_____________________________________________________________________________________________________ *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 s- ovalbumin (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–IgE- mediated 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 non- reactive 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 [35- 41]. 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 egg- propagated 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 semi- quantitative 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 non- reactive 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 follow- up 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-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 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). 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