key: cord-258272-uu6t6tnh authors: dabaghian, farid; khanavi, mahnaz; zarshenas, mohammad m. title: bioactive compounds with possible inhibitory activity of angiotensin-converting enzyme-ii; a gate to manage and prevent covid-19 date: 2020-05-16 journal: med hypotheses doi: 10.1016/j.mehy.2020.109841 sha: doc_id: 258272 cord_uid: uu6t6tnh nan till now, no treatment or vaccine has been characterized for covid-19 (1) . angiotensin-converting enzyme-ii (ace-ii) receptor, most likely the covid-19 target, plays essential roles in virus transmission to the alveolar cells (2) . accordingly, agents with potential inhibition or regulation of ace-ii receptors might be effective in covid-19 management (3) . the averting impact of naringin on pro-inhibitory cytokines (increased in covid-19 infection) including cyclooxygenase-ii, interleukin-6 and -1β, and nitric oxide synthase is considerable (5) . glycyrrhizin can inhibit covid-19 s-protein binding to ace-ii receptors (7) . emodin, a bioactive antiviral agent may prevent s-protein binding to ace-ii receptors. thus, emodin and probably aloe-emodin can stave off the covid-19 infection via competing with s-protein in binding to ace-ii (7) . rheum palmatum l. and aloe vera (l.) burm.f. are rich sources of these compounds (8) . based on this opinion (figure 1) , concerned compounds could be applied in prevention and management of covid-19 solely or combination with conventional interventions. purposing saikosaponins for the treatment of covid-19 covid-19 and the cardiovascular system angiotensin converting enzyme 2: sars-cov-2 receptor and regulator of the renin-angiotensin system potential natural compounds for preventing 2019-ncov infection citrus fruits are rich in flavonoids for immunoregulation and potential targeting ace2 emodin blocks the sars coronavirus spike protein and angiotensin-converting enzyme 2 interaction discovery of anti-2019-ncov agents from chinese patent drugs via docking screening anti-sars coronavirus 3c-like protease effects of rheum palmatum l. extracts the authors of this manuscript have no conflict of interest. key: cord-267612-1h7xpkbe authors: lipsker, dan title: a chilblain epidemic during the covid-19 pandemic. a sign of natural resistance to sars-cov-2? date: 2020-06-06 journal: med hypotheses doi: 10.1016/j.mehy.2020.109959 sha: doc_id: 267612 cord_uid: 1h7xpkbe nan a chilblain epidemic during the covid-19 pandemic. a sign of natural resistance to sars-cov-2? dan lipsker 1,2 , md, phd since the start of the covid-19 pandemic, "epidemics" of chilblains or chilblain-like acral lesions have been reported in the social media, the general press and the medical literature in italy, spain, the uk and france (1) (2) (3) (4) (5) . this was also our experience in the region of alsace which was the earliest to be hit hard by the covid-19 pandemic in france. the great majority of the cases we observed and those reported in the literature share the following common features: -lesions were of sudden onset in young patients without a previous history of chilblains in most of them and they were not necessarily triggered by exposure to cold; -those young patients were otherwise asymptomatic or pauci-symptomatic and only a few had mild clinical signs compatible with covid-19; -none of them had pneumonia or severe manifestations of covid-19; -clinical signs of covid-19 or contact with infected persons occurred a few days to one month before chilblains; -sars-cov-2 infection could be demonstrated by nasopharyngeal swab or by serology only in very limited number of patients. the occurrence of an unusual high number of new cases of chilblain a few weeks after the start of the covid-19 epidemic in many countries is probably not coincidental. chilblains are a prototypical sign of a few inherited disorders of innate immunity, characterized by a strong interferon signature and a severe microangiopathy, called the type i interferonopathies (6, 7) . i therefore hypothesize that in a few genetically predisposed individuals the contact with sars-cov-2 triggers a strong interferon response, of which chilblain are the cutaneous expression. in some of them, this strong interferon response probably allows the clearing of the virus without intervention of the adaptive immune system, explaining the lack of antibodies in the patients who were tested so far. if this hypothesis holds true, that would suggest the possibility of a natural resistance to sars-cov-2 infection in a few individuals. that would also mean that those naturally protected individuals will not develop antibodies and would fail serologic detection, which is important in view of the interpretation of epidemiologic studies based on seroprevalence. chilblains in children in the setting of covid-19 pandemic chilblains are a common cutaneous finding during the covid-19 pandemic: a retrospective nationwide study from france chilblains-like lesions in children following suspected covid-19 infection chilblain-like lesions: a case series of 41 patients during the covid-19 pandemic characterization of acute acro-ischemic lesions in non-hospitalized patients: a case series of 132 patients during the covid-19 outbreak type i interferon-mediated monogenic autoinflammation: the type i interferonopathies, a conceptual overview type i interferonopathies in pediatric rheumatology no conflict of interest "conflict of interest statement" i have no conflict of interest that influenced this work. key: cord-270805-o6rbfmie authors: hussein, osama title: second wave of of covid-19 is determined by immune mechanism date: 2020-09-02 journal: med hypotheses doi: 10.1016/j.mehy.2020.110238 sha: doc_id: 270805 cord_uid: o6rbfmie a second wave of new severe acute respiratory syndrome coronavirus 2 (covid-19) cases is widely feared. in fact resurgence of cases has been clearly observed in several countries that had seen flattening of the epidemic curve. in general, relaxation of community control measures is almost always blamed for the resurgence of cases. in this letter, the author describes an immunological explanation for the double-peaked epidemic curve of new viral diseases including covid-19. according to this hypothesis, a second wave of cases is due to the effective innate immunity in some of the population. these individuals may later develop clinical disease upon repeated exposure. this theory claims that a double-peaked pattern of new cases in a new viral epidemic is intrinsically determined by the pattern of pathogen interaction with the host. according to this hypothesis, relaxation of the community control measures is not responsible; at least in part, for resurgence of cases. countries. in japan, a crisp two-peak incidence curve is clear(2). similar trends are reported from other countries. the notion of epidemics that normally have two peaks of new cases over time is widely accepted(3); although epidemiologists have not characterized this pattern as an established model. in general, the resumption of international travel or the relaxation of the community control measures are are almost always blamed for the resurgence of the disease incidence. in this letter, this author suggests that a double-peaked epidemic curve is an intrinsic feature of viral outbreaks. according to this hypothesis, immune response to virus exposure in different individuals determines the spread pattern in the community. the two principal divisions of the immune response to pathogens are the innate and the adaptive immunity. both divisions play a major role in the body response to viral exposure. together with the development of clonal virus-specific cd8 + t lymphocytes, these individuals will experience subclinical or clinical viral illness and are responsible for the early rise in the epidemic curve. assuming the above hypothesis is true; a viral epidemic curve will show an early upstroke corresponding to persons who pass into viremia upon initial exposure to the virus followed by a second slower rise due to cases who develop the disease after repeated process of inoculation and clearance at the point of entry. it is fairly reasonable to assume that the latter group of patients may be the healthier individuals who will have lower case-fatality rate. together with the notion of the covid-19 cases getting milder with time, objective evidence from japan indicates that the second peak in incidence is not accompanied with a corresponding peak in fatality. the implications of this hypothesis are medically and socially relevant. according to this theory, resurgence of a viral outbreak is intrinsically determined by the nature of the virus interaction with the host. relaxation of the community measures or "reopening" of the economy may not be blamed for a second rise in incidence rates. the theory also provides one explanation for the possible observation of decreased case-fatality over time. in conclusion, the author describes an immunological explanation for a double-peaked epidemic curve of covid-19 and other new viral diseases. e64-e7. 2. who coronavirus disease (covid-19) dashboard. geneva: world health organization beware of the second wave of covid-19 pathogen recognition and innate immunity activation and evasion of type i interferon responses by sars-cov-2 human cell tropism and innate immune system interactions of human respiratory coronavirus emc compared to those of severe acute respiratory syndrome coronavirus the author has no potential conflict of interest to declare. key: cord-258670-dphg8ukj authors: karaahmet, fatih; altan kocaman, sinan title: endothelial progenitor cells and mesenchymal stem cells to overcome vascular deterioration and cytokine storm in critical patients with covid-19 date: 2020-06-10 journal: med hypotheses doi: 10.1016/j.mehy.2020.109973 sha: doc_id: 258670 cord_uid: dphg8ukj nan severe acute respiratory syndrome coronavirus 2 (sars-cov-2 or covid-2019) is an emerging global health threatening viral infection pathogen, originating from wuhan, autoimmunity and infectious agents seems potential immunologic triggers in covi̇d-19. the clinical spectrum of covid-19 infection ranges from asymptomatic to critical patient clinic that requires intensive care management. poorer prognosis is associated with the advanced age, diabetes mellitus and hypertension, which are also vascular risk factors (4) . besides the adequate immune system, sufficient vascular endothelial repair reserve and immune regulatory capacity are needed for resolving the cytokine storm and endothelial damage occurring in this disease. on the other side, activation of cytokine-mediated inflammation, endothelial dysfunction and thrombus formation and this way vascular cardiopulmonary collapse leads to poor prognostic result of the covid-19 process. further, high blood pressure, thrombosis, pulmonary embolism and catastrophic course leads to suggest that the virus is also targeting the endothelium (5, 6) . in light of this information, the sufficient repair of the endothelial lining of blood vessels with endothelial progenitor cells (epcs) treatment may have a crucial role to overcome the vascular collapse driving forces in covid-19 patients, as well as to modulate human immune system. epcs include a heterogeneous population of hematopoietic and nonhematopoietic progenitor cells. expression of specific surface markers (i.e., cd34, vegf-r2, cd133) has been generally accepted as an identifying characteristic of these cells. circulating epc level and function may serve as both biomarkers of vascular function and as prognostic indices for vascular disease. accumulating evidence leads to suggest that bone marrow-derived epcs for repairing endothelial damage is now considered as an important novel potential therapeutic option for vascular repair (7, 8) . in critical patients in the course of covid-19, epcs may have an important contribution to stem cell treatments to maintain vascular endothelin functions. mesenchymal stem cells (mscs) can be derived from various adult tissues with multipotent and self-renewal abilities. the beneficial effects of mscs like tissue repair and immunomodulatory properties have made them a encouraging therapy in different types of disorders. mscs could encourage inflammation when the immune system is underactivated and restrict inflammation when the immune system is overactivated to prevent self-overattack (9) . in critical patients who do not respond to current treatment, the restoration of vascular endothelial function and modulation of immune system by synergistic use of epcs and mscs may have a crucial role to overcome the vascular collapse driving forces in covid-19 patients. potential effect of natural and anabolizan steroids in elderly patient with covid-19. med hypotheses the human coronavirus disease covid-19: its origin, characteristics, and insights into potential drugs and its mechanisms. pathogens covid-19: an update on diagnostic and therapeutic approaches acute myocardial injury is common in patients with covid-19 and impairs their prognosis. heart endothelial cell infection and endotheliitis in covid-19 covid-19 and the cardiovascular system: implications for risk assessment, diagnosis, and treatment options endothelial progenitor cells (cd34+kdr+) and monocytes may provide the development of good coronary collaterals despite the vascular risk factors and extensive atherosclerosis endothelial and cardiac progenitor cells for cardiovascular repair: a controversial paradigm in cell therapy immune modulation by mesenchymal stem cells we hereby declare that all authors have made a substantial contribution to the information submitted for publication; all have read and approved the final manuscript and the manuscript or portionsthereof are not under consideration by another journal key: cord-279084-bbae1qyx authors: liu, bin title: free dna, a reason for severe covid-19 infection? date: 2020-05-05 journal: med hypotheses doi: 10.1016/j.mehy.2020.109812 sha: doc_id: 279084 cord_uid: bbae1qyx the fast-growing outbreak of 2019 novel coronaviruses (sars-cov-2) reached all continents except the antarctica in merely three months. severe sars-cov-2 infection (covid-19) has a bad clinical outcome, and some reports emphasized the role of cytokine storm and dysfunctions of multiple organs. however, the etiology of severe covid-19 has been largely unknown. similar as sars-cov and mers-cov, sars-cov-2 is also thought derived from bat coronaviruses. however, it is not pathogenic for bat at all, because free dna in cytoplasm or blood cannot bring up violent immune response in bat; but it can produce severe inflammations in human. i hypothesized that the damage induced by free dna is a reason for severe covid-19, which can explain many symptoms of this disease, such as cytokine storm, acute respiratory distress syndrome (ards) and muscus plug, acute injuries of heart, liver and kidney, and some special symptoms of covid-19. my hypothesis will be helpful for better understand the etiology of severe covid-19. mers-cov, sars-cov-2 is also thought derived from bat coronaviruses. however, 20 it is not pathogenic for bat at all, because free dna in cytoplasm or blood cannot 21 bring up violent immune response in bat; but it can produce severe inflammations in 22 human. i hypothesized that the damage induced by free dna is a reason for severe 23 covid-19, which can explain many symptoms of this disease, such as cytokine 24 storm, ards and muscus plug, acute injuries of heart, liver and kidney, and some 25 special symptoms of covid-19. my hypothesis will be helpful for better understand 26 the etiology of severe covid-19. cytokines, 5,6 thus risk for "cytokine storm". in addition to these cytokines, free dna 56 can also destroy vascular endothelial cells directly: 7 in vitro study showed it can harm 57 human umbilical vein endothelial cells. the damage of endothelial cells will boost the 58 dysfunctions of multiple organs by cytokine storm, as described as follow. 59 meanwhile, cytokine storm will also promote the permeability of blood vessels. level 60 of lymphocytes is thought as the early identification of risk factors for severe 61 covid-19, [1] [2] [3] 8 while i hypothesized that it was related to free dna-related cytokine 62 storm and blood vessel damage, which can explain many symptoms of this disease, 63 including some "special" symptoms in covid-19, as shown in figure 1 . have no fever at admission to hospital, but most of them will develop a fever during 143 hospital. 2 i thought that the "delayed fever" was partly due to an increasing level of 144 free dna, in addition to the amplification of the virus. third, many clinicians found 145 that some severe covid-19 patients have experienced two or more "attack" even 146 after viral load was reduced. i thought that some attack was due to direct invasion by no research support for this manuscript. clinical features of patients infected with 2019 172 novel coronavirus in wuhan clinical characteristics of 2019 novel coronavirus 174 infection in china clinical course and outcomes of critically ill patients of 177 2019 novel coronavirus pneumonia: a single-centered retrospective observational 178 study from wuhan, china a novel coronavirus from patients with 180 pneumonia in china dampened sting-dependent interferon activation in 182 bats cyclic gmp-amp is an endogenous second messenger 184 in innate immune signaling by cytosolic dna neutrophil-to-lymphocyte ratio predicts severe 186 illness patients with 2019 novel coronavirus in the early stage induces oxidative stress, double-strand dna breaks, and dna damage response 190 in human adipose-derived mesenchymal stem cells aveolar macrophage activation and cytokine 193 storm in the pathogenesis of severe covid-19 high level of neutrophil extracellular traps 196 correlates with poor prognosis of severe influenza a infection lung pathology of severe acute respiratory 199 syndrome (sars): a study of 8 autopsy cases from singapore relation between chest ct findings clinical conditions of coronavirus disease (covid-19) pneumonia: a multicenter 203 national health commission of the people's republic of china. diagnosis and 205 treatment of pneumonia caused by novel coronavirus infection (trial version 7 revised 206 version the pathogenicity of 2019 novel coronavirus in 210 hace2 transgenic mice ebola haemorrhagic fever photolysis of n-hydroxypyridinethiones: a 214 new source of hydroxyl radicals for the direct damage of cell-free and cellular dna clinical course and risk factors for mortality of adult 217 inpatients with covid-19 in wuhan, china: a retrospective cohort study positive rt-pcr test results in patients recovered 10 cell-free nucleic acids and their 222 emerging role in the pathogenesis and clinical management of inflammatory bowel 223 key: cord-253468-pf0xubii authors: emara, mohamed h; mazid, usama; ali atta, mohamed; elshahat, sahar; mohamed mahros, aya title: ketonuria with or without ketoacidosis as the presenting manifestation of sars-cov-2 (covid-19) among uncontrolled type 2 diabetic patients date: 2020-09-02 journal: med hypotheses doi: 10.1016/j.mehy.2020.110226 sha: doc_id: 253468 cord_uid: pf0xubii we present three diabetic patients cases presented with kentonuria as the presenting manifestation of sars-cov-2 infection. we hereby present the data of 3 patients presented to our opd and were admitted as diabetic ketoacidosis (dka) and 2-3 days later they developed manifestations suggestive of and proved by swabbing as positive cases. a 42-year-old male patient who was not fully compliant with oral diabetic medicines over last one month, presented for renewal of monthly medicine without any clinical manifestations. a call from lab about panic rbs value (table 1 ) and positive ketones received. patient admitted as dka, the 2 nd day after admission he developed, and his o 2 saturation was began to drop. chest auscultation and chest x ray were unremarkable and hence chest ct scan was requested ( figure 1 ) and showed picture suggestive of mild-moderate covid-19, swabbing was done and came positive case 2: a 51-year-old male, presented by dizziness over last 2-3 days and when examined found to have high rbs and ketonuria, and hence admitted as kda, and was acidotic (ph 7). second day, o 2 saturation dropped and the patient was afebrile (table 1) . chest x ray showed; bilateral lung infiltration, swabbing was done and came positive for sars-cov-2. a 62-year-old male patient who was not compliant with his medicines over the last 2 months, presented for renewal of medicine without any clinical manifestations, found to have panic rbs measurement, and was positive for urine ketones. patient admitted as dka, on the 3 rd day he begin to report shortness of breath (sob) and low-grade fever. examination showed lowgrade fever, bilateral crepitations, hypoxemia, swabbing was done and was positive for sars-cov-2. patient later deteriorated with severe hypoxemia and connected to mechanical ventilator and unfortunately passed out despite of the correction in the dka. common feature of cases: all the three cases share some common features. all were known type 2 diabetic non-obese males with regular daily activity and without fasting. not all were compliant with their medications over the last 1-2 months; all were free at the time of presentation from fever, cough, sob, or constitutional manifestations. co-morbidities; all the three patients were also hypertensive and dyslipidemias. the most important common feature was the development of fever and hypoxemia by the 2 nd -3 rd day of hospital admission (table 1) which embarked us to investigate for sars-cov-2. the trials to predict the severity of covid-19 patients from different urine parameters has been studied among patients from the sars-cov-2 first focus in wuhan, china. urine occult blood and proteinuria were higher in covid-19 patients than in healthy controls. urine ketonesamong others-was not found to predict the severity in covid-19 infection [1] . however, other reports documented higher frequency of ketoacidosis among diabetic patients infected with sars-cov-2 [2] with variable degrees of severity. in fact, different co-morbidities especially diabetes increase morbidity and mortality among covid-19 patients [3] . the cases presented here deliver many important messages. first, patients with sars-cov-2 may lack all or any of the well-known manifestations as fever and cough; that are listed in the triage checklist of the case definition protocols. second, the importance of continuous tight control of diabetes. all our cases were missed from the regular monthly diabetic follow up and furthermore they were not compliant with medicines. treating physicians and pharmacists should ensure regular dispense of diabetic medicines to well controlled patients while noncontrolled patients should be reached and controlled [4] . in fact, several reports showed not only worsening of diabetic control with sars-cov-2 infection, but also possible sars-cov-2 induced diabetes [5] . this may not be strange due to the widespread presentation of the ace 2 receptors in virtually all organs including the pancreas, which represent the target receptor of the virus to enter the cells [6] . third, and probably the unique finding of this report is that in areas with high sars-cov-2 transmission any diabetic patient with urinary ketones should be meticulously examined and if needed also investigated for covid-19 even if lacking the well-known sars-cov2 manifestations. given the global high prevalence of diabetes, many patients with this presentation would be discovered [4] . why we should focus such cases is not only linked to the morbidity and mortality of patients, but also to the concern about spread of infection outside and inside the hospital particularly most of those patients are treated in the icu with possible contact with many other immunocompromised patients putting them under a real threat.. fourth, the treating team managing patients with dka should increase their level of suspicion during this period of exponential spread of sars-cov-2 around the globe. we realize that, our hypothesis that ketonuria as the presenting manifestations of sars-cov-2 among diabetics may not be one hundred percent convincing because uncontrolled diabetes may result in ketonuria, but it is known that infections are one of the precipitating factors for dka among diabetic patients. hence the clinicians should think in this silent sars-cov infection in this high-risk category particularly when they seems uncontrolled and had ketonuria. the value of urine biochemical parameters in the prediction of the severity of coronavirus disease 2019 covid-19 in diabetic patients: related risks and specifics of management the sars-cov2 (covid-19) pandemic: what clinicians should know caring for patients with diabetes during covid-19 pandemic: important considerations for pharmacists. res social adm pharm new-onset diabetes in covid-19 cell entry mechanisms of sars-cov-2 a written informed consent was obtained from the patients or their first degree relatives key: cord-277931-3hxhsmw8 authors: khitan, zeid j.; khawaja, imran; mufson, maurice a.; sanabria, juan r.; abraham, nader g.; peterson, stephen j.; sundaram, uma; shapiro, joseph i. title: scan charcoal improve outcomes in covid-19 infections? date: 2020-08-10 journal: med hypotheses doi: 10.1016/j.mehy.2020.110176 sha: doc_id: 277931 cord_uid: 3hxhsmw8 covid-19 infection causes considerable morbidity and mortality, especially to those who are aged, have impaired renal function and are obese. we propose to examine the potential utility of oral activated charcoal with the hypothesis that such treatment would lower absorption of microbiome derived toxins and ameliorate systemic oxidant stress and inflammation. covid-19 infection causes considerable morbidity and mortality, especially to those who are aged, have impaired renal function and are obese. we propose to examine the potential utility of oral activated charcoal with the hypothesis that such treatment would lower absorption of microbiome derived toxins and ameliorate systemic oxidant stress and inflammation. while coronaviruses have been known to cause potentially serious disease for ½ a century 1 , covid-19 has created a pandemic with adverse health consequences beyond the experiences of those people living today. as of july 14, 2020, approximately 13 million people have been infected with at least 570,000 dying from this disease and its complications 2 . interestingly, the range of signs and symptoms ranges from those who have essentially no symptoms to those with fatal disease. it appears that age, renal dysfunction and obesity are amongst the most important risk factors for serious or fatal covid-19 infection 3, 4 . while there are multiple mechanisms by which this virus can injure hosts, it appears that increases in systemic cytokines and widespread inflammation may play an important role 5 . our research group has focused on the role that adipocytes play in the pathophysiology of metabolic and cv disease. in particular, we have noted that in experimental models of these diseases, the redox state within adipocytes has profound consequences to systemic oxidant stress, inflammation and disease phenotype [6] [7] [8] [9] . we have specifically identified that products derived from tyrosine and tryptophan which are produced by the intestinal microbiome, specifically p-cresyl sulfate and indoxyl sulfate can directly cause oxidant stress in adipocytes 10 . these substances are excreted by the kidney and are known to accumulate in the plasma with impaired renal function 11 . some workers have hypothesized that the symptoms of uremia itself can be modulated by use of oral activated charcoal to lower absorption of these microbiome products 12 . experimental data also support the concept that uremia potentiates sepsis and that oral activate charcoal can attenuate this 13 . on this background, the adipocyte is a known target for the virus 14 , and as people age there are statistically likely decreases in renal function and increases in visceral adipocity 15 . there are data suggesting that the virus can induce oxidative stress in adipocytes 16 and this oxidative stress can upregulate the expression of the ace-2 protein 17 , the putative receptor for covid-19. in short, the elderly likely have increases in the circulating concentrations of these potentially toxic substances as well as the adipocyte mass which responds to them. 16 administration of activated charcoal has been shown to be well tolerated when administered to a patients with renal dysfunction 18, 19 . this activated charcoal has also been shown to effectively decrease circulating levels of p-cresyl sulfate and indoxyl sulfate 13 . in addition to its ability to scavenge these microbiome derived toxins, activated charcoal may also have non-specific absorptive properties that blunt inflammatory responses to 20 or possibly inactivate viruses 21 . certainly, covid-19 infection may directly involve the gastrointestinal tract in both human and bat 22 . given that the potential toxicity of oral activated charcoal is so limited, we propose that an investigation of this coal-derived substance, widely available in the "mountain" state of wv, to potentially attenuate these adverse outcomes be explored as definitive work seeking effective antivirals and development of a vaccine continues. a schematic summarizing this hypothesis is shown in figure 1. to test this hypothesis, we would suggest first a proof of concept study where a relatively small group of patients at high risk for covid-19 complications are given activated charcoal at doses similar to that used in previous renal failure studies 18, 19 when the diagnosis is first made. cytokine levels, concentrations of indoxyl sulfate and p-cresyl sulfate along with evidence for systemic oxidant stress (e.g., protein carbonylation) and inflammation would be serially monitored. should preliminary outcomes be improved with this strategy, a randomized, prospective blinded study should be performed prior to large scale adaptation of this treatment strategy. coronavirus infection in acute lower respiratory tract disease of infants lifestyle risk factors, inflammatory mechanisms, and covid-19 hospitalization: a community-based cohort study of 387,109 adults in uk risk factors influencing the prognosis of elderly patients infected with covid-19: a clinical retrospective study in wuhan the covid-19 cytokine storm; what we know so far central role for adipocyte na,k-atpase oxidant amplification loop in the pathogenesis of experimental uremic cardiomyopathy the adipocyte na/k-atpase oxidant amplification loop is the central regulator of western diet-induced obesity and associated comorbidities oxidized hdl, adipokines, and endothelial dysfunction: a potential biomarker profile for cardiovascular risk in women with obesity pnaktide inhibits na/k-atpase reactive oxygen species amplification and attenuates adipogenesis uremic toxins activates na/k-atpase oxidant amplification loop causing phenotypic changes in adipocytes in in vitro models protein-bound uremic toxins, inflammation and oxidative stress: a cross-sectional study in stage 3-4 chronic kidney disease altered microbiome in chronic kidney disease: systemic effects of gut-derived uremic toxins oral activated charcoal adsorbent ameliorates chronic kidney disease-induced intestinal epithelial barrier disruption the role of adipocytes and adipocyte-like cells in the severity of covid-19 infections relationship between changes in body fat and a decline of renal function in the elderly enterovirus 71 3c promotes apoptosis through cleavage of pinx1, a telomere binding protein ace2 is expressed in mouse adipocytes and regulated by a high-fat diet relief of idiopathic generalized pruritus in dialysis patients treated with activated oral charcoal oral activated charcoal in patients with uremic pruritus use of activated charcoal for adsorption and elution of ribooligonucleotides investigating the effect of carbon shape on virus adsorption infection of bat and human intestinal organoids by sars-cov-2 this work was supported by national institutes of health grants hl109015, hl071556 and hl105649 (to jis), hl55601 and hl34300 (to nga), cobre accord grant (1p20gm121299) (us), and the brickstreet foundation and the huntington foundation, inc. (to jis). key: cord-253312-bf35afpj authors: unal, gokhan; turan, bahadir; hasan balcioglu, yasin title: immunopharmacological management of covid-19: potential therapeutic role of valproic acid date: 2020-05-27 journal: med hypotheses doi: 10.1016/j.mehy.2020.109891 sha: doc_id: 253312 cord_uid: bf35afpj nan to the editor: severe acute respiratory syndrome-coronavirus-2 (sars-cov-2) which leads to covid-19, was first seen in wuhan province of china in december 2019 and spread globally in a short time. covid-19 was announced as a pandemic by who on march 11th, 2020 and approximately 4 million people infected with the virus and 275.000 died until may 11th, 2020 [1] . symptoms of covid-19 may range from mild such as subfebrile fever to severe symptoms such as respiratory and multiorgan failures [2, 3] . acute respiratory distress syndrome (ards) is a critical consequence of infection, leading to the most of the death due to covid-19. a crucial part of covid-19 patients with ards presents an excess pro-inflammatory cytokine release, which may be related to cytokine storm syndrome in the respiratory system [4] . it has been shown that many patients with covid-19 had increased serum levels of proinflammatory cytokines such as il-6, il-1β, il-2, il-8, il-17, granulocyte-colony-stimulating factor (g-csf), granulocyte-macrophage csf, and tnf-α [5] . however, corticosteroids and other immunosuppressant agents have not been encouraged in covid-19 because of their given deteriorating effects to already vulnerable respiratory tract [6] . at this point, research into novel treatment approaches that focus on direct antiviral effect and immunomodulatory effects has increased to identify the ideal therapeutics for covid-19. we recently read with interest the letter by gómez-bernal entitled "lithium for the 2019 novel coronavirus" [7] . the author postulated that a mood-stabilizing agent, lithium, may be considered in the treatment of covid-19 as it harbors inhibitor functions on the expression of viral rna. another mood stabilizer, valproic acid is a wide spectrum anticonvulsant drug which is commonly used in the treatment of epilepsy and bipolar disorder [8] . despite the mechanism of its actions in different neuropsychiatric disorders is not fully understood, it has been demonstrated that valproic acid has enhancing effects on gabaergic neurotransmission and blockade of voltage-gated sodium channels. it has been indicated that the drug inhibits the production of pro-inflammatory cytokines such as nuclear factor-κb, tumor necrosis factor-alpha, and interleukin-6 in and block the migration of macrophage cells [9] . moreover, it triggers the apoptosis in cd8+ t lymphocytes via caspase-3 activation, but it does not alter the survival of cd8+ t lymphocytes against viral peptides. it has been suggested that histone deacetylase enzyme inhibitory activity of valproic acid plays a role in its immunomodulatory effects [10] . regarding the given immunomodulatory effects of valproic acid, it has been suggested that the drug might provide benefits for ards due to the exaggerated immune response related to covid-19. most of the clinicians have thought that specific antiviral treatment against covid-19 is the best choice to struggle with the current outbreak, however, unfortunately, preclinical and clinical research takes a long time to identify effective and safe novel antiviral therapeutical candidates. therefore, researchers have focused on remodeling or repurposing of current drugs, of which their pharmacological and toxicological properties have been already known and they are in use in clinics at present. it has been demonstrated that valproic acid and its metabolites have direct antiviral activity against several simplex virus-1 (hsv-1) [11] . an unpublished molecular repurposing study showed that valproic acid and especially its metabolite, valproic acid co-a, might be effective against covid-19 [12] . the authors suggested that valproic acid co-a has the potential to inhibit rna dependent rna polymerase (nsp12) of covid-19, which inhibits the replication of virus hosted in the human cells. this study consolidates the findings showing the antiviral activity of valproic acid and gives hope for its potential usage in the covid-19 pandemic. although covid-19 treatment guidelines vary between countries, it commonly consists of polypharmacy primarily based on direct antiviral therapy such as hydroxychloroquine and remdesivir. thus, drug interaction between valproic acid and current antiviral treatment would be important to consider if the potential usage of valproic acid will be at the clinician's plan. it has been shown that valproic acid might affect the cytochrome p450 enzymes, primarily inhibits cyp2c9 in patients. there is no known adverse interaction between valproic acid and hydroxychloroquine, lopinavir, ritonavir, and azithromycin [13] . besides, there is no data about remdesivir and valproic acid interaction. certain studies suggested lopinavir/ritonavir might decrease the blood level of valproic acid [14] . to the best of our knowledge, it does not seem that it will induce a serious drug interaction with current therapy. in conclusion, covid-19 showed a rapid world-wide invasion in a short time and characterized by a lethal ards. currently, it is unlikely to discover novel therapeutic agents to reduce mortality and morbidity related to covid-19 in the short term. in the current situation, in our opinion, it would be feasible to examine the potential usage of valproic acid, a drug that we have been using, and well known in the clinical practice for more than 60 years, against covid-19 pandemic and related ards. we suggest that demonstrating the antiviral effects against some viruses and the immunomodulatory effects of this drug would encourage clinicians for blind studies with valproic acid in further studies. world health organization. who covid-19 dashboard clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study covid-19: consider cytokine storm syndromes and immunosuppression covid-19: immunopathology and its implications for therapy clinical evidence does not support corticosteroid treatment for 2019-ncov lung injury lithium for the 2019 novel coronavirus valproate-associated isolated serum creatine kinase elevation sodium valproate inhibits production of tnf-α and il-6 and activation of nf-κb levetiracetam but not valproate inhibits function of cd8+ t lymphocytes inhibition of herpes virus infection in oligodendrocyte cultured cells by valproic acid virtual screening and molecular dynamics simulation suggest valproic acid co-a could bind to sars-cov2 rna depended repurposing valproate to prevent acute respiratory distress syndrome/acute lung injury in covid-19: a review of immunomodulatory action the authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. the authors received no financial support for the research, authorship, and/or publication of this article. the authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article key: cord-265322-3854ddb9 authors: vavougios, george d. title: a data-driven hypothesis on the epigenetic dysregulation of host metabolism by sars coronaviral infection: potential implications for the sars-cov-2 modus operandi date: 2020-04-23 journal: med hypotheses doi: 10.1016/j.mehy.2020.109759 sha: doc_id: 265322 cord_uid: 3854ddb9 covid-19, the disease caused by the novel sars-cov-2, a betacoronavirus structurally similar to sars-cov. based on both structural and syndromic similarities with sars-cov, a hypothesis is formed on sars-cov-2 potential to affect the host’s metabolism as part of its lifecycle. this hypothesis is evaluated by (a) exploratory analysis of sars-cov / human transcriptomic interaction data and gene set enrichment analysis (b) a confirmatory, focused review of the literature based on the findings by (a). a string viruses (available search for human – sars-cov (ncbi taxonomy id: 9606 vs. ncbi taxonomy id: 694009) genomic interactions reveals ten human proteins, interacting with sars-cov: sgta, fgl2, specc1, stat3, phb, bcl2l1, ppp1ca, cav1, jun, xpo1. gene set enrichment analyses (gsea) with string on this network revealed their role as a putative protein – protein interaction network (ppi; enrichment p-value=0.0296) mediating, viral parasitism, interleukin as well as insulin signaling, diabetes and triglyceride catabolism. in the literature, sars-cov has been known to cause de novo diabetes by ace2-dependent uptake on pancreatic isle cells, and furthermore dysregulate lipid autophagy in favor of the viral lifecycle. conversely, currently there are only non-causative, observational evidence of worse outcomes for covid-19 patients with comorbid diabetes or hyperglycemia. no study has reported on the lipid profiles of covid-19 patients; however, lipid-targeting molecules have been proposed as agents against sars-cov-2. future studies, reporting on lipid and glucose metabolism of covid-19 patients could help elucidate the disease’s seculae and aid drug design. . covid-19 is caused by the novel sars-cov-2, a betacoronavirus structurally similar (approximately 79%) to sars-cov (2) . in a recent study by hoffmann et al. (3) , the similarities between the sars viruses extend to ace2 dependent host cell entry. at present, sars-cov-2 has shown significant similarities with sars-cov, both in clinical characteristics and exploited host intracellular functions (5) . due to these commonalities, sars-cov remains an attractive substitute for the yet to be determined specifics of the sars-cov-2 / human protein interaction (6) and its consequences. among the first studies to report clinical data on covid19 was a recent publication by yang and colleagues (6) . their study provided the foundation for a hypothesis put forth by fang and colleagues indicating that diabetic and hypertensive patients exposed to ace2 inhibitors may be at an increased risk of more severe covid-19 (7) . meta-analyses on sars cohorts have indicated that both a history of diabetes and hyperglycemia were independent factors of worse outcomes including more severe respiratory symptoms and death, regardless of medication (9) . in another study, sars-cov was shown to cause diabetes by ace2-dependent infection of pancreatic isle cells (10) . interestingly, the significantly enriched "camp signaling" pathway is an indirect link between diabetes and ace2 signaling, based on experimental evidence associating camp levels and ace2 activity in diabetic patients (11) (false discovery rate (fdr) <0.05); table 1 . following entry to host cells, lipid metabolism is a subsequent important target of single strand rna viruses, critical for the formation of the viral envelope in subsequent lifecycles (12) . autophagy mediated triglyceride and lipid droplet catabolism is one such mechanism, as identified in denv infection (13) . hijacking the host cells' lipid metabolism has been shown to be a critical step in establishing hcov-22e and mers -coronavirus latency (14) . in sars-cov patients, alterations in lipid metabolism have been detected as far as 12 years after the initial infection (15) . evidence on covid-19's interplay with diabetes is only recently emerging, and can currently only be considered within the context of epidemiological studies, determining diabetes mellitus (dm) as frequent comorbidity. furthermore, dm has recently been characterized as a determinant of more severe respiratory syndrome, along with other comorbidities (16) . aside from dm, novel hyperglycemia was recently associated with an increased with worse outcomes in covid-19 patients, however this association was not independent of other predictors in the multivariate model (17) . epidemiological associations between covid-19 and lipid metabolism are currently not possible, since even large scale cohorts do not report on relevant measurements (18) . on the experimental level, lipid metabolism on the cellular level has been proposed as a treatment target for covid-19; specifically, both interactions between sars-cov's spike protein with lipid rich membrane compartments, as well as the epigenetic modulations in lipid metabolism were considered as the end-point targets for the development of small molecules, aiming to prevent sars-cov-2 infection (19) . barring actual proteomics sars-cov-2, sars-cov based in silico analyses of the sars-cov -human interaction partially support the hypothesis of fang and colleagues, insofar as to warrant further scrutiny on covid19 patient's metabolic states and concomitant medication. while the approach presented here is inherently limited due to setting its basis on the sars-cov proteomic interactions, it nevertheless presents in silico and literature evidence supporting sars-cov-2 potential to affect human metabolism. furthermore, as genes affected by sars-cov infection are significantly enriched for other infections, they may represent a common interface, targeted by viruses. future studies should determine sars-cov-2 interaction and effect on the human transcriptome, further identifying drug targets using pharmacogenomic enrichment analyses. the author confirms that there are no known conflicts of interest associated with this manuscript and there has been no financial support for this work that could have influenced its outcome. sars-cov-2 and covid-19: the most important research questions genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor differences and similarities between severe acute respiratory syndrome (sars)-coronavirus (cov) and sars-cov-2. would a rose by another name smell as sweet? receptor recognition by novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study are patients with hypertension and diabetes mellitus at increased risk for covid-19 infection string v11: protein-protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets plasma glucose levels and diabetes are independent predictors for mortality and morbidity in patients with sars binding of sars coronavirus to its receptor damages islets and causes acute diabetes activation of the ace2/angiotensin-(1-7)/mas receptor axis enhances the reparative function of dysfunctional diabetic endothelial progenitors host lipids in positive-strand rna virus genome replication dengue virus-induced autophagy regulates lipid metabolism modulation of lipid droplet metabolism-a potential target for therapeutic intervention in infections altered lipid metabolism in recovered sars patients twelve years after risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease epidemiological, clinical characteristics of cases of sars-cov-2 infection with abnormal imaging findings prevalence of comorbidities in the novel wuhan coronavirus (covid-19) infection: a systematic review and meta-analysis natural small molecules as inhibitors of coronavirus lipid-dependent attachment to host cells: a possible strategy for reducing sars-cov-2 infectivity? the author confirms that there are no known conflicts of interest associated with this manuscript and there has been no financial support for this work that could have influenced its outcome. george vavougios md, phd 20. 21. key: cord-281354-sa27k8o3 authors: takahashi, harutaka title: role of latent tuberculosis infections in reduced covid-19 mortality: evidence from an instrumental variable method analysis date: 2020-08-26 journal: med hypotheses doi: 10.1016/j.mehy.2020.110214 sha: doc_id: 281354 cord_uid: sa27k8o3 since the outbreak of the coronavirus disease 2019 (covid-19) pandemic, there has been significant interest in the potential protective effect of the bacillus calmette-guerin (bcg) vaccine against covid-19 mortality. this effect has been attributed to innate immune responses induced by bcg vaccination. however, these studies ignore an important fact: according to world health organization estimates, about a quarter of the world's population may have latent tuberculosis infection (ltbi), a condition in which there is no evidence of clinically active tuberculosis but persistent immune responses are stimulated by mycobacterium tuberculosis antigens. thus, both ltbi and bcg induce lifelong immunity and may provide immunological protection against covid-19. in this study, the relationship between ltbi and reduced covid-19 mortality was analyzed using the instrumental variable method. the results showed with robust statistical support that ltbi was also associated with reduced covid-19 mortality. since the outbreak of the coronavirus disease 2019 (covid-19) pandemic, significant attention has been focused on the relationship between bacillus calmette-guerin (bcg) vaccination and covid-19 mortality. in particular, there is interest in whether bcg vaccination is associated with a reduction in covid-19-associated mortality. bcg is the most widespread vaccine against tuberculosis (tb) and also elicits non-specific effects and innate immune memory against non-mycobacterial diseases. a survey of key unpublished and published data regarding the association between bcg vaccination and covid-19 mortality was conducted, and concluded that there was a lack of evidence to support a protective effect of bcg against covid-19 [1] . however, such studies ignore the important fact that about one-quarter of the world's population may have latent tb infection (ltbi), a condition in which there is no evidence of clinically active tb but persistent immune responses are stimulated by mycobacterium tuberculosis antigens. the regional data shown in table 1 illustrate that the number of ltbis far surpasses the number of active tb infections. the number of lbt infections clearly surpasses that of tb infections. ltbi also induces lifelong innate immune immunity [2] , [3] and may confer an immunological protective effect against covid-19. many countries with a relatively high incidence of tb infection, including japan, require bcg vaccination during early childhood. most citizens of these countries also have ltbi, which is highly immunoprotective because of elicited innate immune responses. in fact, tb infection leads to ltbi in 90%-95% of cases, while 5%-10% of individuals develop active tb disease [5] . therefore, the number of tb infections per hundred thousand individuals can be used as a proxy for the number of ltbis. furthermore, m. tuberculosis infection via bcg vaccination can enhance innate immunity. therefore, citizens of countries with high prevalence of tb infection (high tb burden countries) together with high bcg vaccination rates are considered to have enhanced innate immunity compared with the citizens of lower tb burden countries. this high level of natural immunity is thought to be responsible for the lower covid-19 mortality rate. the aim of this study was to test the hypothesis that ltbi is associated with reduced covid-19 mortality. the instrumental variable (iv) method was used to assess causality. all data used in the analysis are publicly available and are described in the appendix. much discussion has centered around the strong correlation between bcg and covid-19 mortality. however, correlation does not imply causation, and can sometimes instead reflect spurious relationships. regression analysis, particularly the iv method, is a statistical method that addresses this problem to assess causality. care must be taken in using covid-19 mortality as a dependent variable [6] . this is because covid-19 mortality is conditionally observed in potentially infected individuals, and can only be detected by testing of symptomatic or asymptomatic individuals. therefore, the case fatality rate (cfr), defined as the ratio of the number of covid-19 deaths per million people to the number of covid-19 infections per million people, is typically used. as explained above, the logarithm of the number of tb infections per 100,000 individuals (lntb10) can be used as a proxy variable for ltbis. for this regression analysis to be statistically accurate, the explanatory variable x must first be correlated with the error term u (i.e., the covariance of x and u must be zero). this condition clearly does not hold in general: besides ltbi, many other co-occurring factors, such as cultural norms, mitigation efforts, health infrastructure, and urban concentration, may influence this relationship [8] . therefore, it is possible that x is correlated with such factors excluded in the regression equation, and that x and the error term may be correlated. this would be an example of a "spurious regression". to overcome such a problem, the iv method can be used. an iv is a variable that is strongly correlated with the explanatory variable x but is not correlated or only weakly correlated with the error term. the ivs used here were as follows. four diagnostic tests were performed to assess whether the estimates were statistically relevant. one test was concerned with the explanatory variables and the other three were concerned with the ivs. the wu-hausman test assesses the endogeneity of the explanatory variables. if the null hypothesis is rejected, one can simply use the standard ordinary least squares regression instead of using the iv. the first test of an instrument is sargan's exogenous test, which assesses whether the right number of ivs are selected and confirms that they are sufficiently uncorrelated with the error term. finally, it is necessary to perform a "weak iv test" to check if the selected ivs are strongly correlated with the explanatory variables. the instrumental variables used here were bcgindex, region and pop65. two models were estimated using the iv method: one with three instruments and the other with two instrument (bcgindex and region). most of the countries with low income levels (annual per capita income less than $825 usd) reported zero deaths attributed to covid-19 [7] . to avoid underreporting bias in these countries, they were excluded. the total number of countries analyzed was thus 104. the results are shown in table 2 . the estimates of the generalized moment method (gmm), which is often used as an alternative to the iv method, are also reported. for the diagnostic tests of the two estimation models, sargan's exogenous test indicated that the selected ivs met the exogenous property. the results of the weak iv test indicated that the ivs were sufficiently and strongly correlated with the explanatory variables. therefore, all the estimation results presented here were statistically robust. all the coefficients of lntb10 were approximately -0.02, indicating a negative association between ltbi and covid-19 mortality. thus, these results lend statistical support to the hypothesis that ltbi can protect against covid-19 mortality. because these estimation models were linear-log type, the estimated coefficient of does bcg vaccination protect against acute respiratory infections and covid-19? a rapid review of current evidence tb prevalence correlation to covid-19 mortality trained immunity from mycobacterium spp. exposure or bcg vaccination and covid-19 outcomes is mycobacterium tuberculosis infection life long? the global burden of latent tuberculosis infection: a reestimation using mathematical modeling is there evidence that bcg vaccination has non-specific protective effects for covid-19 infections or is it an illusion created by lack of testing? doi correlation between universal bcg vaccination policy and reduced morbidity and mortality for covid-19: an epidemiological study bauer (2020) appearent difference in fatalities between central europe and east asia due to sars-cov-2 and covid-19: four hypotheses for possible explanation data appendix  number of covid-19 infections and deaths per million people as of  number of tb infections per 100 the bcg index variable was created to represent the total number of years since 1950 during which bcg vaccination was mandated in a country's immunization schedule. for example, japan made bcg vaccination mandatory in 1942 and continues to do so today, so japan's bcg index is 1. france introduced bcg vaccination in 1950 and discontinued it in  ratio of the population over 65 years old i would like to thank masayuki miyasaka at osaka university for helpful suggestions on the issue and edanz group (https://en-author-services.edanzgroup.com/ac) for editing a draft of this manuscript. this work received no specific funding. the author declares no conflict of interest. i would like to thank masayuki miyasaka at osaka university for helpful suggestions on the issue and edanz group (https://en-author-services.edanzgroup.com/ac) for editing a draft of this manuscript. this work received no specific funding. the author declares no conflict of interest. key: cord-279138-dmhphgp5 authors: deek, sarah a. title: chronic exposure to air pollution implications on covid-19 severity date: 2020-09-28 journal: med hypotheses doi: 10.1016/j.mehy.2020.110303 sha: doc_id: 279138 cord_uid: dmhphgp5 populations in areas with higher levels of air pollution both indoors and outdoors show increased mortality rates when infected with coronavirus disease 2019 (covid-19). the association between air quality and covid-19 is commonly attributed to the risk of transmission. although controlled transmission is crucial, further investigation into air quality traits that contribute to the lethality of covid-19 in infected persons enables risk stratification and optimization of the allocation of resources. there is a need for a valid basis for the proactive identification of indicators of covid-19 severity in air quality that allow for the implementation of systematic environmental improvements aimed at preventing covid-19 mortality. in this paper, fine particulate matter (pm) is identified as a source of disrupted activation of the hypothalamic–pituitary–adrenal (hpa) axis; therefore, a contributable variable to covid-19 mortality. populations in areas with higher levels of air pollution both indoors and outdoors show increased mortality rates when infected with coronavirus disease 2019 . the association between air quality and covid-19 is commonly attributed to the risk of transmission. although controlled transmission is crucial, further investigation into air quality traits that contribute to the lethality of covid-19 in infected persons enables risk stratification and optimization of the allocation of resources. there is a need for a valid basis for the proactive identification of indicators of covid-19 severity in air quality that allow for the implementation of systematic environmental improvements aimed at preventing covid-19 mortality. in this paper, fine particulate matter (pm) is identified as a source of disrupted activation of the hypothalamicpituitary-adrenal (hpa) axis; therefore, a contributable variable to covid-19 mortality. fine particulate matter (pm), coronavirus disease 2019 (covid-19), immune system, hypothalamic-pituitary-adrenal (hpa) axis combating the mortality rate of covid-19, in addition to hygiene measures, must include improving the host environment, which may prevent comorbidity in the immune response of infected hosts. in addition to host factors, the resilience of covid-19 emphasizes the importance of environmental conditions. despite increased interest in air quality associations with covid-19 transmission, many studies pay minimal attention to the impact of air composition on covid-19 severity. notably, populations in highly polluted areas, both indoor and outdoor, are associated with covid-19 pathology and increased likeliness of mortality [1][4] . typical immune response requires activation of the inflammatory pathways of the immune system [5] . however, if the immune system is disrupted, an abnormal response may elevate the impacts from viral infections [5] . the immune system has mechanisms in preparation for various pathogens. a crucial mechanism in the innate immune response regards cytokine production and its interplay with hpa system activation [6] . in this study, the imposition of such severity from covid-19 in highly air polluted environments may be attributable to the recorded effects particulate matter (pm) in air composition has on the hpa axis. this paper discusses the relationship between populations chronically exposed to fine pm and the intensity of covid-19 infection, and its link to the hpa axis interplay with the immune system. populations in environments with elevated fine particulate matter (pm) levels, both indoors and outdoors, are hypothesized to be at increased risk to covid-19 pathogenesis. firstly, we discuss the link between hpa axis disruption and covid-19 symptom severity. secondly, we discuss the chronic exposure to the fine pm role in disruption of the hpa axis. communication and feedback are codependent between the hpa axis and the immune system. the association between chronic immune activation and the pathogenesis of covid-19 has recently been recognized in patients with chronic inflammatory conditions [5] , [6] . the hpa axis modulates immune responses, and cytokines within the immune system such as il-1, il-6, il-10 and tnfα activate the hpa axis [7] [9] . this interplay is aimed to protect the body from immune system dysregulation and minimize tissue damage from associative systemic inflammation [7] , [8] , [10] , [11] . chronic activation of the hpa axis affects the release of glucocorticoids and contributes to immune dysfunction as immune response is signified by glucocorticoid release [7] , [12] [15] . due to the critical interplay of the hpa axis and the glucocorticoid response in maintaining a balance between the beneficial and harmful effects of proinflammatory cytokines and influencing immune responses, there are agreed upon associations between the cytokine-hpa axis are fundamental to the maintenance of the immune system. a disrupted activation of the hpa axis may result in immunosuppression or hyper activation of a particular immune response. the differences in these outcomes are dependent on individual differences among a variety of factors [6][8] . if infected with covid-19, populations with a disrupted hpa system activation and cytokines are at an increased risk for mortality. in people infected with covid-19 higher than standard levels of certain plasma cytokines were recorded. tan's report found that elevations in serum cortisol, taken 48 hours within hospital admission, were predictors of poor outcomes in covid-19 patients. a 42% increase in mortality is shown in patients in cortisol levels measured above 744 nmol/l after confounding variables were adjusted [16] . this suggests deregulation of serum cortisol, typically associated with disrupted hpa system activation which may be predictive of morbidity and mortality from covid-19. building upon the inference that chronic activation of the hpa axis disrupts immune response it is necessary to consider air pollution impacts on hpa baseline function. chronic exposure to fine particulate matter (aerodynamic diameter ≤ 2.5 μm; pm 2.5) is well observed in both disrupted hpa baseline function and acutely increased glucocorticoid secretion [17] [20] . in addition, fine pm exposure has been observed with overexpressed cytokines such as: tnfα, il-1 and il-6, crucial cytokines in the interaction of the hpa axis and immune response [5] , [20] , [21] . chronic exposure to fine pm has a recorded correlation to an increase in the likeliness of mortality from covid-19. wu et al. from harvard, observed populations in counties with higher levels of fine pm are 8% more likely to die from covid-19 than people who live in an area with lower levels of fine pm [3] . in addition, the number of confirmed cases of covid-19 and associated hospital admissions or fatalities are correlated in locations with higher levels of fine pm and pm10 [22] , [23] . hospital admissions imply covid symptoms are severe enough to require professional observation and formalized treatment. therefore, chronic exposure to fine pm may be contributable variable that disrupts hpa system activation typically associated with the altered regulation of circulating glucocorticoids resulting in inefficient or delayed immune response to covid-19 infection. clearly, the implications from fine pm exposure are observed in conduction with covid-19 mortality. in conclusion, chronic fine pm exposure may be attributed to the variation in morbidity and mortality observed in covid-19 infections due to disrupted immune response characterized by hpa system disruption. it is well known as a disrupted immune response is attributable to the severity of covid-19. chronic exposure to elevated fine pm is a contributable variable to the mortality from covid-19, particularly populations who have been chronically exposed to fine pm indoors and outdoors deemed to be at increased risk such as elderly populations, and populations with underlying this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. there are no conflicts of interest for the paper titled: chronic exposure to air pollution implications on covid-19 severity assessing the relationship between surface levels of pm2.5 and pm10 particulate matter impact on covid-19 in indoor air quality and severity of covid-19: where communicable and non-communicable preventive measures meet exposure to air pollution and covid-19 mortality in the united states: a nationwide crosssectional study indoor air pollution (iap) and pre-existing morbidities among under-5 children in india: are risk factors of coronavirus disease (covid-19)? is a 'cytokine storm' relevant to covid-19? covid-19, hypothalamo-pituitary-adrenal axis and clinical implications immune modulation of the hypothalamic-pituitary-adrenal (hpa) axis during viral infection the hpa axis and the immune system: a perspective autonomic nervous system and immune system interactions sympathetic activation: a potential link between comorbidities and covid-19 neuroimmune interaction in inflammatory diseases glucocorticoids : inflammation and immunity glucocorticoids : inflammation and immunity a possible change process of inflammatory cytokines in the prolonged chronic stress and its ultimate implications for health chronic mild stress modulates activity-dependent transcription of bdnf in rat hippocampal slices curcumin reverses the effects of chronic stress on behavior, the hpa axis, bdnf expression and phosphorylation of creb association between high serum total cortisol concentrations and mortality from covid-19 exposure to pm 2.5 activates hypothalamic-pituitary-adrenal axis in mouse models and humans fine particulate matter constituents and stress hormones in the hypothalamus-pituitary-adrenal axis air pollution, stress, and allostatic load: linking systemic and central nervous system impacts effects of fine particulate matter (pm2.5) on systemic oxidative stress and cardiac function in apoe exposure to fine particulate air pollution is associated with endothelial injury and systemic inflammation association of particulate matter pollution and case fatality rate of covid-19 in 49 chinese cities severe air pollution links to higher mortality in covid-19 patients: the 'double-hit' hypothesis this author of this paper is grateful for the support from kara kockelman karim karouta. key: cord-256092-bph9ys72 authors: hussain, aneela n.; hussain, fazal; hashmi, shahrukh k. title: role of testosterone in covid-19 patients a double-edged sword? date: 2020-09-17 journal: med hypotheses doi: 10.1016/j.mehy.2020.110287 sha: doc_id: 256092 cord_uid: bph9ys72 covid-19 affects males twice as frequently as females with significantly increased severity and mortality. current data suggest a direct correlation between the lower level of serum testosterone, inflammatory cytokines, disease severity, and poor clinical outcomes among male patients with covid-19. the gradual decline in total and free testosterone levels has a direct correlation with serious pulmonary complications requiring advanced care (icu, ventilators, ecmo, etc.). sars-cov-2 utilizes angiotensin-converting enzyme ii (ace2) for entry in the host cell, and transmembrane protease, serine 2 (tmprss2) to prime spike protein of sars-cov-2. testosterone induces ace-2 expression, a critical pulmonary protective enzyme. low testosterone levels in males have a direct correlation with the high probability of icu admission and the worse disease outcome (ards, duration of icu stay, mortality). on the contrary, however, high testosterone levels can lead to thrombosis which is also one of the fatal manifestations in covid-19 patients. a critical evaluation of the serum testosterone and its relevance to covid-19 is warranted to re-evaluate strategies to effectively triage, prioritize, and manage high-risk patients for icu admission, survival outcomes, targeted solutions, and operational algorithms. covid-19 affects males twice as frequently as females with significantly increased severity and mortality. current data suggest a direct correlation between the lower level of serum testosterone, inflammatory cytokines, disease severity, and poor clinical outcomes among male patients with covid-19. the gradual decline in total and free testosterone levels has a direct correlation with serious pulmonary complications requiring advanced care (icu, ventilators, ecmo, etc.). sars-cov-2 utilizes angiotensin-converting enzyme ii (ace2) for entry in the host cell, and transmembrane protease, serine 2 (tmprss2) to prime spike protein of sars-cov-2. testosterone induces ace-2 expression, a critical pulmonary protective enzyme. low testosterone levels in males have a direct correlation with the high probability of icu admission and the worse disease outcome (ards, duration of icu stay, mortality). on the contrary, however, high testosterone levels can lead to thrombosis which is also one of the fatal manifestations in covid-19 patients. a critical evaluation of the serum testosterone and its relevance to covid-19 is warranted to re-evaluate strategies to effectively triage, prioritize, and manage high-risk patients for icu admission, survival outcomes, targeted solutions, and operational algorithms. coronavirus disease 2019 (covid-19) affects men significantly more than women (1, 2) . male patients with covid-19 are reported to die at twice the rate of females when they contract the virus (3). lower levels of testosterone result in the upregulation of ace2 and tmprss2 receptors, facilitating sars-cov-1 entry into the alveolar cells, and deregulating a lung-protective pathway (4) . decreased testosterone levels in critically ill males negatively affect endothelial cell functioning, promote defective immune response, impair the ability to clear the virus, and promote systemic inflammation. obesity among males also generates more pro-inflammatory cytokines important in cell signaling, emanating in increased vulnerability, severe disease, and worst outcome. lower serum testosterone level is a poor prognostic indicator for patients with covid-19 by deregulating pulmonary protective pathways (5, 6) . thereby we hypothesize that low testosterone levels in males have a direct correlation with the severity of disease and a worse outcome in covid-19. covid-19 is caused by severe acute respiratory syndrome coronavirus-2 (sars-cov-2) and was declared a global pandemic by the world health organization (who) on march 11 th , 2020 (7, 8) . as of july 10, 2020, there have been more than 12,588,223 confirmed cases of covid-19 and 561,402 deaths, worldwide (9) . covid-19 presentation can range from mild to lifethreatening pneumonia, acute respiratory distress syndrome (ards), septic shock, multi-organ failure, and death (10) (11) (12) (13) (14) . males typically have 7-8 times higher levels of circulating testosterone compared to females (15) . the route of entry of covid-19 is typically through the mucosal membranes, where it enters the lung alveolar epithelial cells using angiotensin-converting enzyme ii (ace-2) receptors and tmprss2 for s protein priming (16) (17) (18) . ace-2 has a pivotal role in pulmonary protection and the lung-protective pathway is deregulated as a result of viral binding to these receptors. additionally, ace-2 is also expressed by the leydig cells of testis as a constitutive product (19) and affects the testosterone secretion in covid-19 patients. tmprss2 is a known target of androgen receptor, the ligand-activated transcription factor; activation of androgen receptor increases tmprss2 levels in various tissues. tmprss2 expression is significantly higher in the male lungs compared to females. testosterone modulates the immune response, and the low serum testosterone is known to impact both of these biological markers negatively (20, 21) . patients with low testosterone have reportedly developed severe manifestations requiring assisted ventilation because of the upregulation of ace-2 receptors in lower respiratory cells, increased risk of lung damage, and respiratory muscle catabolism. lower testosterone levels also inhibit endothelial cell functions as sars-cov-2 reduces ace-2 level by binding, increasing the angiotensin-ii (at-ii), and lowering angiotensin 1-7 (at 1-7) . this process increases superoxide species, leading to endothelial cell dysfunction because of oxidative stress and inflammation (22, 23) . this leads to increased vwf and development of thrombosis in the alveolar capillaries, a precursor of ards (24) (25) (26) . moreover, hypothyroidism also plays a role in lowering male testosterone levels and thyroiditis has reported to be a manifestation of the covid-19 (27) . male covid-19 patients are at increased risk for icu admission and worse outcomes compared to females. therapeutic modalities and vaccine development can be targeted to transcriptionally inhibit lung ace-2 and tmprss2 expression. down-regulation of tmprss2 will result in attenuated spike protein priming, reducing sars-cov-2 interaction with ace-2, and blocking viral entry. it could potentiate the anti-inflammatory effect of tocilizumab in covid-19 ards or cytokine release syndrome (crs). the role of testosterone screening, optimal pharmacotherapeutic administration of dhea, and hormone replacement therapy needs to be considered to minimize the pulmonary syndrome and severity of covid-19. however, exogenous administration of testosterone needs to be carefully monitored as it can exacerbate the benign prostatic hyperplasia (bph) and prostate cancer. moreover, there is already an increased risk of thrombosis in covid-19 patients therefore testosterone use should be contraindicated in covid-19 patients with known thrombosis (28, 29) . the optimal dose of testosterone replacement therapy (trt) needs to be determined to strike the fine balance in varied indications, especially covid-19. careful dose titration and monitoring can help alleviate undesirable side effects (eythrocytosis, thromboembolism, etc.) (30) . baseline hemoglobin and hematocrit levels and periodic monitoring for dose adjustment and risk assessment is pivotal for optimal outcome (31) . thyroid axis also must be corrected if testosterone replacement is sought. if trt is undertaken in covid-19 patients, at least monthly monitoring of hemoglobin should be undertaken given the heightened risk of thrombosis in this population due to erythrocytosis. further longitudinal studies are required to determine the effect of gender and low testosterone levels on the cellular and molecular pathways associated with covid-19 by facilitating a personalized medical approach to risk stratification, prevention, and treatment. gender differences in patients with covid-19: focus on severity and mortality. front public health sex-and gender-specific observations and implications for covid-19 men die of covid-19 at twice the rate of women in england ace-2 expression in the small airway epithelia of smokers and copd patients: implications for covid-19 screening for low testosterone is needed for early identification and treatment of men at high risk of mortality from covid-19 hypogonadism in aged hospitalized male patients: prevalence and clinical outcome who declares covid-19 a pandemic world health organization. coronavirus disease (covid-19 risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease 2019 pneumonia in wuhan, china dysregulation of immune response in patients with covid-19 in wuhan, china. clin infect dis clinical characteristics of coronavirus disease 2019 in china early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72 314 cases from the chinese center for disease control and prevention serum testosterone in women as measured by an automated immunoassay and a ria a pneumonia outbreak associated with a new coronavirus of probable bat origin sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor virtual screening of natural products against type priming agent of coronavirus 2 (sars-cov-2). molecules the novel angiotensin-converting enzyme (ace) homolog, ace2, is selectively expressed by adult leydig cells of the testis worse progression of covid-19 in men: is testosterone a key factor? sex hormones and novel corona virus infectious disease (covid-19) correlation between testosterone and the inflammatory marker soluble interleukin-6 receptor in older men the effect of testosterone on cytokine production in the specific and non-specific immune response pulmonary vascular endothelialitis, thrombosis, and angiogenesis in covid-19 endotheliopathy in covid-19-associated coagulopathy: evidence from a single-centre, cross-sectional study von willebrand factor and endothelial damage: a possible association with covid-19 // ecological genetics subacute thyroiditis after sars-cov-2 infection high risk of thrombosis in patients with severe sars-cov-2 infection: a multicenter prospective cohort study incidence of thrombotic complications in critically ill icu patients with covid-19 erythrocytosis following testosterone therapy the optimal indication for testosterone replacement therapy in late onset hypogonadism key: cord-275569-i5y23mmz authors: de bernardis, e.; busà, l. title: a putative role for the tobacco mosaic virus in smokers’ resistance to covid-19 date: 2020-07-31 journal: med hypotheses doi: 10.1016/j.mehy.2020.110153 sha: doc_id: 275569 cord_uid: i5y23mmz reports from various countries suggest that tobacco smoking might protect from sars-cov-2 infection, since the prevalence of smoking in covid-19 hospitalized patients is lower than in the respective general population. apart from nicotine or other chemicals contained in tobacco smoke, we propose that a single-stranded rna virus that infects tobacco leaves, tobacco mosaic virus (tmv), might be implicated in this effect. tmv, though non-pathogenic, is found in smokers’ airways, and stimulates adaptive and innate immunity, with release of specific antibodies and interferons. the latter may have preventive and/or therapeutic effects against covid-19. if confirmed by epidemiological and interventional studies, this might lead to the use of tmv as an immunological adjuvant against sars-cov-2 infection and covid-19 disease. no funding or other support. 