epitopes (blue), 5 htl epitopes (orange), 5 ctl epitopes (green), and 3 hla-c epitopes (pink). an eaaak linker attaches the adjuvant to the nterminus, while the b-cell, htl, ctl, and hla-c epitopes are connected using kk, gpgpg, and aay linkers naseema; asian j. immunol., vol. 8, no. 1, pp. 269-284, 2025; article no.aji.147582 273 a) b) fig. 2. vaccine construct homology modelling and structural refinement: (a) the rpfe vaccine construct’s three-dimensional structure was first created using swiss-model (b) refined version was produced using castp (binkowski et al., 2003) 11. immune simulation for vaccine efficacy “c-immsim was used to assess the immune response and immunogenicity of the multiepitope vaccine. the server applies a positionspecific scoring matrix and machine learning, along with epitope prediction, to model immune interactions. it contains 6,533 antigenic epitopes and 33 different human hla allele sets, simulating responses by pairing epitope sequences with lymphocyte receptors” (brosch et al., 2007). “the tool predicts immune responses by simulating three important mammalian anatomical regions: the thymus, bone marrow, and a tertiary lymphoid organ, including the spleen, lymph nodes, or tonsils (mangtani et al., 2014). the vaccine construct and the pdb files for tlr4 and mhc i were uploaded to cimmsim, using the default settings for all parameters. 3. results 1. b-cell epitopes prediction: a total of thirty b-cell epitopes were first predicted with abcpred and then evaluated for their antigenic, toxic, and allergenic properties. epitopes identified as toxic, allergenic, or nonantigenic through toxinpred, allertop v.2.0, and vaxijen, respectively, were excluded from further analysis. ultimately, fourteen b-cell epitopes were selected for inclusion in the vaccine construct. 2. htl epitopes prediction: from the initial set of twenty predicted htl epitopes, only those exhibiting antigenic, nontoxic, and non-allergenic properties were considered. using these criteria, five htl epitopes were selected for inclusion in the vaccine construct. 3. ctl epitopes prediction: twenty ctl epitopes with the highest binding affinity to mhc class i molecules were selected for further evaluation of allergenicity, antigenicity, and toxicity. any duplicate epitopes found across multiple hla sets were removed. only epitopes that were non-allergenic, non-toxic, and antigenic were retained for downstream analyses. in total, five ctl epitopes and three hla-c epitopes derived from ppe39, ppe68, rv0310c, pe_pgrs35, pe_pgrs31, cfp10, rv1975, lpqg, esxv, and espj were selected for vaccine construction. 4. vaccine construct preparation: two random vaccine constructs were generated by shuffling the arrangement of the selected epitopes. in each construct, the epitopes were organized in the order of b-cell, htl, ctl, and hla-c. the b-cell, htl, ctl, and hla-c epitopes were connected using kk, gpgpg, and aay linkers, respectively. the adjuvant proteins rpfe and rpfb were attached to the n-terminal end of the vaccine constructs via an eaaak linker. 5. homology modelling and selection of best vaccine construct: homology modelling and structural validation of the vaccine constructs were performed using the swiss-model web server. the generated models were evaluated based on the ramachandran plot statistics, mean score, and naseema; asian j. immunol., vol. 8, no. 1, pp. 269-284, 2025; article no.aji.147582 274 z-score provided by swiss-model reports. among all constructs, vaccine construct 1 exhibited the most favorable results, with residues in the most preferred regions, additionally allowed regions, generously allowed regions, and disallowed regions accounting for 93.06%, 5.17%, 1.77%, and 0.0%, respectively. while the z-score, rmsd, molprobity, clash score for verify3d were -1.10,0.248,1.39 and 6.9 respectively. the final chosen vaccine construct was also analyzed for its antigenicity using vaxijen tool where antigenicity was predicted to be 1.0664. the chosen vaccine construct sequence is shown in supplementary fig.1 and the graphical representation of vaccine construct in order of epitopes utilized can be found in fig. 1. 6. vaccine construct tertiary structure refinement: the homology model generated using swiss model (fig. 2a) is exhibiting the finest ramachandran plot values was exposed to improvement using castp. out of all the five models generated by castp, model 2 (fig. 2b) was found to be most appropriate where values for gdt-ha, rmsd and molprobity were 0.993, 0.248 and 1.378 respectively. after refinement, the most preferred regions of ramachandran increased to 93.72% when compared to the structure obtained from swiss model (fig.3). at last, the refined model 2 was chosen for further investigations like molecular docking and dynamic simulations. fig. 3. ramachandran plot analysis of the refined tertiary structure of vaccine construct obtained from rampage web server. after refinement, most preferred regions increased to 93.72% as compared to the crude 3d structure derived from swiss model where the most favoured regions were 93.06% naseema; asian j. immunol., vol. 8, no. 1, pp. 269-284, 2025; article no.aji.147582 275 7. vaccine constructs physicochemical properties: the selected refined vaccine construct consisted of 623 amino acids with an estimated molecular weight of 61.83 kda. typically, vaccine candidates with molecular masses greater than 50 kda are preferred, as those with lower molecular weights tend to exhibit reduced lymph node accumulation. the construct has a theoretical pi of 9.10, containing 39 negatively and 49 positively charged residues, suggesting a basic nature. its predicted half-life is around 30 hours in mammalian reticulocytes, over 10 hours in e. coli, and over 20 hours in yeast. the aliphatic index was determined to be 60.42, and the instability index was 31.42, suggesting that the protein is thermally stable. the grand average of hydropathicity (gravy) value was calculated as −0.282, indicating the hydrophilic nature of the construct. fig. 4. molecular docking of vaccine construct and tlr4 ligand 8. molecular docking of vaccine construct with tlr4: the castp server was employed to identify the active and passive ligand-binding sites within the refined tertiary structure of the vaccine construct (gülbay et al., 2006). the analysis revealed 62 active and 91 passive ligand-binding residues in tlr4. for the vaccine construct, 129 residues were identified as active and 145 as passive binding sites. both the refined construct and tlr4 monomers were subsequently submitted to the pydock server for molecular docking. pydock provided top 10 models in which model 1 was selected. other calculated parameters are provided in supplementary table 2. the docked vaccine construct and tlr4 receptor can be seen in fig. 4. 9. molecular docking of vaccine construct with mhc i: to predict the active and passive ligand binding sites in the refined tertiary vaccine structure the castp server was used (martinot et al., 2016). it predicted 60 residues as active ligandbinding sites and 92 residues as passive ligandbinding sites for mhc i. the predicted active ligandbinding sites and passive ligand-binding sites for vaccine construct was 129, 145 residues respectively. for molecular docking, the refined vaccine construct and mhc i monomers were uploaded to pydock web server. pydock provided top 10 models in which model 1 was selected. other calculated parameters are provided in supplementary table 3. the docked vaccine draft and mhc i receptor can be seen in fig. 5. fig. 5. molecular docking of vaccine construct and mhc i ligand 10. immune simulation for vaccine efficacy the immune responses predicted by the cimmsim server indicated a robust immunogenic potential for the vaccine construct. the vaccine elicited strong primary and secondary immune responses, as reflected in the simulation graphs. the primary response was characterized by an naseema; asian j. immunol., vol. 8, no. 1, pp. 269-284, 2025; article no.aji.147582 276 fig. 6. in silico immune simulation results of vaccine construct with tlr4 ligand using c-immsim web server (https:// www. iac. cnr. it/ ~filip po/ proje cts/cimmsimonline. html). (a) the rapid expansion of b-cells was accompanied by the formation of memory b-cells. (b) elevated level igg +igm antibodies in peripheral blood cells. (c) elevated b-cell (active) production after a lag phase of 5–7 days after exposure to the vaccine chimera. (d) high level of t helper cells production in response to antigens (e)active t helper cell population within 5 days of exposure to antigen. (f) constant production of th1 cells (g) increased level of cytotoxic t memory cells and (h) evolution of cytotoxic t-cells naseema; asian j. immunol., vol. 8, no. 1, pp. 269-284, 2025; article no.aji.147582 277 fig. 7. in silico immune simulation results of vaccine construct with mhc i ligand using cimmsim web server (https:// www. iac. cnr. it/ ~filip po/ proje cts/cimmsimonline. html). (a) the rapid proliferation of b-cell along with memory b-cells. (b) high level of igg +igm antibodies in peripheral blood cells. (c) increased b-cell (active) production after a lag phase of 5–7 days after exposure to the vaccine chimera. (d) elevated level of t helper cells production in response to antigens (e)active t helper cell population within 5 days of exposure to antigen. (f) constant production of th1 cells (g) higher level of cytotoxic t memory cells and (h) evolution of cytotoxic t-cells naseema; asian j. immunol., vol. 8, no. 1, pp. 269-284, 2025; article no.aji.147582 278 increase in igm antibody levels following a lag period of 5–7 days post-antigen exposure. the secondary response demonstrated enhanced bcell proliferation along with elevated levels of igg+igm, igm, and igg1+igg2 antibodies. in addition to promoting significant b-cell proliferation and memory b-cell formation, the vaccine construct induced strong cytotoxic and helper t-cell activity and maintained elevated ifn-γ levels for an extended duration. 4. discussion mycobacterium tuberculosis (m. tb) is the second deadliest disease which can actively tackle antibiotic treatment (mehla & ramana, 2016). “globally, there is an urge to develop vaccines and treatment for tb, because recently it has been identified that the well-known and only licensed anti-tuberculosis vaccine called bcg is no longer effective in adults” (mustafa, 2002). “in light of ever-growing drug resistance and adverse effects associated with anti-tb drugs such as ototoxicity, hepatotoxicity, neuropsychiatricevents, hyperuricemia, gastrointestinal disturbance, vision loss, skin pigmentation etc, a safe and efficacious vaccine could be an imperious arsenal against this lethal disease” (nagpal et al., 2020). “the different protocols followed by various laboratories over the last few years for bcg culture showed different level of divergence in the efficacy of bcg vaccine” (lee et al., 2014). “several reports suggest that bcg grown in sauton medium elicits a stronger immune response than bcg cultured in middlebrook 7h9 medium” (fleri et al., 2017). “out of the numerous anti-tuberculosis vaccines under development, only vmp1002, mip, and m. vaccae have advanced to phase iii trials, with plans to use them as boosters for bcg in the future. in recent years, the use of immunoinformatics has rapidly gained attention for drug and vaccine development compared to traditional approaches” (jung et al., 2011). in this study, a multi-epitope vaccine was designed using experimentally validated immunogenic exosome vesicle-based antigens in response to the global crisis (carmona et al., 2013). “to assess their immune-stimulating potential, these antigens were examined for b-cell, htl, and ctl epitopes, corresponding to humoral, innate, and cell-mediated responses. additionally, all selected epitopes were evaluated for nontoxicity, non-allergenicity, and ifn-γ induction. the chosen epitopes were linked using appropriate peptide linkers, and the tlr4 agonist peptide rpfe was attached to the nterminal of the construct to serve as an adjuvant and enhance the immune response. during m. tuberculosis infection, both tlr2 and tlr4 are involved in pathogen recognition, activating macrophages and dendritic cells and influencing both innate and adaptive immunity” (hart et al., 1987). “tlr4 was selected as the target receptor for the vaccine construct due to its critical role in infection; tlr4-deficient mice show lower survival rates and higher bacterial loads compared to tlr2-deficient mice when infected with m. tuberculosis” (kim et al., 2018). five vaccine constructs were created and evaluated using homology modeling. one construct was refined in 3d and docked with monomeric tlr4, producing a complex with favorable binding energy and stable structure. finally, an immune simulation study, modeling the innate immune network including t and b cells, was performed to evaluate both humoral and cellular responses. the results indicate that the designed multiepitope vaccine elicits a strong and consistent immune response. 5. conclusion our results show that the vaccine candidate possesses suitable physicochemical qualities, a stable structure, and encouraging immunological attributes capable of promoting both humoral and cellular immunity. building upon these findings, future work will focus on validating the efficacy of the proposed vaccine as both a preventive and therapeutic candidate through in vivo evaluation in a suitable mouse model. therefore, it should be pushed forward as a potential lead candidate for in vivo and in vitro assessments against m. tb. 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(2024). in silico analysis for the development of multiepitope vaccines against mycobacterium tuberculosis. frontiers in immunology, 15, 1474346. naseema; asian j. immunol., vol. 8, no. 1, pp. 269-284, 2025; article no.aji.147582 282 supplementary table vaccine sequence: mknarttliaaaiagtlvtrspagianaddagldpnaaagpdavgfdpnlppapdaapvdtppapeda gfdpnlppplapdflsppaeeappvpvaysvnwdaiaqcesggnwsintgngyygglqftagtwra nggsgsaanasreeqirvaenvlrsqgirawpvcgrrgeaaakaglwgnggsggaggnakkagli gnggaggaggpnkkgspittlyvkidggtpkkggmggsgggigagtttkknvpidpspdydasdeik kggtggaagllgwgangkkrgavtgppspvaaqenkktvqrqagctndvtinpkkvvanvytvqr qagctnkkggvgttggaggngggakkgnsgtantgfgnagnvkktatgiwylqdrvivaekkisty fsalladptttpkknagagntgffdagnyngpgpgyavaeaataqsvqqdgpgpggqeyqavsaq asafhgpgpggnsyavaeaataqsvgpgpgrkdirttrvtvapqygpgpgggnsyavaeaataqs aaystqaktramaayfmligaafyaaysgysgglyyaayqaalamevyaaynfmligaafaayrtye atmslaayaaaasttalaayyvntsttsm adjuvant (rpfe) =1-172 14 b-cell epitopes =178-250 5 mhc ii (htl epitopes) = 433-527 5 mhc i (ctl epitopes) = 530-587 3 mhc i (hla-c epitopes) = 590-623 linkers table 1. comprehensive epitope mapping of mycobacterium tuberculosis antigens for multiepitope vaccine design gene name b-cell epitope ctl epitope hla-c epitope htl epitope ppe 68 stqaktram (b7,b8,b62) qaalamevy (a1,a26,b58,b62) rv0310c tatgiwylqdrvivae fmligaafy (a1,a3,a26,b62) rtyeatmsl (hla-c*01:02 hla-c*03:02 hla-c*03:03 hla-c*03:04 hla-c*04:01 hla-c*05:01 hla-c*07:01 hla-c*07:02 hla-c*07:04 hla-c*08:01 hla-c*08:02 hla-c*12:02 hla-c*12:03 hla-c*15:02 hla-c*16:01 hla-c*17:01) nfmligaaf (a24,b8,b62) pe_pgrs35 aglignggaggaggpn sgysgglyy (a3,a26,b58,b62) gqeyqavsaqasafh (drb1_0101 drb1_0401 drb1_0405 drb1_0701 drb1_0802 drb1_0901 drb1_1101 drb3_0202 gspittlyvkidggtp ggtggaagllgwgang istyfsalladptttp naseema; asian j. immunol., vol. 8, no. 1, pp. 269-284, 2025; article no.aji.147582 283 gene name b-cell epitope ctl epitope hla-c epitope htl epitope hla-dqa10501dqb10301 hla-dqa10301dqb10302 hla-dqa10401dqb10402 hla-dqa10102dqb10602 hla-dpa10201dpb11401) pe_pgrs31 aglwgnggsggaggna ggmggsgggigagttt aaaasttal (hla-c*01:02 hla-c*03:02 hla-c*03:03 hla-c*03:04 hla-c*05:01 hla-c*07:04 hla-c*08:01 hla-c*08:02 hla-c*12:02 hla-c*15:02 hla-c*16:01 hla-c*17:01) yavaeaataqsvqqd (hla-dqa10501dqb10201 hla-dqa10501dqb10301 hla-dqa10301dqb10302 hla-dqa10401dqb10402 hla-dqa10102dqb10602) gnsyavaeaataqsv (drb1_0101 drb1_1101 drb3_0202 hla-dqa10501dqb10301 hla-dpa10201dpb11401) ggvgttggaggnggga ggnsyavaeaataqs (drb1_0405 drb1_0701 drb1_0802 drb1_0901 hla-dqa10501dqb10201 hla-dqa10301dqb10302 hla-dqa10401dqb10402) lpqg rkdirttrvtvapqy (drb1_0401 drb1_0701 drb1_0802 drb4_0101 hla-dqa10301dqb10302 hla-dqa10401dqb10402 hla-dqa10102dqb10602 hla-dpa10201dpb11401) ppe39 gnsgtantgfgnagnv yvntsttsm (hla-c*02:02 hla-c*02:09 hla-c*03:02 hla-c*03:03 hla-c*03:04 hla-c*08:02 hla-c*12:02 hla-c*12:03 nagagntgffdagnyn naseema; asian j. immunol., vol. 8, no. 1, pp. 269-284, 2025; article no.aji.147582 284 gene name b-cell epitope ctl epitope hla-c epitope htl epitope hla-c*15:02 hla-c*16:01 hla-c*17:01) rv1975 nvpidpspdydasdei rgavtgppspvaaqen tvqrqagctndvtinp vvanvytvqrqagctn table 2. docking results (pydock) vaccine construct with tlr4 rmsd from the overall lowest-energy structure 0.248 van der waals energy 42.436 electrostatic energy -10.009 desolvation energy -49.937 z-score -1.10 table 3. vaccine construct with mhc i rmsd from the overall lowest-energy structure 0.248 van der waals energy 81.423 electrostatic energy -19.16 desolvation energy -32.681 z-score -2.89 disclaimer/publisher’s note: the statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of the publisher and/or the editor(s). this publisher and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. _________________________________________________________________________________ © copyright (2025): author(s). the licensee is the journal publisher. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: https://pr.sdiarticle5.com/review-history/147582 https://pr.sdiarticle5.com/review-history/147582 _____________________________________________________________________________________________________ # clinical immunologist; *corresponding author: e-mail: drstoff@imofny.org, jessestoff@gmail.com asian journal of immunology 5(1): 1-39, 2022; article no.aji.77967 a multimodal strategic approach to integrative oncology jesse a. stoff a*# a integrative medicine of new york, 520 franklin ave suite 230 garden city, ny-11530, usa. author’s contribution the sole author designed, analysed, interpreted and prepared the manuscript. article information editor(s): (1) dr. wagner loyola, brazilian agricultural research corporation, concordia. reviewers: (1) yue jia, usa. (2) bhalchandra mirlekar, university of north carolina at chapel hill, usa. complete peer review history, details of the editor(s), reviewers and additional reviewers are available here: https://www.sdiarticle5.com/review-history/77967 received 10 october 2021 accepted 13 december 2021 published 15 january 2022 abstract the term “integrative oncology” can mean particular things to different audiences. for the purposes of this article, i intend it to mean the simultaneous use of multiple anti-cancer strategies that can have a synergistic effect against the tumor, its microenvironment, and its propensity to metastasize. thereby, having a maximum impact against the cancer with a minimum of side effects for the patient to endure. as noted in the references, this paper briefly describes the five prevailing evidence-based systematic approaches in use today. each of these approaches has varying degrees of effectiveness which, in my experience, when clinically integrated, produces exponentially better efficacy, hence shifting progression-free survival into the possibility of increased overall survival and a durable remission. this paper is an attempt to offer some perspective of how to apply these disparate methodologies so that they may be more effectively integrated, resulting in consistently better clinical responses. keywords: integrative oncology; oncology; multimodal strategic; cancer. 1. introduction cancer, as a disease of our time, has been described as a “wound that does not heal” [1] because it has a multitude of biochemical dysfunctions at its core. however, in order for these genetically and phenotypically abnormal cells to survive and thrive, the watchdog of the review article stoff; aji, 5(1): 1-39, 2022; article no.aji.77967 2 body, the immune system, must itself be suffering from a number of serious areas of structural and functional damage. from a perspective of ten thousand feet, there are two basic characteristics of cancer: genetic instability and the propensity to metastasize. from those two basic observational characteristics, the hallmarks of cancer were derived by drs. hanahan and weinberg in 2000 as follows:  self-sufficiency in growth signals. insensitivity to antigrowth signals. evading apoptosis, limitless replicative potential, sustained angiogenesis, tissue invasion and metastasis. and then the following were added in 2011:  deregulating cellular energetics, avoiding immune destruction, genome instability and mutation, tumor-promoting inflammation [2,3]. based upon the characteristics of the cancer, there are five common office-based/out-patient strategies that we can employ. each of these strategies is made up of a number of tactics based upon the unique expressed characteristics of the target cancer and the physiological individuality of the patient. those five strategies are first, to balance and normalize the metabolism of the patient while inhibiting the physiology of the cancer. second, to identify and seek to repair areas of damage within the structure and function of the immune system. third, to identify actionable genetic and protein mutations that are driving the cancer and address them. forth, to employ agents that can inhibit, suppress, or destroy the root of the problem, the cancer stem cells. and fifth, to directly attack the cancer with cytotoxic (chemotherapeutic) agents and radiation therapy. utilizing agents that can decisively accomplish one of these strategies or that can engage and strike two or more of these objectives with less toxicity can lead to greater quality and quantity of life for our patients. 2. metabolic approach 2.1 biochemical support of the patient milieu metabolic targeting is the first anti-cancer strategy because it lays the foundation for the improved response for all of the other strategies that follow. in 1498, albrecht durer created the famous woodcut entitled “the four horsemen, from the apocalypse” which, in a sense, foreshadowed the epidemic of severe and chronic diseases that we now face. from a metabolic, thousand-foot perspective, there are four major processes that are common pathways for the initiation and promotion of cancer. each of which should be assessed for every patient: glycation; which can be easily measured with blood sugar, hemoglobin a1c, and glycomark; inflammation; for which there are a myriad of markers including esr, crp, plasma viscosity, adiponectin, monocyte chemoattractant protein 1 (mcp-1), cd40 ligand and lipoprotein-associated phospholipase a(2) (lp-pla(2)), and ferritin to name but a few; methylation; which can be watched with homocysteine levels; and finally, we come to oxidation; which is largely reflected in levels of lipid peroxides and oxidized ldl. current research demonstrates that cancer is largely a metabolic disease involving disturbances in energy production (deregulating cellular energetics) through a shift from respiration to fermentation, but this is not the only aberrant energy pathway that cancer cells can utilize. the genomic instability observed in tumor cells, leading to the other recognized hallmarks of cancer listed above, is now considered downstream epiphenomena of the initial disturbance of cellular energy metabolism which is often caused by a toxin [4]. the disturbances in tumor cell energy metabolism can be linked to demonstrated abnormalities in the structure and function of and within the mitochondria [5-7]. extrapolating upon this research we come to several conclusions for therapeutic intervention. glycation starts with diet. diet has been recognized and is now accepted by mainstream medicine as a viable strategy for minimizing the risk of getting cancer [8-10]. furthermore, “a higher frequency of organic food consumption was associated with a reduced risk of cancer” [11]. a proper diet can address issues of nutritional deficiency and some toxin exposure while supplying necessary anti-oxidants and helping to manage glycation (while hopefully being delicious). when we apply a dietary strategy to someone with cancer, while recognizing the dysregulated metabolic mitochondrial foundation of the tumors, we come to a low carbohydrate/ketogenic diet. because cancer is biochemically dependant upon sugar to maintain its fermentation metabolism, depriving it as much as possible, of sugar will further destabilize the cancer cells making it easier to trigger apoptosis, with the addition of other therapeutic agents [12,13]. one of those therapeutic agents is metformin. in addition to stoff; aji, 5(1): 1-39, 2022; article no.aji.77967 3 lowering blood sugar levels and thus minimizing the fuel source for cancer cells, metformin interferes directly with cell proliferation and supports apoptosis in a non-insulin-mediated manner. accumulated research and clinical evidence suggest that metformin exerts its positive effect on the clinical course of neoplastic diseases, primarily through the stimulation of 5' amp-activated protein kinase (ampk) in association with the upstream liver kinase b1 (lkb1) [14,15]. ampk is a key cellular energy sensor, activation of which by metformin leads to suppression of energy-consuming processes such as gluconeogenesis [16,17]. in carcinoma cells, the stimulation of ampk, mediated by metformin, resulted in the inhibition of the mtor/ribosomal s6 kinase pathway and thus inhibition of the pathological cell cycle progression, cell growth, and angiogenesis [18,19]. this is a particularly important effect when there is a mutation of phosphatase and tensin homolog (pten) which would normally downregulate this pathway [20]. inflammation is another metabolic pathway that drives cancer, therefore, anti-inflammatory strategies are of critical importance against cancer [21]. inflammation results from or is triggered by virtually every disease known to man and has cellular pathways, plasma cascades, and acute and chronic markers. some of the markers associated with cancer include crp, il-6, il-8, and tnf-alpha. inflammation can stimulate cancer stem cells as well as more mature tumor cells, triggering disease progression. inflammation is a defense mechanism that serves to protect us from acute inflammatory illnesses (infections) and injuries that historically, from an evolutionary point of view, were the main causes of death. inflammatory proteins activate the th2 pathway which generates more inflammation. in doing so, in an apparent effort to focus and conserve resources, when the th2 pathway becomes chronically stimulated by infection, vaccinations, nutritional deficiency, sugar, alcohol, trauma, stress, or toxins, it suppresses the th1 pathway which is a critical pathway of the immune system, needed to protect us from, and fight cancer. cimetidine can help to restore the th1/th2 balance and overcome pro-inflammatory th2 dominance [22]. activation of the th1 pathway and its downstream effector cells results in the production of reactive oxygen species (ros). which are inflammatory molecules that act locally to kill cancer cells, so we don’t want to negate all inflammatory responses [23]. an important enzyme for the inflammatory response is cyclooxygenase (cox), which is critical for the conversion of arachidonic acid to prostaglandins and other eicosanoids. it exists as two isoforms: cox-1 and cox-2; cox-1 is constitutively expressed whereas cox-2 is a highly inducible gene that is activated by cytokines, growth factors, phorbol esters, oncogens, and chemical carcinogens. cox-2 plays a key role in carcinogenesis as has been demonstrated in numerous cancer cell types. multiple pathways have been proposed to explain how increased cox-2 expression might contribute to carcinogenesis including elevated bcl-2 protein levels and inhibition of apoptosis, increased angiogenesis, and enhanced metastatic activity. suppressing cox-2 activity, or at least not stimulating it, which many chemotherapeutic agents do, is an important part of any anticancer strategy. many agents have been identified that suppress cox-2 activity, which can be assayed in vitro and is reflected clinically in decreased levels of crp, interleukin6 (il-6), p-selectin, matrix metalloproteinase-9 (mmp-9), tumor necrosis factor (tnf-α), and other inflammatory markers. some of the antiinflammatory agents that are helpful against cancer include celecoxib, fish oil, (eicosapentaenoic acid (epa) and docosahexaenoic acid (dha)), white willow tree, curcumin, green tea, pycnogenol, boswellia, resveratrol, cats claw, and capsaicin to name just a few of the most common medicaments[24-49]. methylation seems to still be incompletely understood when it comes to cancer. on the one hand, people with the fairly common methylenetetrahydrofolate reductase (mthfr) mutation are at a higher risk of getting certain (and maybe all) cancers [50]. this makes sense because that gene affects some of the liver detox pathways and when it isn’t working well it can lead to known increased risks for cardiovascular heart disease, leukemia, breast cancer, colon cancer, and many other illnesses [51]. the mutation can be tested for and some of its effects monitored with homocysteine blood levels. many of the negative effects can be bypassed with methylated b vitamins. on the other hand, gene expression can be significantly modulated by alterations in dna methylation patterns. methylation within the promoter regions of tumor suppressor genes causes their silencing, and methylation within the gene itself can induce mutational events. these mechanisms may play a fundamental role in precipitating the development of a large and diverse number of stoff; aji, 5(1): 1-39, 2022; article no.aji.77967 4 human cancers[52]. it is important to monitor homocysteine and b vitamin levels while treating people with cancer, because overshooting your mark may make some cancers worse [53]. when a woman is pregnant we give prenatal vitamins which are largely b vitamins to stimulate the cell growth of the fetus, and when you’re fighting cancer you don’t want to do that. however, it is important to note that, b vitamin deficiencies will inhibit many immune functions that are desperately needed for the fight [54]. nothing about fighting cancer is easy, getting biochemical moieties to and maintaining them within the normal range is a safe bet, from a metabolic perspective. oxidation, like the rest of metabolic activity, is also a bit of a double-edged sword when it comes to cancer. oxidative stress is caused by many things including; obesity, high fat diet, sugar, the chemicals in processed foods, radiation, smoking, alcohol, covid-19, environmental pollution and chemicals, etc., etc., etc..