2 reports from various countries suggest that tobacco smoking might protect from sars-cov-2 infection, since the prevalence of smoking in covid-19 hospitalized patients is lower than in the respective general population. apart from nicotine or other chemicals contained in tobacco smoke, we propose that a single-stranded rna virus that infects tobacco leaves, tobacco mosaic virus (tmv), might be implicated in this effect. tmv, though non-pathogenic, is found in smokers' airways, and stimulates adaptive and innate immunity, with release of specific antibodies and interferons. the latter may have preventive and/or therapeutic effects against covid-19. if confirmed by epidemiological and interventional studies, this might lead to the use of tmv as an immunological adjuvant against sars-cov-2 infection and covid-19 disease. covid-19, a new severe acute respiratory syndrome (sars) emerging in late 2019, and due to a new coronavirus (sars-cov-2), has caused a global pandemic, which so far counts more than 13 million cases and more than 570000 deaths worldwide (european centre for disease prevention and control, https://www.ecdc.europa.eu/en/geographical-distribution-2019-ncov-cases, last visit july 14th, 2020). though it is intuitively tempting, on the basis of physiopathological common knowledge, to predict a greater risk of contracting the sars-cov-2 infection in tobacco smokers, an analysis of studies from various countries shows that hospitalized covid-19 patients have a lower, and apparently inversely proportional, rate of current tobacco smoking, in comparison with the respective general population, although once the disease has developed meta-analyses suggest that smoking is associated with a worse prognosis [1] . hence, it has been suggested that tobacco smoking might confer some protection against of the sars-cov-2 infection, at least in its initial phases. the search of a cause for this puzzling finding started from nicotine, the most important pharmacological agent in tobacco smoke, a psychotropic, addictive alkaloid with an antiinflammatory activity and an influence on the biosynthesis of angiotensin conversion enzyme 2 (ace2), the receptor for sars-cov-2 cell adhesion [2] . so far, though, no data are available on the effects of pure nicotine on covid-19. 4 here we suggest that the resistance of tobacco smokers to the sars-cov-2 infection might be immunologically mediated by the chronic exposure to a common tobacco-dwelling virus, the tobacco mosaic virus (tmv). tmv is a single-stranded rna virus that infects several plants of the family of solanaceae, including the tobacco plant, and was the first virus to be discovered, towards the end of the 19th century [3] . though tmv is known to be not pathogenic to humans, it has been found in sputum [4] and saliva specimens from cigarette smokers, as well as in cigarettes, in the form of viable virions, while being absent in non-smokers [5] . in vitro experiments on human epithelial carcinoma hela cells show that after tmv transfection some viral proteins are found in the endoplasmic reticulum, and cleared by autophagy, a defense reaction which actives toll-like receptor 7 and initiates innate antiviral responses [6] . in-vivo experiments show that anti-tmv antibodies are produced both by mice after intratracheal inoculation [7] and by humans after exposure to tobacco products [8] . exogenous rna, including single-stranded rna from non-replicating viral particles, has been shown to induce the production of interferons [9] . accordingly, the oral administration of tmv was found to stimulate the release of endogenous interferon in rhesus monkeys, mice and humans, and exert a marked protective effect in mice against various experimental viral infections [10] . it is conceivable, then, that the oral use of cigarettes, cigars and other derivatives of tobacco leaves, continuously challenges the airways with a inflow of tmv virions, which may colonize the 5 area without replicating and without inducing an overt disorder. the presence of tmv virions and related rna, though, may cause a protracted immune alert, inducing the production of interferons and maybe other cytokines, which will be already induced when the exposition to sars-cov-2 takes place. current knowledge about covid-19 innate immune sensing indicates that the early and properly localized presence of interferon type i can effectively limit coronavirus infections, and initial evidences show that sars-cov-2 is sensitive to interferon type i and type iii pretreatment in vitro; the timing of interferon secretion, though, is critical, because it appears to be protective if early, while on the other hand aggravates the disease if dysregulated, lacking in the early phase and contributing to a cytokines storm later [11] . incidentally, this behavior reminds the proposed effects of tobacco smoking, protective against initial sars-cov-2 infection and deleterious in the florid phase of the covid-19 disease. accordingly, it has been suggested that endogenous or medicinal interferons in the initial phase of the sars-cov-2 infection may have a therapeutic role in preventing or treating covid-19 [12] , and some clinical trials are under way, with interesting results [13] . taken together, all these elements suggest that the oral use of tobacco, continuously exposing to non-pathogenic but immunogenic tmv particles, and chronically stimulating a natural antiviral response, may induce a state of resistance to the initial sars-cov-2 infection. this in turn could be a plausible explanation for the putative protective effect of tobacco smoking observed so far. similarly to the studies on the putative protective effect of smoking, as a first step it could be possible to assess the prevalence of tmv in airways and related immune parameters in covid-19 6 patients vs. the general population, and the findings could be used to infer the effects of tmv on the probability to get infected. this could be completed by searching for a correlation, in selected territories, between the reduction of the rate of smokers among hospitalized covid-19 patients, and the average content of tmv in the most used cigarettes and tobacco derivatives; theoretically, a higher presence of tmv in tobacco products should determine a larger protective effect in smokers against the sars-cov-2 infection. at a greater level of complexity it could be possible to prospectively follow a cohort of tmvpositive individuals in comparison with a comparable tmv-negative cohort, surveying the incidence of sars-cov-2 infection and the development of the covid-19 disease. as a third step, it could be possible to investigate the effects of an interventional tmv exposition on sars-cov-2 infection and development of covid-19 in appropriate animal or human experimental models. tmv has been administered to humans without overt signs of toxicity or disease [10] and like other plant viruses is deemed harmless for mammals, and fit to be used as an immunopotentianting agent [14] . this could lead to the use of tmv virions or tmv-rich plant materials as a cheap, promptly available immune adjuvant to reduce the burden of disease due to sars-cov-2 and covid-19. conflict of interest statement acireale, july 14th, 2020 we hereby declare that there exist no financial or personal relationship with other people or organisations that could inappropriately influence this work. no funding was needed. no sponsor were involved. recent exposure to smoking and covid-19 editorial: nicotine and sars-cov-2: covid-19 may be a disease of the nicotinic cholinergic system the discovery of the causal agent of the tobacco mosaic virus disease in discoveries in plant biology recovery of culturable tobacco mosaic virus from sputum and thoracentesis fluids obtained from cigarette smokers with a history of pulmonary disease tobacco mosaic virus in cigarettes and saliva of smokers the invasion of tobacco mosaic virus rna induces endoplasmic reticulum stress-related autophagy in hela cells tobacco mosaic virus in the lungs of mice following intratracheal inoculation humans have antibodies against a plant virus: evidence from tobacco mosaic virus induction of interferon by nonreplicating single-stranded rna virus comparison of interferon-inducing activities and antiviral effects of tobacco mosaic virus, tilorone and sodium nucleinate immunology of covid-19: current state of the science type i and type iii interferons -induction, signaling, evasion, and application to combat covid-19 an experimental trial of recombinant human interferon alpha nasal drops to prevent coronavirus disease 2019 in medical staff in an epidemic area plant virus particles with various shapes as potential adjuvants key: cord-276715-d1nh2dvb authors: raha, syamal; mallick, rahul; basak, sanjay; duttaroy, asim k. title: is copper beneficial for covid-19 patients? date: 2020-05-05 journal: med hypotheses doi: 10.1016/j.mehy.2020.109814 sha: doc_id: 276715 cord_uid: d1nh2dvb copper (cu) is an essential micronutrient for both pathogens and the hosts during viral infection. cu is involved in the functions of critical immune cells such as t helper cells, b cells, neutrophils natural killer (nk) cells, and macrophages. these blood cells are involved in the killing of infectious microbes, in cell-mediated immunity and the production of specific antibodies against the pathogens. cu-deficient humans show an exceptional susceptibility to infections due to the decreased number and function of these blood cells. besides, cu can kill several infectious viruses such as bronchitis virus, poliovirus, human immunodeficiency virus type 1(hiv-1), other enveloped or nonenveloped, singleor double-stranded dna and rna viruses. moreover, cu has the potent capacity of contact killing of several viruses, including sars‐cov‐2. since the current outbreak of the covid-19 continues to develop, and there is no vaccine or drugs are currently available, the critical option is now to make the immune system competent to fight against the sars‐cov‐2. based on available data, we hypothesize that enrichment of plasma copper levels will boost both the innate and adaptive immunity in people. moreover, owing to its potent antiviral activities, cu may also act as a preventive and therapeutic regime against covid-19. copper (cu) is an essential trace element for humans [1] . dietary cu is absorbed in the small intestine and is rapidly appeared in the circulation. in blood, cu is distributed into a plasma pool associated with larger proteins, an exchangeable fraction of low molecular weight copper complexes, and a red cell pool that is partly nonexchangeable. cu plays an important role in the function and maintenance of the human immune system. cu is involved in the functions of t helper cells, b cells, neutrophils, natural killer cells and macrophages. these cells are involved in the killing of infectious microbes, cell-mediated immunity and production of specific antibodies. cu deficiency symptoms in human include deficiencies in white blood cells, bone and connective tissue abnormalities, and immune reactions [2] . adverse effects of insufficient cu on immune function appear most pronounced in infants and older people. infants with genetic disorders that result in severe cu deficiency suffer from frequent and severe infections [2, 3] . during infection, macrophages can attack invading microbes with high cu load. cu is also elevated at sites of lung infection during infection with a wide array of pathogens [4] . cu deficiency and its excess levels can result in abnormal cellular function or damages that given its central role in host-pathogen interaction. profiling, proteomic analysis, and metabolite profiling, in both data-driven and targeted formats, promise to provide more mechanistic details in animal models that can be tested in human pathology. cu also normalized impaired immunological functions by modulating neutrophil activity, blastogenic response to t helper cell mitogens, the balance between th1 and th2 cells [14] . cu has the potent capacity to neutralize infectious viruses such as bronchitis virus, poliovirus, human immunodeficiency virus type 1(hiv-1), and other enveloped or nonenveloped single-or double-stranded dna and rna viruses [15] . cu can disrupt the lytic cycle of the coccolithovirus, ehv86 with the increase in production of ros [15] . cu 2+ ions can inactivate five enveloped or nonenveloped, single-or double-stranded dna or rna viruses. the virucidal effect of this cu is enhanced by the addition of peroxide as the mixtures of cu 2+ ions and peroxide are more efficient than glutaraldehyde in activating junin and herpes simplex viruses [15] . copper exposure to human coronavirus 229e destroyed the viral genomes and irreversibly affected virus morphology, including disintegration of envelope and dispersal of surface spikes [16] . cupric (ii) chloride dihydrate showed the inhibitory effect on the replication of dengue virus, denv-2 in a cell culture study [17] . cu-chelating agent (atn(ohsv) indicating the importance of cu 2+ ions in this process. thujaplicin-cu chelates inhibit influenza virus-induced apoptosis of mdck cells and also inhibit the virus replication and release from the infected cells [18] . cu 2+ ions inactivate herpes simplex virus by oxidatively damaging its genome [15] . cu surfaces can significantly reduce the number of infectious influenza a virus particles. cu ions can damage the viral genomic dna by binding and cross-linking between and within strands of the genome [19] . replication of influenza a virus was inhibited by cu by damaging the negative-sense rna genome [19] . the contact killing of microbes by cu is mediated by the degradation of genomic and plasmid dna of microbes [20] . human coronavirus was rapidly inactivated on a range of cu alloys cu/zn brasses were very effective at lower cu concentration that cu (i) and cu(ii) moieties were responsible for the inactivation which was enhanced by ros generation on alloy surfaces [21] . novel coronavirus (sars-cov-2), responsible for current covid-19 pandemic is very sensitive to the copper surface [22] . in a cell-based study, cu 2+ was shown to block papain-like protease-2, a protein that sars-cov-1 requires for replication [23, 24] . oxidized cu oxide (cuo) nanoparticles (cuonps) are widely used as catalysts so that the ability of cuonps to reduce virus application is enhanced [25] . nanosized cu(i) iodide particles also show inactivation activity against h1n1 influenza virus. gold/cu sulfide core-shell nanoparticles (au/cus nps) exhibit variable virucidal efficacy against human norovirus (hunov) via inactivation of viral capsid protein [25] . the current outbreak of the novel coronavirus sars-cov-2 (coronavirus disease 2019, , infected around the world. there are nearly 1.9 million confirmed cases of coronavirus in 185 countries, and at least 120,000 people have died, as of april 14, 2020. coronaviruses are enveloped, positive single-stranded large rna viruses that infect humans, but also a wide range of animals. coronaviruses were first described in 1966 by tyrell and bynoe, who cultivated the viruses from patients with common colds [26] . at present, no vaccines exist that protect people against infections by sars-cov-2, which causes covid[29] ; however, it is not known whether they had lowered cu levels too. several studies have shown that lower total cholesterol level may be related in part due to lower cu level in adults [21, 30, 31] . disruption of lipid rafts by cholesterol depletion caused an enhancement of virus particles released from infected cells and a decrease in the infectivity of virus particles [32] . plasma cu may affect all these above processes. cu oxide nanoparticles and cu 2+ ions are involved in the inhibition of viral entry and replication, and degradation of mrna and capsid proteins that are involved in the viral life cycle. cu deficiency is not always about a lack of cu but also could be the result of an imbalance of cu and other minerals in the diet that may often occur in an older population. in older people, cu deficiency can also result from malnutrition, malabsorption, or excessive zinc intake and can be acquired or inherited [28] . copper deficiency could lead a decreased number of circulatory blood cells with a greater susceptibility towards infection in older people in a study of 11 men on a low-cu diet (0.66 mg cu/day for 24 days and 0.38 mg/day for another 40 days) showed a decreased proliferation response of their white blood cells when presented with an immune challenge in cell culture [33] . recent mechanistic studies support a role for cu in the innate immune response against infections [34] . in the condition of specific intestinal malabsorption (such as celiac disease, bowel syndrome, long-term parenteral nutrition) or bone abnormalities or in well genetically determined disease (menkes' disease), cu deficiency is severe with dysfunctions on immune response, antioxidant activity and bone metabolism [35] . altered plasma and tissue levels of cu in acute or chronic inflammation reflect the changes in the metabolism of cu [36, 37] . we hypothesize that copper supplementation can help fight is low copper status immunosuppressive? the yin and yang of copper during infection virus inactivation by copper or iron ions alone and in the presence of peroxide imidazole derivative effectively inhibits replication of denv-2 in vero thujaplicin-copper chelates inhibit replication of human influenza viruses inactivation of influenza a virus on copper versus stainless steel surfaces metallic copper as an antimicrobial surface persistence of coronaviruses on inanimate surfaces and their inactivation with biocidal agents aerosol and surface stability of sars-cov-2 as compared with sars-cov-1 the sars-coronavirus papainlike protease: structure, function and inhibition by designed antiviral compounds papainlike protease 2 (plp2) from severe acute respiratory syndrome coronavirus (sars-cov): expression, purification, characterization, and inhibition antiviral activities of cu2+ ions in viral prevention, replication, rna degradation, and for antiviral efficacies of lytic virus, ros-mediated virus copper deficiency, a new triad: anemia, leucopenia, and myeloneuropathy copper deficiency in humans intechopen.) pp plasma membrane rafts play a critical role in hiv-1 assembly and release effects of low-copper diets on human immune response copper homeostasis at the host-pathogen interface copper deficiency in humans copper and its complexes in medicine: a biochemical approach alterations in lipid profile, zinc and copper levels and superoxide dismutase activities in normal pregnancy and preeclampsia an exposure-response curve for copper excess and deficiency indices of copper status in humans consuming a typical american diet containing either fructose or starch we hereby declare that any of the data contained in the above-mentioned manuscript have not been submitted for publication or under consideration to be published. all the listed authors have seen and approved the manuscript as submitted now key: cord-254094-ed1epul1 authors: mayoral, eduardo pérez-campos; hernández-huerta, maría teresa; pérez-campos mayoral, laura; matias-cervantes, carlos alberto; mayoral-andrade, gabriel; barrios, luis ángel laguna; pérez-campos, eduardo title: factors related to asymptomatic or severe covid-19 infection date: 2020-09-24 journal: med hypotheses doi: 10.1016/j.mehy.2020.110296 sha: doc_id: 254094 cord_uid: ed1epul1 the factors that may contribute to a covid-19 patient remaining in the asymptomatic stage, or to the infection evolving into the more serious stages are examined. in particular, we refer to the tmprss2 expression profile, balance of androgen and estrogen, blood group-a and/or b, nonsynonymous mutations in orf3, and proteins ns7b and ns8 in sars-cov-2. also, we review other factors related to the susceptibility and pathogenicity of sars-cov-2. severe acute respiratory syndrome coronavirus-2 (sars cov-2) has been spreading around the world. as of september 11, 2020, 28,205 ,308 infected subjects and 910,157 deaths have been reported worldwide [1] . thus, researchers are looking for multiple factors that develop efficient antiviral activity in healthy subjects, resulting in asymptomatic infection. also, attention must be drawn to the asymptomatic presence of covid-19 in children, adults and even the elderly. unlike severe pneumonia with hypercoagulopathy and microvascular immunothrombosis [2] , covid-19 is more frequent in older subjects and those with comorbidities, however, it is also presented in young people with and without risk factors [3] . in the first months of the covid-19 pandemic, most authors focused their attention on features such as the high expression of ace2 in the salivary glands in asymptomatic infection [4] , and the maturity and binding capacity of ace2 [5, 6] . nevertheless, there is a possibility that the presumed asymptomatic stage may depend on the virulence of sars-cov-2 and the susceptibility of the subject. susceptibility may be related, in part, to the nasopharynx, salivary glands and other tissues. other factors may also be involved, such as the ace2 gene polymorphisms, which cause variations in the affinity, binding and processing of the sars-cov-2 spike protein [7] , and lower levels of ace-2 and its posterior angiotensin ii up-regulation [8] . moreover, the tmprss2 variation can influence susceptibility [9] because both are expressed in the salivary glands [10] . other genes involved in the different responses between the sexes to sars-cov-2 are sry, sox9 and the tmprss2 gene [11, 12] . based on the balance of androgen and estrogen, a low prenatal testosterone/high prenatal estrogen level is indicated by a high mean 2d:4d. this is expressed in females in the index finger (2d), which is generally equal to or longer than the ring finger (4d), while in males, the 2d is usually shorter than the 4d [13] . a higher 2d:4d ratio is associated with covid-19 severity in men [14] , this means that sex hormones play a role in protection, thus, causing women to develop less serious complications or an asymptomatic covid-19 infection [12] . following sars-cov-2 translation and rna replication, a complex group of glycans is expressed and added to new viruses [15] . these glycans are formed in cells that co-express ace2 [16] . among these new virus glycans, the abo (h), blood group-a and/or b-specific mucin-types [17] may play an important role, i.e., if the subject is blood group "o" and has anti-a and anti-b antibodies, these antibodies may block the attachment and entry of the virus, similar to sars-cov spike protein [18] . this could mean that individuals with blood group o would have a much lower risk of becoming infected, depending on the type of anti-αgal, anti-a or, anti-b antibodies, as reported in an earlier study [19] . although there are still no complete studies related to histo-blood group antigens and susceptibility of low or non-secreting fucosyltransferase 2 salivary status, fucosyltransferase 2 is known to be related to viral infections or complications [20] . alleles of the major histocompatibility complex (mhc) class i may cause vulnerability to a more severe infection, such as hla-b*46:01 and, subsequently, to covid-19, although, hla-b*15:03 may present a better response of t lymphocytes [21] . furthermore, a mineralocorticoid receptor that controls blood pressure may explain cardiac injury in severe cases of covid-19, due to an aberrant cd8+ t cell activation [22] . a case report based on viral kinetics monitoring, shows that clinical evolution could depend on the viral load in the nasopharynx, despite its limitations due to the number of cases studied [23, 24] . on the other hand, the 15-30-bp deletions in the s1/s2 cleavage site region attenuate the ability to cause severe lung disease, as seen in the hamster model [25] . nonsynonymous mutations in orf3a could be related to the pathogenicity of sars-cov-2 [26] . in addition, the mutation of an aspartate (d) at position 614 in the d614g viral spike has a significant correlation with case fatality rates [27] . thus, the deletion of the accessory proteins in ns7b and ns8 could be related to the virus infectivity [28] . other important factors considered useful in keeping a subject at the asymptomatic stage are vitamin d levels in addition to 'essential' amino acids (i, l, k, m, f, t, w, v, h) [29] , zinc, and vitamin e status [30] . the world faces a new disease, named covid-19 [31] . it begins with a lung infection, which we now know is a significant basis in endothelial inflammation and micro thrombosis [32] . it affects numerous systems and organs such as the cardiovascular, central and peripheral nervous, gastrointestinal, reproductive, and vascular, as well as the haematological, renal, and skin [33] . the elderly are known to have a higher death rate from covid-19, moreover, more than 30% of infected subjects have comorbidity, men having a 1.5 times greater probability of dying [34] . the ace2 receptor and the tmprss2 protease facilitate entry of sars-cov-2 are highly expressed in the nasal goblet and ciliated cells [35] . the coexpression of these receptors in these cells suggests that they could be the sites of the original infection and possible reservoirs of dissemination [35] . the coexpression of both cells in specific tissues may explain different phenotypes such as gastrointestinal [36] , neurological [37] , cutaneous [38] and ocular [39] , among others. mechanistically, it is possible that the interaction of factors related to susceptibility or pathogenicity makes a subject asymptomatic or not. an in-depth study of the factors associated with asymptomatic subjects can provide information to limit severe covid-19 as much as possible. the evidence reported to date is shown in table 1 . global initiative on sharing all influenza data -from vision to reality hyperinflammation and derangement of reninangiotensin-aldosterone system in covid-19: a novel hypothesis for clinically suspected hypercoagulopathy and microvascular immunothrombosis predictive factors of severe coronavirus disease 2019 in previously healthy young adults: a single-center, retrospective study asymptomatic infection a systematic review of asymptomatic infections with covid-19 interactions of coronaviruses with ace2, angiotensin ii, and ras inhibitors-lessons from available evidence and insights into covid-19 sanchis-gomar f. do genetic polymorphisms in angiotensin converting enzyme 2 (ace2) gene play a role in coronavirus disease 2019 (covid-19)? the dilemma of coronavirus disease 2019, aging, and cardiovascular disease: insights from cardiovascular aging science first comprehensive computational analysis of functional consequences of tmprss2 snps in susceptibility to sars-cov-2 among different populations systematic analysis of ace2 and tmprss2 expression in salivary glands reveals underlying transmission mechanism caused by sars-cov-2 severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection is likely to be androgen mediated sex-related differences developmental basis of sexually dimorphic digit ratios understanding covid-19: digit ratio (2d:4d) and sex differences in national case fatality rates site-specific glycan analysis of the sars-cov-2 spike harnessing the natural anti-glycan immune response to limit the transmission of enveloped viruses such as sars-cov-2 how blood group a might be a risk and blood group o be protected from sars-cov-2 (covid-19) infections (how the virus invades the human body via abo(h) blood group carbohydrates) inhibition of the interaction between the sars-cov spike protein and its cellular receptor by anti-histo-blood group antibodies relationship between the abo blood group and the covid-19 susceptibility fucosyltransferase 2 non-secretor and low secretor status predicts severe outcomes in premature infants human leukocyte antigen susceptibility map for severe acute respiratory syndrome coronavirus 2 is aberrant cd8+ t cell activation by hypertension associated with cardiac injury in severe cases of covid-19? clinical and virological data of the first cases of covid-19 in europe: a case series understanding covid-19: what does viral rna load really mean? attenuated sars-cov-2 variants with deletions at the s1/s2 junction sars-cov-2 and orf3a: nonsynonymous mutations, functional domains, and viral pathogenesis sars-cov-2 viral spike g614 mutation exhibits higher case fatality rate nonstructural proteins ns7b and ns8 are likely to be phylogenetically associated with evolution of 2019-ncov amino acid catabolism: a pivotal regulator of innate and adaptive immunity nutritional modulation of age-related changes in the immune system and risk of infection covid-19 and the creation of a new disease the emerging threat of (micro)thrombosis in covid-19 and its therapeutic implications extrapulmonary manifestations of covid-19: radiologic and clinical overview immune-epidemiological parameters of the novel coronavirus -a perspective sars-cov-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes microthrombosis associated with gastrointestinal bleeding in covid-19 neurological associations of covid-19 classification of the cutaneous manifestations of covid-19: a rapid prospective nationwide consensus study in spain with 375 cases ocular manifestations and clinical characteristics of 535 cases of covid-19 in wuhan, china: a cross-sectional study ace2 and tmprss2 variants and expression as candidates to sex and country differences in covid-19 severity in italy relationship between abo blood group distribution and clinical characteristics in patients with covid-19 25-hydroxyvitamin d concentrations are lower in patients with positive pcr for sars-cov-2 the authors thank charlotte grundy and eli cruz parada for their assistance. we also thank the national technology of mexico (tecnm) project 8703.20-p. the authors declare that we have no conflicts of interest. key: cord-265724-fdt00qw1 authors: varadarajan, saranya; madapusi balaji, thodur; sarode, sachin c.; sarode, gargi s.; sharma, nilesh k.; gondivkar, shailesh; gadbail, amol; patil, shankargouda title: emmprin/basigin as a biological modulator of oral cancer and covid-19 interaction: novel propositions date: 2020-07-09 journal: med hypotheses doi: 10.1016/j.mehy.2020.110089 sha: doc_id: 265724 cord_uid: fdt00qw1 extracellular matrix metalloproteinase inducer (emmprin), which is also called basigin/cd147, is a cell surface glycoprotein that belongs to the immunoglobulin superfamily and plays a significant role in intercellular recognition in immunology, cellular differentiation and development. apart from ace-2, recently emmprin, has been regarded as a target for the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) attachment and entry into the host cell. since one of the routes of entry for the virus is the oral cavity, it becomes imperative to percept oral comorbidities such oral squamous cell carcinoma (oscc) and oral potentially malignant disorders (opmds) in terms of emmprin as a target for sars-cov-2. in the present paper, it is proposed that oscc, by the virtue of upregulation of emmprin expression, increases the susceptibility to coronavirus disease (covid-19). in turn, covid-19 in oscc patients causes exhaustion of emmprin receptor due to binding with ‘s’ receptor leading to a downregulation of related carcinogenesis events. we proposed that in the ace-2 depleted situation in oscc, emmprin receptor might get high jacked by the covid-19 virus for the entry into the host cells. apart from the anti-monoclonal antibody, it is recommended to explore the use of grape seed and skin containing mouthwash as an adjunct, which could also have anti emmprin effects in patients with oscc and opmds. coronavirus disease (covid-19) pandemic has created a significant global health impact and affected population in developing and developed nations of the world causing significant morbidity and mortality. 1 angiotensin-converting enzyme 2 (ace-2) on the host cells is the attachment protein for the spike receptor present on severe acute respiratory syndrome coronavirus 2 (sars-cov-2). 2 intriguingly, ace-2 expression has been reported at various sites in the oral cavity and is regarded as one of the potential modes of entry for the virus and its infectivity. 3 moreover, differential expression of ace-2 expression in various pathologies prompt researcher to draw many speculative conclusion in pathologies such as oral squamous cell carcinoma (oscc), oral submucous fibrosis (osmf), periodontitis, etc. [4] [5] [6] apart from ace-2, recently extracellular matrix metalloproteinase inducer (emmprin), which is also called basigin/cd147, has been regarded as a target for sars-cov-2 attachment and its entry into the host cell. 7, 8 emmprin is a cell surface glycoprotein that belongs to the immunoglobulin superfamily and plays a significant role in intercellular recognition, which is an important event in immunology, cellular differentiation and development. 9 a research study has demonstrated that meplazumab, an anti-cd147 humanized antibody, was found to prevent the sars-cov-2 invasion into the host cell. 7 an affinity constant of 1.85×10 −7 m was reported on the validation of emmprin and spike ('s') protein interaction. the binding of both the proteins was established by co-immunoprecipitation and elisa technique. immunoelectron-microscopic studies also confirmed the co-localization of emmprin and 's' protein in infected vero e6 cell lines thereby confirming the significance of emmprin as a potential covid-19 receptor. 7 since one of the routes of entry for sars-cov-2 is the oral cavity, it becomes imperative to percept oral comorbidities such as oscc and opmds in terms of emmprin expression as a target for sars-cov-2. in the present paper, efforts have been made to propose a hypothesis based on emmprin role in oral carcinogenesis and covid-19 along with possible ramifications of the complex interaction. oscc, by the virtue of upregulation of emmprin expression (potential and alternative site for 's' receptor), increases the susceptibility to sars-cov-2 infection. in turn, covid-19 in oscc patients causes exhaustion of emmprin receptor leading to downregulation of related carcinogenesis pathways. emmprin being a member of the immunoglobulin superfamily has a diversified role in maintaining tissue homeostasis, growth and development and hence is known to express on a variety of tissues. 9 it is highly expressed in a variety of malignant neoplasms and is involved in many carcinogenesis related events that lead to initiation and progression of malignancy. 10 a meta-analysis published in literature found a significant association between emmprin overexpression and adverse tumor outcomes, such as overall survival, disease-specific survival, progression-free survival, metastasis-free survival or recurrence-free survival, irrespective of the model analysis. in addition, cd147/emmprin overexpression predicted a high risk for chemotherapy drugs resistance. 11 many matrix metalloproteinases molecules such as mmp-1, mmp-3, mmp-9 and membranetype 1-mmp are activated by emmprin thus promoting tumor cell proliferation, invasions and migration. 12 emmprin also upregulates angiogenesis in the tumor microenvironment by virtue of its potential to stimulate vascular endothelial growth factors in tumor and stromal cells. 13 metabolic reprogramming in tumor cells is the hallmark of carcinogenesis need for survival. in this regard, emmprin regulates expression and activity of monocarboxylate transporters-1 (mct-1) and mct-4, and form complexes on the membrane to transport lactic acid produced by anaerobic glycolysis. 14 emmprin has been found to facilitate the activation of phosphatidylinositol 3-hydroxy kinase and mitogen-activated protein kinase pathways, which are important events in carcinogenesis and mainly responsible for chemoresistance in tumor cells. 11 moreover, emmprin interact with α3β1, α6β1 integrins to regulate adhesion with extracellular matrix proteins, collagen, laminin or fibronectin and also promote expression of cyclophilin a to induce cancer cell proliferation. 15 it is noteworthy that the upregulation of emmprin is reported in oral carcinogenesis. 16, 17 increased expression of emmprin was reported in oral premalignant cells and primary and metastatic cell lines of oscc. 16 the prevalence of opmds varies between populations and a recent meta-analysis showed a prevalence of 4.47% (95% ci = 2.43-7.08). 19 among all the opmds, osmf was the most prevalent one (4.96%; 95% ci = 2.28-8.62) followed by leukoplakia (4.11%; 95% ci = 1.98-6.97). 19 with increasing consumption of tobacco and betel nut, the prevalence rate of opmds is likely to increase in future. hence, it is expected that opmd patients might encounter covid-19 with a higher propensity based on the factors previously elucidated. since emmprin was detected as high and low glycosylated forms in opmds, it could have a serious impact on the spread of covid-19. 9 however, in contrast, osmf might cause exhaustion of ace2 receptor by underlying fibrosis and thus might decrease the chances of covid-19 infection. 5 it will be interesting to investigate the expression of emmprin in osmf patients as a marker of an alternative pathway for covid-19 infectivity. to better understand the outcome analysis, it is recommended to take cognizance of all opmds in the form of detailed history in covid-19 positive patients. since both ace-2 and emmprin are potential targets for covid-19's 's' receptor, it is mandatory to consider both the proteins together in any future molecular research. besides in vitro and in vivo studies, in silico approach to understand at the molecular level, crystal structure of the receptor-binding domain of the spike protein of sars-cov-2 in complex with cell receptor ace2 is reported. 20 emmprin abrogation may serve as a potential strategy for the blockade of sars-cov-2. besides the monoclonal antibody approach, peptide mimetics as metalloproteinase inhibitors, pharmacological inhibitors, and natural compounds from plants, small rna interference, and crispr-cas9 based system with precision within the oral epithelial tissue may be speculated. future epidemiological studies are warranted with those patients under the medical for opmd and severity of covid-19 infections. in any case, still a speculation that sars-cov-2 will find a more convenient way to infect normal human epithelial tissue or perturbed epithelial tissues including downregulation of emmprin or other marker proteins in these tissues. a strong possibility arises that if sars-cov-2 is natural, then it will prefer normal epithelial tissues. therefore, any deviations in the molecular landscape of epithelial cells including emmprin will be an obstruction to the entry of sars-cov-2. emmprin directed antibody called meplazumab has been suggested as a treatment modality for covid 19 management. 7 we hypothesize the alternative use of grape seed and skin containing mouthwash as an adjunct, which could also have anti emmprin effect in patients with oscc and opmds. it has been documented that grape seed and grape skin contain resveratrol, which could inhibit emmprin expression in macrophages and monocytes via the inhibition of iκb protein phosphorylation and thus indirectly suppresses the nf-κb pathway. 21 this resveratrol mediated emmprin inhibition could be achieved by the use of mouthwashes containing the grape seed and grape skin extracts in patients with oscc and opmds which could reduce the sars-cov-2 entry and its further infection. current status of epidemiology, diagnosis, therapeutics, and vaccines for novel coronavirus disease physiological and pathological regulation of ace2, the sars-cov-2 receptor high expression of ace2 receptor of 2019-ncov on the epithelial cells of oral mucosa oral cancer and periodontal disease increase the risk of covid 19? a mechanism mediated through furin and cathepsin overexpression oral submucous fibrosis and covid-19: perspective on comorbidity perspective on oral exfoliative cytology and covid-19 sars-cov-2 invades host cells via a novel route: cd147-spike protein structural basis for the recognition of sars-cov-2 by full-length human ace2 emmprin (basigin/cd147): matrix metalloproteinase modulator and multifunctional cell recognition molecule that plays a critical role in cancer progression cd147/emmprin overexpression and prognosis in cancer: a systematic review and meta-analysis prognostic indications of elevated mct4 and cd147 across cancer types: a meta-analysis rna interference targeting cd147 inhibits the proliferation, invasiveness, and metastatic activity of thyroid carcinoma cells by downregulating glycolysis reprogramming energy metabolism and inducing angiogenesis: co-expression of monocarboxylate transporters with vegf family members in cervical adenocarcinomas cd147 required for corneal endothelial lactate transport association of increased ligand cyclophilin a and receptor cd147 with hypoxia, angiogenesis, metastasis and prognosis of tongue squamous cell carcinoma increased emmprin (cd 147) expression during oral carcinogenesis emmprin expression in oral squamous cell carcinomas: correlation with tumor proliferation and patient survival biological behavior of oral squamous cell carcinoma in the background of novel corona virus infection prevalence of oral potentially malignant disorders: a systematic review and meta-analysis structure of the sars-cov-2 spike receptor-binding domain bound to the ace2 receptor resveratrol inhibits macrophage expression of emmprin by activating ppargamma figure caption: figure 1: proposed role of emmprin/basigin as a biological modulator of oral cancer and sars-cov-2 interaction key: cord-254411-e9vvjv8w authors: taghizadeh-hesary, farzad; akbari, hassan title: the powerful immune system against powerful covid-19: a hypothesis date: 2020-04-22 journal: med hypotheses doi: 10.1016/j.mehy.2020.109762 sha: doc_id: 254411 cord_uid: e9vvjv8w on march 11, 2020, the world health organization declared the coronavirus outbreak a pandemic. since december 2019, the world has experienced an outbreak of coronavirus disease 2019 (covid-19). epidemiology, risk factors, and clinical characteristics of patients with covid-19 have been reported but the factors affecting the immune system against covid-19 have not been well described. in this article, we provide a novel hypothesis to describe how an increase in cellular adenosine triphosphate (c-atp) can potentially improve the efficiency of innate and adaptive immune systems to either prevent and fight off covid-19. today, the rapid outbreak of corona virus disease 2019 (covid-19 or sars-cov-2) is the leading health issue. there is a paucity of studies investigating the factors affecting immune response to covid-19. in addition, there has been no detailed report for this immune response. given the genomic similarity of 79% with severe acute respiratory syndrome coronavirus (sars-cov), nearly the same reaction to the immune system is expected for covid-19 (1) . in response to sars-cov, both innate and adaptive immune systems are involved. sars-cov applies several mechanisms to overcome the immune response. first, it inhibits the rapid expression of interferon type 1 (ifn-1) (2). ifn-1 is known as the "initial alarm" upon encounter with the virus that modulates the immune cells to the so-called "antiviral state". moreover, sars-cov interferes with ifn-1 signaling through inhibition of stat-1 phosphorylation (3). the third defensive mechanism of sars-cov is immune exhaustion through exaggerated and prolonged ifn-1 production by plasmacytoid dendritic cells (pdcs). this process leads to the influx of activated neutrophils and inflammatory monocytes/macrophages, that in turn, results in lung immunopathology (e.g. acute respiratory distress syndrome) (4). finally, the resulted so-called "cytokine storm" further weakens the immune system through ifn-1-mediated t cell apoptosis (5) . in this article, we aim to provide a new hypothesis to describe how the repletion of cellular adenosine triphosphate (c-atp) can promote immunity against covid-19. thereafter, we justify the current knowledge regarding the characteristics of covid-19 infection by our hypothesis and give several approaches to improve the c-atp. considering the pivotal role of atp in cellular function, c-atp depletion can lead to cellular dysfunction (6) . immune cells are not an exception. in this article, c-atp is the index of cellular energy. here, we show how c-atp repletion can counteract with defensive mechanisms of covid-19 and promote the immune system to the enhancement pathway. 3.1. atp facilitates ifn production covid-19 interferes with a rapid rise in ifn-1. therefore, it deactivates the so-called "initial alarm" of the innate immune system, by unknown mechanisms. this facilitates its replication. zhang et al. have demonstrated that enhancement in the c-atp can reverse this process. this occurs by the facilitation of ifn secretion through p38/jnk/atf-2 signaling pathway (7) . therefore, atp-depleted cells are more susceptible to this effect of covid-19. following ifn-1 secretion, fundamental changes occur in the immune cells that transform them into the so-called "antiviral state". one of the signaling pathways that take part in this process is the jak/stat pathway. jaks are atp-dependent enzymes that are bound to the cytoplasmic regions of cytokine receptors. following attachment of ifn-1 to the cytokine receptor, jak activates the stat through transphosphorylation (8) . obviously, c-atp depletion interferes with this process and further impairs transformation to "antiviral state". following deactivation of "initial alarm", covid-19 easily proliferates in-situ. among the passive hostcells, there are exceptions that can react to the covid-19, the pdcs. they detect the virus by toll-like receptor 7 (tlr-7). upon attachment to viral nucleic acids, tlr7 induces profound ifn-1 expression. this response recruits other immune cells and causes massive local inflammation (9) . at first glance, this robust immune response is beneficial for the elimination of covid-19. however, two factors prevent it. first, impairment of ifn-1 signaling results in impairment of immune cell transformation to the "antiviral state". therefore, they are not so effective in eliminating existing viruses (8) . second, persistence profound inflammatory responses may lead to immune exhaustion (4) . the depletion of c-atp can potentially enhance these detrimental processes in the following ways. in 2016, rebbapragada et al. demonstrated the effect of atp in the function of tlr7 by controlling the endo-lysosomal ph. they showed that atp-depletion can increase the endo-lysosomal ph and improve the efficacy of tlr7. therefore, atp-depletion can potentially enhance profound ifn-1 secretion. secondly, atp-depletion can potentially prone the recruited immune cells to earlier exhaustion against covid-19. therefore, one may conclude that atp-repletion can prevent the so-called "cytokine storm" and improve the cellular energy to better counteract with covid-19. channappanavar et al. demonstrated that covid-19 can promote t-cells to ifn-induced apoptosis, resulting in reduced numbers of virus-specific cd8 and cd4 t-cells (5) . from the perspective of cellular energy, this process potentially occurs through ifn-mediated t-cell activation that results in c-atp depletion. in line with this hypothesis, perl et al. have shown that following ifn-γ stimulation, mitochondrial hyperpolarization and atp depletion occurs in t-cells that results in apoptosis (10) . therefore, atp-repletion can potentially prevent t-cell apoptosis following "cytokine storm". in the following section, we use our hypothesis to demonstrate why specific groups of people are more susceptible to be infected with covid-19 and why they have a worse prognosis. the case-fatality rate of covid-19 is the highest (14.8%) in elderly-population. in contrast, children have the lowest risk for both infection and mortality rates (11) . this difference can be demonstrated from the cellular energy concept. aging may potentially attenuate the respiratory capacity of mitochondria. this condition may be either due to impairment of peroxisome proliferator-activated receptor-gamma coactivator-1α (pgc-1α) or age-related accumulation of mitochondrial dna mutations (12) . moreover, aging can wane the ability of immune cells to secrete ifn following viral infection (13) . as noted earlier, this may be due to atp-depletion. therefore, one can conclude that a gradual decline in prognosis with age may rely on a gradual decrease in c-atp. the risk of long-lasting and serious covid-19 infection is more among tobacco smokers. apart from a direct effect on lung parenchyma and a decrease in pulmonary capacity, tobacco smoke can potentially induce immune dysfunction through a decrease in atp content of immune cells. this can be due to nicotine-induced mitochondrial dysfunction (14) . the resultant atp-depletion increase the risk of immune dysregulation by covid-19 (refer to the aforementioned defensive mechanisms of covid-19). while men and women have the same susceptibility to covid-19, men are more prone to higher morbidity and mortality independent of age (15) . this difference can be justified by the cell energy hypothesis. estrogens (as the main sex steroid of females) are potent stabilizers of atp production during oxidative stress (e.g. during covid-19-induced inflammation) (16) . therefore, it seems that women are more capable to maintain the c-atp of their immune cells during the immune response to covid-19. with this notion in mind, men are more susceptible to immune dysregulation following covid-19 infection. recent reports have highlighted some chronic illnesses that increase the mortality of covid-19. they include underlying conditions such as hypertension, diabetes, coronary heart disease, chronic obstructive lung disease, cancer, and chronic kidney disease (17) . apart from a decline in cardiovascular reserve, the effect of these chronic conditions on the prognosis of covid-19 can be justified by our hypothesis. human cells need nutrients (including glucose, free fatty acids, essential amino acids, and o 2 ) to maintain their c-atp level. the aforementioned illnesses impede the regular distribution of the nutrients secondary to compromising the function and structure of small and large vessels. therefore, the human cells (including in-situ immune cells) confront atp-depletion and results in further immune dysregulation (as mentioned above). in light of these considerations, the c-atp level can potentially be considered as a crucial component in the infectivity and prognosis of covid-19. with enhancing the c-atp, improvement in both innate and cytotoxic immune systems is expected. moreover, an increase in c-atp can potentially have either preventive and therapeutic effects. the preventive effect through activation of initial ifn-1 secretion and signaling, as "initial alarm" of the innate immune system. the therapeutic effect through the prevention of "cytokine storm" and t-cell apoptosis. there are several approaches to improve c-atp. most of them are easily available through a change in lifestyle. first, regular exercise improves mitochondrial respiratory capacity through an increase in pgc-1α (18) . smoking cessation is the second approach to improve mitochondrial capacity and improvement in c-atp (as mentioned above). consuming foods with low specific dynamic action (sda), as the energetic budget for consuming food, can potentially boost the immune system through improving the c-atp. in 2016, luoma et al. demonstrated the effect of low-sda meals in the up-regulation of the innate immune system in corn snakes (19) . on the other hand, several studies have reported the positive effect of xanthine oxidoreductase inhibitors on c-atp (6). this hypothesis provides a new concept to improve the immune system against covid-19. it demonstrates how an increase in c-atp can decrease the effect of covid-19 on immune dysregulation. considering the strategies to enhance cellular atp, improvement of the immune system against covid-19 is possible. it is hoped that this hypothesis will serve as a stimulus for further investigation into this issue. genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding sars-cov and ifn: too little, too late sars and mers: recent insights into emerging coronaviruses immune responses in covid-19 and potential vaccines: lessons learned from sars and mers epidemic dysregulated type i interferon and inflammatory monocyte-macrophage responses cause lethal pneumonia in sars-cov-infected mice shortage of cellular atp as a cause of diseases and strategies to enhance atp virus-triggered atp release limits viral replication through facilitating ifn-β production in a p2x7-dependent manner the role of jak-stat signaling pathway and its regulators in the fate of t helper cells control of coronavirus infection through plasmacytoid dendritic-cell-derived type i interferon mitochondrial hyperpolarization: a checkpoint of t-cell life, death and autoimmunity characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72 314 cases from the chinese center for disease control and prevention is there a link between mitochondrial reserve respiratory capacity and aging impaired interferon signaling in dendritic cells from older donors infected in vitro with west nile virus mitochondria as a possible target for nicotine action gender differences in patients with covid-19: focus on severity and mortality. medrxiv estrogen receptor signaling and its relationship to cytokines in systemic lupus erythematosus clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study. the lancet endurance training induces muscle-specific changes in mitochondrial function in skinned muscle fibers plasticity of immunity in response to eating the authors have no conflicts of interest. not applicable. key: cord-274715-dcs1rgd0 authors: mani mishra, pushpendra; uversky, vladimir n.; nandi, chayan k. title: serum albumin-mediated strategy for the effective targeting of sars-cov-2 date: 2020-04-24 journal: med hypotheses doi: 10.1016/j.mehy.2020.109790 sha: doc_id: 274715 cord_uid: dcs1rgd0 novel coronavirus (ncov-19), also known as sars cov-2, is a pathogen causing an emerging infection that rapidly increases in incidence and geographic range, is associated with the ever-increasing morbidity and mortality rates, and shows sever economic impact worldwide. the who declares the ncov-19 infection disease (covid-19) a public health emergency of international concern on 30 january 2020 and subsequently, on march 11, 2020, declared it a global pandemic. although some people infected with sars cov-2 have no symptoms, the spectrum of symptomatic infection ranges from mild to critical, with most covid-19 infections being not severe. the common mild symptoms include body aches, dry cough, fatigue, low-grade fever, nasal congestion, and sore throat. more severe covid-19 symptoms are typical of pneumonia, and upon progression, the patient’s condition can worsen with severe respiratory and cardiac problems. currently, there is no drug or vaccine for curing patients. it has been observed that people with challenged immunity are highly prone to sars cov-2 infection and least likely to recover. also, older adults and people of any age with serious underlying medical conditions might be at higher risk for severe forms of covid-19. we are suggesting here a strategy for the covid-19 treatment that could be effective in curing the patients in the current scenario when no efficient medicine or vaccine is currently available, and clinicians solely depend upon the performing trials with drugs with known antiviral activities. our proposed strategy is based on the compilation of published scientific research and concepts. the different published research indicates the success of a similar strategy in different physiological conditions, and such a strategy is widely studied at the cellular level and in animal models. novel coronavirus , also known as sars cov-2, is a pathogen causing an emerging infection that rapidly increases in incidence and geographic range, is associated with the ever-increasing morbidity and mortality rates, and shows sever economic impact worldwide. the who declares the ncov-19 infection disease (covid19) a public health emergency of international concern on 30 january 2020 and subsequently, on march 11, 2020, declared it a global pandemic. although some people infected with sars cov-2 have no symptoms, the spectrum of symptomatic infection ranges from mild to critical, with most covid-19 infections being not severe. the common mild symptoms include body aches, dry cough, fatigue, low-grade fever, nasal congestion, and sore throat. more severe covid-19 symptoms are typical of pneumonia, and upon progression, the patient's condition can worsen with severe respiratory and cardiac problems. currently, there is no drug or vaccine for curing patients. it has been observed that people with challenged immunity are highly prone to sars cov-2 infection and least likely to recover. also, older adults and people of any age with serious underlying medical conditions might be at higher risk for severe forms of covid-19. we are suggesting here a strategy for the covid-19 treatment that could be effective in curing the patients in the current scenario when no efficient medicine or vaccine is currently available, and clinicians solely depend upon the performing trials with drugs with known antiviral activities. our proposed strategy is based on the compilation of published scientific research and concepts. the different published research indicates the success of a similar strategy in different physiological conditions, and such a strategy is widely studied at the cellular level and in animal models. ncov-19 or sars cov-2 first emerges in the wuhan city of hubei province of china in december 2019, and since then, this virus is rapidly spreading to the different parts of the world and causing the fatalities at a devastating rate particularly in the italy, spain, and united state [1, 2] . the virus is given name coronavirus due to the similarity of its spike protein to the crown (latin corona) [3] . sars cov-2 virus belongs to group iv coronaviruses and is the member of the order nidoviriales, family coronaviridae, subfamily coronavirinae, and genus betacoronavirus [4] . the virus genome is a~30 kb positivesense single-stranded rna that encodes structural, non-structural and accessory proteins. the virion is enveloped and spherical with a diameter of 60-140 nm and with the spike length of 9-12 nm. the phylogenetic study suggested a closer relation of this virus to bat-sl-covzxc21 and bat-covzc45 than to human sars [5] . the early cases reported infection in people after attending the seafood and animal market, thereby suggesting the animal to human transmission of the virus. the later cases indicated the sustained human to human transmission of sars cov-2 [6, 7] . human to human transmission occurs through respiratory droplets and fomites. the primary site of infection is in epithelial cells of respiratory or enteric tracts while secondary sites are kidney and heart though in question [8] . currently, there is no cure for the covid-19, and clinicians are solely depending upon the drugs that could provide symptomatic relief. the clinical trials are performed using drugs that have been known to treat other viral infections/related diseases. our purpose in writing this hypothesis article is to shed some light on the well-known facts and highlight the strategy that can be used for covid-19 treatment. the drugs or therapeutic materials are likely to be https://doi.org/10.1016/j.mehy.2020.109790 received 9 april 2020; accepted 23 april 2020 effective in the covid-19 treatment if they either block the entry of the viral particles to cells or block the intracellular viral components [9] . the latter property plays one of the important roles in halting the propagation of viruses from infected cells while blocking the virus particles at the extracellular site inhibits the cell infection. many factors define the effectiveness of the drug molecules, including their sustainability in the environment and cell penetration capacity. to efficiently treat the sars-cov-2 infection, we are proposing the strategy of simultaneous targeting the viral particle extracellularly and intracellularly. to ensure the effective internalization of the potential drugs with demonstrated activity against the viral protease, polymerase, and rna, serum albumin could be used as an excellent delivery vehicle. serum albumin is a multifunctional protein known to interact with a range of exogenous and endogenous compounds. it is predominately present in the extracellular space, with high concentration being reported in the skin, guts, muscles, fluids (cerebrospinal, pleural), and secretions such as sweat, tear, milk, and saliva [10] . the wellknown fact about albumin is that there is a complex relationship between the inflammation and albumin level in the extracellular matrix under various physiological and pathological conditions [11] [12] [13] . the earlier studies indicated that the stressed and inflamed cells increase the uptake of albumin [11] [12] [13] . we are suggesting the use of combinatorial drugs therapy, in which one drug inhibits the fusion and entry of the virus into the cells and another drug internalizes and targets multiple viral components inside the cell and/or cell signaling to halt the viral propagation (see fig. 1 ). table 1 shows the list of drugs that have been shown to improve the state of the sars-cov-2 infected patients [8, 14] . the drug combination has been chosen in such a way that it can effectively neutralize both extracellular and intracellular viral components. furthermore, the serum albumin should be used to maximize the cellular internalization and enhance the pharmacological effects. a similar strategy based on the proposed concept was utilized in the influenza virus-infected mouse liver cells, where it yielded a positive outcome [15] . a recent search for the treatment of sars-cov-2 infection in pubmed revealed the usefulness of traditional medicine in treating the covid-19 in the current pandemic [16] . for example, research indicated the high potential of the epigallocatechin gallate (egcg) and curcumin in neutralizing the range of viruses [17] [18] [19] [20] [21] [22] . the recent studies support the albumin therapy of covid-19 patients since there is the complex relation of albumin concentration and ace2 receptor expression in the cells, ace2 receptors are crucial in mediating the virus infection [23] . if the albumin is used to stabilize and deliver the egcg and curcumin for targeting the intracellular virus components in combination with the drug that could block the virus fusion and/or entry to a cell, this strategy might represent an effective way of treating the sars cov-2 infection. egcg and curcumin combination is proven to be very effective in treating various pathological conditions by augmenting the key cellular signaling [24] [25] [26] . the inhibition of these key cellular pathways also helps in inhibiting the virus multiplication inside the cells as reported in the case of sars-cov [27] [28] [29] . in addition to their antiviral outcomes, curcumin and egcg are known for their health-boosting properties [30, 31] . in light of presented facts complemented by the referenced publications, it is highly recommended to use albumin as a therapeutic material, stabilizer, and deliverer of the drugs (such as known antiviral drugs and traditional molecules) that could effectively target the extracellular and intracellular viral components in the therapy of patients infected with sars-cov-2. none declared. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. decrease tlr signaling thus reduce activation of dendritic cells and inflammatory processes 2 interferon alfacon-1 induce the synthesis of key antiviral mediators and activate cytotoxic t cells 3 lopinavir inhibit the activity of an enzyme critical for the hiv lifecycle 4 remdesivir interfere with the activity of rna polymerase thus could be effective in treating the variety of rn viruses. 5 ritonavir protease inhibitor activity against hiv-1 6 sofosbuvir inhibit the hepatitis c ns5b protein thus used in the treatment of hepatitis c infection 7 umifenovir used in the prophylaxis and treatment of respiratory virus infection 8 alfa-interferon broad-spectrum of antiviral and immune-regulatory activity with the subtypes acting synergistically to give a wide range of response 9 corticosteroids class of steroid hormone use in the treatment of various physiological and pathological conditions 10 ribavirin interfere in the rna metabolism, such as blocking the viral rna synthesis and mrna capping require for viral replication phase-adjusted estimation of the number of coronavirus disease covid-19) map n genomic study points to natural origin of covid-19 -nih director's blog n severe acute respiratory syndrome coronavirus 2 isolate wuhan-hu-1, co -nucleotide -ncbi n virus pathogen database and analysis resource (vipr) -coronaviridae -genome database with visualization and analysis tools n mystery deepens over animal source of coronavirus a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster therapeutic strategies in an outbreak scenario to treat the novel coronavirus originating in wuhan unraveling the mysteries of serum albumin-more than just a serum protein nutrient scavenging in cancer serum albumin is associated with higher inflammation and carotid atherosclerosis in treated human immunodeficiency virus infection clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study covid-19 drug therapy-potential options therapeutic impact of human serum albumin-thioredoxin fusion protein on influenza virus-induced lung injury mice updated approaches against sars-cov-2 epigallocatechin gallate inhibits hepatitis b virus infection in human liver chimeric mice antiviral effect of epigallocatechin gallate via impairing porcine circovirus type 2 attachment to host cell receptor curcumin inhibits influenza virus infection and haemagglutination activity curcumin inhibits zika and chikungunya virus infection by inhibiting cell binding antiviral potential of curcumin inhibition of enveloped viruses infectivity by curcumin the potential use of albumin in covid-19 patients | the bmj n curcumin and epigallocatechin gallate inhibit the cancer stem cell phenotype via down-regulation of stat3-nfκb signaling combination curcumin and (-)-epigallocatechin-3-gallate inhibits colorectal carcinoma microenvironment-induced angiogenesis by jak/stat3/il-8 pathway combination of low concentration of (-)-epigallocatechin gallate (egcg) and curcumin strongly suppresses the growth of non-small cell lung cancer in vitro and in vivo through causing cell cycle arrest targeting coronaviral replication and cellular jak2 mediated dominant nf-κb activation for comprehensive and ultimate inhibition of coronaviral activity severe acute respiratory syndrome coronavirus orf3a protein activates the nlrp3 inflammasome by promoting traf3-dependent ubiquitination of asc inhibition of nf-b-mediated inflammation in severe acute respiratory syndrome coronavirus-infected mice increases survival curcumin: a review of its' effects on human health n green tea extracts epigallocatechin-3-gallate for different treatments the authors acknowledge the funding support from the council of scientific and industrial research (csir), india. the file no. for the same is 09/1058(0013)/2019-emr-i. the authors also acknowledge the host support and facilities provided by the indian institute of technology mandi, himachal pradesh, india. supplementary data to this article can be found online at https:// doi.org/10.1016/j.mehy.2020.109790. key: cord-286038-a62k3lma authors: klimke, a.; hefner, g.; will, b.; voss, u. title: hydroxychloroquine as an aerosol might markedly reduce and even prevent severe clinical symptoms after sars-cov-2 infection date: 2020-04-27 journal: med hypotheses doi: 10.1016/j.mehy.2020.109783 sha: doc_id: 286038 cord_uid: a62k3lma covid-19 is a new coronavirus disease first described in december 2019. this respiratory illness is severe and potentially fatal. severe cases make up to 15%, lethality ranges between 1.5 and more than 10 %. what is urgently needed is an efficient pharmacological treatment for the treatment of severe cases. during the infection of alveolar epithelial cells of the lung, the ace2 receptor has a central function. the antimalarial drugs chloroquine phosphate (cq) and hydroxychloroquine (hcq) impair in vitro the terminal glycosylation of ace2 without significant change of cell-surface ace2 and, therefore, might be potent inhibitors of sars-cov-2 infections. starting inhibition at 0.1 µm, cq completely prevented in vitro infections at 10 µm, suggesting a prophylactic effect and preventing the virus spread 5 hours after infection. in a first clinical trial, cq was effective in inhibiting exacerbation of pneumonia, improving lung imaging findings, promotion of virus-negative conversion, and shortening the disease. in addition, hcq, which is three times more potent than cq in sars-cov-2 infected cells (ec50 0.72 µm), was significantly associated with viral load reduction/disappearance in covid-19 patients compared to controls. theoretically, cq and hcq could thus be effectively used in the treatment of sars-cov pneumonia. from a pharmacological standpoint, however, the major problems of oral treatment with these drugs are possible severe side effects and toxicity. concretely, this relates to (a) the inconsistent individual bioavailability of these drugs at the alveolar target cells, depending on intestinal resorption, hepatic first-pass metabolism and accumulation in liver, spleen and lung, and (b) the need for a relatively high concentration of 1-5 µm at the alveolar surface. therefore, we propose in a first dose estimation the use of hcq as an aerosol in a dosage of 2-4 mg per inhalation in order to reach sufficient therapeutic levels at the alveolar epithelial cells. by using a low-dose non-systemic aerosol, adverse drug reactions will markedly be reduced compared with oral application. this increase in tolerability enables a broader use for prevention and after contact with an infected person, which would be an advantage especially for the high-risk, often multi-morbid and elderly patients. empirical data on self-medication with a one-week aerosol application by two of the authors is presented. inhalation was well tolerated without relevant side effects. the 2019-ncov pandemic is rapidly spreading with currently more than a million reported cases and 57.000 deaths worldwide (1). while the majority of infections with covid-19 are asymptomatic or show a mild course (2, 3), up to 15% of patients develop a severe and potentially fatal respiratory illness with an estimated lethality ranging between 1.5 and more than 10 % (4). currently, as no effective antiviral pharmacological treatment is available, lethality seems to depend on individual risk factors including age, gender and hypertonia and regarding the course of the disease in cases of severe pneumonia, on the availability of large-scale intensive care units and extracorporeal membrane oxygenation (ecmo) (5, 6). regarding pharmacological treatment, several clinical trials have recently been initiated, e.g. with lopinavir/ritonavir (7), remdesivir peptide (ek1) (8) and interferon alpha (9). the latter originally being used for the treatment of hepatitis b (novaferon, beijing genova biotech). however, the clinical efficacy of these new drugs for covid-19 has not been proven and their safety has not been extensively tested. further, cq and hcq have been discussed as promising, cost-effective and easily available agents in the treatment of covid-19 (10-12). these are relatively old agents, introduced primarily for prophylaxis and treatment of malaria and also for rheumatic diseases (13). in vitro cell cultures, hcq seems to be a more potent inhibitor of infection with sars-cov-2 than chloroquine (14, 15). both drugs, taken orally, may cause severe side effects (16) ranging from psychiatric symptoms (17) to ocular toxicity (18) to myocardial dysfunction (19) , even at only low overdosage (20) . these side effects may be severe and, therefore, limit widespread application in vivo, at least at the present time. as drosten (21) pointed out, a major limiting factor of oral hydrochloroquine treatment lies in the fact that the drug may not reach the target, i.e. the surface of the lungs. instead, it is metabolized internally, requiring treatment dosages which, in turn, may cause toxic and sometimes even lethal side effects. here, we propose a different approach to the investigation of the effectiveness of the hydrochloroquine drug that would imply a direct application of the active agent to the respiratory tract. we hypothesize that hcq especially as an aerosol application will prevent or at least markedly reduce the replication rate of the sars-cov-2 virus in the early phase of the infection and subsequently substantially lower the number of severe pneumonias and casualties. this hypothesis is new since the major assumption in ongoing clinical studies and actual recommendations is that hcq and cq should be used in oral application form in patients with severe covid-19 pneumonia and only when other treatment strategies have failed. however, the typical clinical course of this infection suggests that the virus load in the respiratory tract increases stepwise starting with mild symptoms and ending in up to 15 % of patients with severe and potentially life-threatening pneumonia (4). therefore, the treatment with a drug which inactivates the cell receptor for the virus should start after exposition with high risk, e.g., when one person was infected very recently with the virus or is in the early phase of the disease. moreover, our hypothesis differs from the standard recommendation to try hcq/cq in a late phase of the disease when other antiviral drugs failed. we believe that a respiratory virus infection should be treated very early because the severe acute respiratory syndrome is caused by ion channel activity of the viroporin 3a which activates the nlrp3 inflammasome (22) . unfortunately, as of now, there is no evidence yet that hcq/cq has any inhibiting effect on this inflammasome activation. the idea to propose application of hcq/cq as aerosol is generated because one major objection against the clinical efficacy of these drugs is that they have to be administered in relatively high oral dosages. such high dosages may have several toxic side effects, strongly limiting their utilizability as preventive treatment. an aerosol application of drugs which are primarily intended to act on the respiratory system is well established for several drugs, e.g. in the treatment of asthma with corticosteroids, e.g. budesonide (23, 24) and beta mimetica, e.g. fenoterol (25) , and in the early treatment of influenza (during the first 48 hours) with neuraminidase blockers like zanamivir (26) . moreover, there are reports of undergoing clinical studies of aerosol interferon alpha (novaferon) for treatment of covid-19 (27) leading us to advocate a clinical trial to evaluate also hcq/cq in this application form. it has been demonstrated that the sars-cov-2 virus enters ace2-expressing cells including alveolar epithelial cells of the lung and in other organs (28) (29) (30) , which has been shown before also for sars-cov-1. therefore, during the infection of alveolar epithelial cells of the lung, the ace2 receptor has a central function (31) . the antimalarial drugs cq and hcq impair the terminal glycosylation of ace2 without significant change of cell-surface. ace2 increases the local ph value, which reduces the activity of cathepsin l needed for hydrolysis of the viral s protein and might influence the generation of pro-inflammatory cytokines (32) . therefore cq might be a potent inhibitor of sars-cov infection (29, 33, 34) . in vitro, cq, starting with 0.1 µm inhibited and, moreover, completely prevented sars-cov infections in cell cultures at 10 µm, suggesting a prophylactic effect and preventing the virus spread 5 hours after infection. hcq is threefold more potent than cq in sars-cov-2 infected cells, resulting in the clinical recommendation to treat orally with 800 mg at the first day and at 400 mg on the following four days (27) . two major points of action are postulated for cq/hcq in the treatment of covid-19: (1) they perturb the terminal glycosylation of the ace2 protein and inhibit cell-binding from a pharmacological standpoint, one major problem of oral treatment with chloroquine phosphate or hydroxychloroquine is that relatively high concentrations between 1-10 µm are needed to inhibit the glycosylation of the soluble ace2 receptor at the alveolar surface in the lung. therefore, the bioavailability of these drugs in the pulmonic target regions depends individually on intestinal resorption, hepatic first-pass metabolism and diffusion from the blood to the alveolar cells. this could in part be compensated by higher oral dosages, which are, however, limited by adverse and potentially toxic side effects. another problem can be that cq and hcq are accumulated in the liver, spleen, kidney, lungs and other organs. 100 to 500 times of the chloroquine plasma concentration is found in parenchyma cells and 1000 times in pigmented cells. around 55% of it is bound to plasma proteins (pharmaceutical product information). from a clinical standpoint, potential fatal arrhythmias and drug-induced sudden cardiac death in rare cases or in case of overdose can be a resulting problem. our hypothesis prompts to be tested in controlled randomized clinical studies. currently, an aerosol preparation of hcq is not commercially available. therefore, experimental data is needed to examine whether it is possible to achieve and maintain an antiviral concentration of 1-5 µm in the alveolar cells which has been proven effective in vitro. in a first approximation, one can assume that in the normal adult, the lungs weigh approximately 1.000 g which is 0.014 of the whole body weight of 70 kg. although it is difficult to predict in the lung the effects of blood perfusion and on the other hand accumulation of hcq, it seems reasonable to substitute 2-400 mg since hcq/cq is an old drug with a known profile of side effects, also for much higher dosages, it might also be appropriate to perform open pilot studies with higher numbers of cases and compare (in a non-randomized study) the clinical course and final result in patients with similar symptoms but without hcq inhalation. for objective testing of the preventive potential, persons at risks like therapeutic staff following direct unprotected contact with corona patients as well as relatives of patients who have tested positive for sars-cov-2 and who are living in the same household should inhale hcq in comparison to placebo during the incubation period of 5-7 days without symptoms. we would predict that the number of subsequent infections of the contact persons will be significantly lower in the verum group. to test whether hcq as aerosol lowers the severity of the course, a randomized comparison should be performed including patients with beginning mild symptoms who were actually tested sars-cov-2 positive in order to evaluate the effect on clinical symptoms including fever, cough, beginning pneumonia and other complications. finally, hcq as aerosol could be tested preferentially as co-medication in more severely ill patients where an indication for oral hcq treatment is given. the prediction would be that those with the co-medication will have a better treatment outcome than those in oral monotherapy with hcq. since there is currently no commercial aerosol application available, two of the authors (a.k., clinical director and b.w., medical student working at an icu with acute covid-19 patients) decided to test tolerability and possible side effects of the inhalation of hcq, starting with a dosage of 1 mg b.i.d (dissolved in 2 ml of 0.9 % nacl solution and using a commercial nebulizer generating a droplet size < 5 µm which can reach the alveolar space) which was stepwise increased up to 4 mg per day over a period of one week. inhalation was well tolerated without relevant side effects. the only observation was after 4 days the feeling of a transient bitter taste in the mouth which lasted 2-3 hours after the inhalations. if our hypothesis is true, hcq as an aerosol might not only reduce the side effect potential of the oral application form but can also be clinically used as an efficient antiviral agent in the early phase of covid-19 and eventually lower the rate of severely ill patients and fatalities. this might have great relevance for further prognosis and treatment of this often fatal disease. (1) https://coronavirus.jhu.edu/map.html, downloaded april 3, 2020) ( are the current recommendations for chloroquine and hydroxychloroquine screening appropriate revisiting the cardiotoxic effect of chloroquine chloroquine serum concentration and side effects: evidence for dose-dependent kinetics severe acute respiratory syndrome coronavirus viroporin 3a activates the nlrp3 inflammasome adjunctive corticosteroid therapy for patients with severe novel coronavirus pneumonia (covid-19): a randomized controlled trial effect of inhaled formoterol and budesonide on exacerbations of asthma effect of aerosol and oral fenoterol on histamine and methacholine challenge in asthmatic subjects pharmacokinetics of zanamivir after intravenous, oral, inhaled or intranasal administration to healthy volunteers outcome reporting from protocols of clinical trials of coronavirus disease 2019 (covid-19): a review covid-19: perspectives on the potential novel global threat ace2 receptor expression and severe acute respiratory syndrome coronavirus infection depend on differentiation of human airway epithelia therapeutic effect of hydroxychloroquine on novel coronavirus pneumonia (covid-19 a multiscale and comparative model for receptor binding of 2019 novel coronavirus and the implication of its life cycle in host cells new insights on the antiviral effects of chloroquine against coronavirus: what to expect for covid-19? chloroquine as a potential treatment and prevention measure for the 2019 novel coronavirus: a review in vitro inhibition of severe acute respiratory syndrome coronavirus by chloroquine severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and corona virus disease-2019 (covid-19): the epidemic and the challenges bioavalibility of hydroxychlorquine tablets in patients with rheumatoid arthritis the authors confirm that they have no conflict of interest. key: cord-271785-vmnc8yg6 authors: minetti, giampaolo title: mevalonate pathway, selenoproteins, redox balance, immune system, covid-19: reasoning about connections date: 2020-07-21 journal: med hypotheses doi: 10.1016/j.mehy.2020.110128 sha: doc_id: 271785 cord_uid: vmnc8yg6 it has been proposed that a degraded immune system is (one of) the condition(s) that predispose certain subjects to fatal consequences from infection by sars-cov-2. it is unknown whether therapeutic regimens to which these patients may have been subjected to in the months/years preceding the infection could be immunocompromising. statins are among the most widely prescribed cholesterol-lowering drugs. as competitive inhibitors of hmg-coa-reductase, the key enzyme of the “mevalonate pathway” through which essential compounds, not only cholesterol, are synthesized, statins decrease the levels of cholesterol, and thus ldls, as an innate defense mechanism, with controversial results in decreasing mortality from cardiovascular disease. moreover, statins have pleiotropic, mostly deleterious effects on many cell types, including immune cells. in the attempt to decipher the enigma of sars-cov-2 infectivology, the hypothesis should be tested whether the population of subjects who succumbed to covid-19 may have developed a compromised immunity at sub-clinical levels and have become more susceptible to fatal consequences from sars-cov-2 infection due to statin therapy. it has been suggested that the presence of a degraded immune system is (one of) the condition(s) for the susceptibility of certain predisposed subjects, usually the elderly and those presenting with various co-morbidities, to infection by sars-cov-2 and its often fatal consequences. but a significant number of young and otherwise apparently healthy subjects has succumbed, and still is succumbing, to covid19 . immunodepression has been linked to several possible environmental factors, such as pollution, electromagnetic fields, global warming, but also to drug induced side effects, such as in oncologic patients on chemotherapy. no attempts have been made, however, to carry out an anamnestic evaluation of the subjects, especially the younger ones, who have died from the consequences of covid-19. in particular it is unknown whether a weakened immune system could be the result of possible adverse effects from therapeutic regimens to which these patients may have been subjected to in the months/years preceding the infection. several drugs have been considered as potential factors that predispose (anti-hypertensive, in particular angiotensin ii receptor blockers, mineralocorticoid-receptor antagonists, anti-diabetics) or protect (hydroxychloroquine, corticosteroids) from sars-cov-2 infection. however, to the best of our knowledge, no systematic studies have been carried out to evaluate the role played by one family of drugs that has been largely prescribed to the general population over the past couple of decades: the statins. a couple of studies have indeed been published that exclude a statistically significant correlation between covid-19 outcome and statin routine treatment of patients, but no data on compliance, or cholesterol levels were provided. 1,2 one of the articles has been retracted by the authors almost immediately. 1 statins are cholesterol-lowering drugs that act as competitive inhibitors of the enzyme -hydroxy--methyl-glutaryl-coenzyme-a (hmg-coa)-reductase, the key enzyme of the "mevalonate pathway". starting from acetic acid as the building block, this pathway leads to the biosynthesis of isoprenic units, which are in turn assembled to produce such various and essential compounds for both plant and animal cells: dolichol, coenzyme q, isopentenyl for the modification of trnas, farnesyl and geranyl-geranyl for the prenylation of proteins, rubber, plant hormones, phytol chain of chlorophyll, carotenoids, vitamin a, vitamin e, vitamin k, vitamin d, bile acids, steroid hormones and cholesterol. since hmg-coa reductase is at the very root of the pathway, its inhibition will prevent the production of not only cholesterol, but also of all the essential compounds listed above. the theory enforcing cholesterol-lowering therapies holds that high levels of serum cholesterol, in the form of low density lipoproteins (ldl), are positively correlated with the prevalence of cardiovascular disease and mortality from myocardial infarction and stroke. however, metaanalyses of the outcome of several clinical trials have revealed that the correlation between cholesterol levels and mortality is either non-existing or inverse: the lower the cholesterol levels the higher the mortality from all causes, especially those related to infection. [3] [4] [5] [6] furthermore, the levels of ldl cholesterol are inversely associated with longevity in the elderly, 5 and low serum cholesterol is even a negative prognostic factor in patients with advanced heart failure, independent from lipid-lowering therapy. 7 ldl cholesterol as part of the innate immunity the role of lipoproteins as first line defense against microbial infection is well established. 8, 9 prolonged infection with hepatitis b virus has been show to correlate with low blood cholesterol levels and cancer, 10 and hypocholesterolemic men have significantly fewer circulating lymphocytes, total t cells and cd8 + cells compared with hypercholesterolemic subjects. 11 in spite of this evidence, the prevailing theory has not been could not be overturned, and statins are still widely prescribed for the "treatment of blood cholesterol". 12 indeed, although statins are ineffective in the secondary prevention of cardiovascular events, they appear to be beneficial in the primary prevention of mortality from myocardial infarction. however, the benefit is very modest, as one hundred subjects need to be treated to prevent one single infarction-related casualty. 3 moreover, this efficacy appears to be related to one of the multiple pleiotropic effects of statins (see below), i.e. an anti-inflammatory effect, rather than a cholesterol-lowering action. a number of different cell types can be targeted by the adverse effects of statins, the best known being the skeletal muscle cell. severe rhabdomyolysis, leading to more than 30 deaths, was reported in 2001 for subjects on treatment with "first generation" statins. 13 all muscle cells, including the myocardium, are especially dependent on mitochondrial respiration for the production of atp. coenzyme q is an essential component of the electron transport chain in the inner mitochondrial membrane and it is synthesized in the mevalonate pathway. one of the most diffusely reported adverse effects of "new generation" statins is myopathy, which is linked to mitochondrial damage likely resulting from deficiency of coenzyme q. not surprisingly, coenzyme q supplementation has been found to ameliorate myopathy in subjects on statins. 14 deleterious side-effects of statins on the central and peripheral nervous systems, including cognitive disorders have also been reported. 3 because statins inhibit the biosynthesis of substances that are required for the post-translational modification of proteins (and the maturation of trnas, see below), virtually every cellular metabolic and signaling pathway may be affected. thus, statins exert a general inhibitory action on immune cells, probably by interfering with prenylation of g proteins: growth, proliferation, adhesiveness and chemotaxis are inhibited in monocytes. 15 lymphoid cell function is suppressed by statins in vitro, although the mechanism has not been characterized. 16 given their depressive action on immune cells, statins have been proposed as immunomodulators with milder effects than the conventional immunosuppressive drugs. 17 it can be concluded that statin therapy may contribute to generalized immunosuppression. less investigated is the impact of statins on redox metabolism. an important contribution to the redox balance in cells and tissues comes from selenoproteins, which contain one or more selenocysteine (sec) residues in their sequence. the majority of seleoproteins are selenoenzymes, as the sec residue(s) are located in the catalytic site, with antioxidant and oxidative damage-repairing activities. the human selenoproteome comprises 25 different selenoproteins. 18 the best characterized are glutathione peroxidases, thioredoxin reductases, iodothyronine deiodinases, and selr, or methionine-r-sulfoxide reductase 1 (msrb1) which reduces the l-methionine-r-sulfoxide and has an important role in repairing proteins that have lost function because of oxidation of lmethionine residues. of the less characterized selenoproteins, some are molecular chaperones in the endoplasmic reticulum, others (seln) are important for muscle function, as their mutation is associated with muscular disease. 19 for a third class of all selenoproteins the role is still unknown. 18 glutathione peroxidases are antioxidant enzymes in plasma and blood cells. msrb1 is highly expressed in neutrophils. 20 all selenoproteins are synthesized through a mechanism of translational recoding of the stop codon tga (uga in the messenger) as a codon for sec insertion. key factor in this mechanism is the special trna sec , which must carry an isopentenyladenosine at position 37 for being functional. 21 the isopentenyl moiety required for this modification is produced in the mevalonate pathway, hence the link with a potential inhibitory effect of statins on selenoprotein expression. 22 increased oxidative stress is one of the factors that explain statin-induced myopathy. this effect has been until now mainly linked to decreased levels of coenzyme q and associated increased production of reactive oxygen species in the mitochondrion. 23 however, a contribution of selenoproteins downregulation to the generation of a statin-induced pro-oxidant state in muscle and other tissues has not been evaluated so far. pro-or anti-inflammatory effects of statins? as statins have been claimed to have anti-inflammatory effects, it may seem contradictory that they could contribute to the hyper-inflammatory state observed in covid-19 patients. however, statins interfere with the proper modification of essential g proteins in the signal transduction pathways that control innate immunity. inhibition of prenylation of ras-family g-proteins is associated with the generation of a hyper-inflammatory conditions with hyper-production of pro-inflammatory cytokines. 24 interestingly, mevalonate-kinase-deficiency, a hereditary disorder associated with a defective mevalonate pathway, is typically characterized by an auto-inflammatory state of the patients. 24 statins may also perturb acquired immunity by altering antigen presentation by antigenit may be concluded that an intact redox balance is essential for white blood cells and that it would be worth investigating whether downregulation of selenoproteins may contribute to compromise leukocyte function in subjects on statin therapy. this study cannot be a retrospective analysis of pre-existing data, because, to the best of our knowledge, no quantification of selenoprotein activity has ever been carried out in vivo in subjects on statin treatment compared to normal subjects. a recent nutritional study reports on a beneficial effect of selenium supplementation on markers of muscle damage in subjects on statin therapy. 26 selenoprotein expression levels were not changed, except for an increase in glutathione peroxidase activity, which correlated with a decrease in serum creatine kinase activity, in subject supplemented with selenium. however, the study lacked a control of normal subjects not on statins. therefore, until now it is not known how the modulation of the mevalonate pathway activity impacts on the levels and availability of the various intermediates produced in the pathway and in particular on selenoprotein activity. also lacking is the information whether hypercholesterolemia is associated with increased levels of mevalonate pathway intermediates. therefore, this hypothesis could only be tested within an "ad hoc" research project, possibly in a placebo controlled, double blind type of study. a second prediction of the hypothesis could be tested retrospectively, as discussed below. hmg-coa reductase as well as most enzymes for sterol biosynthesis are all integral membrane proteins in the endoplasmic reticulum of eukaryotic cells. all cells require cholesterol and actively synthesize it as an essential structural constituent of the plasma membrane. for the purpose of replicating, a virus heavily engages the host cell's molecular machinery for protein synthesis and vesicular trafficking and in this process it deranges the cell's metabolism. it is expected that any cellular reaction that takes place at the level of the endoplasmic reticulum membrane, like the mevalonate pathway, will be affected by the tremendous burden that an actively replicating virus poses on the intracellular membranous compartments. this view seems to be supported by a report revealing a sharp decrease in neutrophil, lymphocyte and cholesterol levels in covid-19 patients. 27 thus, subjects with already low ldl levels and possibly weakened leukocyte function because of statin treatment would be even more susceptible to infection and its fatal consequences. this prediction of the hypothesis could be tested by retrospectively evaluating whether a correlation exists between the cholesterol levels of subjects infected by sars-cov-2 and the severity of the consequences suffered with the progression of the disease. the long list of deleterious effects that statins exert at all levels of biochemical cellular processes should be sufficient to outweigh the minor potential benefits that these drugs may show in preventing cardiovascular disease. the minimal benefit in reducing the absolute risk of myocardial infarction by one mere percent point may be linked to statins' an anti-inflammatory action, which, however, is associated with a plethora of adverse effects that include dysregulation of immune cells. therefore, the suggestion that statins should be used for the prevention of covid-19 is questionable. 28 by decreasing the levels of ldls as an innate defense mechanism against pathogens and by unleashing the power of a drug that acts at multiple basic cellular levels with deleterious effects, we may have reared a population of subjects who have over time developed a compromised immune system. a new perspective ad alternative approaches are mostly wanted to address an issue of such general interest as a global infective pandemic. serious attempts to decipher the enigma of the sars-cov-2 infectivology must certainly include the analysis of factors that could compromise the immune system, among which statin therapy has to be included. hydroxychloroquine or chloroquine with or without a macrolide for treatment of covid-19: a multinational registry analysis phenotypic characteristics and prognosis of inpatients with covid-19 and diabetes: the coronado study how statistical deception created the appearance that statins are safe and effective in primary and secondary prevention of cardiovascular disease lack of an association or an inverse association between low-density-lipoprotein cholesterol and mortality in the elderly: a systematic review ldl-c does not cause cardiovascular disease: a comprehensive review of the current literature low serum total cholesterol is associated with marked increase in mortality in advanced heart failure plasma lipoproteins are important components of the immune system infections may be causal in the pathogenesis of atherosclerosis prolonged infection with hepatitis b virus and association between low blood cholesterol concentration and liver cancer immune system differences in men with hypo-or hypercholesterolemia acc/aha guideline on the treatment of blood cholesterol to reduce atherosclerotic cardiovascular risk in adults: a report of the american college of cardiology/american heart association task force on practice guidelines bayer decides to withdraw cholesterol lowering drug effects of coenzyme q10 on statin-induced myopathy: an updated meta-analysis of randomized controlled trials antiinflammatory and immunomodulatory properties of statins suppression of lymphoid cell function in vitro by inhibition of 3-hydroxy-3-methylglutaryl coenzyme a reductase by lovastatin immune modulatory effects of statins characterization of mammalian selenoproteomes mutations in sepn1 cause congenital muscular dystrophy with spinal rigidity and restrictive respiratory syndrome neutrophil granulocytes uniquely express, among human blood cells, high levels of methionine-sulfoxide-reductase enzymes inhibition of selenoprotein synthesis by selenocysteine trna[ser]sec lacking isopentenyladenosine selenoprotein synthesis and side-effects of statins oxidative stress as a possible mechanism of statin-induced myopathy control of the innate immune response by the mevalonate pathway simvastatin inhibits the mhc class ii pathway of antigen presentation by impairing ras superfamily gtpases association between creatine kinase activity, oxidative stress and selenoproteins mrna expression changes after brazil nut consumption of patients using statins low serum cholesterol level among patients with covid-19 infection in wenzhou statin therapy in covid-19 infection key: cord-270740-3su8pc3f authors: sultan, sherif; sultan, mohamed title: covid-19 cytokine storm and novel truth date: 2020-05-22 journal: med hypotheses doi: 10.1016/j.mehy.2020.109875 sha: doc_id: 270740 cord_uid: 3su8pc3f nan while the world is facing an unprecedented crisis with novel coronavirus disease (ncov) 2019/ covid-19, there are no proven effective pharmacological agents (1) . following the detection of severe acute respiratory syndrome coronavirus(2) (sars-cov-2) in december 2019 in wuhan, china, we witnessed a massive surge in the number of cases globally, until who declared a pandemic on march 11, 2020 (2). the search for a 'novel' agent is ongoing and as of 7 april 2020, 'trialstracker' project(3) had listed 883 clinical trials on covid-19, including 344 intervention trials. during an evolving pandemic the real-world evidence can only be obtained from respected sources. unfortunately, by the time a manuscript is produced and accepted for publication, we might have missed the boat. who failed us and some governments opted to protect the economy rather than controlling the spread of the covid 19 pandemic. investigational journalism had the upper hand in exposing the problem with china and later on, in south korea and italy. during the same time who was assuring the world that there was nothing to worry about. when the death rate mushroomed in italy, everything had exploded, the world knew that a new era had arrived with the greatest lockdown of all time. corona's correction will allow us to get rid of all the dinosaurs for every top position in any organization and to evolve to a world of trust and confidence. based on observations in the usa, spain, italy, france and the uk, and from the postmortem of lung involvement in covid 19, all revealed pulmonary thrombosis, which is not typical ards. more alarming was that patient hypoxemia was not responding to peep but high oxygen flow(4). a chinese scientist(5) used a bio-informatics model to describe the hypothesis of covid 19 as methemoglobin, where the covid-19 virus structural protein sticks to heme -displaces oxygen -which alters the iron-free ion, leading to inflammation of alveolar macrophages, which culminate in a systemic response ending in a cytokine storm . they suggested that if free radical scavengers and iron chelating agents are added to the protocol of management, these might ameliorate the inflammatory response. what we must focus on is that covid19 attacks rbcs. patients have frequently been found covid-19, sars2 is not 'pneumonia' nor ards through the current experience across the world, invasive ventilation is becoming the last resort, as emergency intubation from the chinese, italian and american experience evidences higher mortality, not to mention complications from tracheal scarring and stiff lung during the duration of intubation. furthermore, a new treatment protocol needs to be established in order to control the prolonged and progressive hypoxia of covid19(4). people are desaturating due to failure of the blood to carry oxygen. this will lead to multiorgan failure and high mortality. the lung damage seen on ct scans is due to the oxidative stress released from the hemolysed red blood cells, which in turn overwhelm the natural defenses against pulmonary oxidative stress and cause a cytokine storm. there is alwaysbilateral ground-glass opacity in the lungs. recurrent admission for post-hypoxic leukoencephalopathy strengthens the findings of the italians, spanish and americans(4) that covid-19 patients are suffering from metabolic hypoxia due to blood capacity failure. the chinese hypothesis(5) that had been publicized, theoretically, via bioinformatics modeling, postulated that the catastrophic cascade of oxidative stress explains the vicious cycle as follows: 1) without the iron ion, hemoglobin can no longer bind to oxygen. once the hemoglobin is impaired, the red blood cell is essentially unable to carry and deliver oxygen to any tissues. drop significantly. in covid-19 patients, unlike with co poisoning in which eventually the co can break off, the affected hemoglobin is permanently stripped of its ability to carry oxygen. the body compensates by secreting excess erythropoietin to stimulate the bone marrow to secrete new red blood cells. this is the reason we find thrombocytosis, or thrombocytopenia depending on the stage and severity of covid-19, a high ferritin level and d-dimer with decreased blood oxygen saturation as the primary indicators of the covid-19 severity score. 2) the free-floating iron ions(5) are highly reactive and cause oxidative damage. this always happens physiologically and is natural to a limited extent in our bodies, and such cleanup is a defense mechanism to keep the balance. of the three primary lung defenses to maintain "iron homeostasis", two are in the alveoli. the first of the two are macrophages that roam around and scavenge the free radicals of the oxidative iron. the second is a lining on the epithelial surface which has a thin layer of fluid packed with high levels of antioxidant molecules such as ascorbic acid. when too much iron is in circulation, it begins to overwhelm the lungs' countermeasures and the process of pulmonary oxidative stress begins. this leads to damage and inflammation, which leads to the so-called cytokine storm (5) ; this can be documented on high-resolution ct scans of covid-19 patients' lungs. the liver attempts its best to remove the iron, but only becomes overwhelmed too. it is starved of oxygen. the liver function will deteriorate with elevated alanine aminotransferase (alt), which is one of the primary covid-19 severity score indicators. through extensive discussion with some of our colleagues in beijing, milan, sienna, paris, barcelona, london and new york(6), we have reached a consensus to recommend that the patient might be better managed on maximum oxygen flow through a hyperbaric chamber (7, 8) on 100% oxygen at double or multiple atmospheres of pressure for 90 minutes twice per day for five days. the randomized clinical trial of hyperbaric medicine started in usa in the first week of april (7) . this is in order to give what is left of a patient's functioning hemoglobin a chance to carry enough oxygen to the organs and keep them alive. as we do not have nearly enough of those hyperbaric chambers, we might use all the parked grounded airplanes as a ready-made functional hyperbaric chamber with the advantage of providing double atmospheric pressure with an aerosol of prostacyclin as a pulmonary hypertension modulator. some chinese physicians tried blood transfusion with packed fresh red blood cells to patients after plasmapheresis(9,10) , and witnessed some amelioration of the cytokine storm. the main point is that patients will require ventilators if they present late with multi-organ system failure to tide them over this life or death scenario. however, intubation is futile unless the patient's immune system modulates the situation (11) . we must address the root of the illness and avoid using traditional teachings to manage a failing system (12, 13) . armchair pseudo-physicians an no longer sit in their ivory towers proclaiming "chloroquine use is stupid as malaria is a bacterium, covid-19 a virus: anti-bacterial drugs do not work on a virus!". a drug does not need to act on the pathogen to be effective. a few key opinion leaders advised that chloroquine lowers the blood ph and interferes with replication of the virus. however, a publication in press at the new england journal of medicine advises against the use of chloroquine (14) . sweden has decided to halt its use, as have some parts of france. we recommend that if covid-19 positive patients are conscious, alert and compliant, they should be kept on maximum oxygen. a prone position allows better lung perfusion, and we may have to initiate hyperbaric oxygen as early as possible (8, 9, 10) . if we reach the inevitable and need to ventilate, usa pulmonologists (15) have recommended that this should be performed at low pressure but with maximum oxygen flow. we must avoid tearing up the lungs with maximum peep as this does more harm to the patient because we may be addressing the wrong organ (16) . last week, a discussion on medscape about covid 19 virus and diabetes mellitus illustrated that the virus may be attacking the islets of langerhans in the pancreas and causing insulin deficiency (17) . as those patients had good reserve they present late with diabetic ketoacidosis. the main focus of management i these cases needs to be high flow oxygen plus insulin supplementation. it is crucial not to underestimate covid-19 severity in patients with diabetes even in the absence of classical worrisome signs and symptoms, and we must develop different clinical early severity scores for patients with diabetes. although acute inflammatory states and acute stress responses will certainly raise glucose levels, the sars-cov-2 virus is actually damaging the pancreatic islet cells. a study was done during the sars 2003 coronavirus outbreak in china in which diabetes developed within two weeks of hospitalization in patients who did not previously have diabetes. immuno-staining in patients who had died after contacting covid-19 showed strong staining of the angiotensin-converting enzyme 2 (ace2) protein -the coronavirus binding site -in the islets but not the exocrine pancreatic tissue, which means coronavirus causes diabetes by damaging pancreatic islets. covid-19 causes insulin deficiency, meaning many patients require markedly elevated insulin drip rates. this is not just pressor/steroid related. something else is going on here. we've not seen this pattern of glycaemia with associated insulin requirements before. this is a new beast. the degree of glucose toxicity is profound and independent of preadmission diabetes control. patients with seemingly well-controlled diabetes at home with hba1cs in the single digits experience severe dysglycaemia. there is a subset of patients presenting with euglycaemic dka with normal lactate, with other causes of ketoacidosis ruled out. diabetics had higher levels of biomarkers due to an inflammatory "cytokine storm" preceding rapid deterioration of covid-19 (17, 20) . there is a small village in northern italy where the majority of its population suffers from thalassemia. these individuals have had no deaths and no cross-community spread, due to the abnormal shape of the red blood cells. moreover, parts of nepal that are 1km above sea level are covid-19 free, the same logic applies, abnormal shape of the red blood cell. all the evidence suggests that we are chasing the wrong organ: it is not the lungs, it is a blood problem. we advised a few international organizations that al & ml must be utilized in analyzing big data. ai, big data analysis and bioinformatics must be harnessed to allow us to scrutinize how can we provide the best options for our patients, creating bioinformatics modelling that can surpass any rct. adaptive platform designs must be structured to promote maximum learning from around the world to adjust how we deliver the best care to our patients (21, 22, 23, 24, 25, 26, 27) . people are desaturating due to failure of the blood to carry oxygen (17) . this will lead to multi-organ failure and high mortality due to cytokine storm. we believe that management protocols for ards should not be applied for covid-19 patients (18, 19, 21, 22, 23, 24, 25, 26, 27) . we recommend the following: 1. inhibit viral growth and replication by any proven means. currently the adjuvant use of chq+zpak+zinc appears indicated, or the rising star of ramedesivir, which has proven effectiveness in ameliorating covid-19 mortality. other retroviral therapies are being studied. the less viral load we have, the less severity of the damage with the prevention of cytokine storm. 2. theoretical use of hyperbaric medicine or pressurized grounded airplanes may prevent rapid ascent into the abyss. 3. plasmapheresis and blood transfusions may give supportive symptomatic relief when indicated. 4. no international travel until an effective vaccine is available. 5. cessation of tobacco, vaping, and the use of alcohol products. until we create a sound protocol for managing our sick patients and understand why chloroquine and invasive ventilation have failed, as both have been unable to bail out our oxygen-starved patients, the only available option is symptomatic relief. pharmacologic treatments for coronavirus disease 2019 (covid-19): a review director-general's opening remarks at the media briefing on covid-19 -11 mike stobbe -why some doctors are now moving away from ventilator treatments for coronavirus patients attacks the 1-beta chain of hemoglobin and captures the porphyrin to inhibit human heme metabolism a bridge between life and death: most covid-19 patients put on ventilators will not survive hyperbaric oxygen for covid-19 patients vasculogenic stem cells, and wound healing -antioxid redox signal a novel coronavirus from patients with pneumonia in china adam wells -a novel treatment approach to the novel coronavirus: an argument for the use of therapeutic plasma exchange for fulminant covid-19 sharon worcester -is protocol-driven covid-19 ventilation doing more harm than good? sharon begley -with ventilators running out, doctors say the machines are overused for covid-19 xihui lin -clinical outcomes of hydroxychloroquine in hospitalized patients with covid-19: a quasi randomized comparative study paul marik -critical care covid-19 management protocol why some doctors are moving away from ventilators for virus patients more than 65% of people put on ventilators will die, nhs data shows 80% of nyc's coronavirus patients who are put on ventilators ultimately die, and some doctors are trying to stop using them over 80% of covid patients placed on ventilators in new york city have died covid-19 and diabetes: known mechanisms and a 'new beast hrct imaging features in representative imported cases of 2019 novel coronavirus pneumonia. precision clinical medicine epidemiologic and clinical characteristics of novel coronavirus infections involving 13 patients outside wuhan, china clinical features of patients infected with 2019 novel coronavirus in wuhan potential of large 'first generation'human-to-human transmission of 2019-ncov clinical characteristics of 138 hospitalized patients with 2019 novel coronavirusinfected pneumonia in wuhan early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia a novel coronavirus from patients with pneumonia in china professor of vascular & endovascular surgeon chairman of the western vascular institute university college hospital galway ireland re: cover letter & conflict of interest covid19 cytokine storm and novel truth dear chief editor, i wish to submit this editorial to the journal of medical hypothesis about 'covid19 cytokine storm and novel truth'. we declare no conflict of interest and have received no such funding to write the editorial. i hope the journal reviewer board and the readers find this interesting. yours sincerely key: cord-261574-zcazhkad authors: garcía, néstor h.; porta, daniela j.; alasino, roxana v.; muñoz, sonia e.; beltramo, dante m. title: ibuprofen, a traditional drug that may impact the course of covid-19 new effective formulation in nebulizable solution date: 2020-07-07 journal: med hypotheses doi: 10.1016/j.mehy.2020.110079 sha: doc_id: 261574 cord_uid: zcazhkad the traditional formulation of ibuprofen is poorly soluble in water, so the administered dose must be 10 times higher than the dose required for a therapeutic effect. the development of a hydrosoluble form of ibuprofen can be a strategy to reach a high concentration in the lungs by using modern inhalation devices. therefore, the development of an inhalable formulation with high bioavailability in the lungs was the leitmotiv of our investigation. the hypertonic ibuprofen solution to be nebulized (nih) presents great relevant characteristics: bactericidal, virucidal, mucolytic and has a known anti-inflammatory property. bactericidal and virucidal effects are related to the physico-chemical properties of na-ibuprofenate as an amphipathic molecule. it has the capability to insert into the bilayer membranes destabilizing the structure, altering its biological properties and avoiding the duplication or infection. our preliminary results indicate that the presence of this high ionic strength solution reduces 10 times the amount of ibuprofen necessary to kill bacteria, but also the time to kill 1x10(6) bacteria, from 4 hours (in its absence) to only three minutes (in its presence). that was observed using pseudomona aeruginosa, methicillin-resistant staphylococcus aureus and burkholderia cepacia. also, “in vitro'' ibuprofen demonstrated virucidal activity against the so-called enveloped virus, a family that includes coronavirus strain (2019-ncov). we observed too, the markedly reduced local inflammation in the airways after administering nih lays on its ability to inhibit the enzyme cyclooxygenase and to markedly diminish reactive oxygen species (ros). other investigators also showed the importance of actin in the rapid spread of virus infection. furthermore, reorganization of the actin filaments is a key step in lung inflammation induced by systemic inflammatory responses caused by sars-cov-2. these findings suggest that the interaction between actin proteins and s1 is involved in the 2019-ncov infection and pathogenesis. consequently, the possibility of interfering in this interaction could represent a valid hypothesis for the development of promising therapeutic and prevention strategies. in conclusion, we consider that treating people with covid-19 with nih may be beneficial and an opportunity to contribute for the current global health emergency. the traditional formulation of ibuprofen is poorly soluble in water, so the administered dose must be 10 times higher than the dose required for a therapeutic effect. the development of a hydrosoluble form of ibuprofen can be a strategy to reach a high concentration in the lungs by using modern inhalation devices. therefore, the development of an inhalable formulation with high bioavailability in the lungs was the leitmotiv of our investigation. the hypertonic ibuprofen solution to be nebulized (nih) presents great relevant characteristics: bactericidal, virucidal, mucolytic and has a known anti-inflammatory property. bactericidal and virucidal effects are related to the physico-chemical properties of na-ibuprofenate as an amphipathic molecule. it has the capability to insert into the bilayer membranes destabilizing the structure, altering its biological properties and avoiding the duplication or infection. our preliminary results indicate that the presence of this high ionic strength solution reduces 10 times the amount of ibuprofen necessary to kill bacteria, but also the time to kill 1x10 6 bacteria, from 4 hours (in its absence) to only three minutes (in its presence). that was observed using pseudomona aeruginosa, methicillin-resistant staphylococcus aureus and burkholderia cepacia. also, "in vitro'' ibuprofen demonstrated virucidal activity against the so-called enveloped virus, a family that includes coronavirus strain (2019-ncov). we observed too, the markedly reduced local inflammation in the airways after administering nih lays on its ability to inhibit the enzyme cyclooxygenase and to markedly diminish reactive oxygen species (ros). other investigators also showed the importance of actin in the rapid spread of virus infection. furthermore, reorganization of the actin filaments is a key step in lung inflammation induced by systemic inflammatory responses caused by sars-cov-2. these findings suggest that the interaction between actin proteins and s1 is involved in the 2019-ncov infection and pathogenesis. consequently, the possibility of interfering in this interaction could represent a valid hypothesis for the development of promising therapeutic and prevention strategies. in conclusion, we consider that treating people with covid-19 with nih may be beneficial and an opportunity to contribute for the current global health emergency. the importance of ibuprofen in the respiratory tract has been described previously in children with pulmonary cystic fibrosis but besides the beneficial effect observed in this scenario, it is not frequently prescribed, and the main reason is the high dosage required to produce the desired effect in the lungs. the traditional formulation of ibuprofen is poorly soluble in water, so the administered dose must be 10 times higher than the dose required for a therapeutic effect (1) . in this sense, the distribution rate of currently available traditional dosage forms is limited, leading to poor bioavailability from a high oral dosage (2) . the development of a hydrosoluble form of ibuprofen can be a strategy to reach a high concentration in the lungs by using modern inhalation devices, without the potential systemic effects of high dosages. this approach would allow us to deliver micro or nanometer sized particles to reach a large surface area, such as the pulmonary alveolar. the other benefit of this size is that particles from 0.5 to 3 µm are captured by macrophages through phagocytosis, but not those < 0.25 µm (1). having a formulation with these properties would lead to a high potential for the treatment of lung infections, such as pulmonary cystic fibrosis, pneumonia, and bronchiectasis (21) . therefore, the development of an inhalable formulation with high bioavailability in the lungs was the leitmotiv of our investigation. considering the characteristics of available ibuprofen formulations, a solution with hypertonic ibuprofeno solution to be nebulized (nih) was developed with great relevant characteristics: it is bactericidal, virucidal, mucolytic and has a known anti-inflammatory property. this formulation was developed for the treatment of serious lung conditions, such as cystic pulmonary fibrosis, pneumonia and bronchiectasis, because complications due to opportunistic infections are of great importance in these lung pathologies. additionally, since administration via inhalation was decided, it overcomes the limitations of the currently available administrative routes to achieve a better therapeutic action at a very low dose (5) . recently irvine et al. (20) explained the challenge of ibuprofen solubilization in water. these researchers describe the solubility of this drug as the major problem. so, different technologies particle engineering or crystallization were applied in improving formulation strategies. finally, an attempt has been made to improve the delivery of this drug into the pulmonary route for achieving better therapeutic action at a very low dose, thus overcoming the limitations of currently available delivery routes. nanotechnology is the science that recently appeared to solve this problem. in this case, several strategies have been developed; however, the appropriate delivery of ibuprofen to the lung using nanocarriers such as liposomes, dendrimers, emulsions or polymers has been achieved, because there is an inability to reach enough lung deposit (3). the bactericidal effect was possibly confounded with the anti-inflammatory effect initially. during the 80's, ibuprofen's ability to limit bacterial damage from "in vivo'' was evaluated (12) (13) (14) . in 1990 konstan et al., (15) interestingly, this interaction is strongly reinforced and stabilized in the presence of a high ionic strength solution, such as hypertonic sodium chloride. the presence of this high ionic strength solution reduces 10 times the amount of ibuprofen necessary to kill bacteria, but also the time to kill 1x10 6 bacteria, from 4 hours (in its absence) to only three minutes (in its presence). that was observed using pseudomona aeruginosa, methicillin-resistant staphylococcus aureus and burkholderia cepacia. other investigators have reported similar results (22) . but the effect does not end here, also "in vitro'' ibuprofen demonstrated virucidal activity against the socalled enveloped virus, a family that includes coronavirus strain (2019-ncov) ( table 1) . using an "in silico'' approach, ibuprofen has been predicted as an entry inhibitor of ebola virus (6) . this prediction has then been experimentally confirmed (7, 8) , showing that ibuprofen reduced 50% of virus title in vero cells. this virucidal effect has also been reported "in vitro'' for zika virus (zikv), where ibuprofen plunged axl expression, a zikv entry cofactor. this data highlights the worth of drugs with a high safety profile, which may even be indicated during pregnancy, to prevent zikv infection. although by different mechanisms, clear viral replication regulatory effects have also been shown "in vitro'' and in humans for other viruses like hsv-1, hsv-2, rotavirus, rhinovirus (9) (10) (11) . the markedly reduced local inflammation in the airways after administering nih lays on its ability to inhibit the enzyme cyclooxygenase and to markedly diminish reactive oxygen species (ros). (table 2 ) these data are consistent with previous publications (18) . on the other hand, the mucolytic properties of nih was detected in healthy volunteers, and also in patients with copd, a disease characterized by significant chronic lung inflammation (19) ; this response shows the effectiveness of the compound in pathologies with respiratory intervention and the broad spectrum demonstrated in the treatment of conditions with similar characteristics. the safety of inhaled nsaids has been previously demonstrated (20) , evaluating the response of inhaled sodium metaphosphate and adenosine monophosphate in the absence of prostaglandins. they used inhaled l-aspirin, indomethacin and sodium salicylate in asthmatic patients. in this report, none of the inhaled nsaids changed fev 1 in sensitive patients. in another study, tamaoki j et al., reported that inhaled indomethacin decreased fluid and mucus in the respiratory tract after 14 days of treatment without alterations in lung function. no adverse effects were observed (21) . other investigators found similar results, inhalted aines are safe (22) . further confirmation of these findings can be found in a recent cochrane review (22) . our laboratory evaluated the effect of hypertonic ibuprofen in healthy adults and we found an increase on fev1/fvc ratio suggesting a bronchodilator effect, perhaps intrinsic to the anti-inflammatory effect (unpublished data), during this experiment, serum ibuprofen increased from 0 to 1.09±0.7 μg/ml and also the reactive oxygen species decreased from 1043±252 to 441±48 λu determined 30 minutes after the nebulization, demonstrating in spite of the low serum concentration, this presentation still maintains the antiinflammatory property. we also evaluated the effect of hypertonic ibuprofen using compassion and in copd patients, after 4 months of treatment, their fev1, and the walking test improved compared to basal, while the daily oxygen requirement was reduced. another patient, with idiopathic pulmonary fibrosis, after 1.5 years of treatment, he improved his fev 1 , and the ground glass opacity observed ct scan disappeared after 4 months of treatment. although these may be anecdotal findings, they may also indicate the beneficial effects of inhaled ibuprofen. the new pathogen belonging to a family of viruses with a lipid envelope in its structure. using confocal microscopy and molecular biology tools, (28) , studied how the human coronavirus oc43 enters the susceptible cell, visualizing that the virus uses endocytosis and that the internalization process of vesicles that require actin, so it can play an important role in early entry events during human coronavirus infections (29) . other investigators also showed the importance of actin in the rapid spread of virus infection (35). furthermore, reorganization of the actin filaments is a key step in lung inflammation induced by systemic inflammatory responses caused by sars-cov-2 (30) . these findings suggest that the interaction between actin proteins and s1 is involved in the 2019-ncov infection and pathogenesis. on the other hand, the angiotensin-converting enzyme 2 (ace2) is the functional receptor for the spike glycoprotein of the sars-cov-2, therefore it would play a crucial role in the entry of the virus into the cell to cause the final infection (31) . consequently, the possibility of interfering in this interaction could represent a valid hypothesis for the development of promising therapeutic and prevention strategies. nebulized hypertonic ibuprofen has antibiotic, viricidal and anti-inflammatory effects that are expected to prevent viral replication in the respiratory tract, minimize bacterial superinfections and reduce inflammatory cytokines which appear to be the major problems in covid19 [41] . ibuprofen with the highest fda performance side effect profile, would be a promising adjunctive treatment for covid-19 patients with moderate-severe respiratory symptoms. additional research is needed to further define its efficacy and safety in treating covid-19. solubility of ibuprofen, phytosterol, salicylic acid, and naproxen in aqueous solutions ibuprofen extrudate, a novel, rapidly dissolving ibuprofen formulation: relative bioavailability compared to ibuprofen lysinate and regular ibuprofen, and food effect on all formulations solubility of ibuprofen in some ethanolþwater cosolvent mixtures at several temperatures formulation and delivery strategies of ibuprofen: challenges and opportunities high concentrations of sodium chloride improve microbicidal activity of ibuprofen against common cystic fibrosis pathogens pharmaceuticals in silico analysis suggests repurposing of ibuprofen for prevention and treatment of ebola virus disease toremifene interacts with and destabilizes the ebola virus glycoprotein ibuprofen as a template molecule for drug design against ebola virus inhibitors of signal peptide peptidase (spp) affect hsv-1 infectivity in vitro and in vivo inhibition of rotavirus infection in cultured cells by n-acetyl-cysteine, pparγ agonists and nsaids combined antiviral-antimediator treatment for the common cold ibuprofen modifies the inflammatory response of the murine lung to pseudomonas aeruginosa effects of ibuprofen on a pig pseudomonas ards model ibuprofen and methylprednisolone in a pig pseudomonas ards model effect of high-dose ibuprofen in patients with cystic fibrosis progression of cystic fibrosis lung diseases a function of serum immunoglobulin g levels: a 5-year longitudinal study antimicrobial activity of ibuprofen against cystic fibrosis-associated gram-negative pathogens ibuprofen causes photocleavage through ros generation and intercalates with dna: a combined biophysical and molecular docking approach comparison of three inhaled non-steroidal anti-inflammatory drugs on the airway response to sodium metabisulphite and adenosine 5'-monophosphate challenge in asthma effect of indomethacin on bronchorrhea in patients with chronic bronchitis, diffuse panbronchiolitis, or bronchiectasis in vivo study of indomethacin in bronchiectasis: effect on neutrophil function and lung secretion analgesic effect from ibuprofen nanoparticles inhaled by male mice early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia capsid protein structure in zika virus reveals the flavivirus assembly process nuevo brote de coronavirus 2019: un nuevo desafío first case of 2019 novel coronavirus in the united states early events during human coronavirus oc43 entry to the cell repulsion of superinfecting virions: a mechanism for rapid virus spread actin filament reorganization is a key step in lung inflammation induced by systemic inflammatory response syndrome high expression of ace2 receptor of 2019-ncov on the epithelial cells of oral mucosa this paper represents a collaborative effort among members of córdoba center for products and processes (ceprocor), the national council for scientific and technical research (conicet) and the private pharmaceutical company luar chemistry (química luar ®). we wish to acknowledge jessica buck for her technical assistance with english grammar and spelling.8 the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. key: cord-275353-ezrmuw48 authors: donma, mustafa metin; donma, orkide title: the effects of allium sativum on immunity within the scope of covid-19 infection date: 2020-06-02 journal: med hypotheses doi: 10.1016/j.mehy.2020.109934 sha: doc_id: 275353 cord_uid: ezrmuw48 the severity of coronavirus disease 2019 (covid‐19) infection is quite variable and the manifestations varies from asymptomatic disease to severe acute respiratory infection. fever, dry cough, dyspnea, myalgia, fatigue, loss of appetite, olfactory and gustatory dysfunctions are the most prevalent general symptoms. decreased immune system cells such as suppressed regulatory t cells, cytotoxic and helper t cells, natural killer cells, monocytes/macrophages and increased proinflammatory cytokines are the characteristic features. compounds derived from allium sativum (garlic) have the potential to decrease the expression of proinflammatory cytokines and to reverse the immunological abnormalities to more acceptable levels. allium sativum is suggested as a beneficial preventive measure before being infected with sars‐cov‐2 virus. allium sativum is a functional food well-known for its immunomodulatory, antimicrobial, antiinflammatory, antimutagenic, antitumor properties. its antiviral efficiency was also demonstrated. some constituents of this plant were found to be active against protozoan parasites. within this context, it appears to reverse most immune system dysfunctions observed in patients with covid-19 infection. the relations among immune system parameters, leptin, leptin receptor, adenosin mono phosphate-activated protein kinase, peroxisome proliferator activated receptor-gamma have also been interpreted. leptin’s role in boosting proinflammatory cytokines and in appetite decreasing suggest the possible beneficial effect of decreasing the concentration of this proinflammatory adipose tissue hormone in relieving some symptoms detected during covid-19 infection. in conclusion, allium sativum may be an acceptable preventive measure against covid-19 infection to boost immune system cells and to repress the production and secretion of proinflammatory cytokines as well as an adipose tissue derived hormone leptin having the proinflammatory nature. coronavirus disease 2019 , is related to a rna virus of the coronavirus family, severe acute respiratory syndrome coronavirus (sars-cov), which is renamed as sars-cov-2 [1, 2] . the severity of covid-19 infection is quite variable and the manifestations varies from asymptomatic disease to severe acute respiratory infection. persons with obesity are also at high risk due to chronic diseases associated with obesity. quarantine in obese subjects should likely be longer than normal weight individuals [3] . on the other hand, obese people already avoid social contact and experience high rates of depression. health care providers are expected to fight obesity more than ever. the covid-19 pandemic and the obesity epidemic threat the world in unprecedented ways [4] . this pandemic disease has also destroyed the lives of children and their families [5] . world health organization declares that although all age groups are at risk of covid-19, older people face significant risk of developing severe illness, due to physiological changes, especially in immune system, associated with ageing and presence of chronic diseases [6]. the profiles of some immune cells in covid-19 have also been clarified. decreased regulatory t (treg) cells, cytotoxic and helper t cells, natural killer (nk) cells, monocytes/macrophages have been pointed out. on the other hand, proinflammatory cytokines such tumor necrosis factor-alpha (tnf-α), interleukin-1 (il-1), interleukin-2 (il-2), interleukin-6 (il-6), interferon gamma (ifn-γ), as well as leptin are among those exhibiting an increasing tendency [12] [13] [14] . the beneficial effects of allium sativum (garlic) on health have been emphasized for centuries. garlic contains numerous compounds that have the potential to influence immunity [15, 16] . in recent reports, garlic and its complex constituents have been investigated as promising candidates for improving immune system. garlic extracts and compounds isolated were examined in terms of their immunoregulatory functions in detail. it is a well-known fact that immune dysfunction plays an important role in the development and progress of several diseases and this functional food may contribute to the prevention and treatment of pathologies such as obesity, metabolic syndrome, cardiovascular disorders, gastric ulcer, and even cancer [17, 18] . aged garlic extract (age) might be used as herbal medicine with few side effects as compared to chemotherapy in treating cancers caused by substances like aflatoxin b1 [19] . garlic participates in cytokine secretion modulation, which may provide a mechanism of action for many of its therapeutic effects. alliin is the main organosulfur compound in garlic and has been shown to induce a decrease in the expression of proinflammatory cytokines [17, 18] . there are some suggestions towards increasing hemoglobin production and increasing hemoglobin availability for oxygen binding [20] . it is also hypothesized that patients with severe covid-19 infection may have a severely compromised immune response and could be treated by monalizumab, interferon α, chloroquine, and other antiviral agents [21] . in the current emergency state, there are some suggestions related to the use of all available therapeutic tools and the potential antiretroviral activity of hesperidin as well as routine as co-treatment or as preventive treatment in patients with covid-19 infection [22] . today, it is well understood that prevention of this infection will be much more plausible than treatment to get rid of the outbreak of this epidemic disease. allium sativum seems to counteract most of the negativities caused by covid-19 infection. within the scope of preventive measures, this functional food may prevent this viral agent from spreading over the body. we suggest that administration of this plant will contribute to the immune system elements during the fight against this pathogen. t tumor cells [23] [24] [25] [26] . in patients with covid-19 infection, supression of these cells were noted. in severe cases, they are even much lower. as one of the results of this profile, a reduction in ifn-γ was also observed [12] [13] [14] . garlic supplementation causes significant increases in cd4+ and cd8+ cells. this plant also stimulates nk cells [18, 27] . decreased leptin, leptin receptor, peroxisome proliferator activated receptor-gamma (ppar-γ) and il-6 concentrations were also detected as the other beneficial immunological and hormonal effects of the garlic [17, 28, 29] . leptin leads to appetite decrease [30] . decreased leptin concentrations caused by garlic may be helpful to alleviate appetite loss observed in patients with covid-19 infection. when all these findings including antimicrobial and antiviral features of garlic were evaluated together, our hypothesis is that it is plausible to consider this plant as a preventive measure to alleviate harmful effects of the disease. the recently emerged coronavirus mainly act on lymphocytes. the sars-cov-2 infection affects primarily t lymphocytes particularly cd4+ t and cd8+ t cells, resulting in decrease in ifn-γ production. absolute number of t lymphocytes, cd4+ t and cd8+ t cells decreased in nearly all the patients, and were markedly lower in severe cases than moderate cases. patients with covid-19 exhibit lower level of treg cells, and more obviously damaged in severe cases. natural killer cells decreased in covid-19 patients, and severe cases had a lower level than mild cases. these immunological markers may be of importance due to their correlation with disease severity in covid-19 [12] [13] [14] . figure 2 shows alterations observed during covid-19 infection. on the other hand, significant increases in cd4+ and cd8+ t cells were observed following a short-term garlic extract supplementation. this finding showed that using garlic extract supplementation promoted cellular immune system of karate athletes [27] . allicin is the major biologically active component of garlic and shows anti-microbial activity. in recently performed studies, it was also reported that this functional food also combats against malaria parasite, tumor growth and cytomegalovirus [15, [31] [32] [33] [34] . allicin is also active against protozoan parasites including plasmodium, which is thought to be mediated by inhibiting cysteine proteases. it was demonstrated that allicin reduced parasitaemia in malaria. the absolute numbers of cd4+ t cells, dendritic cells (dc) and macrophages were significantly higher in allicin-treated mice with malaria infection [31] . in a recent report, the use of antimalarial drugs have been recommended for prophylaxis and treatment of high-risk individuals with sars-cov-2 infection [32] . allitridin (allyl trisulfide) can promote cytomegalovirus (cmv)-induced treg expansion and treg-mediated anti-cmv immunosuppression. therefore, allitridin may be useful as a therapeutic agent fighting against cmv [34] . garlic has antiinflammatory, antimutagenic and antitumor properties. intra-tumor injection of a protein fraction purified from fresh garlic bulbs significantly increased cd8+ t lymphocytes in the peripheral blood, augmented cd8+ t cell infiltration into the tumor site, decreased tumor size and inhibited tumor growth in experimental studies [15, 33] . garlic seems to increase the immune system functions. it stimulates macrophages, lymphocytes, nk cells, dc and eosinophils, by mechanisms including modulation of cytokine secretion, immunoglobulin synthesis, phagocytosis and macrophage activation [18] . after 45 days of age consumption, γδ-t and nk cells proliferated better and were more activated than cells of the placebo group [16] . rats. this showed the immune system boosting capability of garlic [35] . in a study, salad lowered participants' desire to eat and caused satiety compared to the garlic bread. dieters were less hungry after the salad compared to the garlic bread [36] . this data showed the appetite increasing effect of garlic. it has been reported decreases in gene expression and serum protein levels of the adipocytokines leptin and resistin, as well as decreased serum il-6 concentrations. treatment with alliin reduces metaflammation markers in diet induced obesity mice and improves some metabolic parameters without affecting others [17] . the effects of allium sativum on various systems are shown in figure 3 . aged garlic extract suppresses the production of proinflammatory cytokines such as tnf-α and crp in the liver [37] . in the hypothalamus, aged black garlic (abg) treatment induced a decrease in leptin receptor (lepr) mrna levels. in subcutaneous adipose tissue, abg treatment decreased adipose weight and downregulated the gene expression of ppar-γ and lepr. in brown adipose tissue, ppar-γ mrna levels were significantly decreased in abg-treated rats [28] . leptin activates adenosin mono phosphate-activated protein kinase (ampk) and activated ampk inhibits ppar-γ transcriptional activity in hepatoma cells [29] . peroxisome proliferator activated receptor-γ is also suggested to be involved in the treatment protocols of obesity and depression [38] . leptin secreted from adipose tissue stimulates ampk in the liver. ampk phosphorylates acetyl coa carboxylase. the phosphorylation inactivates this enzyme, which converts acetyl coa to malonyl coa. inhibition of this reaction prevents the synthesis of fatty acids. the increase in ampk activity stimulates fatty acid oxidation enzymes and leads to reduced triacylglycerols. depending upon these effects, fatty acid synthesis will be inhibited and fatty acid oxidation (fao) will take action. this leads to reductions in triacylglycerol (tag) levels [39] . the physiological, biochemical and immunological effects of leptin are shown in the investigations related to the developmental trials of the vaccine, which may require a long period of time are still in progress. isolation and social distancing are primary interventions [3] . however, under the light of these findings, garlic may also be proposed during the prevention of covid-19. garlic was known as a powerful antibacterial agent for years, and also has been shown to be beneficial in cancer and hypertension therapies [15, 31, 34] . it is known that both cancer and hypertension are also the main morbidity and mortality risk factors for covid-19 infection. in this viral infection, most of the immune-compromised patients have losted their lives during the medical treatments in intensive care units because of bacterial sepsis exhibiting high c-reactive protein levels, septic shock and multiorgan failures. this information concerning garlic has a special importance on this issue. this food may be helpful 1. to reverse some signs and symptoms observed in these patients, 2. to reincrease or regain the decreased or lost gustatory sense, 3. to increase the number of treg cells, 4. to increase cytotoxic and helper t cells, 5. to decrease the levels of leptin, leptin receptor and ppar-γ 6. to increase appetite, 7. to prevent the inhibition of cd4 + cd25 + foxp3 + , treg cells, 8. to decrease il-6 concentrations, 9. to stimulate nk cells, 10. to suppress tnf-α and creactive protein. immuno-compromised clinical status is life-threatening for patients with covid-19 infection, therefore, these beneficial effects of allium sativum on immune system are quite important. novel coronavirus pneumonia emergency response epidemiology team. the epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (covid-19) in china the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2 influenza and obesity: its odd relationship and the lessons for covid-19 pandemic covid 19 and the patient with obesity -the editors speak out coronavirus disease (covid-19) unicef" olfactory and gustatory dysfunctions as a clinical presentation of mild-to-moderate forms of the coronavirus disease (covid-19): a multicenter european study unique epidemiological and clinical features of the emerging 2019 novel coronavirus pneumonia (covid-19) implicate special control measures covid-19 and anosmia in tehran clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study dysregulation of immune response in patients with covid-19 in wuhan, china characteristics of peripheral lymphocyte subset alteration in covid-19 pneumonia clinical and immunologic features in severe and moderate coronavirus disease chemical constituents and pharmacological activities of garlic (allium sativum l.): a review aged garlic extract modifies human immunity alliin, an allium sativum nutraceutical, reduces metaflammation markers in dio mice immunomodulation and antiinflammatory effects of garlic compounds evaluation of the number of cd4(+) cd25(+) foxp3(+) treg cells in normal mice exposed to afb1 and treated with aged garlic extract respiratory conditions in coronavirus disease 2019 (covid-19): important considerations regarding novel treatment strategies to reduce mortality innate immunity in covid-19 patients mediated by nkg2a receptors, and potential treatment using monalizumab, chloroquine, and antiviral agents phytotherapeutics and sars-cov-2 infection: potential role of bioflavonoids foxp3 (+) t regulatory cell levels in obese, asthmatic, asthmatic obese, and healthy children the molecular mechanism of natural killer cells function and its importance in cancer immunotherapy effect of short-term garlic supplementation on cd4 and cd8 factors in young karate athletes after intense exercise session beneficial effects of an aged black garlic extract in the metabolic and vascular alterations induced by a high fat/sucrose diet in male rats activated ampk inhibits ppar-{alpha} and ppar-{gamma} transcriptional activity in hepatoma cells physiology, obesity neurohormonal appetite and satiety control allicin enhances host pro-inflammatory immune responses and protects against acute murine malaria infection can it be a novel drug for covid-19 purified protein fraction of garlic extract modulates cellular immune response against breast transplanted tumors in balb/c mice model allium sativum-derived allitridin inhibits treg amplification in cytomegalovirus infection effect of allium cepa and allium sativum on some immunological cells in rats slimming starters. intake of a dietcongruent food reduces meal intake in active dieters mechanisms underlying the attenuation of chronic inflammatory diseases by aged garlic extract: involvement of the activation of amp-activated protein kinase (review) promising link between selenium and peroxisome proliferator activated receptor gamma in the treatment protocols of obesity as well as depression role of leptin and adiponectin in insulin resistance key: cord-276564-o21ncldx authors: miller, r.; wentzel, a.r.; richards, g. title: covid-19: nad(+) deficiency may predispose the aged, obese and type2 diabetics to mortality through its effect on sirt1 activity date: 2020-06-29 journal: med hypotheses doi: 10.1016/j.mehy.2020.110044 sha: doc_id: 276564 cord_uid: o21ncldx the sars-cov-2 hyperinflammatory response is associated with high mortality. this hypothesis suggests that a deficiency of nicotinamide adenine dinucleotide (nad(+)) may be the primary factor related to the sars-cov-2 disease spectrum and the risk for mortality, as subclinical nutritional deficiencies may be unmasked by any significant increase in oxidative stress. nad(+) levels decline with age and are also reduced in conditions associated with oxidative stress as occurs with hypertension, diabetes and obesity. these groups have also been observed to have high mortality following infection with covid-19. further consumption of nad(+) in a pre-existent depleted state is more likely to cause progression to the hyperinflammatory stage of the disease through its limiting effects on the production of sirt1. this provides a unifying hypothesis as to why these groups are at high risk of mortality and suggests that nutritional support with nad(+) and sirt1 activators, could minimise disease severity if administered prophylactically and or therapeutically. the significance of this, if proven, has far-reaching consequences in the management of covid-19 especially in third world countries, where resources and finances are limited. covid-19: nad + deficiency may predispose the aged, obese and type2 diabetics to mortality through its effect on sirt1 activity we hypothesize that reduced nicotinamide adenine dinucleotide (nad + ) levels with consequent deficient activity of the nad + dependent molecule sirt1, which modulates cytokine production, may be the factor that predisposes the aged, obese, type 2 diabetics and other vulnerable groups to an increased mortality. the sars-cov-2 hyperinflammatory response is associated with high mortality. this hypothesis suggests that a deficiency of nicotinamide adenine dinucleotide (nad + ) may be the primary factor related to the sars-cov-2 disease spectrum and the risk for mortality, as subclinical nutritional deficiencies may be unmasked by any significant increase in oxidative stress. nad + levels decline with age and are also reduced in conditions associated with oxidative stress as occurs with hypertension, diabetes and obesity. these groups have also been observed to have high mortality following infection with covid-19. further consumption of nad + in a pre-existent depleted state is more likely to cause progression to the hyperinflammatory stage of the disease through its limiting effects on the production of sirt1. this provides a unifying hypothesis as to why these groups are at high risk of mortality and suggests that nutritional support with nad + and sirt1 activators, could minimise disease severity if administered prophylactically and or therapeutically. the significance of this, if proven, has far-reaching consequences in the management of covid-19 especially in third world countries, where resources and finances are limited. background covid-19 may be asymptomatic or manifest in 3 clinical phases, an initial upper respiratory tract infection, with a few patients thereafter progressing to a pneumonic phase, and an even smaller number to the hyperinflammatory phase which may be lethal. (1) the aim of any therapy would be to intervene at an early stage, either prophylactically or therapeutically, to prevent progression of the disease to a point where mechanical ventilation (mv) is required, or significant organ dysfunction occurs. (2) risk factors for a poor outcome include older age, comorbidity (in particular diabetes, hypertension and cardiac disease), non-asthmatic respiratory disease, obesity, immunosuppression and male sex. (2, 3) the independent associations of advancing age, male sex, chronic respiratory conditions (though not well controlled asthma), chronic cardiac and chronic neurological disease with in-hospital mortality, are in line with other international reports. (4) it is difficult however to determine why these conditions specifically are linked to mortality. docherty et al. report that severe sars-cov-2 infections are rare in those under 18 years of age, comprising only 1.4% of those admitted to hospital. only 0.8 % of those in this study were under 5 years of age, and this "j" shaped age distribution was starkly different from the "u" shaped distribution seen in seasonal influenza. (5) it has not been clear from observational studies however, why sars-cov-2 mostly spared children, but it has been speculated that it is due to differential expression of the ace2 receptor in the developing lung. (6) similarly, pregnancy has not been reported to be associated with mortality, in contrast to the situation with influenza. (4, 7) while the general concept of an excessive or uncontrolled release of pro-inflammatory cytokines is well known, an actual definition of what a hyperinflammatory response or "cytokine storm" is, is lacking. furthermore, there is a poor understanding of the molecular events that precipitate this response and the contribution it makes to pathogenesis. it is also not known what therapeutic strategies might be used to prevent this catastrophic progression of the disease or lessen its severity once initiated. (8) in this phase, there is an unbalanced and exacerbated inflammatory response with the release of inter alia, tumor necrosis factor (tnf-α), interleukin 1β (il-1β), interleukin 6 (il-6), as pro-inflammatory mediators together with interleukin 10 (il-10) and interferon β as anti-inflammatory mediators. the complex interactions between tnf-α, the interleukins, chemokines and interferons in sars-cov-2 are currently poorly understood; however, they are associated with and related to a significant viraemia. (9, 10) the fact that most international studies have indicated that certain risk factors were common suggested that a similar systemic abnormality might be present in those at high risk, predisposing to severe illness or mortality. in this context, the nicotinamide adenine dinucleotide (nad + )-and zincdependent molecule, the silent information regulator 1 (sirt1) represents a potential common thread in the aetiology of the hyperinflammatory response and increased mortality. sars-cov-2 targets and binds to the angiotensin-converting enzyme 2 receptor (ace2r), a membrane-associated aminopeptidase also expressed in the vascular endothelium, renal and cardiovascular tissue, and small intestine, testis, and respiratory epithelia. (11) the ace2r acts as the host cell receptor for the virus which binds via the spike protein on the viral capsid. (12) this stimulates clathrin-dependent endocytosis of both the ace2r and virus, events that are essential for infectivity. this process induces adam 17 activity which reduces expression of ace2 on the cell surface. (13) nicotinamide adenine dinucleotide (nad + ) nad + is a cofactor found in every cell of the body, and it is involved in multiple metabolic pathways. it is a fundamental housekeeping molecule that catalyses electron transfer in metabolic reductionoxidation reactions, functioning as an electron shuttle in the production of adenosine triphosphate (atp). increased age is a strong predictor of sars-cov-2-associated in-hospital mortality after adjusting for comorbidity. (6) older patients have also been identified as having the lowest levels of nad + , (14) while, conversely, those with the lowest risk, infants and children have the highest levels. the decline in nad + levels with ageing is mainly dependent on cd38, a 45kda transmembrane molecule, encoded on chromosome 4. in leukocytes, it acts as a receptor in adhesion and signalling pathways. (15) cd38 expression is increased with insulin resistance, and may exacerbate the age-dependent decline of nad + .