(i.e. life) [55]. damage from oxidative stress occurs when the cells and tissues are unable to keep up with harm done by the free radicals that are generated from the above, and other causes. a free radical is an atom, molecule, or ion that has an unpaired valence electron that can bind to, and damage, dna and messenger molecules but is normally kept in check by the body’s store of anti-oxidants. this damage is carcinogenic. anti-oxidants can mitigate oxidative stress and are found in vegetables such as broccoli, spinach, carrots, cabbage, and vitamins c, e, d, a, etc., etc., etc... uncontrolled oxidative stress can activate a variety of transcription factors including nuclear factor kappa-light-chainenhancer of activated b cells (nf-κb), activator protein 1 (ap-1), tumor protein 53 (p53), hypoxia-inducible factor-1a (hif-1α), peroxisome proliferatoractivated receptor gamma (ppar-γ), β-catenin/wnt, and nuclear factor erythroid 2–related factor 2 (nrf2). activation of these transcription factors can lead to the expression of over 500 different genes, including those for growth factors, inflammatory cytokines, chemokines, and cell cycle regulatory molecules. activation of these molecules by oxidative stress stimulates inflammatory pathways leading to the transformation of a normal cell to a tumor cell, increases tumor cell survival, proliferation, chemoresistance, radioresistance, invasion, angiogenesis and supports cancer stem cell survival [56]. on the other side of the sword, the production of oxidative reactive oxygen species (ros) is required for the function of another major effector of the innate immune system, natural killer (nk) cells—hydroxyl radical production is responsible for nk cells-mediated cytolysis, by promoting the secretion of cytotoxic factors from nk cells granules [57-62]. ros can promote carcinogenesis by inducing genetic mutations, activating oncogenes, and raising oxidative stress, which can stimulate cell proliferation, survival, and reduce apoptosis. over the past few decades, natural compounds have attracted attention as potential cancer therapies because of their ability to maintain cellular redox homeostasis with minimal toxicity. research and clinical studies show that bioactive dietary polyphenols exert antitumor effects by inducing ros-mediated cytotoxicity in cancer cells. these bioactive compounds also regulate cell proliferation, survival, and apoptotic and antiapoptotic signaling pathways [63]. amongst many other examples, this is demonstrated in the ability of aronia melanocarpa to increase the expression and activity of p53 and p73 that leads to the down-regulation of the expression of cyclin b1, which contributes to the cell cycle arrest of cancer cells [64-66]. some commonly used anti-cancer agents include: glycation metformin [67,68] berberine [69,70] azadirachta indica (neem) [71,72] inflammation celebrex [73] curcumin [74,75] grape seed extract [76,77] cimetidine [78] viscum [79] methylation decitabine [80] egcg [81] oxidation dmso [82] raloxifene [83] vitamin c and many other vitamins and minerals [84] 2.2 biochemical targets of the tumor: the micro-environment a closer look at the cancer itself demonstrates that by corrupting and recruiting non-malignant cells in the area, the tumor has created its own biochemical tumor microenvironment (tme) stoff; aji, 5(1): 1-39, 2022; article no.aji.77967 5 suitable for its self-protection and propagation [85]. many different cell types, besides the cancer, can be found in the tme including; adipocytes, fibroblasts, dendritic cells, lymphocytic endothelial cells, myeloid-derived suppressor cells, pericytes, tumor-associated macrophages, tumor-associated neutrophils, and vascular endothelial cells, to name but a few [86]. other cells to be found in tme include t-cells, nk cells, nkt cells, and some b-cells that are there to fight the good fight. dendritic cells and macrophages found just outside of the tme are also engaged in the battle against the cancer, but if consumed by the tumor, seem to become corrupted and aid in its survival [87]. the dynamic interactions of the cancer cells with the stromal cells (cellular part) and extracellular matrix (ecm) components (non-cellular structures) are essential for stimulating the multiple cell lines of the cancer, as well as its clonal evolution and propensity to develop multidrug resistance, resulting in cancer cell progression and metastasis [88]. the ecm holds cells, tissues, and organs together and maintains the three-dimensional structure of the body. for a cancer cell to metastasize from the primary tumor to other organs, it must locally degrade the ecm components that are the physical barriers to cell motility. cancer cells build, shape, and recruit support for their tme through the release of many different types of molecules including; matrix remodeling enzymes, mediators of inflammation (which can stimulate the activity of the matrix metalloproteinases), cytokines, cellfree dna (cfdna, which we can now test for [89] chemokines, growth factors, exosomes, and can facilitate horizontal gene transfer [90]. the reciprocal and self-supporting cell-cell/ecm interaction forces the expression of new phenotypes of non-cancer cells that promotes the development and invasion of the cancer at large [91]. trying to bring order back to such a maelstrom is the focus of a great deal of research at this time as it is an important piece of a durable remission. targeting the tme has proven to be no easy feat and has been based upon trying to undermine the major components of it. starting with the milieu itself, it has long been noted that the tme is more acidic than normal tissue in the local area, while the intracellular ph is often more alkaline [92]. however, just trying to inject the tme with sodium bicarbonate does not seem to be a reliably effective treatment because the active biological processes that created the abnormal gradient, to begin with, will rebound and increase its level of activity to re-establish its supportive environment. combining sodium bicarb with cytotoxic agents seems to offer a better strategy [93,94]. changing the electrochemical gradient of the tme, and thus the ph, with an electrical current has also shown to be effective in some cases [95-97]. another strategy that targets the tme is to inhibit the matrix metalloproteinases(mmps) that degrade the extracellular matrix (ecm) components that then allows for local metastasis. research has identified many substances that can inhibit these mmp’s. resveratrol, a polyphenol present in various plants, food products, red wine, and grapes, has been demonstrated to suppress mmp-2 and mmp-9 [98]. additionally, resveratrol can inhibit the expression of vascular endothelial growth factor (vegf) and act as an anti-inflammatory agent [99]. agents derived from marine sources have also been found to inhibit mmp’s including various plant and animal sources of saccharoids, flavonoids, polyphones, and fatty acids but thus far, few have been shown to be reliably clinically effective [100]. one agent that did make it into clinical trials was ae-941/neovastat. according to the national cancer institute, “shark cartilage extract, ae-941 competitively inhibits the binding of pro-angiogenic vascular endothelial growth factor (vegf) to its cell receptor, thereby inhibiting endothelial cell proliferation”. this agent also inhibits matrix metalloproteinases (mmps), but at this time, there are no clinical studies involving its use [101]. chlorella, a protein extract from algae has also been shown to have mmp’s inhibitory properties [102]. tissue inhibitors of metalloproteinases (timp’s) naturally impede the activity of the mmp’s and its the imbalance between the activation and inhibition of them that is largely responsible for the progression of a tumor’s growth [103]. other polyphenols that can inhibit the mmp’s are quercetin, epigallocatechin gallate, and curcumin. they act as dynamic antioxidants, and are able to interact with cell-signaling pathways, modulating gene expression in three different ways: i) influencing the activity of transcription factors, ii) epigenetically, modulating micrornas, and iii) having an anti-inflammatory effect, all of these actions serve to down-regulate mmp activity [104]. combining polyphenols with amino acids that are used in the production of the ecm along with vitamin c which, too, is critical for ecm support, has been shown to be an effective strategy for slowing the progression of tumors. their combined anti-cancer effects include stoff; aji, 5(1): 1-39, 2022; article no.aji.77967 6 inhibition of: metastasis, tumor cell growth, matrix metalloproteinase secretion and activity, local invasion, angiogenesis, as well as induction of apoptosis of the cancer cells [105-108]. this combination has been effective both in vitro and in vivo against many different cancers and cancer cell lines including ovarian, melanoma, squamous cell, and others in a dose-dependant curve [109]. a chief problem for the use of polyphenolic compounds against cancer is their low bioavailability [110]. different types of formulations have been designed to address this issue, nanonization being one of the most notable approaches among them. variations of nanoformulations are designed to be effectively transported through the relevant biological barriers to the targeted organs, tissues, and cells. research on natural polyphenols as bioactive agents include: resveratrol, curcumin, quercetin, epigallocatechin-3-gallate, chrysin, baicalein, luteolin, honokiol, silibinin, and coumarin derivatives, which have shown efficacy in a dose-dependent manner and may result in better treatments of cancer [111]. the tumor microenvironment, created by the cancer, further supports its dysfunctional growth and ability to metastasize through epigenetic changes. epigenetic changes are heritable but, unlike genetic nucleotide modification, are reversible and therefore targetable as part of an oncolytic therapy. a variety of epigenetic mechanisms have been identified that can induce and/or support a cancer, such as: silencing of tumor suppressor genes, activation of oncogenes by altered cytosine phosphorus guanine (cpg) island methylation patterns, histone modifications, and dysregulation of dna binding proteins. a quick search in https://www.ncbi.nlm.nih.gov/pmc for “epigenetic toxins and human cancers” will yield thousands of articles about substances both natural and synthetic that can create such changes [112]. not only are the epigenetic changes potential targets for treatment, but their inducers are as well [113,114]. the tme creates and supports epigenetic biochemical reactions that dynamically regulate dna expression. nucleotide methylation and histone modification induce the dysregulation of genes related to proliferation, apoptosis, and metastasis [115]. dna methylation is strongly associated with the repression of transcription of tumor suppressor genes through adding a methyl group by dna methyltransferases (dnmts) [116]. other reactions by histone deacetylases (hdacs) contribute to transcriptional repression by removing the acetyl group at lysine residues leading to tumorigenesis, as a result of the research, both biochemical pathways have become the focus of intense research to develop dnmt and hdac inhibitors [117]. histone acetyltransferases (hat), erasers (histone demethylases (hdm) and histone methylases (hmt) have also become epigenetic targets of research [118]. some drugs are currently available and used for their epigenetic effects such as decitabine, which can prevent dna remethylation and re-activate silenced suppressor genes [119]. azacitidine is considered a global dnmt inhibitor and upon treatment of breast cancer cells, dna re-methylation was inhibited for 23 out of 26 tested hypermethylated genes, allowing 5 tumor suppressor genes to be reexpressed [120]. the problem with these and the many other drugs, thus far developed for their epigenetic effects, are the unintended consequences (side effects) on normal, healthy cells elsewhere in the body. reducing and pulsing the doses given has helped somewhat to reduce the side effects while maintaining efficacy [121]. the tactics for targeting the tme have a certain amount of overlap with those that seek the destruction of the cancer stem cells because they are the major architects of their immediate environment. however, the chief goal of an antimetabolic cancer therapy is to disrupt the energy production pathways that support the cancer cells. for example, enox2 is a gene located on the long arm of the x chromosome and encodes for the ecto-nox protein. it is a member of the nox family of nadph oxidases which are important for energy production and the growth of the cell. tumor-associated nadh oxidase (tnox), has been identified as a target for lowdose cell killing (apoptosis) of cancer cells by green tea catechins and capsicum vanilloid combinations [122-125]. this protein is uniquely associated with all forms of cancer and is absent from normal cells and tissues. another anticancer metabolic strategy is to use the combination of two nutraceuticals: lipoic acid and calcium hydroxycitrate, which target two cancer energy enzymes atp citrate lyase and pyruvate dehydrogenase kinase. experimentally, this treatment was as efficient as chemotherapy in the mouse cancer models that were tested [126,127]. clinically, in combination with lowdose naltrexone, this anti-metabolic tactic has also been effective even against advanced chemo-resistant carcinomas [128, 129]. stoff; aji, 5(1): 1-39, 2022; article no.aji.77967 7 2.3 genetic molecular targeting targeting the abnormal genetics of cancer is the second anti-cancer strategy. before we even begin to specifically target aberrant genetics, there are therapies that can be instituted for generally stabilizing genes which buys time for more definitive testing that allows for precise targeting of the cancer genome. each of these agents has a multitude of beneficial effects, but chief among them is their ability to stabilize aberrant genetics, creating the time and opportunity for an integrative anticancer strategy. during the course of oncogenesis and tumor progression, cancer cells constitutively upregulate signaling pathways relevant to cell proliferation as a result of any number of genetic mutations. it is that genetic instability that leads to cancer cells acting as cancer in the first place, and it is those unstable mutations that account for the multiple cell lines that compose every tumor. many agents have been identified that can aid in cell differentiation and reversion back to a phenotypically normal cell type [130]. some of them are considered as follows. first on our list of genetic stabilizers is dimethyl sulfoxide. dimethyl sulfoxide (dmso) is an organosulfur compound with polar, aprotic, and amphiphilic properties. it serves as a solvent for many polar and nonpolar molecules and continues to be one of the most used solvents (vehicle) in medical applications and scientific research. it also displays a diversity of antitumor activities[131]. previous studies have demonstrated that dmso can modulate ap-1 activity and lead to cell cycle arrest at the g1 phase [132]. the fibrinolytic system, more appropriately referred to as the plasminogen activator (pa) system, controls not only the intravascular fibrin deposition (with its effects on cardio-vascular disease) but also participates in a wide variety of other physiologic and pathologic processes. in cancer, the components of this system are involved in tumor growth, invasion, and metastasis through their effect on angiogenesis and cell migration, by way of its effects on the extracellular matrix of the tumor microenvironment. plasminogen activator components are found in most tumors and their expression signifies not only their function but also carries a prognostic value. their expression is in turn modulated by cytokines and growth factors, many of which are up-regulated in cancer and found throughout the tme. dmso can inhibit the activity of pa’s which helps to stabilize the tme and reduce the risk of metastasis [133]. furthermore, dmso can cause the maturation/differentiation, of at least some cancers, into a benign, more normal cell type [134,135]. vitamin d is next on our list of genetic stabilizers. in addition to enhancing dna repair, vitamin d also induces growth arrest and apoptosis of tumor cells and their nonneoplastic progenitors. cell-based studies show that the active metabolite 1,25 dihydroxyvitamin d is the biologically active form that works through the vitamin d receptor to regulate gene transcription. vitamin d (d3) is produced from 7dehydrocholesterol when skin is directly exposed to uvb light which, in more northern locations, is largely filtered out by the atmosphere. vitamin d is readily sourced from various foods including fish, eggs, caviar (for the gourmets among us), some mushrooms, beef liver, and cheese. regardless of whether vitamin d comes from the skin or the diet, vitamin d3 is transported through the blood by the vitamin d binding protein (dbp). once delivered to the liver, vitamin d is hydroxylated on its side chain to form 25 hydroxyvitamin d (25oh d). this is a stable metabolite whose serum levels are commonly used to assess vitamin d status. as needed and if available, d3 circulates to the kidneys which is the primary site where the active form of vitamin d, 1,25(oh)2 d, is produced through the genomic actions of 1α,25(oh)2 via the vitamin d receptor (vdr), and its analogs inhibit cell cycle progression and tumor cell growth. mechanisms of action range from preventing cell proliferation (through cell cycle arrest) in cancer cells to inducing apoptosis, or suppressing cell adhesion molecules and growth factors that promote cellular homing and metastasis. it also affords important antioxidant protection and serves as an immunomodulatory for both the innate and adaptive arms of the immune system [136-140]. next comes indole-3-carbinol and its metabolite 3,3′-diindolylmethane (dim). they target multiple aspects of cancer cell cycle regulation and survival including akt-nfκb signaling, caspase activation, and cyclin-dependent kinase activities. furthermore, they stabilize estrogen metabolism, normalize estrogen receptor signaling, reduce endoplasmic reticulum stress, and limit brca gene expression. dna hypermethylation is a common feature of cancer genetics. when methylation detox pathways start to fail, certain regions of the genome will accumulate too many methyl groups, such as at cpg promoter regions (segments of the dna involved in dna and rna stoff; aji, 5(1): 1-39, 2022; article no.aji.77967 8 transcription); this can lead to increased mutagenesis and eventual cancer development. much research has demonstrated how dim reduced methylation at 5 cpg promoter regions. for example, in split population studies, mice given transgenic adenocarcinoma of the mouse prostate (tramp) prostate cancer cells were also given dim. the mice given the dim showed a much lower incidence of cancer and metastasis than controls, as well as much higher expression of antioxidant/anticarcinogen protective enzymes nqo1 and nfr2 in prostate tissues [141-146]. curcumin, a component of turmeric (curcuma longa), is a low molecular weight molecule that has antiproliferative activity and inhibits tumor initiation and propagation through a variety of pathways. it accomplishes this through several epigenetic effects that result in genetic modulation that then changes the expression of several key proteins, some of which are the cysteine-aspartic acid proteases (caspases). caspases are a family of enzymes that play an essential role in apoptosis, necrosis, and inflammation. research demonstrates that curcumin activates caspases-3 and -8 but not caspase-9, indicating that the apoptosis induced in cancer cells occurs via a membrane-mediated mechanism. membrane-bound enzymes play other important roles in the perpetuation of cancer cells such as the ecto-nox 2 system. p53 (tp53) is a tumor suppressor gene and is responsible for protecting cells from tumorigenic alterations. mutational inactivation of p53 is frequently observed in many cancers. curcumin selectively increases p53 expression during the g2 phase of the cell cycle of carcinoma cells and releases cytochromes from the mitochondria, which is an essential requirement for then inducing p53-dependent apoptosis in the cancer cell. another protein affected by curcumin is akt (protein kinase b), a serine/threonine kinase. it is a critical enzyme in signal transduction pathways involved in cell proliferation, apoptosis, and angiogenesis. curcumin inhibited the phosphorylation of akt in a dose-dependent manner leading to another pathway of apoptosis. curcumin also induces the upregulation of carcinogen-detoxifying enzymes, such as glutathione s-transferases, which have antioxidation effects and suppresses cyclooxygenase that in turn lead to the reduction of the level of inflammation and the stimulation of cancer stem cells. real-time animal model studies have demonstrated that curcumin also decreased the expression of dna damage response genes, including serine/threonine kinase (atm), ataxia telangiectasia and rad3related protein (atr), breast cancer 1 (brca1), 14-3-3σ, dna-dependent protein kinase (dnapk), and o6-methylguanine-dna methyltransferase (mgmt); thus, the reduction of a dna damage response is but a part of the reason for curcumin-induced growth inhibition of cancer cells [147-149]. sulforaphane (sfn) is an isothiocyanate found in cruciferous vegetables such as broccoli, brussel sprouts, cauliflower, and cabbage. experimentally, in cell cultures and animal models, sfn was shown to be a highly effective chemoprotective against carcinogen-induced, and genetic animal cancer models, as well as in xenograft transplant models of cancer. the early research focused on the detoxification ability of sfn to induce phase 2 enzyme pathways. later studies showed that sfn could cause cancer cells to enter g2/m phase arrest and result in apoptotic cell death, with the latter being evidenced by caspase-mediated cleavage of poly(adp-ribose) polymerase and increased release of histone-associated dna fragments from the tumor downstream. furthermore, it leads to the transcriptional activation of genes including tumor suppressor genes. the effect on cancer genetics is profound and therapeutically beneficial [150-153]. fish oil rounds out our list of top genetic stabilizing supplements. one of many changes that occur to the cancer cell’s biochemistry and genetics is in the production, metabolism, and expression of micrornas (mirnas). first discovered in the early 1990s, their importance as a distinct class of biological regulators was not appreciated for another decade. micrornas (mirnas) are short molecules, just 21–25 nucleotides long, but can have powerful effects on gene expression. more than 2000 mirnas have been identified including many specific mirnas that have been found to be associated with cancer and the risk of metastasis. the identification of circulating mirna specific to metastatic cancer presents a unique opportunity for early disease identification and for monitoring disease burden as a circulating biomarker. for example, abnormal activation of some mirnas found in the blood, let-151a, mir-21, mir-155, mir-,145 mir-18a, and mir-16, as well as tissue-specific mirnas, mir-182, mir-145, mir stoff; aji, 5(1): 1-39, 2022; article no.aji.77967 9 21, mir-155/154, mir-203, mir-213, and mir-7, are often found in patients affected by breast cancer. furthermore, there is a growing body of evidence on the value of mirnas associated with the development of drug resistance, suggesting their values, once targeted, as a potential approach to overcoming chemoresistance. mirnas that are absorbed into our bloodstream from genetically modified organism (gmo) foods are currently being studied for their oncogenic potential, as they are normally foreign to human biology. fish oil can modulate the expression of the mirnas which can significantly reduce the risk of metastasis while having many other anticancer effects [154159]. the science of genetics studies the genes that make up our chromosomes, which lead to inherited characteristics; think gregor mendle and his peas. genomics is the study of large groups of genes or even the entire genome, looking for mutations, fusions, deletions, loss of heterozygosity, etc. variations and alterations that lead to the expression of cancer. aberrations of the genome lead to the transcription of the faulty gene into a messenger rna (mrna) molecule, that leaves the cell nucleus and enters the cytoplasm, where it directs the synthesis of the protein for which it encodes. abnormal genes lead to abnormal proteins. the study of proteins is called proteomics; it’s primarily the functional proteins, which includes enzyme activities, as well as protein/protein interactions (such as immune receptor sites) and post-translational modifications of the proteins that are of the most interest because they are the most antigenic and hence the most targetable/treatable. the sciences of genomics and proteomics have opened the door to what is being referred to as “precision medicine” [160]. precision medicine is "an emerging approach for disease treatment and prevention that takes into account individual variability in genes, environment, and lifestyle for each person". this personalized approach allows clinicians to predict more accurately which treatment is more likely to work for which person [161]. this is in contrast to a one-size-fits-all approach, in which cancer treatment strategies are developed for the average person based upon group clinical trials (cohorts), with little consideration for the differences between individuals. precision medicine works by profiling live or preserved cells for biomarkers that indicate an abnormal process, gene or protein is present that is carcinogenic and/or cancer-promoting [162]. cancer inhibiting genes that have been abnormally silenced are also tested for. cutting edge technologies that can detect these clinically useful aberrations include: next-generation sequencing (which allows for whole-exome dna sequencing and whole transcriptome rna sequencing), dna sequencing (sequence analysis reliably detects dna mutations, copy number variations, and gene fusions across the entire exome), rna sequencing (is done to detect clinically relevant aberrations, in particular, gene-fusion events and splice variants), immunohistochemistry biomarkers, in situ hybridization (to detect amplification and fusion events), and methylation analysis. when evaluated as a group, disease-specific lab test panels are developed which can then zero in on the aberrations for any individual patient to improve the therapeutic response [163 165]. based upon the biomarkers found, clinically beneficial therapies may be employed including the use of; hormone therapies, signal transduction inhibitors, gene expression modulators, apoptosis inducers, angiogenesis inhibitors, immunotherapies, and toxin delivery molecules [166,167]. some of the commonly used small molecules include the “nibs” ( tyrosine-kinase and serine/threonine kinase inhibitors) and the “mabs” (monoclonal antibodies). monoclonal antibodies have been developed to attack growth receptors of the cancer cells, growth factors for new blood vessels that feed the tumor, and targets on cells of the immune system to dis-inhibit their activity [168]. natural substances too can be used to target specific aberrations; a few, of many, examples [169,170]: p53, p73 aronia melanocarpa [171,172] vegf omega-3 fatty acids [173] jak/stat3 astaxanthin [174,175] pi3k/akt viscum alb [176] kdr artemisinin [177] mmp2, mmp9 curcumin [178] her2-neu quercetin [179] egfr silymarin [180] ve-cadherin camellia sinensis [181] hif-1α ginseng [182] nf-κb liquorice [183] bcl2 scutellaria baicalensis[184] kras coffee [185] mcl-1 piperlongumine [186] stoff; aji, 5(1): 1-39, 2022; article no.aji.77967 10 2.4 immunotherapy immuno-therapy is the third anti-cancer strategy. edward jenner was a pioneering english physician and scientist who improved the procedure of smallpox inoculation to create the smallpox vaccine in 1796 [187,188]. in the scientific fashion, he purposely exposed people that he had vaccinated against smallpox to prove that the cowpox pus that he used did indeed confer protective resistance. although others before him had used cowpox to provide protection from smallpox, he was the first to then demonstrate that it did in fact work and that the effect was reproducible in subsequent patients [189-191]. although not understanding the intricacies of the immune system, because he applied the scientific method, as it existed in his time, he is referred to as the “father of immunology” [192]. since jenner’s time, imuno-therapy has evolved into an office-based strategy for which they write very thick books and, in the broadest terms possible, it is a treatment that uses the immune