(9) nad + and nadp profoundly affect age-influencing factors such as oxidative stress and mitochondrial activity, and nad + -dependent sirtuins also mediate the ageing process. (10) as humans, age, antioxidant defence mechanisms such as glutathione production are also depleted and the associated increase in reactive oxidative species (ros) causes all cells (16) to enter a state of pseudohypoxia in which the ratio of nad + /nadp declines. (17) (18) (19) oxidative stress also activates the nad + -dependent enzyme, poly adp ribose polymerase 1 (parp1). (20) hyperactivity of parp1 results in depletion of cellular nad + pools, leading to atp deficiency, energy loss, and subsequent cell death. these processes have the potential to enhance the pro-inflammatory cascade. nad + deficiency impairs sirt1 function (21) and its successful activation. whereas extreme niacin deficiency is associated with the development of pellagra, more subtle decreases occur in diabetes, ageing and hypertension with resultant attenuation of responsiveness to inflammatory stimuli. (22) silent information regulator 1 (sirt1) sirtuins are an ancient family of seven nad + -dependent deacylase and mono-adp-ribosyl transferase signalling proteins that are intrinsically involved in metabolic regulation and cellular homeostasis. of particular interest is sirt1, which downregulates adam 17 (a disintegrin and metalloproteinase domain 17), also called tnf-α converting enzyme (tace), by increasing expression of timp3 the gene that encodes for tissue metalloproteinase inhibitor 3. (23) in so doing it decreases levels of tnf-α, il-1b and il-6. an increase in tnf-α causes sirt1 to down-regulate adam 17, thereby controlling tnf-α formation in a negative feedback loop that secondarily influences il-1b and il-6 production, which are dependent on tnf-α. (23) sirt1 is known to play a crucial role in obesity-associated metabolic diseases, cancer, ageing, cellular senescence, cardiac ageing and stress, prion-mediated neurodegeneration, inflammatory signalling in response to environmental stress, embryonal development of the heart, brain, spinal cord and dorsal root ganglia, and placental cell survival. (24) in its inactive or open state, it contains a zn ++ module and an nad +binding site. (25) whereas certain conditions such as ulcerative colitis, crohn's disease, short bowel syndrome, renal failure, alcoholism, and inadequate meat intake are specifically associated with zinc deficiency there is evidence that zinc intake among older adults might be marginal. an analysis of the third national health and nutrition examination survey (nhanes iii), 1988-1994 data found that 35-45% of adults aged 60 years or older had a zinc intake below estimated average requirements. (26) when nad + binds to the sirt1 molecule in the presence of the zn ++binding module, it undergoes a structural change, enveloping the nad + molecule and causing it to be "closed" or activated. (25) the presence of both the zn ++ and the nad + moieties are imperative for its function. sirt1 downregulates adam17 and cytokine production adam17 is a proteinase encoding gene. tnf-α and the cytokine receptor for il-6 must be proteolytically cleaved in order to be systemically active, and adam17 provides this function. if adam17 expression is not downregulated by sirt1, tnf-α and il-6 are released, resulting in an uncontrolled hyperinflammatory response as may occur with covid-19. (23, 27-30) sirt 1, by inhibition of adam17 and thereby tnf-α and il-6, performs an anti-inflammatory function. (31) (32) (33) (34) (35) (36) (37) (38) if oxidative stress is severe, increased adam17 attempts to ameliorate tissue injury by converting active iron (fe2 + ) to its inert form (fe3 + ) which is stored in hepatocytes and macrophages and as ferritin by means of the fenton reaction (fe2 + +h 2 o 2 →fe 3+ + ho • + oh − ), (fe 3+ + h 2 o 2 →fe 2+ + ho 2 • + h + ). this also potentially transforms haemoglobin to methaemoglobin, reducing its capacity to bind to oxygen. (39) covid-19 replication and sirt1 sirt1 not only controls and modifies the inflammatory response, but along with the sirtuin family (sirt1-7) is also a primary defence against dna and rna viral pathogens. (40) in some respiratory infections and cardiovascular conditions, sirt1 promotes autophagy (the destruction of damaged or redundant cellular components occurring in vacuoles within the cell), and in so doing inhibits apoptosis and provides protection against hypoxic stress. [37] [38] [39] [40] upregulation of sirt1 directly decreases viral replication and inhibits the activation of adam17, thereby decreasing tnf-α, il-1b and il-6. conversely depletion of sirt1 allows for increased viral replication with little or no inhibition of adam17 activity, causing uncontrolled increases in tnf-α, il-6 and il-1b. whereas an increase in tnf-α would usually increase sirt1 activity to downregulate adam17, in the presence of a deficiency of nad + or zn ++ , this would not occur due to insufficient activation of sirt1, causing an unchecked increase in tnf-α. in both obesity and type 2 diabetes mellitus, intracellular nad + levels are decreased in multiple tissues, including adipose tissue, skeletal muscle, liver and the hypothalamus. (41) furthermore, both conditions are characterised by low-grade inflammation associated with activation of both il6 and tnf-α. (42, 43) obesity or type 2 diabetes mellitus would increase the risk for cytokine storm due to an inability to activate sirt1. sirt1 maintains vascular endothelial function, preventing or reducing the potential for the metabolic syndrome, ischaemia-reperfusion injury and inflammation in obesity. with increasing age however, nad + levels and sirtuin activity decline and this is exacerbated by obesity and sedentary lifestyles (22) . sirt1 is an effective inhibitor of oxidative stress in vascular endothelial cells (ec) (44) via various signalling pathways. (45) the endothelial glycocalyx (eg) is a web of membrane-bound glycoproteins on the luminal side of endothelial cells, associated with various glycosaminoglycans that cover the vascular endothelium. (46) the eg separates cellular blood components from the endothelium and maintains osmotic tension of the intravascular compartment. (44, 45) conditions causing damage to, and shedding or fragmentation of the eg, (as seen in sarscov-2 under severe oxidative stress induced by the hyperinflammatory response), exposes the endothelium, allowing adhesion, clumping and activation of platelets with degranulation and release of vasoactive substances. the eg has anticoagulant properties as it is a binding site for mediators such as heparin cofactor ii, antithrombin, thrombomodulin and tissue factor pathway inhibitor (tfpi). heparin cofactor ii and dermatan sulphate inhibit thrombin, and antithrombin activity is enhanced when bound to heparan sulphate. conversely, exposure of the endothelial cell surface protein, thrombomodulin, which contains a cofactor for thrombin, chondroitin sulphate, promotes coagulation via activation of tissue factor (46) as seen in sar-cov-2. the eg is already compromised in systemic inflammatory states, such as diabetes, hyperglycaemia, surgery, trauma and sepsis. (46) under conditions of more severe oxidative stress, as in the hyperinflammatory response, widespread damage may lead to its destruction, with the occurrence of capillary leak and oedema formation, accelerated inflammation, platelet aggregation, hypercoaguability and a loss of vascular responsiveness. (47) inflammatory mediators that are implicated in this process are tnf-α, bradykinin, c-reactive protein and mast cell tryptase. given the above, it is possible that activation of sirt1 may be a crucial factor in the prevention of the hyperinflammatory response and may be necessary for a successful defence against viral attack. vulnerable patient groups would potentially be less likely or unable to ensure sufficient activation of sirt1 due to low nad + levels or associated nutritional deficiencies including zn ++ , and as such contribute to an inability to control viral replication and reduce the uncontrolled expression of pro-inflammatory cytokines. the sars-cov-2 hyperinflammatory response is associated with high mortality. a deficiency of nad + , in the context of an elevated cd38, may be the primary factor related to the sars-cov-2 disease spectrum and the risk of mortality, as subclinical nutritional deficiencies may be unmasked by any significant increase in oxidative stress. nad + levels decline with age and are also reduced in conditions associated with oxidative stress as occurs with hypertension, diabetes and obesity. these same groups have also been observed to have high mortality following infection with covid-19. further consumption of nad + in a pre-existent depleted state is more likely to cause progression to the hyperinflammatory stage of the disease through its limiting effects on the production of sirt1. given that activation of sirt1 is dependent on the availability of nad + and zinc and that high levels of oxidative stress deplete nad + , thereby decreasing sirt1 activity, nutritional support with nad + precursors and sirt1 activators, could minimise disease severity if administered prophylactically and or therapeutically. the significance of this hypothesis, if proven, has far-reaching consequences in the management of covid-19 especially in third world countries, where resources and finances are limited. robert miller is managing director of battle brew tactical nutrition covid-19 illness in native and immunosuppressed states: a clinical-therapeutic staging proposal covid-19 and the rationale for pharmacotherapy: a south african perspective opensafely: factors associated with covid-19-related hospital death in the linked electronic health records of 17 million adult nhs patients host susceptibility to severe influenza a virus infection predictors of clinical outcome in a national hospitalised cohort across both waves of the influenza a/h1n1 pandemic 2009-2010 in the uk features of 16,749 hospitalised uk patients with covid-19 using the isaric who clinical characterisation protocol risk factors for hospitalisation and poor outcome with pandemic a/h1n1 influenza: united kingdom first wave into the eye of the cytokine storm why nad(+) declines during aging: it's destroyed age-associated changes in oxidative stress and nad+ metabolism in human tissue ace2 receptor expression and severe acute respiratory syndrome coronavirus infection depend on differentiation of human airway epithelia focus on receptors for coronaviruses with special reference to angiotensin-converting enzyme 2 as a potential drug target -a perspective angiotensin-converting enzyme 2: the first decade why does covid-19 disproportionately affect the elderly? new additions to antibody panels in the characterisation of chronic lymphoproliferative disorders aging-related correlation between serum sirtuin 1 activities and basal metabolic rate in women, but not in men aging and cigarette smoking are associated with decreased glutathione levels in humans nad+/nadh and nadp+/nadph in cellular functions and cell death: regulation and biological consequences investigating mitochondrial redox state using nadh and nadph autofluorescence calorie restriction and sirtuins revisited sirtuins and nad(+) in the development and treatment of metabolic and cardiovascular diseases mmps, adams and their natural inhibitors in inflammatory bowel disease: involvement of oxidative stress mammalian sirt1: insights on its biological functions structural and functional analysis of human sirt1 analysis of zinc data from the second national health and nutrition examination survey (nhanes ii) timp3 is reduced in atherosclerotic plaques from subjects with type 2 diabetes and increased by sirt1 tnf-α and lps activate angiogenesis via vegf and sirt1 signalling in human dental pulp cells negative regulation of inflammation by sirt1 interferon β protects against lethal endotoxic and septic shock through sirt1 upregulation sirt1 mediates a primed response to immune challenge after traumatic lung injury sirtuins link inflammation and metabolism induction of sirtuin-1 signaling by resveratrol induces human chondrosarcoma cell apoptosis and exhibits antitumor activity negative regulation of nlrp3 inflammasome by sirt1 in vascular endothelial cells sirt1mediated regulation of oxidative stress induced by pseudomonas aeruginosa lipopolysaccharides in human alveolar epithelial cells sirt1 activators suppress inflammatory responses through promotion of p65 deacetylation and inhibition of nf-κb activity sirt1 exerts anti-inflammatory effects and improves insulin sensitivity in adipocytes sirt1 inhibits inflammatory pathways in macrophages and modulates insulin sensitivity iron-overload triggers adam-17 mediated inflammation in severe alcoholic hepatitis intricate roles of mammalian sirtuins in defense against viral pathogens implications of altered nad metabolism in metabolic disorders inflammation as a link between obesity, metabolic syndrome and type 2 diabetes obesity and inflammation: the linking mechanism and the complications sirt1 inhibits oxidative stress in vascular endothelial cells revised starling equation and the glycocalyx model of transvascular fluid exchange: an improved paradigm for prescribing intravenous fluid therapy the role of endothelial glycocalyx in health and disease derangement of the endothelial glycocalyx in sepsis robert miller is managing director of battle brew tactical nutrition key: cord-274698-i3mzzxwq authors: stief, thomas w title: the physiology and pharmacology of singlet oxygen date: 2003-02-19 journal: med hypotheses doi: 10.1016/s0306-9877(03)00026-4 sha: doc_id: 274698 cord_uid: i3mzzxwq reactive oxygen species (ros) are generated by many different cells. singlet oxygen (1o2) and a reaction product of it, excited carbonyls (co(*)), are important ros. 1o2 and co(*) are nonradicalic and emit light (one photon/molecule) when returning to ground state oxygen. especially activated polymorphonuclear neutrophil granulocytes (pmn) produce large amounts of 1o2. via activation of the respiratory burst (nadph oxidase and myeloperoxidase) they synthesize hypochlorite (naocl) and chloramines (in particular n-chlorotaurine). chloramines are selective and stable chemical generators of 1o2. in the human organism, 1o2 is both a signal and a weapon with therapeutic potency against very different pathogens, such as microbes, virus, cancer cells and thrombi. chloramines at blood concentrations between 1 and 2 mmol/l inactivate lipid enveloped virus and chloramines at blood concentrations below 0.5 mmol/l, i.e. at oxidant concentrations that do not affect thrombocytes or hemostasis factors, act antithrombotically by activation of the physiologic pmn mediated fibrinolysis; this thrombolysis is of selective nature, i.e. it does not impair the hemostasis system of the patient allowing the antithrombotic treatment in patients where the current risky thrombolytic treatment is contraindicated. the action of 1o2 might be compared to the signaling and destroying gunfire of soldiers directed against bandits at night, resulting in an autorecruitment of the physiological inflammatory response. chloramines (such as the mild and untoxic oxidant chloramine t(®) (n-chloro-p-toluene-sulfonamide)) and their signaling and destroying reaction product 1o2 might be promising new therapeutic agents against a multitude of up to now refractory diseases. the human redox state is a balanced system of pro-and anti-oxidants. the main cellular reactive oxygen species (ros) are hydrogen peroxide ðh 2 o 2 þ, superoxide anion ðo åà 2 þ, hydroxyl radical ðho å þ, and singlet oxygen ð 1 o 2 þ. singlet oxygen -in contrast to the other oxidants -is nonradicalic and excited, i.e. 1 o 2 or the reaction product of 1 o 2 with a c@c group, i.e. an excited carbonyl, emits 1 photon when returning to ground state oxygen (1) . whereas the radicalic oxygen species are harmful for the organism, nonradicalic 1 o 2 is rather mild and untoxic for mammalian tissue. this mild oxidative character has been used for diagnostic purposes, such as the radiohalogenation of proteins (2) (3) (4) . ros are generated by pro-oxidative enzyme systems or by redox-cycling of pro-oxidative compounds. pro-oxidative enzymes are the nadph-oxidase (5), myeloperoxidase (6) , no-synthase (7, 8) , or the cytochrome p-450 chain (9) (10) (11) . physiologic activation of these pro-oxidative enzymes results into the normal oxidative state. nadph-oxidase is mainly found in polymorphonuclear leukocytes (pmn). the membranous nadph-oxidase generates superoxide anions that dismute to hydrogen peroxide. h 2 o 2 can react with superoxide anions or with hocl or chloramines to form the nonradicalic 1 o 2 (10, 11) . since nadph-oxidase is present in many (5), diverse cells seem to generate the signal/messenger 1 o 2 for inter-or intra-cellular signaling. 1 o 2 is a cell signal and messenger (12) (13) (14) : redox active agents regulate ion channel activity in animals and plants (15) . 1 o 2 activates large-conductance, ca 2þ -activated (maxi) k þ channels (16): monochloramine ðnh 2 clþ -in contrast to tau-cl -is membrane permeating and at 3-30 lmol=l it increases outward currents more than 8-fold (17, 18) . 1 o 2 , generated by chloramine-t â (n-chloro-p-toluene-sulfonamide), also inactivates the na þ currents from skeletal or heart muscle fibers, presumably by oxidation of methionine residues (19) (20) (21) . chloramine-t â has also been shown to modulate dose dependently outward currents in rabbit atrial cells (22, 23) or potassium channels (24) (25) (26) (27) . chloramine-t â is known to abolish inactivation of na þ and k þ channels (28) (29) (30) (31) (32) (33) . potential receptors for excited oxygen species/ light are cryptochromes (34) , that consist of flavin-and pteridine-prosthetic groups. pteridines seem to interact with excited oxygen (35-37). chloramine-t â is bactericidal (38, 39) . n-chloramines exhibit low toxicity and skin irritation and are superior to chlorhexidine in preventing the expansion of the normal skin flora in vivo (40) . chloramine-t â is better than hocl in inactivation of staphylococcus aureus (41) and monochloramine is superior to n-chlorotaurine in inactivation of mycobacterium terrae (42) . naocl shows higher activity than chloramine-t â against bacillus subtilis spores, coat and cortex material was degraded by chloramine-t â (43) . because of their untoxicity and antimicrobial power (44), chloramines -especially chloramine-t â -is used for disinfection of drinking water, dialysate, or ice cream machines (45) (46) (47) (48) . chloramine t â is also a therapeutic drug for treating bacterial gill disease, a predominant disease of a variety of fish species (49) . however, chloramine-t â at 10 g/l (35 mm) has been shown to be ineffective as fungicide (50) . chloramines are virucidal, too (51) (52) (53) (54) (55) (56) . even such dangerous viruses as the marburg virus (57), or the ebola virus (58, 59) are inactivated by chloramines. bhanja virus (60), lymphocytic choriomeningitis virus (61) , simian rotavirus (62), or poliovirus (63) (64) (65) are sensible to naocl/chloramines. even replicating agents of the creutzfeldt-jakob disease show some sensibility to naocl (74, 75) . poliovirus on whole hands is inactivated (reduction factor >100) by 35 mm chloramine t â (63, 67) . coxsackievirus b3, adenovirus type 5, parainfluenza virus type 3 and coronavirus 229e are inactivated (reduction factor >1000) by a 100 mm chloramine-t â solution (68) . naocl inactivates hiv-1 (66, (69) (70) (71) (72) . the 1.5 mm naocl inactivated more than 10 000 fold hiv in serum and 7.5 mm more than 10 fold in blood (73) . own experiments show that chloramine-t â at blood concentrations that are tolerable for normal hemostasis function inactivate the lipid enveloped model virus vsv (vesicular stomatitis virus): 1 mmol/l chloramine-t â inactivates 90% of added vsv, 2 mmol/l chloramine-t â inactivate 99% of added vsv, i.e. there seems to exist a narrow therapeutical window for 1 o 2 treatment of human infections by enveloped viruses. intravenous infusions of 1-1.5 mmol/l (blood concentration) chloramine (chloramine-t â or the physiologic n-chlorotaurine) once a week for several weeks might be a potent treatment modality for infections with lipid enveloped viruses, such as human immunodeficiency virus (hiv) (74) . singlet oxygen is tumoricidal (75) . in photodynamic therapy (pdt) high concentrations of singlet oxygen are generated by illumination of a photosensitizer, resulting in a cytostatic action of pdt (76, 77) . however, excessive oxidant concentrations are carcinogenic (78) (79) (80) (81) (82) . 1 o 2 mediates pmn adherence to the endothelium (12, 83, 84) and subsequently selective thrombolysis (10, 11) . 1 o 2 activates the complement cascade, transforming c5 into a c5b-like molecule (85) ; activation of the complement cascade results in increased pmn adhesion to endothelial cells (86, 87) . since cholesterol is an inhibitor of 1 o 2 , the atherogenic action of cholesterol might be explained by insufficient thrombolytic capacity of a hypercholesterolemic individuum (10, 11, 88) . however, and according to paracelsus (dosis sola venenum facit (only the dosage makes the poison)), high concentrations of chloramines can act toxic to normal tissue (89) . 3 mm monochloramine induced dna breakage (90) . pmn are the main cells that use singlet oxygen as a weapon. they also dispose of an enzyme that reverses methionine oxidation -the methionine sulfoxide-peptide reductase (91) . taurine-chloramine is the major chloramine generated in activated pmn as a result of the reaction between hocl (92) and taurine, an abundant free amino acid in their cytosol (93) (94) (95) (96) . also other plasma proteins react with hypochlorite to chloramines (97). hocl ð25 lmþ or nh 2 cl ð10 lmþ -but not tau-cl ð100 lmþ -increase endothelial permeability (98) or epithelial cell injury (99) . nh 2 cl, the reaction product of hypochlorite with ammonia (nh 3 ), seems to be more toxic than tau-cl (100, 101) . the 60 mm nh 2 cl (about 10 times the concentration generated by activated pmn!) is ulcerogenic in rat stomachs, taurine application (1 ml 200 mm) attenuates the deleterious action of nh 2 cl (102), nh 2 cl induces apoptosis in gastric mucosa (103) . tau-cl selectively modulates the ability of dendritic cells to induce the release of il-2 and il-10 from t cells (104) . tau-cl inhibits monocyte chemoattractant protein-1 and macrophage inflammatory protein-2 production in glioma cells (105) . tau-cl inhibits the production of no and superoxide anions (106) (107) (108) (109) , prostaglandin e2 (110, 111) , interleukin 6, and tumor necrosis factor-a and it has been suggested that tau-cl may regulate the balance between protective, microbicidal and toxic effect of pmn, tau-cl at 0.1-0.3 mm inhibits interleukin-2 release of purified t cells (112) . chloramines -in contrast to sodium chlorite -do not induce detectable hematologic (! methemogloblin) or hepatic (! elevation of serum alanine-amino-transferase) in african green monkeys (113) . however, a chloramineinduced haemolysis and erythropoietin resistance occurred when the dialysate chloramine levels rose from <0.1 to 0.3 p.p.m. (about 1 mm) resulting in an increase in mean methaemoglobin of 23% and a 21% fall in mean haptoglobin during haemodialysis; only one patient with glucose-6-phosphate-dehydrogenase deficiency had heinz bodies (114, 115) . dogs treated with 1 mmol/l blood concentration of chloramine t â 3 times a week for several months did not show toxic side effects (116) . singlet oxygen is a major agent generated by many different cell types, especially by neutrophil granulocytes. 1 o 2 is nonradicalic and emits light when returning to ground state oxygen. like the gunfire of soldiers directed against bandits, 1 o 2 is both a signal and a weapon, directed against multiple pathogens -including microbes, virus, cancer cells, thrombi -and resulting in an autorecruitment of the physiological inflammatory response. chloramines are stable chemical generators of 1 o 2 . n-chlorotaurine is an important physiological chloramine, for therapeutic purposes chloramine-t â seems to be a promising new therapeutic agent against a multitude of up to now refractory diseases. chemiluminescence of abei-labelled igg triggered by the n-chloramine-h 2 o 2 -p-iodophenol system iodination of biological samples without loss of functional activity a universal and simple chloramine t version for hormone iodination chloramine-t in high-specific-activity radioiodination of antibodies using n-succinimidyl-3-(trimethylstannyl) benzoate as an intermediate nadph-oxidase: an update singlet oxygen ((1)d(g)o(2)) as the principal oxidant in myeloperoxidase-mediated bacterial killing in neutrophil phagosome endothelium-dependent relaxation, endothelium-derived relaxing factor and photorelaxation of blood vessels fad and gsh participate in macrophage synthesis of nitric oxide spectral charcterization of brain and macrophage nitric oxide synthases. cytochrome p-450 like hemeproteins that contain a flavin semiquinone radical the antithrombotic factor singlet oxygen/light ð 1 o 2 =hmþ the blood fibrinolysis/deep-sea analogy: a hypothesis on the cell signals singlet oxygen/photons as natural antithrombotics activation of transcription factor ap-2 mediates uva radiation-and singlet oxygen induced expression of the human intercellular adhesion molecule1 gene current perspectives of singlet oxygen detection in biological environments toxic and signaling effects of photochemically or chemically generated singlet oxygen in biological systems redox agents regulate ion channel activity in vacuoles from higher plant cells activation of the nf-jb transcription factor in a t-lymphocytic cell line by hypochlorous acid monochloramine directly modulates ca(2+)-activated k(+) channels in rabbit colonic muscularis mucosae detachment of cultured cells from the substratum induced by the neutrophil-derived oxidant nh2cl: synergistic role of phosphotyrosine and intracellular ca 2þ concentration oxidation of methionyl residues in proteins: tools, targets, and reversal involvement of methionine residues in the fast inactivation mechanism of the sodium current from toad skeletal muscle fibers the activation gate of cardiac na+ channel modulates voltage-and ph-dependent unbinding of disopyramide modulation by chloramine-t of 4-aminopyridine-sensitive transient outward current in rabbit atrial cells the inactivation of sodium channels in the node of ranvier and its chemical modification an endogenous inactivating inward-rectifying potassium current in oocytes of xenopus laevis modification of c-type inactivating shaker potassium channels by chloramine-t inactivation of the cloned potassium channel mouse kv1.1 by the human kv3.4 'ball' peptide and its chemical modification modulation of voltagedependent inactivation of the inwardly rectifying k+ channel by chloramine-t abolition with chloramine-t of inactivation in barnacle muscle fibers results in stimulation of the ouabain-insensitive sodium efflux chloramine-t effect on sodium conductance of neuroblastoma cells as studied by whole-cell clamp and single-channel analysis modification of electrophysiological and pharmacological properties of k channels in neuroblastoma cells induced by the oxidant chloramine-t some properties of sodium channels in neuroblastoma cells modified with scorpion toxin and chloramine-t. single channel measurements irreversible modification of sodium channel inactivation in toad myelinated nerve fibres by the oxidant chloramine-t removal of sodium channel inactivation in squid axon by the oxidant chloramine-t cryptochromes: blue light receptors for plants and animals effects of pteridines on chloramine-t-induced growth inhibition in e. coli strains: correlations with molecular structure effects of pteridines on luminol-dependent chemiluminescence induced by chloramine-t. free radic a possible origin of chemiluminescence in phagocytosing neutrophils. reaction between chloramines and h2o2 evaluation of bactericidal activity and lag of regrowth (postantibiotic effect) of five antiseptics on nine bacterial pathogens efficacy of a variety of disinfectants against actinobacillus pleuropneumoniae serotype 1 comparative antimicrobial activity, in vitro and in vivo, of soft n-chloramine systems and chlorhexidine model tests for the efficacy of disinfectants on surfaces. iv. communication: dependence of test results on the amount of contamination and the kind of active substance rapid killing of mycobacterium terrae by n-chlorotaurine in the presence of ammonium is caused by the reaction product monochloramine interaction of bacillus subtilis spores with sodium hypochlorite, sodium dichloroisocyanurate and chloramine-t neopterin derivatives modulate toxicity of reactive species on escherichia coli effect of monochloramine disinfection of municipal drinking water on risk of nosocomial legionnaires' disease erythropoietin resistance due to dialysate chloramine: the two-way traffic of solutes in haemodialysis chloramine, a sneaky contaminant of dialysate spectrodensitometric determination of chloramine-t in ice cream liquid chromatographic determination of para-toluenesulfonamide in edible fillet tissues from three species of fish fungicidal effect of 15 disinfectants against 25 fungal contaminants commonly found in bread and cheese manufacturing inactivation of lactobacillus helveticus bacteriophages by thermal and chemical treatments inactivation of feline calicivirus, a norwalk virus surrogate inactivation of respiratory syncytial virus by detergents and disinfectants a surface test for virucidal activity of disinfectants: preliminary study with herpes virus sodium hypochlorite in the treatment of herpes simplex virus infections inactivation of coxsackieviruses b3 and b5 in water by chlorine disinfecting action of chloramine b on marburg virus the effect of some physical and chemical factors on inactivation of the ebola virus ebola virus: what the practitioner needs to know some physical and chemical properties of bhanja virus v. influence of several chemical reagents on lymphocytic choriomeningitis and tacaribe viruses inactivation of simian rotavirus sa11 by chlorine, chlorine dioxide, and monochloramine inactivation of poliovirus by chloramine-t virucidal activity of disinfectants: studies with the poliovirus inactivation of poliovirus i (brunhilde) single particles by chlorine in water cupric and ferric ions inactivate hiv two in-vivo protocols for testing virucidal efficacy of handwashing and hand disinfection chemical disinfection of non-porous inanimate surfaces experimentally contaminated with four human pathogenic viruses mash d. c. preliminary laboratory studies of inactivation of hiv-1 in needles and syringes containing infected blood using undiluted household bleach evaluation of hypochlorite-releasing disinfectants against the human immunodeficiency virus (hiv) chemical inactivation of human immunodeficiency virus in vitro stability and inactivation of htlv-iii/lav under clinical and laboratory environments inactivation of hiv-1 by chemical disinfectants: sodium hypochlorite singlet oxygen ð 1 o 2 þ-generating chloramines at concentrations that are tolerable for normal hemostasis function inactivate the lipid enveloped vesicular stomatitis virus in human blood efficacy of tumoricidal agents in vitro and in vivo jr photodynamic therapy: a review evidence for an important role of neutrophils in the efficacy of photodynamic therapy in vivo attenuation by methionine of monocloramine-enhanced gastric carcinogenesis induced by n-methyl-n 0 -nitro-n-nitrosoguanidine in wistar rats results of long-term carcinogenicity studies of chlorine in rats assessment of the carcinogenic potential of chlorinated water: experimental studies of chlorine, chloramine, and trihalomethanes monochloramine induced dna fragmentation in gastric cell line mkn45 singlet molecular oxygen evolution upon simple acidification of aqueous hypochlorite: application to studies on the deleterious health effects of chlorinated drinking water neutrophil-derived oxidants promote leukocyte adherence in postcapillary venules mpo) may mediate neutrophil adherence to the endothelium through upregulation of cd 11b expression -an effect downregulated by taurine oxidants generated by the myeloperoxidase-halide system activate the fifth component of human complement, c5 neutrophil adhesion to human endothelial cells is induced by the membrane attack complex: the roles for p-selectin and platelet activating activating factor complement and atherogenesis: the unknown connection molecular chlorine generated by the myeloperoxidase-hydrogen peroxide-chloride system of phagocytes converts low density lipoprotein cholesterol into a family of chlorinated sterols severe lung injury after exposure to chloramine gas from household cleaners natural antioxidant, chlorogenic acid, protects against dna breakage caused by monochloramine human methionine sulfoxide-peptide reductase, an enzyme capable of reactivating oxidized a-1-proteinase inhibitor in vitro mechanisms of hypochlorite injury of target cells myeloperoxidase, hydrogen peroxide, chloride antimicrobial system: nitrogen-chlorine derivatives of bacterial components in bactericidal action against escherichia coli tissue destruction by neutrophils oxidative regulation of neutrophil elastase-a-1-proteinase inhibitor interactions evidence of oxidant-induced injury to epithelial cells during inflammatory bowel disease hypochlorite-induced oxidation of proteins in plasma: formation of chloramines and nitrogen-centred radicals and their role in protein fragmentation hypochlorous acid and chloramines increase endothelial permeability: possible involvement of cellular zinc taurine modulation of hypchlorous acid-induced lung epithelial cell injury in vitro. role of anion transport monochloramine-induced cytolysis to cultured rat gastric mucosal cells: role of glutathione and iron in protection and injury effect of monochloramine on recovery of gastric mucosal integrity and blood flow response in rat stomachs -relations to capsaicin-sensitive sensory neurons ulcerogenic and healing impairing actions of monochloramine in rat stomachs: effects of zinc l l -carnosine, polaprezinc monochloramine-induced cell growth inhibition and apoptosis in a rat gastric mucosal cell line regulation of murine dendritic cell functions in vitro by taurine chloramine, a major product of the neutrophil myeloperoxidase-halide system monocyte chemoattractant protein-1 and macrophage inflammatory protein-2 production is inhibited by taurine chloramine in rat c6 glioma cells preactivation exposure of raw 264.7 cells to taurine chloramine attenuates subsequent production of nitric oxide and expression of inos mrna taurine chloramine inhibits the production of superoxide anion, il-6 and il-8 in activated human polymorphonuclear leukocytes monochloramine inhibits phorbol ester-inducible neutrophil respiratory burst activation and t cell interleukin-2 receptor expression by inhibiting inducible protein kinase c activity the production of superoxide anion and nitric oxide by cultured murine leukocytes and the accumulation of tnf-a in the conditioned media is inhibited by taurine chloramine taurine chloramine inhibits production of nitric oxide and prostaglandin e2 in activated c6 glioma cells by suppressing inducible nitric oxide synthase and cyclooxygenase-2 expression taurine chloramine inhibits prostaglandin e2 production in activated raw 264.7 cells by post-transcriptional effects on inducible cyclooxygenase expression modulation of antigen-specific t-cell activation in vitro by taurine chloramine subchronic toxicity of chlorine dioxide and related compounds in drinking water in the nonhuman primate chloramine-induced haemolysis presenting as erythropoietin resistance a hemodialysis patient with chloramine-induced hemolysis. a discussion of the mechanism a model of decreased functional a-1-proteinase inhibitor. pulmonary pathology of dogs exposed to chloramine t key: cord-289049-4ozwhcyi authors: roncati, luca; gallo, graziana; manenti, antonio; palmieri, beniamino title: renin-angiotensin system: the unexpected flaw inside the human immune system revealed by sars-cov-2 date: 2020-03-21 journal: med hypotheses doi: 10.1016/j.mehy.2020.109686 sha: doc_id: 289049 cord_uid: 4ozwhcyi nan medical hypotheses journal homepage: www.elsevier.com/locate/mehy renin-angiotensin system: the unexpected flaw inside the human immune system revealed by sars-cov-2 in humans the renin-angiotensin system is the hormone system which regulates blood pressure and vascular resistances, as well as electrolytic balance; within this important system, the angiotensinconverting enzyme (ace), present on the surface of vascular endothelial cells, in particular those of the lungs, is deputed to the conversion of angiotensin i to angiotensin ii (aii), a potent vasoconstrictive peptide [1] . for this, both ace and aii have been widely exploited as pharmacological targets in the treatment of hypertension, heart failure or diabetic nephropathy by means of ace inhibitors and aii receptor antagonists, respectively [1] . in addition, homologous ace2 receptors have been identified on the oral mucosa, in type-ii pneumocytes, along the intestine and on the kidney and heart endothelia [1, 2] ; these receptors have been found overexpressed in course of ace inhibitors and aii receptor antagonists administration in murine models [3, 4] . surprisingly, the surface spike proteins of the 'severe-acute-respiratory-syndrome-coronavirus-2′ (sars-cov-2), the etiological agent of the ongoing 'coronavirus disease 2019' (covid-19) , are able to bind the ace2 receptors [2] . therefore, a hypothesis arises: could a chronic therapeutic assumption of ace inhibitors or aii receptor antagonists have induced several elderly and middle-aged patients to be more vulnerable to the virus by upregulating ace2 receptors? if we translate the murine model to the human cells, the expected result would be that shown in the figure (fig. 1) . in our opinion, during the covid-19 pandemic, it would be more prudent to replace these drugs, when possible, with calcium channel blockers, adrenergic receptor blockers, diuretics or vasodilators. none. angiotensin-converting enzyme 2-a new cardiac regulator angiotensin-converting enzyme 2 (ace2) as a sars-cov-2 receptor: molecular mechanisms and potential therapeutic target upregulation of angiotensin-converting enzyme (ace) 2 in hepatic fibrosis by ace inhibitors effect of angiotensin-converting enzyme inhibition and angiotensin ii receptor blockers on cardiac angiotensin-converting enzyme 2 no conflict of interest. key: cord-268425-xg8xnjf9 authors: dinicolantonio, james j.; barroso-aranda, jorge title: harnessing adenosine a2a receptors as a strategy for suppressing the lung inflammation and thrombotic complications of covid-19: potential of pentoxifylline and dipyridamole date: 2020-07-02 journal: med hypotheses doi: 10.1016/j.mehy.2020.110051 sha: doc_id: 268425 cord_uid: xg8xnjf9 counterproductive lung inflammation and dysregulated thrombosis contribute importantly to the lethality of advanced covid-19. adenosine a2a receptors (a2ar), expressed by a wide range of immune cells, as well as endothelial cells and platelets, exert camp-mediated anti-inflammatory and anti-thrombotic effects that potentially could be highly protective in this regard. the venerable drug pentoxifylline (ptx) exerts both anti-inflammatory and antithrombotic effects that reflect its ability to boost the responsiveness of a2ar to extracellular adenosine. the platelet-stabilizing drug dipyridamole (dip) blocks intracellular uptake of extracellularly-generated adenosine, thereby up-regulating a2ar signaling in a way that should be functionally complementary to the impact of ptx in that regard. moreover, dip has recently been reported to slow the cellular replication of sars-cov-2 in clinically feasible concentrations. both ptx and dip are reasonably safe, well-tolerated, widely available, and inexpensive drugs. when covid-19 patients can be treated within several days of symptom onset, using ptx + dip in conjunction with hydroxychloroquine (hcq) and an antibiotic azithromycin (azm) or doxycycline – might be warranted. hcq and azm can suppress sars-cov-2 proliferation in vitro and may slow the cell-to-cell spread of the virus; a large case series evaluating this combination in early-stage patients reported an impressively low mortality rate. however, whereas hcq and azm can promote qt interval lengthening and may be contraindicated in more advanced covid-19 entailing cardiac damage, doxycycline has no such effect and exerts a potentially beneficial anti-inflammatory action. in contrast to hcq, we propose that the combination of ptx + dip can be used in both early and advanced stages of covid-19. concurrent use of certain nutraceuticals – yeast beta-glucan, zinc, vitamin d, spirulina, phase 2 inducers, n-acetylcysteine, glucosamine, quercetin, and magnesium – might also improve therapeutic outcomes in covid-19. generation, expression of adhesion molecules, trans-endothelial migration of neutrophils, opening of the endothelial barrier, tissue factor generation, and platelet aggregation in a2ar-responsive cells. 5 downregulation of nf-kappab activation and jak-stat signaling pathways contribute importantly to these effects of camp. 5 importantly, neutrophils, whose activation and transit into lung interstitial tissue and alveolar space is a key mediator of the respiratory distress syndrome associated with covid-19, are highly responsive to the functionally suppressive effects of a2ar, as are the endothelial cells whose activation attracts and enables transendothelial passage of activated neutrophils. [8] [9] [10] [11] these considerations suggest that selective agonists of a2ar may have important potential for blunting the lethality of covid-19. as may be expected, such agents have shown protective effects in rodent models of inflammatory lung injury. [12] [13] [14] [15] unfortunately, these agents are not yet clinically available. however, at least two drugs are currently available -venerable, reasonably safe and well tolerated, and inexpensive -that can function to up-regulate a2ar signaling: pentoxifylline (ptx) and dipyridamole (dip). although ptx is known to have broad anti-inflammatory activity, it is employed primarily in the treatment of intermittent claudication; by lessening neutrophil activation, ptx renders these cells more distensible, so that they can more readily pass through narrow capillaries in affected legs. 16, 17 (upstream stenotic obstructions decrease the transcapillary pressure gradient, rendering the passage of bulky neutrophils through narrow capillaries more difficult in this syndrome.) although the clinical effects of ptx have usually been ascribed to the ability of this drug to inhibit camp phosphodiesterase -thereby boosting camp levels -this effect is only significant in vitro at millimolar concentrations that are orders of magnitude higher than the low micromolar concentrations of this drugs achieved clinically. 16 ironically, however, it does appear that camp mediates ptx's clinical effects. within the last decade, ptx's anti-inflammatory effects have been shown to be contingent on activation of a2ar. 18-20 whether ptx can act as a direct agonist for a2ar is currently unclear, and some data argue against this. 18 what is clear is that ptx can potentiate the responsiveness of this receptor to adenosine. the latter is produced extracellularly from atp released into the extracellular space, which is then converted to adenosine by the sequential activity of the cd39 and cd73 ecto-phosphatases expressed on the plasma membranes of a2ar-expressing cells. 5, 21 the signaling activity of extracellularly-generated adenosine is terminated by intracellular uptake of the adenosine. the platelet-stabilizing agent dip is distinguished by its ability to block this re-uptake by platelets. 22, 23 hence, dip up-regulates the adenosine-mediated activation of platelet a2ar, thereby boosting platelet levels of camp, which functions to suppress platelet aggregation. moreover, dip blocks adenosine uptake by a range of other a2ar-expressing cell types, including endothelial cells, neutrophils, and monocytes. [23] [24] [25] it is evident that ptx and dip have the potential to work in a complementary fashion to boost a2ar signaling -dip can be expected to boost the extracellular levels of adenosine whose signaling activity ptx potentiates. surprisingly, only a handful of studies have evaluated this combination experimentally or clinically -with encouraging results -likely because the mechanism of action of ptx has been clarified only recently. 26-28 pre-administration of ptx is protective in rodent models of acute respiratory distress syndrome (ards) evoked by lipopolysaccharide (lps) administration or severe hemorrhage. [29] [30] [31] clinically, it was found to reduce mortality, lower plasma tumor necrosis factor, and achieve clinical and radiological improvements in ards associated with cancer. 32 a meta-analysis of clinical studies found that ptx therapy is associated with a decrease in plasma levels of both tumor necrosis factor and c-reactive protein. 33 in chimpanzees, it was shown to blunt lps-induced activation of coagulation and fibrinolysis. 34 in isolated lungs, ptx pre-treatment reduces the tissue injury induced by neutrophil infusion. 35 in endothelial cells, ptx counteracts the ability of pro-inflammatory cytokines to stimulate expression of adhesion factors and chemokine production. 36 these findings are expectable in light of the known effects of a2ar signaling, and encourage the speculation that ptx could have potential for blunting the exuberant lung inflammation and pro-thrombotic effects of advanced covid-19. not surprisingly, the use of ptx for treatment of ards associated with sars infection was suggested in 2003. 37 (presumably, this was not studied because the syndrome rapidly disappeared.) in seeming contradiction, a large multi-center study of lisofylline therapy in ards patients failed to show benefit. 38 lisofylline is the r-isomer of a reductive metabolite of ptx, notable for its protective impact in rodent models of type 1 diabetes. 39 conceivably, the impact of this agent on a2ar signaling -which has not been reported -is different than that of ptx. alternatively, this finding may reflect the fact that, for unclear reasons, a2ar agonism is more effective for controlling ards when implemented before the syndrome becomes florid. konrad and colleagues, in interpreting this result, suggest that adenosine levels may be too low in the context of advanced sepsis. 19 if so, the concurrent use of dip would make logical sense. most studies with dip have focused on its platelet-stabilizing effects -which presumably could provide some protection from sars-cov-2's pro-thrombotic effects -but experimental studies also show that dip can act on neutrophils to suppress superoxide production, adhesion to endothelial cells, and, in a mouse model of anti-phospholipid syndrome (a sometime feature of covid-19), netosis formation. [40] [41] [42] [43] and dip has been shown to suppress superoxide production and tissue factor expression in monocytes. 44 of particular pertinence is this new discovery: chinese researchers have reported that, in clinically relevant concentrations as low as 100 nm, dip slows the replication of sars-cov-2 in vero e6 cells; this effect may be mediated in part but not entirely by the binding to dip to the sars-cov-2 protease mpro. 45 in a controlled pilot study, 31 hospitalized covid-19 patients with respiratory difficulties were treated with either dip (50 mg 3 times daily) or placebo; of the 14 patients who received dip, including 8 that were severely ill, all but one recovered, and the remaining patient was in remission at time of the report. of 12 severely ill patients in the control group, 2 patients died and 2 were in remission. 45 the difference in therapeutic outcome just missed traditional statistical significance (p=0.06). the response in d-dimer levels was significantly better in the treated than in the control patients. an anti-viral effect of dip is not unprecedented -cell culture studies have reported that this agent can slow the proliferation of various rna viruses, and a russian clinical report some decades ago concluded that dip administered prophylactically was effective for reducing risk for influenza and upper respiratory infections. [45] [46] [47] [48] [49] [50] these considerations suggest that a ptx/dip regimen might have considerable potential for control of the progression and complications of covid-19. provisionally, we recommend dosage schedules for ptx and dip typically used for their approved indications: ptx 400 mg 3 times daily, and dip 50 mg 3 times daily. another venerable drug which has been suggested for use in covid-19 management is the anti-parasitic agent ivermectin. evidence that it can suppress proliferation of sars-cov-2 in cell culture is likely of little pertinence, as the ic50 concentration that achieves this -about 2 um -is vastly higher than the plasma concentrations achievable by doses approved for clinical use. 51, 52 nonetheless, anecdotal claims of its apparent effectiveness in late-stage covid-19 are encouraging clinical trials with this agent. largely overlooked is the fact that, in oral doses that are roughly analogous to the standard clinical dose in humans, ivermectin pre-administration can protect mice from a lethal dose of lps. 53, 54 hence, if ivermectin proves useful in covid-19, an anti-inflammatory mechanism may underlie this benefit. however, it should be acknowledged that a2ar agonism also has potential for suppressing the dendritic cell activity that provides the antigen presentation necessary for developing an antiviral antibody response. 4 the authors have been unavailable to find any studies suggesting that ptx increases infection risk -in marked contrast to the well-known literature on anti-inflammatory corticosteroids -so perhaps this is a relatively minor consideration. indeed, some studies fail to find an effect of a2ar agonists on dendritic cell antigen presentation. 55 and ptx has actually been suggested as an adjuvant to vaccination, as it boosts memory response to vaccination by increasing survival of activated t cells. 56 nevertheless, it would seem prudent to complement ptx/dip therapy with agents such as yeast beta-glucan that specifically boost dendritic cell activity, as a compensatory measure. [57] [58] [59] curiously, beta-glucan administration has been found to be protective in rodent models of sepsis-induced ards. 60, 61 supplemental zinc could be another worthwhile adjuvant measure, as it has been found to decrease incidence of infection while lowering systemic markers of inflammation in elderly subjects. 62 in early-stage ambulatory patients with covid-19, it would be appropriate to consider using ptx + dip in conjunction with hydroxychloroquine (hcq). currently, this agent is the most commonly used drug for treatment of early-stage covid-19. 63 hcq decreases replication of sars-cov-2 in vitro in clinically relevant concentrations. 64, 65 studies examining the molecular biology of sars-cov-2 cell-tocell spread have found that endosomal cathepsin l protease activity markedly expedites such spread, presumably because it enables sars-cov-2 virions taken up into cellular endosomes to fuse their membranes with that of the endosome, thereby allowing the virion to enter the cytoplasm and begin replication. 66 it is well known that hcq functions to alkalinize endosomes, and this would be expected to inhibit cathepsin l activity. 67, 68 moreover, hcq has recently been shown to inhibit activation of nadph oxidase complexes in endosomes; this would be expected to exert an anti-inflammatory effect that might complement the anti-viral activity of this agent. 69 this model makes evident the desirability of employing hcq as early during the clinical course of covid-19 as feasible, as the ability of this agent to slow cell-to-cell spread may be of less utility once the lung epithelium is already widely colonized by the virus. pharmacokinetic modeling, combined with in vitro data, suggest a regimen for hcq of 400 mg twice daily for one day, and 200 mg twice daily for a further 4 days; this is predicted to maintain antiviral plasma levels of hcq for at least 10 days. 64 hcq therapy often induces modest increases in qt interval, and concurrent administration of azithromycin can amplify this effect. such increases can increase chances for dangerous torsade de pointes arrhythmias; studies examining ecgs in hospitalized covid-19 patients treated with this combination reported 2 cases of torsade de pointes in 640 such patients. [70] [71] [72] [73] although it is very rare for these drugs to induce arrhythmias when used for their current indications, sars-cov-2 can directly attack the heart, and conceivably this could potentiate the pro-arrhythmic impact of hcq. 74, 75 hence, monitoring of qt interval appears to be prudent when using hcq in covid-19. fortunately, neither ptx nor dip have been linked to qt prolongation or torsade de pointes arrhythmias. adding an antibiotic such as azithromycin or doxycycline to early-stage treatment of covid-19 to prevent bacterial super-infection -as many doctors have done when employing hcq in covid-19 therapy -would also be a reasonable option. 76 in addition to their antibiotic activities, azithromycin exerts anti-viral effects in vitro against various viruses, and doxycycline has anti-inflammatory properties that likely would be beneficial in sars-cov-2-induced cytokine storm. 77, 78 however, azithromycin might be inappropriate for late-stage therapy, as it has a greater tendency than hcq to prolong qt intervals; 75 doxycycline does not have this effect. nutraceutical adjuvant measures that support the antigen-specific immune response -such as yeast glucan and zinc -would also appear to be indicated. nutraceuticals that might be expected to boost the interferon response evoked by sars-cov-2 while lessening the contribution of oxidants to lung inflammation have been proposed, including spirulina, phase 2 inducers, n-acetylcysteine. 79 supplemental glucosamine may likewise up-regulate the type 1 interferon responses to viruses, while exerting anti-inflammatory effects that render it protective in rodent models of sepsis and lung inflammation induced by lps or cigarette smoke. [80] [81] [82] [83] [84] this anti-inflammatory effect might reflect upregulated activity of the de-ubiquitinase a20, which opposes traf6 signaling. 85 up-regulation of type 1 interferon induction may also play a role in the anti-viral effects of quercetin. 86, 87 theoretical considerations as well as epidemiological findings suggest that good vitamin d status may also be protective. [88] [89] [90] in the context of inflammation, lung epithelium and alveolar macrophages can convert circulating 25-hydroxyvitamin d to the active hormone calcitriol; this in turn can boost expression of the antimicrobial protein cathelicidin, which is destructive to many enveloped viruses. [91] [92] [93] [94] [95] intracellular magnesium supports effective function of the a2ar. 96 lower serum magnesium has been associated with increased thrombotic risk and slowed fibrinolysis. [97] [98] [99] [100] [101] hypomagnesemia predicts poor outcomes in icu patients, and its correction may improve their prognosis. 102, 103 moreover, magnesium deficiency up-regulates nf-kappab activation and hmbg1 secretion in lps-treated macrophagesconsistent with other evidence that it may up-regulate inflammation. 104, 105 in light of the pro-thrombotic and pro-inflammatory diathesis associated with covid-19, magnesium supplementation might be prudent. high risk of thrombosis in patients with severe sars-cov-2 infection: a multicenter prospective cohort study endothelial cell infection and endotheliitis in covid-19 severe covid-19 infection associated with endothelial activation a(2b) adenosine receptors in immunity and inflammation anti-inflammatory and immunosuppressive effects of the a2a adenosine receptor a2a adenosine receptor agonists and their potential therapeutic applications. an update molecular mechanisms involved in adenosine-induced endothelial cell barrier enhancement adenosine a2a receptors mediate the inhibitory effect of adenosine on formyl-met-leu-phe-stimulated respiratory burst in neutrophil leucocytes neutrophil a2a adenosine receptor inhibits inflammation in a rat model of meningitis: synergy with the type iv phosphodiesterase inhibitor, rolipram identification of adenosine a2 receptor-camp system in human aortic endothelial cells adenosine regulates the proinflammatory signaling function of thrombin in endothelial cells bench-to-bedside review: adenosine receptors--promising targets in acute lung injury? effects of dipyridamole, pentoxifylline or dipyridamole plus pentoxifylline on platelet reactivity in patients with ischemic cerebrovascular insufficiency pentoxifylline pretreatment decreases the pool of circulating activated neutrophils, in-vivo adhesion to endothelium, and improves survival from hemorrhagic shock pentoxifylline pretreatment decreases neutrophil activation during endotoxic shock and improves survival pentoxifylline attenuates lung injury and modulates transcription factor activity in hemorrhagic shock respiratory distress syndrome in patients with advanced cancer treated with pentoxifylline: a randomized study effects of pentoxifylline on inflammatory markers and blood pressure: a systematic review and meta-analysis of randomized controlled trials inhibition of endotoxin-induced activation of coagulation and fibrinolysis by pentoxifylline or by a monoclonal anti-tissue factor antibody in chimpanzees pentoxifylline reduces injury to isolated lungs perfused with human neutrophils pentoxifylline inhibits icam-1 expression and chemokine production induced by proinflammatory cytokines in human pulmonary epithelial cells pentoxifylline and severe acute respiratory syndrome (sars): a drug to be considered placebo-controlled trial of lisofylline for early treatment of acute lung injury and acute respiratory distress syndrome lisofylline: a potential lead for the treatment of diabetes inhibition of active oxygen generation by dipyridamole in human polymorphonuclear leukocytes inhibition by dipyridamole of neutrophil adhesion to vascular endothelium during coronary bypass surgery modification of neutrophil adhesion to human endothelial cell line in acute ischemic stroke by dipyridamole and candesartan adenosine receptor agonism protects against netosis and thrombosis in antiphospholipid syndrome dipyridamole inhibits o2-release and expression of tissue factor activity by peripheral blood monocytes stimulated with lipopolysaccharide potential therapeutic effects of dipyridamole in the severely ill patients with covid-19 inhibition of herpes simplex virus reactivation by dipyridamole in a mouse model inhibition of herpes simplex virus reactivation by dipyridamole dipyridamole reversibly inhibits mengovirus rna replication the fda-approved drug ivermectin inhibits the replication of sars-cov-2 in vitro the approved dose of ivermectin alone is not the ideal dose for the treatment of covid-19 ivermectin inhibits lps-induced production of inflammatory cytokines and improves lps-induced survival in mice ivermectin may be a clinically useful antiinflammatory agent for late-stage covid-19 dendritic cells tolerized with adenosine a(2)ar agonist attenuate acute kidney injury pentoxifylline functions as an adjuvant in vivo to enhance t cell immune responses by inhibiting activation-induced death beta glucan: supplement or drug? from laboratory to clinical trials baker's yeast beta-glucan supplement reduces upper respiratory symptoms and improves mood state in stressed women improvement of immune responses to influenza vaccine (h5n1) by sulfated yeast beta-glucan protective effect of beta-glucan on lung injury after cecal ligation and puncture in rats beta-glucan attenuates inflammatory cytokine release and prevents acute lung injury in an experimental model of sepsis zinc supplementation decreases incidence of infections in the elderly: effect of zinc on generation of cytokines and oxidative stress efficacy of chloroquine and hydroxychloroquine in the treatment of covid-19 in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov-2 infection in vitro characterization of spike glycoprotein of sars-cov-2 on virus entry and its immune cross-reactivity with sars-cov mechanism of action of hydroxychloroquine as an antirheumatic drug delineation of chicken cathepsin l secondary structure; relationship between ph dependence activity and helix content hydroxychloroquine inhibits proinflammatory signalling pathways by targeting endosomal nadph oxidase the effect of chloroquine, hydroxychloroquine and azithromycin on the corrected qt interval in patients with sars-cov-2 infection risk of qt interval prolongation associated with use of hydroxychloroquine with or without concomitant azithromycin among hospitalized patients testing positive for coronavirus disease 2019 (covid-19) qt interval prolongation and torsade de pointes in patients with covid-19 treated with hydroxychloroquine/azithromycin experience with hydroxychloroquine and azithromycin in the covid-19 pandemic: implications for qt interval monitoring association of cardiac injury with mortality in hospitalized patients with covid-19 in wuhan, china cardiac safety of off-label covid-19 drug therapy: a review and proposed monitoring protocol early outpatient treatment of symptomatic, high-risk covid-19 patients that should be ramped-up immediately as key to the pandemic crisis clinical pharmacology perspectives on the antiviral activity of azithromycin and use in covid-19 doxycycline, a widely used antibiotic in dermatology with a possible anti-inflammatory action against il-6 in covid-19 outbreak nutraceuticals have potential for boosting the type 1 interferon response to rna viruses including influenza and coronavirus mavs o-glcnacylation is essential for host antiviral immunity against lethal rna viruses acute increase in o-glcnac improves survival in mice with lps-induced systemic inflammatory response syndrome glucosamine improves survival in a mouse model of sepsis and attenuates sepsis-induced lung injury and inflammation glucosamine attenuates cigarette smoke-induced lung inflammation by inhibiting ros-sensitive inflammatory signaling attenuation of lps-induced lung inflammation by glucosamine in rats o-linked beta-n-acetylglucosamine modification of a20 enhances the inhibition of nf-kappab (nuclear factor-kappab) activation and elicits vascular protection after acute endoluminal arterial injury targeting casein kinase 2 with quercetin or enzymatically modified isoquercitrin as a strategy for boosting the type 1 interferon response to viruses and promoting cardiovascular health. med hypotheses melanoma suppression by quercein is correlated with rig-i and type i interferon signaling evidence that vitamin d supplementation could reduce risk of influenza and covid-19 infections and deaths editorial: low population mortality from covid-19 in countries south of latitude 35 degrees north supports vitamin d as a factor determining severity does vitamin d status impact mortality from sars-cov-2 infection? med drug discov vitamin d increases the antiviral activity of bronchial epithelial cells in vitro tumor necrosis factor-alpha induces vitamin d-1-hydroxylase activity in normal human alveolar macrophages the human cathelicidin ll-37 inhibits influenza a viruses through a mechanism distinct from that of surfactant protein d or defensins antiviral activities of human host defense peptides vitamin d and the anti-viral state mechanistic insights into allosteric regulation of the a2a adenosine g protein-coupled receptor by physiological cations reduced plasma magnesium levels in type-1 diabetes associate with prothrombotic changes in fibrin clotting and fibrinolysis deficit of magnesium and states of hypercoagulation: intellectual analysis of data obtained from a sample of patients aged 18-50 years from medical and preventive facilities in russia magnesium as a predictor of acute stent thrombosis in patients with st-segment elevation myocardial infarction who underwent primary angioplasty antithrombotic effects of magnesium sulfate in in vivo experiments low intracellular magnesium levels promote plateletdependent thrombosis in patients with coronary artery disease does hypomagnesemia impact on the outcome of patients admitted to the intensive care unit? a systematic review and meta-analysis hypomagnesemia in the magnesium deficiency promotes secretion of high-mobility group box 1 protein from lipopolysaccharide-activated macrophages in vitro magnesium deficiency and increased inflammation: current perspectives key: cord-288009-8i3gsq9p authors: javor, s.; salsano, a. title: why not consider an endothelin receptor antagonist against sars‐cov‐2? date: 2020-04-25 journal: med hypotheses doi: 10.1016/j.mehy.2020.109792 sha: doc_id: 288009 cord_uid: 8i3gsq9p nan medical hypotheses journal homepage: www.elsevier.com/locate/mehy why not consider an endothelin receptor antagonist against sars-cov-2? background on 12 january 2020, the world health organization named a new 2019-ncov as severe acute respiratory syndrome coronavirus 2 (sars-cov-2) [1] . about 15% of cases progress to a life-threatening severe phase with lung inflammation and a cytokine storm, driven by interleukin (il)-6 [2, 3] . bilateral interstitial pneumonia in chest computed tomography were found in 98% of cases [1] and high percentage of patients develop lung fibrosis after recovery from respiratory syndrome [4] . since there are no registered vaccines for the disease, the straight control of the sources of infection remains crucial. antivirals and anti-malarian drugs have been proved to be effective for 2019-ncov treatment. chinese guidelines in the latest 6th edition, recommend: interferon (ifn)-α, lopinavir/ritonavir, ribavirin, chloroquine phosphate, arbidol [5] [6] [7] [8] . in addition, tocilizumab has been used as a new therapeutic opportunity results targeting il-6 [9] . in the last two decades, many publications have shown that endothelin-l (et-1) has a key role in inflammatory cascade [10] . bosentan is a dual endothelin-receptor antagonist approved for the treatment of pulmonary arterial hypertension (pah) in new york heart association functional classification (nyha) ii-iv and in scleroderma patients [10] . bosentan significantly reduced profibrotic and proinflammatory cytokines: il-2, il-6, il-8 and ifn-γ levels in scleroderma patients, slowing progression to fibrosis and vascular damage [10] . il-6 has been identified as the main cytokine in the genesis of pah lesions, even in human immunodeficiency virus (hiv) patients [11] . bosentan was also studied for its antiviral effect. an important reduction of viral rna copy number (70-90%) was detected in human umbilical vein endothelial cells pretreated with 56 cmax bosentan or 106 cmax valsartan, even with low dosages [12] . guo et al [13] described a case of 57-year-old man with influenza a (h7n9) virus infection initially treated with empirical antibacterial therapy and oseltamivir with progression to acute respiratory distress syndrome and mechanical ventilation. after bosentan administration patient's right ventricular systolic pressure improved rapidly with successful weaning from mechanical ventilation [13] . in patients with hiv infection, pah is a life-threatening complication [14] . these patients present high levels of et-1 that also correlates with severity of the disease [14] . ritonavir and lopinavir, given together to sup-press hiv-replication, have been associated with bosentan to treat pah in hiv-infected patients without dosage adjustment of protease inhibitors with good tolerability [14] . in conclusion, we think bosentan could be considered, in association with other approved drugs, in the treatment of sars-cov-2 to improve hemodynamics, potentiate antiviral effects and to prevent lung fibrosis. none. understanding of covid-19 based on current evidence covid-19 infection: the perspectives on immune response pathological findings of covid-19 associated with acute respiratory distress syndrome follow-up chest radiographic findings in patients with mers-cov after recovery first case of 2019 novel coronavirus in the united states remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro discovering drugs to treat coronavirus disease 2019 (covid-19) new therapeutic opportunities for covid-19 patients with tocilizumab: possible correlation of interleukin-6 receptor inhibitors with osteonecrosis of the jaws evaluation of the effect of bosentan treatment on proinflammatory cytokine serum levels in patients affected by systemic sclerosis pulmonary arterial hypertension related to hiv: is inflammation related to il-6 the cornerstone? antiviral effect of bosentan and valsartan during coxsackievirus b3 infection of human endothelial cells bosentan as rescue treatment in refractory hypoxemia and pulmonary hypertension in a patient with ards and h7n9 influenza virus infection mutual pharmacokinetic interactions between bosentan and lopinavir/ritonavir in healthy participants the authors have no funding sources to disclose. the authors declare that they have no financial or personal relationships with other people or organisations that could inappropriately influence this work. key: cord-261370-jp5sqqwc authors: bollag, wendy b.; gonzales, joyce n. title: phosphatidylglycerol and surfactant: a potential treatment for covid-19? date: 2020-09-16 journal: med hypotheses doi: 10.1016/j.mehy.2020.110277 sha: doc_id: 261370 cord_uid: jp5sqqwc a hypothesis concerning the potential utility of surfactant supplementation for the treatment of critically ill patients with covid-19 is proposed, along with a brief summary of the data in the literature supporting this idea. it is thought that surfactant, which is already approved by the food and drug administration for intratracheal administration to treat neonatal respiratory distress syndrome in infants, could benefit covid-19-infected individuals by: (1) restoring surfactant damaged by lung infection and/or decreased due to the virus-induced death of the type ii pneumocytes that produce it and (2) reducing surface tension to decrease the work of breathing and limit pulmonary edema. in addition, a constituent of surfactant, phosphatidylglycerol, could mitigate covid-19-induced lung pathology by: (3) decreasing excessive innate immune system activation via its inhibition of toll-like receptor-2 and -4 activation by microbial components and cellular proteins released by damaged cells, thereby limiting inflammation and the resultant pulmonary edema, and (4) possibly blocking spread of the viral infection to non-infected cells in the lung. therefore, it is suggested that surfactant preparations containing phosphatidylglycerol be tested for their ability to improve lung function in critically ill patients with covid-19. introduction covid-19, caused by the novel coronavirus sars-cov-2, has resulted in massive morbidity and mortality, as well as profound economic difficulties due to the necessity for quarantining to contain and mitigate the pandemic. although many people who become infected exhibit only mild or moderate symptoms, others develop severe symptoms, and covid-19 appears to be more deadly than influenza, especially in older individuals and those with preexisting conditions. treatment to date is mainly symptomatic supportive care including invasive or non-invasive ventilation. in a recent retrospective study of 52 chinese patients with covid-19 requiring intensive care, more than 60% of the patients died [1] . of the non-survivors about 80% of the patients developed acute respiratory distress syndrome (ards) [1] , and respiratory failure associated with ards is the leading cause of covid-19 mortality [2] . ards is characterized by lung inflammation and pulmonary edema, which reduces gas exchange and leads to hypoxemia and dyspnea, often requiring mechanical ventilation to provide sufficient oxygenation. ards is also accompanied by enhanced secretory phospholipase a 2 (spla 2 ) activity in the lungs [3, 4] ; spla 2 degrades the phospholipids that are components of surfactant, including phosphatidylglycerol (pg) (reviewed in [5] ). indeed, a recent study has demonstrated an increase in the activity of an spla2 that preferentially hydrolyzes pg, as well as a significant decrease in pg in the bronchoalveolar lavage fluid of ards patients versus normal control subjects [3] . the impairment of surfactant function not only can increase surface tension and reduce lung compliance but may also further exacerbate pulmonary edema, since surfactant helps to reduce fluid infiltration into the alveoli through its reduction of surface tension [6, 7] . knowledge about this sequence of events has led to studies in humans testing the efficacy of exogenous surfactant in the treatment of ards, and some results have been promising [8] [9] [10] [11] ; although meta-analyses have largely failed to show an effect of exogenous surfactant administration on the survival of adult ards patients (e.g., [12] and [13] ). on the other hand, another meta-analysis determined a benefit of surfactant administration on oxygenation levels and mortality in those patients with severe ards caused by pneumonia or aspiration of gastric contents [14] , suggesting that co-morbidities other than ards may potentially determine the effect of surfactant administration on survival. in contrast, the pulmonary failure induced by covid-19 seems to differ in many respects from other types of ards [15, 16] . for example, many covid-19 patients initially present with hypoxemia with maintained lung compliance and low elastance, termed the l type presentation [17] . these l-type patients can often be treated with oxygen supplementation and prone positioning, or if they are intubated due to worsening hypoxia, mechanical ventilation at low positive end expiratory pressures (peep). however, l-type patients often transition into the second or h-type clinical presentation [17] , in which they exhibit high elastance and low compliance and usually require mechanical ventilation at higher peep [18] ; low pulmonary compliance portends worsening lung disease manifested by atelectasis and increasing hypoxia. therefore, only the h-type mimics the lung parameters observed in pre-term infants that produce minimal surfactant, suggesting that at least in the early stages, covid-19 patients exhibiting l-type disease may retain some pulmonary surfactant activity. a key role of pulmonary surfactant is to reduce surface tension and prevent alveolar atelectasis at end expiration. in the absence of active surfactant, high surface tension at the airliquid interface in the alveoli creates collapsing forces [19] . in addition, since surface tension draws fluid from the capillaries into the alveolar spaces, surfactant decreases pulmonary fluid accumulation by reducing surface tension to maintain airway dryness [20] . increased surface tension related to surfactant dysfunction also alters alveolar capillary shape and pulmonary blood flow to exacerbate hypoxemia [19] . on the other hand, during severe respiratory distress with mechanical ventilation, the role of higher peep is to keep the alveoli "recruited" or, in other words, to prevent end-expiratory alveolar collapse. however, mechanical ventilation and high peep represent a double-edged sword: maintaining or improving oxygenation while causing alveolar lung injury. mechanical ventilation with high peep may also reduce the ability of surfactant to lower surface tension, since compression of surfactant to an area of less than 50% of its original surface area by higher pressures can result in rupturing of the film on reexpansion, resulting in compromised surface tension-reducing capacity [21, 22] . in addition, the stresses of mechanical ventilation also stimulate the inflammatory response [21] . thus, it seems likely that another function of surfactant may be important: its ability to dampen the inflammatory response to microbial components. thus, at least one of the surfactant phospholipids, pg, has been shown to inhibit activation of toll-like receptors (tlr) of the lung innate immune system by microbial components (reviewed in [23] ). voelker and colleagues have shown that pg inhibits tlr2 and tlr4 activation by microbial components, also known as pathogen-associated molecular patterns (pamps), such as acylated lipopeptides and lipopolysaccharide [23] . this inhibition then results in reduced production of inflammatory mediators and decreased lung inflammation and damage [24] [25] [26] . these results are also consistent with the results of wu at al. [27] , who demonstrated that pg inhibits endotoxinstimulated activation of nuclear factor-kappab (nfb), a transcription factor associated with inflammation, to reduce type iia secretory phospholipase a 2 levels/activity in macrophages. the mechanism of action of pg seems to be related to the ability of the tlr2 and tlr4 coreceptor cd14 to bind this phospholipid and somehow prevent tlr activation [26, 28] . indeed, martin et al. [29] have recently suggested blocking cd14, but with inhibitory antibodies, to control inflammation in covid-19. of note, pamps are not the only molecules that can activate tlrs. tlr activation can also be induced by endogenous proteins that are released by damaged or stressed cells, the so-called danger-or damage-associated molecular patterns (damps) (reviewed in [30] ). many such tlr-stimulating damps have been identified (reviewed in [31] ), including several heat shock proteins, high mobility group b1 (hmgb1) and fibrinogen [31] , and these can be released extracellularly upon cell damage. we recently showed that pg can inhibit damp-induced inflammatory mediator production [32] and skin inflammation [32, 33] . specifically, pg inhibits tlr2 and tlr4 activation by the damps s100a9 and beta-defensin-2 [32] . pg can also inhibit tlr2 and tlr4 activation in response to pamps in several cell types and in tlr2 and tlr4 reporter cell lines [34] , with minimal effects on the activation of, or stimulation of inflammatory mediator expression by, other pattern recognition receptors, such as the tlr7/8 that recognizes single-stranded rna. in addition, supplementation of surfactant with additional pg (to a molar percentage of 6%) preserves lung function and prevents alveolar epithelial injury and the expression of pro-fibrotic mediators in a neonatal pig triple injury model of ards [35] . the ability of pg to protect against cell injury would be expected to be beneficial in covid-19 ards, furthermore, covid-19 patients have been reported to exhibit elevated levels of tumor necrosis factor-alpha (tnf) [2] , and drugs targeting pro-inflammatory mediators, such as interleukin (il)-1 and il-6 have been proposed or are in use for the treatment of covid-19 [36, 37] . pg has been shown to inhibit the expression of il-1, il-1, il-6, and/or tnf, as well as il-8, interferon-gamma and/or macrophage inflammatory protein-2, in response to tlr activation by pamps and damps [24-26, 28, 32, 34, 38, 39] . in turn, several of these inflammatory mediators (e.g., il-6 and tnf) are also known to increase the levels of certain spla 2 s, in particular that encoded by the gene pla2g2a [3] , which would decrease pg levels even further. finally, excessive inflammation (e.g., markedly increased c-reactive protein and d-dimer levels) is associated with the hypercoagulopathy sometimes seen in covid-19 patients [40] . therefore, the ability of pg to inhibit pamp-and damp-induced might also decrease these covid-19 sequelae as well. collectively, these results have led to the current hypothesis that pg, in the form of exogenous surfactant, might be efficacious in treating the symptoms of covid-19. by analogy with sars-cov [41, 42] , sars-cov-2 is thought to target alveolar type-ii cells [43, 44] , the lung cells that produce surfactant; the resulting release of endogenous molecules by these damaged cells would presumably activate tlrs and stimulate inflammatory mediator production and inflammation. these effects likely would, together with the gradual reduction in surfactant resulting from the death of these type ii pneumocytes (and possibly the increased activity of spla 2 ) [3] [4] [5] , promote the pulmonary edema that is a hallmark of covid-19. the pulmonary edema, in turn, further impairs gas exchange and leads to ards with further hypoxemia and dyspnea. however, the initial presentation might be expected to show differences from the respiratory distress seen in pre-term infants: phosphatidylcholine represents approximately twothirds to three-quarters of pulmonary surfactant lipid content [45, 46] and thus provides the majority of its surfactant activity. therefore, gradual loss of phosphatidylcholine would allow maintenance of compliance despite enhanced inflammation resulting from decreased levels of pg, which comprises only 9-12% of surfactant phospholipid [45, 46] , and the resultant pulmonary edema. presumably, loss of the anti-surface tension effects of surfactant would only occur once large numbers of type ii alveolar cells were destroyed and phosphatidylcholine was severely depleted. at this point, then, patients would transition to the h-type clinical presentation, with the low compliance more typical of neonatal respiratory distress syndrome. pulmonary administration of exogenous surfactant would be expected to counter this sequence of events in multiple ways: (1) it would restore the levels of surfactant to protect against increased surface tension in the lung; (2) it would inhibit activation of the innate immune system by released damps to reduce inflammation and inflammatory damage; and (3) it would decrease pulmonary edema through the combination of the first two effects. in addition, it is thought that in some individuals, covid-19-related morbidity and mortality may be related to an over-reaction of the immune system and a "cytokine storm" [2, 37, 47, 48] . by inhibiting innate immune system activation and release of pro-inflammatory mediators that recruit and activate additional immune cells, including those of the adaptive immune system, pg would likely interrupt this process of immune system hyper-responsiveness, acting as a dampening mechanism, or rheostat, to regulate lung inflammation [23] . finally, it is known that pulmonary surfactant can facilitate recruitment of collapsed airways and offer protection from mechanical ventilation-induced lung injury. thus, exogenous surfactant therapy may restore or replenish insufficient or dysfunctional endogenous surfactant activity and improve outcomes in covid-19. thus, we are proposing that pg-containing surfactant medications that are already approved by the food and drug administration for the treatment of neonatal respiratory distress syndrome (table 1) be administered intratracheally via bronchoscopy to covid-19 patients with severe acute respiratory distress syndrome. it should be noted that natural and second-generation synthetic surfactant preparations have been found to exhibit increased efficacy for improving neonatal respiratory distress syndrome relative to first-generation protein-free surfactant medications like exosurf®. these results are consistent with data indicating the importance of certain surfactant proteins to improve the effect of surfactant on surface tension [49] and others to reduce microbial infection ( [50, 51] and reviewed in [52] ). the histologic description of covid 19 pathology at autopsy shows diffuse alveolar damage with cellular fibromyxoid exudates, acute fibrinous, hyaline membrane formation, organizing pneumonia and desquamation of pneumocytes, all consistent with ards [53, 54] . hyaline membrane formation has been observed in histological samples at both the early and later stages of the disease, suggesting early type ii pneumocyte injury with surfactant dysfunction [53] . although not all covid-19 patients progress to a low-compliance phenotype, evidence in histological specimens highly suggests that there is surfactant dysfunction and hyaline membrane formation comparable to that observed in the non-covid-19 ards-mediated alveolar damage described by matthay and zemans [55] . one approach to improve the dysfunctional surfactant in this disease is to treat with exogenous surfactant, thereby allowing maintenance of its function in the alveoli. indeed, it seems likely that covid-19-affected lungs will require functioning surfactant to fully recover. exogenous bronchial surfactant instillation has been a feasible and safe approach in infants, although a higher dose and repeated administration may be required to restore dysfunctional alveoli impacted by covid-19. it should also be noted that despite its ability to inhibit tlr activation and inflammation, pg in surfactant does not seem to be globally immunosuppressive. in fact, in animal models in vivo it protects against infection resulting from several viruses, including respiratory syncytial virus, influenza a (h3n2) and h1n1 [38, 39, 56] , by inhibiting the interaction of these viruses with their receptors on host cells. although it is not known whether pg has a similar inhibitory effect on the infectious capacity of sars-cov-2, a positive-sense single-strand rna virus, the minimal effect of pg on the activation of tlr7/8 [32, 34] would suggest that this phospholipid would likely not suppress innate immune system responses to the virus. therefore, surfactant might be useful in preventing the spread of sars-cov-2 viral infection between infected and naïve cells within the lung without affecting the response to this infection, in addition to protecting against the damage caused by excessive inflammation and edema and the increased surface tension that eventually results from loss of surfactant. on the other hand, the surfactant lipids phosphatidylcholine, in particular disaturated phosphatidylcholine (dipalmitoylphosphatidylcholine), and phosphatidylserine are reported to potentially promote infection by viral pathogens [57] . however, the mechanisms are thought to involve facilitation of viral entry via the ability of the virus to bind lipid and co-opt reuptake/recycling pathways in the case of pc and promotion of viral fusion by mimicking of an apoptotic signal in the case of ps [57] . since sars-cov-2 purportedly gains entry into cells through angiotensin-converting enzyme 2 (ace2) [40] , these mechanisms used by other viruses seem unlikely to be relevant to sars-cov-2 and covid-19 pneumonia. it should also be noted that certain conditions that increase the risk of a severe response to sars-cov-2 infection are also known to reduce surfactant and/or surfactant phospholipid levels or to impair surfactant function. thus, phospholipid levels inversely correlate with age, at least in horses [58] . smoking also reduces phospholipid levels in pulmonary surfactant [59] and is thought to increase the risk of adverse outcomes from covid19 [48] . diabetes causes increased serum levels of high mobility group-b1 (hmgb1) [60] , a known damp [31] that activates tlr4 [31] , which would be expected to enhance inflammation. similarly, in some cases hypertension has also been proposed to result from enhanced serum damp levels (reviewed in [61] ), which again could possibly lead to a chronic low-level inflammation. obesity is also thought to be accompanied by inflammation (reviewed in [62] ). indeed, serum levels of c-reactive peptide, a marker of inflammation, have been observed to correlate well with sars-cov-2 viral load and the murray score, which assesses the severity of lung injury in individuals with ards [63] . diabetes, hypertension and obesity have been suggested to predispose individuals to worse outcomes from covid19 [64] . in addition, serum levels of angiotensin ii, which is also reported to increase inflammation through tlr4 [31, 65] , are reported to be elevated in patients with covid-19 [63] . finally, a recent report has suggested that the corticosteroid dexamethasone may improve survival in patients with severe covid-19 [66] . if confirmed, this result would be consistent with the hypothesis described here, since glucocorticoids are known not only to suppress inflammation but also to increase lung surfactant synthesis [67, 68] . by stimulating any remaining type ii alveolar cells to produce more surfactant phospholipids including pg, dexamethasone could both directly and indirectly decrease lung inflammation. surfactant has already been used in studies to treat ards [8] [9] [10] [11] in adult patients, although with less than impressive results. it should be noted that walmrath et al. [9] discussed the likelihood that higher doses and/or more frequent administration of surfactant might be necessary in the case of ards (versus neonatal respiratory distress syndrome) to overcome the ongoing surfactant-inactivating conditions (increased spla 2 levels, inflammation and oxidative stress) often present in ards lungs. we would also like to point out that not all surfactant medications contain pg (for example, exosurf® does not), which could potentially be another explanation, in addition to potentially inadequate dosing and inactivation of surfactant function by shearing [13] , for why not all studies of surfactant administration in ards have found a benefit [12] . despite the mixed results concerning exogenous surfactant medication in adult ards [12, 13] , it is approved by the food and drug administration for intratracheal administration to pre-term infants to treat neonatal respiratory distress syndrome. in infants there are few side effects, and infants who receive surfactant have shorter hospital stays and better survival [69] . similarly, eleven clinical trials in adults have indicated that surfactant therapy is both feasible and safe, with no significant adverse effects reported [70] . therefore, it is postulated that because sars-cov-2 is thought to target and damage/destroy surfactantproducing type ii pneumocytes, covid-19 may be more like neonatal ards than are other types of ards. indeed, like pre-term infants, patients with covid-19 have been found to exhibit hyaline membrane formation [53] . therefore, it is proposed that investigative studies to administer pg-containing surfactant, either synthetic (e.g., surfaxin®) or isolated from bovine (alveofact®, survanta®, beraksurf® and infasurf®) or porcine lungs (curosurf®), to critically ill covid-19 patients be initiated, particularly in view of the fact that few therapies for severe covid-19 have been shown to be effective to date [37] , and such treatments are actively being sought. clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study covid-19: consider cytokine storm syndromes and immunosuppression secretory phospholipase a2-mediated depletion of phosphatidylglycerol in early acute respiratory distress syndrome clinical and biological role of secretory phospholipase a2 in acute respiratory distress syndrome infants phospholipase a2 subclasses in acute respiratory distress syndrome increased surface tension favors pulmonary edema formation in anesthetized dogs' lungs lung morphology and surfactant function in cardiogenic pulmonary edema: a narrative review bronchoscopic surfactant administration in patients with severe adult respiratory distress syndrome and sepsis bronchoscopic administration of bovine natural surfactant in ards and septic shock: impact on gas exchange and haemodynamics bronchoscopic administration of bovine natural surfactant in ards and septic shock: impact on biophysical and biochemical surfactant properties exogenous surfactant in acute respiratory distress syndrome: more is better exogenous pulmonary surfactant for acute respiratory distress syndrome in adults: a systematic review and meta-analysis recombinant surfactant protein c-based surfactant for patients with severe direct lung injury a search for subgroups of patients with ards who may benefit from surfactant replacement therapy: a pooled analysis of five studies with recombinant surfactant protein-c surfactant (venticute) covid-19 pneumonia: different respiratory treatments for different phenotypes? intensive care med covid-19 does not lead to a "typical" acute respiratory distress syndrome ventilation of covid-19 patients in intensive care units intensive care management of coronavirus disease 2019 (covid-19): challenges and recommendations pulmonary surfactant surface tension influences alveolar capillary shape and oxygenation respiratory physiology: a clinical approach the role of ventilation-induced surfactant dysfunction and atelectasis in causing acute respiratory distress syndrome altered surface tension of lung extracts and lung mechanics phospholipid regulation of innate immunity and respiratory viral infection pulmonary surfactant phosphatidylglycerol inhibits mycoplasma pneumoniae-stimulated eicosanoid production from human and mouse macrophages anionic pulmonary surfactant phospholipids inhibit inflammatory responses from alveolar macrophages and u937 cells by binding the lipopolysaccharide-interacting proteins cd14 and md-2 surfactant protein-a and phosphatidylglycerol suppress type iia phospholipase a2 synthesis via nuclear factor-kappab structural analogs of pulmonary surfactant phosphatidylglycerol inhibit toll-like receptor 2 and 4 signaling targeting innate immunity by blocking cd14: novel approach to control inflammation and organ dysfunction in covid-19 illness danger signals in the icu endogenous ligands of tlr2 and tlr4: agonists or assistants? journal of leukocyte biology phosphatidylglycerol inhibits toll-like receptor-mediated inflammation by danger-associated molecular patterns soy phosphatidylglycerol reduces inflammation in a contact irritant ear edema mouse model in vivo pathogen-associated molecular patterninduced tlr2 and tlr4 activation increases keratinocyte production of inflammatory mediators and is inhibited by phosphatidylglycerol 18:1/18:1-dioleoyl-phosphatidylglycerol prevents alveolar epithelial apoptosis and profibrotic stimulus in a neonatal piglet model of acute respiratory distress syndrome phosphatidylglycerol provides short-term prophylaxis against respiratory syncytial virus infection covid-19: towards understanding of pathogenesis sars-cov replicates in primary human alveolar type ii cell cultures but not in type i-like cells innate immune response of human alveolar type ii cells infected with severe acute respiratory syndrome-coronavirus pathogenesis of covid-19 from a cell biology perspective role of lung surfactant in respiratory disease: current knowledge in large animal medicine understanding the principle biophysics concepts of pulmonary surfactant in health and disease. arch dis child fetal neonatal ed contentti ec, correa j. immunosuppression during the covid-19 pandemic in neuromyelitis optica spectrum disorders patients: a new challenge surfactant preparations for preterm infants with respiratory distress syndrome: past, present, and future pulmonary surfactant proteins and lipids as modulators of inflammation and innate immunity infection of human alveolar macrophages by human coronavirus strain 229e the role of collectins and galectins in lung innate immune defense pathological findings of covid-19 associated with acute respiratory distress syndrome pulmonary and cardiac pathology in african american patients with covid-19: an autopsy series from new orleans the acute respiratory distress syndrome: pathogenesis and treatment pulmonary surfactant lipids inhibit infections with the pandemic h1n1 influenza virus in several animal models surfactant and its role in the pathobiology of pulmonary infection. microbes infect influence of age on surfactant isolated from healthy horses maintained on pasture smoking reduces surfactant protein d and phospholipids in patients with and without chronic obstructive pulmonary disease the role of hmgb1 in the pathogenesis of type 2 diabetes the toll of the gridiron: damage-associated molecular patterns and hypertension in american football inflammatory mechanisms in obesity clinical and biochemical indexes from 2019-ncov infected patients linked to viral loads and lung injury angiotensin ii induces inflammatory response partly via tolllike receptor 4-dependent signaling pathway in vascular smooth muscle cells environmental health perspectives chronic hyperglycemia with secondary hyperinsulinemia inhibits the maturational response of fetal lamb lungs to cortisol role of exogenous surfactant in acute lung injury tensiometric and phase domain behavior of lung surfactant on mucus-like viscoelastic hydrogels surfactant inhibition by plasma proteins: differential sensitivity of various surfactant preparations toll-like receptors in antiviral innate immunity the authors would like to acknowledge and thank dr. william davis (augusta university) for helpful feedback on this work. this work received no specific funding. wbb was supported in part by veterans affairs merit award #cx001357 and in part by national institutes of health/national eye institute award #r01ey030576. these sponsors had no role in the research, the writing of the manuscript or the decision to publish. the contents of this article do not represent the official views of the department of veterans affairs or the united states government. the authors declare no conflicts of interest. key: cord-274510-fo7p98np authors: spadera, lucrezia; spadera, maria title: potential role of gcmaf in suppressing the severity of covid-19-induced immune responses: lesson learned from hiv date: 2020-09-24 journal: med hypotheses doi: 10.1016/j.mehy.2020.110293 sha: doc_id: 274510 cord_uid: fo7p98np nan over the last six months, there have been increasing numbers of reports that struggle to understand the pathogenesis of the coronavirus disease 2019 (covid19) pandemic. as of august 16, 2020, the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has been responsible for more than 21 294 000 infections and about 760 000 deaths worldwide [1] , but the mechanisms of virus-induced host damage remain a mystery. understanding the pathways behind viral pathogenicity just like cellular and tissue tropism, counteracting host defence processes and immunological responses are crucial to find new therapeutic strategies. based on clinical reports, it is noteworthy that covid-19 causes various degree of illness ranging from asymptomatic or milder symptomatic cases to severe lung injury or even multi-organ dysfunction with liver and kidney impairment [2] [3] [4] . even in not severe patients, the heterogeneity of symptoms is consistent with the increasing evidence that sars-cov-2 shows a broad tissue tropism, being able to attack almost anything in the body [2] [3] [4] [5] . to date, the most commonly investigated hypothesis about the underlying mechanisms of multi-organ failure may be summarized into three main targets: microcirculation dysfunction, overwhelming inflammation and abnormal coagulation [7] . radiologic and laboratory findings as well as preliminary autopsy studies seem to support this hypothesis. the most common patterns seen on chest ct were ground-glass opacity, interlobular septal thickening, air bronchogram, bilateral patchy shadowing, crazy-paving pattern, and thickening of the adjacent pleura, resembling an interstitial involvement in viral pneumonia [2] [3] 7] . under the light of microscope, the lungs revealed diffuse alveolar damage with formation of numerous hyaline membranes, very patchy and sparse interstitial chronic inflammation composed mainly of lymphocytes, thrombi within a few small pulmonary artery branches, congestion of alveolar septal capillaries, focal edema fluid, and macrophage infiltration within the airspaces [9] [10] [11] [12] . the more significant laboratory abnormalities were metabolic acidosis, lymphocytopenia, leukopenia, thrombocytopenia, elevated levels of creactive protein (crp), interleukin-6 (il-6), lactate dehydrogenase (ldh) and d-dimer [2] [3] [4] [5] [6] [7] . as firstly suggested by huang c et al. [4] , the systemic cytokine storm could play a key role in the virusinduced tissue damage. however, the question "what the link for the overproduction of proinflammatory mediators and the immune suppression, on the hand, and microvascular injury and thromboembolism, on the other, is", remains unclear. being the knowledge of this issue very scarce, lessons learned from other human pathogenic viruses, with specific reference to human immunodeficiency virus (hiv), could be diriment. based on the aforementioned findings and on documented analogies between sars-cov-2 and hiv [13] , we hypothesized that the reduced conversion activity of the gc protein (human groupspecific component (gc)) into the macrophage activating factor (maf) could have a key role in the dysregulate immune response induced by sars-cov-2, just like for hiv infected patients [14] [15] . if this hypothesis is correct, it might help to set a valid strategy of immunotherapy also based on an off-label use of gcmaf in critically ill covid-19 patients. gc globulin, dbp and gcmaf: three in one. serum gc protein, also known as vitamin d-binding protein (dbp), is a multifunctional protein present in plasma/serum at concentrations of 300-600 mg/l [16] . it carries a trisaccharide consisting of n-acetylgalactosamine with dibranched galactose and sialic acid termini at 420 threonine residue [17] . stepwise hydrolysis of gc protein by the inducible membranous βgalactosidase of stimulated b-lymphocytes, and by the neu-1 sialidase of t-lymphocytes converts it into the active gcmaf [17] [18] [19] . on the contrary, deglycosilation of gc protein by action of the enzyme alpha-n-acetylgalactosaminidase, named nagalase, secreted from hiv-infected cells leads to lack of macrophage activation and to immunosuppression, as a consequence [14] [15] . it is remarkable that nagalase was demonstrated to be an intrinsic component not only of the envelope glycoproteins gp120 and gp160 of hiv but also of the hemagglutinin (he) of influenza virus [15, 20] and even produced by neoplastic cells [21] [22] [23] . indeed, flu-like symptoms with serum nagalase activity similar to the influenza acute state were reported in the early stage of hiv-infection, so that the serum enzyme activity may be detectable at all phases of hiv-infection [14] [15] . similarly, most covid-19 patients complained of flu-like symptoms in the early stages of the disease [2] [3] [4] [5] . it is now well known that dbp gc-globulin plays a crucial role in immune system regulation as a primary defense against infections [14] [15] [16] [17] [18] [19] [20] . in addition to the storage and transport of active vitamin d3, gcmaf's effects include macrophage modulation, osteoclast activation, facilitation of neutrophil chemotaxis mediated by c5 derived peptide, superoxide activity, scavenging of circulating g-actin, anti-angiogenetic and anti-tumor properties [24] [25] [26] [27] [28] . thus, this multifunctional protein, released into the blood stream, acts as a systemic immune modulator without pro-inflammatory activities. this means that any function impairment of gc-globulin could result in a state of both immunosuppression and uncontrolled inflammation, just like in severe covid-19. interestingly, hiv viremia was associated with higher level of biomarkers of inflammation (measured by il-6), monocyte activation (soluble cd14), and coagulation (d-dimer), leading to increased mortality, as compared with uninfected people [29] . meanwhile, in covid-19 patients, in addition to the reduced peripheral lymphocyte counts, mainly cd4 + t and cd8 + t cells, there were found significant high levels of pro-inflammatory cytokines and chemokines [2] [3] [4] [5] [6] [7] . indeed, gcmaf is not only a simple potent activator for macrophages, but more specifically is able to turn macrophage activity on at the sites of infection/inflammation and then to induce their apoptosis by upregulating caspase activity via the p38 and jnk1/2 pathways when no longer needed [30] . post-mortem lung observations of patients died of covid-19 showed the presence of mononuclear cells and macrophages infiltrating air spaces by autopsy [9] [10] [11] [12] . with regards to the anti-oxidant properties, it was assessed that gcmaf promotes the superoxide generating capacity of activated macrophages and the production of nitric oxide (no) [31] . an article by nozik-grayck et al. [32] pertinently and interestingly showed that the expression of extracellular superoxide dismutase (ec-sod) mrna and protein is cell-and tissue-specific and is prominent in lung, heart, blood vessels, placenta and kidney. in particular, high levels of ec-sod are present in lung macrophages, alveolar type ii cells, fibroblasts, vascular smooth muscle cells, and endothelial cells. ec-sod limits oxidative stress and preserves no bioactivity, thus protecting against a number of lung and cardiovascular diseases [32] . even though only in a minority of cases, covid-19 may progress to life-threatening complications, including respiratory failure, acute cardiac injury, acute kidney injury, septic shock, disseminated intra-vascular coagulation (dic), and multi-organ dysfunction [2] [3] [4] [5] . hypoxemia was found to be associated with interstitial pneumonia and, in 10% to 20% of cases, developed into acute respiratory distress syndrome (ards) [2] [3] [4] [5] . in this connection, it was documented that ards as well as organ dysfunction and septic shock is characterized by actin release which is involved in microvascular impairment [33] [34] . dbp has an additional function in binding monomeric globular (g)-actin with high affinity. thereby, rapidly removing polymeric actin fibrils from the blood stream, it prevents actin polymers from clogging the micro vessels not unlike fibrinogen/fibrin and consequently platelet aggregation and micro thrombi formation [35] [36] . what we postulated could also explain hypercoagulability with elevated concentrations of d-dimer, fibrin degradation products increase, pt and aptt prolongation, observed in covid-19 patients [37] [38] . tang n et al. [38] reported that 71.4% of the non survivors of covid-19 matched the grade of overt-dic according to the international society on thrombosis and haemostasis (isth) diagnostic criteria for dic. murine models deficient in dbp showed lung damage caused by actin polymerization, developing severe acute lung inflammation with vascular leakage, hemorrhage and thickening of the vascular wall after actin injection [39] . interestingly, the lung was the only organ that showed inflammatory injury after intravenous actin injection. the observed lung inflammation was consistent with alterations to lung microvascular endothelial cells. indeed, when lung endothelial cells were exposed to dbp-actin complexes in vitro showed enhanced cell death [39] . reduced levels of dbp were even observed in sepsis and organ dysfunction of trauma patients as well as complete depletion of free dbp in those affected by septic shock [33] [34] . these data could provide support for pathogenic explanations of cellular and tissue damage by sars-cov-2 and, at the same time, for the therapeutic use of dbp to bind extracellular actin and counteract microcirculatory alterations. whereas dbp also binds free fatty acids, it was shown that the administration of gcmaf complexed with oleic acid (oa) via nebulisation or subcutaneous injection led to rapid decrease of blood pressure and increase in splenic blood flow, as a result of a verisimilar synergistic no release by oa-gcmaf-activated alveolar and splenic macrophages [31] . severe or critically ill covid-19 patients developed clinical typical manifestations of shock, even in the absence of overt hypotension [7] . furthermore, it was found that gcmaf can inhibit the angiogenesis induced by proinflammatory prostaglandin e1 [40] , which serves roles in the promotion of vascular endothelial growth factor (vegf) expression [41] . a key role of vegf in acute lung injury and ards was confirmed [42] . reflecting the fact that clinical features and severity of symptoms vary widely between and within each covid-19 patient, with older males more likely to be affected and in a more severe manner [43] , we sought to relate it with some special feature of dbp. several studies showed that the polymorphisms of dbp were associated with susceptibility or resistance to disease states including chronic obstructive pulmonary disease [44] [45] . moreover, whereas androgens were not found to have any effect on circulating levels of dbp, exposure to high levels of estrogens increased them by up to 50%, suggesting a potential protective role of estrogens against covid-19 [28] . on the other hand, in relation to vitamin d status, advanced age was recognized as one of the major risk factors for vitamin d deficiency [46] . animal-based studies also demonstrated that deficiencies in both dietary protein-and energy-intake decreased the concentration of dbp in the circulation [47] . these data seem to be in line with the growing evidence that vitamin d supplementation could reduce the risk of covid-19 infections and deaths [48] [49] [50] . to date, a pharmaceutical grade gcmaf agent that can be administered to patients with covid-19 is not developed yet, but, as our hypothesis suggests that gcmaf, an activator of macrophage, a key player of innate immunity, may be effective in suppressing the severity of covid-19-induced immune responses, it would be advisable to proceed in this way. in the gcmaf development timeline, there have been three major types of gcmaf until now: purified (i.e. first-generation) gcmaf, serum (i.e. second-generation) gcmaf, and oral colostrum maf (i.e. third-generation gcmaf). the first-generation gcmaf is produced from gc protein isolated from human serum by an artificial enzymatic method using an affinity column modified with 25-hydroxy-vitamin d3 [51] . the second-generation gcmaf is prepared from degalactosylated/desialylated human serum without isolation of gc protein using vitamin d affinity chromatography, leading to a higher concentration, stability and activity of the final gcmaf, without the risk of cross-contamination between different serum samples [52] . in addition, the second-generation gcmaf has been shown to have increasing activity of macrophages, superoxide radical generation, anti-angiogenetic effects, and antitumor effects. in 2014, saisei mirai clinics (cell processing center, clinic in kobe, osaka and tokyo), in collaboration with tokushima university, developed a new form of gcmaf made from bovine colostrum [53] . colostrum maf has the advantage that it can be orally administered, namely, in an acid-resistant enteric capsule to activate macrophages in the gut-associated lymphoid tissue (galt) [54] . this is considered to be the largest macrophage pool in the body playing a very important role in maintaining and regulating mucosal immunity [54] . macrophages in the gastrointestinal mucosa also could modulate the respiratory tract mucosal immunity through immune regulation, the so-called "gut-lung axis". additionally, colostrum maf is administered as a powder in the mouth to activate macrophages in the lymphoid tissue of the waldeyer's tonsillar ring. in this regard, it is remarkable that angiotensin converting enzyme 2 (ace2), an entry receptor for sars-cov-2, was found to be highly expressed in gastrointestinal epithelial cells, providing a prerequisite for sars-cov-2 infection [55] . on this base, if we consider gcmaf as an off-label immunomodulating agent for the treatment of covid-19, oral administration should be preferred to all the other ones. although the administration of gcmaf is a yet an unapproved therapy, data from previous studies and clinical practice reported its effectiveness in the treatment of many pathologies such as hiv infection [56] and other infectious diseases [54] , some types of cancer [57] [58] [59] [60] [61] , juvenile osteopetrosis [62] , immunological (systemic erythematous lupus) [63] and neurological (multiple sclerosis, autism) diseases [58, [64] [65] . in the same conditions, it was found an inverse correlation between the maf precursor activity and serum levels of nagalase (reference ranges from 0,32 to 0,65-0,95 nm/min/ng), therefore showing to be other than pathogenicity or cancer biomarkers, also good prognosticators of illness and response to therapy [55] [56] [57] [58] [59] [60] [61] [62] [63] [64] [65] . however, it's clear that only well-designed clinical trials will be able to properly evaluate the therapeutic use of gcmaf. the main targets of the pharmacologic approaches to covid-19, especially for the complicated cases, are addressed to modulate the immune system and counteract the overwhelming inflammation. notably, the mechanisms we have hypothesized about the possible pathogenesis of the cell and tissue damage induced by sars-cov-2 seem to provide a common denominator in explaining the effects of most drugs currently considered for the treatment of covid-19 : these include antivirals (i.e. remdesivir, lopinavir/ritonavir, darunavir, cobicistat) and immunomodulating and/or antiinflammatory drugs [66] [67] . in particular, based on their antiviral activity [68] , chloroquine and hydroxychloroquine, initially conceived as antimalarial therapeutics, were proposed to treat patients hospitalized with covid-19, better if associated to azithromycin, showing promising efficacy in "inhibiting the exacerbation of pneumonia, improving lung imaging findings, promoting a virus negative conversion and shortening the disease course" [69] [70] . on the other hand, hydroxychloroquine is the cornerstone of medical therapy in lupus, where it acts as an immunomodulatory without immunosuppressive effects [71] . however, in light of ongoing serious cardiac adverse events [72] and other serious side effects, the known and potential benefits of chloroquine and hydroxychloroquine no longer outweigh the known and potential risks for the authorized use in covid-19 patients. in addition, the covidtocilizumab, an il-6 antagonist, approved for the treatment of rheumatoid arthritis and juvenile idiopathic arthritis, also had therapeutic application in critical covid-19 patients, providing encouraging results [74] . however, the phase iii clinical trial (covacta) [75] for evaluating tocilizumab in hospitalized patients with severe covid-19 pneumonia found no difference between tocilizumab versus placebo in intensive care requirements or mortality. therefore, there are insufficient data for the panel to recommend either for or against the use of the il-6 inhibitors for the treatment of covid19 [73] . the rationale basis for the use of monoclonal antibodies in patients affected by sars-cov-2 seems to lie in the so-called systemic cytokine storm. taking into account the key role of vegf in enhancing angiogenesis in acute lung injury and ards [76] , two trials, evaluating the efficacy of bevacizumab as vegf antagonist in the treatment of covid-19 (best-pc and best-rct), were started [77] [78] . less than two months after the declaration of pandemic state by the world health organization, every effort by the entire scientific community has been made to face this worldwide emergency of covid-19 in the best possible way. however, at the present days, the underlying mechanisms of pathophysiology remain unknown. nowadays, although a number of preliminary clinical trials are underway and scientific evidence is growing on this topic, neither specific drugs nor effective preventive measures are yet available for the treatment of covid-19. anyway, it seems we still have a great deal of work to find the "miracle care". however, due to the risk of serious drug-related adverse events, the immunomodulatory and anti-inflammatory drugs currently used for covid-19, are still restricted to carefully selected and complicated cases [72, [79] [80] [81] [82] . so, in sight of this, given its multifunctional properties, we believe that gcmaf could have a very important role in the pathophysiology of organ damage induced by sars-cov-2, providing explanations which are consistent with the clinical, radiological and histopathological findings observed in patients with covid-19. despite burgeoning data from case series of various pathological conditions demonstrated the potential clinical benefits of gcmaf as above mentioned [56] [57] [58] [59] [60] [61] [62] [63] [64] [65] , no randomized controlled clinical trials verifying these preliminary results have been made till now. so, there are still unresolved controversies about the possibility of an its therapeutic application. to date, no other researcher has investigated the possibility of a potential linkage between gcmaf and covid-19. however, in view of the immunomodulatory potential and the high safety profile of gcmaf and because covid-19 remains a life-threatening condition in many cases, despite currently recommended therapies, we think it is worth explorating our hypothesis further by: in conclusion, even though our hypothesis might be not fully correct, we still wanted to give our own contribution to the research on covid-19 pandemic which had the only underserved merit to give all humans across the world a common goal. so, as we still see life from a rosy perspective from this small italian hospital facing the sea, our last thought and gratitude are for all the researchers and health care professionals which are committed to the forefront of the war against sars-cov-2. world health organization clinical characteristics of coronavirus disease 2019 in china epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study clinical features of patients infected with 2019 novel coronavirus in wuhan, china. lancet coronavirus disease 2019 (covid-19): a clinical update longitudinal characteristics of lymphocyte responses and cytokine profiles in the peripheral blood of sars-cov-2 infected patients sars-cov-2 and viral sepsis: observations and hypotheses radiological findings from 81 patients with covid-19 pneumonia in wuhan, china: a descriptive study covid-19 autopsies pulmonary pathology of early-phase 2019 novel coronavirus (covid-19) pneumonia in two patients with lung cancer: special report pathological findings of covid-19 associated with acute respiratory distress syndrome pathological study of the 2019 novel coronavirus disease (covid-19) through post-mortem core biopsies a tug-of-war between severe acute respiratory syndrome coronavirus 2 and host antiviral defence: lessons from other pathogenic viruses. emerg microbes infect structural modification of serum vitamin d3-binding protein and immunosuppression in aids patients pathogenic significance of alpha-n-acetylgalactosaminidase activity found in the envelope glycoprotein gp160 of human immunodeficiency virus type 1 turnover of the plasma binding protein for vitamin d and its metabolites in normal human subjects vitamin d3 binding protein (group-specific component) is a precursor for the macrophage-activating signal factor from lysophosphatidylcholine -treated lymphocytes vitamin d3-binding protein as a precursor for macrophage activating factor in the inflammation-primed macrophage activation cascade in rats regulation of inflammation-primed activation of macrophages by two serum factors, vitamin d3-binding protein and albumin pathogenic significance of alpha-n-acetylgalactosaminidase activity found in the hemagglutinin of influenza virus. microbes infect deglyco-sylation of serum vitamin d3-binding protein leads to immunosuppression in cancer patients is α-n-acetylgalactosaminidase the key to curing cancer? a mini-review and hypothesis promising role for gc-maf in cancer immunotherapy: from bench to bedside gc protein (vitamin d-binding protein): gc genotyping and gcmaf precursor activity is chondroitin sulfate responsible for the biological effects attributed to the gc protein-derived macrophage activating factor (gcmaf)? med hypotheses vitamin d-binding protein (gc-globulin) binds actin effects of vitamin d(3)-binding protein-derived macrophage activating factor (gcmaf) on angiogenesis plasma vitamin d-binding protein (gc-globulin): multiple tasks do biomarkers of inflammation, monocyte activation, and altered coagulation explain excess mortality between hiv infected and uninfected people? mitogen-activated protein kinase pathway mediates dbp-maf-induced apoptosis in raw 264.7 macrophages oleic acid, deglycosylated vitamin d-binding protein, nitric oxide: a molecular triad made lethal to cancer extracellular superoxide dismutase plasma concentration of gc-globulin is associated with organ dysfunction and sepsis after injury the extracellular actin-scavenger system and actin toxicity vitamin d-binding protein (gc-globulin) binds actin coordinated inhibition of actin-induced platelet aggregation by plasma gelsolin and vitamin d-binding protein hematological findings and complications of covid-19 abnormal coagulation parameters are associated with poor prognosis in patients with novel coronavirus pneumonia circulating complexes of the vitamin d binding protein with g-actin induce lung inflammation by targeting endothelial cells effect of paricalcitol and gcmaf on angiogenesis and human peripheral blood mononuclear cell proliferation and signaling can angiogenesis be exploited to improve stroke outcome? mechanisms and therapeutic potential vascular endothelial growth factor in acute lung injury and acute respiratory distress syndrome sex and gender disparities in the covid-19 pandemic vitamin d binding protein gene polymorphisms and chronic obstructive pulmonary disease: a meta-analysis investigation of vitamin d-binding protein polymorphism impact on coronary artery disease and relationship with longevity: own data and a review vitamin d physiology changes with malnutrition in the concentration of plasma vitamin d binding protein in growing rats evidence that vitamin d supplementation could reduce risk of influenza and covid-19 infections and deaths sex-specific sars-cov-2 mortality: among hormone-modulated ace2 expression, risk of venous thromboembolism and hypovitaminosis d early nutritional supplementation in noncritically ill patients hospitalized for the 2019 novel coronavirus disease (covid-19): rationale and feasibility of a shared pragmatic protocol affinity purification of human plasma vitamin d-binding protein gcmaf: our next-generation immunotherapy degalactosylated/desialylated bovine colostrum induces macrophage phagocytic activity independently of inflammatory cytokine production oral colostrum macrophage-activating factor for serious infection and chronic fatigue syndrome: three case reports immunotherapy of hiv-infected patients with gc proteinderived macrophage activating factor (gcmaf) immunotherapy for prostate cancer with gc protein-derived macrophage-activating factor therapeutic effects of highly purified de-glycosylated gcmaf in the immunotherapy of patients with chronic diseases clinical experience of integrative cancer immunotherapy with gcmaf case report: a breast cancer patient treated with gcmaf, sonodynamic therapy and hormone therapy the use of gc protein-derived macrophage activating factor for management of thyroid cancer defective lymphocyte glycosidases in the macrophage activation cascade of juvenile osteopetrosis deglycosylation of serum vitamin d3-binding protein by alpha-n-acetylgalactosaminidase detected in the plasma of patients with systemic lupus erythematosus case report: gcmaf treatment in a patient with multiple sclerosis initial observations of elevated alpha-nacetylgalactosaminidase activity associated with autism and observed reductions from gc protein--macrophage activating factor injections current pharmacological treatments for covid-19: what's next? treatment options for covid-19: the reality and challenges comparison of the antiviral activity in vitro of some non-steroidal anti-inflammatory drugs of chloroquine and covid-19 breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid-19 associated pneumonia in clinical studies current and emerging treatment options in the management of lupus chloroquine intoxication tocilizumab: a review of its use in the management of rheumatoid arthritis -identifier: nct04320615, a study to evaluate the safety and efficacy of tocilizumab in patients with severe covid-19 pneumonia (covacta) biological activity of bevacizumab, a humanized anti-vegf antibody in vitro -identifier nct04275414, bevacizumab in severe or critical patients with covid-19 pneumonia (best-cp) national library of medicine (us) ocular toxicity of hydroxychloroquine use of tocilizumab for covid-19 infection-induced cytokine release syndrome: a cautionary case report letter to the editor: lipemic serum in patients with covid-19 undergoing treatment rational use of tocilizumab in the treatment of novel coronavirus pneumonia key: cord-290803-v5ndlk9c authors: khan, imran; aziz hatiboglu, mustafa title: can covid-19 induce glioma tumorogenesis through binding cell receptors? date: 2020-06-19 journal: med hypotheses doi: 10.1016/j.mehy.2020.110009 sha: doc_id: 290803 cord_uid: v5ndlk9c the outbreak of novel coronavirus 2019 (covid-19) represents a global threat to the public healthcare. the viral spike (s) glycoprotein is the key molecule for viral entry through interaction with angiotensin converting enzyme 2 (ace2) receptor molecules present on the cell membranes. moreover, it has been established that covid-19 interacts and infects brain cells in humans via ace2. therefore in the light of these known facts we hypothesized that viral s protein molecule may bind to the other overexpressed receptor molecules in glioma cells and may play some role in glioma tumorogenesis. thus we leverage docking analysis (hex and z-dock) between viral s protein and epidermal growth factor receptors (egfr), vascular endothelial growth factor receptors (vegfr) and hepatocyte growth factor receptors (hgfr/c-met) to investigate the oncogenic potential of covid-19. our findings suggested higher affinity of viral s protein towards egfr and vegfr. although, the data presented is preliminary and need to be validated further via molecular dynamics studies, however it paves platform to instigate further investigations on this aspect considering the aftermath of covid-19 pandemic in oncogenic perspective. the outbreak of novel coronavirus 2019 (covid-19) represents a global threat to the public healthcare. the viral spike (s) glycoprotein is the key molecule for viral entry through interaction with angiotensin converting enzyme 2 (ace2) receptor molecules present on the cell membranes. moreover, it has been established that covid-19 interacts and infects brain cells in humans via ace2. therefore in the light of these known facts we hypothesized that viral s protein molecule may bind to the other overexpressed receptor molecules in glioma cells and may play some role in glioma tumorogenesis. thus we leverage docking analysis (hex and z-dock) between viral s protein and epidermal growth factor receptors (egfr), vascular endothelial growth factor receptors (vegfr) and hepatocyte growth factor receptors (hgfr/c-met) to investigate the oncogenic potential of covid-19. our findings suggested higher affinity of viral s protein towards egfr and vegfr. although, the data presented is preliminary and need to be validated further via molecular dynamics studies, however it paves platform to instigate further investigations on this aspect considering the aftermath of covid-19 pandemic in oncogenic perspective. considering the importance of these surface receptors on glioma cells, we studied the interaction between egfr, vegfr and c-met receptor proteins with s protein of covid-19. since wan et al. 4 already analyzed the binding of the human ace2 protein with rbd of covid-19 s protein, we used this binding as reference for protein-protein interaction in our analysis. the 3d structures of egfr, vegfr, c-met, ace2 and s protein were extracted from uniprot, protein data bank (pdb) id numbers -1m14, 1vpf, 2uzx, 2ajf and 2ghv, respectively. in order to test our hypothesis, we utilized two different molecular protein-protein docking platforms: hex 5.1 and z dock server 5,6 . z-dock was performed on the protein-protein docking server using fast fourier transform algorithm. our results from hex docking showed that covid-19 s protein has binding affinity towards egfr, vegfr and c-met receptor proteins, which was comparable to the binding affinity between ace2 and s protein (figure 1) . these findings were further substantiated by z dock binding scores of s -egfr, s -c-met, s -vegfr and s -ace2 interacting protein complexes which were found to be 1697.528 for s -egfr, 1958.115 for s -c-met, 1690.053 for s -vegfr and 1843.070 for s -ace2. the mechanism through which viruses exploit the presence of selective receptors on cells to interact with and infect the cells is well known 7, 8 . for instance, complement receptor 2 (cr2), which is found on astrocytes, facilitates the entry of epstein bar virus (ebv) through interaction between ebv surface protein gp350 and cr2 7, 8 . similarly, cytomegalovirus (cmv) immediate-early (ie) proteins bind to retinoblastoma (rb), p53 and p21, which subsequently leads to alteration in telomerase activity and cell cycle regulation 9 . overall, there are several known receptor mediated entry and oncogenesis of different viruses. our preliminary findings suggested that covid-19 s protein might have a binding affinity to egfr, c-met and vegfr on glioma cells. therefore, we speculate that covid-19 can induce glioma tumorogenesis through the s protein, this may increase the risk of developing glioma in covid-19 infected individuals, and may amplify tumor growth in covid-19 infected glioma patients. finally, our findings do not provide a definitive model for establishing the oncogenic potential of s protein and warrant further investigation. this could be accomplished via molecular dynamic simulation methods in the future. (7) correlation type-shape + electrostatics. coronavirus infections-more than just the common cold evidence of the covid-19 virus targeting the cns: tissue distribution, host-virus interaction, and proposed neurotropic mechanisms targeting cellular pathways in glioblastoma multiforme receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus protein docking using spherical polar fourier correlations docking unbound proteins using shape complementarity, desolvation, and electrostatics epstein-barr virus infection of human astrocyte cell lines progress and problems in understanding and managing primary epstein-barr virus infections role of infectious agents in the carcinogenesis of brain and head and neck cancers the authors are grateful to dr. a. matteen rafiqi for language editing of key: cord-264916-c4n0kyog authors: zimmerman, keith; kearns, fiona; tzekov, radouil title: natural protection of ocular surface from viral infections – a hypothesis date: 2020-07-09 journal: med hypotheses doi: 10.1016/j.mehy.2020.110082 sha: doc_id: 264916 cord_uid: c4n0kyog a pandemic outbreak of a viral respiratory infection (covid-19) caused by a coronavirus (sars-cov-2) prompted a multitude of research focused on various aspects of this disease. one of the interesting aspects of the clinical manifestation of the infection is an accompanying ocular surface viral infection, viral conjunctivitis. although occasional reports of viral conjunctivitis caused by this and the related sars-cov virus (causing the sars outbreak in the early 2000s) are available, the prevalence of this complication among infected people appears low (∼1%). this is surprising, considering the recent discovery of the presence of viral receptors (ace2 and tmprss2) in ocular surface tissue. the discrepancy between the theoretically expected high rate of concurrence of viral ocular surface inflammation and the observed relatively low occurrence can be explained by several factors. in this work, we discuss the significance of natural protective factors related to anatomical and physiological properties of the eyes and preventing the deposition of large number of virus-loaded particles on the ocular surface. specifically, we advance the hypothesis that the standing potential of the eye plays an important role in repelling aerosol particles (microdroplets) from the surface of the eye and discuss factors associated with this hypothesis, possible ways to test it and its implications in terms of prevention of ocular infections. ocular surface infections caused by viruses are a considerable public health problem worldwide. for example, an analysis of the 1985 national ambulatory medical care survey found that ~1% of all primary care office visits in the united states were related to conjunctivitis [1] . if one assumes that the proportion remained the same, which would translate to ~2.3m visits in 2019. similar proportion has been reported for uk [2] . infectious conjunctivitis 1 is more prevalent than the other types (e.g. allergic, chemical, etc.) and viral conjunctivitis is estimated to be the most common cause of infectious conjunctivitis, at up to 80% of all cases [3] . of all different types of viruses as candidates of causing infectious conjunctivitis, adenoviruses have been found to be the most common pathogen, in up to 90% of all cases [4] . however, it has to be kept in mind that exhaustive testing related to the causation of conjunctivitis is not always possible, which may introduce bias in reported results. apart from adenoviruses, other types of viruses reported to be isolated from the conjunctiva and implicated in the development of conjunctivitis are herpes simplex virus, enterovirus 70 and coxsackie a24 variant virus [5] . somewhat surprisingly, the majority of the viruses responsible for respiratory viral infections are not a major cause of infectious conjunctivitis [6] . thus, even the adenovirus causing the majority of infectious conjunctivitis cases is subtype d, while strains that cause respiratory infections are subtypes b, c and e and they rarely cause conjunctivitis ( table 1 in [6] ). similarly, only one of the influenza viruses subtypes (h7, causing avian influenza), is a significant cause of conjunctivitis, with ~80% of the cases with this infection presenting with conjunctivitis [7] , while the much more common subtypes causing human infection with influenza virus subtypes that are more commonly associated with respiratory illness are an infrequent cause of the disease [6] . recently, the worldwide attention has been focused on a viral infection caused by a coronavirus, resulting in a global pandemic. this infection is most often associated with respiratory symptoms and its major complication is overwhelmingly a disease of the lower respiratory tract, a bilateral viral pneumonia [8] and, therefore, can be classified as a respiratory viral infection. it is caused by a coronavirus (sars-cov-2) [9] , while the disease has been named covid-19 [10] . this virus belongs to the subfamily of coronaviruses, a term proposed in 1968 to describe a group of enveloped, positive-sense, single-stranded rna viruses with similar form, including a characteristic appearance of the envelope glycoproteins in electron microscopy observation, recalling the solar corona [11] . this proposal was accepted and currently this type of viruses are classified as subfamily orthocoronavirinae (of family coronaviridae) and contains 4 major groups (genera): alpha-, beta-, gamma-and deltacoronaviruses, a total of 25 species, the majority of which are found in animals, but not humans [12] . the viruses found either exclusively or not in humans typically cause respiratory tract illnesses [13] . seven types of coronaviruses are known to cause disease in humans. the two types that were discovered in the 1960s, hcov-oc43 (a betacoronavirus) and hcov229e (an alphacoronavirus), both causing mostly mild upper respiratory tract illnesses in adults. they are still in circulation, together with three types identified in the 2000s: hcov-nl63, hcov-hku1 (both alphacoronaviruses) and mers-cov (a betacoronavirus), the first two casing mild upper and lower respiratory tract infections in adults, while the third one causing more severe infections. another virus, sars-cov (a betacoronavirus), identified also in the early 2000s, causing severe acute respiratory syndrome (sars), is no longer circulating in humans after strict and coordinated public health measures [13] . the new virus, sars-cov-2, has closest similarity (40-90% identity) to the first sars-cov virus [14] and shows similar clinical manifestations, although with a lower mortality rate [15] . generally, coronaviruses infecting humans have been rarely associated with ocular surface infections [6] . only rare reports of conjunctivitis have been associated with hcov-nl63 [16] and no reports have been presented for sars-cov [17] . whether and to what extent these types of viruses can be spread through ocular surface exposure remains a subject of debate and uncertainty. for example, the sars-cov virus was detected in the conjunctiva from 3 probable cases (out of 36) in one study [18] , but not in two other studies in tears and conjunctival scraping samples [19, 20] , triggering questions about the mechanism and details of identification of the virus in ocular tissues and fluids [21, 22] . two recent studies by xia et al. [23] and wu et al. [24] , showed also a low rate of virus detection in conjunctiva (4%). a recent meta-analysis including 1,167 patients, indicates that frequency of conjunctivitis associated with sars-cov-2 infection (covid-19) was generally low: ~1.1% (3% in severe and 0.7% nonsevere covid-19 patients) [25] . on the other hand, two recent reports showed expression of established sars-cov [26] and sars-cov-2 [27] receptors, the angiotensin-converting enzyme 2 (ace2) and transmembrane protease serine subtype 2 (tmprss2) in human conjunctiva, limbus and cornea [28, 29] . this finding indicates that ocular surface cells including in the conjunctiva could be susceptible to the virus and theoretically serve as a point of entry for the viral infection. therefore, a question arises: if the receptors for the virus are present in ocular surface tissues, why the incidence of ocular surface infection is so low and isolating the virus from ocular surfaces presents such a challenge? our hypothesis is the standing potential of the eye interacts with microdroplets carrying the virus and prevents -either partially or in whole -microdroplets from landing and attaching to the ocular surface. such an interaction would greatly reduce the probability of virus presence on, and in turn reduce infections in, ocular surface tissues. the fact the eye is electrically charged was first demonstrated in an animal eye by du bois-reymond in 1849 [30] and re-discovered by frithiof holmgren in 1865 in a fish eye [31]. the first rigorous confirmation of the standing potential of the eye as a corneo-retinal potential in humans was provided by mowerer et al. in 1935 [32] . in the 1960s, detailed investigation of the magnitude of the standing potential under standardized light-adapted conditions showed that it is on average ~+0.7 mv (range 0.25 to 1.1 mv), with the cornea having a positive charge compared to the back of the eye [33] and it was shown that this potential is relatively stable on an hourly, daily and weekly basis, but shows some diurnal variation. it has to be noted that these measurements were made indirectly with electrodes placed on both sides of the eyeball, at the outer and inner canthus of the eye, and, therefore, the true electrical potential in front of the cornea is likely to be higher (+5 mv), which was suspected since the 1940s based on animal studies and confirmed in humans in the 1970s with more direct laboratory measurements [34] . it was estimated further that if the corneal potentials were measured relative to the skin on the forehead or cheek, the potential difference would be even higher at +10 to +15 mv [35] . this potential could be further enhanced by electrostatic charges on the eyelid skin. it is well-documented that human oily skin can become highly positively electrostatically charged, as it is usually listed near the top of the triboelectric series [36] . bioaerosols generated by human exhalation are considered a possible route for sars-cov-2 spread [37] [38] [39] [40] . in support of this possibility, previous studies testing of air samples showed the presence of another coronavirus (mers-cov) have found the virus in 4 of 7 air samples [41] . studies of the sars-cov virus also suggested airborne transmission [42] and infective droplet inhalation [43] , although some uncertainty remains [44] . influenza virus rna was also recovered from air sampled in a hospital [45] . finally, a recent study identified seasonal human coronaviruses, influenza viruses and rhinoviruses in exhaled breath and coughs of patients with respiratory disease [46] . currently, it is generally accepted that bioaerosols as an infectious disease transmission medium should be divided into three groups based on aerodynamic droplet diameter: small particles (less than 10 µm), that can remain airborne, larger droplets (more than 20 µm), which settle relatively quickly to the ground and an intermediate size (10-20 µm) may either settle or remain airborne [47] . apart from the consensus built around this reasonable classification, there is a great deal of variation within the range and distribution of particle size reported by different studies, which likely depends largely on the methodology used to count and classify the particles. thus, the following discussion of this topic should not be treated as a comprehensive analysis of this subject, but rather as one specific perspective on the reported results in view of the matter discussed here. the measurement of the size of exhaled particles in a 2011 study showed that more than 82% of all exhaled particles from three healthy and 16 human rhinovirus (hrv)-infected subjects were within 0.3-0.5 µm diameter range, placing it firmly in the small droplets category [48] . given that the average diameter of the sars-cov-2 virus is ~0.12 µm [49, 50] and assuming that the maximal viral concentration per droplet should be an occupancy of ~30% of the whole surface of the droplet 2 , the maximal viral load per particle can be estimated between 8 and 21 virions/droplet 3 . although this appears like a small load per droplet, the same study found that the droplet production varied dramatically between subjects, with some subjects (4 out of 17) producing ~3,500 particles/liter (range ~1,000 to ~7,000), equivalent to ~28,000 droplets/min, with the potential to spread more than 500,000 viral particles/min via airborne particles. in contrast, the rest of the subjects produced on average ~7.4 particles/liter (equivalent to ~52 droplets/min with some participants generating 0 droplets), thus, supporting the hypothesis that some people could be "high spreaders" of the viral infection. it should be emphasized that this study estimated the droplet size and production under regular breathing. the result is likely different when sneezing. a study estimating the droplet sizes from sneezing found almost exclusively large droplets with diameter larger than 50 µm (and up to 1,000 µm) [51] . for comparison, cough in healthy volunteers appeared to generate particles with less than 1 µm in diameter [52] . some studies suggest that large droplets only originate from the oral cavity [53] . although studies with coronavirus are not available, one study measured influenza virus in cough samples and found that 35% of the influenza rna was isolated from particles >4 µm diameter, while 23% of influenza rna was isolated from particles 1 to 4 µm diameter, and 42% in particles <1 µm [54] . high spreaders for the influenza virus were confirmed in another study [55] . the size and viral load have potential importance for the probability of landing on ocular surfaces. thus, larger droplets, loaded with more viral particles are expected to have poorer aerodynamic characteristics and be more susceptible to gravity forces and quicker landing. given lower airflow speed under normal breathing conditions, the main type of viral airborne spread seems to be small droplets, which can stay afloat for many minutes to hours, but carry relatively little viral load per droplet. as the intensity of exhaled airflow increases, as in loud talking, singing, coughing and sneezing, the droplet formation becomes more and more dominated by larger and larger droplets with high viral load per droplet and high initial velocity, but short airborne time. water. although it is assumed that bioaerosol contains some amount of water, the exact water content of bioaerosol microdroplets is difficult to determine and probably varies a lot, depending on the relative humidity and temperature of ambient air. most physiological models of human breathing assume 100% relative humidity and 36-37° c in the air of the lower respiratory tract, in accordance with experimental data [56, 57] . with expiration (tidal breathing and room temperature air), some of this water content is reabsorbed in the upper respiratory tract, where the temperature is assumed to be ~32°c below the glottis [57] and vary in the naso-pharyngeal cavity, from ~25°c in the nasal vestibule, to ~30°c in the internal nasal valve area and ~32°c in the middle turbinate [58, 59] , with some differences (~2°c) between the air and nasal mucosa, allowing for an efficient water reabsorption during exhaling, but also providing favorable conditions for condensation and droplet formation. these values change with change in ambient air temperature, as inhalation of cold air lowers the temperature in both the upper [60] and lower respiratory tract [57] , thus further improving the conditions for water condensation during exhalation. once the bioaerosol leaves the human body, several factors will affect the water content, probably the most important one being change in droplet size, influenced by the rate of evaporation [61] , interaction with each other (coalescence or fragmentation [62] ) and relative position in relation to the center of airflow [63] . these effects are complex and still insufficiently understood in the case of exhaled aerosol. organic components. the main organic component of exhaled human air is an airway lining fluid component, which is highly diluted in water, with a consensus estimate for dilution between 2,000 to 10,000 times [64, 65] . the airway lining fluid likely contains various types of lipids and electrolytes. thus, a recent study identified 75 glycerophospholipids, 13 sphingolipids, 5 sterol lipids and 12 neutral glycerolipids in samples from healthy volunteers [66] , with a large variation in composition between subjects, underscoring the complexity of organic content of exhaled air condensate. within this context, another phenomenon should be pointed out, namely the effect of temperature and relative humidity. two aspects should be mentioned. first, natural evaporation from the lungs depends on temperature and humidity -at relatively low temperature (~2° c), water loss depends very little on relative humidity (2.0-2.2 ml h 2 o per l/breath/min), but at higher temperature (~27° c), the range increases dramatically (0.5-2.1 ml h 2 o per l/breath/min) with much less water loss in more humid air [67] . the second aspect to be mentioned is the water loss at low relative humidity, which is substantial [68] . both factors would apply to most modern air-conditioned indoor environments, where the temperature is kept typically in the range of 20-23° c and humidity at ~40%. that electrical potential value does not seem very strong as a generator of an electrostatic repelling force (coulombic force), but it has to be kept in mind that the volume of space to be protected from particles in front of the cornea is not large, particles need only be deflected less than 1 cm in front of the corneal surface, as the thickness of the eyelid margins is only about 2 mm [69] . considering that ocular surface area exposed to air is typically 1.25-1.75 cm 2 [70] [71] [72] (although it can reach intermittently up to 3 cm 2 , depending on the visual task), the "protected volume of air" 4 in front of the cornea is probably less than 0.4 cm 3 and does not exceed 0.75 cm 3 . furthermore, human eyelashes act as passive dust controlling system and reduce evaporation and particle deposition up to 50% [73] , further facilitating the defense against foreign particles (including microdroplets). what is the effect of blinking on the airflow within the "protected volume of air" is currently unknown, however, it can be speculated that it would create some change in low-velocity airflow, as it was estimated that blinking increases corneal temperature by 1.3° c [74] and this could create convection air microcurrents away from the corneal surface. the speed and direction of local airflow can be further modified by artificial objects in eye vicinity. for example, it is highly likely that even regular eyeglasses can restrict the airflow around the eyes, although we are not aware of a quantitative evaluation of this phenomenon. another factor to be considered under the scenario of direct, close human-to-human communication is that the majority of microdroplets would not reach the eye. a recent study using a 3d-printed realistic face and airway model, with an outlet of a particle jet centered at the nose of the model at a distance of 20 cm, found that more 80% of the generated aerosol particles with an initial velocity of 0.94 m/s was deposited on the lips rather than on the eyes [75] . electrostatic force 4 the "protected volume of air" in this context can be defined as the volume of air enclosed in the space between the upper and lower eyelids when the eyelids are open, and the gaze is directed forward in primary position. electrostatic (coulombic) forces can play considerable part in aerosol deposition [76] [77] [78] [79] , especially for particle size range 0.01 µm to 5 µm [80] , which, as discussed below is the most important range of water microdroplet particles carrying viruses. additionally, it was shown that even relatively low voltage (12v) ionizer device effectively captured airborne transmitted calicivirus, rotavirus and influenza virus and prevented airborne transmitted influenza a between animals [81] . however, it needs to be emphasized that, according to hoque (2010) : "no specific distribution has been identified in the literature to describe charge distribution in bioaerosols" [82] , and, thus further work is needed to clarify the role of electrical charge of bioaerosols for deposition on human tissue in the real world. to the best of our knowledge, direct measurements of the electrical charge of bioaerosols generated by human exhalation have not been published to date. therefore, we would advance some theoretical considerations supported by some indirect experimental data to explore what would be the more likely overall change of the droplets comprising the bioaerosol generated by exhalation and will focus on bioaerosol containing mostly coronaviruses. one such consideration is that the ph of exhaled breath condensate is slightly alkaline, e.g. in normative database from 404 subjects, the mean ph was 7.83, and the median ph was 8.0 [83] and similar results were obtained in another report and a meta-analysis [84, 85] . however, the role of bioaerosol de-aeration with co 2 -free gas is unclear. therefore, it is possible that the net charge of bioaerosol droplets generated by breathing and other activities is positive. if this would be the case, it would explain the low probability of airborne particles to land in sufficient numbers to the ocular surfaces and remain there for long enough time to attach to receptors and cause inflammation. of note, the overall isoelectric point of coronaviruses has not been reported in the literature. most of the factors discussed above would apply to relatively similar indoor controlled environments, including air conditioning, central heating, etc., with relatively slow airflow and limited temperature and humidity range. the production rate, spread and infective potential of bioaerosol from exhaled air would be very different in outdoor environments. it is likely that the outdoor infectious potential of bioaerosol would be much more dependent on environmental factors, such as temperature, humidity, wind speed and direction, air ionization, solar irradiance, etc. currently, most people in industrialized nations spend most of their time indoors. a large (n= 9,196), 2-year probability-based telephone survey in the us (1992) (1993) (1994) found that the respondents reported spending an average of ~87% of their time in enclosed buildings and ~7.6% outdoors, confirming similar findings from earlier surveys in industrialized countries [86] . however, the exact proportion of infectious events for viral respiratory diseases occurring in indoor environments vs. outdoors is unknown and it is likely to be different for different types of viruses. this hypothesis could be tested by measuring the electrical charge of bioaerosol generated by normal breathing in healthy subjects and in patients with viral infections caused by different viruses, causing respiratory infections or with suspected aerosol transmission pathway. an established reliable isoelectric point for all studied viruses would be very helpful in modeling the relationship between virion electrical charge and droplet electrical charge. the discrepancy between the theoretically expected high rate of concurrence of viral ocular surface inflammation and its observed relatively low occurrence in covid-19 is surprising. natural protective factors related to anatomical and physiological properties of the eyes could prevent the deposition of large number of virus-loaded particles on the ocular surface and play a protective role. it is possible that the standing potential of the eye plays an important role in repelling aerosol particles (microdroplets) near the surface of the eye and serve as major contributing factor in securing either complete protection or fast elimination of certain type of bioaerosols containing viruses. a comparison of eye problems in primary care and ophthalmology practices management of ophthalmic disease in general practice conjunctivitis: a systematic review of diagnosis and treatment acute conjunctivitis: truth and misconceptions a twenty-one year surveillance of adenoviral conjunctivitis in sapporo ocular tropism of respiratory viruses past, present, and possible future human infection with influenza virus a subtype h7 clinical characteristics of coronavirus disease 2019 in china sars-cov-2 is an appropriate name for the new coronavirus editorial -differences and similarities between severe acute respiratory syndrome (sars)-coronavirus (cov) and sars-cov-2. would a rose by another name smell as sweet? human coronaviruses: what do they cause a sequence homology and bioinformatic approach can predict candidate targets for immune responses to sars-cov-2 comparison of confirmed covid-19 with sars and mers cases -clinical characteristics, laboratory findings, radiographic signs and outcomes: a systematic review and meta-analysis human coronavirus nl63 the severe acute respiratory syndrome the severe acute respiratory syndrome coronavirus in tears tears and conjunctival scrapings for coronavirus in patients with sars ocular screening in severe acute respiratory syndrome sars virus in tears? lancet infect dis the severe acute respiratory syndrome coronavirus in tears evaluation of coronavirus in tears and conjunctival secretions of patients with sars-cov-2 infection characteristics of ocular findings of patients with coronavirus disease conjunctivitis and covid-19: a meta-analysis efficient activation of the severe acute respiratory syndrome coronavirus spike protein by the transmembrane protease tmprss2 sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor expression of sars-cov-2 receptor ace2 and tmprss2 in human primary conjunctival and pterygium cell lines and in mouse cornea ace2 and tmprss2 are expressed on the human ocular surface, suggesting susceptibility to sars-cov-2 infection untersuchingen uber die thierische elektrizitat. reimer holmgren f (1865) method att objective effekten av ljusintryck pa retina the corneo-retinal potential difference as the basis of the galvanometric method of recoirding eye movements changes in the electro-oculogram potential level the directly recorded standing potential of the human eye ocular artifacts in recording eegs and event-related potentials. ii: source dipoles and source components transmission of 2019-ncov infection from an asymptomatic contact in germany rapid expert consultation on the possibility of bioaerosol spread of sars-cov-2 for the covid-19 pandemic aerosol and surface transmission potential of sars-cov-2 singapore novel coronavirus outbreak research t (2020) detection of air and surface contamination by sars-cov-2 in hospital rooms of infected patients consideration of the aerosol transmission for covid-19 and public health extensive viable middle east respiratory syndrome (mers) coronavirus contamination in air and surrounding environment in mers isolation wards evidence of airborne transmission of the severe acute respiratory syndrome virus viral infections in workers in hospital and research laboratory settings: a comparative review of infection modes and respective biosafety aspects airborne transmission of communicable infection--the elusive pathway influenza virus rna recovered from droplets and droplet nuclei emitted by adults in an acute care setting respiratory virus shedding in exhaled breath and efficacy of face masks recognition of aerosol transmission of infectious agents: a commentary origin of exhaled breath particles from healthy and human rhinovirus-infected subjects properties of coronavirus and sars-cov-2 viral architecture of sars-cov-2 with post-fusion spike revealed by characterizations of particle size distribution of the droplets exhaled by sneeze cough aerosol in healthy participants: fundamental knowledge to optimize droplet-spread infectious respiratory disease management airborne spread of infectious agents in the indoor environment measurements of airborne influenza virus in aerosol particles from human coughs exposure to influenza virus aerosols during routine patient care physiology humidification: basic concepts in: esquinas am (ed) humidification in the intensive care unit thermal mapping of the airways in humans temperature profile in the nasal cavity simultaneous in vivo measurements of intranasal air and mucosal temperature nasal mucosal temperature after exposure to cold, dry air and hot, humid air kinetics and evaporation of water drops in air interaction of water droplets in air flow at different degrees of flow turbulence evaporation and movement of fine water droplets influenced by initial diameter and relative humidity exhaled breath condensate: methodological recommendations and unresolved questions exhaled breath condensate: an overview lipid phenotyping of lung epithelial lining fluid in healthy human volunteers water lost through breathing dynamics of airborne influenza a viruses indoors and dependence on humidity basic conceptsdiseases and disorders of the orbit and ocular adnexa e-book ocular surface-area as an informative index of visual ergonomics the exposed ocular surface and its relationship to spontaneous eyeblink rate in elderly caucasians digital photography as a novel technique of measuring ocular surface dimensions eyelashes divert airflow to protect the eye mathematical model of thermal effects of blinking in human eye measuring the administered dose of particles on the facial mucosa of a realistic human model aerosol wall losses in electrically charged chambers combined effects of electrophoresis and thermophoresis on particle deposition onto flat surfaces an eulerian model for particle deposition under electrostatic and turbulent conditions direct numerical-simulation of particle entrainment in turbulent channel flow particle deposition in a nearly developed turbulent duct flow with electrophoresis ionizing air affects influenza virus infectivity and prevents airborne-transmission development of computational fluid dynamics based multiple linear and neural network metamodels for bioaerosol fate and normative data for ph of exhaled breath condensate exhaled breath condensate ph in adult croatian population without respiratory disorders: how healthy a population should be to provide normative data? a review of the usefulness of non-invasive exhaled breath condensate ph analysis for diseases diagnosis with elements of meta-analysis: an update from 2012 the national human activity pattern survey (nhaps): a resource for assessing exposure to environmental pollutants no conflict of interest. key: cord-287824-zg5akivn authors: chan, yinghan; ng, sin wi; mehta, meenu; anand, krishnan; kumar singh, sachin; gupta, gaurav; chellappan, dinesh kumar; dua, kamal title: advanced drug delivery systems can assist in managing influenza virus infection: a hypothesis date: 2020-09-24 journal: med hypotheses doi: 10.1016/j.mehy.2020.110298 sha: doc_id: 287824 cord_uid: zg5akivn outbreaks of influenza infections in the past have severely impacted global health and socioeconomic growth. antivirals and vaccines are remarkable medical innovations that have been successful in reducing the rates of morbidity and mortality from this disease. however, the relentless emergence of drug resistance has led to a worrisome increase in the trend of influenza outbreaks, characterized by worsened clinical outcomes as well as increased economic burden. this has prompted the need for breakthrough innovations that can effectively manage influenza outbreaks. this article provides an insight into a novel hypothesis that describes how the integration of nanomedicine, with the development of drugs and vaccines can potentially enhance body immune response and the efficacies of anti-viral therapeutics to combat influenza infections. apart from covid-19, influenza is another infectious disease that ranks high as one of the 62 deadliest, characterized by a remarkably high rate of transmission that could cause a rapid 63 spread. it is estimated that influenza kills approximately 500 thousand people yearly [4, 8] . 