system, in some way, to identify, attack, and destroy the cancer. using the immune system against cancer can be accomplished through active or passive pathways. as a quick overview, there are over one hundred and eighty-seven cell types identified within the immune system. it includes the lymphatic system along with the bone marrow, spleen, thymus, and, of course, lymph nodes. cells known as cd4 t cells are the “apparent brains” of the immune system and they coordinate the central immune response to any serious health threat. activated b cells become plasma cells and, in most instances, generate an antibody response against bacterial and viral invasion. lymphokine activated killer cells and cytotoxic t cells respond to viruses and cancer. suppressor t cells are used to downregulate the actions of the immune system after the threat has been eliminated through negative feedback. macrophages are voracious amoeboid-like lymphocytes that eat foreign substances and send a message back to the rest of the immune system indicating what further immune response is necessary. macrophages and their cousins, the dendritic cells, are involved in all aspects of the immune response by initially sending out the alarm that something is amiss. natural killer cells are preprogrammed at their birth to destroy virally infected cells, cancer cells, some bacteria, and parasites on contact without the need for further direction from the cd4 cells. some mushroom extracts and the bioengineered nutraceutical aie10 tm can increase the concentration and activity of natural killer cells. the immune response cascade operates according to three directives. the first is to recognize that which is foreign and sound the alarm soon enough to thwart the invader. molecules and cell surfaces that are identified as foreign are referred to as antigens and have the ability to elicit an immunogenic response. the second directive is to respond to the alarm with enough of a counterattack to effectively neutralize the invader quickly. the third directive is to remember what happened so that if the same situation were to arise again, an effective response could be generated faster. the length and efficacy of the immune response depends upon the “intactness” of the underlying biochemistry. the immune response cascade is the ultimate biological information processing and transfer vehicle designed to define, defend, and integrate oneself relative to the environment that surrounds us. when there is a miscommunication, disease ensues due to corruption, misdirection, or a lack of that informational flow. active immune therapies include, among other agents, il-2, ifn-g, and ifn-a cytokines to stimulate the th1 cells, pathways, and natural killer cells. monoclonal antibodies, checkpoint inhibitors, that disinhibit an immune response are also an active immunotherapy that seems to be most effective when some support has been given to the immune system first. other active therapies include creating primed dendritic cells (for example, sipuleucel-t (provenge)) and natural killer cells and then infusing them into the patient to “patch” holes and reconstitute an effective anticancer response. passive immunotherapies include the use of infused antibodies to bind to cancer cells. then, when natural killer cells encounter antibodycoated cells, the latter’s fc regions interact with their fc receptors, releasing perforin and granzyme b to kill the tumor cell. adoptive t cell therapy is another passive immunotherapy. several ways of producing and obtaining tumortargeted t cells have been developed. t cell infiltrating lymphocytes (tils), specific to the tumor antigens, can be removed from a tumor sample with a core biopsy and then purified with a cell separator or filtered from the blood. subsequent activation with cytokines and cell culturing is performed ex vivo, and then the results were reinfused into the patient. activation stoff; aji, 5(1): 1-39, 2022; article no.aji.77967 11 can take place through gene therapy or by exposing the t cells to tumor antigens in the presence of cytokines. tils can also be stimulated in vivo with hypofractionated sbrt to induce an abscopal effect, which when achieved can have miraculous results. according to the national cancer institute, the abscopal effect “describes the shrinking or disappearance of tumors in parts of the body that were not the direct target of local therapy” [193]. it was first described in 1953, by a researcher named r. h. mole who demonstrated that radiation could shrink a tumor on one side of a mouse and lead to the regression of an untreated tumor on the other side of the animal[194]. he coined the term “abscopal effect” from the latin ab (position away from) and scopus (mark or target). since the phenomena was first observed using radiation, it was assumed that that was the key to the response. a lot of research has been done on varying different parameters of the radiation in order to reliably reproduce the effect [195]. tumor-infiltrating lymphocytes are seen as an important part of the “recipe” as they can signal and initiate a counter-tumor cascade and bode well prognostically [196]. further research has demonstrated that cryotherapy, too, can trigger an abscopal response [197]. in my experience, it seems that any locally applied treatment that causes the sudden death of a large number of cancer cells, thus quickly releasing antigens, without suppressing normal systemic physiology (i.e. the immune system), can trigger a cascade of events that can lead to the death of tumors far removed from the point of the therapy. this includes; radio frequency ablation (rfa), hyperthermia, hi frequency ultrasound (hifu), cryotherapy, and of course fractional dose radiation therapy [198-204]. combining these “mildly” oncolytic therapies with checkpoint inhibitors has yielded better results [205,206]. the checkpoint inhibitors are helpful only to the point that the immune system is still basically structurally intact and functionally effective. when the principles of immune reconstitution are followed, the therapeutic response can be much more beneficial [207]. a “new”, state-of-the-art, passive immunotherapy is the use of chimeric antigen receptors (cars, also known as chimeric immunoreceptors, chimeric t cell receptors, or artificial t cell receptors) which are bioengineered cell receptors that combine a new specificity with an immune cell to target cancer cells. essentially, what is done is that specific monoclonal antibody fractions are grafted onto t cell receptors. the receptors are referred to as chimeric because they are a fusion of proteins and receptors from different immune sources. car-t cell therapy refers to an infusion of such specifically transformed cells for targeted cancer therapy. car-t cells destroy the cancer cells through several mechanisms such as having a direct cytotoxic effect and/or stimulating other cells of the immune system through the release of various cytokines and growth factors. due to the car-t cells very narrow specificity, less prominent tumor cell lines can be missed allowing for a future recurrence [208-218]. overall, immunotherapy is designed to correct, stimulate, direct, or reconstitute an effective anticancer response. immune reconstitution is the evolving clinical science of restoring immune competence by correcting biochemical imbalances, augmenting cytokines, restoring cascade pathways, and/or implanting specific stem cells to bridge areas of damage. all severe or chronic diseases are known to have one or more significant defects in the immune system adversely affecting the immunological imperatives of recognition, response, and memory which, in the case of cancer, leads to anergy and tolerance. testing for and breaking anergy is the first step in immune reconstitution [219]. correcting anergy is of critical importance because it directly correlates to the stage of cancer: over 90% of patients with stage 4 disease are found to be anergic [220]. the immune system cannot fight something that it doesn’t know is there, and that’s exactly what arises with anergy. anergy manifests when there is a failure of signal transmission at any point in the immune response cascade. before we can break anergy, and wake up the immune system, we must make sure that we are dealing with true anergy as opposed to another pathology. of historical interest, some years ago the mérieux company manufactured a skin test called the “multitest mérieux” or “cmi multitest” system (istituto merieux italia, rome, italy). it is used as a general test for the level of the cellular immune response. it is an intradermal test of skin reactivity (similar to allergy tests) in which a control (glycerol) is used with seven common antigens of bacterial or fungal origin (tetanus toxoid, tuberculin, diphtheria, streptococcus, candida, trichophyton, and proteus). in this test, reactions are categorized according to the number of antigens provoking a response, and the summed magnitude of the skin response to stoff; aji, 5(1): 1-39, 2022; article no.aji.77967 12 all seven antigens. based on the chart and information supplied with the simple skin test, basic anergy can be quickly assessed and quantified [221]. unfortunately, this test cannot tell us where in the recognition/response chain of events the problem lies. however, specialized blood tests are now generally able to do that. once this state of unresponsiveness has been confirmed, the next step in breaking anergy is with an immunotherapy protocol specific to the area(s) of immune response dysfunction as briefly described above. 2.5 cancer stem cells as a short overview, cancer stem cells (cscs) are cancer cells (found within tumors or hematological cancers) that possess characteristics associated with normal stem cells, specifically the ability to give rise to all of the heterogeneous cancer cell lines found in a particular tumor. targeting them is the fourth anti-cancer strategy. until recently, attacking them would have been included in the general biochemical targeting strategy but recent research has shown them to be a separate and viable strategy. cscs mediate tumor initiation, progression, and metastasis, and inhibiting them is an emerging new area of research to prevent, stop, and reverse tumor growth. they play key roles in tumor metastasis, drug resistance, and cancer relapse. oncologic research has established that subpopulations of cells, identified by monoclonal antibodies to specific cell surface markers, behaved like developmental stem cells in their capacity to regrow the human tumors, for multiple generations, in experimental immune-deficient animal hosts. in all of the cancers studied so far, there is good evidence that cscs are relatively resistant to radiation therapy and chemotherapy because of their slow rate of growth, indicating that novel csc-targeted therapies are needed. several pathways are promising targets against cscs including inducing their apoptosis, inhibiting stem cell self-renewal to either stop their division or to promote their differentiation, or targeting the csc milieu in the tme that supports them. the anti-csc agents are categorized under two broad headings: small and macromolecules with different subclasses such as kinase inhibitors and polypeptides. one of the first and safest agents to be recognized as a csc inhibitor is metformin [222-235]. tumor initiation can either be driven by transformed differentiated cells or transformed tissue-resident stem cells forming the new cancer stem cells [236,237]. as tissue stem cells already possess unlimited growth potential, it is believed that the transformation into a csc requires only a very small number of genomic changes [238]. multiple biomarkers that characterize cscs have been identified and correlated to diagnosis, therapy, and prognosis [239]. they have been shown to display a high degree of plasticity, which changes their phenotypic and functional appearance making it harder to track them and predict their activity. such shifts in their expression can be induced by chemoand radiation therapy as well as by senescent tumor cells. therapeutic regimens such as chemoor radiation therapy shift the composition of tumor cell subpopulations changes by first killing some of the tumor cells, those with the highest proliferative capacity, causing a decrease in tumor size, while cscs which multiply at a much slower rate survive. instead of dying, some of the tumor cells will become senescent which causes further alterations in the tumor microenvironment that better support the cscs [240]. the transformation to a cancer stem cell can take place during tissue regeneration or can be initiated and/or accelerated as a response to infections, toxins, radiation, or metabolic epigenetic influences that cause mutations [241]. during the process of transformation, oncogenes are overexpressed and tumor suppressor genes are inactivated, hence promoting the creation of, and uncontrolled growth of the cscs. as a consequence of the transformative process, the affected cells “de-differentiate” and acquire stem cell self-renewal characteristics forming the nucleus of the cancer [242,243]. the mutations of the csc progeny have a higher rate of replication which leads to the formation of a tumor [244]. anti-csc research has yielded some very promising therapeutic approaches. nutraceuticals derived from herb and vegetable extracts show anti-csc activity at various levels of clinical utility. cruciferous vegetables belong to the cruciferae family such as arugula, bok choy, broccoli, broccoli rabe, brussel sprouts, cabbage, and others, contain anti-cancer isothiocyanates including sulforaphane, that are enzymatically hydrolyzed from glucosinolates [245]. normal dietary “doses” of isothiocyanate sulforaphane have been shown to have anti-csc activity by altering phosphorylation of several kinases and their substrates including glycogen synthase stoff; aji, 5(1): 1-39, 2022; article no.aji.77967 13 kinase 3 (gsk3), jun n-terminal kinase (jnk), and protein kinase c (pkc) [246]. soy isoflavones like genistein have been shown to have a potent anti-csc effect by inhibiting the phosphorylation of protein kinase b (akt) and forkhead box o 3a (foxo3a) [247]. polyphenolic catechins including epigallocatechin-3-gallate (egcg) found in green tea extracts have demonstrated anti-csc activity, against a variety of cancers, through its ability to inhibit nf-κb activity, mitogen-activated protein kinase (mapk) pathway, activator protein-1 (ap1) activity, and egfr-mediated downstream signaling pathways [248,249]. other anti-csc nutraceuticals are derived from quercetin, curcumin, resveratrol, and ginger [250]. more potent anti-csc pharmaceuticals are also derived from plant sources. taxol is derived from the bark of the pacific yew tree (taxus brevifolia) and was discovered, through a random screening of approximately 15,000 species of plants, to have powerful anti-csc activity against many different cancers [251]. vinca alkaloids are a group of drugs that were originally extracted from the madagascar periwinkle plant, catharanthus roseus g. don, and have anti-csc activity as well as cytotoxic effects. there are three major vinca alkaloids in clinical use: vinblastine (vbl), vinorelbine (vrl), and vincristine (vcr) [252]. several pharmaceuticals have been “repurposed” as anti-cancer agents when their anticsc “side-effect” was noticed. studies have demonstrated that metformins’ anti-cancer, anticsc effects go far beyond its ability to lower blood sugar in that metformin impairs cellular metabolism and suppresses oncogenic signaling pathways, including tyrosine kinase receptors, pi3k/akt, and mtor pathways [253]. furthermore, it suppressed the self-renewal ability of cancer stem cells and induced g0/g1 phase arrest by blocking the activity of cyclindependent kinases. in studies on osteosarcoma cancer cells metformin-induced apoptosis through a mitochondria-dependent pathway, leading to the collapse of the mitochondrial transmembrane potential and the production of oxidatively destructive reactive oxygen species [254]. this destruction of the already compromised mitochondria further decreased atp synthesis and triggered autophagy cancer cell death [255]. thus, metformin is an excellent addition to a metabolic strategy in that its activity is synergistic with the goals of further degrading the already damaged mitochondria in cscs and cancer cells [256]. when combined with a betablocker and aspirin the anti-csc and antimetastasis effects are even more pronounced [257,258]. doxycycline too has been “re-purposed” for the fight against cancer. one of the fda recognized side effects of doxycycline is the inhibition of mitochondrial biogenesis. currently, in the https://www.anticancerfund.org/en/redo-db database, there is a listing of 356 “non-cancer drugs” which have shown some evidence of anticancer activity. amongst them, doxycycline is high on the list of beneficial activity [259]. sorting them out into a generally useful algorithm is a research project for watson, but for now, we have some important clues. doxycycline is a known inhibitor of the small mitochondrial ribosome (28s) and, as a consequence, is an inhibitor of mitochondrial protein translation necessary for biogenesis [260]. in vitro and in vivo evidence demonstrates the inhibitory effects of doxycycline on cancer growth through mitochondrial destruction and the consequence of csc suppression [261]. azithromycin inhibits the large mitochondrial ribosome (39s) and enhanced the tumor necrosis factor-related apoptosis-inducing ligand (trail) activity, which selectively targets tumor cells without damaging healthy cells as an offtarget “side-effect” [262]. together, these two antibiotics have a synergistic, anti-metabolic effect on cscs that is greater than either one alone. we can add to the targeted anti-metabolic csc effect with vitamin c that, in high dose iv’s (greater than 15 grams), acts as a pro-oxidant, which can produce free radicals further stressing the already dysfunctional mitochondria and hastening their destruction [263]. this combination of antibiotics and vitamin c has shown efficacy both in vitro and in vivo [264]. further compromising the cscs mitochondria leading to their destruction, seems to be an important process for stopping cancer. a shift from respiration to fermentation (glucose pathway) is not the only energy production shift that cancer cells, and in particular that cscs can use, but it does seem to be an important and common one. to an extent, cscs are metabolically flexible so multiple pathways may need to be blocked, often simultaneously, to minimize the risk of selective metabolic escape. in addition to inhibiting the major glucose pathway, as described above, fatty acids and glutamine can also be used by many cscs to support their deranged metabolism. lipophilic statins can inhibit the use of fatty acids as an stoff; aji, 5(1): 1-39, 2022; article no.aji.77967 14 energy source for many types of cancer [265]. hydroxychloroquine blocks cancer cells' ability to scavenge nutrients, including glutamine, through macropinocytosis, thereby closing yet another pathway for energy generation by the mutated cells [266]. research on new anti-csc strategies focuses on their signaling pathways and cellular communication system including; hedgehog, wnt/beta-catenin, notch, toll-like receptor 4 (tlr-4), toll-like receptor 9 (tlr-9), integrins, egfr, il-1, il-6, peroxisome proliferatoractivated receptor (ppar), and others. cscs and tumor cells, like all eukaryotic cells, produce structures called exosomes in their endosomal compartment [267]. they are approximately 100 nm in diameter and are surrounded by a lipid bilayer membrane. exosomes have been recognized as potent vehicles of intercellular communication due to their capacity to transfer proteins, lipids, and nucleic acids; thereby influencing various physiological and pathological functions of both recipient and parent cells [268]. the involvement of exosomes in the phenotype transformation from non-csc to csc has been recently evidenced by research on the x chromosome long non-coding rna, which is overexpressed in malignant tumor tissues and activates toll-like receptor (tlr) 7 and nf-κb inflammatory signaling [269]. exosomes have been reported to interact with the cells of the immune system, modulating and downregulating its response thus allowing for tumor progression [270]. tumor-derived exosomes (texs) induce apoptosis of the activated cd8+ (cluster of differentiation 8) t cells (which have an anti-cancer surveillance function), suppress natural killer (nk) cell activity, promote the induction of regulatory t cells (tregs) and myeloid-derived suppressor cells, and interfere with monocyte differentiation into anti-cancer dendritic cells [271]. while texs form the immunosuppressive microenvironment, tregderived exosomes inhibit the induction of cytotoxic t lymphocytes, further allowing for the growth and spread of the tumor [272]. as a result of the research on tumor cell exosomes, they have become a promising target for the treatment of cancer but as of yet this therapy is not available [273-276]. 2.6 cytotoxic cancer therapy cytotoxic cancer treatments are traditionally covered by the term chemotherapy and have come to imply the use of agents that act, separately from the body at large, as intracellular poisons to inhibit mitosis or directly induce some sort of cell death and is the fifth anti-cancer strategy. substances that accomplish this as a secondary effect through blocking extracellular signals or which act through a specific genetic, enzymatic, or hormonal pathway are excluded from this therapeutic strategy as they are referred to as targeted therapies and elsewhere described. chemotherapeutic agents are characteristically purified to the point of being a chemical, and may be of natural origin or synthetically created. the deliberate use of natural, herbal source agents began in the early 1920s, including viscum alkaloids and lectins, and they are still used today, whereas the first synthetic agent, nitrogen mustard, was “discovered accidentally” during world war ii when it was observed that it could shrink lymphoma tumors in mice. a few years later, it was discovered that alkaloids extracted from the vinca rosea plant were useful in treating hodgkin’s disease, and so a multibillion dollar industry was birthed creating and extracting new substances helpful in the “war on cancer”. unfortunately, many of these substances have similar effects on healthy cells, thus creating a multitude of side effects and limiting their usefulness. newer protocols that use lower doses of these agents, such as metronomic or insulin potentiated chemotherapy, are showing good results with far fewer side effects. metronomic, low dose chemotherapy has been in development for two decades and seems to act through several mechanisms including inhibiting the growth of new blood vessels, the restoration of an anticancer immune response, and the induction of tumor dormancy. whereas insulin potentiated therapy (ipt), another low dose strategy, takes physiologic advantage of the excessive number of insulin receptors found on the cell surface of cancer cells. giving insulin just prior to the infusion of low dose chemotherapeutic agents, usually in a combination designed to intervene at several sites of the cell cycle, causes much fewer side effects and can hold the cancer at bay, while buying time to reconstitute an effective immune response or integrate another anti-cancer therapeutic strategy. ipt has been in development and clinical use since the 1930s and has been used to help treat other chronic diseases as well [277-290]. cytotoxic chemotherapy is usually administered as per a protocol derived by treating a group of stoff; aji, 5(1): 1-39, 2022; article no.aji.77967 15 people (cohorts) with a similar diagnosis. these clinical trials are done to determine the general dose and combination of agents that are most effective and least toxic according to the structure of the trial for that disease diagnosis. this approach is designed to attack different genetic targets and the aspects of dna transcription of rapidly dividing cells [291-293]. a more contemporary way for using cytotoxic agents is to target them so that the agents affect biochemical factors or cellular pathways that are unique to the malignant cells or characteristic of tumors based upon genetic studies. cytotoxic chemotherapy can be very good at killing rapidly dividing cells, such as those found in a growing tumor, but are not good at killing the slow-growing cancer stem cells that are at the root of the problem. this leads to the observed high risk for recurrence and to eventual multidrug resistance [294]. there is a great deal of research currently engaged in addressing this problem and certain drug combinations have been found that can lessen this issue by making the cytotoxic response more effective [295]. at this point, we know that p-glycoprotein (also known as multidrug resistance protein 1 (mdr1) or atp-binding cassette sub-family b member 1 (abcb1)) plays a crucial role in determining the neutralizing response against chemotherapy. pglycoprotein (p-gp) acts as an atp-dependent pump that pumps out small molecules from cells, including chemotherapeutic agents, before they can exert their cytotoxic effects. research demonstrates that p-glycoprotein expression levels correlate inversely with drug efficacy, which suggests the rationale for developing pglycoprotein inhibitors for treatment against cancer [296]. while new agents are being developed and going through drug trials, some currently available pharmaceuticals are helpful against multi-drug resistance. verapamil is an ltype calcium channel blocker with antiarrhythmic, antianginal, and antihypertensive activity, but is also a p-gp inhibitor. unfortunately, the dose required to achieve this effect can cause significant hypotension thus limiting its usefulness [297]. other agents that may be helpful are to be found within the class of drugs known as proton pump inhibitors (ppis) [298]. proton pump inhibitors (ppis) given before the chemotherapy have been shown to inhibit mrna levels of vacuolar-type atpase (v-atpases), multidrug resistance mutation 1 (mdr1), multidrug resistance protein 1 (mrp1), phosphatidylinositol 3-kinases (pi3k), akt, mtor, and hif-1α. ppis inhibited v-atpases and down-regulated the expressions of p-gp and mrp1 in a dose-dependent manner both in vitro and in vivo [299]. there are also low concentration natural agents that are cytotoxic to cancer cells that may be used with low doses of cytotoxic chemotherapy to reduce side effects and improve the quality of life. in use for a 100 years, research has shown that viscum album (mistletoe) extracts (vae) have a significant positive impact on the quality of life and the reduction of the side effects from conventional therapies (chemotherapy, radiation) in experimental trials as well as in routine daily application [300,301]. furthermore, pooled data from multiple clinical studies demonstrate that adjuvant treatment of cancer patients with the vae is associated with better overall survival [302]. in some cases not only is vae effective as an additional cytotoxic, anti-cancer agent, but it may succeed when more conventional chemotherapy and immunotherapy have failed [303-310]. another important benefit of vae is its ability to overcome some scenarios of chemoresistance. axl protein is a receptor for tyrosine kinases. some reports showed that targeting anexelekto (axl) enhanced egfr tki response in selected egfr wild type (wt) nsclc patients. studies have found that when targeting axl in egfr wt nsclc cells, the cells showed a more sensitive response to erlotinib than those that overexpressed axl thereby overcoming chemoresistance [311,312]. chemoresistance is a big problem with chemotherapy as the agents seem to have an almost evolutionary effect on tumors, selecting for the most sensitive cancer cells and leaving the rest, including the cscs untouched, or more mutated, therein setting the stage for a recurrence. radiation therapy has similar drawbacks. doxycycline is helpful with both treatment modalities to reduce chemoresistance, largely through interrupting oxidative phosphorylation by inhibiting moieties of the electron transport chain [313,314]. this brings us back to the necessity for targeting cscs to help overcome chemoresistance [315-319]. another cytotoxic strategy that generates much less chemoresistance because it also targets cscs is the use of vitamin c (vc) and vitamin k(3) (vk(3). administered intravenously in a vc:vk(3) ratio of 100:1 exhibit synergistic antitumor activity and preferentially kills tumor stoff; aji, 5(1): 1-39, 2022; article no.aji.77967 16 cells without harming normal cells [320]. this natural vitamin combination accomplishes this through a process of autoschizis, a novel type of necrosis characterized by exaggerated membrane damage and progressive loss of organelle-free cytoplasm through a series of selfexcisions [321]. this combination also attacks the dna of cancer cells since a deficiency of alkaline and acid dnase is a hallmark in all living cancer cells and they are reactivated in the early stages of cancer cell death by vitamin c (acid dnase) and vitamin k(3) (alkaline dnase) leading to dna fragmentation [322]. vitamin c by itself can regulate the proliferation and differentiation of cancer stem cells, the amount and stability of collagen, extracellular matrix remodeling, anti-cancer immunity, and hypoxia in the tumor microenvironment, hence epigenetically suppressing the cscs and downregulating chemoresistance [323-326]. when combined with doxycycline, the effect is even more pronounced [327,328]. 