64 killed virus vaccine as an intramuscular injection and attenuated live vaccine as a nasal spray, 65 are the two most widely known vaccines for this deadly virus [9] . in the recent years, an 66 increasing trend of influenza outbreaks have been observed, prompting medical researchers to 67 design and develop suitable vaccines and novel therapeutic modalities [10] . despite the 3 68 availability of vaccines that may protect individuals from well-matched strains, it is well-69 known that the influenza virus has high mutation rates, resulting in frequent mismatches due 70 to antigenic drift and shift, thereby, necessitating the development of a new vaccine every few 71 years [11] . however, the development of a new vaccine is time-consuming. in addition, 72 vaccine-development remains mostly applicable to developed countries, attributing to the cost 73 factor involved. moreover, long term use of standalone anti-influenza drugs and vaccines are 74 often associated with adverse reactions and other shortcomings, that limit their effective 75 clinical applications [12] . for instance, although the neuraminidase inhibitor oseltamivir has 76 been widely employed as an anti-influenza drug, it was found that the drug does not offer 77 benefits in patients with pre-existing medical conditions [12, 13] predict possible chronic and other unforeseen in-vivo effects [61, 62] . hence, it is hoped that 327 this hypothesis will trigger further exploration into nanomedicine-based approach to elucidate 328 the in-depth mechanisms involved, along with their safety, to pave way for a paradigm shift in 329 influenza management approaches. the authors declare that they have no known competing financial interests or personal 338 relationships that could have appeared to influence the work reported in this paper. single-371 dose mucosal immunization with a candidate universal influenza vaccine provides 372 rapid protection from virulent h5n1, h3n2 and h1n1 viruses exploiting nanotechnology to target viruses universal influenza vaccines: from viruses to 378 nanoparticles nanotherapeutic anti-influenza solutions: current knowledge and future challenges influenza antivirals and resistance: the next 10 384 years? influenza viruses -antiviral therapy and resistance influenza vaccines: evaluation of the 389 safety profile immunogenicity and 392 protection of oral influenza vaccines formulated into microparticles celastrol-loaded liquid crystalline nanoparticles as an anti-inflammatory intervention 396 for the treatment of asthma nanomaterials designed for antiviral drug delivery transport across biological barriers dramatically increase zanamivir absolute bioavailability in rats: implications for an 403 nanotechnologies for boswellic acids the potential 408 of sirna based drug delivery in respiratory disorders: recent advances and progress targeting 411 neutrophils using novel drug delivery systems in chronic respiratory diseases emerging 414 complexity and the need for advanced drug delivery in targeting candida species albumin nano-encapsulation of piceatannol enhances its anticancer potential in 418 colon cancer via downregulation of nuclear p65 and hif-1α. cancers (basel) nanomedicine for infectious disease applications: 421 innovation towards broad-spectrum treatment of viral infections nanoparticles in influenza 424 subunit vaccine development: immunogenicity enhancement. influenza other respi 425 nanoparticle-based 427 vaccines against respiratory viruses organic and inorganic nanoparticle vaccines for prevention of 430 infectious diseases formulation and characterization of glibenclamide and quercetin-434 loaded chitosan nanogels targeting skin permeation patented therapeutic 437 drug delivery strategies for targeting pulmonary diseases increasing 440 complexity and interactions of oxidative stress in chronic respiratory diseases: an 441 emerging need for novel drug delivery systems nanotechnology-based antiviral therapeutics rutin loaded 446 liquid crystalline nanoparticles inhibit lipopolysaccharide induced oxidative stress and 447 apoptosis in bronchial epithelial cells in vitro biopolymer 450 encapsulated live influenza virus as a universal cd8+ t cell vaccine against influenza 451 virus. vaccine anti-455 bacterial activity of inorganic nanomaterials and their antimicrobial peptide conjugates 456 against resistant and non-resistant pathogens perspectives and 459 advancements in the design of nanomaterials for targeted cancer theranostics microparticles as vaccine adjuvants and delivery systems investigation of 465 tunable acetalated dextran microparticle platform to optimize m2e-based influenza 466 vaccine efficacy molecular assembly and application of biomimetic microcapsules hybrid inorganic-organic capsules for eficient intracellular delivery of novel sirnas 472 against influenza a (h1n1) virus infection biodegradable polyelectrolyte/silica composite microcapsules as carriers for small 476 application of dendrimers for the treatment of 479 infectious diseases nanoparticle vaccines against infectious diseases delivery vehicles for active phytoconstituents emerging trends 492 in nanomedicine for topical delivery in skin disorders: current and translational 493 oligonucleotide 495 therapy: an emerging focus area for drug delivery in chronic inflammatory respiratory 496 diseases emerging trends in 498 the novel drug delivery approaches for the treatment of lung cancer interactions 501 with the macrophages: an emerging targeted approach using novel drug delivery 502 systems in respiratory diseases gene delivery by pamam dendrimer 505 conjugated with the nuclear localization signal peptide derived from influenza b 506 identification of biomarkers and genetic approaches toward chronic obstructive 510 pulmonary disease inhibition of influenza a virus infection in vitro by saliphenylhalamide-loaded 513 porous silicon nanoparticles enhanced inhibition of influenza virus infection 516 by peptide-noble-metal nanoparticle conjugates development of an adjuvanted nanoparticle vaccine against influenza virus, an 520 in vitro study pulmonary surfactant-biomimetic 523 nanoparticles potentiate heterosubtypic influenza immunity protein nanoparticle immunization induces broad cross-protection against 527 different influenza viruses in mice virus-mimetic polymer 530 nanoparticles displaying hemagglutinin as an adjuvant-free influenza vaccine porous gold nanoparticles for 533 attenuating infectivity of influenza a virus inhibition of h1n1 influenza virus infection by zinc oxide nanoparticles: 537 another emerging application of nanomedicine phase 3 pivotal trial of nanoflu tm in older adults editorial: advances and challenges in nanomedicine nanoethics: from utopian dreams and apocalyptic nightmares towards a 543 more balanced view the authors declare no conflict of interest, financial or otherwise. key: cord-278271-rpq62xhl authors: lyu, jinglu; miao, tianyu; dong, jiajia; cao, ranran; li, yan; chen, qianming title: reflection on lower rates of covid-19 in children: does childhood immunizations offer unexpected protection? date: 2020-05-15 journal: med hypotheses doi: 10.1016/j.mehy.2020.109842 sha: doc_id: 278271 cord_uid: rpq62xhl the incidence of covid-19 in children and teenagers is only about 2% in china. children had mild symptoms and hardly infected other children or adults. it is worth considering that children are the most vulnerable to respiratory pathogens, but fatal sars-like virus had not caused severe cases among them. according to the pathological studies of covid-19 and sars, a sharp decrease in t lymphocytes leads to the breakdown of the immune system. the cellular immune system of children differs from that of adults may be the keystone of atypical clinical manifestations or even covert infection. the frequent childhood vaccinations and repeated pathogens infections might be resulting in trained immunity of innate immune cells, immune fitness of adaptive immune cells or cross-protection of antibodies in the children. therefore, due to lack of specific vaccine, some vaccines for tuberculosis, influenza and pneumonia may have certain application potential for the front-line health workers in the prevention and control of covid-19. however, for high-risk susceptible populations, such as the elderly with basic diseases such as hypertension and diabetes, it is necessary to explore the acclimatization effect of the planned immune process on their immunity to achieve the trained immunity or immune fitness, so as to improve their own antiviral ability. the pathogen causing covid-19 is a newly identified pathogen, bioinformatic analyses indicated that the virus had features typical of the coronavirus family and belonged to the β -coronavirus lineage (5) , named severe acute respiratory syndrome coronavirus 2 (sars-cov-2). covid-19 is mainly transmitted through respiratory droplets and close contact, although virus particles have been detected in patients' feces, lacrimal secretions, and aerosols, there is no clear evidence that these secretions are infectious. currently, the main source of infection is confirmed patient, including asymptomatic infections. in theory, humans have no pre-existing immunity to this newly identified pathogen. so everyone is assumed to be susceptible, although there are a variety of factors that can increase the risk of infection. whether there is life-long neutralizing immunity after infection requires further study. there have been recent reports of "re-positive" cases of nucleic acid testing in discharged patients, which may be related to "false positive" tests at discharge, rather than actual recurrence (6, 7) . the latest follow-up study of sars survivors found that specific igg antibodies persisted for up to 12 years (8) . other earlier observations have also reported maintenance ranging from 2 to 4 years of sars-cov specific antibodies (9, 10) . like mers and sars, it is a cross-species infectious disease. since mers did not occur on a large scale in china, we compared it with the epidemiological and pathological characteristics of sars in the following content. at present, most of the reported cases in children are clustered cases caused by close contact, known as "second generation" infection. the sporadic features of the disease is particularly obvious in the areas outside of wuhan (11) . recently, as the epidemic has been gradually brought under control, especially after the release of some statistics of hubei province, we can see more intuitively that the incidence of children is significantly lower than that of adults (12) , newborns and infants are less susceptible than adolescents (2) . the incidence of covid-19 in children 2.4% according to 2019 report, and it is worth noting that some cases of covert infection may not be detected (3, 13) . very few neonatal deaths had been reported (14) . the epidemiological investigations of sars are also consistent with this conclusion. according to the statistics of 2003, the confirmed cases of children under 14 years old accounted for only 2.7% (as of may 4) of the total cases of sars in beijing municipality and 4.88% (as of april 27) in guangdong province. 10 sars children were admitted to a hong kong hospital in 2003, eight of whom had been in school before they were confirmed but had not spread the disease to other students(4). children and adolescents might be susceptible to sars-cov infection if they had close contact with confirmed patients, but the clinical course and outcome are more favorable in children younger than 12 years of age compared with adolescents and adults. transmission of sars from pediatric patients appears to be uncommon but is not impossible (15) . only a few cases of child deaths or transmission of the disease to adults as a source of infection have been reported. these observations raise the question of whether children have natural resistance to these two coronaviruses. covid-19 was divided into four clinical subtypes according to the severity(16): light type, normal type, severe type and critical type. fever, dry cough, fatigue are the main symptoms. however, patients with severe or critical illness may have moderate or low fever, or even no significant fever (17) . candida isolated from 4 airway specimens in a case report of patients with new coronavirus pneumonia compared with adult cases, children tend to have milder symptoms, shorter disease course and generally better prognosis. fever is the main symptom in most children, the fever duration is 2-3 days, up to 8 days, most within 1 week. some children came to see the doctor due to gastrointestinal symptoms. according to 2019 report, a very small proportion of those aged under 19 years especially with some basic diseases, have developed severe (2.5%) or critical disease (0.2%). children with fever account for a relatively large number of the cases, which is convenient for early identification and diagnosis and contribute to fewer severe cases. there are no death cases in children under 9 years of age (18) . as the coverage of the nucleic acid test became wider, more and more asymptomatic patients were diagnosed, and the majority of them were young people (19, 20) . routine blood tests in most patients show typical signs of viral infection, with normal or reduced white blood cell counts and reduced lymphocyte counts in early stage. in severe cases, d-dimer increased and peripheral blood lymphocytes progressively decreased, and cytokines were apparently accumulated (21) . compared with 120 healthy controls, the absolute value of lymphocytes (0.87 vs 2.13)×10 9 /l and the percentage (19.5% vs 33.7%) were significantly reduced (22) . another study showed that 25% and 63% of the patients had leukopenia and lymphocytopenia, respectively (11) . most viruses induce cell-mediated immune responses as well as humoral immunity. the main cells involved in cellular immune response include cd4+t cells and cd8+t cells. the virus activates primary cd4+t cells which are involved in cell-mediated immune response. cd8+t cells kill virus-infected target cells mainly by direct contact (23) . however, the vast majority of covid-19 patients showed reduced lymphocytes, the proportion of cd4+t decreased in mild and severe patients was 52.90% and 95.24%, and cd8+t decreased was 28.40% and 61.90%, respectively (24) . further anatomical and pathological findings showed that the state of t lymphocytes was overactivated, and cd8+t cells had a high concentration of cytotoxic particles, thus causing serious immune damage (25) . in addition, compared with the mild group, the increase of serum inflammatory factors in the severe group significantly increased the risk of further tissue damage caused by cytokine storms (11) . the progressive increases in il-6 and creactive protein as warning indicators of critical illness in adults (16) . single cell rna sequencing the bronchoalveolar lavage fluid of covid-19 patients, the proportion of macrophages in severe patients is higher, subtype is spp1+ and fabp4+ rather than fcn1+ macrophages. while the proportion of cd8+t cell and nk cells in mild patients is higher (26) . however, in most children patients, the white blood cell count and absolute number of lymphocytes in the peripheral blood were normal. in a few severe cases, the total number of white blood cells decreased, with decreased absolute number of t lymphocytes, and changes in t lymphocytes subsets, c-reactive protein was usually normal or temporarily increased (13) . in children with covid-19 and sars, the maintenance of t cell function may be related to thymic development. the thymus is the site of t cell development, and its absolute weight is the largest between 6 and 13 years old. more evidence is needed to confirm that differences in cellular immunity between children and adults are related to this phenomenon. frequent childhood vaccinations and repeated pathogens infections have resulted in children who are exposed to various antigens. immunization is considered one of the greatest public health achievements of the 20 th century due to its important interventions for human health. the popularity of smallpox vaccine, mycobacterium tuberculosis vaccine (bcg), oral polio vaccine (opv) and measles vaccine has reduced morbidity and mortality worldwide. human smallpox has been eradicated and polio is also a disease to be overcome by immunization. china has officially become a polio-free country in 2000. china's basic immunization policy mainly targets children (hepb, bcg, ipv, opv, dtap, dt, mr, mmr, je, mpsv, hepa) to receive mandatory vaccines. these vaccines will prevent 12 diseases in total. children should complete basic immunization at 6 years old and compensate the missing vaccines before 14 years of age (27) . infectious diseases rank second in the global cause of death. children and the elderly are the most susceptible groups to infectious diseases, especially infectious diseases of the respiratory system. common infectious diseases in children are divided into viral infections and bacterial infections, some of which have been covered by mandatory vaccines. preventive vaccines have also been developed for influenza, hand-foot-mouth disease, and streptococcus pneumoniae (s.p)-related respiratory diseases, although which are currently non-mandatory vaccines in china. with the gradual increase up-take rates, the prevention of these diseases will become increasingly apparent. in this state of mixed infection, the immune response tends to be more complicated. take rsv infection as an example, n protein of rsv is one of the target antigens for cytotoxic t cells (ctl), whereas the g protein was not recognized and can at best represent a minor target antigen for ctl (28) . if sars-cov-2 co-infects with other pathogens, the immune disorders will be more difficult to correct as can be expected. clinical data shows the prevention and treatment of coinfections in hospitalized covid-19 patients, with 60~70% of receiving antimicrobial treatment and 15% receiving anti-fungal treatment (31, 32) . candida was isolated from 4 airway specimens in a case report of patients with covid-19 (33) . these candida might come from the environment or the oral microenvironment (34) . thus preventing invasive fungal infections should be an important part of treatment (35) . during the sars epidemic in 2003, therapeutic regimen in hong kong recommended the use of second-generation cephalosporin plus macrolide antibiotics in the pediatric patients as the first step in response to coinfection (36) . whether the relatively weak function or low expression of ace2 receptor in children leads to the limited invasion pathway of the virus? lower expression of ace2 in children will limit virus invasion pathway, which will be responsible for avoiding large-scale outbreak in children. previous studies have found that the expression of ace2 receptor is inhibited in the infection of rsv, h5n1 and h7n9 avian influenza viruses (37) (38) (39) . on the other hand, ambroxol, which is selected as a potential ace2 receptor binding agent by an artificial intelligence drug screen system, may block the entering pathways of sars-cov-2, which provides a valuable therapeutic approach for the prevention or reduction of acute lung injury (ards), or even kidney and heart injury caused by sars-cov-2(40). ambroxol is a common drug in pediatric respiratory department due to the high incidence of respiratory tract diseases among children. whether a lasting effect on ace2 of ambroxol maintains and plays an immunoregulatory role faced with the invasion of sars-cov-2 is worth exploring. the high frequency of infection and vaccination in children, together with the special immune system in childhood, make the children's immune response to sars-cov-2 in a state of high immune clearance and low immune response. in addition, various immune adjuvants in vaccines can directly promote the body's non-specific and cellular immune function, make its innate immune trained, leading to immune fitness(41). the concept of trained immunity was first proposed in 2011 (42) and elaborated the characteristics of the innate immune system exhibiting immune memory or non-specific effects (nse) (43) . g.b. mackaness first proposed that bacterial infections may cause "cross-protection" of other pathogens. this classic form of cross-protection is mediated by lymphocytes, which release ifn-γ, thereby activating bystander macrophages, resulting in a temporarily enhanced innate immune status against secondary infections, and this cross-protection status will quickly disappear once the major pathogens have been eliminated (44) . contrary to this, berg et al. found that memory lymphocytes can also mediate longer-term cross-protection as a byproduct of adaptive immunity: cd8 + memory t cells can be activated by cytokines (il-12 and il-18) in early stages of infection in an antigen-independent manner, leading to the production of ifn-γ and enhanced response to subsequent infectious agents (45) . memory t cells that are specific for unrelated pathogens might have roles in protective immunity and immunopathology caused by heterologous infectious agents (46) . administration of bcg induces a trained phenotype in circulating monocytes with increased capacity on secreting proinflammatory mediators, leading to non-specific protection against unrelated pathogens. bcg vaccination also upregulates the production of proinflammatory cytokines, such as il-1β and il-6 by nk cells in response to the stimulation of m. tuberculosis and other pathogens. these characteristics of trained immunity indicate innate immune cells (nk, dc and macrophage) to be the alternative target for vaccination in order to generate more effective immune response. bcg, which can live on the skin for up to a few months, not only triggers the memory b and t cells characteristic of mycobacterium, but also stimulates the blood cells for a long time. bcg prevents about 60% of childhood tb cases, and may also enhance the immune to fight off 30% of all known pathogens (including viruses). the world health organization concluded that bcg appeared to reduce overall mortality of children. latest research proposed that national differences in covid-19 impact could be partially explained by the different national bcg childhood vaccination policies. according to their investigation, bcg vaccination reduced the number of national reported covid-19 cases and outbreaks in countries without universal bcg vaccination are more critical (47) . this result is consistent with our hypothesis, and makes bcg vaccination a potential new tool in the fight against covid-19. immune cells are able to clear the pathogen without causing a severe inflammatory response, resulting in covert infection or mild to moderate symptoms with short course, which is called immune fitness (41) .the severity of the infection is not necessarily related to the load of infection, but rather to host fitness, the balance between killing the pathogen and tolerance. the low incidence and mild symptoms of covid-19 in children suggest that the immune system in children (including neonates and infants) is well developed and functional. in the past, the so-called immature infant immune system is actually that the infant immune system has not been exposed to the corresponding antigen and lacks immune memory, not that the immune function is low. multiple respiratory tract infections in childhood, among which a large proportion are covert infections, apply the host with opportunities of immune adaptation. at present, many cases of children with atypical symptoms or covert infections had been reported. this phenomenon did not only occur in children. after pathogen screening in close contacts and suspected contacts, a large number of confirmed patients were found with a high proportion of typical patients (3, 11, 12, 19, 48, 49) . a covert infection, like the effect of vaccination, is the best clinical phenotype for immune fitness. the reason why the lymphocyte function of these patients maintains a moderate response and the immune homeostasis is maintained might be involved in immune fitness (41) . children may have lots of memory t cells that target various antigens. the study on sars t cell subsets in children showed that the decrease of cd4+ and cd8+t cells was not obvious. studies have confirmed that the decrease of cd4+ and cd8+ cells is closely related to the degree of lung lesions, so the absorption of inflammatory lesions in the lung in children is relatively mild. at the same time, b cells with t cell help also accelerate sars specific antibody production, with higher positive rate than adults. bcg promotes the production of the th1 phenotype in cd4+t cells, which secrete high levels of ifn-γ and have activity against intracellular pathogens. again, it produces cd8+t cell activation, which mediates the cytotoxic response. however, the tlr signal transduction stimulated by the components of mycobacteria induces b cells to secrete il-10, and thus inhibits the activity of th1 and th17, which helps to suppress the autoimmune response. the macrophages and especially dendritic cells, recognize and phagocytic pathogens and destroy them, passing the information to the t cells. immune cells secrete cell factors to induce t cells to differentiate into different functional subsets. different viral infections cause different t cell responses. in hiv patients, the cd4+ cytotoxic t lymphocytes have a protection role on virus infection (50) . cd45ra+ memory t cells is highly enriched in dengue fever patients (51) . lymphocytic choriomeningitis virus results in depletion of t follicular helper(tfh) co-expressed by il-10 and il-21, which impaired humoral immunity and viral control (52) . bacterial infection also stimulates a strong t cell response, such as mycobacterium tuberculosis. children's vaccines might have the potential to vary subsets of immune cells and molecular markers, force the host to find a balance between killing pathogens and immune tolerance. sars-cov-2 is 79% similar to sars-cov and 55% similar to middle east respiratory syndrome coronavirus (mers-cov). by comparing the sequences of several coronavirus genomes, the homology of nonstructural proteins and structural proteins in the coding region was 58% and 43%, respectively. sars-cov-specific antibodies were able to prevent sars-cov-2 from entering cells, indicating the presence of antibody cross-protection between the two viruses (53) . in a latest long-term follow-up investigation to 2015, the serum specific igg levels in health care workers participating in the treatment of infected sars patients for up to 12 years (8) . at the late stage of the epidemic and after the outbreak of sars, more and more cases of covert infection or asymptomatic infection with specific antibodies were reported (54, 55) . since the incidence of sars in children was relatively lower, many studies at that time investigated the specific antibody levels of children and speculated that children's innate antibodies contributed to the the positive rate decreased gradually with age and down to zero in healthy adult control group, which led to the speculation that there were real differences between adults and children (58) . another survey found that the antibody positive rate of healthy children and children was 2%, and the antibody positive rate of healthy adults and patients was only 0.2%, which also agreed that there was a significant difference between children and adults(56). in the current situation of virus outbreak, bcg vaccine, influenza vaccine and pneumonia vaccine may have certain application potential in the prevention and control of covid-19. emergency injections could be considered for front-line health workers. however, for high-risk susceptible populations, such as the elderly with basic diseases such as hypertension and diabetes, it is necessary to explore the acclimatization effect of the planned immune process on their immunity to achieve the immune fitness, so as to improve their own antiviral ability. we suggest a detailed immunization plan should be set for the elderly, after several years and multiple vaccines inoculated. different pathogens have different attack effects on immune cells, so it is necessary to map the detailed immune cells against the planned vaccine, so as to achieve precise regulation of immune cells and inflammatory factors. eleven t and b lymphocyte tests were used to analyze the changes of specific immune cells. single cell rna sequencing could demonstrated dynamically change the gene spectrum, host cell antiviral factors, and immune cell subsets that might cause cytokine storms. finally, an appropriate immunization program was found that resulted in trained immune and immune fitness to protect the elderly population from lethal infection. emergency immunization, also known as contingency vaccination (cv), is aiming at protecting vulnerable populations in case of an infectious disease begins to spread or there is a sign of epidemic. it follows the basic theories of vaccination and carry out emergency vaccination on specific populations in epidemic areas to achieve the purpose of preventing epidemic disease. for example, students and staffs must be equipped with contingency vaccination in case of the outbreak of measles in schools. emergency vaccination is of special significance to curb the spread of infectious diseases. unfortunately, sars-cov-2-targeted vaccines have not yet been developed, but innovation proposals or hypothesis of new therapies, such as blocking agents of ace2 receptor or ace2 immunoadhesin strategy have been reported (61) . here we assume that some provisional substitute candidates will work. due to the same pathogenic mechanism(62), adjusted sars-cov-targeted vaccines can come in handy timelier. vaccines for influenza may also make a difference in the orientation of th1/2 response. the sglycoprotein of sars-cov-2 has certain similarities with the ha glycoprotein of several influenza viruses. it is not optimal, but the safety of commercial influenza vaccines has been confirmed (63) . pneumococcal conjugate vaccine and 23-valent pneumococcal polysaccharide vaccine may have some preventive effects on coinfection. equipping confirmed covid-19 patients with these vaccinations as emergent prophylaxis may prevent severe illness caused by secondary infection, in the meantime, it may mobilize the host's lymphocyte response to the opposite direction in response to sars-cov-2. researchers in four countries (the netherlands, australia, germany, and the united kingdom) will soon begin clinical trials of vaccines for sars-cov-2 using unconventional methods. they will test whether a century-old bcg can broadly improve the body's immune system, making it better able to fight sars-cov-2, or prevent its symptoms from getting worse. the study will be administered to people at high risk, including doctors and nurses, and to older adults (64) . data-driven discovery of clinical routes for severity detection in covid-19 pediatric cases a retrospective study of the clinical characteristics of covid-19 infection in 26 children a novel coronavirus from patients with pneumonia in china positive rt-pcr test results in patients recovered from covid-19 discharged patients with covid-19 virus returning, medrxiv (2020) long-term persistence of igg antibodies in sars-cov infected healthcare workers, medrxiv consecutive five-year follow-up analysis of specific igg antibody of 22 cases of sars patients after recovery prospective 2-year clinical study of patients with positive igg-antibodies after recovering from severe acute respiratory syndrome, zhongguo wei zhong bing ji jiu yi xue = chinese critical care medicine clinical features of patients infected with 2019 novel coronavirus in detection of covid-19 in children in early diagnosis and treatment recommendations for pediatric respiratory infection caused by the 2019 novel coronavirus clinical analysis of 10 neonates born to mothers with 2019-ncov pneumonia severe acute respiratory syndrome in children national recommendations for diagnosis and treatment of pneumonia caused by 2019-ncov (7th trial version clinical characteristics of coronavirus disease 2019 in china characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72 314 cases from the chinese center for disease control and prevention clinical characteristics of 24 asymptomatic infections with covid-19 screened among close contacts in nanjing novel coronavirus pneumonia emergency response epidemiology, the epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (covid-19) in china restoration of leukomonocyte counts is associated with viral clearance in covid-19 hospitalized patients a rapid advice guideline for the diagnosis and treatment of 2019 novel coronavirus (2019-ncov) infected pneumonia (standard version) editorial: orchestration of an immune response to respiratory pathogens characteristics of lymphocyte subsets and cytokines in peripheral blood of 123 hospitalized patients with 2019 novel coronavirus pneumonia (ncp), medrxiv pathological findings of covid-19 associated with acute respiratory distress syndrome the landscape of lung bronchoalveolar immune cells in covid-19 revealed by single-cell rna sequencing procedures and instructions of national immunization programm for children human and murine cytotoxic t cells specific to respiratory syncytial virus recognize the viral nucleoprotein (n), but not the major glycoprotein (g), expressed by vaccinia virus recombinants frequency of circulating cd4+ rsv+ t cells as a marker of disease severity in young children cytokines and cd8 t cell immunity during respiratory syncytial virus infection sars-cov-2 viral load in upper respiratory specimens of infected patients epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study salivary mycobiome dysbiosis and its potential impact on bacteriome shifts and host immunity in oral lichen planus experts opinion on the laboratory diagnosis and treatment of invasive fungal infection secondary to severe novel coronavirus pneumonia severe acute respiratory syndrome (sars) in neonates and children angiotensin-converting enzyme 2 inhibits lung injury induced by respiratory syncytial virus angiotensin-converting enzyme 2 protects from lethal avian influenza a h5n1 infections angiotensin-converting enzyme 2 (ace2) mediates influenza h7n9 virus-induced acute lung injury prospect of ambroxol in the treatment of covid-2019 (in chinese, epub ahead of print associativity from children with 2019 novel coronavirus disease (in chinese, epub ahead of print trained immunity: a memory for innate host defense trained immunity: a program of innate immune memory in health and disease the influence of immunologically committed lymphoid cells on macrophage activity in vivo memory cd8+ t cells provide innate immune protection against listeria monocytogenes in the absence of cognate antigen no one is naive: the significance of heterologous t-cell immunity correlation between universal bcg vaccination policy and reduced morbidity and mortality for covid-19: an epidemiological study characteristics of pediatric sars-cov-2 infection and potential evidence for persistent fecal viral shedding clinical findings in a group of patients infected with the 2019 novel coronavirus (sars-cov-2) outside of wuhan, china: retrospective case series single-cell analysis identifies cellular markers of the hiv permissive cell precursors of human cd4(+) cytotoxic t lymphocytes identified by single-cell transcriptome analysis single-cell rna sequencing unveils an il-10-producing helper subset that sustains humoral immunity during persistent infection structure, function, and antigenicity of the sars-cov-2 spike glycoprotein some medical staff positive for serum sars coronavirus antibody igg have only mild symptoms, nan fang yi ke da xue xue bao study on the dynamic prevalence of serum antibody against severe acute respiratory syndrome coronavirus in employees from wild animal market in guangzhou determination and comparison of anti-sars antibody in children and adults a preliminary investigation on the serological and epidemiological characteristics of severe acute respiratory syndrome in children detection and analysis of sars coronavirus-specific antibodies in sera from non-sars children serological analysis of sars coronavirus in children diagnosed clinically as severe acute respiratory syndrome cases during sars epidemic in beijing new research from scientific research unit shows that sars antibodies are found in 40% of children therapeutic strategies in an outbreak scenario to treat the novel coronavirus originating in wuhan, china structure, function, and antigenicity of the sars-cov-2 spike glycoprotein exploring preventive measures for covid-19 based on the existing virus vaccines (in chinese, epub ahead of print tb vaccine steel the immune system against the new coronavirus? | science | aaas this study was supported by national natural science key: cord-269563-2979u47a authors: caetano silva-filho, josé; germoglio farias de melo, cynthia; lima de oliveira, janaína title: the influence of abo blood groups on covid-19 susceptibility and severity: a molecular hypothesis based on carbohydrate-carbohydrate interactions date: 2020-08-02 journal: med hypotheses doi: 10.1016/j.mehy.2020.110155 sha: doc_id: 269563 cord_uid: 2979u47a the world is experiencing one of the most difficult moments in history with the covid-19 pandemic, a disease caused by sars-cov-2, a new type of coronavirus. virus infectivity is mediated by the binding of spike transmembrane glycoprotein to specific protein receptors present on cell host surface. spike is a homotrimer that emerges from the virion, each monomer containing two subunits named s1 and s2, which are related to cell recognition and membrane fusion, respectively. s1 is subdivided in domains s1a (or ntd) and s1b (or rbd), with experimental and in silico studies suggesting that the former binds to sialic acid-containing glycoproteins, such as cd147, whereas the latter binds to ace2 receptor. recent findings indicate that the abo blood system modulates susceptibility and progression of infection, with type-a individuals being more susceptible to infection and/or manifestation of a severe condition. seeking to understand the molecular mechanisms underlying this susceptibility, we carried out an extensive bibliographic survey on the subject. based on this survey, we hypothesize that the correlation between the abo blood system and susceptibility to sars-cov-2 infection can be presumably explained by the modulation of sialic acid-containing receptors distribution on host cell surface induced by abo antigens through carbohydrate-carbohydrate interactions, which could maximize or minimize the virus spike protein binding to the host cell. this model could explain previous sparse observations on the molecular mechanism of infection and can direct future research to better understand of covid-19 pathophysiology. in december 2019, a new type of coronavirus was discovered in wuhan province, china, causing a condition of severe respiratory failure called covid-19. sars-cov-2, the technical name of this new infectious agent, is genetically linked to sars-cov-1 and mers-cov, two other human coronaviruses that have caused severe lower respiratory tract infections in china in 2002-2003 and in the middle east since 2012, respectively [1] . from china, sars-cov-2 quickly spread across the world, making the world health organization (who) decree this pathology a pandemic in mid-march 2020 (https://www.who.int/dg/speeches). as of the 13 th july 2020, more than 13 the rapid transmissibility of sars-cov-2 has led several countries to adopt strategies to mitigate contagion spread, which includes social isolation/distancing, lockdown and temporary closure of educational institutions [2] , [3] . such measures aim to prevent an accumulation of a large number of people with the severe form of the disease in need of hospital care simultaneously, which could lead to a collapse in health systems worldwide [4] . however, the great concern and need for such governmental policies has led part of the affected nations to an unprecedented economic and social crisis, since unemployment rates, the number of companies' bankruptcies and reports of depressive conditions and other mental disorders have greatly increased in this period [5] [8] . in this context, the global scientific community has engaged in a worldwide effort to understand the pathophysiology of covid-19, in order to develop an effective treatment as quickly as possible. results obtained so far have proved this journey very fruitful. for instance, it has been demonstrated that although sars-cov-2 is directly related to respiratory tract dysfunction due to an intense inflammatory process called "cytokine storm", which promotes the accumulation of fluid in alveoli [9] , the infection can also be systemic, with evidence of the presence of viral genetic material in the gastrointestinal tract and the central nervous system [10] , [11] . these findings can potentially explain why symptoms unrelated to the respiratory tract, like vomiting and diarrhea [12] , as well as neurological disorders [13] , are found in covid-19 patients. studies developed so far have also started to shed a light on the molecular mechanisms mediating the process of viral infection in human cells. the entry of viral particles in host cells depends on the specific binding of virus spike protein to the human membrane receptor ace2 -from angiotensin-convertor enzyme 2 [14] , [15] . this receptor is distributed all over the surface of a large diversity of cell types, such as those from the central nervous system, upper airways and lungs, liver, kidneys, pancreas, heart and endothelial cells [16] . its function is equally diverse depending on the cell type where it lies, ranging from blood pressure regulation, through the renin-angiotensin-aldosterone system, to controlling blood glucose and renal activity [17] , [18] . despite the central role of ace2 in the virus-human cell interaction, other host molecules have also been recognized as important players in the infectious process, such as the transmembrane proteins cd147 [19] , [20] and tmprss2 [21] . cd147 (also known as emmprin, from extracellular matrix metalloproteinase inducer, basigin, m6, neurothelin and hab18g) is believed to function as a coreceptor for the novel coronavirus attachment to host cells [20] . it is a type i transmembrane protein heavily glycosylated that plays roles in spermatogenesis and fertilization, neural network formation and development, tumor metastasis and angiogenesis, and cardiovascular disease [22] , [23] . in turn, tmprss2 (from transmembrane protease serine type 2) has been reported to promote cleavage of spike protein in two different sites to induce sars-cov-1 and sars-cov-2 invasion [24] [26] , since application of a clinically available inhibitor of its protease activity blocks sars-cov-2 entry in cell culture [21] . tmprss2 is lightly glycosylated, playing important roles in human and mammal development and homeostasis, as well as in several diseases, such as cancer [27] and infecton by influenza [28] . a couple of preprint reports and a study using large-scale genetic data from infected and non-infected patients have identified a significant association between type a blood individuals (from the abo blood system) and higher susceptibility to both infection with sars-cov-2 and development of more severe forms of covid-19 [29] [31] . although a correlation between abo blood type and infection susceptibility is not unique of the new coronavirus, since it was previously reported also for the infection by the protozoan that causes malaria [32] [34] , the molecular mechanism underlying this relationship is still poorly understood. supported by an extensive bibliographic review highlighting (i) the mode of binding of sars-cov-2 to cell receptors, as well as (ii) the biochemical aspects of abo blood group system and its association to infection and some circulatory conditions, we hypothesize that the influence of blood type on covid-19 severity relies on the differential clustering of glycoproteins receptors to sars-cov-2 on host cell surface, induced by abh antigens through carbohydrate-carbohydrate interactions with the glycan portions of these receptors, which could modulate virus binding to the target cell. although sars-cov-2 is a new virus, much has been studied about its biochemical characteristics, especially regarding its spike protein, within less than a year since its discovery [35] [38] . in this way, because our focus is on trying to understand how an individual's blood type turns him more or less susceptible to being infected, it is important to address the main known aspects of the virus-host cell interaction. from the pathogen perspective, the interaction is mediated by spike (s) -a structural glycoprotein that emerges from the viral envelope in a homotrimeric arrangement, i.e., through the non-covalent association of three equal monomers or polypeptide chains. each monomer is composed of two subunits called s1 and s2, which are essential for the binding of the virus to receptors present on host cells surface and for the fusion of the viral coat with the plasma membrane, respectively. the two subunits are connected by an amino acid segment that in some coronaviruses species, including sars-cov-2, is cleaved by tmprss2 at a stage prior to the membrane fusion [39] . however, it is suggested that the high pathogenicity of the novel coronavirus is attributed to the additional spike protein priming by furin, a specialized serine endoprotease that cleaves multibasic motifs, a peculiar characteristic not seen in any coronavirus species up to date [40] , [41] . s1 is subdivided into two domains named s1a (or s a ) and s1b (or s b ), arranged in a "v" conformation. the former corresponds to the n-terminal region of the polypeptide chain (hereafter called ntd, from n-terminal domain) and, in most coronaviruses, interacts with glycoproteins and glycolipids that have sialic acid molecules at the distal end of the glycan portion, especially if the monosaccharide is in the modified form of 5-n-acetyl-9-o-acetyl-sialoside [42] . s1b (hereafter named rbd -from receptor-binding domain), on the other hand, binds to the ace2 receptor, which is largely recognized as the main entry route for some coronaviruses into host cells [37] . for other coronaviruses, instead, this function is played by diptidyl-peptidase 4 -dpp4 [43] , [44] or aminopeptidase n -apn [45] , [46] receptors. in some coronaviruses species, like mers-cov, the likelihood interaction of its rbd domain to dpp4 receptor seems to be increased by prior binding of ntd domain to sialosides [47] , [48] . for most coronaviruses, however, it is not known if both domains are used for viral entry [49] , [50] , altought studies from others mammalian coronaviruses indicate that sialosides may facilitate the interaction between spike protein and transmembrane receptors, and can be even essential in more advanced stages of infection [51] , [52] , suggesting that both domains are equally necessary for virus attachment, entry and spread. interestingly, it is reported that sars-cov-1 does not bind sialic acid, a feature that could be extended to the novel coronavirus [53] . in fact, a recent preprint study using glycan microarray did not detect significant fluorescent signals when sars-cov-2 spike protein was incubated with sialic acid-containing oligosaccharides [54] . however, in silico preprint analyses through molecular docking simulations and electronic density mapping surface predict the existence of a sialic acidbinding site in sars-cov-2 ntd domain similar to that one in mers-cov [55] , [56] . surprisingly, sialoside moieties are present in glycans attached to both ace2 [57] and cd147 [58] receptors, which are potentially necessary for the virus anchoring to host cells [59] . taken together, these conflictant reports urge more deeply studies to clarify the sialoside participation in sars-cov-2 infectivity and pathogenicity. the translated product of fut1 recognizes and adds an l-fucose residue to the terminal disaccharide of a glycan precursor anchored to lipids or proteins on cell surface [62] . the disaccharide is formed by a terminal galactose residue connected to an n-acetylglucosamine molecule, either through a β3 or β4 glycosidic bond. addition of l-fucose to galactose creates the antigen h, the chemical determinant of the o phenotype [62] . antigens a and b, on the other hand, arise from enzymatic modification of the h epitope through the attachment of the d isomer of n-acetylgalactosamine or galactose to the terminal galactosil residue, which is mediated by enzymes a and b, respectively. the ab phenotype arises from the simultaneous expression of the two transferases, promoting the formation of their respective antigens in the same cell [62] . abh antigens are not restricted to the erythrocyte membrane, being found in a wide variety of other cells such as lymphocytes, platelets, venular and arterial capillary endothelium, spleen sinusoid cells, bone marrow, gastric mucosa, in addition to secretions and other fluids such as saliva, urine and milk [63] , a feature involved with numerous physiological and pathological processes, as discussed below. individuals with certain types of abo blood groups are more susceptible to diverse kinds of infections [64] . for instance, blood types a and ab predisposes individuals to severe malaria, while type o confers resistance to the protozoan agent. additionally, this blood system is directly or indirectly associated to some cardiovascular conditions. groot et al [65] observed that type a individuals are more likely to have an unhealthier aging than those bearing the o phenotype. these authors also reported that people with a, b and ab blood types are more likely to develop thrombosis and myocardial infarction, while those with type o are more likely to develop hypertensive conditions. the a antigen also seems to predispose individuals to a greater risk of thromboembolism and metabolic disorders, such as hyperlipidemia, hypercholesterolemia and diabetes mellitus [66] . the mechanisms involved in these relationships are, however, poorly understood, with speculative explanations offered to most cases, whereas solid explanations are rare. for malaria infection, for example, it has been reported that type a blood induces rosette formation of erythrocytes, a known virulence factor and contributor to microvascular ischemia and thrombosis [65] . for cardiovascular effects, it is suggested that individuals carrying the a antigen have a higher prevalence of thromboembolism due its association with high levels of von willebrand factor [67] , a glycoprotein synthesized and secreted by endothelial cells and megakaryocytes that stimulates coagulation [68] . in this particular case, it is speculated that antigen a, as well as b, act by increasing the secretion of the factor or by decreasing its clearance, or both [66] . these explanations, however, do not take into account a molecular perspective for the abo blood group participation in the pathological conditions discussed. in other words, they do not respond the question "how exactly do abh antigens correlate with infection and cardiovascular pathophysiology?". a more detailed answer can be found in a work carried out in 2009 by cohen, hurtado-ziola and varki [69] . using three proteins that specifically bind to sialosides, the group analyzed the pattern of interaction between these molecules and human erythrocytes representing the four abo blood phenotypes. they observed that proteins interacted more strongly with a, b and ab cell types, with a certain preference for the first, and less with the o cell type. when using specific glycosidases that converted determinants a and b to h, they found that interactions between all three proteins included in the study and the antigens decreased to the levels observed for the original type o red blood cells. to explain these findings, the authors proposed a model where blood antigens modulate the distribution of sialosides in the plasma membrane, with antigens a and b (specially the former) stimulating the formation of carbohydrate clusters, whereas the h antigen would not promote such effect. the mechanistic causal explanation to these observations was attributed to the presence of carbohydrate-carbohydrate interactions (ccis) between abo blood determinants and sialic acidcontaining glycans, which could influence, even indirectly, cell recognition and communication [70] , [71] . ccis can occur in cis, when the interacting carbohydrates are anchored on the same cell, or in trans, when they are anchored on different cells [69] . ccis have been reported since 1963, when humphreys [72] demonstrated the participation of proteoglycans in cell adhesion of marine sponge. since then, other studies have supported these findings, showing that self-association of surface carbohydrates guides cell aggregation in marine sponge and mouse embryo [73] [75] . the first two decades of the 21st century have been characterized by great advances on this subject, specially concerning methodological developments for studying ccis [76] [82] , as well as by showing their participation in antibody-receptor binding [83] , neonatal immunity stimulation [84] , oncogenesis [85] and potential application in drug delivery approaches [86] . interestingly, divalent ions, mainly ca 2+ , seem to be very important to promote ccis. several studies have showed that this kind of interaction only occur in the presence of these ions [87] [90] , possibly because they coordinate forces to mediate interactions, although ionic forces could also be involved [91] . it is in light of this whole picture that we formulate the hypothesis that the abo blood system correlates to covid-19 severity due to ccis. we speculate that the antigens that determine a, b, ab and o blood cell phenotypes can modulate the distribution of sialic acid-containing receptors in the plasma membrane of host cells. specifically, we hypothesize that mostly antigen a, but also antigens b and ab at a lesser extent, can stimulate the formation of sialoside clusters in target cells through cis ccis. this would maximize the interaction of the cells with sars-cov-2 by increasing the likelihood of binding of the ntd and (possibly) rbd domains to cd147 and ace2 receptors, respectively, through multivalency and avidity. the participation of rbd is proposed based on a recent preprint report showing that ace2 is also decorated with sialoside glycans [57] . additionally, trans ccis cannot be neglected, since spike can be decorated with glycans from host cells [92] . in this case, natural or monoclonal anti-histo-blood group antibodies could bind to spike glycans, inhibiting its interaction to host cell glycoprotein receptors, as reported previously for sars-cov-1 [93] and recently proposed for sars-cov-2 [94] . it is important to note that a recent preprint study reporting the inability to detect the interaction between the novel coronavirus spike protein and sialic acid through glycan microarray [54] does not argues against the proposed model of trans cci, since it is based on the modulation of the distribution of sialoside-containing receptors in plasma membrane. in this perspective, even the possibility of occurrance of trans ccis in the reported assay presumably would not induce detectable fluorescent signals due the immobilization of the tested glycans on the array chip, therefore not allowing the formation of carbohydrate clusters, which would accordingly increase the spike binding. the present hypothesis becomes more interesting when we take into account that (i) covid-19 increases the risk of coagulopathies and venous thromboembolism in those patients who develop a severe condition [95] and (ii) a recent proposition that these traits can be related to deregulatory balance of von willebrand factor levels [96] : two features more prevalent in individuals with type a blood, as mentioned early. a last point to be addressed is that some reports have proposed the use of zinc as a coadjuvant component in the treatment of covid-19 [97] , [98] . although a rigid body of evidence for its efficacy is missing, it is suggested that zinc supplementation performs antiviral activity by various mechanisms, such as restoration of depleted immune function, blocking of virus attachment and infection, and inhibition of virus replication [99] . the hypothesis described here can be considered in future (pre)clinical studies to understand the possible role of this micronutrient in this context. as ccis are commonly mediated by ca 2+ , zinc ions (zn 2+ ) could disrupt its proposed coordinated forces and consequently break the interactions between abh antigens and sialoside moieties, blocking or at least diminishing sars-cov-2 anchoring to host cells. this idea arises from studies with hydroxyapatite crystals, where substitution of zn 2+ for ca 2+ cause remarkable rearrangement of the unit cells [100] , [101] . extending this observation to our model, it is possible that similar changes also occur in the molecular environment of ccis. in summary, this work proposes that the molecular mechanism underlying the influence of abo blood groups on covid-19 susceptibility and severity relies on carbohydrate-carbohydrate interactions between abh antigens and sialoside glycans present on host cell receptors. it is important to highlight that as a review work, its conclusions should be seen and interpreted carefully as an attempt to contribute to a better understand of the pathophysiology of covid-19, which may be further supported or not with experimental and clinical studies. this work has not receveid funding research. the authors declare no conflict of interest. credit authorship contribution statement josé caetano silva-filho: conceptualization, writing -original draft, writing -review and editing. cynthia germoglio farias de melo: writing -original draft. janaína lima de oliveira: writing -review and editing. genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan estimating the effects of non-pharmaceutical interventions on covid-19 in europe susceptibility to and transmission of covid-19 amongst children and adolescents compared with adults: a systematic review and meta-analysis effect of nonpharmaceutical interventions to contain covid-19 in china social consequences of covid-19 in a low resource setting in sierra leone, west africa mental health consequences during the initial stage of the 2020 coronavirus pandemic (covid-19) in spain the socio-economic implications of the coronavirus pandemic (covid-19): a review the impact of the covid-19 pandemic on suicide rates a comprehensive review of manifestations of novel coronaviruses in the context of deadly covid-19 global pandemic clinical insights into the gastrointestinal manifestations of covid-19 neurological manifestations and complications of covid-19: a literature review manifestations and prognosis of gastrointestinal and liver involvement in patients with covid-19: a systematic review and meta-analysis central nervous system manifestations of covid-19: a systematic review structure of the sars-cov-2 spike receptor-binding domain bound to the ace2 receptor structural and functional basis of sars-cov-2 entry by using human ace2 angiotensin-converting enzyme 2: sars-cov-2 receptor and regulator of the renin-angiotensin system: celebrating the 20th anniversary of the discovery of ace2 ace2 deficiency reduces β-cell mass and impairs β-cell proliferation in obese c57bl/6 mice ace2 receptor polymorphism: susceptibility to sars-cov-2, hypertension, multi-organ failure, and covid-19 disease outcome cd147 as a target for covid-19 treatment: suggested effects of azithromycin and stem cell engagement sars-cov-2 invades host cells via a novel route: cd147-spike protein sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor extracellular matrix metalloproteinase inducer emmprin (cd147) in cardiovascular disease crystal structure of hab18g/cd147: implications for immunoglobulin superfamily homophilic adhesion evidence that tmprss2 activates the severe acute respiratory syndrome coronavirus spike protein for membrane fusion and reduces viral control by the humoral immune response structural basis of sars-cov-2 spike protein priming by tmprss2. biorxiv 2020 enhanced isolation of sars-cov-2 by tmprss2-expressing cells tmprss2, a serine protease expressed in the prostate on the apical surface of luminal epithelial cells and released into semen in prostasomes, is misregulated in prostate cancer cells tmprss2: a potential target for treatment of influenza virus and coronavirus infections relationship between the abo blood group and the covid-19 susceptibility testing the association between blood type and covid-19 infection, intubation, and death genomewide association study of severe covid-19 with respiratory failure the abo blood group system and plasmodium falciparum malaria effect of the abo blood group on susceptibility to severe malaria: a systematic review and meta-analysis plasmodium falciparum malaria and abo blood group: is there any relationship? characterization of spike glycoprotein of sars-cov-2 on virus entry and its immune cross-reactivity with sars-cov cell entry mechanisms of sars-cov-2 structural basis for the recognition of sars-cov-2 by full-length human ace2 a highly conserved cryptic epitope in the receptor binding domains of sars-cov-2 and sars-cov structure, function, and antigenicity of the sars-cov-2 spike glycoprotein potential therapeutic targeting of coronavirus spike glycoprotein priming structural features of coronavirus sars-cov-2 spike protein: targets for vaccination structural basis for human coronavirus attachment to sialic acid receptors molecular basis of binding between novel human coronavirus mers-cov and its receptor cd26 the receptor binding domain of the new middle east respiratory syndrome coronavirus maps to a 231-residue region in the spike protein that efficiently elicits neutralizing antibodies the human coronavirus hcov-229e s-protein structure and receptor binding structural bases of coronavirus attachment to host aminopeptidase n and its inhibition by neutralizing antibodies identification of sialic acid-binding function for the middle east respiratory syndrome coronavirus spike glycoprotein structures of mers-cov spike glycoprotein in complex with sialoside attachment receptors recombination, reservoirs, and the modular spike: mechanisms of coronavirus cross-species transmission stabilized coronavirus spikes are resistant to conformational changes induced by receptor recognition or proteolysis human coronaviruses oc43 and hku1 bind to 9-o-acetylated sialic acids via a conserved receptor-binding site in spike protein domain a distinct roles for sialoside and protein receptors in coronavirus infection structural analysis of sars-cov-2 and prediction of the human interactome 2020. biorxiv 2020 binding of the sars-cov-2 spike protein to glycans. biorxiv 2020 n-terminal domain (ntd) of sars-cov-2 spike-protein structurally resembles mers-cov ntd sialosidebinding pocket in-silico evidence for two receptors based strategy of sars-cov-2. arxvi 2020 comprehensive characterization of n-and o-glycosylation of sars-cov-2 human receptor angiotensin converting enzyme 2. biorxiv 2020 importance of n-glycosylation on cd147 for its biological functions distribution of ace2, cd147, cd26 and other sars-cov-2 associated molecules in tissues and immune cells in health and in asthma, copd, obesity, hypertension, and covid-19 risk factors the discovery of the abo blood group system and its value for teaching medical students an integrative evolution theory of histo-blood group abo and related genes biological and clinicel aspects of abo blood group system blood groups: the past 50 years blood groups in infection and host susceptibility genetically determined abo blood group and its associations with health and disease biologic roles of the abh and lewis histo-blood group antigens part ii: thrombosis, cardiovascular disease and metabolism measurement of von willebrand factor activity: relative effects of abo blood type and race biochemistry and genetics of von willebrand factor abo blood group glycans modulate sialic acid recognition on erythrocytes modulation of glycan recognition by clustered saccharide patches notable aspects of glycan-protein interactions chemical dissolution and in vitro reconstruction of sponge cell adhesions. i. isolation and functional demonstration of the components involved specific interaction between le(x) and le(x) determinants. a possible basis for cell recognition in preimplantation embryos and in embryonal carcinoma cells involvement of carbohydrates as multiple low affinity interaction sites in the self-association of the aggregation factor from the marine sponge microciona prolifera involvement of a carbohydrate group in the active site for surface guided reassociation of animal cells synthesis of glycosylated metal complexes for probing carbohydratecarbohydrate interactions multivalent glycoliposomes and micelles to study carbohydrate-protein and carbohydrate-carbohydrate interactions studying carbohydrate self-recognition in marine sponges using synthetic aggregation factor epitopes analysis of carbohydrate-carbohydrate interactions using sugar-functionalized silicon nanoparticles for cell imaging studies of the carbohydrate-carbohydrate interaction between lactose and gm 3 using langmuir monolayers and glycolipid micelles triggerable multivalent glyconanoparticles for probing carbohydrate-carbohydrate interactions carbohydrate-coated fluorescent silica nanoparticles as probes for the galactose/3-sulfogalactose carbohydrate-carbohydrate interaction using model systems and cellular binding studies unique carbohydrate-carbohydrate interactions are required for high affinity binding between fcγriii and antibodies lacking core fucose human milk oligosaccharides influence neonatal mucosal and systemic immunity tn and stn are members of a family of carbohydrate tumor antigens that possess carbohydrate-carbohydrate interactions exploiting the lactose-gm3 interaction for drug delivery carbohydrate-carbohydrate interactions mediated by sulfate esters and calcium provide the cell adhesion required for the emergence of early metazoans self-recognition and ca 2+ -dependent carbohydrate-carbohydrate cell adhesion provide clues to the cambrian explosion carbohydrate-carbohydrate interaction as a major force initiating cell-cell recognition quantitative and qualitative approach of glycan-glycan interactions in marine sponges carbohydrate-carbohydrate interaction provides adhesion force and specificity for cellular recognition deducing the n-and o-glycosylation profile of the spike protein of novel coronavirus sars-cov-2 inhibition of the interaction between the sars-cov spike protein and its cellular receptor by antihisto-blood group antibodies coronavirus disease 2019 (covid 19) and malaria. have anti glycoprotein antibodies a role? thromboembolic risk and anticoagulant therapy in covid-19 patients: emerging evidence and call for action willebrand factor and endothelial damage: a possible association with covid-19 does zinc supplementation enhance the clinical efficacy of chloroquine/hydroxychloroquine to win todays battle against covid-19? can zn be a critical element in covid-19 treatment? potential role of zinc supplementation in prophylaxis and treatment of covid-19 formation and structure of zinc-substituted calcium hydroxyapatite synthesis and characterization of zn-doped hydroxyapatite: scaffold application, antibacterial and bioactivity studies key: cord-302212-zt4lv5g4 authors: patruno, cataldo; paul nisticò, steven; fabbrocini, gabriella; napolitano, maddalena title: covid-19, quarantine, and atopic dermatitis date: 2020-05-19 journal: med hypotheses doi: 10.1016/j.mehy.2020.109852 sha: doc_id: 302212 cord_uid: zt4lv5g4 nan atopic dermatitis (ad) is a chronic inflammatory skin disease affecting up to 20% of children and up to 10% of adults (1) . western lifestyle, pollution, stress, and atmospheric elements can affect the prevalence and severity of the disease (1, 2) . its pathogenesis involves environmental factors that interact with genetic skin barrier defects and immune th2 adverse psychological effects induced by the covid-19 quarantine have been associated with increase of itch in some chronic skin disease, also due to neuroendocrine modulation of skin inflammation (4). lockdown can lead to a diet rich in saturated fats and refined carbohydrates, and reduction of physical activity, increasing pathogenic th2 phenotype (1). obviously, quarantine is associated with less exposure to sunlight that, together with high temperature and low humidity, may exert an immunosuppressive effect on skin inflammation of ad (2) . moreover, ad has been associated with pollution that may induce both epigenetic changes in utero and worsening of postnatal ad (1). the requirement to stay home leads to greater exposure to indoor pollutants, such as tobacco smoke or volatile organic compounds (1) . on the other hand, a paradoxical positive consequence of lockdown is the significant reduction of outdoor air pollution subsequent to the reduction of urban traffic; ad has been associated with traffic-related air pollution, probably for oxidative damage of skin barrier (1). similarly, as mentioned, the reduction of physical activity on the one hand is proinflammatory, while on the other the reduction of profuse sweating induces a reduction in the flares of dermatitis in affected subjects (2) . furthermore, during quarantine, routine dermatologic visits are difficult to access; indeed, teledermatology programs increase during lockdown (4), and some patients stop taking immunosuppressive drugs on their own initiative for the fear of contracting covid-19. all these considerations suggest the hypothesis that covid-19 pandemic may be associated with changes in lifestyle of ad patients that can significantly influence their clinical disease activity. obviously, epidemiological studies are needed to confirm this hypothesis, which, if confirmed, could also lead to more information on the pathogenesis of ad. the exposome in atopic dermatitis effects of climate change on skin diseases the role of occupational dermatology in the covid-19 outbreak mass quarantine measures of covid-19 pandemic: psychosocial implications for chronic skin conditions and a call for qualitative studies all authors have no interests to report key: cord-288733-c51lfwd6 authors: kavanagh, oisín; marie healy, anne; dayton, francis; robinson, shane; o'reilly, niall j.; mahoney, brian; arthur, aisling; walker, gavin; farragher, john p. title: inhaled hydroxychloroquine to improve efficacy and reduce harm in the treatment of covid-19 date: 2020-07-15 journal: med hypotheses doi: 10.1016/j.mehy.2020.110110 sha: doc_id: 288733 cord_uid: c51lfwd6 current formulations and dose regimens of hydroxychloroquine (hcq) put patients at risk of harm. an analysis of clinical trials registered on clinicaltrials.gov revealed that this may continue as many studies combine hcq with agents that prolong the qt interval. further, almost all of the trials registered do not consider dosage adjustment in the elderly, a patient population most likely to require hcq treatment. here we describe an inhaled formulation of hcq which has passed safety studies in clinical trials for the treatment of asthma and discuss how this approach may reduce side-effects and improve efficacy. as this simple formulation progressed to phase ii studies, safety data can be used to immediately enable phase ii trials in covid-19. 2 25 the covid-19 (sars-cov-2) pandemic has had devastating health consequences 26 worldwide 1 , and life has been brought to a standstill as the world awaits a vaccine. meanwhile, 27 many patients continue to fall critically ill and, in the absence of a vaccine, antivirals and other 28 treatments to alleviate the effects of the disease are urgently sought. 29 30 in recent months, since the outbreak of covid-19, a large number of clinical trials have been 31 registered on clinicaltrials.gov with the aim of repurposing medicines to reduce the severity 32 of the disease 2 . chloroquine and hydroxychloroquine (hcq) were two of the earliest drugs to 33 receive attention as possible repurposable treatment options for covid-19 3 . indeed, a number 34 of studies investigated their anti-sars-cov activity as early as 2003 4-6 . however, the 35 potential efficacy of hcq must be balanced against its side-effects, particularly those 36 associated with qt elongation, which is exacerbated by age, comorbidities and administration 37 with other agents (such as azithromycin) that prolong the qt interval 7 . a recent analysis of 38 nhs (uk) electronic health records revealed that the most striking risk factors for covid-19 39 death were age and male gender 8 , and those same risk factors have been identified previously 40 in the context of drug induced qt elongation 9,10 . concerns associated with severe side effects 41 are such that the fda and ema now formally recommend against taking hcq for covid effects of chloroquine on 178 viral infections: an old drug against today's diseases? new insights into the 181 antiviral effects of chloroquine hydroxychloroquine in sle: old drug, new perspectives. nat coronavirus disease 185 2019, and qt prolongation opensafely: factors associated 189 with covid-19-related hospital death in the linked electronic health records of 17 190 million adult nhs patients detailed analysis of the impact of age on the 192 qt interval aging effects on qt interval: implications for cardiac safety of 194 antipsychotic drugs covid-19: reminder of risk of serious side effects with 196 chloroquine and hydroxychloroquine fda drug safety communication: fda cautions 199 against use of hydroxychloroquine or chloroquine for covid-19 outside of the 200 hospital setting or a clinical trial due to risk of heart rhythm problems dosing design of hydroxychloroquine for the treatment of severe acute respiratory 204 syndrome coronavirus 2 (sars-cov-2) hydroxychloroquine for treatment of sars-cov-2 infection? 