2.7 supportive tactics there are other therapeutic tactics that may be synergistic, but they are not considered strategies unto themselves because their anticancer effects are less reliable. this includes energy-based therapies such as homeopathy and acupuncture, and psychodynamic tactics like psychoneuroimmunology, visualization, prayer, and meditation practices. other tactics employ the use of oncolytic viruses, oxidative therapies (ozone), traditional chinese medicine, naturopathy, ayurvedic herbal medicines, and detoxification protocols, to name but a few. there is no doubt that some of them can be extraordinarily beneficial to the patient by reducing physical symptoms, psychological stress and by engendering a feeling of more selfcontrol against a disease that threatens total chaos. indeed hundreds of books and articles have been scribed as to their use and intermittent efficacy by notable physicians and practitioners; to this day the well-documented phenomena of the spontaneous remission of cancer remains a medical mystery. early in my career, the immunotherapeutic strategy that i employed against cancer was still considered something just short of quackery, and the clinical responses to it were often attributed to this phenomenon. 3. discussion an evidence-based medical practice is established through the application and integration of the best clinical research available at the time, balanced with the clinicians’ expertise and the patients’ life values and beliefs [329]. evidence-based medicine may be considered as an interdisciplinary approach itself, which uses techniques from science, engineering, biostatistics, and epidemiology, such as meta-analysis, decision analysis, riskbenefit analysis, verified case reports, and randomized controlled trials to deliver “the right care at the right time to the right patient” [330]. every time that a physician treats a patient for anything, it is essentially a clinical trial with an “n” of 1. thus the practice of evidence-based medicine tacitly dictates that physicians should make "conscientious, explicit, and judicious use of their understanding of the best medical information available" for the validation of the proposed treatment and its application in patient care in partnership with the patient and their expectations [331]. cancer is a serious and often life-threatening disease that deserves careful consideration of the benefits and long-term risks of each of these five strategic approaches, evolving beyond cohort-based trials into an integrated, personalized approach for the treatment of each patient. champions for the exclusive use of any one of these strategies abound, but it is my belief and experience that the best answer for cancer, for any one patient, will be found in their integration into an individualized cohesive clinical protocol. research is needed to codify the algorithm necessary for applying the best integrative therapeutic strategy given the dynamics of the cancer and the patient’s biochemistry, genetics, and immunology at any given point in their treatment. 4. conclusion cancer, as a disease of our time, has been described as a “wound that does not heal” and is characterized as having numerous biochemical dysfunctions at its core, which lead to a multitude of genetic aberrations and pathologic phenotypic expression. however, in order for these genetically and phenotypically abnormal cells to survive and thrive, the watchdog of the body, the immune system, must itself be suffering from a number of serious areas of structural and functional damage. there are many tactics that can support the therapeutic strategies briefly described above, and they should be applied in an integrative manner as they are nonexclusive stoff; aji, 5(1): 1-39, 2022; article no.aji.77967 17 with each other and can be mutually supportive [332]. however, more aggressive cytotoxic strategies, i.e., full-dose chemotherapy and radiation, can render many of these strategies null and void but may be considered for possible later use. when treating cancer, the physician and patient should make the decisions together, since the stakes can be no higher, using the best information available at the time, and then never look back. when treating cancer there is but one direction to go and that is forward, striving for ever-increasing improvements in the quality and quantity of life with the hope of achieving a durable remission [333-336]. consent it is not applicable. ethical approval it is not applicable. acknowledgements i would like to thank summer a. stoff, b.a., and colleen a. murtha-stoff, n.p. for their highly valued editing skills in preparing this manuscript. competing interests author has declared that no competing interests exist. references 1. byun, gardner k. wounds that will not heal. am j pathol. 2013 apr;182(4):1055– 1064. doi: 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this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: https://www.sdiarticle5.com/review-history/77967 http://creativecommons.org/licenses/by/4.0 _____________________________________________________________________________________________________ *corresponding author: e-mail: celso@alergoimuno.med.br; cite as: olivier, celso eduardo, daiana guedes pinto, ana paula monezzi teixeira, cibele silva miguel, jhéssica letícia santos santana, regiane patussi santos lima, nicole sartoreto rocha, and raquel acácia pereira gonçalves santos. 2025. “endotyping cellular and humoral immunoreactivity against formaldehyde in patients with atopic and/or contact dermatitis”. asian journal of immunology 8 (1):70-81. https://doi.org/10.9734/aji/2025/v8i1161. asian journal of immunology volume 8, issue 1, page 70-81, 2025; article no.aji.134825 endotyping cellular and humoral immunoreactivity against formaldehyde in patients with atopic and/or contact dermatitis celso eduardo olivier a*, daiana guedes pinto a, ana paula monezzi teixeira a, cibele silva miguel a, jhéssica letícia santos santana b, regiane patussi santos lima c, nicole sartoreto rocha d and raquel acácia pereira gonçalves santos a a instituto alergoimuno de americana, brazil. b instituto de ensino e pesquisa do hospital de amor de barretos, brazil. c lavoisier laboratórios, são paulo, brazil. d faculdade de americana, são paulo, brazil. authors’ contributions this work was carried out in collaboration among all authors. author ceo conceptualized the study, did data curation, formal analysis, literature review, and wrote the original draft. authors dgp, apmt, csm, nsr, jlss and rpsl performed the laboratory procedures. author rapgs performed the cutaneous tests. all authors read and approved the final manuscript. article information doi: https://doi.org/10.9734/aji/2025/v8i1161 open peer review history: this journal follows the advanced open peer review policy. identity of the reviewers, editor(s) and additional reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here: https://pr.sdiarticle5.com/review-history/134825 received: 19/02/2025 published: 25/04/2025 original research article https://doi.org/10.9734/aji/2025/v8i1161 https://pr.sdiarticle5.com/review-history/134825 olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 70-81, 2025; article no.aji.134825 71 abstract background: several publications report that formaldehyde is responsible for hypersensitivity reactions in patients with contact dermatitis, as diagnosed by "in vivo" provocation tests. there is no standardized lab exam that can endotype the mechanisms responsible for these phenotypes, aim: to evaluate the potential of the tube titration of precipitins (ttp) and the leukocyte adherence inhibition test (lait) to discriminate and endotype cellular and humoral immunoreactivity against formaldehyde in patients with contact dermatitis. methods: we retrospectively examined the medical charts of two cohorts (n = 100, each) of patients diagnosed with atopic dermatitis and/or contact dermatitis with clinical suspicion of formaldehyde hypersensitivity, who were investigated with the help of ttp or ex vivo challenge tests monitored by lait against formaldehyde. the registered results were distributed in ranges through cascade distribution charts. the statistical characteristics of these cohorts were calculated. results: ttp showed a distribution concentrated over the more diluted titrations with no negative result. the mean was estimated at 1:385; the median at 1:512; and the standard deviation at 1:166. the lait results demonstrated a wide range. the lai ranged from 0% to 100%. the mean was 57.5%; the median was 65.5%; the standard deviation was 37.4%. conclusion: our preliminary results support that the ttp and lait performed with formaldehyde solution may discriminate diverse degrees of humoral and cellular immunoreactivity in patients suffering from atopic and/or contact dermatitis. by evaluating the utility of ttp and lait as diagnostic tools, the study provides preliminary evidence for endotyping immunoreactivity, which could advance precision medicine in allergy management. the findings may guide clinicians in identifying hidden formaldehyde exposure in products and inform safer therapeutic strategies for sensitized patients. it is worthwhile conducting more in-depth studies to evaluate the usefulness of ttp and lait in endotyping non–ige-mediated hypersensitivity to formaldehyde. keywords: atopic dermatitis; contact dermatitis; endotype; hypersensitivity; formaldehyde; leukocyte adherence inhibition test; precipitins; precision medicine. abbreviations lai: leukocyte adherence inhibition lait: leukocyte adherence inhibition test ttp: tube titration of precipitins 1. introduction the american contact dermatitis society elected formaldehyde as the "allergen of the year 2015" (pontén & bruze 2015). formaldehyde (known as methanal, methylene oxide, oxymethyline, methylaldehyde, and oxomethane) is the simplest aldehyde, with the chemical structure h2c=o (gerberich & seaman 2013). usually stored as an aqueous solution containing variable amounts of methanol (formalin), it turns on a colorless, pungent, suffocating, flammable gas when liberated into the atmosphere (commission, 2024). formaldehyde in aqueous solutions spontaneously hydrates to h(h2c=o) oh and aggregates to form mixtures of hydrated oligomers: h(h2c=o)n oh depending on formaldehyde concentration. methanol stabilizes aqueous formaldehyde solutions by decreasing the average value of n (dankelman & daemen 1976). the "plastic age" was inaugurated in 1910 by polymerizing formaldehyde and phenol, producing bakelite (braun et al., 2013). nowadays, several industrial uses have been discovered for formaldehyde, turning it into an intermediate chemical tool for the production of adhesives, fabrics, polymers, resins, plastics, paints, lacquers, dyes, explosives, and so forth, stimulating the industrial research of diverse patented productions methods worldwide (walker, 1964 disponible at "internet archive": https://archive.org/details/formaldehyde0000walk ). formaldehyde may also be formed and liberated in the atmosphere by the incomplete combustion of tobacco, wood, coal, gasoline, diesel, and ethanol in internal combustion engines (dias et al., 2012). gaseous formaldehyde at higher concentrations can irritate the eyes and mucous membranes of the respiratory tract, even producing asthma (bardana & montanaro 1991; pougnet et al., 2025; zhang et al.; 2025). olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 70-81, 2025; article no.aji.134825 72 initially used as a disinfectant, embalming (anatomy dissection classrooms), and viral inactivator for the production of vaccines, nowadays, formaldehyde is listed as a human carcinogen with restricted uses (commission, 2021). formaldehyde is prohibited from use in cosmetic products in most countries. however, the so-called formaldehyde releasers are usually allowed (commission, 2019). to avoid the inconveniences of formalin, chemists developed the formaldehyde releasers (or formaldehyde donors), reversible linear or cyclic polymers of formaldehyde that slowly release free formaldehyde at levels suppressing microbial growth but (theoretically) sufficiently low not to harm humans (de groot et al., 2009). these formaldehyde releasers (such as quaternium-15, diazolidinyl urea, dmdm hydantoin, imidazolidinyl urea, 2-bromo-2-nitropropane-1,3diol (bronopol), germall-115, preventol, and so forth) are found in cosmetics (creams, lotions, make-up removers, soaps, shampoos, deodorants, toiletries, nail products), cleaning household products (detergents) and industrial chemical products (flyvholm & andersen, 1993). soon, it was realized that formaldehyde releasers were also causes of contact dermatitis in patients sensitized to formaldehyde (dahlquist & fregert 1978). recently, it was reported that about threequarters of tested us tattoo inks analyzed by the chromotropic acid method resulted in a positive for formaldehyde releasers (liou et al., 2021). until recently, textile finishes released an elevated level of free formaldehyde (textileformaldehyde resins), causing frequent textile dermatitis in individuals sensitive to formaldehyde. however, nowadays, clothing finishes release much less free formaldehyde, and allergic contact dermatitis from clothing due to formaldehyde releasers is much less frequent than in the past decades. (lazarov et al., 2003). nowadays, the main question about formaldehyde hypersensitivity is not about the known products with the declared presence of formaldehyde or formaldehyde releasers in their composition but the undeclared presence of formaldehyde in industrial products. in a recent sampling, it was demonstrated by highperformance liquid chromatography (hplc) that 23 of 130 cosmetic products (18%) (without formaldehyde or formaldehyde releasers on the package ingredient list) were presenting variable amounts (0.5–507 ppm) of formaldehyde (søgaard et al., 2024). the undeclared presence of formaldehyde in industrial products results from the air oxidation of ethoxylated alcohols, such as polyethylene glycols (bergh et al., 1998). polyethylene glycols are polymers of ether monomers such as ethylene glycol, ethylene oxide, or oxyethylene, usually available as mixtures of different chain lengths polymers, used as emulsifiers in industrialized food and food supplements (e 1521), medicines (macrogol), cosmetics and housecleaning products (olivier et al. 2024e). even corticoid creams may present formaldehyde, mainly when presenting macrogol in their composition (dahlquist et al., 1980). the methyl ester of the aspartic acid/phenylalanine dipeptide (aspartame) may also be degraded to formaldehyde and produce systemic allergic dermatitis (veien & lomholt 2012, hill & belsito 2003, castanedo-tardan et al., 2009). besides producing their characteristic hypersensitivity conditions (generalized and localized allergic contact dermatitis, airborne symptoms such as rhino-conjunctivitis and asthma, immediate-type allergies such as urticaria and anaphylaxis), hypersensitivity to formaldehyde may also aggravate preexisting dermatoses, producing flares of atopic dermatitis, stasis dermatitis, and rosacea (goossens & aerts 2022). formaldehyde is unanimous among the diverse batteries recommended for composing diagnostic cutaneous contact test kits (patch tests) (bruynzeel et al., 1995). when cutaneous tests are inconclusive, the best way to diagnose formaldehyde hypersensitivity is the exclusion/provocation test, when the patient interrupts the use of the suspected allergen until the symptoms disappear. then, the allergen is re-introduced to observe reactions. however, this is particularly difficult when polysensitization dominates the clinical picture. in order to shorten the list of suspected allergens, we performed triage tests to elect the allergens that will be emphasized in the exhaustive in vivo exclusion/provocation tests. cellular immunoreactivity against haptens and hapten-carrier conjugates had been classically olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 70-81, 2025; article no.aji.134825 73 evaluated by the leukocyte adherence inhibition test (lait) (kuratsuji, 1981). humoral immunoreactivity against haptens has been classically evaluated by precipitin research (rittenberg & amkraut 1966). to evaluate cellular immunoreactivity, we employ in our facilities the lait (olivier et al. 2022b, olivier et al. 2022a, olivier et al. 2022c, olivier et al. 2023b, olivier et al. 2023a). to evaluate the humoral immunoreactivity, we employ at our facilities the tube titration of precipitins (ttp) (olivier et al. 2021c, olivier et al. 2021e, olivier et al. 2023c, olivier et al. 2024f, olivier et al. 2025b). the present study hypothesizes that the lait and the ttp may help differentiate diverse endotypes and degrees of immunoreactivity against formaldehyde among patients suffering from non–ige-mediated atopic and/or contact dermatitis. to evaluate the potential of the lait and the ttp to endotyping non-ige-mediated immunoreactivity against formaldehyde, we retrospectively compiled the electronic medical charts of patients with these conditions who were investigated with these procedures in our outpatient clinic. 2. materials and methods 2.1 subjects after receiving institutional review board approval from the instituto alergoimuno de americana (brazil; 03/2025), we proceeded with the electronic chart review of 10,270 outpatients who attended our facility from january 2018 to april 2025. a cohort of 100 outside patients had been submitted to ttp with formaldehyde solution for presenting atopic and/or contact dermatitis. this cohort counted 29 males; mean age 38.8 years; sd 19.6 years; range 3 to 90 years; median 38 years; modes: 7; 9; and 70 years (each appeared 4 times); geometric mean = 32 years. a cohort of 100 outside patients had been submitted to an ex vivo allergen challenge test with formaldehyde solution monitored with lait for presenting non–ige-mediated atopic and/or contact dermatitis. this cohort counted 29 males; mean age 44.7 years; sd 19.1 years; range 9 to 91 years; median 45 years; modes = 35; 48 and 58 years (each appeared three times); geometric mean = 39.5 years. this study did not include patients under biological and/or systemic anti-inflammatory therapy. these procedures were offered to patients with clinical suspicion of formaldehyde hypersensitivity who demonstrated a nonreactive or inconclusive skin test performed with formaldehyde solution (olivier et al. 2013). 2.2 formaldehyde solution the formaldehyde solution was prepared with 1.5 ml of a solution of formaldehyde 10% (perfyl tech®) diluted to 15 ml with distilled water to finalize a 1 mg/ml solution to perform the allergic skin tests, ttp and lait. 2.3 ex vivo investigation: leukocyte adherence inhibition test 2.3.1 procedure for allergen ex vivo challenging we performed the lait as previously described (olivier et al. 2012, olivier et al. 2014, olivier et al. 2021a, olivier et al. 2021b, olivier et al. 2021d). shortly, each donor's fresh plasma was divided into two parts and used in paralleled ex vivo challenging tests with formaldehyde solution and the unchallenged plasma assay. we collected plasma with high leukocyte content (buffy coat) from the heparinized tube after one hour of sedimentation at 37 °c. then, we distributed aliquots of 100 μl into eppendorf tubes kept under agitation for 30 minutes (200 rpm at 37 °c) with formaldehyde solution (10μl) or without formaldehyde solution (when used as control). 2.3.2 procedure for adherence assay after incubation, the plasma was allocated into a standard neubauer hemocytometer counting chamber with a plain, non-metallic glass surface and left to stand for 2 hours at 37 °c in the humidified atmosphere of the covered water bath to allow leukocytes to adhere to the glass. next, we counted the leukocytes, removed the coverslip, and washed the chamber by immersion in a beaker with pbs (phosphatebuffered saline) at 37 °c. then, we added a drop of pbs to the hemocytometer's chamber and allocated a clean coverslip over it. the remaining cells were counted in the same squares as previously examined. olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 70-81, 2025; article no.aji.134825 74 2.3.3 procedure for calculation the percentage of leukocyte adherence (la) of each assay was estimated as: (the number of leukocytes observed on the hemocytometry chamber after washing divided by the number of leukocytes observed on the hemocytometry chamber before washing) and multiplied by 100 (%). the leukocyte adherence ratio (lar) was estimated based on the ratio between the la from the antigen-specific challenged plasma and the la from the unchallenged control plasma: lar = la of the challenged sample divided by la of unchallenged control plasma multiplied by 100 (%). to further calculate the leukocyte adherence inhibition (lai), we subtracted the lar from 100 (%). we employed the lai results for the cascade distribution chart and the statistics calculations, both performed with the help of the microsoft excel® statistical package. 2.4 in vitro investigation: tube titration of precipitins (ttp) as previously reported, a transparent vitreous tube array performed the semi-quantitative ttp against the formaldehyde solution (olivier et al. 2024b, olivier et al. 2024c, olivier et al. 2024a, olivier et al. 2024d, olivier et al. 2025a). shortly, the patient's blood was collected in a clotactivator collecting tube. after separation, the serum was centrifugated at 2,000 rpm for 10 minutes. the allergen extracts were allocated in sets of eleven glass tubes at progressive duplicated serum dilutions. the progressive dilutions were combined with the 15 μl of the antigen solution with 250 μl of the patient's serum, progressively diluted into physiological saline solution (nacl 0,9%) in the dilution ratios of 1:1; 1:2; 1:4; 1:8; 1:16; 1:32; 1:64; 1:128; 1:256; and 1:512. one tube was a blank control done with the water and serum to observe occasional spontaneous precipitation (sia test). after 24 hours, the tubes were examined, and the titers (the highest dilution factor that yields a positive reading) were recorded (williams & chase. 1971). 3. results as a retrospective survey, there was no research protocol; therefore, we report the incidental immune investigation as registered in the digital medical charts. the ttp showed a distribution concentrated over the more diluted titrations (fig 1). there was no negative result. the mean was estimated at 1:385; the median was 1:512; the standard deviation was estimated at 1:166; the mode was 1:512 (appeared 61 times). all sia tests were negative. the cascade distribution of lai results demonstrated a wide range (fig.2). the lai ranged from 0% to 100%. the mean was 57.5%; the median was 65.5%; the standard deviation was 37.4%; the mode was 0% (appeared eleven times). about half the patients presented high immunoreactivity during the ex vivo challenge test (lai > 60%). fig. 1. cascade distribution chart of the tube titration of precipitins (x-axis %) resulting from the formaldehyde solution against the serum of a cohort of 100 tests/subjects (y-axis) olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 70-81, 2025; article no.aji.134825 75 fig. 2. cascade distribution chart of the range groups of leukocyte adherence inhibition (lai) results (x-axis %) of ex vivo formaldehyde solution monitored by the leukocyte adherence inhibition test (lait), according to the respective number of outcomes over a cohort with 100 tests/subjects (y-axis) all patients evaluated with ttp demonstrated some degree of humoral immunoreactivity, and most presented positivity by the more diluted titrations. eleven patients did not present cellular immunoreactivity against formaldehyde (lai = zero%), while others presented an extensive range of inhibition of the leukocyte adherence after the ex vivo provocation test. 4. discussion despite being universally recognized by its sensitizing properties, little is known about the intrinsic physiopathology of the immunoreactivity (or immunoreactivities) against formaldehyde. formaldehyde can establish cross-linking reactions among several amino acids, producing deformation in the proteins' tertiary structure and altering their immunoreactivity (sompuram et al. 2004, fraenkel-conrat et al., 1947; fraenkelconrat & olcott, 1948b; fraenkel-conrat & olcott 1948a). when absorbed into human blood current, formaldehyde may conjugate with serum proteins, such as the human serum albumin (has), forming hapten-carrier complexes able to develop the production of antibodies (ige, igm, igg, and iga) against their non-self-conjugates (patterson et al. 1986, patterson et al. 1989). ige-mediated sensitization against formaldehyde is not an easy diagnosis but has already been described in children exposed to gaseous formaldehyde (kramps et al. 1989, wantke et al. 1996a, wantke et al. 1996b, mizuki & tsuda 2001). additionally, to the capacity to produce antibodies against the formaldehyde-has conjugated, analysis of individuals occupationally exposed to formaldehyde demonstrated elevated t antigen memory cells and lymphocyte subpopulations of t-helper/suppressor (h/s) ratios ranging from 0.8 to 3.3, suggesting chronic antigenic stimulation by formaldehyde (thrasher et al., 1988). there is no standardized lab examination to (unequivocally) diagnose non–ige-mediated formaldehyde hypersensitivity or to suggest formaldehyde immunoreactivity. however, endotyping the mechanisms responsible for allergic phenotypes is crucial for diagnosing and supervising treatments under personalized medicine and recognizing differential diagnoses among phenotypes (olivier. 2024). several phenotypes have been described; however, the endotypes have been poorly explored since the main clinical tools to verify hypersensitivity against formaldehyde are still the cutaneous tests. as a proof-of-concept, we submitted formaldehyde to an ex vivo challenge monitored by the lait to demonstrate cellular immunoreactivity. in the same proof-of-concept mentality, we titrated precipitins against formaldehyde to demonstrate humoral olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 70-81, 2025; article no.aji.134825 76 immunoreactivity. despite non-reactive or inconclusive skin tests, we propose these procedures to patients with atopic dermatitis and/or contact dermatitis with a strong clinical suspicion of hypersensitivity to formaldehyde. the clinical reasoning to indicate these tests is in the assumption that these immunoassays may function as triage tests to reinforce the need for a more exhaustive diagnostic exclusionprovocation test (when the patient excludes the suspected allergen until the symptoms disappear and then re-introduces the allergen to observe the reactions). as a retrospective cohort analysis, there was no prospective plan. we spreadsheeted a compilation of registered results produced by ttp and lait, exploring humoral and cellular immunoreactivity against formaldehyde. these assays provide clues about humoral and cellular immunoreactivity, and the results are distributed in an extensive spectral range, presumably between immune tolerance and symptomatic hypersensitivity. results provided by lait and ttp were interpreted as markers of the immune response after contact with the specific antigen, configuring themselves as techniques to identify exposition to the antigen, immune stimulation, and immunoreactivity, as proposed by the exposome-wide association study (chung et al. 2024). at the clinical set, the diagnosis of formaldehyde allergy is accomplished by anamnesis, skin tests, and in vivo provocation tests; however, when employing a multi-omics approach, several clinical phenotypes and endotypes may be differentiated (yoon & bunyavanich 2025). this retrospective survey demonstrated that the ttp and the ex vivo challenge test monitored by lait against formaldehyde can demonstrate significant cellular and humoral immunoreactivity in patients diagnosed with atopic and/or contact dermatitis. however, these immunoassays did not prove per se that hypersensitivity to formaldehyde is responsible for these patients' symptoms. this association may only be confirmed by further in vivo provocation studies. none of our patients presented an exclusive reaction to formaldehyde. we assessed every patient simultaneously with several chemical and biological allergens, demonstrating positive results for some of them, according to clinical suspicions. the most vital suggestion driven by the results is that allergic patients may impair their symptoms by using creams or cosmetics contaminated with formaldehyde. in our practice, when preceding in vivo cutaneous tests with formaldehyde, we observe immediate cutaneous reactions (obtained by the skin scrape test) and delayed reactions obtained by a forty-eight-hour contact test or a photosensitized ninety-six-hour contact test (patch test). based on this clinical experience, we can hypothesize that at least three endotypes are associated with formaldehyde hypersensitivity, which may be produced by at least three mechanisms: a predominantly humoral, a predominantly cellular, and a compound of both. integrating ttp and lait into clinical workflows may guide clinicians in identifying hidden hypersensitivity due to formaldehyde exposure in products and inform safer therapeutic strategies for sensitized patients. 5. conclusion our preliminary results show that the lait and ttp may differentiate diverse cellular and humoral immunoreactivity degrees against formaldehyde in patients clinically diagnosed with non–ige-mediated allergies. lait and ttp are inexpensive, can be performed with minimum laboratory equipment, and can be incorporated into strategies to address health disparities in respiratory and food allergies (anagnostou et al., 2025). as a preliminary report, the propaedeutic meaning of the presented results and the possibility of interferents must be yet established (anouar et al., 2024). more studies focused on the quality-by-design approach with prospective larger double-blind cohorts need to evaluate the potential contribution of lait and ttp for endotyping immunoreactivity of patients suspected of symptomatic hypersensitivity against formaldehyde and other similar preservatives (chiarentin et al., 2023). 