206 improving our confidence in a model-based approach to dose selection optimizing hydroxychloroquine 210 dosing for patients with covid-19: an integrative modeling approach for effective 211 drug repurposing the aging kidney: physiological changes. adv. chronic 214 kidney dis the species severe acute respiratory 216 syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2 219 the novel coronavirus 2019 (2019-ncov) uses the sars-coronavirus receptor ace2 220 and the cellular protease tmprss2 for entry into target cells hydroxychloroquine as an aerosol might 224 markedly reduce and even prevent severe clinical symptoms after sars-cov-2 225 infection development of an inhaled hydroxychloroquine 229 sulfate product using the aerx ® system to treat asthma clinical study report: a two-part, randomised, 231 double-blind, placebo-controlled, ascending-dose study to evaluate the safety, 232 tolerance and pharmacokinetics of orally inhaled hydroxychloroquine sulfate via the evaluation of an immediate-244 release formulation of hydroxychloroquine sulfate with an interwoven pediatric 245 taste-masking system comprehensive evidence-based review on european antitussives effect of taste sensation on cough reflex sensitivity sweet taste and menthol increase cough reflex 251 thresholds development and palatability 253 assessment of norvir® (ritonavir) 100 mg powder for pediatric population key: cord-291722-xnfdjriz authors: meenakshisundaram, ramachandran; senthilkumaran, subramanian; thirumalaikolundusubramanian, ponniah title: protective effects of vaccinations and endemic infections on covid-19: a hypothesis date: 2020-05-26 journal: med hypotheses doi: 10.1016/j.mehy.2020.109849 sha: doc_id: 291722 cord_uid: xnfdjriz nan severe acute respiratory distress syndrome (sars) -coronavirus (cov) 2 infection is in the pandemic state with inter-country variations in the occurrence and case fatality. we believe that the low occurrence/fatality of covid-19 may be due to the existing vaccination status and endemicity of other infections which might have enhanced their immune system to face the challenges of covid-19. we would like to highlight on possible reasons and the extended evolutionary synthesis (ees) to support our views. a few vaccines including bacille calmette-guerin (bcg) vaccination offer nonspecific immune effect (1)and this trained immunity gives protection against several other pathogens (2, 3) and reduces viremia, it's virulence, sepsis and mortality (4, 5) . so, a clinical trial has been initiated to ascertain the above fact (6) . interestingly, bcg produces persistent conformational changes in innate and adaptive immune cells and increases interleukin-1b secretion, which enhances anti-microbial immunity (7) (8) (9) . also, antibodies generated against live attenuated measles vaccine provide neutralising effect on sars-cov and also induce antibodies against sars-cov antigen (10) . since bcg and measles vaccines may provide some non-specific protection against covid-19, these may be considered at least for the susceptible population before the development covid-19 specific vaccine. countries with a high prevalence of infections with dengue, chikungunya, malaria etc., have a low occurrence of covid-19 makes one to hypothesize that endemic infections may protect through interferon which retard subsequent illness/disease through viral interference (11, 12) . this viral interference happens via multifactorial manners such as immune response, cellular response, rna interference and defective interfering particles or genomes of the host (13) . the concept of cross-protection offered by previous herpetic infections against vaccinia lesions (14) was first brought to light by edward jenner two centuries ago. previous experimental studies have revealed that beta coronaviruses are capable of inducing immune responses against one another by way of generating neutralizing antibodies which cross-react against other sars-cov viruses (15, 16) . based on the above, we believe that the above principles may apply for the less occurrence of covid-19 in regions where other viral infections are prevalent. overall the organisms and individuals based on previous exposure to vaccines and infections come from the environment, internal sensors, memorized experience, and genome prefer to develop pathways in a goal-directed manner and improve the behavioural traits and phenotypic variability so as to protect and survive from infectious agents. all these are considered under ees by pigliucci and müller(17) and ees brings out the hidden morphogenetic capabilities and protect the organisms/cells. cross immunity though helps, the questions to be considered are: "will crossimmunity enhance career status for covid-19 and make the disease endemic or contribute to mutations of viruses and hamper vaccine research/vaccination against covid-19?", "will the low levels of cross-immunity produced from the other beta coronaviruses make sars-cov-2 to die out, and/ or will it contribute to a resurgence of the same after a few years (18) ?". further studies are warranted to answer the above. till then, we accept that nature and science strengthen the immune system through repeated infections and vaccinations respectively, and prepares living organisms to face the challenges of existing, emerging and re-emerging infections. during this process, infectious agents may mutate, and attack the living organisms differently. all are aware that we can't fight against nature, but live in a symbiotic/harmonious manner with other infectious agents as much as possible with and/ or without vaccinations. thus, the successful one survives and life propagates forever. considering bcg vaccination to reduce the impact of covid-19 connecting bcg vaccination and covid-19: additional data. medrxiv correlation between universal bcg vaccination policy and reduced morbidity and mortality for covid-19: an epidemiological study. medrxiv non-specific effects of bcg vaccine on viral infections routine vaccinations and child survival: follow up study in reducing health care workers absenteeism in covid-19 pandemic through bcg vaccine (bcg-corona) bcg vaccination may be protective against covid-192020 trained immunity: a program of innate immune memory in health and disease long-term in vitro and in vivo effects of gamma-irradiated bcg on innate and adaptive immunity induction of neutralising antibodies and cellular immune responses against sars coronavirus by recombinant measles viruses investigating viral interference between influenza a virus and human respiratory syncytial virus in a ferret model of infection evidence for viral interference and cross-reactive protective immunity between influenza b virus lineages viral interference and persistence in mosquito-borne flaviviruses on the effects of cutaneous eruptions an outbreak of human coronavirus oc43 infection and serological cross-reactivity with sars coronavirus cross-reactive antibodies in convalescent sars patients' sera against the emerging novel human coronavirus emc (2012) by both immunofluorescent and neutralizing antibody tests elements of an extended evolutionary synthesis projecting the transmission dynamics of sars-cov-2 through the postpandemic period key: cord-301619-0ojayw16 authors: adamowicz, jan; juszczak, kajetan; drewa, tomasz title: may patients receiving 5-alpha-reductase inhibitors be in higher risk of covid-19 complications ? date: 2020-04-22 journal: med hypotheses doi: 10.1016/j.mehy.2020.109751 sha: doc_id: 301619 cord_uid: 0ojayw16 covid-19 pandemic is a major challenge for global and national healthcare providers. number of new cases is continuously increasing with an emerging trend showing worse prognosis in males in comparison to females. based on this observation, our proposed hypothesis is that 5-alpha-reductase inhibitors, that are commonly used for bph treatment, may be one of the factors contributing to poorer prognosis in males. background: with increasing number of covid-19 cases, an evident sexdependent difference in disease outcomes can be observed. based on published studies with short term follow-up, males have 65% higher mortality rate (1). the question remains, whether long term observational studies will confirm improved recovery in females. jan adamowicz 1 2* , kajetan juszczak 1 , tomasz drewa 1 abstract covid-19 pandemic is a major challenge for global and national healthcare providers. number of new cases is continuously increasing with an emerging trend showing worse prognosis in males in comparison to females. based on this observation, our proposed hypothesis is that 5-alpha-reductase inhibitors, that are commonly used for bph treatment, may be one of the factors contributing to poorer prognosis in males. with increasing number of covid-19 cases, an evident sex-dependent difference in disease outcomes can be observed. based on published studies with short term follow-up, males have 65% higher mortality rate (1). the question remains, whether long term observational studies will confirm improved recovery in females. the major alleged cause of this phenomenon, is a general poorer male health condition, related mostly to a higher cigarette consumption and more common heart disease (2) . in spite of these reports, there have been other published opinions, raising the possibility of an intrinsic protective immunomodulation mediated by estrogen receptor pathway. indeed, the sex dependent susceptibility to covid-19 infection, is a result of many not yet identified factors. these however, should be recognized as soon as possible, in order to improve an accurate management of the disease. from urologist's perspective, a potential explanation might be related to the bph (benign prostatic hyperplasia) treatment with finasteride and dutasteride. both of these members of 5-alpha-reductase inhibitors are used to reduce prostate volume pharmacologically (3) . therapeutic effect is mediated by decreasing intraprostatic level of dht (dihydrotestosterone). dht is more potent than testosterone at maintaining normal prostate weight and stroma volume (4). the influence of 5-alpha-reductase inhibitors on prostate is well established but 5-alpha-reductase is also an enzyme associated with androgen metabolism in many organs. consequently, we introduce hypothesis that 5-alpha-reductase inhibitors may disrupt androgens metabolism in lungs, which in turn may have a negative impact on course of covid-19 infection. finasteride and less selective dutasteride both block 5-alpha-reductase isoform 3, which is expressed in respiratory epithelium and fibroblasts (5) . the regulative role of androgens in adult human lungs function is an underdeveloped field. nevertheless, there is limited scientific data indicating that androgens are involved in proper function of respiratory epithelium. importantly, maintenance and restoration of surfactant layer may be controlled by androgens' metabolism, where 5-alpha-reductase plays a key role (6) . during alveolar fetal development and alveolar repair after inflammatory lung disease close contacts needs to be established between fibroblasts and lung epithelial cells through gaps in the basement membrane (7). it was documented that androgens including dht disrupt communication between fibroblasts and alveolar type ii cells by a mechanism involving tgfβ (transforming growth factor beta) and egf (epidermal growth factor) receptor signaling pathways (8) (9). during human fetal lung development, dht slows down epithelial layer maturation. in animal studies supplementation with dht inhibited surfactant phospholipid production during fetal lung development whereas application of antiandrogen flutamide increased surfactant phospholipid production (8) (10) . it is likely that in mature lung tissue, the androgen regulative pathways analogously to fetal ones are also active. the expression of major regulative enzymes 17 beta-hydroxysteroid dehydrogenase and 5 alpha-reductase was detected in adult lung tissue. interestingly, it was shown that balanced interplay of both enzymes is physiologically adjusted to obtain precisely regulated androgen inactivation within lungs. therefore, in contrast to prostate, the physiological function of 5 alpha-reductase in lungs is to locally minimalize androgen potential (11) . this action profile is a result of low 5 alphareductase affinity to testosterone and high to androstadiene with respiratory epithelium. the respiratory epithelium is characterized with capacity of spontaneous regeneration. despite being quiescent tissue, lungs' regeneration mechanism are activated in pneumonia related injuries (12) . regeneration processes are universal and mimic organogenesis stages, in this situation, restoration of proper respiratory epithelial layer may be dependent on androgens metabolism. under this assumption, 5-alpha-reductase inhibitors might increase androgen concentration in lungs hampering their regeneration. interstitial pneumonia is the main cause of life-threatening respiratory disorders at the severe stage of covid-19 infection (13) . complex lung damage including lung alveolar epithelial cells and fibroblasts is a major hurdle to recovery in those patients. therefore, inhibition of 5 alpha-reductase might result in impairment of spontaneous regeneration capacity and prolonged or deteriorated recovery prognosis. due to high prevalence of 5-alpha-reductase inhibitor in bph treatment, its potential negative influence on recovery after covid-19 infection, should be established. according to presented hypothesis, patients receiving 5-alpha-reductase inhibitors, might be vulnerable to covid-19 infection with poorer prognosis. in such dynamic situation as covid-19 pandemic, an interdisciplinary analysis of patients may deliver plenty of clues for development of treatment protocols optimization. sex difference and smoking predisposition in patients with covid-19 the use of 5-alpha reductase inhibitors in the treatment of benign prostatic hyperplasia the role of dihydrotestosterone in benign prostatic hyperplasia the 5 alpha-reductase isozyme family: a review of basic biology and their role in human diseases a link between lung androgen metabolism and the emergence of mature epithelial type ii cells alveolar type ii cellfibroblast interactions, synthesis and secretion of surfactant and type i collagen gene expression profile of androgen modulated genes in the murine fetal developing lung transdifferentiation of alveolar epithelial type ii cells to type i cells involves autocrine signaling by transforming growth factor β1 through the smad pathway hormonal influences on lung function and response to environmental agents: lessons from animal models of respiratory disease androgen inactivation in human lung fibroblasts: variations in levels of 17β-hydroxysteroid dehydrogenase type 2 and 5α-reductase activity compatible with androgen inactivation epithelial regeneration and lung stem cells covid-19 infection: the perspectives on immune responses may patients receiving 5 alpha-reductase inhibitors be in higher risk of covid-19 complications this is a pdf file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. this version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. key: cord-291781-gs81g1db authors: caruso, arturo armone; prete, antonio del; lazzarino, antonio ivan title: hydrogen peroxide and viral infections: a literature review with research hypothesis definition in relation to the current covid-19 pandemic date: 2020-06-01 journal: med hypotheses doi: 10.1016/j.mehy.2020.109910 sha: doc_id: 291781 cord_uid: gs81g1db we reviewed the literature concerning the innate response from nasal and oral epithelial cells and their reaction to hydrogen peroxide (h(2)o(2)). hydrogen peroxide is produced physiologically by oral bacteria and plays a significant role in the balance of oral microecology since it is an important antimicrobial agent. in the epithelial cells, the enzyme superoxide dismutase catalyzes a reaction leading from hydrogen peroxide to the ion superoxide. the induced oxidative stress stimulates a local innate response via activation of the toll-like receptors and the nf-κb. those kinds of reactions are also activated by viral infections. virus-induced oxidative stress plays an important role in the regulation of the host immune system and the specific oxidant-sensitive pathway is one of the effective strategies against viral infections. therefore, nose/mouth/throat washing with hydrogen peroxide may enhance those local innate responses to viral infections and help protect against the current coronavirus pandemic. we strongly encourage the rapid development of randomized controlled trials in both sars-cov-2 positive and negative subjects to test the preliminary findings from the in-vitro and in-vivo observational studies that we identified. the current coronavirus outbreak appears to be characterized by respiratory transmission and respiratory infection, although the routes of transmission and the pathophysiology of the disease have not been fully clarified yet. however, it is proven that the virus resides in the nasal and oral mucosa. [1] 2/6 while impeding person-to-person transmission is key to limiting the outbreak, so far little importance has been given to the events taking place after a transmission has happened, when innate immunity plays a crucial role. the main purpose of the innate immune response is to immediately prevent the spread and movement of foreign pathogens throughout the body. there is a close interplay between innate immunity and oxidative stress, and the molecule hydrogen peroxide may play a central role. [2] we reviewed the literature concerning the innate response from nasal and oral epithelial cells to evaluate the role of hydrogen peroxide (h 2 o 2 ). we searched the databases embase and pubmed for relevant papers using the following entry terms: ("hydrogen peroxide" or h 2 o 2 ) and (virus or viral infection or innate response). we have not used any other constrains. after duplicates were removed, we identified 2119 papers. two independent observers scrutinized those articles in their titles and abstracts to apply eligibility criteria and filter the relevant ones. disagreements were resolved by discussion. we defined the eligibility criteria using a pico model as follows. population: no specific reference population. intervention: administration of hydrogen peroxide within clinical studies or animal studies or in-vitro studies. comparison: placebo controlled or intra-individual pre-post comparison. outcome: duration of current viral infection or protection against viral infections or enhancement of innate immunity reactions. all references from all eligible articles were assessed for eligibility, with no success. finally, the papers were read, criticized, and narratively summarized. figure 1 shows a flow chart of the literature screening method used. (figure 1 ), none of which was a randomized controlled trial or a clinical observational study with a control group. hydrogen peroxide is produced physiologically by oral bacteria and plays a significant role in the balance of oral microecology since it is an important antimicrobial agent. [3] in the epithelial cells, the enzyme superoxide dismutase catalyzes a reaction leading from hydrogen peroxide to the ion superoxide. the induced oxidative 3/6 stress stimulates a local innate response via activation of the toll-like receptors and the nf-κb. [4] those kinds of reactions are also activated by viral infections. [5] virus-induced oxidative stress plays an important role in the regulation of the host immune system and the specific oxidant-sensitive pathway is one of the effective strategies against viral infections. [6] [7] [8] many viruses have been found to be sensitive to hydrogen peroxide, including swine flu, rubella, rabies, and others. [7, [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] we therefore hypothesize that nose and mouth washing with hydrogen peroxide may enhance those local innate responses to viral infections and help protect against viral infections, including the current coronavirus pandemic. moreover, a hydrogen peroxide solution of a concentration as little as 0.5% efficiently inactivates coronaviruses (e.g. sars, mers) on inanimate surfaces within 1 minute. [22] this may justify the use of hydrogen peroxide washes in infected people too: the solution may decrease the viral load of their respiratory droplets, hence help tackle the spread in the community. in the british nationally formulary, h 2 o 2 is indicated for oral hygiene at the concentration of 6%. hydrogen peroxide is safe to use for gargling or as a nasal spray: its 3% solution is commonly used off-label in otolaryngology to treat many viral conditions, [23] [24] [25] [26] and moderate concentrations are present in drinks including tea and instant coffee. [27] there are no randomized controlled trials or clinical observational studies concerning the curative or preventive effect of hydrogen peroxide against viral infections. however, the literature from in-vitro immunological studies clearly points out that the application of hydrogen peroxide on the epithelial cells of nose, throat and mouth may well be extremely effective against viruses, including coronaviruses. we strongly encourage the rapid development of randomized controlled trials in both sars-cov-2 positive and negative subjects to study the effects that we have hypothesized. those studies included in quantitative synthesis (meta-analysis) (n = 0) the origin, transmission and clinical therapies on coronavirus disease 2019 (covid-19) outbreak -an update on the status the origin of hydrogen peroxide in oral cavity and its role in oral microecology balance oxidative stress modulates the expression of toll-like receptor 3 during respiratory syncytial virus infection in human lung epithelial a549 cells influenza a virus and tlr7 activation potentiate nox2 oxidase-dependent ros production in macrophages the role of oxidative stress in influenza virus infection rubella virus strain-associated differences in the induction of oxidative stress are independent of their interferon activation oxidative stress enhances the expression of il-33 in human airway epithelial cells inactivation of rabies virus by hydrogen peroxide effectiveness of hydrogen peroxide and electron-beam irradiation treatment for removal and inactivation of viruses in equine-derived xenografts the detection of hydrogen peroxide involved in plant virus infection by fluorescence spectroscopy hydrogen peroxide induces la cytoplasmic shuttling and increases hepatitis c virus internal ribosome entry site-dependent translation virucidal efficacy of a hydrogen peroxide nebulization against murine norovirus and feline calicivirus, two surrogates of human norovirus efficacy of accelerated hydrogen peroxide® disinfectant on foot-and-mouth disease virus, swine vesicular disease virus and senecavirus a efficacy of an accelerated hydrogen peroxide disinfectant to inactivate porcine epidemic diarrhea virus in swine feces on metal surfaces roles of superoxide anion and hydrogen peroxide during replication of two unrelated plant rna viruses in nicotiana benthamiana evaluation of an accelerated hydrogen peroxide disinfectant to inactivate porcine epidemic diarrhea virus in swine feces on aluminum surfaces under freezing conditions evaluation of hospital-grade disinfectants on viral deposition on surfaces after toilet flushing pcr prevalence of murine opportunistic microbes and their mitigation by using vaporized hydrogen peroxide hydrogen peroxide vapour is an effective replacement for formaldehyde in a bsl4 foot and mouth disease vaccine manufacturing facility sensitivity of african swine fever virus (asfv) to heat, alkalinity and peroxide treatment in presence or absence of porcine plasma persistence of coronaviruses on inanimate surfaces and their inactivation with biocidal agents hydrogen peroxide: a review of its use in dentistry treatment of pharyngitis and laryngitis with an improved hydrogen peroxide glycerite of hydrogen peroxide; a correlative review of laboratory and clinical data glycerite of hydrogen peroxide; its use in various inflammatory aural conditions hydrogen peroxide in the human body the authors received no funding from any source. all authors declare no conflict of interest of any kind. all authors contributed to the conception of the study. ail wrote the manuscript. all authors approved the manuscript before submission. key: cord-289332-hvakv08t authors: chen, guoqian; chen, da-zhi; li, jianhua; czura, christopher j.; tracey, kevin j.; sama, andrew e.; wang, haichao title: pathogenic role of hmgb1 in sars? date: 2004-04-30 journal: med hypotheses doi: 10.1016/j.mehy.2004.01.037 sha: doc_id: 289332 cord_uid: hvakv08t high mobility group box 1 protein (hmgb1) is released by necrotic cells or activated macrophages/monocytes, and functions as a late mediator of lethal systemic and local pulmonary inflammation. passive immunization with anti-hmgb1 antibodies confers significant protection against lethal endotoxemia, sepsis, and acute lung injury, even when antibodies are administered after the onset of these diseases. in light of observations that three chinese herbal formulations recommended for treatment of severe acute respiratory syndrome (sars) specifically inhibited the release of hmgb1 from innate immune cells, we hypothesize that hmgb1 might occupy a pathogenic role in sars by mediating an injurious pulmonary inflammatory response. severe acute respiratory syndrome (sars) has killed more than nine hundreds of people around the world and infected thousands more. it has created international anxiety because of its rapid progression and high mortality. in china, several clinical trials revealed efficacy of traditional chinese herbal remedies in reducing symptoms and mortality of patients with sars [1] . high mobility group box 1 protein (hmgb1, formerly known as hmg-1 or amphoterin) has recently been identified as a new proinflammatory cytokine and a late mediator of inflammation, sepsis, and acute lung injury. in light of observations that several chinese herbal remedies recommended for treatment of sars specifically inhibited the release of hmgb1 from activated innate immune cells, we hypothesize that hmgb1 might occupy a pathogenic role in sars by mediating an injurious pulmonary inflammatory response. hmgb1 as a proinflammatory cytokine hmgb1 has been for a long time described as a nuclear dna-binding protein in mammalian cells [2] . recent evidence has revealed that hmgb1 can be released by necrotic cells or activated innate immune cells (such as macrophages and monocytes), and functions as a proinflammatory cytokine [3] [4] [5] . in response to exogenous stimuli (such as bacterial endotoxin, lipopolysaccharide, lps) or endogenous cytokines (such as tnf, il-1b or ifn-c), cultures of macrophages, monocytes or pituicytes actively release hmgb1 in a time-and dose-dependent manner [3, 6, 7] . the kinetics of hmgb1 release from activated macrophages/monocytes is significantly delayed, with hmgb1 accumulation first detectable at 8 h after stimulation [3, 7] . this delayed release of hmgb1 distinguishes it from tnf and other early mediators of systemic inflammatory responses [5] . extracellular hmgb1 is able to stimulate the production of tnf and other proinflammatory cytokines from macrophages, endothelial cells or neutrophils, triggering an inflammatory response [8] . in murine models of endotoxemia or sepsis, or patients with sepsis or rheumatoid arthritis [8] [9] [10] , serum hmgb1 levels are significantly elevated [3] . administration of hmgb1 via various (e.g., focal, intraperitoneal, intracerebroventricular, and intratracheal) routes induces marked inflammatory responses in vivo [5, 11, 12] . taken together, these studies indicate that accumulation of hmgb1 can amplify the cytokine cascade, and mediate injurious inflammatory responses. the pathogenic role of hmgb1 as a late mediator of lethal systemic inflammation has been defined in animal models of endotoxemia and sepsis using anti-hmgb1 antibodies or inhibitors [2, 3, 13] . anti-hmgb1 antibodies confer a dose-dependent protection in animal models of sepsis, even when the first dose of anti-hmgb1 antibodies is delayed for 24 h after the onset of sepsis [2] . administration of a novel anti-inflammatory agent, ethyl pyruvate, dose-dependently inhibits hmgb1 release, and confers significant protection against the lethality of sepsis [14] . in animal models of collagen-induced arthritis, administration of anti-hmgb1 antibodies also confers significant protection [15] . thus, therapeutic agents capable of inhibiting hmgb1 activities (such as anti-hmgb1 antibodies) or release (such as ethyl pyruvate) may hold potential for the clinical management of sepsis and other inflammation diseases [16] . note: all herbal ingredients were obtained from ny-tongrentang, inc. (flushing, ny, usa), mixed, and boiled in water for 40 min. the water-soluble fraction (recalibrated to a total volume of 100 ml with sterile water) was cleared by centrifugation (4000 rpm, 20 min, 4°c) and filtration (through 0.2 lm filter), and the clear water-soluble herbal extract was evaluated for the immunomodulatory properties. intratracheal administration of hmgb1 to lps-resistant c3h/hej mice stimulates lung neutrophil accumulation, and increases pulmonary levels of proinflammatory cytokines such as tnf, il-1b, and mip-2 [13] . histological examination of lung tissue reveals evidence of an acute diffuse inflammatory response, with accumulation of neutrophils in the interstitial and intraalveolar areas, interstitial edema, and protein exudation into the alveolar space [13] . in a widely used animal model of acute lung injury induced by intratracheal administration of bacterial endotoxin, administration of anti-hmgb1, either before or after endotoxin treatment, significantly decreases endotoxin-induced neutrophil infiltration, and lung edema [13] . despite the ameliorative effects of anti-hmgb1 antibodies on the development of lung injury and neutrophil accumulation, this treatment does not affect endotoxin-induced increases in pulmonary levels of tnf, il-1b, or mip-2, establishing the important role of endogenous hmgb1 itself as a mediator of acute lung injury [13] . after the outbreak of the sars in china, the state administration of traditional chinese medicine recommended the use of several traditional chinese herbal remedies for prevention and treatment of sars [17] . clinical observations suggest that chinese herbal remedies are beneficial in the reduction of (i) fever; (ii) air-space shadowing of the chest radiographs; (iii) the use of corticosteroids; and (iv) the death rate of severe sars patients [1] . to evaluate the immunomodulatory properties of traditional chinese herbal remedies recommended for sars, we examined their potential effect on the release of several proinflammatory mediators in cultures of macrophages and human peripheral blood mononuclear cells (hupbmcs). three chinese herbal formulations were prepared according to the recipes released by the state administration of traditional chinese medicine [17] (table 1) . at clinically relevant doses (5-15 ll of extract/ml culture medium), none of these herbal formulations inhibited endotoxin-induced production of tnf or il-1b by either macrophages or hupbmcs (data not shown). two out of three herbal remedies significantly attenuated endotoxin-induced production of nitric oxide ( fig. 1(b)) . surprisingly, all three chinese herbal remedies dose-dependently (5-15 ll/ml) suppressed endotoxin-induced hmgb1 release by figure 1 effects of three traditional chinese herbal formulations on endotoxin-induced release of nitric oxide and hmgb1. murine macrophage-like raw 264.7 cells (panels a, b) or huupbmcs (panel c) were stimulated with lps (500 ng/ml) either alone, or in the presence of herbal formulations (f1, f2, and f3), and levels of hmgb1 and nitric oxide in the culture medium were determined 16 h later by western blotting analysis or the griess reaction as previously described [7] . shown in the bar graphs (panels a, b, and c) are means ae sem. of three experiments in duplicates (n ¼ 6). student's t-test was performed and a p < 0:01 was considered significant (*). shown in the bottom of panel c is a representative western blot. cultures of both macrophages ( fig. 1(a) ), and hu-pbmcs ( fig. 1(c) ). an excessive immune response to acute coronavirus infection appears to be the fatal factor in patients who die of sars [18] [19] [20] [21] . autopsy of sars patients revealed signs of diffuse alveolar damage, airspace edema, and bronchiolar fibrin [19] [20] [21] . our hypothesis is that initial acute coronavirus infection of alveolar endothelial cells or macrophages leads to cell injury via virus-mediated cytolysis, which is accompanied by hmgb1 release from damaged cells (fig. 2) . extracellular hmgb1, as a mediator of acute lung injury [13] , subsequently mediates injurious pulmonary inflammatory responses including neutrophil infiltration, derangement of epithelial barrier, lung edema, and lung injury. collectively, these injurious pulmonary inflammatory responses may consequently lead to respiratory failure, and death. this hypothesis can be tested using anti-hmgb1 antibodies or hmgb1 inhibitors (such as ethyl pyruvate) in future studies. for instance, if immunoassays using anti-hmgb1 antibodies indicate a potential elevation of pulmonary hmgb1 levels in sars patients, it will support the above hypothesis. similarly, if hmgb1-specific antibodies or inhibitors attenuate the progression of sars in animal models or clinical settings, it will support an important role of hmgb1 in the pathogenesis of sars. figure 2 hypothetical role of hmgb1 in the pathogenesis of sars. hmgb1 is released from coronavirus infected cells, and then stimulates excessive inflammatory responses. is traditional chinese medicine useful in the treatment of sars? reversing established sepsis with antagonists of endogenous hmgb1 hmg-1 as a late mediator of endotoxin lethality in mice release of chromatin protein hmgb1 by necrotic cells triggers inflammation hmgb1 as a late mediator of lethal systemic inflammation proinflammatory cytokines (tumor necrosis factor and interleukin 1) stimulate release of high mobility group protein-1 by pituicytes ifn-gamma induces high mobility group box 1 protein release partly through a tnf-dependent mechanism high mobility group box chromosomal protein 1: a novel proinflammatory mediator in synovitis high mobility group box chromosomal protein 1 plays a role in the pathogenesis of rheumatoid arthritis as a novel cytokine high mobility group box chromosomal protein 1, a dna binding cytokine, induces arthritis the cytokine handbook extracellular role of hmgb1 in inflammation and sepsis hmg-1 as a mediator of acute lung inflammation ethyl pyruvate prevents lethality in mice with established lethal sepsis and systemic inflammation successful treatment of collagen-induced arthritis in mice and rats by targeting extracellular high mobility group box chromosomal protein 1 activity lipid unites dissipate syndromes of sepsis herbal supplement severe immune response kills sars victims lung pathology of severe acute respiratory syndrome (sars): a study of 8 autopsy cases from singapore the clinical pathology of severe acute respiratory syndrome (sars): a report from china pathological study on severe acute respiratory syndrome we thank drs. ping wang, man yu, and xiaoling qiang for critical reading of the manuscript. this research was supported in part by the north shore-lij research institute, and the national institutes of health (nih, nigms, gm063075). key: cord-254162-tu81j66h authors: bai, xiyuan; hippensteel, joseph; leavitt, alida; maloney, james p.; beckham, david; garcia, cindy; li, qing; freed, brian m.; ordway, diane; sandhaus, robert a.; chan, edward d. title: hypothesis: alpha-1-antitrypsin is a promising treatment option for covid-19 date: 2020-11-12 journal: med hypotheses doi: 10.1016/j.mehy.2020.110394 sha: doc_id: 254162 cord_uid: tu81j66h no definitive treatment for covid-19 exists although promising results have been reported with remdesivir and glucocorticoids. short of a truly effective preventive or curative vaccine against sars-cov-2, it is becoming increasingly clear that multiple pathophysiologic processes seen with covid-19 as well as sars-cov-2 itself should be targeted. because alpha-1-antitrypsin (aat) embraces a panoply of biologic activities that may antagonize several pathophysiologic mechanisms induced by sars-cov-2, we hypothesize that this naturally occurring molecule is a promising agent to ameliorate covid-19. we posit at least seven different mechanisms by which aat may alleviate covid-19. first, aat is a serine protease inhibitor (serpin) shown to inhibit tmprss-2, the host serine protease that cleaves the spike protein of sars-cov-2, a necessary preparatory step for the virus to bind its cell surface receptor ace2 to gain intracellular entry. second, aat has anti-viral activity against other rna viruses hiv and influenza as well as induces autophagy, a known host effector mechanism against mers-cov, a related coronavirus that causes the middle east respiratory syndrome. third, aat has potent anti-inflammatory properties, in part through inhibiting both nuclear factor-kappa b (nfκb) activation and adam17 (also known as tumor necrosis factor-alpha converting enzyme), and thus may dampen the hyper-inflammatory response of covid-19. fourth, aat inhibits neutrophil elastase, a serine protease that helps recruit potentially injurious neutrophils and implicated in acute lung injury. aat inhibition of adam17 also prevents shedding of ace2 and hence may preserve ace2 inhibition of bradykinin, reducing the ability of bradykinin to cause a capillary leak in covid-19. fifth, aat inhibits thrombin, and venous thromboembolism and in situ microthrombi and macrothrombi are increasingly implicated in covid-19. sixth, aat inhibition of elastase can antagonize the formation of neutrophil extracellular traps (nets), a complex extracellular structure comprised of neutrophil-derived dna, histones, and proteases, and implicated in the immunothrombosis of covid-19; indeed, aat has been shown to change the shape and adherence of non-covid-19-related nets. seventh, aat inhibition of endothelial cell apoptosis may limit the endothelial injury linked to severe covid-19-associated acute lung injury, multi-organ dysfunction, and pre-eclampsia-like syndrome seen in gravid women. furthermore, because both nets formation and the presence of anti-phospholipid antibodies are increased in both covid-19 and non-covid pre-eclampsia, it suggests a similar vascular pathogenesis in both disorders. as a final point, aat has an excellent safety profile when administered to patients with aat deficiency and is dosed intravenously once weekly but also comes in an inhaled preparation. thus, aat is an appealing drug candidate to treat covid-19 and should be studied. no definitive treatment for covid-19 exists although promising results have been reported with remdesivir and glucocorticoids. short of a truly effective preventive or curative vaccine against sars-cov-2, it is becoming increasingly clear that multiple pathophysiologic processes seen with covid-19 as well as sars-cov-2 itself should be targeted. because alpha-1-antitrypsin (aat) embraces a panoply of biologic activities that may antagonize several pathophysiologic mechanisms induced by sars-cov-2, we hypothesize that this naturally occurring molecule is a promising agent to ameliorate covid-19. we posit at least seven different mechanisms by which aat may alleviate covid-19. first, aat is a serine protease inhibitor (serpin) shown to inhibit tmprss-2, the host serine protease that cleaves the spike protein of sars-cov-2, a necessary preparatory step for the virus to bind its cell surface receptor ace2 to gain intracellular entry. second, aat has anti-viral activity against other rna viruses hiv and influenza as well as induces autophagy, a known host effector mechanism against mers-cov, a related coronavirus that causes the middle east respiratory syndrome. third, aat has potent anti-inflammatory properties, in part through inhibiting both nuclear factor-kappa b (nfb) activation and adam17 (also known as tumor necrosis factor-alpha converting enzyme), and thus may dampen the hyperinflammatory response of covid-19. fourth, aat inhibits neutrophil elastase, a serine protease that helps recruit potentially injurious neutrophils and implicated in acute lung injury. aat inhibition of adam17 also prevents shedding of ace2 and hence may preserve ace2 inhibition of bradykinin, reducing the ability of bradykinin to cause a capillary leak in covid-19. fifth, aat inhibits thrombin, and venous thromboembolism the number of covid-19 cases worldwide is approaching 30 million as of september 2020. one of the most serious manifestations of covid-19 is acute respiratory distress syndrome, especially in the elderly and those with cardiopulmonary disorders (1) . curiously, the onset of respiratory compromise often occurs > 7 days following known or suspected exposure or 5-7 days after symptom onset (2) . this delayed onset of respiratory compromise has been attributed to various pathophysiologic processes including diffuse alveolar damage, in situ microthrombi formation, venous thromboembolism, immunothrombosis, cardiac dysfunction, and hyper-inflammatory cytokine responses (3) (4) (5) (6) (7) . there is currently no definitive treatment for covid-19 (8) . no efficacy was seen with combined lopinavir and ritonavir (9) . despite initial optimism with hydroxychloroquine, a recent observational study found that it had no significant impact on the composite end point of endotracheal intubation or death in hospitalized covid-19 patients (10) . remdesivir initially showed a trend in reducing the time to clinical improvement (11) . a more recent, double-blind, placebo-controlled study showed that remdesivir significantly reduced the recovery time from covid-19 by approximately four days and there was a trend toward improved mortality (12) . glucocorticoid was initially not recommended by some during the early period of the covid-19 pandemic (13, 14) . a plausible rationale -which may still be true -is that a potent, initial pro-inflammatory response is necessary for viral clearance. however, in the more delayed severe cases, where an overzealous inflammatory response ("cytokine storm") may result in lung tissue damage, there is increasing evidence that glucocorticoids are therapeutic. thus, timing of administration and severity of disease are likely important factors in whether glucocorticoids are effective or not (15) . the large recovery trial showed that compared to placebo, daily intravenous or oral dexamethasone 6 mg -beginning ≥ 7 days into the symptomatic phase for up to 10 days of treatment -reduced death rate by one-third in ventilated patients and by 20% in patients who required supplemental oxygen only (16) . this benefit of delayed glucocorticoid administration coincides with the belated onset of respiratory insufficiency and lends credence to the notion that a delayed hyper-inflammatory response is implicated in the oxygenation failure. in contrast, the use of dexamethasone in milder covid-19 cases showed a trend toward increased mortality in the recovery trial (16) . in a meta-analysis of 7 randomized clinical trials of systemic glucocorticoid use in critically ill covid-19 patients, glucocorticoid was associated with a lower 28-day allcause mortality (17) . hydrocortisone for 7 days was also linked to reduced number of days requiring icu-based respiratory or cardiovascular support for those with severe covid-19 (18) . other, more targeted anti-inflammatory drugs are also being investigated as treatments for covid-19, including inhibitors / antagonists to janus kinase, interleukin-1 (il-1), il-6, il-6 receptor, and tumor necrosis factor-alpha (tnf) in the hope of further limiting the hyper-inflammatory response and resultant multi-organ damage (7) . despite initial optimism with the use of neutralizing agents against il-6 signaling (19) , the recent multicenter, randomized, double-blind, placebo-controlled covacta trial of hospitalized patients with severe covid-19 pneumonia found that tocilizumab (anti-il-6 receptor antibody) had no significant efficacy as analyzed by clinical status, mechanical ventilation use, or mortality (20) . until definitive anti-viral treatments are developed against sars-cov-2 and an effective prophylactic vaccine comes to fruition, the scientific community should continue to investigate existing drugs -with acceptable side effect profiles -that may target sars-cov-2 and the pathophysiologic mechanisms of covid-19. alpha-1-antitrypsin (aat) is a serine protease inhibitor (serpin) and the third most abundant protein in circulation. aat plasma level can increase 3-to 5-fold in states of systemic inflammation and / or infection, perhaps indicative of a homeostatic role of aat but which may be deficient or overwhelmed in severe cases of covid-19 (21, 22) . while the best described function of aat is that it irreversibly inhibits the serine protease elastase (22) , it has a panoply of biological activities that may be independent of its serpin activity. thus, because aat possesses several biological functions that may antagonize both sars-cov-2 infection and the array of pathophysiologic processes that have been ascribed to covid-19, we hypothesize that aat is a promising candidate for the successful treatment of covid-19. in the u.s., it is estimated that there are ~300,000 individuals with frank aat deficiency and the vast majority are undiagnosed. even in the absence of aat shortage, the aat response to a systemic infection may be inadequate in some individuals. an estimated 9% of individuals in the u.s. (>20 million) are carriers of a single aat gene mutation (with over 400 different mutations identified) with most having no ill effects but an uncertain number have inadequate aat response to infections or inflammation (21) . vianello and braccioni (23) showed that in italy there was geographic co-localization between those with aat deficiency and the number of covid-19 cases. furthermore, since oxidation of methionine 351 and/or 358 residues of normal aat may lead to loss of its serpin activity, the increased oxidative stress seen with covid-19 may render even normal or elevated levels of aat ineffective (24, 25) . our central hypothesis is that aat, a naturally occuring molecule which has been utilized at pharmacologic doses for decades, is a promising agent against covid-19. we describe below seven potential mechanisms by which aat could antagonize both sars-cov-2 and some of the known pathogenic processes of covid-19. the seven mechanisms discussed coincide with the numbers shown in figure 1 . aat augments host immunity against a wide variety of pathogens including influenza (26) , hiv (27) (28) (29) (30) (31) , pseudomonas aeruginosa (32) , and mycobacterium intracellulare (33) . aat antagonizes hiv by several mechanisms including interacting with gp41 to block hiv entry into cd4 + lymphocytes, inhibiting hiv replication through alteration of ib ubiquitination and inhibition of nuclear factor-kappa b (nfb) activation (a transcription factor that induces hiv replication), and inducing prostaglandin synthase-2, which inhibits hiv replication (27) (28) (29) (30) (31) . a mechanism by which aat reduces the burden of cell-associated m. intracellulare is through sequential inhibition of nfb, reduction in the expression of a20 (a deubiquitinating enzyme that inhibits autophagosome maturation by inhibiting traf6 from ubiquitinating a key autophagic protein beclin-1), and induction of autophagy (33) . autophagy has been implicated in controlling mers-cov, a related coronavirus that causes the middle east respiratory syndrome (mers) (34) . given the similarities between the highly pathogenic coronaviruses, we posit that aat augmentation of autophagy is likely important in the host immune response to sars-cov-2. the coronaviruses that may cause fatal disease -sars-cov, mers-cov, and sars-cov-2 -all utilize the host cell serine protease tmprss-2 to process the viral spike protein so that it may bind to the cell surface receptor ace2 (or dpp4 in the case of mers-cov) on host cells to gain intracellular entry. tmprss-2 may also process ace2 to facilitate entry of sars-cov (35) but whether this applies to sars-cov-2 is not known. the serpin camostat inhibits tmprss-2 and entry of sars-cov and sars-cov-2 into cells (36) (37) (38) . camostat was also shown to inhibit influenza replication and cytokine production in airway epithelial cells, likely due to inhibition of the host serine protease hepsin (39) . because aat is a potent serpin, it also has the potential to inhibit viral entry into (42) reported that sera from sars-cov-2 naïve individuals inhibited cellular entry of sars-cov-2 and identified aat as the molecule responsible. de loyola and co-workers (43) also showed that aat inhibits disintegrin/metalloproteinase 17 (adam17), a protease that can cause shedding of ace2 (which would decrease viral entry) (35) but may also process membranebound ace2 and enhance sars-cov entry (44) although this latter finding is controversial (35) . to the best of our knowledge, whether adam17 processing of ace2 to enhance sars-cov-2 cellular entry is not known. while much has been written of the injurious role the delayed hyper-inflammatory response may play in covid-19 and evinced by the numerous clinical trials being undertaken to counter inflammation, this concept is not completely established because a recent trial of neutralizing antibodies to the il-6 receptor showed no significant efficacy (20) . furthermore, genome-wide association studies have not implicated any targetable inflammatory pathways as linked to covid-19 risk (45) . but negative studies in which only one pro-inflammatory cytokine was targeted does not rule out the potential injurious role an array of cytokines may play as evinced by the benefit of delayed administration of glucocorticoid in severe covid-19. aat also has potent anti-inflammatory properties (46) (47) (48) . a mechanism by which aat attenuates inflammation is by inhibiting nfb activation, a prototypical proinflammatory transcription factor, through binding of ib and/or altering ib ubiquitination (31, 47, 49) . aat also binds extracellular il-8, preventing the chemokine from binding to its receptor cxcr1 and activating akt signaling pathway (46) . because akt signaling inhibits the early stages of autophagy, perhaps this binding of aat to il-8 attenuates akt activation, thereby inducing autophagy (50) . in addition, since neutrophilia is associated with worse outcome in covid-19 (51) , the ability of aat to sequester il-8, a chemokine for neutrophils, may limit both neutrophil influx and acute lung injury. covid-19 is also associated with increased oxidative stress (25) and aat has been shown to inhibit neutrophil superoxide production (52) . another anti-inflammatory mechanism of aat is inhibition of adam17 (43) . also known as tnf-converting enzyme, adam17 is a cell surface metalloprotease that is activated by the spike protein of coronaviruses and cleaves membrane-bound tnf to soluble tnf. but as previously mentioned, adam17 also causes shedding of ace2 (figure 1 ) (35) . in the blood compartment or within tissues, shedding of ace2 by adam17 would increase inflammation since ace2 normally converts angiotensin i and pro-inflammatory angiotensin ii (53) to "resolution peptides" -angiotensin-(1-7) and angiotensin-(1-9) -that are anti-inflammatory, anti-fibrotic, and vasodilatory (figure 2a ) (54) . while these biochemical functions of ace2 may also occur in the airway lumen, the specific ligand(s) other than sars-cov or sars-cov-2 that bind ace2 in the airways is not known (figure 2b) . neverthless, normal ace2 expression has been shown to protect the lungs from injury -by reducing both bradykinin production and neutrophil infiltration (55) as well as catalyzing angiotensin ii to the anti-inflammatory angiotensin-(1-7) and angiotensin-(1-9); both these two metabolic products also protect lung epithelial cells from death (56) . consequently, adam17-induced shedding of ace2 may cause excessive inflammation, resulting in lung injury whereas aat would dampen these host-deleterious responses (35) . to summarize these complex interactions, ace2, while being a receptor for sars-cov-2, also has anti-inflammatory properties ( figure 2c) . adam17, by inducing ace2 shedding, would decrease the cell surface receptor for sars-cov-2 but also negate ace2 inhibition of overzealous inflammation, acute lung injury, and lung edema ( figure 2c) . aat inhibition of adam17 would reduce inflammation by decreasing both soluble tnf formation and ace2 shedding. pharmacologic inhibition of the renin-angiotensin-aldosterone (raa) axis (e.g., ace inhibitors) appear to have a neutral effect on the course of covid-19. this neutrality may be due to off-setting effects in that raa inhibition induces ace2 cell surface expression (potentially increasing viral entry) but decreases angiotensin-ii (which would decrease overzealous inflammation) (57) . as shown in figure 2c community-acquired pneumonia that there is a blunted aat acute-phase response in the critically-ill covid-19 patients but not in non-covid-19 patients requiring intensive care. in addition, they found that an increased il-6:aat ratio predicted prolonged icu stay and mortality, while relative reduction in il-6:aat ratio was associated with clinical resolution (58) . thus, their findings support our hypothesis that an inadequate aat response may be responsible for the hyper-inflammatory response associated with covid-19 and predict a worse outcome. neutrophil elastase at sites of acute inflammation is known to mediate acute lung injury (59) . elastase contributes to excessive inflammation by inducing the release of il-8 from neutrophil vesicles and facilitating conversion of pro-il-1 to il-1 (60). aat is a potent and irreversible inhibitor of elastase. in a rat model of lipopolysaccharide / ventilator-induced lung injury, pre-treatment with aat resulted in improved outcome, as evinced by increased pao 2 /fio 2 ratio and decreased wet/dry lung weight ratio as well as decreased protein levels, pro-inflammatory cytokines, and cell count in the bronchoalveolar lavage fluid (61) . recently, mehraban and colleagues (62) showed that broken down elastic fibers as a result of elastase plus another insult such as lipopolysacchardide was pro-inflammatory. however, there have been mixed results with the use of neutrophil elastase inhibitors in various forms of non-covid-19-related acute lung injury (63) . this may be due, in part, to the fact that neutrophil elastase may not only incite excessive inflammation but is also required for optimal intracellular killing of gram-negative bacteria (64) . acute lung injury is also manifested by non-cardiogenic pulmonary edema due to leakage of exudate through the alveolar-capillary membrane (figure 2) . bradykinin may play a role in this leakage (55, 65) . since ace2 inactivates bradykinin by cleaving a single amino acid from its carboxyl terminus, ace2 can protect against this mechanism of acute lung injury. given that adam17 causes shedding of ace2 (35), adam17 would theoretically exacerbate the capillary leakage by attenuating ace2 inhibition of bradykinin. but since aat inhibits adam17, aat would mitigate the pulmonary edema caused by the virus-induced bradykinin pathway (figure 2c ) (55) . while aat inhibition of adam17 would inhibit adam17 shedding of ace2 and in theory enhance viral binding, aat was found to inhibit sars-cov-2 viral entry into cells through inhibition of tmprss-2 (41, 42). both venous thromboembolism as well as in situ micro-and macro-thrombi are increasingly recognized with covid-19 and likely contribute to the hypoxemia seen with the acute lung injury (6, 66, 67) . aat has been shown to antagonize thrombin, a serine protease (68) . because most of the enzymes in the coagulation cascade are also serine proteases, aat has the potential to inhibit other pro-coagulant proteins in addition to thrombin. thus, while aat itself is unlikely to be thrombolytic in those with established thrombi, it may help retard thrombus formation (42). increased absolute neutrophil number, percentage of neutrophils, and neutrophil:lymphocyte ratio in the blood of covid-19 patients are predictive of progression to severe disease (51) . while this association may simply be a non-specific reflection of increased severity, there is increasing evidence that aberrant formation of neutrophil extracellular traps (nets) -essentially comprised of neutrophil-derived decondensed chromatin (cell-free dna) and proteins such as elastase, cathepsin g, and histones to trap and kill extracellular pathogens -play a pathogenic role in the immunothrombosis, mucous secretions, and cytokine production seen with covid-19 based on autopsy results, ex vivo nets formation studies, and blood biomarkers for nets (cell-free dna, myeloperoxidase dna, and citrullinated histone h3) (5, (69) (70) (71) (72) (73) . since elastase plays a key role in nets formation by degrading specific histones and promoting chromatin decondensation (74) , aat has the potential to inhibit nets formation and reduce the excessive inflammation and immunothrombosis seen with covid-19. indeed, frenzel and co-workers (75) showed that while aat did not decrease the formation of phorbol myristate acetate-induced nets, it changed the shape and adherence of the nets in ex vivo experiments using blood neutrophils. sars-cov-2 infects endothelial cells and covid-19 lungs demonstrate endothelial cell injury, microthrombi and angiogenesis (66) . this endothelial injury seen with covid-19 may be part of a spectrum of pulmonary pathology observed that includes acute lung injury, microthrombi formation, and extra-pulmonary multi-organ dysfunction associated with the most severe cases. another disorder increasingly recognized in gravid patients with severe covid-19 is pre-eclampsia or pre-eclampsia-like syndrome (76, 77) . one obvious common denominator for both pre-eclampsia and severe covid-19 is endothelial injury / pathology as a hallmark of pre-eclampsia is disrupted placentation which leads to endothelial dysfunction and end-organ damage (78) . an indirect support for a similar underlying pathophysiology of pre-eclampsia and severe covid-19 is that both disorders may manifest with non-cardiogenic pulmonary edema, venous thromboembolism, and/or multi-organ dysfunction. two other specific pathogenic elements that are increased in both covid-19 and non-covid-19 pre-eclampsia is further evidence that the two disorders are linked pathogenically. one is that nets -as previously mentioned with mounting evidence of a pathologic role in covid-19 -have also been implicated in the pathogenesis of non-covid-19-related severe preeclampsia (79) . another is that the presence of anti-phospholipid antibodies (apla) is a major risk factor for pre-eclampsia (80) and one study found that 52% of covid-19 patients had elevated apla levels (81) . this very high prevalence of pro-thrombotic apla in covid-19 is most likely a consequence of its induction by sars-cov-2 rather than being a pre-existing state. aat inhibits endothelial cell apoptosis and thus may antagonize the endothelial injury seen with covid-19 (82, 83) . more specifically, murine models revealed that in endothelial cells, aat inhibits caspase-3, an executioner caspase in the classical apoptotic pathway (83) . in addition, aat treatment of endothelial cells decreased oxidative stress, inflammation, and cell wall deterioration (83) . interestingly, low levels of plasma aat have been associated with severe non-covid-19-related pre-eclampsia (84) . furthermore, aat suppresses oxidative stress in both murine and molecular models of non-covid-19 pre-eclampsia (85, 86) . thus, aat may also be a promising agent against the pre-eclampsia-like syndrome seen in pregnant women with severe covid-19 and should be studied. while large prospective studies have not evaluated aat therapy in pregnancy, there are case reports of safe use in pregnancy with normal neonatal outcomes (87) . before administering aat -as part of a study or as augmentation therapy in those with aat deficiency -screening for iga deficiency should be done since those with iga deficiency are more likely to develop hypersensitivity reactions due to the potential presence of antibodies directed against iga. in summary, given its anti-viral, serpin (anti-tmprss-2 and anti-elastase), antiinflammatory, anti-thrombin, anti-nets, and anti-apoptotic activities, aat is a promising therapeutic for covid-19. it is also important to note that aat is routinely prescribed to those with aat deficiency, has an excellent safety profile, and normal plasma aat levels may be achieved with once weekly intravenous administration. moreover, an inhaled aat formulation is available although its efficacy remains to be fully determined. it is important to also be cognizant that even if a truly effective prophylactic vaccine is developed against sars-cov-2, there will continue to be barriers, including the challenge of administering one or more doses of the vaccine to each person in the world, vaccine efficacy likely will not be 100%, vaccine refusal by a significant number of individuals, and the looming specter of mutations of sars-cov-2, rendering the vaccine less effective, vis-à-vis what is seen with influenza. if mounting evidence shows that aat does have significant activity against sars-cov-2 infection, it can be studied by randomized, placebo-controlled trials in which is aat administered by different means depending on the severity of the sars-cov-2 infection (figure 3) . for example, aat administered intravenously (iv) along with an anti-oxidant to protect aat from oxidation in critically-ill covid-19 subjects (figure 3a) , by nebulization in noncritically-ill patients with covid-19 pneumonia (figure 3b we hypothesize that aat is a promising therapeutic against covid-19 via at least seven mechanisms (see accompanying text for full description). in brief, we posit that aat will: (1) augment host immunity against sars-cov-2 by enhancing autophagy, (2) inhibit tmprss-2 activity, mitigating a key and necessary step prior to sars-cov-2 entry into cells, (3) antagonize inflammation, (4) inhibit neutrophil elastase and ameliorate acute lung injury, (5) inhibit thrombin, retarding microthrombi formation, (6) inhibit neutrophil extracellular traps (nets) adherence, limiting immunothrombosis seen with covid-19, and (7) protect against endothelial cell apoptosis, curbing covid-19-associated endothelial injury. whereas tmprss-2 may also process ace2 to facilitate binding and entry of sars-cov, it is not known whether such activity also enhances sars-cov-2 binding to ace2; this uncertainly is denoted by the question mark. ace2 = receptor for sars-cov-2; tmprss-2 = serine protease necessary to "activate" sars-cov-2; t-shaped "arrows" = inhibition; red heptagon = sars-cov-2. angiotensin-(1-9), with the latter metabolites also known as pro-resolution peptides because, unlike a-ii, they have anti-inflammatory, anti-fibrotic, and vasodilatory properties. (b) in the nasal and lung epithelium, ace2 is the receptor for sars-cov-2 after the viral spike protein is processed by the serine protease tmprss-2. however, ace2 is also anti-inflammatory and protects against various forms of acute lung injury through metabolism of pro-inflammatory a-ii to anti-inflammatory angiotensin-(1-7) and angiotensin-(1-9), inhibition of bradykinin production, and preservation of cell viability but the precise ligand in the airways that ace2 catalyzes is not known. adam17 is proinflammatory in that it converts membrane tnf to soluble tnf as well as causes shedding of ace2, reducing the latter's anti-inflammatory effects. thus, (c) the two faces of ace2 are that it is the receptor for sars-cov-2 and yet is anti-inflammatory and protects against lung injury. adam17 causes ace2 shedding, reducing cell surface expression of the sars-cov-2 receptor but also induces a pro-inflammatory state. inhibition of the renin-angiotensin-aldosterone (raa) axis is also known to induce clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study dexamethasone in the management of covid-19: preliminary trial results are mostly good news, but timing is everything china medical treatment expert group for covid-19. clinical characteristics of coronavirus disease 2019 in china cardiovascular complications in covid-19 neutrophil extracellular traps (nets) contribute to immunothrombosis in covid-19 acute respiratory distress syndrome online ahead of print tissue plasminogen activator (tpa) treatment for covid-19 associated acute respiratory distress syndrome (ards): a case series the cytokine release syndrome (crs) of severe covid-19 and interleukin-6 receptor (il-6r) antagonist tocilizumab may be the key to reduce the mortality covid-19) treatment guidelines a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 observational study of hydroxychloroquine in hospitalized patients with covid-19 remdesivir in adults with severe covid-19: a randomised, double-blind, placebo-controlled, multicentre trial actt-1 study group members. remdesivir for the treatment of covid-19 -preliminary report clinical evidence does not support corticosteroid treatment for 2019-ncov lung injury risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease the role of cytokines including interleukin-6 in covid-19 induced pneumonia and macrophage activation syndrome-like disease effect of dexamethasone in hospitalized patients with covid-19 -preliminary report who rapid evidence appraisal for covid-19 therapies (react) working group. association between administration of systemic corticosteroids and mortality among critically ill patients with covid-19: a metaanalysis writing committee for the remap-cap investigators. effect of hydrocortisone on mortality and organ support in patients with severe covid-19: the remap-cap covid-19 corticosteroid domain randomized clinical trial tocilizumab for treatment of mechanically ventilated patients with covid-19 tocilizumab for severe covid-19 pneumonia genetic epidemiology of alpha-1 antitrypsin deficiency in north america and australia/new zealand: australia, canada, new zealand and the united states of america alpha-1-antitrypsin deficiency: genetic variations, clinical manifestations and therapeutic interventions geographical overlap between alpha-1 antitrypsin deficiency and covid-19 infection in italy: casual or causal? oxidation of either methionine 351 or methionine 358 in alpha 1-antitrypsin causes loss of anti-neutrophil elastase activity an anti-oxidative therapy for ameliorating cardiac injuries of critically ill covid-19-infected patients transcriptome profiling and protease inhibition experiments identify proteases that activate h3n2 influenza a and influenza b viruses in murine airway alpha-1-antitrypsin interacts with gp41 to block hiv-1 entry into cd4+ t lymphocytes hiv infection is associated with reduced serum alpha-1-antitrypsin concentrations hiv replication in cd4+ t lymphocytes in the presence and absence of follicular dendritic cells: inhibition of replication mediated by α-1-antitrypsin through altered iκbα ubiquitination serpin induced antiviral activity of prostaglandin synthetase-2 against hiv-1 replication alpha-1-antitrypsin inhibits human immunodeficiency virus type 1 alpha-1 antitrypsin reduces severity of pseudomonas pneumonia in mice and inhibits epithelial barrier disruption and pseudomonas invasion of respiratory epithelial cells alpha-1-antitrypsin enhances primary human macrophage immunity against non-tuberculous mycobacteria skp2 attenuates autophagy through beclin1-ubiquitination and its inhibition reduces mers-coronavirus infection tmprss2 and adam17 cleave ace2 differentially and only proteolysis by tmprss2 augments entry driven by the severe acute respiratory syndrome coronavirus spike protein sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor simultaneous treatment of human bronchial epithelial cells with serine and cysteine protease inhibitors prevents severe acute respiratory syndrome coronavirus entry enhanced isolation of sars-cov-2 by tmprss2-expressing cells the serine protease inhibitor camostat inhibits influenza virus replication and cytokine production in primary cultures of human tracheal epithelial cells alpha 1 antitrypsin is an inhibitor of the sars-cov2-priming protease tmprss2 alpha-1 antitrypsin inhibits sars-cov-2 infection in plain sight: the role of alpha-1-antitrypsin in covid-19 pathogenesis and therapeutics alpha-1-antitrypsin: a possible host protective factor against covid-19 tace antagonists blocking ace2 shedding caused by the spike protein of sars-cov are candidate antiviral compounds severe covid-19 gwas group. genomewide association study of severe covid-19 with respiratory failure online ahead of print α-1 antitrypsin regulates human neutrophil chemotaxis induced by soluble immune complexes and il-8 alpha-1-antitrypsin inhibits nitric oxide production alpha-1-antitrypsin is an endogenous inhibitor of pro-inflammatory cytokine production in whole blood immune-modulating effects of alpha-1 antitrypsin akt-mediated regulation of nfkb and the essentialness of nfkb for the oncogenicity of pi3k and akt early warning indicators of severe covid-19: a single-center study of cases from shanghai inhibition of neutrophil superoxide production by human plasma alpha 1-antitrypsin angiotensin ii and inflammation: the effect of angiotensin-converting enzyme inhibition and angiotensin ii receptor blockade devil and agel in the renin-angiotensin system: ace-angiotensin ii-at1 receptor axis vs. ace2-angiotensin-(1-7)-mas receptor axis attenuation of pulmonary ace2 activity impairs inactivation of des-arg(9) bradykinin/bkb1r axis and facilitates lpsinduced neutrophil infiltration ace2, much more than just a receptor for sars-cov-2 cardiovascular considerations for patients, health care workers, and health systems during the covid-19 pandemic characterization of the inflammatory response to severe covid-19 illness neutrophil elastase contributes to acute lung injury induced by bilateral nephrectomy neutrophil elastase promotes interleukin-1β secretion from human coronary endothelium alpha 1-antitrypsin for treating ventilator-associated lung injury in acute respiratory distress syndrome rats the proinflammatory activity of structurally altered elastic fibers the role of neutrophil elastase inhibitors in lung diseases mice lacking neutrophil elastase reveal impaired host defense against gram negative bacterial sepsis a mechanistic model and therapeutic interventions for covid-19 involving a ras-mediated bradykinin storm pulmonary vascular endothelialitis, thrombosis, and angiogenesis in covid-19 thrombosis and coagulopathy in covid-19: an illustrated review α1-antitrypsin, an inhibitor for thrombin and plasmin do neutrophil extracellular traps contribute to the heightened risk of thrombosis in inflammatory diseases? neutrophil extracellular traps in covid-19 neutrophil extracellular traps (nets) as markers of disease severity in covid-19 neutrophil extracellular traps and thrombosis in covid-19 targeting potential drivers of covid-19: neutrophil extracellular traps neutrophil elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps does augmentation with alpha1-antitrypsin affect neutrophil extracellular traps formation? a snapshot of the covid-19 pandemic among pregnant women in france pre-eclampsia-like syndrome induced by severe covid-19: a prospective observational study pre-eclampsia: pathophysiology and clinical implications increased neutrophil activation and plasma dna levels in patients with pre-eclampsia prothrombotic antiphospholipid antibodies in covid-19 a novel antiapoptotic role for alpha1-antitrypsin in the prevention of pulmonary emphysema alpha-1 antitrypsin inhibits caspase-3 activity, preventing lung endothelial cell apoptosis lower circulation levels and activity of α-1 antitrypsin in pregnant women with severe preeclampsia alpha-1-antitrypsin functions as a protective factor in preeclampsia through activating smad2 and inhibitor of dna binding 4 alpha-1-antitrypsin suppresses oxidative stress in preeclampsia by inhibiting the p38mapk signaling pathway: an in vivo and in vitro study alpha-1 antitrypsin deficiency and pregnancy aat administered by nebulization in non-critically-ill patients with covid-19 pneumonia, or (c) aat administered prophylactically by a nasal spray (yet-to-be-developed) in asymptomatic persons with multiple risk factors for severe covid-19 aat = alpha-1-antitrypsin this work is dedicated to the patients who have suffered from or lost their lives to severe covid-19 as well as to all the health-care workers who have risk their lives in taking care of them. dr. robert a. sandhaus is the medical director of alphanet. key: cord-282899-kp114q7n authors: biswas, saurav; thakur, vikram; kaur, parneet; khan, azhar; kulshrestha, saurabh; kumar, pradeep title: blood clots in covid-19 patients: simplifying the curious mystery date: 2020-11-06 journal: med hypotheses doi: 10.1016/j.mehy.2020.110371 sha: doc_id: 282899 cord_uid: kp114q7n the universal phenomenon of blood clotting is well known to be protective in external cellular/ tissue injury. however, the emergence of unusual thrombotic presentations in covid-19 patients is the real concern. interaction of the spike glycoprotein with ace2 receptor present in the host cell surface mediates the entry of sars-cov-2 causing covid-19 infection. new clinical findings of sars-cov-2 pathogenesis are coming out every day, and one such mystery is the formation of mysterious blood clots in the various tissues and organs of covid-19 patients, which needs critical attention. to address this issue, we hypothesis that, high ace2 expression in the endothelium of blood vessels facilitates the high-affinity binding of sars-cov-2 using spike protein, causing infection and internal injury inside the vascular wall of blood vessels. this viral associated injury may directly/indirectly initiate activation of coagulation and clotting cascades forming internal blood clots. however, the presence of these clots is undesirable as they are responsible for thrombosis and need to be treated with anti-thrombotic intervention. , since its emergence from wuhan province, china in december 2019, now spreading to 213 countries worldwide, forcing the world health organization to declare this outbreak a global pandemic on 11 march 2020 (cucinotta and vanelli, 2020) . covid-19 is caused by highly infectious, severe acute respiratory syndrome coronavirus (sars-cov-2), infecting more than 36 million individuals, with 1,060,563 reported deaths as of 8 october 2020 (worldometer, 2020) . the understanding of this novel virus and disease evolves sequentially in the past seven months. initially, thought to transmit by droplets or aerosols causing fever as classical clinical symptoms, mainly in old age or immunocompromised individuals. however, the dynamics of covid-19 keeps on evolving with the emergence of different sars-cov-2 strains. evidence of airborne mode of sars-cov-2 transmission (klompas, baker and rhee, 2020), asymptomatic clinical presentations along with extra-pulmonary manifestations (gupta et al., 2020) are the real concern. sars-cov-2 is a single-stranded, positive-sense rna virus having envelope, glycoprotein, and spike protein. being a respiratory virus, sars-cov-2 enters inside the human body and infect the lungs as a primary and predominant organ (jain, 2020) . the entry is mediated by the binding of the receptor-binding domain (rbd) of the s1 subunit of the viral spike protein with the host angiotensin-converting enzyme 2, (ace2) receptor primarily expressed in the type ii pneumocytes, serving as a viral reservoir (hoffmann et al., 2020) (lin et al., 2020) . usually, covid-19 presented with fever, sore throat, dry cough, and shortness of breath as common clinical manifestations (kakodkar, kaka and baig, 2020), however asymptomatic cases are also being reported which are more critical to diagnose. ace2 is also found to be expressed in the oral, nasal mucosa, epithelial cells of lungs, kidney, and heart, enterocytes of the small intestine, and in the endothelial cells of blood vessels (hamming et al., 2004) . the sars-cov-2 associated extra-pulmonary manifestations are encephalitis, rashes on the skin, meningitis, conjunctivitis, acute hepatic, and renal injury. surprisingly, autopsies of covid-19 patients have revealed clots in the small vessels of the lungs, heart, liver, and kidney which are responsible for strokes and heart attacks (tang et al., 2020) . more than 33% of critical covid-19 patients' are reported with critically high levels of blood clotting or elevated levels of d-dimer (levi et al., 2020) . the development of these mysterious clots causing coagulation abnormalities and thrombosis is the real concern and needs to be addressed. so, we hypothesis the possible mechanism for the formation of the vascular blood clots in covid-19 patients. the hypothesis for the formation of blood clots in the vessels is retrieved from the classical concept of blood clotting. the mechanism of blood clotting is like a two-edged sword, wherein the case of severe external injury, clotting is very crucial for preventing the blood loss, whereas, in the case of internal blood vessels injury, it, unfortunately, leads to the formation of blood clots, causing vascular blockades and thrombosis, expanding to every organ leading to severe and fatal outcomes. the above concept is substantiated by the following three facts: a.) recognition of ace2 receptor-expressing endothelial cells of the blood vessels by spike protein of sras-cov-2. b.) direct injury to the endothelial cell by sars-cov-2 infection resulting in vascular damage. c.) indirect damage due to immune-mediated cytokine release syndrome (crs) causing inflammation and cell death. to protect the direct and/or indirect damage caused by sars-cov-2 to endothelial cells lining the blood vessels, host innate defensive mechanism of blood clotting activates, recruiting the activated platelets at the injury site to form a clot which although reduce the damage but negatively may lead to obstruction of blood vessels and vasoconstriction causing more damage. the virus enters into the peripheral blood causing viremia and gets transmitted to the different organs such as the heart, kidney, gastrointestinal tract resulting in multiple organ failure. as it is evident that blood clots were observed in both live and dead covid-19 patients, so it is out most interesting to decode the mystery. considering the above facts and recent unusual reports, a hypothesis develops for the blood clots formation in the covid-19 patients (figure 1) , states that "due to an internal injury in the endothelium of blood vessels, either directly by sars-cov-2 infection (coexpression and binding of the spike protein with the ace2) or my virus-mediated inflammatory immune response, may result in vasoconstriction and the activation of coagulation and blood clotting pathways, resulting in the formation of blood clots". during