6. limitations this study is a retrospective analysis of data collected over six years. there was no protocol research, no control group, and the subject's data were limited to the essentials available on our electronic sheets. therefore, we could not establish a cross-comparison between positive and negative controls to validate the results. the number of subjects is appropriate for a preliminary study; however, future studies must be more comprehensive. the lack of a research olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 70-81, 2025; article no.aji.134825 77 protocol implies the possibility of a bias produced by the physician's point of view, who suggested the exam barely on clinical suspicion led purely by the anamnesis and physical examination. the study lost many of these patients to follow-up, so assuring the relationship between the immunoassays' results and the patient's clinical outcome is impossible. 7. future directions and recommendations for clinical practice the primary intended use of in vitro or ex vivo allergen challenge tests is to spare the patients from being submitted to unnecessary, exhaustive, and dangerous in vivo challenge tests. exploring the humoral and the cellular arms of immune systems, the ttp and lait alone or combined may represent, in the near future, a tool for allergists to construct an etiologic diagnosis from their patients, as well as determine the endotypes (mechanisms) of hypersensitivity, in order to choose more convenient and personalized therapies for them. adding data provided by ttp and lait may also contribute to streamlining biomedical research and improving tools such as large language models, usually used by clinicians as a decision support system to enhance diagnostic accuracy (abers & mathias 2025). consent as a retrospective survey of results recorded in cognito, consent was given collectively by the institution's ethics committee following the principles of the declaration of helsinki (wma, 2013). ethical approvals the authors have collected and preserved written ethical approval per international standards. disclaimer (artificial intelligence) author(s) hereby declare that no generative ai technologies such as large language models (chatgpt, copilot, etc.) and text-to-image generators have been used during the writing or editing of this manuscript. acknowledgements the instituto alergoimuno de americana funded this work. we want to thank the laboratory technicians alessandra vieira de oliveira and luciana sacilotto carvalho for their help with the exams. competing interests authors have declared that they have no known competing financial interests or non-financial interests or personal relationships that could have appeared to influence the work reported in this paper. references abers, m. s., & mathias, r. a. 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(2025). innovative formaldehyde adsorption with optimized deep eutectic solvents: an experiment and multilevel computational chemistry approach. environmental research, 266, 120593. disclaimer/publisher’s note: the statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of the publisher and/or the editor(s). this publisher and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. _________________________________________________________________________________ © copyright (2025): author(s). the licensee is the journal publisher. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: https://pr.sdiarticle5.com/review-history/134825 https://pr.sdiarticle5.com/review-history/134825 _____________________________________________________________________________________________________ *corresponding author: e-mail: celso@alergoimuno.med.br; cite as: olivier, celso eduardo, daiana guedes pinto, ana paula monezzi teixeira, cibele silva miguel, jhéssica letícia santos santana, regiane patussi santos lima, nicole sartoreto rocha, raquel acácia pereira gonçalves santos, and everton salgado monteiro. 2025. “endotyping cellular and humoral cross-reactivity among canine, feline, and swine allergens in patients with allergic multimorbidity”. asian journal of immunology 8 (1):142-62. https://doi.org/10.9734/aji/2025/v8i1168. asian journal of immunology volume 8, issue 1, page 142-162, 2025; article no.aji.138781 endotyping cellular and humoral cross-reactivity among canine, feline, and swine allergens in patients with allergic multimorbidity celso eduardo olivier a*, daiana guedes pinto a, ana paula monezzi teixeira a, cibele silva miguel a, jhéssica letícia santos santana b, regiane patussi santos lima c, nicole sartoreto rocha d, raquel acácia pereira gonçalves santos a and everton salgado monteiro e a instituto alergoimuno de americana, brazil. b instituto de ensino e pesquisa do hospital de amor de barretos, brazil. c lavoisier laboratórios, são paulo, brazil. d faculdade de americana, são paulo, brazil. e faculty of medicine, são paulo university, brazil. authors’ contributions the authors conducted this work in collaboration with each other. the author ceo is responsible for conceptualization, data curation, formal analysis, literature review, and writing the original draft. authors dgp, apmt, csm, nsr, jlss and rpsl performed laboratory procedures. author esm extracted the proteins from the fur of cats and dogs. author rapgs performed cutaneous tests. all authors read and approved of the final manuscript. article information doi: https://doi.org/10.9734/aji/2025/v8i1168 open peer review history: this journal follows the advanced open peer review policy. identity of the reviewers, editor(s) and additional reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here: https://pr.sdiarticle5.com/review-history/138781 https://doi.org/10.9734/aji/2025/v8i1168 https://pr.sdiarticle5.com/review-history/138781 olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 142-162, 2025; article no.aji.138781 143 received: 16/06/2025 published: 03/07/2025 abstract background: the cat-pork syndrome and the cross-reactivity between cat dander and dog dander are particular situations associated with the allergic multimorbidity phenotypes. the perception of this cross-reactivity must benefit the allergist dealing with these conditions. aim: to evaluate the capacity of the tube titration of precipitins (ttp) and the leukocyte adherence inhibition test (lait) to discriminate cellular and humoral immunoreactivity against cat fur and dog fur extracts as well as pork meat extract in patients with non–ige-mediated multimorbidity phenotypes. study design: we examined retrospectively the medical charts of two cohorts of patients clinically diagnosed with non–ige-mediated multimorbidity allergic phenotypes related to inhalation or contact with cat fur, dog fur, and/or consumption of pork meat, who were investigated with the help of ttp or lait. methodology: the correlation between the paired assays was calculated using pearson's methodology and demonstrated by dispersion graphs. results: pearson's correlation indicated a significant positive relationship between the cat fur and dog fur extracts in lait results: r (98) = 0.395, p-value < 0.001. the paired-t test indicated a significant difference between the ttp results of cat fur and pork meat extracts (p-value = 0.009). pearson correlation analysis indicated a non-significant, negative relationship between the ttp results of cat fur and pork meat (p-value = 0.644). the paired t-test indicated a significant difference between the ttp results of dog fur and pork meat extracts (p < 0.001). conclusion: the preliminary results suggest that the ttp and lait may discriminate between diverse humoral and cellular immunoreactivity levels in patients with various allergic phenotypes, as observed in cat and dog fur and pork meat extracts. a significant association was found between the immunoreactivity of cat fur and dog fur. there was no clear association between pork meat and fur immunoreactivity. keywords: endotype; hypersensitivity; cat fur; dog fur; leukocyte adherence inhibition test; non–igemediated immunoreactivity; pork meat; precipitins. abbreviations lai : leukocyte adherence inhibition. lait : leukocyte adherence inhibition test ttp : tube titration of precipitins 1. introduction hypersensitivity conditions, such as allergic rhinoconjunctivitis and allergic bronchitis caused by sensitization to allergens from furry pets, affect more than 10% of the worldwide population, producing a deleterious impact on patients' quality of life (chan and leung 2018; van hage et al. 2023; konradsen et al. 2015). allergic multimorbidity (defined in patients with concomitant or consecutive allergic phenotypes) is the subject study for the "mechanisms of the development of allergy" (medall) project to understand the links between multimorbidity and polysensitization in allergic phenotypes that may be ige-mediated, partly ige-mediated, or nonige-mediated (bousquet et al. 2015; bousquet et al. 2025). allergic multimorbidity is a concept associated with the "atopic march", a pattern of allergic disease development recognized in children with sequential and concomitant sensitization to food allergens and inhalant allergens (hahn and bacharier 2005; kulig et al. 1999). allergic sensitization may happen at any age. in general, no clinical symptoms are detectable at birth; however, the earlier sensitization is usually directed against food proteins (hen's egg and cow's milk, occurring via the mother's milk), while sensitization to environmental allergens and other food allergens (such as soy and wheat original research article olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 142-162, 2025; article no.aji.138781 144 allergens) are increasingly progressing after the first birthday (wahn 2000). initial signs of allergic disease are atopic dermatitis and food allergies, which typically have their most significant incidence peaks during the first two years of life. nearly half of children with atopic dermatitis develop symptoms within the first six months of life, and approximately 85% of individuals with eczema have symptoms onset by the age of five years (kay et al. 1994). allergic multimorbidity represents a group of conditions that deleteriously impair the quality of life and usually involves polysensitization in which cat and dog dander may be particularly associated (li et al. 2025). cross-reactivities among related and unrelated allergens are standard features in allergic patients for whom allergen immunotherapy represents the appropriate treatment, necessitating the identification of responsible allergens (liang et al.; 2024; alvarez-cuesta et al.; 2007; patel et al.; 2013; varney et al.; 1997; worm; et al. 2013). some murine models have been developed to study cross-reactivity among multiple food allergens (musa et al. 2024). cross-reactivity between furry animals and animal-derived food allergens is under review (gromek et al., 2025). the allergen nomenclature sub-committee of the world health organization and international union of immunological societies (who/iuis) has recognized so far eight allergens weighing from 11 to 1,000 kda, identified from the domestic cat (felis domesticus) named after fel d 1 (uteroglobin) to fel d 8 (latherin-like protein) (sub-committee 2025b). the same subcommittee has recognized eight allergens so far, weighing from 14 to 29 kda, identified in the domestic dog (canis familiaris), named after can f 1 (lipocalin) to can f 8 (cystatin) (subcommittee, 2025a). the allergen nomenclature sub-committee of the world health organization and the international union of immunological societies (who/iuis) has recognized one allergen from the domestic pig (sus scrofa) to date: a serum albumin of 60 kda, named sus s 1 (sub-committee, 2025c). cross-reactivity between cat fur and dog fur is a particular situation, as dog extracts contain a fel d 1-like allergen that is cross-reactive to fel d 1 (hellu et al., 2024). molecular mimicry between unrelated mammalian proteins from different animal species has been associated with unexpected cross-reactive allergic reactions, particularly between proteins from cat dander and pork meat (kile et al., 2023). the first reports of a direct cross-reactivity between cat dander and pork meat came from france in 1994, manifested as urticaria associated with abdominal symptoms, and were named the "catpork syndrome" (also referred to as pork-cat syndrome) (drouet et al. 1994; drouet; et al 1994a; drouet et al. 1994). the first case report was accompanied by an extensive investigation performed with skin tests, specific ige quantification, electrophoresis, western blot, and chromatography that confirmed a crossed reaction against a common epitope from pork meat and cat extract, a protein with a molecular weight of 67 kda (sabbah et al. 1994a; sabbah et al. 1994b). after this initial description, several cases in france were further reported, associating the syndrome with cross-reactivity against similar allergens, such as dog dander, boar meat, and heparin (couturier et al. 1999; drouet et al. 2001; drouet; et al. 1997; drouet and sabbah 1996). the first description of the so-called cat-pork syndrome in the united states of america was published in 2013, reporting eight patients with elevated specific ige against cat dander, dog dander, and pork meat allergens. the more illustrative cases presented abdominal cramping, nausea, itching, and hives after ingestion of pork (posthumus et al. 2013). in 2014, spanish physicians also described a case of occupational asthma in a patient sensitized to cat dander, dog dander, and pork meat (alvarez-perea et al., 2014). soon, in 2015, the cat-pork syndrome became just one more example of crossreactivity among aeroallergens and food allergens (popescu 2015). further, in 2019, japanese investigators reported a case of early childhood onset pork-cat syndrome associated with dog sensitization. a 6year-old girl presented with recurrent episodes of urticaria after consumption of pork meat with specific ige (≥ 50 ui/ml) against cat dander, dog dander, pork meat, sus s 1, fel d 2, can f 1, can f 2, and can f 3. western blotting analysis demonstrated specific ige activity against a 67kda protein in pork meat and cat dander extract. cross-reactivity between these two proteins was confirmed by an inhibition test (yamada et al. 2019). more recently, component resolved diagnosis associated fel d 2 and sus s 1 as the main molecular allergens involved in crossreactivity (barradas lopes et al. 2022). olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 142-162, 2025; article no.aji.138781 145 the alpha-gal syndrome is another condition that warrants careful diagnosis, as it may present cross-reactive allergens in cat and dog dander, as well as pork meat (commins and platts-mills, 2013). first put in evidence in 2009, the alphagal syndrome is a cross-reactive allergic multimorbidity condition elicited by ige and nonige antibodies against the galactose-α-1,3galactose (α-gal), a carbohydrate moiety commonly expressed on non-primate mammalian proteins (such as beef, pork, and lamb), and in the cat iga (commins et al. 2009). allergy against α-gal is usually triggered by tick bites (van nunen et al. 2009). besides the classic gell and coombs igemediated type i hypersensitivity mechanism, several types and subtypes of non-ige-mediated hypersensitivity mechanisms are being studied, associated with various allergic phenotypes (jutel et al., 2023). humoral immunoreactivity against food allergens and aeroallergens has been traditionally evaluated through research on precipitins (augustin, 1953; augustin, et al. 1960; cunningham-rundles et al., 1978; ferguson and carswell, 1972; heiner, et al. 1962). we also routinely employ the tube research of precipitins (ttp) in our facilities as a triage to evaluate humoral non–ige-mediated immunoreactivity against suspected allergens before performing more exhaustive in vivo provocation tests (olivier et al. 2023e; olivier et al. 2021e; olivier et al. 2021d; olivier et al. 2024e; olivier et al. 2024c). the leukocyte adherence inhibition test (lait) and its similar assay, the leukocyte migration inhibition test (lmit), have traditionally been used to differentiate non–ige-mediated immunoreactivity against microorganisms and aeroallergens (fink et al. 1987; kallen and nilsson 1979; kuratsuji 1981; thomson 1982). the lait and the lmit have also been used to differentiate non–ige-mediated immunoreactivity against food allergens (allardyce and shearman 1975; george and vaughan 1962; ashkenazi et al. 1980; butler et al. 1981; papageorgiou et al. 1983). non–ige-mediated cellular immunoreactivity against food allergens had also been reported by our group with the help of the lait (olivier et al. 2022b, olivier et al. 2022a, olivier et al. 2022c, olivier et al. 2023a). non–ige-mediated cellular immunoreactivity against aeroallergens and microorganisms had also been reported by our group with the help of the lait (olivier et al. 2023d, olivier et al. 2023f, olivier et al. 2023b, olivier et al. 2023c, olivier et al. 2024f). we also routinely employ the tube research of precipitins (ttp) in our facilities as a triage to evaluate non–ige-mediated immunoreactivity against suspected allergens before performing more exhaustive in vivo provocation tests (olivier et al. 2024b, olivier et al. 2024d, olivier et al. 2024g, olivier et al. 2025). to evaluate the potential of the lait and ttp to endotype non–ige–mediated cellular and humoral immunoreactivity against cat dander, dog dander, and pork meat extract, we retrospectively compiled the electronic medical charts of patients diagnosed with non–ige– mediated allergic multimorbidity who were investigated for immunoreactivity simultaneously using one of these assays. the present study serves as a proof-of-concept, hypothesizing that lait and the ttp may demonstrate a correlation between cellular and/or humoral immunoreactivity against cat dander, dog dander, and pork meat proteins in patients suffering from non–ige–mediated allergic multimorbidity. 2. materials and methods 2.1 subjects after receiving institutional review board approval from the instituto alergoimuno de americana (brazil; 03/2025), we reviewed the electronic chart of 10,500 outpatients who attended our facility from january 2018 to june 2025 selecting patients diagnosed with allergic multimorbidity who were evaluated simultaneously with lait or ttp against cat dander, dog dander and pork meat extracts. a cohort of 100 consecutive outside patients (ttp cohort) had been submitted to ttp with cat dander, dog dander, and pork meat extract for presenting non–ige-mediated allergic multimorbidity as defined by the concomitant or consecutive presence of at least two allergic phenotypes (allergic rhinoconjunctivitis, allergic bronchitis, atopic dermatitis, urticaria, gastrointestinal hypersensitivity, and/or anaphylaxis). this cohort counted 39 males; mean age 32.2 years; sd 20.2 years; range 3 to 88 years; median 31 years; mode = 7 (appeared six times); geometric mean = 24.6 years. olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 142-162, 2025; article no.aji.138781 146 a cohort of 100 consecutive outside patients (lait cohort) had been submitted to lait with cat dander, dog dander, and pork meat extract for presenting non–ige-mediated allergic multimorbidity as defined by the concomitant or consecutive presence of at least two allergic phenotypes (allergic rhinoconjunctivitis, allergic bronchitis, atopic dermatitis, urticaria, gastrointestinal hypersensitivity, and/or anaphylaxis). this cohort counted 29 males; mean age 30.3 years; sd 17.2 years; range 5 to 86 years; median 30 years; mode = 32 (appeared five times); geometric mean = 24.9 years. inclusion criteria: these procedures were offered to patients with allergic multimorbidity and had an undetectable specific ige and had demonstrated a non-reactive or inconclusive skin test against cat fur, dog fur, and pork meat extracts (olivier et al. 2013a). exclusion criteria: this study excluded patients receiving biological and/or systemic antiinflammatory therapy. 2.2 pork meat extract pork meat (shank) acquired from the local market was crushed, homogenized, and then left for 48 hours in a coca-based extractor solution (propylparaben 0.5g, methylparaben 1g, sorbitol 30g, nacl 5g, nahco3 2.5g, 1,000ml h2o) at 4 °c for protein extraction before centrifugation and separation of the water-soluble fraction from solid particles and oily fraction (coca 1922). the protein quantification of the allergen extracts was done according to bradford's protein-dye binding methodology (bradford 1976). the solution was diluted in an antigen dilution solution (nacl, 10g; kh2po4, 0.72g; na3po4, 2.86g; methylparaben, 1g; propylparaben, 0.5g; glycerin, 400ml; h2o, 600ml) to an estimated protein concentration of 1 mg/ml and stored at 4 °c in amber, opaque glass vials. the pork extract solution was used to perform allergic skin tests, ttp, and lait. all relevant and mandatory laboratory health and safety measures have been complied with during the experiments. 2.3 dog and cat fur extracts the dog and cat fur's protein extraction was performed as follows: the material was furnished by a veterinarian who took special care not to mix cat fur with dog fur collected from several animals. the fur was treated with acetone to remove the fat. after this, the acetone was removed from the sample using the autoclave. the sample was ground for 48 hours at 4 °c with a coca-based extractor solution added to cover the amount of antigen. the sample was centrifuged (4,500 rpm for 10 min) and filtered. the protein concentration was estimated spectrophotometrically and diluted to 1 mg/ml in antigen dilution solution to perform allergic skin tests, ttp, and lait. 2.4 lait: ex vivo investigation: leukocyte adherence inhibition test 2.4.1 lait: procedure for allergen ex vivo challenging we performed the lait as previously described (olivier et al. 2012, olivier et al. 2014, olivier et al. 2021a, olivier et al. 2021b, olivier et al. 2021c). shortly, each donor's fresh plasma was divided into two parts and used in parallel ex vivo challenging tests with the three allergen extracts (cat fur, dog fur, and pork meat) and the unchallenged plasma (added with antigen dilution solution as a control). we collected plasma with high leukocyte content (buffy coat) from the heparinized tube after one hour of sedimentation at 37 °c. then, we distributed aliquots of 100 μl into eppendorf tubes with (or without) the challenging extract and kept them under agitation for 30 minutes (200 rpm at 37 °c). 2.4.2 lait: procedure for adherence assay after incubation, the challenged plasma was allocated into a standard neubauer hemocytometer counting chamber with a plain, non-metallic glass surface and left to stand for 2 hours at 37 °c in a humidified atmosphere of the covered water bath, allowing leukocytes to adhere to the glass. next, we counted the leukocytes, removed the coverslip, and washed the chamber by immersing it in a beaker containing phosphate-buffered saline (pbs) at 37 °c. then, we added a drop of pbs to the hemocytometer's chamber and covered it with a clean coverslip. the remaining cells were counted in the same squares as previously examined. 2.4.3 lait: procedure for calculation the percentage of leukocyte adherence (la) of each assay was estimated as: (the number of leukocytes observed on the hemocytometry olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 142-162, 2025; article no.aji.138781 147 chamber after washing divided by the number of leukocytes observed on the hemocytometry chamber before washing) and multiplied by 100 (%). the leukocyte adherence ratio (lar) was estimated based on the ratio between the leukocyte adherence (la) from the antigenspecific challenged plasma and the la from the unchallenged control plasma: lar = la of the challenged sample divided by la of the unchallenged control plasma, multiplied by 100 (%). to further calculate the leukocyte adherence inhibition (lai), we subtracted the lar from 100 (%). we utilized the lai results for the cascade distribution chart and the statistical calculations, both of which were performed using the microsoft excel statistical package. 2.5 ttp: in vitro investigation: tube titration of precipitins as previously reported, the semi-quantitative ttp was performed in a transparent vitreous tube array (olivier et al. 2024a). shortly, the patient's blood was collected in a clot-activator collecting tube. after separation, the serum was centrifuged at 2,000 rpm for 10 minutes. each allergen extract was allocated in sets of eleven glass tubes at progressively diluted serum concentrations. the progressive dilutions were combined with separated aliquots of 15 μl of the allergen extract with 250 μl of the patient's serum, progressively diluted into physiological saline solution (nacl 0,9%) in the dilution ratios of 1:1; 1:2; 1:4; 1:8; 1:16; 1:32; 1:64; 1:128; 1:256; and 1:512. one tube was a blank control, performed with water and serum, to observe occasional spontaneous precipitation (sia test). after 24 hours, the tubes were examined, and the titers (the highest dilution factor that yields a positive reading) were recorded (williams and chase, 1971). 3. results as a retrospective survey, no research protocol was in place; therefore, we report the incidental immune investigation as documented in the digital medical charts. the ttp for the cat fur extract showed a distribution concentrated on the higher dilutions (fig. 1). there was no negative result. the mean was estimated at 1:352; the median was 1:512; the standard deviation was estimated at 1:170; the mode was 1:512 (appeared 51 times); the geometric mean was estimated at 1:296 (see fig. 1). the ttp for the dog fur extract showed a distribution concentrated on the higher dilutions (fig. 2). there was no negative result. the mean was estimated at 1:318; the median was 1:256; the standard deviation was estimated at 1:176; the mode was 1:512 (appeared 42 times); the geometric mean was estimated at 256 (see fig. 2). the ttp for the pork meat extract showed a distribution concentrated on the higher dilutions (fig. 3). there was no negative result. the mean was estimated at 1:413; the median was 1:512; the standard deviation was estimated at 1:144; the mode was 1:512 (appeared 67 times); the geometric mean was estimated at 1:377 (see fig. 3). the lait for the cat fur extract showed a wide distribution range of results. the lai ranged from 0% to 100%. the mean was 56.2%; the median was 57.5%; the standard deviation was 27.0%; the mode was 68% (appeared six times). the cascade distribution demonstrates a wide range of lai results. most patients exhibited strong immunoreactivity, which could reflect the participation of cat fur allergens in a non–igemediated hypersensitivity condition in these patients (see fig. 4). the lait for the dog fur extract showed a wide distribution range of results. the lai ranged from 0% to 100%. the mean was 56.1%; the median was 60.5%; the standard deviation was 29.1%; the mode was 0% (appeared seven times). the cascade distribution demonstrates a wide range of lai results. most patients exhibited strong immunoreactivity, which may reflect the involvement of dog fur allergens in a non–igemediated hypersensitivity condition in these patients (see fig. 5). the lait for the pork meat extract showed a wide distribution range of results. the lai ranged from 0% to 100%. the mean was 46.8%; the median was 48%; the standard deviation was 27.6%; the mode was 0% (appeared six times). the cascade distribution demonstrates a wide range of lai results. most patients showed low or moderate immunoreactivity during the ex vivo challenge test. in contrast, others displayed strong immunoreactivity, which could reflect the involvement of pork meat allergens in a non– ige-mediated hypersensitivity condition in these patients (see fig. 6). olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 142-162, 2025; article no.aji.138781 148 fig. 1. cascade distribution chart of the tube titration of precipitins (x-axis %) resulting from the cat dander extract against the serum of the ttp cohort of 100 tests/subjects (y-axis). the graph shows a concentration of results in higher titrations olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 142-162, 2025; article no.aji.138781 149 the paired t-test indicated a non-significant, slight difference between the ttp results of cat fur and dog fur (p-value = 0.165). pearson's correlation indicated a non-significant small positive relationship between ttp results of cat fur and dog fur; r(98) = 0.0337; p-value = 0.739 (see fig. 07). the paired t-test indicated a significant small difference between the ttp results of cat fur and pork meat (p-value = 0.009). pearson correlation indicated a non-significant, minimal negative relationship between ttp results of cat fur and pork meat; r(98) = 0.0468; p-value = 0.644 (see fig. 08). the paired t-test indicated a significant small difference between the ttp results of dog fur and pork meat (p < 0.001). pearson's correlation indicated a significant small negative relationship between ttp results between dog fur and pork meat; r(98) = 0.248; p-value = 0.013) (see fig. 09). the paired t-test indicated no significant difference between cat fur and dog fur lait results (p-value = 0.9949). pearson's correlation indicated a significantly moderate positive relationship between the cat fur and dog fur lait results: r(98) = 0.395, p-value < 0.001 (see fig. 10). the paired t-test indicated a significant small difference between cat fur and pork meat lait results (p-value = 0.009082). pearson's correlation indicated a non-significant, small positive relationship between cat fur and pork meat lait results: r(98) = 0.193, p-value = 0.054 (see fig. 11). the paired t-test indicated a significant small difference between dog fur and pork meat lait results (p-value = 0.01968). however, pearson's correlation indicated a non-significant, minimal positive relationship between dog fur and pork meat lait results: r(98) =0.0519, p-value = 0.608 (see fig. 12) fig. 2. cascade distribution chart of the tube titration of precipitins (x-axis %) resulting from the dog dander extract against the serum of the ttp cohort of 100 tests/subjects (y-axis). the graph shows a concentration of results in higher titrations fig. 3. cascade distribution chart of the tube titration of precipitins (x-axis %) resulting from the pork meat extract against the serum of the ttp cohort of 100 tests/subjects (y-axis). the graph shows a concentration of results in higher titrations olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 142-162, 2025; article no.aji.138781 150 fig. 4. cascade distribution chart of the range groups of leukocyte adherence inhibition (lai) results (x-axis %) of the ex vivo challenge test against cat fur extract monitored by the leukocyte adherence inhibition test (lait), according to the respective number of outcomes over the lait cohort with 100 tests/subjects (y-axis). the graph shows a wide distribution of results fig. 5. cascade distribution chart of the range groups of leukocyte adherence inhibition (lai) results (x-axis %) of the ex vivo challenge test against dog fur extract monitored by the leukocyte adherence inhibition test (lait), according to the respective number of outcomes over the lait cohort with 100 tests/subjects (y-axis). the graph shows a wide distribution of results olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 142-162, 2025; article no.aji.138781 151 fig. 6. cascade distribution chart of the range groups of leukocyte adherence inhibition (lai) results (x-axis %) of the ex vivo challenge test against pork meat extract monitored by the leukocyte adherence inhibition test (lait), according to the respective number of outcomes over the lait cohort with 100 tests/subjects (y-axis). the graph shows a wide distribution of results olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 142-162, 2025; article no.aji.138781 152 fig. 7. dispersion chart of the tube titration of precipitins (ttp) against cat fur extract (x-axis %), plotted against the ttp against dog fur extract (y-axis %). the paired t-test indicated a nonsignificant, slight difference between the ttp results of cat fur and dog fur (p-value = 0.165). pearson's correlation indicated a non-significant small positive relationship between ttp results of cat fur and dog fur; r(98) = 0.0337; p-value = 0.739 fig. 8. dispersion chart of the tube titration of precipitins (ttp) against cat fur (x-axis %), plotted against the ttp against pork meat extract (y-axis %). the paired t-test indicated a significant small difference between the ttp results of cat fur and pork meat (p-value = 0.009). pearson correlation indicated a non-significant, minimal negative relationship between ttp results of cat fur and pork meat; r(98) = 0.0468; p-value = 0.644 olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 142-162, 2025; article no.aji.138781 153 fig. 9. dispersion chart of the tube titration of precipitins (ttp) against dog fur extract (x-axis %), plotted against the ttp against pork meat extract (y-axis %). the paired t-test indicated a significant small difference between the ttp results of dog fur and pork meat (p < 0.001). pearson's correlation indicated a significant small negative relationship between ttp results between dog fur and pork meat; r(98) = 0.248; p-value = 0.013) fig. 10. dispersion chart of the leukocyte adherence inhibition (lai) results of the ex vivo challenge test against cat fur extract (x-axis %), plotted against the lai results of the ex vivo challenge test against dog fur extract (y-axis %). the paired t-test indicated no significant difference between cat fur and dog fur lait results (p-value = 0.9949). pearson's correlation indicated a significantly moderate positive relationship between the cat fur and dog fur lait results: r(98) = 0.395, p-value < 0.001 olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 142-162, 2025; article no.aji.138781 154 fig. 11. dispersion chart of the leukocyte adherence inhibition (lai) results of the ex vivo challenge test against cat dander (x-axis %), plotted against the lai results of the ex vivo challenge test against pork meat extract (y-axis %). the paired t-test indicated a significant small difference between cat fur and pork meat lait results (p-value = 0.009082). pearson's correlation indicated a non-significant, small positive relationship between cat fur and pork meat lait results: r(98) = 0.193, p-value = 0.054 fig. 12. dispersion chart of the leukocyte adherence inhibition (lai) results of the ex vivo challenge test against dog dander extract (x-axis %), plotted against the lai results of the ex vivo challenge test against pork meat extract (y-axis %). the paired t-test indicated a significant small difference between dog fur and pork meat lait results (p-value = 0.01968). however, pearson's correlation indicated a non-significant, minimal positive relationship between dog fur and pork meat lait results: r(98) =0.0519, p-value = 0.608 olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 142-162, 2025; article no.aji.138781 155 4. discussion currently, efforts to understand the innate immune system's role in allergic diseases are increasing, accompanied by a growing arsenal of tools (lee and cohen, 2025). the correlation and distribution of simultaneous positive specific ige against food allergens and inhalant allergens are usually weak; however, polysensitization and cross-reactivity are more the rule than the exception (zhang et al., 2025; čelakovská et al., 2024; hasnain, et al. 2017). the treatment of monosensitized allergic patients through subcutaneous shots raised the paradigm of allergen-specific immunotherapy (olivier 2017). however, despite the use of precision techniques such as the component resolved diagnosis to identify allergens at a molecular level, several allergens (such as the major cat allergen fel d 1) present great diversity (dissimilar substitutions in the protein sequence) among cat species reflecting the genetic evolution of felidae (cleveland et al. 2024). therefore, it is common to see patients who report allergic symptoms elicited by some cats but not by others. the same happens concerning dogs. in the context of polysensitization, multimorbidity, and cross-sensitization, where several allergens appear to be clinically relevant, a more rational treatment strategy consists of the use of groupspecific multiallergen desensitization immunotherapy, which subcutaneous shots cannot administer due the extension of the local inflammatory response, but rather by the sublingual-swallow route, where the collateral effects are minimal (olivier et al. 2013b, khan 2016). assessing diverse ways of immunoreactivity and hypersensitivity against allergens responsible for clinical symptoms is a multi-omics approach to evaluate both the diagnosis and treatment of allergic patients (czolk et al., 2021). alternative approaches, such as the lait and the ttp, are proposed not to pinpoint the molecular allergen responsible for the allergic phenotype but to obtain an overall view of the patient's immunoreactivity against the whole extract. however, executing these immunoassays with molecular allergens to demonstrate a specific reaction is also possible. we usually do not use molecular allergens purely due to a lack of resources. to contour the difficulties in diagnosing non–igemediated hypersensitivity, some scientists have committed to evaluating the utility of the specific igg in helping clinically diagnose their patients (alkhateeb 2020). this is controversial, as igg may function as both a hypersensitivity trigger and an allergen blocker, depending on the reaction of the other immune players (atwah and koshak, 2024). igg antibodies can participate in type ii (antibody-dependent cell-mediated) and type iii (immune complex disease) gell and coombs hypersensitivity reactions, which may be theoretically reproduced by the lait and the ttp assays, respectively (olivier et al., 2021a; olivier et al., 2021d). the semi-quantitative titration of precipitins is a pioneering laboratory exam that laid the fundamental basis of immunology (wells 1911). precipitating antibodies indicate the presence of a humoral immune response against the tested antigens (gell, harington, and rivers, 1946). before the discovery of ige, research on precipitins was the leading method for in vitro diagnosis of immunoreactivity against allergenic agents (augustin and hayward, 1960). the lait is an ex vivo challenge test performed with a viable leukocyte buffy coat, which can theoretically explore the most well-known immune pathways, as it allows the interaction of all immune-circulating participants with the allergens (olivier et al., 2021a). several immune pathways can inhibit leukocyte adherence (tong et al., 1979; halliday, et al. 1974). the present study is a proof-of-concept that hypothesizes that lait and the ttp may differentiate diverse degrees of cellular and humoral immunoreactivity against cat dander, dog dander, and pork meat allergens among patients suffering from non–ige-mediated allergic multimorbidity. as the tests were performed simultaneously with the same venous sample with the three allergens, it was possible to calculate a correlation to distinguish some order of cross-reactivity between them. the retrospective compilation of our data revealed a wide distribution of results when we assessed the outcomes of ttp and lait to explore humoral and cellular immunoreactivity against the studied allergens. these immunoassays did not precisely identify the olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 142-162, 2025; article no.aji.138781 156 mechanisms responsible for clinical conditions. instead, they provide evidence about cellular and humoral immunoreactivity distributed across an extensive spectral range, which may suggest immune tolerance or hypersensitivity. this preliminary retrospective survey demonstrated extensive results from the ttp and the ex vivo challenge test monitored by lait against cat fu, dog fur, and pork meat in two cohorts of non–ige-mediated allergic multimorbidity patients. ttp and lait are complementary triage tests used at our facilities to select worthwhile antigens to proceed with more laborious in vivo provocation tests when the specific ige is undetectable. none of our patients presented an exclusive reaction to these allergens. every patient was simultaneously evaluated for several chemical and biological allergens, demonstrating positive results for some of them. our results suggest that dog and cat dander allergy patients may experience additional symptoms when consuming pork meat and vice versa. 5. limitations this study is a retrospective analysis of data collected over seven years since our facility began employing laboratory immune assays. there was no protocol research, and the subject's data was limited to the essentials available on our electronic sheets. therefore, we could not establish a cross-comparison between the positive and negative controls to validate the results. the number of subjects is suitable for a preliminary study; however, future studies should be more comprehensive. the lack of a research protocol implies the possibility of bias introduced by the physician's point of view (ceo) based on a clinical suspicion driven solely by the anamnesis and physical examination. the study lost many of these patients to follow-up, so it is not yet possible to ensure the relationship between the immunoassay results and the patient's clinical outcome. unfortunately, comparing the two procedures using paired ttests was impossible because they were obtained from distinct patient groups. 6. conclusion the preliminary results suggest that the ttp and lait, when applied to cat and dog fur extracts and pork meat extract, may discriminate between diverse humoral and cellular immunoreactivity levels in patients with various allergic phenotypes. a significant association was found between the immunoreactivity of cat fur and dog fur. the association between pork meat and fur immunoreactivity was not clear. lait and ttp are inexpensive, can be performed with minimum laboratory equipment, and can be incorporated into strategies to address health disparities in respiratory and food allergies (anagnostou et al. 2025). as a preliminary report, the propaedeutic significance of the presented results and the potential interfering factors must be further established (anouar, et al. 2024). more studies focused on the quality-by-design approach with larger, prospective, double-blind cohorts are needed to evaluate the potential contribution of lait and ttp for endotyping cellular and humoral immunoreactivity in patients suspected of hypersensitivity to cat fur, dog fur, and pork meat allergens (chiarentin et al., 2023). 7. future directions and recommendations for clinical practice the primary intended use of in vitro or ex vivo allergen challenge tests is to spare patients from undergoing unnecessary, exhaustive, and potentially hazardous in vivo challenge tests. exploring the humoral and the cellular arms of immune systems, the ttp and lait alone or combined may represent, in the near future, a tool for allergists to elaborate etiologic diagnosis for their patients, as well as determine the endotypes (mechanisms) of hypersensitivity, in order to choose more convenient and personalized therapies (adly e. al., 2021). adding data provided by ttp and lait may also contribute to streamlining biomedical research and improving tools, such as large language models, which clinicians often use as decision support systems to enhance diagnostic accuracy (abers and mathias, 2025). concent it is not applicable. ethical approval the authors have obtained and documented written ethical approval in accordance with international standards.as a retrospective compilation of results recorded in cognito, consent was obtained collectively by the institution's ethics committee, following the principles of the declaration of helsinki (wma, 2013). olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 142-162, 2025; article no.aji.138781 157 disclaimer (artificial intelligence) authors hereby declare that no generative ai technologies such as large language models (chatgpt, copilot, etc.) and text-to-image generators have been used during the writing or editing of this manuscript. acknowledgements the instituto alergoimuno de americana funded this work. we want to thank the laboratory technicians, alessandra vieira de oliveira and luciana sacilotto carvalho, for their assistance with the exams competing interests authors have declared that no competing interests exist. references abers, m. s., mathias, r. a. 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(2025). investigating allergen-specific ige distribution and correlations in chronic urticaria: a retrospective study in shanghai, china. european journal of medical research, 30(1): 182. disclaimer/publisher’s note: the statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of the publisher and/or the editor(s). this publisher and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. _________________________________________________________________________________ © copyright (2025): author(s). the licensee is the journal publisher. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: https://pr.sdiarticle5.com/review-history/138781 https://pr.sdiarticle5.com/review-history/138781 _____________________________________________________________________________________________________ *corresponding author: e-mail: celso@alergoimuno.med.br; cite as: olivier, celso eduardo, daiana guedes pinto, ana paula monezzi teixeira, cibele silva miguel, raquel acácia pereira gonçalves santos, nicole sartoreto da rocha, jhéssica letícia santos santana, and regiane patussi santos lima. 2025. “endotyping cellular and humoral immunoreactivity against pollen and citrus fruits in patients with non–ige-mediated rhinoconjunctivitis”. asian journal of immunology 8 (1):8-25. https://doi.org/10.9734/aji/2025/v8i1156. asian journal of immunology volume 8, issue 1, page 8-25, 2025; article no.aji.131451 endotyping cellular and humoral immunoreactivity against pollen and citrus fruits in patients with non–ige-mediated rhinoconjunctivitis celso eduardo olivier a*, daiana guedes pinto a, ana paula monezzi teixeira a, cibele silva miguel a, raquel acácia pereira gonçalves santos a, nicole sartoreto da rocha b, jhéssica letícia santos santana c and regiane patussi santos lima d a instituto alergoimuno de americana, brazil. b faculdade de americana, são paulo, brazil. c instituto de ensino e pesquisa do hospital de amor de barretos, brazil. d lavoisier laboratórios, são paulo, brazil. authors' contributions the authors conducted this work in collaboration. the author ceo is responsible for the conceptualization, data curation, formal analysis, literature review, and writing the original draft. authors dgp, apmt, csm, nsr, jlss and rpsl performed laboratory procedures. author rapgs performed cutaneous tests. all authors read and approved of the final manuscript. article information doi: https://doi.org/10.9734/aji/2025/v8i1156 open peer review history: this journal follows the advanced open peer review policy. identity of the reviewers, editor(s) and additional reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc., are available here: https://pr.sdiarticle5.com/review-history/131451 received: 15/12/2024 published: 20/02/2025 original research article https://doi.org/10.9734/aji/2025/v8i1156 https://pr.sdiarticle5.com/review-history/131451 olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 8-25, 2025; article no. aji.131451 9 abstract background: allergies to citrus fruits are often associated with pollinosis due to cross-reactivity among pollen and food allergens (fruit-pollen syndrome) displayed in patients with several conditions, such as allergic rhinoconjunctivitis. aim: to evaluate the potential of the tube titration of precipitins (ttp) and the leukocyte adherence inhibition test (lait) to discriminate cellular and humoral immunoreactivity against two citrus spices (orange and lemon) and a pollen extract in patients with non–ige-mediated allergic rhinoconjunctivitis. study design: we retrospectively examined the medical charts of two cohorts of patients clinically diagnosed with non–ige-mediated rhinoconjunctivitis with clinical suspicion of hypersensitivity against citrus spices and pollen, who were investigated with the help of ttp or lait, simultaneously tested against individual extracts of orange, lemon and pollen. methodology: the registered results of ttp and lait were distributed in ranges through a cascade distribution chart to outline the variability of the results. dispersion graphs plotting the results of lait between the results of each pair of allergens were presented. the statistical significances were calculated. results: the ttp for the pollen, orange, and lemon extracts showed a distribution concentrated on the higher dilutions, precluding an adequate differentiation among patients’ immunoreactivities. on the contrary, the lait results showed a wide distribution of results, demonstrating a better potential to differentiate patients and predict hypersensitivity. while the ttp results showed a slight correlation between the paired tests (pearson’s correlation coefficient between r = 0.007 to 0.11), the lait results demonstrated a significant moderate correlation between the paired assays, projecting a better potential to predict cross-reactivity among the allergens (pearson’s correlation coefficient between r = 0.43 to 0.56). conclusion: our preliminary results support that the ttp and lait performed with orange, lemon, and pollen extracts can potentially discriminate diverse degrees of humoral and cellular immunoreactivity in non–ige-mediated allergic rhinoconjunctivitis patients. keywords: conjunctivitis; hypersensitivity; lemon; leukocyte adherence inhibition test; orange; pollen; precipitins; rhinitis. abbreviations lai : leukocyte adherence inhibition lait : leukocyte adherence inhibition test ttp : tube titration of precipitins 1. introduction citrus is a genus of wild and domesticated fruit plants in the rutaceae family in which several cultivars are classified, such as oranges (e.g., citrus sinensis), mandarins (e.g., citrus reticulata), grapefruits (e.g., citrus paradisi), pomelos (e.g., citrus maxima), limes (e.g., citrus latifolia), and lemons (e.g., citrus limon) (wu et al. 2018). a survey performed by the good housekeeping institute pointed citrus fruits among the top ten foods perceived by food-allergic people as responsible for their symptoms (sloan and powers 1986). allergies to citrus fruits are often associated with pollinosis due to a phenomenon of crossreactivity among pollen and food allergens (fruitpollen syndrome), displayed in patients with combined conditions such as allergic rhinitis, allergic conjunctivitis, allergic bronchitis, oral allergy syndrome, urticaria, angioedema, digestive symptoms and anaphylaxis (iorio et al. 2013, li et al. 2022, canonica et al. 2013, armentia et al. 2016, hasnain, alqassim and alfrayl 2017, bartra et al. 2009). nasal provocation with pollen extracts is an uncomplicated way to diagnose pollen hypersensitivity (doyen et al. 2018). fruit-pollen syndrome is also frequently associated with latex allergy, sometimes called latex-pollen-fruit syndrome or latex-fruit-pollen syndrome (čelakovská et al. 2024, olivier et al. 2022b). pollen-derived products such as honey and royal jelly were described as triggers for anaphylaxis and urticaria in patients with pollinosis (fuiano et al. 2006, lombardi et al. 1998). the allergen nomenclature sub-committee of the world health organization and international union of immunological societies (who/iuis) olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 8-25, 2025; article no. aji.131451 10 has recognized so far four allergens weighting from 8 to 23 kda, identified from the sweet orange (citrus sinensis), according to their official nomenclature: cit s 1 (germin-like protein), cit s 2 (profilin), cit s 3 (non-specific lipid-transfer protein type 1) and cit s 7 (gibberellin regulated protein) (sub-committee 2024c). the same subcommittee listed one allergen from lemon (citrus limon): cit l 3, a 9.6 kda non-specific lipidtransfer protein type 1 (sub-committee 2024a), as well as one allergen from mandarin (citrus reticulata): cit r 3, a 9 kda non-specific lipidtransfer protein type 1 (sub-committee 2024b). the orange profilin, cit s 2, has an amino acid sequence similar to pollen profilins, such as the birch bet v 2 (73% identity) (lópez-torrejón et al. 2005). profilins are plant pan-allergens responsible for cross-sensitization between pollen and plant-derived foods (valenta et al. 1992). pollen and plant food profilin allergens show equivalent ige and igg reactivity, are quickly inactivated by gastric digestion, and are commonly involved in polysensitization of allergic patients (sirvent et al. 2011). profilin hypersensitivity is common in patients with crossreactivity to pollen and fruits such as oranges, pineapples, melons, watermelons, tomatoes, and bananas (asero et al. 2003, asero, monsalve and barber 2008). gibberellin-regulated proteins (grp) are a group of emergent allergens described in orange, japanese apricots, sweet cherries, pomegranates, bell peppers, strawberries, and also in pollen from the cupressaceae tree (cypress) family (iizuka et al. 2022). grp hypersensitivities are clinically associated with severe adverse reactions, such as a case of orange-induced anaphylaxis (kàtcheff et al. 2024, özdemir 2024). cystatin-like proteins found in freshly squeezed orange juice also produced angioedema, dysphonia, and dyspnea (solórzano-zepeda et al. 2021). cystatins are enzymes playing several roles in microorganisms, plants, pollens, animals, and humans already considered autoallergens since they were implicated in autoallergies through cross-reaction ige-mediated and t-cell mediated hypersensitivities (rogers et al. 1993, roesner et al. 2022). citrus seeds also contain reaginic proteins that may produce allergic reactions when mixed with whole-fruit-crushed juices or when accidentally ingested, producing anaphylaxis, urticaria, and respiratory and digestive symptoms in patients who otherwise tolerate squeezed juices (kayode et al. 2020, glaspole et al. 2007, wang 2008). reactions to citrus seeds are related to hypersensitivity to citrin, an 11s globulin belonging to the cupin superfamily, which crossreact with cashew and pistachio allergens (konstantinou et al. 2023). citrus-induced phytophotodermatitis is associated with the presence of coumarins and furocoumarins (psoralens, xanthotoxins, and bergaptens) in the peel or the juice of the fruit, involving cutaneous photosensitivity, phototoxicity, and/or photoallergy (jiang et al. 2024, belcadi, oulad ali and senouci 2024). sensory hypersensitivity (photophobia and osmophobia) in patients with migraine are also related to citrus fruits (vitalisilva et al. 2024). non–ige-mediated cellular immunoreactivity against food allergens had already been reported by our group with the help of the leukocyte adherence inhibition test (lait), as well as humoral immunoreactivity against food allergens with the help of tube titration of precipitins (ttp) (olivier et al. 2021d, olivier et al. 2022a, olivier et al. 2022c, olivier et al. 2024c, olivier et al. 2024b, olivier et al. 2024a). we routinely employ the lait and the ttp in our facilities as a triage to evaluate non–non–ige-mediated immunoreactivity against suspected allergens before performing more exhaustive in vivo provocation tests (kuratsuji 1981, olivier et al. 2023e, olivier et al. 2023g, olivier et al. 2023c, olivier et al. 2023b, olivier et al. 2023d, olivier et al. 2024f). to evaluate the potential of the lait and ttp to endotyping non–ige-mediated cellular and humoral immunoreactivity against orange, lemon, and pollen extracts, we retrospectively compiled the electronic medical charts of patients diagnosed with non–igemediated rhinoconjunctivitis who were investigated simultaneously for immunoreactivity against these three allergens by one of these assays. the present study is a proof-of-concept that hypothesizes that lait and the ttp may differentiate diverse degrees of cellular and humoral immunoreactivity against orange, lemon, and pollen allergens among patients suffering from non–ige-mediated rhinoconjunctivitis. as the tests were performed simultaneously with the same venous sample with the three allergens, it is possible to calculate two-sample paired t-tests between each pair of lait results (since they refer to the same quantitative variable), as well to present a dispersion graph between them to olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 8-25, 2025; article no. aji.131451 11 distinguish some order of correlation suggesting (or not) cross-reactivity (gosset-student 1908). 2. materials and methods 2.1 subjects after receiving institutional review board approval from the instituto alergoimuno de americana (brazil; 01/2025), we reviewed the electronic chart of 9,980 outpatients who attended our facility from january 2018 to february 2025. a cohort of 100 consecutive outside patients (ttp cohort) had been simultaneously submitted to ttp with orange extract, lemon extract, and pollen extract for presenting non–ige-mediated allergic rhinoconjunctivitis. this cohort counted 28 males; mean age 37.8 years; sd 20.4 years; range 4 to 88 years; median 32.5 years; modes = 29 (appeared seven times); geometric mean = 29.4 years. a cohort of 100 consecutive outside patients (lait cohort) had been simultaneously submitted to tial with orange extract, lemon extract, and pollen extract for presenting non–ige-mediated allergic rhinoconjunctivitis. this cohort counted 35 males; mean age 42.9 years; sd 18.7 years; range 8 to 76 years; median 38 years; modes = 9 and 38 years (each appeared five times); geometric mean = 32.2 years. this study did not include patients under biological and/or systemic anti-inflammatory therapy. these procedures were offered to patients with clinical suspicion of citrus spices hypersensitivity who demonstrated a nonreactive or inconclusive skin test against pollen, orange, and lemon extracts (olivier et al. 2013). 2.2 extracts 2.2.1 orange extract the whole orange (pulp, peel and seeds) was crushed, homogenized, and then left for 48 hours in a coca-based extractor solution (propylparaben 0.5g, methylparaben 1g, sorbitol 30g, nacl 5g, nahco3 2.5g, 1,000ml h2o) at 4 °c for protein extraction before centrifugation and separation of the water-soluble fraction from solid particles and oily fraction (coca 1922). the protein quantification of the allergen extracts was done according to bradford’s protein-dye binding methodology (bradford 1976). the solution was diluted in the antigen dilution solution (nacl 10g; kh2po4 0.72g; na3po4 2.86g; methylparaben 1g; propylparaben 0.5g; glycerin 400ml; h2o 600ml) to an estimated protein concentration of 1 mg/ml and stored at 4 °c into amber opaque glass vials. the orange extract solution was used to perform allergic skin tests, ttp, and lait. all relevant and mandatory laboratory health and safety measures have been complied with during the experiments. 2.2.2 lemon extract the lemon extract solution was prepared using a similar technique employed for the orange extract. 2.2.3 pollen extract the pollen’s protein extraction was performed as follows: in a beaker, 5g of dehydrated beekeeping pollen, acquired from a local provider, was added to the coca-based extractor solution to cover the amount of pollen. the sample was crushed and then left for 48 hours at 4 °c. the sample was centrifuged (4,500 rpm for 10 min) and filtered. the protein concentration was estimated spectrophotometrically and diluted to 1 mg/ml in antigen dilution solution (nacl 10g, kh2po4 0.72g, na3po4 2.86g, methylparaben 1g, propylparaben 0.5g, glycerin 400 ml, h2o 600ml) and used to perform the lait and allergic skin tests. 2.3 lait: ex vivo investigation: leukocyte adherence inhibition test 2.3.1 lait: procedure for allergen ex vivo challenging we performed the lait as previously described (olivier et al. 2012, olivier et al. 2014, olivier et al. 2021b, olivier et al. 2021c, olivier et al. 2021e, olivier et al. 2022b, olivier et al. 2022a, olivier et al. 2022c, olivier et al. 2023e, olivier et al. 2023g, olivier et al. 2023a, olivier et al. 2024e). shortly, each donor's fresh plasma was divided into two parts and used in parallel ex vivo challenging tests with the orange or lemon extracts and the unchallenged plasma (added with antigen dilution solution as a control). we collected plasma with high leukocyte content (buffy coat) from the heparinized tube after one hour of sedimentation at 37 °c. then, we distributed aliquots of 100 μl into eppendorf tubes with (or without) the challenging extract olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 8-25, 2025; article no. aji.131451 12 and kept them under agitation for 30 minutes (200 rpm at 37 °c). 2.3.2 lait: procedure for adherence assay after incubation, the plasma was allocated into a standard neubauer hemocytometer counting chamber with a plain, non-metallic glass surface and left to stand for 2 hours at 37 °c in the humidified atmosphere of the covered water bath to allow leukocytes to adhere to the glass. next, we counted the leukocytes, removed the coverslip, and washed the chamber by immersion in a beaker with phosphate buffer saline (pbs) at 37 °c. then, we added a drop of pbs to the hemocytometer's chamber and allocated a clean coverslip over it. the remaining cells were counted in the same squares as previously examined. 2.3.3 lait: procedure for calculation the percentage of leukocyte adherence (la) of each assay was estimated as: (the number of leukocytes observed on the hemocytometry chamber after washing divided by the number of leukocytes observed on the hemocytometry chamber before washing) and multiplied by 100 (%). the leukocyte adherence ratio (lar) was estimated based on the ratio between the la from the antigen-specific challenged plasma and the la from the unchallenged control plasma: lar = la of the challenged sample divided by la of unchallenged control plasma multiplied by 100 (%). to further calculate the leukocyte adherence inhibition (lai), we subtracted the lar from 100 (%). we employed the lai results for the cascade distribution chart and the statistics calculations, both performed with the help of the microsoft excel® statistical package. 2.4 ttp: in vitro investigation: tube titration of precipitins as previously reported, the semi-quantitative ttp against the aluminum solution was performed in a transparent vitreous tube array (olivier et al. 2021f, olivier et al. 2024g, olivier et al. 2024d, olivier et al. 2024b, olivier et al. 2024a). shortly, the patient’s blood was collected in a clotactivator collecting tube. after separation, the serum was centrifugated at 2,000 rpm for 10 minutes. each allergen extract was allocated in sets of eleven glass tubes at progressive duplicated serum dilutions. the progressive dilutions were combined with the 15 μl of the antigen (1 mg/ml) with 250 μl of the patient’s serum, progressively diluted into physiological saline solution (nacl 0,9%) in the dilution ratios of 1:1; 1:2; 1:4; 1:8; 1:16; 1:32; 1:64; 1:128; 1:256; and 1:512. one tube was a blank control done with the water and serum to observe occasional spontaneous precipitation (sia test). after 24 hours, the tubes were examined, and the titers (the highest dilution factor that yields a positive reading) were recorded (williams and chase 1971). 3. results as a retrospective survey, there was no research protocol; therefore, we report the incidental immune investigation as registered in the digital medical charts. the ttp for the orange extract showed a distribution concentrated on the higher dilutions (fig. 1). there were no negative results. there were no positive results in the range from 1:1 to 1:32 dilutions. the mean was estimated at 1:347; the median was 1:256; the standard deviation was estimated at 1:169; the mode was 1:512 (appeared 49 times). the ttp for the lemon extract showed a distribution concentrated on the higher dilutions (fig. 2). there were no negative results. there were no positive results in the range from 1:1 to 1:32 dilutions. the mean was estimated at 1:358; the median was 1:256; the standard deviation was estimated at 1:157; the mode was 1:512 (appeared 49 times). the ttp for the pollen extract showed a distribution concentrated on the higher dilutions (fig. 3). there were no negative results. there were no positive results in the range from 1:1 to 1:32 dilutions. the mean was estimated at 1:387; the median was 1:512; the standard deviation was estimated at 1:155; the mode was 1:512 (appeared 59 times). the lait for the orange extract showed a wide distribution range of results (fig. 4). most results were concentrated in the more immunoreactive groups. there were five negative results. the lai ranged from 0% to 98%. the mean was 62.6%; the median was 68.5%; the standard deviation was 26.9%; the mode was 0% (appeared five times). some patients showed low or moderate immunoreactivity during the ex vivo challenge test. in contrast, others displayed strong immunoreactivity, which could reflect the participation of orange allergens in a non–ige olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 8-25, 2025; article no. aji.131451 13 mediated hypersensitivity condition in these patients. the lait for lemon extract showed a wide distribution range of results (fig. 5). most were concentrated in the more immunoreactive groups. there were six negative results. the lai ranged from 0% to 99%. the mean was 54%; the median was 56.5%; the standard deviation was 27.7%; the mode was 0% (appeared six times). some patients showed low or moderate immunoreactivity during the ex vivo challenge test. in contrast, others displayed strong immunoreactivity, which could reflect the participation of lemon allergens in a non–igemediated hypersensitivity condition. the lait for pollen extract showed a wide distribution range of lai results (fig. 6). there were three negative results. the lai ranged from 0% to 98%. the mean was 55.9%; the median was 60.5%; the standard deviation was 27.5%; and the mode was 79% (appeared four times). some patients showed low or moderate immunoreactivity in response to the ex vivo challenge test. most displayed strong immunoreactivity, which could reflect the participation of pollens in the non–ige-mediated hypersensitivity of these patients. the paired-t test indicated a non-significant slight difference between the results of lemon ttp and orange ttp (p = .640). pearson’s correlation indicated a non-significant small positive relationship between lemon ttp and orange ttp results: r(98) = .00798, p-value = .937. the paired-t test indicated a non-significant slight difference between pollen ttp and orange ttp results (p-value = .077). pearson’s correlation indicated a non-significant small positive relationship between pollen ttp and orange ttp results: r(98) = .0761, p-value = .452. the paired-t test indicated a non-significant slight difference between pollen ttp and lemon ttp results (p-value = .172). pearson’s correlation indicated a non-significant small positive relationship between pollen and lemon ttp results: r(98) = .114, p-value = .259. the paired t-test indicated a significant difference between orange and lemon lait results (pvalue = 0.02741). however, pearson’s correlation indicated a significantly moderate positive relationship between the orange and lemon lait results: r(98) = .45, p-value < .001. the paired t-test indicated a significant difference between pollen and orange lait results (pvalue = 0.08241). however, pearson’s correlation indicated a significant positive relationship between pollen and orange lait results: r(98) = .56, p-value < .00. the paired t-test indicated a significant difference between pollen and lemon lait results (pvalue =. 0.6322). however, pearson’s correlation indicated a significant positive relationship between pollen and lemon lait results: r(98) = .43, p-value < .001. fig. 1. cascade distribution chart of the tube titration of precipitins (x-axis %) resulting from the orange extract against the serum of the ttp cohort of 100 tests/subjects (y-axis) olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 8-25, 2025; article no. aji.131451 14 fig. 2. cascade distribution chart of the tube titration of precipitins (x-axis %) resulting from the lemon extract against the serum of the ttp cohort of 100 tests/subjects (y-axis) fig. 3. cascade distribution chart of the tube titration of precipitins (x-axis %) resulting from the pollen extract against the serum of the ttp cohort of 100 tests/subjects (y-axis) fig. 4. cascade distribution chart of the range groups of leukocyte adherence inhibition (lai) results (x-axis %) of the ex vivo challenge test against orange extract monitored by the leukocyte adherence inhibition test (lait), according to the respective number of outcomes over the lait cohort with 100 tests/subjects (y-axis) olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 8-25, 2025; article no. aji.131451 15 fig. 5. cascade distribution chart of the range groups of leukocyte adherence inhibition (lai) results (x-axis %) of ex vivo ex vivo challenge test against lemon extract monitored by the leukocyte adherence inhibition test (lait), according to the respective number of outcomes over the lait cohort with 100 tests/subjects (y-axis) fig. 6. cascade distribution chart of the range groups of leukocyte adherence inhibition (lai) results (x-axis %) of ex vivo challenge test against pollen extract monitored by the leukocyte adherence inhibition test (lait), according to the respective number of outcomes over the lait cohort with 100 tests/subjects (y-axis) 4. discussion charles blackley reported the first diagnosis of pollinosis when conceiving the skin provocation test to associate the respiratory symptoms of his patients to pollen inhalation (blackley 1873). pollinosis is preferably referred to as pollen allergy or, by extension, allergic rhinoconjunctivitis due to the frequent association of nasal and conjunctival symptoms. except for desertic or icy regions, pollinosis is a disease that occurs worldwide (peternel et al. 2007, ferreiro, dopazo and aira 2002, xu et al. 2000, nakamura 1996, tilandyová et al. 1989, wüthrich et al. 1986, noferi, ferrante and testa 1965, negrini and belloni 1963, bean and glaser 1960). pollinosis in brazil has regional characteristics dependent on local flora and regional crops (oliveira et al. 2020, rosario 1990, taketomi et al. 2006, taketomi et al. 2005). our facility borders a city called “limeira” due to its excellent production of lime olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 8-25, 2025; article no. aji.131451 16 oranges, from where several of our patients come. the two major phenotypes related to allergic rhinoconjunctivitis are “seasonal” and “perennial.” perennial allergic rhinoconjunctivitis is usually related to house dust mites, and seasonal allergic rhinoconjunctivitis is usually related to pollinosis (at least in regions with well-defined climatic stations) (lluch-bernal et al. 2024). atmospheric pollen concentration is a standard parameter regularly measured by air controller agencies and is strongly related to pollinosis symptoms (sheng et al. 2022). allergic rhinoconjunctivitis is a prototype model of disease that may be classically produced by igemediated hypersensitivity and non–ige-mediated hypersensitivity (olivier et al. 2023f, yamana, yamana and uchio 2022). longitudinal clustering analysis has recently characterized novel rhinoconjunctivitis phenotypes; however, several questions have not yet been answered (togias et al. 2025). fig. 7. dispersion chart of the leukocyte adherence inhibition (lai) results of the ex vivo challenge test against lemon extract (x-axis %), plotted against the lai results of the ex vivo challenge test against orange extract (y-axis %) fig. 8. dispersion chart of the leukocyte adherence inhibition (lai) results of the ex vivo challenge test against pollen extract (x-axis %), plotted against the lai results of the ex vivo challenge test against orange extract (y-axis %) olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 8-25, 2025; article no. aji.131451 17 fig. 9. dispersion chart of the leukocyte adherence inhibition (lai) results against pollen extract (x-axis %), plotted against lai results of the ex vivo challenge test against lemon extract (y-axis %) a well-studied non–ige-mediated hypersensitivity endotype responsible for pollen-related allergic conjunctivitis and allergic blepharitis is the macrophage migration inhibitory factor, a cytokine responsible for eosinophil accumulation in the conjunctivas and eyelid dermis exposed to pollen (nagata et al. 2015). migration inhibition factors (mifs) were the first lymphokines related to delayed hypersensitivity (bloom and bennett 1966). mifs are pluripotent cytokines essential in non–ige-mediated allergic inflammation, recruiting reaginic cells, such as macrophages and eosinophils, to the inflammatory site (das et al. 2011). mifs are essential cytokines for t cell activation and sustainment of innate proinflammatory responses (mitchell et al. 2002). the lait is an easy and affordable way to put in evidence the possible participation of leukocyte (or macrophage) inhibition cytokines in ex vivo challenge tests with allergens (rocklin 1974, dunn and halliday 1980). despite being considered a respiratory condition, proteins bearing correlated pollen epitopes are usually eaten through fruits and vegetables, producing cross-reacting allergic reactions (andersen, hall and dragsted 2011, worm et al. 2014). endotyping biomarkers of cellular and humoral immunoreactivity and cross-reactivity are essential to build better strategies to impersonate treatments for allergic patients (agache and akdis 2020). at the clinical set, diagnosis of igemediated hypersensitivity is an easy task, accomplished by anamnesis, skin tests, and the laboratory research of specific ige; however, to diagnose non–ige-mediated hypersensitivity, it is necessary to employ a multi-omics approach to differentiate the particularities of the variety of clinical phenotypes and immune endotypes responsible for allergic diseases (macowan et al. 2025, yoon and bunyavanich 2025, khan 2016). the concept of immune dysregulation is evolving, and besides the major primary immunodeficiencies, there are secondary immunodeficiencies following inflammatory conditions raised by immune hypersensitivities, clinically known as allergies (henrickson 2025). the semi-quantitative research and titration of precipitins is a pioneering laboratory exam upon which the fundamental bases of immunology were constructed (wells 1911). precipitating antibodies suggest the presence of a humoral immune response against the tested antigens (gell, harington and rivers 1946). before the discovery of ige, the research of precipitins against pollen and mold allergens was the leading way to realize in vitro diagnostic of immunoreactivity against these agents (augustin and hayward 1960, augustin, hayward and longbottom 1960). precipitins to pollen allergens are obtained after sensitization of guinea pigs with phleum pratense and dactylis glomerata pollens, producing antiserum reactive against these pollens and cross-reactive against festuca pratensis and cynodon dactylon (augustin 1953). olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 8-25, 2025; article no. aji.131451 18 the lait is an ex vivo challenge test performed with a viable leukocyte buffy coat that can theoretically explore most known immune pathways as it allows the interaction of all immune-circulating participants with the allergens several immune pathways can produce the final leukocyte adherence inhibition (olivier et al. 2021a). the present study is a proof-of-concept that hypothesizes that lait and the ttp may differentiate diverse degrees of cellular and humoral immunoreactivity against orange, lemon, and pollen allergens among patients suffering from non–ige-mediated rhinoconjunctivitis. as the tests were performed simultaneously with the same venous sample with the three allergens, it was possible to calculate a paired t-test to distinguish some order of cross-reactivity between them. the retrospective compilation of our data showed a large distribution of results when we ascertained the results of ttp and tial to explore humoral and cellular immunoreactivity against two citrus allergens and a pollen extract. these immunoassays did not precisely identify the mechanisms responsible for clinical conditions. instead, they provide evidence about cellular and humoral immunoreactivity distributed into an extensive spectral range that may suggest immune tolerance or hypersensitivity. the ttp for the pollen, orange, and lemon extracts showed a distribution concentrated on the higher dilutions, precluding an adequate differentiation among patients’ immunoreactivities. further studies performed with assays extended to higher dilutions are needed to achieve more reliable conclusions. on the contrary, the lait results showed a wide distribution of results, demonstrating a better potential to differentiate patients and predict hypersensitivity. while the ttp results showed a slight correlation between the paired tests, the lait results demonstrated a moderate correlation between the paired assays, projecting a better potential to predict cross-reactivity among the allergens. on the contrary, the lait results showed a wide distribution of results, demonstrating a better potential to differentiate patients and predict hypersensitivity. while the ttp results showed a slight correlation between the paired tests (pearson’s correlation coefficient between r = 0.007 to 0.11), the lait results demonstrated a significant moderate correlation between the paired assays, projecting a better potential to predict cross-reactivity among the allergens (pearson’s correlation coefficient between r = 0.43 to 0.56). this preliminary retrospective survey demonstrated extensive results from the ttp and the ex vivo challenge test monitored by lait against pollen and two citrus species in two cohorts of non–ige-mediated rhinoconjunctivitis patients. ttp and lait are complementary triage tests used at our facilities to select worthwhile antigens to proceed with more laborious in vivo provocation tests when the specific ige is undetectable. none of our patients presented an exclusive reaction to these allergens. every patient was simultaneously tested for several chemical and biological allergens, demonstrating positive results for some of them. our results suggest that pollen-allergic patients may impair their symptoms by additional crossimmunoreactivity against citrus allergens. 5. limitations this study is a retrospective analysis of data collected over six years and nine months. there was no protocol research, and the subject's data was limited to the essentials available on our electronic sheets. therefore, we could not establish a cross-comparison between positive and negative controls to validate the results. the number of subjects is appropriate for a preliminary study; however, future studies must be more comprehensive. the lack of a research protocol implies the possibility of a bias produced by the point of view of the physician who indicated the exam (ceo) based on a clinical suspicion led purely by the anamnesis and physical examination. the study lost many of these patients to follow-up, so assuring the relationship between the immunoassays’ results and the patient's clinical outcome is not possible yet. unfortunately, it was impossible to compare the two procedures with paired tests because they were taken from distinct groups of patients. 6. conclusion our preliminary results show that the lait and ttp may differentiate diverse degrees of immunoreactivity against pollen, orange, and lemon extracts in patients clinically diagnosed with non–ige-mediated allergic rhinoconjunctivitis. tial and ttp are inexpensive, can be performed with minimum laboratory equipment, and can be incorporated into strategies to address health disparities in olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 8-25, 2025; article no. aji.131451 19 respiratory and food allergies. as a preliminary report, the propaedeutic meaning of the presented results and the possibility of interferents must be yet established. more studies focused on the quality-by-design approach with prospective larger double-blind cohorts need to evaluate the potential contribution of lait and ttp for endotyping cellular and humoral immunoreactivity in patients suspected of hypersensitivity against citrus fruits and pollen allergens. 7. future directions and recommendations for clinical practice the primary intended use of in vitro or ex vivo allergen challenge tests is to spare the patients from being submitted to unnecessary, exhaustive, and dangerous in vivo challenge tests. exploring the humoral and the cellular arms of immune systems, the ttp and tial alone or combined may represent, in the near future, a tool for allergists to construct an etiologic diagnosis from their patients, as well as determine the endotypes (mechanisms) of hypersensitivity, in order to choose more convenient and personalized therapies for them. adding data provided by ttp and tial may also contribute to streamlining biomedical research and improving tools such as large language models, usually used by clinicians as a decision support system to enhance diagnostic accuracy. consent as a retrospective survey of results recorded in cognito, consent was given collectively by the institution's ethics committee following the principles of the declaration of helsinki. ethical approvals the authors have collected and preserved written ethical approval per international standards. disclaimer (artificial intelligence) authors hereby declare that no generative ai technologies such as large language models (chatgpt, copilot, etc.) and text-to-image generators have been used during the writing or editing of this manuscript. acknowledgements the instituto alergoimuno de americana funded this work. we want to thank the laboratory technicians alessandra vieira de oliveira and luciana sacilotto carvalho for their help with the exams. competing interests the authors have declared that no competing interests exist. references agache, i., & akdis, c. a. 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(2025). multi-omic approaches for endotype discovery in allergy/immunology. journal of allergy and clinical immunology. in press. published online december 31, 2024. doi:10.1016/j.jaci.2024.12.1083 disclaimer/publisher’s note: the statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of the publisher and/or the editor(s). this publisher and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. _________________________________________________________________________________ © copyright (2025): author(s). the licensee is the journal publisher. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: https://pr.sdiarticle5.com/review-history/131451 https://pr.sdiarticle5.com/review-history/131451 _____________________________________________________________________________________________________ *corresponding author: e-mail: celso@alergoimuno.med.br cite as: olivier, celso eduardo, daiana guedes pinto, ana paula monezzi teixeira, cibele silva miguel, jhéssica letícia santos santana, regiane patussi santos lima, nicole sartoreto rocha, raquel acácia pereira gonçalves santos, and everton salgado monteiro. 2025. “endotyping cellular and humoral cross-reactivity between blomia tropicalis and farfantepenaeus brasiliensis in patients with allergic multimorbidity”. asian journal of immunology 8 (1):163-78. https://doi.org/10.9734/aji/2025/v8i1169. asian journal of immunology volume 8, issue 1, page 163-178, 2025; article no.aji.139203 endotyping cellular and humoral cross-reactivity between blomia tropicalis and farfantepenaeus brasiliensis in patients with allergic multimorbidity celso eduardo olivier a*, daiana guedes pinto a, ana paula monezzi teixeira a, cibele silva miguel a, jhéssica letícia santos santana b, regiane patussi santos lima c, nicole sartoreto rocha d, raquel acácia pereira gonçalves santos a and everton salgado monteiro e a instituto alergoimuno de americana, brazil. b instituto de ensino e pesquisa do hospital de amor de barretos, brazil. c lavoisier laboratórios, são paulo, brazil. d faculdade de americana, são paulo, brazil. e faculty of medicine, são paulo university, brazil. authors’ contributions this work was carried out in collaboration among all authors. author ceo conceptualized the study, did data curation, formal analysis, literature review, and wrote original draft of the manuscript. authors dgp, apmt, csm, nsr, jlss and rpsl performed laboratory procedures. author esm cultured the blomia tropicalis specimens and prepared their extract. author rapgs performed cutaneous tests. all authors read and approved the final manuscript article information doi: https://doi.org/10.9734/aji/2025/v8i1169 open peer review history: this journal follows the advanced open peer review policy. identity of the reviewers, editor(s) and additional reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here: https://pr.sdiarticle5.com/review-history/139203 received: 20/04/2025 published: 03/07/2025 original research article https://doi.org/10.9734/aji/2025/v8i1169 https://pr.sdiarticle5.com/review-history/139203 olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 163-178, 2025; article no.aji.139203 164 abstract background: panallergens, such as tropomyosin, produce cross-reactivity between indoor allergens and food allergens, including the house dust mite blomia tropicalis and the brazilian pink shrimp farfantepenaeus brasiliensis, and are responsible for the clinical symptoms present in patients with allergic multimorbidity phenotypes. aim: to evaluate the potential of the tube titration of precipitins (ttp) and the leukocyte adherence inhibition test (lait) to discriminate and correlate cellular and humoral immunoreactivity against protein extracts of b. tropicalis and f. brasiliensis in patients with non– ige-mediated allergic multimorbidity phenotypes. study design: we examined retrospectively the medical charts of two cohorts of patients clinically diagnosed with non–ige-mediated allergic multimorbidity phenotypes related to inhalation or contact with house dust and/or consumption of shrimps, who were concomitantly investigated for these allergens with the help of ttp or lait. methodology: the registered results of the ttp and lait against protein extracts of b. tropicalis and f. brasiliensis were distributed in ranges through cascade distribution charts. the correlation between the paired assays was calculated using pearson's methodology and demonstrated by dispersion graphs. results: the ttp for the b. tropicalis extract showed a distribution concentrated on the higher dilutions. the mean was 1:353; the median was 1:256; the standard deviation was 1:163; the mode was 1:512 (appeared 49 times). the ttp for the f. brasiliensis extract showed a distribution concentrated on the higher dilutions. the mean was 1:380; the median was 1:512; the standard deviation was 1:157; the mode was 1:512 (which appeared 57 times). the lait for the b. tropicalis extract showed a wide distribution range of results. the lai ranged from 0% to 100%. the mean was 49.8%; the median was 51.5%; the standard deviation was 30.0%; the mode was 0% (appeared eleven times). the lait for the f. brasiliensis extract showed a wide distribution range of results. the lai ranged from 0% to 100%. the mean was 55%; the median was 56%; the standard deviation was 28.8%; the mode was 0% (appeared five times). there was no significant correlation between b. tropicalis and f. brasiliensis when analyzed by ttp or lait results. conclusion: our preliminary results suggest that the ttp and lait may reveal humoral and cellular immunoreactivity in patients with allergic multimorbidity phenotypes using protein extracts of b. tropicalis and f. brasiliensis. keywords: blomia tropicalis; farfantepenaeus brasiliensis; hypersensitivity; house dust mite; leukocyte adherence inhibition test; non–ige-mediated immunoreactivity; shrimp; precipitins. abbreviations lai : leukocyte adherence inhibition lait : leukocyte adherence inhibition test ttp : tube titration of precipitins 1. introduction blomia tropicalis is an arthropod (phylum) of the subphylum chelicerata; class arachnida; subclass acari; superorder acariformes; order sarcoptiformes; suborder astigmata; superfamily glycyphagoidea; family echimyopodidae (schoch et al. 2020a). farfantepenaeus brasiliensis (penaeus brasiliensis) is an arthropod (phylum) of the subphylum crustacea; class malacostraca; order decapoda; suborder dendrobranchiata; superfamily penaeoidea; family penaeidae (schoch et al. 2020b). the first suspicion and evidence that house dust mites (hdm), from the genus dermatophagoides, were causing respiratory allergies were publicized in the mid-1960s (voorhorst et al., 1964; fain, 1966). the allergenicity of hdm from the blomia genus and its cross-reactivity with other hdm species were studied using in vitro neutralization of skin-sensitizing antibodies, as described in japan in the late 1960s (miyamoto et al. 1969). the species blomia tropicalis was described in the early 1970s as a dust and storage mite found in tropical and subtropical regions (van bronswijk et al., 1973). soon, olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 163-178, 2025; article no.aji.139203 165 several reports on the allergenicity of blomia tropicalis in western tropical countries were also published (fernández-caldas et al., 1990; fernández-caldas et al., 1993; chew et al., 1999; baqueiro et al., 2006). almost six decades after the discovery of their causality in respiratory allergies, several reports have indicated that food contamination with hdm, including blomia tropicalis, also produces urticaria and anaphylaxis and is associated with hypersensitivity to non-steroidal antiinflammatory drugs (sánchez-borges et al., 2013; barrera-de-pino, murgas, and miranda, 2012). after that, it was noticed that the hdm, including blomia tropicalis, also produced atopic dermatitis (emran et al. 2019). the allergen nomenclature sub-committee of the world health organization and the international union of immunological societies (who/iuis) has recognized twenty-six allergens to date, ranging in molecular weight from 7 to 110 kda, which have been identified in blomia tropicalis (sub-committee, 2025a; chua et al., 2007). tropomyosin is a phylogenetically conserved heat-stable alpha-helical coiled-coil dimeric protein found in vertebrates and invertebrates that interacts with actin, producing muscle contraction (bailey 1946) (reese et al., 1999). tropomyosin homologs are defined as the group 10 hdm allergens, and blo t 10 is the blomia tropicalis tropomyosin, sharing amino acid sequence homology with several forms of tropomyosins that are involved in cross-reactivity with mites, cockroaches, shrimps, snails, oysters, crabs, lobsters, squids, and other invertebrates (martínez et al. 2024, papia et al., 2021). tropomyosin is the major allergen of crustaceans from the genus penaeus (daul et al. 1994). tropomyosin is considered a panallergen, a sensitizing protein acquired from different fonts, responsible for (apparently) unrelated clinical allergic reactions such as respiratory symptoms (elicited by inhalation of house dust mites) or systemic symptoms (elicited by the ingestion of edible invertebrates such as shrimps) (wong et al., 2016, yang et al. 2010). farfantepenaeus brasiliensis (pink shrimp) is the most exploited shrimp species along the brazilian coast (carvalho et al., 2019). shrimp's tropomyosin is a 34to 38-kda heat-stable allergen that can provoke ige-mediated immediate-type hypersensitivity reactions after ingestion (shanti et al., 1993). tropomyosin is considered the major allergen of shrimp and is usually classified as a group 1 allergen, following the group 1 allergens (crac c 1, exo m 1, lit v 1, met e 1, pan b 1, pen a 1, pen i 1, pen m 1) (sub-committee, 2025b). nevertheless, uncharacterized, the farfantepenaeus brasiliensis tropomyosin should receive the allergen nomenclature far b 1). besides the common major allergen, tropomyosin, several species-specific shrimp allergens may also participate in allergic reactions (morgan et al. 1989). several symptoms have been attributed to shrimp allergy (urticaria, angioedema, chest tightness, shortness of breath, nausea, vomiting, diarrhea, fainting with documented hypotension, chills, fever, abdominal discomfort, abdominal pain, and finger stiffness) (waring et al. 1985). allergic multimorbidity is defined as the presence of concomitant or consecutive allergic phenotypes that may be ige-mediated, partly ige-mediated, or non-ige-mediated (bousquet et al., 2015; bousquet et al., 2025). besides the ige-mediated hypersensitivity mechanism, several types of hypersensitivity mechanisms are associated with allergic phenotypes (jutel et al., 2023). the humoral-dependent non–igemediated allergic phenotypes may be evaluated by the research of precipitins (augustin 1953; augustin et al., 1960; cunningham-rundles et al. 1978; ferguson & carswell 1972; heiner et al., 1962). the tube titration of precipitins (ttp) is routinely used at our facilities to evaluate humoral immunoreactivity against suspected allergens as a triage test prior to the exhaustive in vivo provocation tests, which define hypersensitivities diseases (olivier et al. 2023e, olivier et al. 2021e, olivier et al. 2021d, olivier et al. 2024e, olivier et al. 2024c). the leukocyte adherence inhibition test (lait) and its similar assay, the leukocyte migration inhibition test (lmit), have traditionally been used to differentiate non–ige-mediated immunoreactivity against microorganisms and aeroallergens (fink et al. 1987, kallen & nilsson 1979, kuratsuji 1981, thomson, 1982). the lait and the lmit have also been classically used to differentiate non–ige-mediated immunoreactivity against food allergens (allardyce & shearman 1975, george & vaughan 1962, ashkenazi et al. 1980, butler et al. 1981, papageorgiou et al. 1983). olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 163-178, 2025; article no.aji.139203 166 non–ige-mediated cellular immunoreactivity against food allergens had also been reported by our group using lait (olivier et al. 2022b, olivier et al. 2022a, olivier et al. 2022c, olivier et al. 2023a). non–ige-mediated cellular immunoreactivity against aeroallergens and microorganisms had also been reported by our group with the help of the lait (olivier et al. 2023d, olivier et al. 2023f, olivier et al. 2023b, olivier et al. 2023c, olivier et al. 2024f). we also routinely employ the lait and ttp to endotype cellular and humoral non–ige-mediated immunoreactivity against allergens already proved to produce allergic symptoms by in vivo provocation tests (olivier et al. 2024b, olivier et al. 2024d, olivier et al. 2024g, olivier et al. 2025) to evaluate the potential of the lait and ttp to endotyping non–ige-mediated cellular and humoral immunoreactivity against blomia tropicalis and farfantepenaeus brasiliensis extract, we retrospectively compiled the electronic medical charts of patients diagnosed with non–ige-mediated allergic multimorbidity who were investigated for immunoreactivity simultaneously by one of these assays. the present study provides proof of concept that hypothesizes lait and the ttp may demonstrate cellular and/or humoral immunoreactivity against blomia tropicalis and farfantepenaeus brasiliensis proteins in patients suffering from non–ige–mediated allergic multimorbidity. this paper is a retrospective study of the results of the immunoassays, so we do not have access to the nutritional background or the genetic constitution of the patients. however, we are planning to address these issues in further prospective studies. 2. materials and methods 2.1 subjects after receiving institutional review board approval from the instituto alergoimuno de americana (brazil; 04/2025), we reviewed the electronic chart of 10,500 outpatients who attended our facility from january 2018 to june 2025 selecting patients diagnosed with allergic multimorbidity who were evaluated simultaneously with lait or ttp against blomia tropicalis or farfantepenaeus brasiliensis extracts. a cohort of 100 consecutive outside patients (ttp cohort) had been submitted to ttp with blomia tropicalis extract and farfantepenaeus brasiliensis extract for presenting non–igemediated allergic multimorbidity as defined by the concomitant or consecutive presence of at least two allergic phenotypes (allergic rhinoconjunctivitis, allergic bronchitis, atopic dermatitis, urticaria, gastrointestinal hypersensitivity, and/or anaphylaxis). this cohort counted 42 males; mean age 41.2 years; sd 18.4 years; range 10 to 82 years; median 39 years; mode = 36 (appeared five times); geometric mean = 36.5 years. a cohort of 100 consecutive outside patients (lait cohort) had been submitted to ttp with blomia tropicalis extract and shrimp extract for presenting non–ige-mediated allergic multimorbidity as defined by the concomitant or consecutive presence of at least two allergic phenotypes (allergic rhinoconjunctivitis, allergic bronchitis, atopic dermatitis, urticaria, gastrointestinal hypersensitivity, and/or anaphylaxis). this cohort counted 30 males; mean age 36.4 years; sd 23.5 years; range 2 to 91 years; median 35 years; modes = 5 (appeared five times); geometric mean = 26.4 years. this study did not include patients under biological and/or systemic anti-inflammatory therapy. these procedures were offered to patients with allergen multimorbidity related to house dust and/or shrimp consumption who had indetectable specific ige against these suspected agents and demonstrated non-reactive or inconclusive skin tests against blomia tropicalis and farfantepenaeus brasiliensis extracts (olivier et al., 2013). 2.2 preparation of the blomia tropicalis extract 2.2.1 origen of the blomia tropicalis strain the specimens of blomia tropicalis were kindly provided by prof. dr. jorge martínez quesada from the faculty of pharmacy at the university of the basque country, spain. the culture medium was placed in 50 ml thermofisher® cell culture flasks (15 g per flask) in a sterile environment. these flasks were placed in an airtight container containing a saturated solution of distilled water and sodium chloride for at least 24 hours to maintain the humidity of the culture medium. they were brought to brazil inside airtight bags containing cotton soaked in the saturated nacl solution, thus maintaining humidity during the flight. olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 163-178, 2025; article no.aji.139203 167 2.2.2. preparation of the culture medium the culture and extraction of blomia tropicalis proteins were performed at the bioallergy laboratory (álvaro de carvalho, são paulo, brazil). the culture media (yeast extract and tetramin®) were weighed on thermomix's built-in scale (vorwerk, tm6®), then crushed using the device's primary function for 30 seconds at maximum speed (10), and this step was repeated three times until a fine, homogenized powder was obtained. after the grinding step in the thermomix®, all culture media were sieved through a 60-mesh sieve (250 μm opening) to ensure standardization and granulometry of the medium. the sieved culture medium was then weighed (10 g) and placed in erlenmeyer flasks with a cotton plug and gauze to be transferred to a sterilization and drying oven (ethik technology, 400d®), previously heated, where it remained for 1 hour at 110ºc. after cooling, the contents of the erlenmeyer flasks were transferred to 50 ml cell culture flasks using a previously sterilized plastic funnel, which was performed under unidirectional airflow. the flasks had been previously treated with ultraviolet light for 15 minutes to prevent contamination by microorganisms and other mite species. the 50 ml culture flasks containing only the culture medium were kept in airtight boxes with a saturated sodium chloride solution and distilled water for at least 48 hours to humidify them before inoculating the mites. the population peak (71,000 mites/g of culture) was reached after 90 days of culture. 2.2.3 production of blomia tropicalis allergenic extract the samples of whole culture (mites and culture medium) underwent the grinding process to improve the exposure of allergens to the extraction buffer. next, they were subjected to the defatting process (using ethyl ether), which favored the loss of non-protein molecules. the material was sonicated to facilitate disruption. the sample was solubilized in phosphatebuffered saline (ph 7.2; sodium chloride (nacl) – 81.9 g/l; potassium chloride (kcl) – 1.87 g/l; dibasic sodium phosphate (anhydrous na2hpo4) – 14.19 g/l; monobasic potassium phosphate (kh2po4) 2.38 g/l). the extraction was performed at a rate of 10 ml of buffer per gram of raw material (10%), using gentle magnetic stirring (1,000 rpm) for 4 hours at 4ºc. the material was centrifuged at 5,000 rpm (3,020g for 30 minutes, and the supernatant was preserved. the sediment was resuspended under the same conditions as in the first step and kept under stirring for 2 hours. the centrifugation was then repeated, and the supernatants were mixed. the final extract was dialyzed against the buffer to eliminate low-molecular-weight proteins (<5,000 da) for 24 hours, with three buffer changes and a 1:50 dilution ratio. the process was continued by centrifugation at 10,000 rpm (12,100 g) for 30 minutes at 4 °c. protein concentration was determined using the bradford method (bradford, 1976). the solution was diluted in an antigen dilution solution (nacl, 10g; kh2po4, 0.72g; na3po4, 2.86g; methylparaben, 1g; propylparaben, 0.5g; glycerin, 400ml; h2o, 600ml) to an estimated protein concentration of 1 mg/ml and stored at 4 °c in amber, opaque glass vials. 2.3 shrimp extract farfantepenaeus brasiliensis was acquired from the local market and identified based on its morphological characteristics (silveira et al., 2022). the sample was grounded for 48 hours at 4 °c with a coca-based extractor solution added to cover the amount of antigen. the sample was centrifuged (4,500 rpm for 10 min) and filtered. the protein concentration was estimated spectrophotometrically and diluted to 1 mg/ml in antigen dilution solution to perform allergic skin tests, ttp, and lait. 2.4 lait: ex vivo investigation: leukocyte adherence inhibition test 2.4.1 lait: procedure for allergen ex vivo challenging we performed the lait as previously described (olivier et al. 2012, olivier et al. 2014, olivier et al. 2021a, olivier et al. 2021b, olivier et al. 2021c). shortly, each donor's fresh plasma was divided into two parts and used in parallel ex vivo challenging tests with the allergen extract and the unchallenged plasma (added with antigen dilution solution as a control). we collected plasma with high leukocyte content (buffy coat) from the heparinized tube after one hour of sedimentation at 37 °c. then, we distributed aliquots of 100 μl into eppendorf tubes with (or without) the challenging extract and kept them under agitation for 30 minutes (200 rpm at 37 °c). 2.4.2 lait: procedure for adherence assay after incubation, the challenged plasma was allocated into a standard neubauer olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 163-178, 2025; article no.aji.139203 168 hemocytometer counting chamber with a plain, non-metallic glass surface and left to stand for 2 hours at 37 °c in a humidified atmosphere of the covered water bath, allowing leukocytes to adhere to the glass. next, we counted the leukocytes, removed the coverslip, and washed the chamber by immersing it in a beaker containing phosphate-buffered saline (pbs) at 37 °c. then, we added a drop of pbs to the hemocytometer's chamber and allocated a clean coverslip over it. the remaining cells were counted in the same squares as previously examined. 2.4.3 lait: procedure for calculation the percentage of leukocyte adherence (la) of each assay was estimated as: (the number of leukocytes observed on the hemocytometry chamber after washing divided by the number of leukocytes observed on the hemocytometry chamber before washing) and multiplied by 100 (%). the leukocyte adherence ratio (lar) was estimated based on the ratio between the leukocyte adherence (la) from the antigenspecific challenged plasma and the la from the unchallenged control plasma: lar = la of the challenged sample divided by la of the unchallenged control plasma, multiplied by 100 (%). to further calculate the leukocyte adherence inhibition (lai), we subtracted the lar from 100 (%). we utilized the lai results for the cascade distribution chart and the statistical calculations, both of which were performed using the microsoft excel® statistical package. 2.5 ttp: in vitro investigation: tube titration of precipitins as previously reported, the semi-quantitative ttp was performed in a transparent vitreous tube array (olivier et al. 2024a). shortly, the patient's blood was collected in a clot-activator collecting tube. after separation, the serum was centrifugated at 2,000 rpm for 10 minutes. each allergen extract was allocated in sets of eleven glass tubes at progressively diluted serum concentrations. the progressive dilutions were combined with separated aliquots of 15 μl of the allergen extract with 250 μl of the patient's serum, progressively diluted into physiological saline solution (nacl 0,9%) in the dilution ratios of 1:1; 1:2; 1:4; 1:8; 1:16; 1:32; 1:64; 1:128; 1:256; and 1:512. one tube was a blank control, performed with water and serum, to observe occasional spontaneous precipitation (sia test). after 24 hours, the tubes were examined, and the titers (the highest dilution factor that yields a positive reading) were recorded (williams & chase, 1971). 3. results as a retrospective survey, no research protocol was in place; therefore, we report the incidental immune investigation as documented in the digital medical charts. fig. 1. cascade distribution chart of the tube titration of precipitins (x-axis %) resulting from the blomia tropicalis extract against the serum of the ttp cohort of 100 tests/subjects (y-axis) olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 163-178, 2025; article no.aji.139203 169 fig. 2. cascade distribution chart of the tube titration of precipitins (x-axis %) resulting from the farfantepenaeus brasiliensis extract against the serum of the ttp cohort of 100 tests/subjects (y-axis) fig. 3. dispersion chart of the tube titration of precipitins results of the ex vivo challenge test against blomia tropicalis extract (x-axis %), plotted against the tube titration of precipitins results of the ex vivo challenge test against farfantepenaeus brasiliensis extract (y-axis %) the ttp for the blomia tropicalis extract showed a distribution concentrated on the higher dilutions (fig 1). there was no negative result. the mean was estimated at 1:353; the median was 1:256; the standard deviation was estimated at 1:163; the mode was 1:512 (appeared 49 times). the ttp for the farfantepenaeus brasiliensis extract showed a distribution concentrated on the higher dilutions (fig 1). there was no negative result. the mean was estimated at 1:380; the median was 1:512; the standard deviation was estimated at 1:157; the mode was 1:512 (which appeared 57 times). pearson's correlation indicated a non-significant, small positive relationship between ttp results for blomia tropicalis and farfantepenaeus brasiliensis; r(98) = 0.0629, p-value = 0.534 (see fig. 3). the lait for the blomia tropicalis extract showed a wide distribution range of results. the lai ranged from 0% to 100%. the mean was 49.8%; the median was 51.5%; the standard deviation was 30.0%; the mode was 0% (appeared eleven times). the cascade distribution demonstrates a wide range of lai results (fig. 4). some patients showed low or moderate immunoreactivity during olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 163-178, 2025; article no.aji.139203 170 the ex vivo challenge test. in contrast, others displayed strong immunoreactivity, which could reflect the participation of blomia tropicalis allergens in a non–ige-mediated hypersensitivity condition in these patients (see fig. 4) the lait for the farfantepenaeus brasiliensis extract showed a wide distribution range of results. the lai ranged from 0% to 100%. the mean was 55%; the median was 56%; the standard deviation was 28.8%; the mode was 0% (appeared five times). the cascade distribution demonstrates a wide range of lai results (fig. 5). some patients showed low or moderate immunoreactivity during the ex vivo challenge test. in contrast, others displayed strong immunoreactivity, which could reflect the participation of shrimp allergens in a non–igemediated hypersensitivity condition in these patients (see fig. 5). pearson's correlation indicated a non-significant, small positive relationship between lait results for blomia tropicalis and f. brasiliensis; r(98) = 0.0713, p-value = 0.481 (see fig. 6). fig. 4. cascade distribution chart of the range groups of leukocyte adherence inhibition (lai) results (x-axis %) of the ex vivo challenge test against the blomia tropicalis extract monitored by the leukocyte adherence inhibition test (lait), according to the respective number of outcomes over the lait cohort with 100 tests/subjects (y-axis) fig. 5. cascade distribution chart of the range groups of leukocyte adherence inhibition (lai) results (x-axis %) of the ex vivo challenge test against the farfantepenaeus brasiliensis extract monitored by the leukocyte adherence inhibition test (lait), according to the respective number of outcomes over the lait cohort with 100 tests/subjects (y-axis) olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 163-178, 2025; article no.aji.139203 171 fig. 6. dispersion chart of the leukocyte adherence inhibition (lai) results of the ex vivo challenge test against blomia tropicalis extract (x-axis %), plotted against the lai results of the ex vivo challenge test against farfantepenaeus brasiliensis extract (y-axis %) 4. discussion polysensitization and cross-reactivity are related phenomena frequently observed in patients with allergic multimorbidity (zhang et al., 2025; čelakovská et al., 2024; hasnain et al., 2017). when investigating the etiology of allergic reactions, it is advisable to maintain an open mind regarding cross-reactivity among panallergens, especially in polysensitized patients presenting with allergic multimorbidity (miltner et al., 2024). the specificity and facility for researching precipitins transformed this immunoassay into a pioneering examination upon which the fundamentals of immunology were established (hunter, 1905). precipitins are yet used nowadays to monitor emerging diseases when more precise and sophisticated assays are inexistent or unavailable (bellanger et al., 2022). precipitating antibodies indicate a humoral immune response against the tested antigens (gell, harington, and rivers, 1946). before the discovery of ige, the research of precipitins was the only viable way to demonstrate immunoreactivity against allergenic agents (augustin & hayward, 1960) (wells, 1911). precipitin antibodies may belong to any immunoglobulin class (except for igg4), can mount immune complexes, and can participate in type ii (antibody-dependent cell-mediated) and type iii (immune complexes disease) gell and coombs hypersensitivity reactions, which may (theoretically) be demonstrated by the lait and the ttp assays, respectively (gell & coombs, 1968). the lait is an ex vivo challenge test performed with live leukocytes, which allows the interaction of all immune-circulating cells with allergens. several immune pathways can inhibit leukocyte adherence (tong et al., 1979; halliday et al., 1974). the present study is a proof-of-concept that hypothesizes that lait and the ttp may differentiate diverse degrees of cellular and humoral immunoreactivity against blomia tropicalis and farfantepenaeus brasiliensis extracts among patients suffering from non–igemediated allergic multimorbidity. as the tests were performed simultaneously using the same venous sample with both allergens, it was possible to calculate a correlation to distinguish any order of cross-reactivity between them. the retrospective compilation of our data revealed a wide distribution of results when analyzing the lait results to explore cellular immunoreactivity. on the contrary, there was no such variability in the results of ttp, which demonstrated the presence of high titers of precipitin antibodies predominantly in the higher dilutions, suggesting the presence of humoral activity against these extracts in all selected patients, thereby limiting the analytical value of this immunoassay. despite these immunoassays not precisely identifying the mechanisms responsible for the clinical condition, they provide general evidence about cellular and humoral immunoreactivity that may be involved in immune olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 163-178, 2025; article no.aji.139203 172 tolerance, sensitization, and clinical hypersensitivity. this preliminary retrospective survey yielded diverse results from the ttp and the ex vivo challenge test, monitored by lait, against blomia tropicalis and farfantepenaeus brasiliensis extracts in two cohorts of patients diagnosed with non–ige–mediated allergic multimorbidity. ttp and lait are complementary triage tests used at our facilities to select worthwhile antigens to proceed with more laborious in vivo provocation tests when the specific ige is undetectable. none of our patients presented an exclusive reaction to these allergens. every patient was simultaneously evaluated for several chemical and biological allergens, demonstrating positive results for some of them. our results suggest that patients with allergies to hdm may exacerbate their symptoms by experiencing additional crossimmunoreactivity when consuming shrimp. assessing diverse pathways of immunoreactivity and hypersensitivity through tools such as tial and ttp against the whole extract of suspected allergens, as well as suspected molecular allergens, is a multi-omics approach that evaluates both diagnosis and treatment effectiveness in allergic patients (hubert et al., 2023). 5. limitations this study is a retrospective analysis of data collected over a seven-year period since our facility began employing laboratory immune assays. there was no protocol research, and the subject's data was limited to the essentials available on our electronic sheets. therefore, we were unable to establish a cross-comparison between the positive and negative controls to validate the results. the number of subjects is suitable for a preliminary study; however, future studies should be more comprehensive. the lack of a research protocol implies the possibility of bias introduced by the physician's point of view (ceo) based on a clinical suspicion driven solely by the anamnesis and physical examination. the study lost many of these patients to follow-up, so it is not yet possible to ensure the relationship between the immunoassay results and the patient's clinical outcome. unfortunately, it was impossible to compare the two procedures using paired statistics methodologies because they were derived from two distinct patient groups. 6. conclusion our preliminary results suggest that the lait and ttp may distinguish between varying degrees of immunoreactivity against blomia tropicalis and farfantepenaeus brasiliensis extracts in patients clinically diagnosed with non–ige–mediated allergic multimorbidity. despite requiring trained personnel for execution, lait and ttp are inexpensive, can be performed with minimal laboratory equipment, and can be incorporated into strategies to address health disparities in diagnosing respiratory and food allergies (anagnostou et al., 2025). as a preliminary report, the propaedeutic significance of the presented results and the potential interfering factors must be established (anouar et al., 2024). more studies focused on the quality-by-design approach with larger, prospective, double-blind cohorts are needed to evaluate the potential contribution of lait and ttp for endotyping cellular and humoral immunoreactivity in patients suspected of hypersensitivity to aeroallergens and food allergens (chiarentin et al., 2023). 7. future directions and recommendations for clinical practice the primary intended use of in vitro or ex vivo allergen challenge tests is to spare patients from undergoing unnecessary, exhaustive, and potentially dangerous in vivo challenge tests. exploring the humoral and the cellular arms of immune systems, the ttp and lait alone or combined may represent, in the near future, a tool for allergists to construct an etiologic diagnosis from their patients, as well as determine the endotypes (mechanisms) of hypersensitivity, in order to choose more convenient and personalized therapies for them. adding data provided by ttp and lait may also contribute to streamlining biomedical research and improving tools, such as large language models, which clinicians often use as a decision support system to enhance diagnostic accuracy (abers & mathias, 2025). ethical approval the authors have obtained and documented written ethical approval following international standards. as a retrospective survey of results recorded in cognito, consent was obtained collectively by the institution's ethics committee, following the principles outlined in the declaration of helsinki (world medical association, 2013). olivier et al.; asian j. immunol., vol. 8, no. 1, pp. 163-178, 2025; article no.aji.139203 173 disclaimer (artificial intelligence) authors hereby declare that no generative ai technologies such as large language models (chatgpt, copilot, etc.) and text-to-image generators have been used during the writing or editing of this manuscript. acknowledgements the instituto alergoimuno de americana funded this work. we want to thank the laboratory technicians, alessandra vieira de oliveira and luciana sacilotto carvalho, for their assistance with the exams. competing interests authors have declared that they have no known competing financial interests or non-financial interests or personal relationships that could have appeared to influence the work reported in this paper. references abers, m. s., & mathias, r. a. 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(2025). investigating allergen-specific ige distribution and correlations in chronic urticaria: a retrospective study in shanghai, china. european journal of medical research, 30(1), 182. disclaimer/publisher’s note: the statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of the publisher and/or the editor(s). this publisher and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. _________________________________________________________________________________ © copyright (2025): author(s). the licensee is the journal publisher. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peer-review history: the peer review history for this paper can be accessed here: https://pr.sdiarticle5.com/review-history/139203 https://pr.sdiarticle5.com/review-history/139203 