covid-19 infection, sars-cov-2 enters into the systemic circulation and binds with the ace2 expressing endothelial cells (endothelium) lining the blood vessels. binding facilitates the virus internalization, causing infection and injury in the vascular wall of blood vessels. the vascular injury might result in vasoconstriction, which may further reduce the blood flow at the site of injury. also, injury to endothelial cells reduces the expression of fibrinolytic heparin and thrombomodulin. conversely, high expression and secretion of the von willebrand factor enhance the aggregation of platelets at the site of injury in blood vessels and activate the coagulation cascade for the formation of blood clots. immediately within a few seconds of endothelial cells injury in a blood vessel, platelets along with collagen, recruit at the injury site to swell and aggregate. soon the coagulation is initiated and fibrin strands begin to intersperse among the wound to form a complete platelet plug. viruses like dengue and herpes simplex virus (hsv) are known to infect endothelial and circulating blood cells, activating coagulation cascade by promoting tissue factor (antoniak and mackman, 2014) . interestingly, lysis of fibrin known as fibrinolysis by fibrinolytic molecule plasminogen and plasmin proved to induce deleterious inflammation and defective fibrin clot formation in influenza a viruses (iav) (berri et al., 2013) . in addition to viral stimulation, binding of an extracellular proteolytic enzyme known as urokinase-type plasminogen activator (upa) with its receptor, upar is crucial for the activation of plasminogen to plasmin as reported in various malignancies (mahmood, mihalcioiu and rabbani, 2018) . similarly, the sars-cov-2 induced vascular trauma/injury, activates the platelets exposed endothelium or collagen following the intrinsic pathway. following the injury to blood vessels, near-by platelets stick to the vascular proteins, get degranulated, releasing prothrombin activator, serotonin, adenosine diphosphate (adp), and thromboxane a2 for further activation of platelets. the mechanism for the formation of clots in the blood vessels may start sequentially by 12 clotting factors, i.e. activating the factor xii, converting prothrombin to thrombin and later fibrinogen to long and insoluble fibrin which may entangle with platelets by covalent cross-linking, forming a stable interlocking fibrous network of a fibrin clot at the site of injury. this may slow down the blood flow, but platelets in the clot begin to shrink to initiate the wound healing process with intrinsic and extrinsic coagulation pathways. however, the upregulation of the expression of fibrinogen subunits fga, fgb, and fgg also plays a very significant role in the formation of the clot. various thrombotic complications and coagulation abnormalities are associated with covid-19 like sepsis-induced coagulopathy (sic), disseminated intravascular coagulation (dic), venous thromboembolism (vte), pulmonary embolism (pe), micro-thrombosis, microvascular thrombosis, and thrombotic micro-angiopathy. intense endothelial inflammation may be the involved mechanism leading to microvascular thrombosis as suggested in lung pathology contributing to ards (wang et al., 2020) . sic is hypercoagulability, characterized by elevated fibrinogen and d-dimer levels causing endothelial dysfunction, micro-thrombosis, and stroke (iba et al., 2019) . additionally, vasoconstriction has an inverse relation with ace2. during systemic infection, the ace2 expressed blood vessels' endothelial cells, might attach to the viral spike protein resulting in the unavailability of ace2. renin-angiotensin-aldosterone system (raas) may drive the formation of micro-thrombin in covid-19 patients stimulating angiotensin ii (ag ii) to induce tissue factor (tf) and plasminogen activator inhibitor-1 (pai-1) expression by endothelial cells. ag ii acts on the cns to increase vasopressin production which causes vasoconstriction. depletion of ace2 by sars-cov-2, pai-1/tpa imbalance, and a hypercoagulable state may favor tissue injury and stroke (hess, eldahshan and rutkowski, 2020) . this may explain the unresolved mystery of unresolved fibrin deposits in the alveoli of patients with general ards. (vaughan, lazos and tong, 1995) . ace2 converts ag ii to ag (1-7), protecting endothelial cell function and prevent early atherosclerosis which can be caused by a clot in the blood vessels (zhang et al., 2015) . profound hypoxemia in the pulmonary capillaries may result in the activation of hypoxiainducible factors (hifs) resulting in inducing or inhibition of tf and pai-1 respectively, and vasoconstriction thereby promoting vascular occlusion (yan et al., 1999 ; gupta, zhao and evans, 2019) (grimmer and kuebler, 2017) . the deposition of the c56-9 component of the complement system in the damaged vessel results in micro thrombosis as an anti-phospholipid syndrome (magro et al., 2020) . neutrophil extracellular traps (net) are associated with elevated levels of histones, activating increased thrombin production (barnes et al., 2020) . in a study including 362 closed covid-19 cases from italy, high mortality attributed to pulmonary embolism (pe) and pulmonary thrombosis with vte and ischemic stroke as primary and dic as a secondary outcome. thromboembolic events occurred in 28 (7.7%) cases, with vte in 16 (36%), pe in 10 (33%) and, dic in 8 (2.2%) patients (lodigiani et al., 2020) . high-risk coagulation abnormalities are associated with an elevated concentration of d-dimer and a reduction in platelet count (levi et al., 2020) . thrombotic microangiopathy in the lungs autopsies showing aggregates of cd4 around thrombosed small vessels and are significantly associated with hemorrhage. (fox et al., 2020) . vte may develop via immunologic activation of intravascular and platelet-releases thrombin. severe derangement of hemostasis, accompanied by reduced platelet count and fibrinogen, may explain the events of venous thromboembolism supporting hypercoagulability in covid-19 positive patients (panigada et al., 2020) . incidence of vt increase between 25% and 49% in severe covid-19 patients, with pulmonary embolism being the most common thrombotic complication (cui et al., 2020) (klok et al., 2020) . microvascular thrombotic complications are indicative of a strong interaction between the sars-cov-2 and coagulation. more than 80% of lung autopsies reported platelet-fibrin thrombi, especially in the small pulmonary vasculature (carsana et al., 2020) . the presence of antiphospholipid antibodies is a serious complication causing thrombotic stroke in young patients (zhang et al., 2020) . so, in covid-19 patients, the sars-cov-2 mediated endothelial inflammation, thrombin generation, platelet, and leukocyte recruitment, complement activation, and the initiation of innate and adaptive immune responses, forming clots, culminate in immunothrombosis, ultimately resulting in thrombotic complications, stroke, and finally death. based on the literature and clinical observation of mysterious clots reported in the covid-19 patients, expression of ace2 in the endothelium of blood vessels, blood clotting pathways, interaction of the sars-cov-2 spike protein with host ace2, the pathogenesis of sars-cov-2, and the role of ace2 in ras, we can hypothesize and end with the conclusion that the mysterious clots reported in the covid-19 patients may be due to the binding of the spike protein of sars-cov-2 with the ace2 receptor expressed in the endothelial cells of blood vessels which may cause, vasoconstriction and activation of the intrinsic pathway of coagulation and eventually results in the formation of blood clots. multiple roles of the coagulation protease cascade during virus infection targeting potential drivers of covid-19: neutrophil extracellular traps plasminogen controls inflammation and pathogenesis of influenza virus infections via fibrinolysis', plos pathogens pulmonary post-mortem findings in a large series of covid-19 cases from northern italy who declares covid-19 a pandemic prevalence of venous thromboembolism in patients with severe novel coronavirus pneumonia pulmonary and cardiac pathology in covid-19: the first autopsy series from new orleans', medrxiv the endothelium in hypoxic pulmonary vasoconstriction extrapulmonary manifestations of covid-19', nature medicine the stimulation of thrombosis by hypoxia tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis covid-19-related stroke sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor diagnosis and management of sepsis-induced coagulopathy and disseminated intravascular coagulation covid-19 and lung pathology a comprehensive literature review on the clinical presentation, and management of the pandemic coronavirus disease 2019 (covid-19) confirmation of the high cumulative incidence of thrombotic complications in critically ill icu patients with covid-19: an updated analysis', thrombosis research airborne transmission of sars-cov-2: theoretical considerations and available evidence coagulation abnormalities and thrombosis in patients with covid-19', the lancet haematology hypothesis for potential pathogenesis of sars-cov-2 infection-a review of immune changes in patients with viral pneumonia', emerging microbes and infections venous and arterial thromboembolic complications in covid-19 patients admitted to an academic hospital in complement associated microvascular injury and thrombosis in the pathogenesis of severe covid-19 infection: a report of five cases', translational research multifaceted role of the urokinase-type plasminogen activator (upa) and its receptor (upar): diagnostic, prognostic, and therapeutic applications', frontiers in oncology hypercoagulability of covid-19 patients in intensive care unit: a report of thromboelastography findings and other parameters of hemostasis abnormal coagulation parameters are associated with poor prognosis in patients with novel coronavirus pneumonia angiotensin ii regulates the expression of plasminogen activator inhibitor-1 in cultured endothelial cells: a potential link between the renin-angiotensin system and thrombosis tissue plasminogen activator (tpa) treatment for covid-19 associated acute respiratory distress syndrome (ards): a case series coronavirus cases hypoxia/hypoxemia-induced activation of the procoagulant pathways and the pathogenesis of ischemia-associated thrombosis', arteriosclerosis, thrombosis, and vascular biology coagulopathy and antiphospholipid antibodies in patients with covid-19 ace2 and ang-(1-7) protect endothelial cell function and prevent early atherosclerosis by inhibiting inflammatory response', inflammation research special thanks to prof. prem kumar khosla, vice-chancellor, shoolini university, solan for providing financial support and necessary facilities. key: cord-291037-19csqq89 authors: janssen, rob; walk, jona title: vitamin k epoxide reductase complex subunit 1 (vkorc1) gene polymorphism as determinant of differences in covid-19-related disease severity date: 2020-08-25 journal: med hypotheses doi: 10.1016/j.mehy.2020.110218 sha: doc_id: 291037 cord_uid: 19csqq89 covid-19, caused by sars-cov-2, has major world-wide health-related and socio-economic consequences. there are large disparities in the burden of covid-19 with an apparent lower risk of poor outcomes in east asians compared to populations in the west. a recent study suggested that covid-19 leads to a severe extrahepatic vitamin k insufficiency, which could lead to impaired activation of extrahepatic proteins like endothelial anticoagulant protein s in the presence of normal hepatic procoagulant activity. this would be compatible with the enhanced thrombogenicity in severe covid-19. the same study showed that vitamin k antagonists (vka) that inhibit vitamin k recycling, had a greater impact on procoagulant activity than on the activation of extrahepatic vitamin k-dependent proteins during sars-cov-2 infections. a genetic polymorphism in the vitamin k epoxide reductase complex 1, vkorc1 -1639a, is particularly prevalent in east asia and associates with low vitamin k recycling rates. carriage of the allele may be regarded as bioequivalent to low-dose vka use. we speculate that vkorc1 -1639a confers protection against thrombotic complications of covid-19 and that differences in its allele frequency are partially responsible for the differences in covid-19 severity between east and west. 1 abstract covid-19, caused by sars-cov-2, has major world-wide health-related and socio-economic consequences. there are large disparities in the burden of covid-19 with an apparent lower risk of poor outcomes in east asians compared to populations in the west. a recent study suggested that covid-19 leads to a severe extrahepatic vitamin k insufficiency, which could lead to impaired activation of extrahepatic proteins like endothelial anticoagulant protein s in the presence of normal hepatic procoagulant activity. this would be compatible with the enhanced thrombogenicity in severe covid-19. the same study showed that vitamin k antagonists (vka) that inhibit vitamin k recycling, had a greater impact on procoagulant activity than on the activation of extrahepatic vitamin k-dependent proteins during sars-cov-2 infections. a genetic polymorphism in the vitamin k epoxide reductase complex 1, vkorc1 -1639a, is particularly prevalent in east asia and associates with low vitamin k recycling rates. carriage of the allele may be regarded as bioequivalent to low-dose vka use. we speculate that vkorc1 -1639a confers protection against thrombotic complications of covid-19 and that differences in its allele frequency are partially responsible for the differences in covid-19 severity between east and west. 2 the emergence and global spread of severe acute respiratory syndrome coronavirus 2 (sars-cov)-2 has led to far-reaching changes in modern societies, reminiscent of descriptions of the 14 th century plague in europe. although it is generally assumed that the bacterium yersinia pestis is responsible for the 'black death', others have argued that evidence pleads against this and hold a yet unidentified virus responsible for the medieval catastrophe [1] . we recently found severely reduced extrahepatic vitamin k status in dutch patients with coronavirus disease 2019 (covid19) , and particularly in those who needed invasive ventilation and/or died [2] . activation of procoagulant factor ii by vitamin k in the liver, however, was hardly affected and not different between covid-19 patients with good and poor outcomes [2] . there are remarkably lower covid-19-related morbidity and mortality rates in east asia than in the americas and western europe [3] . we hypothesize that this may be in part due to genetic differences in vitamin k metabolism. vitamin k is a cofactor for the activation of various pro-and anticlotting factors as well as proteins outside the coagulation cascade [4] . in contrast to other vitamin k-dependent coagulation factors, a significant proportion of anticoagulant protein s is extrahepatically synthesized [5] . protein s produced in endothelial cells seems to be an important inhibitor of local thrombosis formation [5] . vitamin k1, representing the main source of vitamin k in for most people, is preferentially transported to the liver [4] . evidence indicates that during times of mild vitamin k insufficiency hepatic factors coagulation factors are preferentially activated over extrahepatic ones [6, 7] . vitamin k-dependent matrix gla protein (mgp) is a critical inhibitor of soft tissue mineralization and elastic fiber degradation [8] . unpublished data from our research group suggest that mgp synthesis is strongly enhanced in the lungs of patients with sars-cov-2 pneumonia, plausibly to protect the pulmonary extracellular matrix from inflammation-induced degradation. accelerated vitamin k utilization in the lungs may readily deplete extrahepatic vitamin k stores and prevent endothelial protein s from being activated [4, 5] . vitamin k1 consumed during covid-19 hospitalization will preferentially be directed to the liver for activation of hepatic coagulation factors [4] . this suggests that activation of endothelial protein s in these patients will be more severely compromised than activation of hepatic procoagulant factors [4] , which is compatible with enhanced thrombogenicity in covid-19 [9] . a greater proportion of vitamin k2 than of k1 is directly transported to extrahepatic tissues after absorption from the gut [10]. vitamin k2 may therefore be more suitable than vitamin k1 for supplementation in covid-19 patients to activate pulmonary mgp and endothelial protein s. natto is a japanese breakfast dish of fermented soybeans containing extremely high amounts of vitamin k2 [11] . incidence of sars-cov-2 infections was remarkably low in japanese regions where average natto intake is high [12] . although fascinating, this does not provide an explanation for the low incidence of covid-19-related morbidity and mortality in parts of east asia where less natto is consumed [11] . after being utilized during vitamin k-dependent protein activation, vitamin k is repeatedly reactivated in the vitamin k cycle [13] . the rate of vitamin k recycling depends on a polymorphism in the promoter region of the vitamin k epoxide reductase complex subunit 1 (vkorc1) gene [13] . in east asia, positive selection for an allele that is associated with low vitamin k recycling rates started around 4,500 years ago [14] . allele frequency of this derived -1639a vkcorc1 allele is around 75-100% in east asians, whereas it is below 10% in africans and intermediate in europeans [15] . the near complete selective sweep in east asian populations is remarkable due to the recent and rapid occurrence, and strongly suggests a survival benefit rather than the loss of redundant function [14] . however, it is an enigma why a polymorphism associated with reduced vitamin k recycling would carry a significant survival benefit, given that even high-dose vitamin k intake is not associated with adverse side effects [16] . based on our recent data demonstrating pronounced extrahepatic vitamin k insufficiency and normal hepatic vitamin k status in covid-19 [2] , we suggest a hypothesis by which this allele might give a survival benefit in covid-19 patients. vitamin k antagonists (vkas) are anticoagulant drugs that inhibit vkorc1, constraining vitamin k recycling rates and leading to vitamin k deficiency [13] . when this deficiency is severe enough, it impairs the activation of hepatic coagulation factors (e.g. factor ii) increasing clotting time. while levels of inactive factor ii were low in most covid-19 patients, unsurprisingly, covid-19 patients using vka had strongly increased levels of inactive factor ii. however, there was much less difference in activation of mgp between covid-19 patients with and without vkas, suggesting that covid-19 had already induced complete extrahepatic vitamin k deficiency without any additional effect from vkas [2] . since protein s is also significantly extrahepatically produced [5] , we expect that the relative effect of vka use on protein s activation in covid-19 patients is also less pronounced than on factor ii activation. given the high incidence of thrombotic complication in covid-19 [9] , the simultaneous decrease of both pro-and anticoagulant factors in patients using vka might be advantageous compared with a decrease in only anticoagulant protein s activation. the vkorc1 -1639a allele is associated with low vitamin k recycling rates, which may be regarded as a natural equivalent of using low-dose vka. therefore, we hypothesize that vkorc1 -1639a 5 protects against thrombosis during sars-cov-2 infection and thereby from poor disease outcomes. if this is indeed the case, the high incidence of vkorc1 -1639a in east asia may form an explanation for the lower covid-19-related morbidity and mortality rates. strong genetic selection in a short period of time appears to be extremely rare in human populations [17] and has to be caused by a cataclysmic event. driving a gene to near fixation, as appears to be the case for the vkorc1 -1639a allele in east asia, also requires some degree of sustained selection pressure [18] . endemic diseases with a moderate or high mortality rate can rapidly drive such genetic selection in humans. some of the most wellknown examples of this are the influences of the malaria parasites plasmodium vivax on the frequency of the duffy blood group fy*o allele [19] and plasmodium falciparum on the frequency of the sickle-cell allele [20] in sub-saharan africa. we speculate that the vkorc1 -1639a allele might have given a survival advantage to a previous infectious disease with a higher mortality rate than covid-19. an interesting candidate for this infection is the disease known as the 'black death' that raged across europe starting in the 14 th century and was thought to have emerged from asia. several researchers have theorized that this pandemic plague was caused by a virus, and medieval writings suggest the disease had coagulopathic manifestations [1] . for instance, hemorrhagic purplish splotches (i.e. god's tokens) typically appeared in black death victims shortly before their demise [1] . unfortunately, no specimens have remained from victims to assess whether organs were affected by microvessel thrombosis, like those found in postmortem lung examinations of covid-19 patients [21] . if black death was indeed accompanied by coagulopathic manifestations, then vkorc1 -1639a might have conferred a survival benefit. considering the epidemic also emerged from east asia and was probably endemic in those regions for extended periods of time before arriving in europe, we speculate this could be the reason for the near fixation of the vkorc1 -1639a allele in east asian 6 populations and intermediate prevalence in european populations. black death probably did not pass the sahara, which would be compatible with low carriage of vkorc1 -1639a in african populations [15] . we hypothesize that the vkorc1 -1639a allele may be protective against thrombotic disease manifestations and disease-related mortality of covid-19. the most straightforward method of testing our hypothesis is by determination of vkorc1 -1639 in european patients who have been admitted with covid-19. our hypothesis would be supported if the vkorc1 -1639a allele frequency were higher in controls than in individuals who developed respiratory failure and/or died due to covid-19. it would also be relevant to assess whether the vkorc1 -1639a allele has a lower frequency among hospitalized covid-19 patients with thrombotic complications than those without. we speculate that the disparity in morbidity and mortality from covid-19 between east and west may be at least partially explained by differences in the allele distribution of a vkorc1 polymorphism determining the rate of vitamin k recycling. if the vkorc1 -1639a allele is shown to be associated with decreased thrombotic complications and/or death, it would further support the hypothesis that vitamin k metabolism is an important determinant of covid-19-related disease severity. rj discloses application of a patent on vitamin k in covid-19. rj and jw have a scientific collaboration with kappa bioscience as, a manufacturer of vitamin k2 (mk-7). what caused the black death? reduced vitamin k status as a potentially modifiable prognostic risk factor in covid-19 researchers ponder why covid-19 appears deadlier in the u.s. and europe than in asia. the washington post vitamin k, an example of triage theory: is micronutrient inadequacy linked to diseases of aging? human endothelial cells synthesize protein s dietary phylloquinone depletion and repletion in older women effect of vitamin k intake on the stability of oral anticoagulant treatment: dose-response relationships in healthy subjects vitamin k-dependent carboxylation of matrix gla-protein: a crucial switch to control ectopic mineralization incidence of thrombotic complications in critically ill icu patients with covid-19 low-dose menaquinone-7 supplementation improved extrahepatic vitamin k status, but had no effect on thrombin generation in healthy subjects japanese fermented soybean food as the major determinant of the large geographic difference in circulating levels of vitamin k2: possible implications for hip-fracture risk this japanese region has no coronavirus. the wall street journal vkorc1 and the vitamin k cycle positive selection in the chromosome 16 vkorc1 genomic region has contributed to the variability of anticoagulant response in humans worldwide allele frequency distribution of four polymorphisms associated with warfarin dose requirements institute of medicine (us) panel on micronutrients classic selective sweeps were rare in recent human evolution the genetics of human adaptation: hard sweeps, soft sweeps, and polygenic adaptation detection of the signature of natural selection in humans: evidence from the duffy blood group locus malaria continues to select for sickle cell trait in central africa pulmonary vascular endothelialitis, thrombosis, and angiogenesis in covid-19 conflict of interest statements rj discloses application of a patent on vitamin k in covid-19. rj and jw have a scientific collaboration with kappa bioscience as key: cord-296253-bxyzhsfs authors: elham, elzat; wumaier, reziya; wang, chengji; luo, xiangying; chen, tao; zhong, nanshan title: anatomic evidence shows that lymphatic drainage exists in the pituitary to loop the cerebral lymphatic circulation date: 2020-05-30 journal: med hypotheses doi: 10.1016/j.mehy.2020.109898 sha: doc_id: 296253 cord_uid: bxyzhsfs respiratory infections can result in intracranial infections and unknown neurological symptoms. the central nervous system lacks classical meningeal lymphatic (circulation) drainage, and the exact underlying mechanisms of how immune cells from the peripheral lymphatic system enter the central nervous system (cns) remain unknown. to determine whether the perinasal lymphatic system or lymphatic vessels are involved in cerebral immune defence and play a role in causing cns infections (especially respiratory tract-related infections), we performed an anatomic study to investigate the drainage differences between the perinasal and intracerebral lymphatic systems by using injection of evans blue and anatomic surgery, together with immunohistochemistry and immunofluorescence assays. surprisingly, we found that (1) the pituitary (adenohypophysis) is involved and is rich in lymphatic vessels and (2) perinasal tissue could communicate with central pituitary lymphatic vessels in a specific and unidirectional manner. taken together, our study may be the first to anatomically demonstrate the existence of novel lymphatic vessel structures in the pituitary, as well as their communication with the perinasal (lymphatic) tissue. our findings suggest the existence of an ultimate loop for “classical” meningeal lymphatic drainage and are relevant to cerebral infection and immune defence. respiratory infections can result in intracranial infections and unknown neurological symptoms. the central nervous system lacks classical meningeal lymphatic (circulation) drainage, and the exact underlying mechanisms of how immune cells from the peripheral lymphatic system enter the central nervous system (cns) remain unknown. to determine whether the perinasal lymphatic system or lymphatic vessels are involved in cerebral immune defence and play a role in causing cns infections (especially respiratory tract-related infections), we performed an anatomic study to investigate the drainage differences between the perinasal and intracerebral lymphatic systems by using injection of evans blue and anatomic surgery, together with immunohistochemistry and immunofluorescence assays. surprisingly, we found that (1) the pituitary (adenohypophysis) is involved and is rich in lymphatic vessels and (2) perinasal tissue could communicate with central pituitary lymphatic vessels in a specific and unidirectional manner. respiratory infections (e.g., fungi, bacteria, and coronavirus) can result in unknown intracranial infections and consequent neurological symptoms (1) (2) (3) . for example, in the current covid-19 epidemic in china, 78 (36.4%) of 214 patients with covid-19 were admitted with neurological symptoms to wuhan union hospital (2) , and we observed 2 cases of diabetes insipidus (di) related to pituitary disorder in patients with severe covid-19 (in the first affiliated hospital of guangzhou medical university). it is generally believed that pathogens cause intracranial infection by entering the subarachnoid space via nasopharyngeal or middle ear passages, blood flow, blood-brain, and cerebrospinal fluid (csf) barriers, although we still cannot explain the existence of pathogens in the csf, as the blood-brain barrier (bbb) can prevent the transmission of pathogens to the meninges(1). csf originates from the choroid plexus of the intracranial lateral ventricle (4) . the reflux of csf to the lymphatic system plays an important role in cerebral immunity (4, 5) . csf is drained through meningeal lymphatic vessels, which allow immune cells to enter draining lymph nodes (dlns) and play an important role in cerebral immune defence. however, the exact underlying mechanisms of how immune cells from the peripheral lymphatic system enter the central nervous system (cns) remain unknown (4, 6) . the perinasal lymphatic system is the first-line barrier of respiratory immunity against pathogen invasion of the respiratory tract and body (7) . respiratory infections can lead to cns infections, but it is unclear whether the perinasal lymphatic system and lymphatic vessels are involved in cerebral immune defence and play a role in cns infections caused by respiratory pathogens (1, 7) . to elucidate the roles of the perinasal lymphatic system during cerebral infection (especially respiratory-related infections) and cerebral immune defence, we carried out an anatomic study to investigate the drainage differences between the perinasal and intracerebral lymphatic systems. under an anatomic (20× magnification) microscope, we dissected the mouse intracranial nervous system after injection of evans blue (perinasal lymphatic reflux assay) and found that lymphatic vessels that exist in the pituitary and loop the cerebral lymphatic circulation are responsible for the perinasal-pituitary lymphatic drainage. the lyve1-alexa 488 antibody was purchased from ebioscience (catalogue # 53-0443-80) and used at a 1:250 dilution. anti-cd31 was purchased from abcam (catalogue # ab222783) at a 1:100 dilution. anti-rabbit igg (h+l) highly cross-adsorbed secondary antibody was purchased from invitrogen co., ltd., (catalogue # a32740) and used at a 1:1000 dilution. evans blue and other reagents were purchased from sangon biotech (shanghai) and were of high analytical grade. mice (balb/c, 7 weeks old) were divided into different groups (5 for each group). for the treated groups, mice were anaesthetized with pentobarbital sodium (70 mg/kg) by intraperitoneal injections and then subcutaneously injected with 0.1 ml of evans blue (5%) by microsyringe via either or both limbs, the tail and the perinasal area (e.g., bilateral the hindlimbs, the second toe of the dorsal feet, both flanks, the dorsal sides of the bilateral forelimbs, the bilateral retroauricular regions, the parietal midpoint between both ears, the tip of the nose, and the bilateral ventral mucosae of the tongue). the control groups received saline instead. after injection, the mice were placed on a heating pad at a stable (25°c) temperature for 4 hours and then euthanized (350 mg/kg) for anatomic analysis. in brief, with the abdomen facing down, the dorsal fur of the mouse was moistened with saline, after which the dorsal skin was cut transversely and then longitudinally to the mouse nose to fully expose the skull with scissors. then, the cervical muscles were cut off the skull from the foramen magnum to expose the brain. the brain was then removed with tweezers; nerves connecting the brain were also cut off to expose the pituitary for observation under a zeiss operating microscope (zeiss opmi primo ceiling-mount microscope, 20× magnification). photos were taken with a canon 5dsr camera (micro lens: canon ef 100 mm f/2.8l is usm; micro flash: canon mr-14ex ii). the pituitary was removed from the mouse, immersed in 4% pfa for fixation, and embedded in paraffin. the pituitary was transversely sectioned (leica cm 1950) and adhered to a glass slide. the sections were then deparaffinized, after which they underwent antigen retrieval, 3% hydrogen peroxide solution blocking for endogenous peroxidase and 3% bsa blocking. the blocking buffer was discarded, and primary antibody in pbs was added to the section and incubated in a wet box at 4°c overnight. the glass slides were washed with pbs three times (5 min each) after incubation. then, the slides were incubated with secondary antibody (hrp-labelled) for 50 min at room temperature. the sections then underwent dab and harris haematoxylin staining after three washes in pbs. then, the section was mounted using neutral balsam, and the blue staining was reversed in ammonia water. photos were taken under a fluorescence microscope (leica dmi4000b). the sections and slide preparation, antigen retrieval, bsa blocking, and primary and secondary antibody incubation processes were the same as the aforementioned conditions used for immunohistochemistry. after staining with secondary antibodies, the glass slide was washed in pbs three times (5 min each). after the section dried slightly, dapi was added to the sample, followed by 10 min of room temperature incubation in the dark. the glass slide was then washed again three times and mounted using an antifluorescence quenching mounting agent. photos were taken under a confocal microscope (lsm710 laser confocal microscope, zeiss). adobe photoshop, fiji image analysis, and graphpad prism software were chosen for image processing. two to four hours after the subcutaneous injection of evans blue into the mouse limbs, tail, and perinasal area, we unexpectedly observed a light-blue, well-discriminated, regular "birds eye" region in the central pituitary (8) (fig. 1a; we then dissected other untreated mice and found that the "birds eye" region exists in the central pituitary, which was identifiable but can be carefully discriminated from peripheral white matter ( fig. 1c ; see green arrow and circle). we further performed pathological staining and confirmed that the "birds eye" region was actually the posterior pituitary or adenohypophysis(8) (fig. 1d) . moreover, lymphatic endothelial growth factor was found to be highly expressed in the "birds eye" region ( fig. 1e and we acknowledge that the brain lacks classic lymph circulation according to our current understanding. however, it remains unclear how respiratory pathogens cause intracranial infection, how immune cells enter the brain for immune defence and whether the peripheral lymphatic system communicates with the central nervous system in addition to the meningeal lymphatic draining system. a, colocalization analysis of lyve-1 and cd31 under the scope in figure 2d (upper right, the most colocalized area in figure 2d was selected and marked with a yellow line; scale bar: 100 μm). b, immunofluorescence analysis showed no colocalization of lyve-1 and cd31 (fiji image analysis). funding: the study was financially supported by grants from the national natural science we declare that none of the authors has any potential conflicts of interest with regard to this manuscript. a, colocalization analysis of lyve-1 and cd31 under the scope in figure 2d (upper right, the most colocalized area in figure 2d was selected and marked with a yellow line; scale bar: 100 μm). b, immunofluorescence analysis showed no colocalization of lyve-1 and cd31 (fiji image analysis). nasal lymphatics as a novel invasion and dissemination route of bacterial meningitis neurological manifestations of hospitalized patients with covid-19 in wuhan, china: a retrospective case series study pituitary aspergillus infection structural and functional features of central nervous system lymphatic vessels implications of the discovery of brain lymphatic pathways current understanding of lymphatic vessels in the central nervous system rapid transepithelial transport of prions following inhalation neuronal m<sub>3</sub> muscarinic acetylcholine receptors are essential for somatotroph proliferation and normal somatic growth none of the authors has any potential conflicts of interest. key: cord-304658-vxu33v7i authors: brenner, steven r. title: covid-19, tmprss2, and whether android regulation affects pandemic virus gender incidence and age distribution of disease date: 2020-04-22 journal: med hypotheses doi: 10.1016/j.mehy.2020.109773 sha: doc_id: 304658 cord_uid: vxu33v7i nan medical hypotheses journal homepage: www.elsevier.com/locate/mehy covid-19, tmprss2, and whether android regulation affects pandemic virus gender incidence and age distribution of disease the likely reason that covid19 has male predominance is that it appears to be an androgen driven pathogen. sars-cov2, coronavirus spike protein undergoes proteolytic activation by transmembrane serine protease 2 (tmprss2), to enable sars-cov-2 to utilize the ace2 receptor for cellular entry [1] . tmprss2 is highly expressed primarily in prostate epithelium, highgrade prostate cancers and is androgen regulated [2] . androgen regulation may explain the paucity of cases of covid19 in preadolescents, since they don't have the androgens to prime the tmprss2 cell surface protease. covid19 has affected men, smokers and the elderly more than other groups. smoking appears to increase the ratio of androgens to estrogen, which may prime the tmprss2 cell surface protease [3] . possibly, benign prostatic hypertrophy being more common in elderly men, may contribute to increased tmprss2 as well, leading to increased severity of viral infection in the older age group. inhibitors of tmprss2 include bromohexine [2] and camostat [1] . inhibiting androgens could also be another approach to managing the covid19 virus. tmprss2 is also a host cell factor necessary for viral spread of h1n1 and h3n2 influenza a viruses, indicating a similar phenomena may occur with other pandemic viruses, since male predominance has been observed in pandemic influenza a [4] . no grant support. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. sars-c0v-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor the androgenregulated protease tmprss2 activates a proteolytic cascade involving components of the tumor microenvironment and promotes prostate cancer metastasis cigarette smoking, androgen levels, and hot flushes in midlife women tmprss2 is essential for influenza h1n1 virus pathogenesis in mice supplementary data to this article can be found online at https:// doi.org/10.1016/j.mehy.2020.109773. key: cord-282853-l0c69uul authors: massad, eduardo; burattini, marcelo n.; lopez, luis f.; coutinho, francisco a.b. title: forecasting versus projection models in epidemiology: the case of the sars epidemics date: 2005-03-30 journal: med hypotheses doi: 10.1016/j.mehy.2004.09.029 sha: doc_id: 282853 cord_uid: l0c69uul in this work we propose a simple mathematical model for the analysis of the impact of control measures against an emerging infection, namely, the severe acute respiratory syndrome (sars). the model provides a testable hypothesis by considering a dynamical equation for the contact parameter, which drops exponentially with time, simulating control measures. we discuss the role of modelling in public health and we analyse the distinction between forecasting and projection models as assessing tools for the estimation of the impact of intervention strategies. the model is applied to the communities of hong kong and toronto (canada) and it mimics those epidemics with fairly good accuracy. the estimated values for the basic reproduction number, r(0), were 1.2 for hong kong and 1.32 for toronto (canada). the model projects that, in the absence of control, the final number of cases would be 320,000 in hong kong and 36,900 in toronto (canada). in contrast, with control measures, which reduce the contact rate to about 25% of its initial value, the expected final number of cases is reduced to 1778 in hong kong and 226 in toronto (canada). although sars can be a devastating infection, early recognition, prompt isolation, and appropriate precaution measures, can be very effective to limit its spread. public health is the technological arm of epidemiology and its actions are based essentially on prevention and intervention. to prevent and to intervene, however, one must predict. to predict the natural course of a system in the absence of intervention and to predict what is going to happen to such a system after the proposed intervention. prediction, on its side, is strongly dependent on the scientific foundations of the subject in question. in this sense, mathematical models have provided a precise template against which new observations or theories can be tested as understanding of the phenomenon develops [1] . 0306 three major aims of mathematical models in epidemiology can be identified: the first centres on the need for scientific understanding and precision in the expression of current theories and concepts; a second aim, linked to the first, is the role of theory in identifying areas in which better epidemiological data is required to refine prediction and improve understanding; and the third, and in many instances, the most difficult objective is that of prediction [1] . in addition to these three aims of modelling we propose a fourth objective: the generation of testable hypotheses by providing a theoretical framework on which plausible scenarios can be simulated in a computer environment (in silico experiments). prediction in general science can be divided into two components: forecasting and projections [2] . a forecast is an attempt to predict what will happen. a projection is an attempt to describe what would happen, given certain hypotheses [3] . among the tools available to the modern epidemiologists for both forecasting and projection are the mathematical (or dynamical) models, which, when well structured, can provide predictive capacity to the public health professional, helping in the design, and assessment of the impact, of control strategies [4] [5] [6] [7] . for instance, by projecting what would happen with a given population if individuals were not vaccinated, it is possible to quantify the relative impact of a specific vaccination program. the aim of this work is to provide a projection of what would have happened with the course of severe acute respiratory syndrome (sars) epidemic if the universal procedures to reduce contact were not implemented in the affected areas. we do this by the application of a dynamical system approach, as described below. the sars epidemics: a case in point sars is a recently discovered infectious disease with high potential for transmission [8] , transmitted by droplet and direct contact and caused by a new strain of corona virus [9] . on 5 july 2003, who announced that the last known chain of human-to-human transmission of the sars corona virus had been broken. a cumulative number of 8422 cases have been reported worldwide to the who [10] , with 908 deaths, as of august 2003. in the end of 2002, reports from china suggested that a new, highly contagious, and very severe atypical pneumonia of unknown cause was occur-ring in the guangdong province. as it reached southeastern asian countries, the condition appeared to be particularly prevalent among health care workers and their household members. in response to that threat, on 13 march 2003, who issued a global alert, for the first time on more than a decade, and instituted worldwide surveillance. on march 27, scientists in the who laboratory network reported major progress in the identification of the causative agent, a new member of the corona virus family. by that time, sars has already become a global health hazard, and its high infectivity was alarming. early recognition, prompt isolation, and appropriate precaution measures were considered to be key factors in combating this infection [11] . however, while much has been learnt about sars since it was brought to international attention in march 2003, there remain many unanswered questions about where it came from, how it spreads, and the effectiveness of public health and other measures employed to control the disease [12] . this paper is an attempt to answer the last question. for this, we propose a dynamical model to assess the expected burden of the epidemic in the absence of control measures and the impact of adopting well-known precautions methods, like the set of measures included in the air-borne and contact precautions recommended by cdc [9] , on the epidemic course. some affected communities, like hong kong and toronto (canada), have a detailed record of the epidemics, allowing a deeper analysis of its dynamics. we designed a simple mathematical model to describe the spread of sars and to predict the impact of control measures. we calculated the basic reproduction number, r 0 , for those communities and simulated the temporal evolution of the epidemics with and without control measures. the model is deterministic of the susceptibleinfected-recovered (sir) [13] type, assuming homogeneously mixing contacts and considering the known parameters (duration of the infectious period and mortality rate of the infection). the model is described by the following set of differential equations, representing the temporal evolution of the number of individuals in each of the possible states, susceptibles x(t), infectious, y(t) and recovered, z(t): dxðtþ=dt ¼ àbxðtþyðtþ=nðtþ à lxðtþ þ l½yðtþ þ zðtþ; where b is the contact rate, c is the recovery rate, l is the natural mortality rate and a is the infection mortality rate. the cumulative number of cases, c(t), was calculated according to the contact rate parameter was estimated from the exponential initial phase of the epidemics [13] [14] [15] [16] . as we mentioned in a previous work [17] , r 0 can be estimated from the initial exponential growing phase of the number of cases. for this, we begin by fitting an exponential curve to the initial growing phase of the number of human cases of sars, y(t) having r 0 it is possible to estimate the contact rate, b, the most difficult parameter to be obtained from epidemiological field research, through the relation we then simulated the model numerically under two hypothetical scenarios, the natural course of the infection (the contact parameter is kept constant in time) and under control measures (the contact parameter drops exponentially with time). in fig. 1 we show the simulation for the hong kong community. the parameters applied were l = 0.00004 days à1 ; c = 0.1 days à1 ; and a = 0.08 days à1 , adapted from the data described by the who [18] . the initial value of b was calculated according to eq. (4) and we used the relation b ¼ 2:16 â 10 à1 for t 6 15 days; and bðtþ ¼ ð2:16 â 10 à1 þ expðàjtþ for t > 15 days; ð5þ with j = 0.0158 days à1 , for simulating control. that is, for the first 15 days b was assumed as constant and equal to 0.216 potentially infective contacts per day, dropping exponentially with rate j = 0.0158 days à1 thereafter. in order to compare the actual curve of cases with the theoretical projection of the number of cases without any control we kept the rate j as equal to zero for the entire epidemic period, as shown by the continuous line in fig. 1 . in fig. 2 we show the simulation for toronto (canada). the canadian case has some peculiarities worth commenting. besides being the only western country with a significant autochthonous sars epidemic, after about 65 days the beginning of the first outbreak, when the number of new cases was almost zeroed, a second outbreak began, as can be easily noted by the sudden rise in the number of new cases occurring between days 65 and 80 (see fig. 2 below) . the parameters used were b ¼ bðtþ ¼ ð2:9 â 10 à1 þ expðàjtþ; with j = 0.0145 days à1 , simulating control; l = 0.00004 days à1 ; c = 0.1 days à1 ; and a = 0.12 days à1 . the second outbreak was simulated by assuming the number of the last cases before the outbreak as initial condition and applying the same equation for b(t), with b (0) = 2.9 · 10 à1 but with j = 0.45 days à1 . we therefore assumed a very quick response of the canadian health authorities to this second outbreak. the calculated value for r 0 for hong kong was 1.20 and for toronto (canada) was 1.32. in fig. 3 we show the simulation of the reproduction number of both epidemics. at time equals zero we have the basic reproduction number, r 0 , and the time evolution of the number of secondary cases. we note that this value drops below the threshold due to the exponential reduction in the contact parameter b, as explained above. it can be noted also the recrudescence of the canadian epidemic, when the value of b re-turned to its basal value. when the reproduced number crosses the threshold line (r = 1), the number of new cases reaches its maximum, dropping thereafter. the model mimics real data with good accuracy for both communities when considering adoption of control measures. the model's prediction demonstrated an epidemic that is, by far, milder than expected without control measures. the model projects that, in the absence of control, the final number of cases would be 320,000 in hong kong and 36,900 in toronto (canada). by contrast, with control measures, which reduce the contact rate model's prediction "natural course" of the epidemics figure 2 results of the simulation for toronto (canada). the parameters applied were: b = b(t) = (2.9 · 10 à7 ) exp (àjt), with j = 0.0145 days à1 , simulating control; l = 0.00004 days à1 ; c = 0.1 days à1 ; and a = 0.12 days à1 . the second outbreak was simulated by assuming the number of the last cases before the outbreak as initial condition and applying the same equation for b(t) but with j = 0.45 days à1 . we therefore assumed a very quick response of the canadian health authorities to this second outbreak. the same community was simulated with the line expressing the ''natural course'' of the epidemics calculated with j = 0, simulating the absence of control. to about 25% of its initial value, the expected final number of cases is reduced to 1778 in hong kong and 226 in toronto (canada). in fact, the stability level predicted by the model was indeed attained in both hong kong and toronto (canada) by the end of the outbreaks. note that the model's performance should be taken with some caution, since there are some constraints that could influence our results. the first one is the assumption of homogeneously mixing pattern of transmission. in fact, this is a simplification that does not take into account the fact that sars occurred mainly as focal outbreaks, with a concentration of cases in small communities somewhat related. this simplification may overestimate the projections of the natural course of the epidemics. however, the qualitative results are not affected, that is, by introducing in the model the parameter j in eq. (5), which reduced the contact rate, we were able to reproduce the change in the natural impetus of the epidemics. the second constraint of the model is the deterministic nature of the model, that is, we considered only the average values of each variable and parameter (the mean field approach). however, the size of the epidemics and the affected populations justify the deterministic approximation. finally, as our objective was not to forecast the real size of the epidemics but rather to analyse the possible impact of implementing control measures, the above-mentioned constraints do not compromise the final conclusions. this work proposes a theoretical framework of the sars epidemic dynamics on which a hypothesis, namely, the spread of the infection can be checked by simple, universal procedures that reduces contacts, was tested. the test consisted in reproducing the course of the epidemic along its temporal evolution by mimicking the contact reduction in the parameter b, as compared with the absence of intervention. the model reproduces the temporal evolution of the epidemics with good accuracy and suggests what would have happen if the control measures were not implemented. therefore, our model is not a forecasting model but rather a projection one. this distinction is an important one, in particular concerning the concept of validation. forecasting models can be tested by comparing their predictions with actual data. projection models should be seen in a counterfactual way, that is, their projections cannot be tested because they will never happen. the main purpose of projection models is, therefore, to provide support by plausible hypotheses on the efficacy of intervention policies. this is the role of the present model, to provide support to the hypothesis that simple control procedures interrupted the transmission chain of the sars virus in the areas analysed. in spite of the fact that sars is indeed a very contagious disease, public health authorities have succeeded in restricting its spread, by isolating patients and using measures to block exposure to coughed-up droplets, such as face-masks, which also avoid indirect contact by limiting hand-nosehand-environment contamination. those are the same well known measures applied to limit transmission of other directly or air-borne transmitted infections such as tuberculosis or influenza. the novel aspect of our approach is the proposition of a method for mimicking control measures, represented by the function that describes the reduction in the contact probability with time. the model reproduced the epidemic in hong kong and the toronto case, where the epidemic recrudesced. no explanation for this second outbreak has been proposed. we suggest that it was probably due to a relaxing of public health measures (b immediately returned to b 0 ), which were promptly corrected with a greater rigour than that applied during the first outbreak (j increased 30fold). this phenomenon can be seen in the curves of fig. 3 , which display the time evolution of the number of secondary cases. as mentioned above, this number starts with the basic reproduction number, r 0 , dropping according with the reduction in the proportion of susceptibles and, in our case, also with the reduction in the contact parameter b. another aspect of our model worth commenting is the absence of a latency compartment. although variable, short incubation periods of less than a week has been considered to be dominant in sars [19, 20] . in addition, most of the incubating cases evolved to clinical cases and were probably infectives before clinical recognition, what reinforces our sir structure for the model. in fact, recent publications [21, 22] consider that sub clinical sars is not an important feature of the disease. but perhaps the most important contribution of this work is that our approach allows the projections of what would have happened if control measures had not been implemented with the appropriate speed and efficacy. as seen in figs. 1 and 2, the sars epidemics had, indeed, the potential of reaching a huge number of cases, spreading havoc among the affected populations. however, super spreading events are suspected to have a pivotal role in the global spread of sars [21] [22] [23] . in fact, it is suspected to have played major roles in epidemics of hong kong and toronto (canada). therefore, it is possible that our projections of the size of those epidemics can be overestimated since we assumed homogenously mixing and our calculations could not be generalized for the entire population of those communities due to the heterogeneity in spreading potential of the affected clusters of individuals. although sars can be a devastating infection, our model shows that simple measures can be very effective to limit its spread. simple dynamic models can indeed be very helpful in providing insights to public health authorities, in particular in situations where very little is known about the course of emerging or re-emerging epidemics. epidemiological models and predictions on future population matrix population models vaccination against rubella: analysis of the temporal evolution of the age-dependent force of infection and the effects of different contact patterns modelling the dynamics of leishmaniasis considering human, animal host and vector populations assessing the efficacy of a mixed vaccination strategy against rubella in são paulo, brazil dynamics of malarial transmission based on serological data severe acute respiratory syndrome (sars) severe acute respiratory syndrome: what everyone should know about sars cumulative number of reported probable cases of severe acute respiratory syndrome (sars). world health organization a major outbreak of severe acute respiratory syndrome in hong kong severe acute respiratory syndrome (sars) -report by the secretariat. world health organization executive board, eb113/33 infectious diseases of humans. oxford the basic reproduction number for dengue fever in são paulo state, brazil: 1990-1991 epidemics fuzzy logic and measles vaccination: designing a control strategy the risk of yellow fever in a dengue infested area dengue and the risk of urban yellow fever reintroduction in são paulo, brazil severe acute respiratory syndrome (sars) -status of the outbreak and lessons for the immediate future. world health organization. communicable disease surveillance and response multiple contact dates and sars incubation periods healthcare worker seroconversion in sars outbreak the authors thank the financial support of fapesp, cnpq, pronex and lim01/hcfmusp. key: cord-293059-2iwzieqm authors: tao, huaqiang; ge, gaoran; li, wenming; liang, xiaolong; wang, hongzhi; li, ning; sun, houyi; zhang, wei; geng, dechun title: dysimmunity and inflammatory storm: watch out for bone lesions in covid-19 infection date: 2020-10-06 journal: med hypotheses doi: 10.1016/j.mehy.2020.110332 sha: doc_id: 293059 cord_uid: 2iwzieqm at the end of 2019, a new kind of pneumonia which was proven to be supported by novel coronaviruses named sars-cov-2 emerges and it seems to be more complicate in its clinical course and management. related researches have demonstrated that sars-cov-2 serves roles in respiratory, intestinal and neuronal diseases. given the growing cases of covid-19, analyzing the relevance between covid-19 and fragile patients who suffer from bone destruction is entirely indispensable. accordingly, the recapitulatory commentary is necessary to advance our knowledge on covid-19 and orthopedics. in this article, we particularly clarify the possible relationship between the newly covid-19 infection and bone lesions from the standpoints of dysimmunity and inflammatory storm. it has been approved that inflammation-induced pathogenesis in covid-19 infection has a strong correlation with incidence of cardiovascular metabolic diseases and gastrointestinal injury (1) . processes that co-exist in the inflame milieu (2) . the pneumonia patients constantly suffer from hypoxaemia. senile patients with sars-cov-2 infection always stand perennial disability, such as pulmonary fibrosis and respiratory insufficiency (3) . bone owns a quite hypoxic microenvironment, the hypoxia of organism will affect heterogeneous po 2 in bone marrow in a degree. not only those inflammatory responses, but also the innate immune system is biologically intertwined with the processes of bone homeostasis. when subjected to sars-cov-2, host cellmediated and humoral immune responses are rapidly activated and take defensive measures(4). massive immune cells were recruited in alveolar cells as the disease progresses. to initiate an antiviral response, pattern recognition receptors (prrs) were involved in innate immune responses to identify the invasion of the virus. this recognition event results in the activation of downstream signaling cascade, including nf-κb, activator protein1 (ap-1) and interferon regulatory factor-3 (irf3). concomitantly, these transcription proteins translocate into nuclear and prompt expression of type 1 ifns and other pro-inflammatory cytokines to defend against viral infection and dissemination at the entry site(5). as for severe covid-19 infection, huang et.al found that patients who need icu monitoring had much higher concentrations of cytokines and chemokines than other ones, such as il-1β, ifnα, il-1ra and il-8, suggesting that the inflammatory storm was involved in infection severity (6) . significantly high release of blood pro-inflammatory mediators also responds to lung injury and viral replication, including tnf-α, il-2, il-10 and ip-10. however, studies on the correlation between pro-inflammatory cytokine responses and bone metabolism in covid-19 patients are still lacking. in this special background, will inflammatory disorder and immune imbalance affect bone metabolism after covid-19 infection? the role of inflammatory factors has been closely associated with bone loss and early osteoclastogenesis (7) . it has been reported that the deficiency of ace2 in mesenchymal stem cells (mscs) increases the expression of tnf-α, which may be responsible for skeleton dysfunction and adverse structure outcomes (8) . as is accepted, inflammatory cytokines promote osteoclastogenesis by regulating rank/rankl/opg axis in direct (9) . concretely, they drive up bone resorption by promoting rank expression on monocytes. at the same time, they downregulate osteoblastic production by restraining opg. it's conjecturable that, if rankl is not resolved by normal homeostatic system, the inflammation stimuli initiated by sars-cov-2 can become chronic in nature and lead to the secretion of a plenty of pro-inflammatory cytokines. in these signaling cascades of osteoclastogenesis, it is quite evident that tnf-α regulate the activation of calcium signaling and the auto-amplification of nfatc1, which has an active acceleration in osteoclast expression (10) . some other pro-inflammatory cytokines, such as il-1β and il-2, have also been studied in the context of bone loss or arthritis (11, 12) . in parallel, inflammation storm seems to share the commonness in the progress of bone healing. dysregulated inflammation responses lead to increased bone resorption, thereby leading to subsequent bone destruction and arthritis. hypoxia signaling is an intervention factor for osteoclast differentiation and osteoblast formation. literature indicates that hypoxia boosts the overproduction of pro-osteoclastogenic cytokines, including receptor activator of nuclear factor-b ligand (rankl), vascular endothelial growth factor (vegf), macrophage colony-stimulating-factor (m-csf), leading to osteoclasts activation (13) . simultaneously, hypoxia inducible factor (hif-1) was proven to facilitate osteoclast differentiation by overexpressing rankl and nuclear factor of activated t cells cytoplasmic 1 (nfatc1) (14) . as for osteoblasts, hypoxia signaling seems to exhibit inactive effects on osteogenesis capacity, which was linked to angiogenesis-induced bone-forming and the intervention of canonical osteoblastic wnt signaling (15, 16) . therefore, sars-cov-2 induced hypoxaemia is most likely to mediate bone destruction and disturb bone matrix. the lack of oxygen reduces the energy supply of cell membrane. at the same time, the metabolic disorder of oxygen evokes intracellular free radicals to damage membrane transport proteins (17) . the deleterious effects of oxidative stress in bone metabolism have elicited much attention in recent years. oxygen free radicals, especially reactive oxygen species, maintain homergy in bone biology. an extensive variety of intracellular signaling events are involved in osteoclast activation, including the regulation of mitogen-activated protein kinases (mapks) and intracellular ca 2+ levels (18) . in addition, excessive free radicals hamper osteoblast adhesion to worsen bone homeostasis in further. especially, hypoxaemia can also give rise to ca 2+ metabolism disorder, which may injury osteocyte. with the imbalance between oxygen delivery and consumption in covid-19 patients, not only do we need to search novel treatment for minimizing lung damage, but also attract attention on succeeding hypoxia-induced cascade reactions in the whole biological system. as osteoblasts and osteoclasts exist in approach with immune cells in medullary cavity, it's no wonder that immune system shares massive regulatory cytokines, signaling molecules and transcription factors with bone biology. osteoclasts take responsibility for bone resorption, which share a collaborative precursor with macrophages and dendritic cells (19) . in the antiviral innate immune, irf3 emerges as a key molecule in regulating immune responses. on the other hand, it has been found to be associated with the expression of c-jun and mark, leading to nfatc1 activation and bone loss (20) . the activation of nf-κb signaling is directly associated with osteoclast differentiation and function. at the same time, it impairs both the differentiation of mscs towards the osteogenesis and osteoblast-mediated bone-forming capacity (21) . apart from that, nf-κb and ap-1 stimulate the expression of many elements which required for inflammatory cytokines, driving up osteoclast activity and usually implicated inhibition on proliferation and differentiation of osteoblasts (22) . in patients who suffered from sars-cov-2, lymphopenia is the most common property, along with that is the drastically reduced numbers in cd4+ t cells, cd8+ t cells and b cells (23) . such (25) . analyzing the inner link, we can infer that immune imbalance could disturb bone metabolism in a large extent, which exhibit a tendency to bone destruction in immunization, although the truth remains to be verified. for infected patients who are free from bone lesions, health education is a major method of controlling risk factors. we need to inform the patients about the possible bone destruction and joint inflammation through the health education. similarly, the mental health of the patient cannot be ignored. meanwhile, regular exercise or physical activity practice should be carried on, not only can it strengthen the immune system to defend infection, but also have a positive effect on bone quality and strength. for the infected elderly who have taken a turn for the better, it's important to routinely supplement calcium and vitamin d3 to prevent decreased bone matrix. apart from that, combining to the therapeutic experiences from sars-cov, we especially need to pay attention to the prescription and avoide the occurrence of femoral head necrosis steroids (26) . it's conjecturable that cytokine storm-induced systemic inflammation and immunologic dissonance ultimately increase bone resorption and restrain bone formation in bone marrow microenvironment. various kinds of pathological factors seem to share undesirable commonness in the process of bone healing. in clinic, it's essential to take precautions against the incidence of bone destruction and arthritis associated with covid-19. standard interventions to manage is the foundation of nursing and we need a team-based care model to resolve orthopedic problem in this special background. the authors declare that they have no known competing financial interestsor personal relationships that could have appeared to influence the work reported in this paper. covid-19: consider cytokine storm syndromes and immunosuppression oxidative stress and inflammation in cardiovascular disease current status of cell-based therapies for respiratory virus infections: applicability to covid-19 immune responses in covid-19 and potential vaccines: lessons learned from sars and mers epidemic immune responses and pathogenesis of sars-cov-2 during an outbreak in iran: comparison with sars and mers clinical features of patients infected with 2019 novel coronavirus in inflammation in bone physiology and pathology deficiency of ace2 in bone-marrow-derived cells increases expression of tnf-α in adipose stromal cells and augments glucose intolerance in obese c57bl/6 mice activation of rank/rankl/opg pathway is involved in the pathophysiology of fibrous dysplasia and associated with disease burden il-4 administration exerts preventive effects via suppression of underlying inflammation and tnf-α-induced apoptosis in steroidinduced osteonecrosis monosodium urate in the presence of rankl promotes osteoclast formation through activation of c-jun n-terminal kinase circulating microparticles in acute diabetic charcot foot exhibit a high content of inflammatory cytokines, and support monocyte-to-osteoclast cell induction hypoxic microenvironment and metastatic bone disease interleukin 17 under hypoxia mimetic condition augments osteoclast mediated bone erosion and expression of hif-1α and mmp-9 hypoxia inhibits the growth, differentiation and bone-forming capacity of rat osteoblasts the hif signaling pathway in osteoblasts directly modulates erythropoiesis through the production of epo hypoxia, oxidative stress and inflammation reactive oxygen species and oxidative stress in osteoclastogenesis, skeletal aging and bone diseases bone and the immune system hesperetin prevents bone resorption by inhibiting rankl-induced osteoclastogenesis and jnk mediated irf-3/c-jun activation nf-κb as a therapeutic target in inflammatory-associated bone diseases ap-1 and mitf interact with nfatc1 to stimulate cathepsin k promoter activity in osteoclast precursors covid-19: immunopathology and its implications for therapy physiological and pathophysiological bone turnover -role of the immune system b cells and t cells are critical for the preservation of bone homeostasis and attainment of peak bone mass in vivo clinical and basic research on steroid-induced osteonecrosis of the femoral head in japan declaration of competing interest the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work this study was supported by the national natural science foundation of china (81873991, 81771885, 81672238, 81472077, 81572183, 91849114 and 81472105) key: cord-303022-9hqoq7tf authors: madapusi balaji, thodur; varadarajan, saranya; vishal rao, u.s.; thirumal raj, a.; patil, shankaragouda; arakeri, gururaj; brennan, peter a title: oral cancer and periodontal disease increase the risk of covid 19? a mechanism mediated through furin and cathepsin overexpression date: 2020-06-01 journal: med hypotheses doi: 10.1016/j.mehy.2020.109936 sha: doc_id: 303022 cord_uid: 9hqoq7tf nan covid-19 was first reported in wuhan, china in december 2019 [1] . the infectious disease has spread rapidly and was upgraded by who to be a pandemic. it commonly presents as fever, dry cough, and dyspnea. in a minor proportion of patients as the disease progresses, it may lead to severe alveolar damage-causing respiratory distress, which can culminate in mortality [2] . the main route of human to human transmission has been suggested to occur by respiratory droplets released by the infected [3] , which has necessitated social distancing. despite numerous measures by health agencies the disease continues to afflict the human race. understandably, the past couple of months have seen a surge in the number of articles published on sars-corona virus-2. these articles largely consist of clinical case reports, molecular profiling of the virus, bio-informatic analysis, and hypothesis. among the hypothesis, the focus has largely been on identifying highrisk group, decoding pathogenic pathways, and in formulating therapeutic strategies. we present a hypothesis to recognize a potentially high-risk group and to strategize a prophylactic measure to reduce the risk of virus infection in the specific group. studies including bio-informatic analysis have shown the presence of angiotensin-converting enzyme 2 (ace2) receptors in oral mucosa, including the tongue, buccal mucosa, and gingiva [4] . similar to sars-corona virus-1, even the sars-corona virus-2 exhibits affinity towards ace2 receptors [2] . thus, oral mucosa could be a possible route for sars-corona virus-2 infection. oral mucosa in pathological states such as chronic periodontitis/oral cancer has shown to exhibit higher levels of osteopontin, which in turn can activate the p38 mitogen-activated protein kinase, stimulating nuclear factor-kappa b signaling and elevating the level of the protease furin [5] [6] [7] . in addition to furin, another protease cathepsin l is also elevated in chronic periodontitis and oral cancer, which in turn could be a result of the interleukin 6 mediated activation of the caveolin -1 mediated jnk-ap-1 signaling pathway [8] [9] [10] . both furin and cathepsin play a major role in enabling the sars-corona virus-2 to infect the host cells as elaborated in the following steps: 1) furin pre-cleaves the s glycoprotein of the sars-corona virus-2 into s1 and s2 subunits [11, 12] . 2) following the pre-cleavage of the s glycoprotein, the receptor-binding domain (rbd) of the s1 subunit attaches itself to the angiotensin-converting enzyme 2 (ace2) present in the host cells [13] . 3) following binding of the s1 subunit to the ace-2 receptors, the virus fuses with the host cell in two mechanisms: (a) endosomal fusion which is mediated by cysteine proteases cathepsin b/l and (b) plasma membrane fusion mediated by the serine protease tmprss2. the heptad repeat (hr) 1 and the hr2 of the s2 subunit form a six-helix bundle (6-hb) fusion core. the formation of this core brings the cell membrane of the virus and the host close allowing cell fusion and infection [12, 13] . based on the above-mentioned data, it can be hypothesized that the increased protease levels in chronic periodontitis and oral cancer could potentially increase the risk of an oral mucosa mediated sars-corona virus-2 infection (figure 1). in addition to increasing proteases, chronic periodontitis, and oral cancer patients have also reported having a low melatonin level [14, 15] . melatonin possesses anti-inflammatory, anti-oxidant properties. 13 also, melatonin has shown to inhibit cathepsin l [16] . thus, exogenous supplementation of the melatonin could aid in reducing the virus-induced inflammation, oxidative stress, and disrupting the cathepsin mediated fusion of virus and host cell. a novel coronavirus outbreak of global health concern a pneumonia outbreak associated with a new coronavirus of probable bat origin surface environmental, and personal protective equipment contamination by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) from a symptomatic patient high expression of ace2 receptor of 2019-ncov on the epithelial cells of oral mucosa plasma and crevicular fluid osteopontin levels in periodontal health and disease immunohistochemical analysis of osteopontin expression in oral squamous cell carcinoma p38 kinase is crucial for osteopontin-induced furin expression that supports cervical cancer progression expression of proteolytic cathepsins b, d, and l in periodontal gingival fibroblasts and tissues quantitative analysis of cathepsin l mrna and protein expression during oral cancer progression enhances cathepsin pathway in human gingival caveolin-1-mediated jnk-ap-1 b and l production via fibroblasts significant expression of furin and ace2 on oral epithelial cells may facilitate the efficiency of 2019-ncov entry sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor inhibition of sars-cov-2 (previously 2019-ncov) infection by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion salivary and gingival crevicular fluid melatonin in periodontal health and disease clinical significance of serum melatonin in predicting the severity of oral squamous cell carcinoma effect of melatonin administration on activities of some lysosomal enzymes in the mouse key: cord-310217-p9nqcz5d authors: nikolina, basic-jukic title: can hyperimmune anti-cmv globuline substitute for convalescent plasma for treatment of covid-19? date: 2020-05-31 journal: med hypotheses doi: 10.1016/j.mehy.2020.109903 sha: doc_id: 310217 cord_uid: p9nqcz5d information on treatment of covid-19 infection in renal transplant recipients is scarce, especially in symptomatic patients and patients with recent major clinical events. this group of patients suffers from different opportunistic infections which may coexist with covid-19. currently available expert opinions suggest reduction of immunosuppression therapy for renal transplant recipients with symptomatic covid-19 infection with either antiviral drugs, hydroxychloroquine and/or azithromycin. inspired by our experience in treatment of cmv pneumonia and literature data on the potential benefit of convalescent plasma for treatment of different viral diseases we suggest use of the hyperimmune anti-cmv gamma globulins in addition to other available therapies. besides the immunosuppression reduction which is supposed to be beneficial, immunoglobulins with their immunomodulatory effects and possible antiviral role, may increase a possibility for favorable outcome. since december 2019, the coronavirus covid-19 pandemic has affected almost 2,5 million people worldwide with more than 170.000 proven deaths by april 21th 2020 (1). renal transplant recipients are at increased risk for development of infection due to their immunocompromised state, but may also have more severe forms of the disease and an increased mortality risk due to numerous comorbidities. information on treatment of covid-19 infection in renal transplant recipients is scarce, especially in symptomatic patients and patients with recent major clinical events. current epidemiologic situation with the covid-19 pandemic present a great challenge for transplant physitians. lack of experience and well known fact that even in the simplest cases "one size does not fit all", we should more than ever focus on the individual approach to each patient. currently available expert opinions suggest reduction of immunosuppression therapy for renal transplant recipients with symptomatic covid-19 infection. however, a huge gap in knowledge exists for patients with additional problems besides the covid-19 infection. inspired by our experience in treatment of cmv pneumonia and literature data on the potential benefit of convalescent plasma for treatment of different viral diseases we suggest use of the hyperimmune anti-cmv gamma globulins in addition to other available therapies. besides the immunosuppression reduction which is supposed to be beneficial, immunoglobulins with their immunomodulatory effects and possible antiviral role, may increase a possibility for favorable outcome. hyperimmune anti-cmv immunoglobulin is a cmv-specific polyclonal immunoglobulin preparation that binds to cmv surface antigens neutralizing the potential of cmv from entering host cells. additionally, it presents the cmv particle for phagocytosis by binding to the cmv surface. finally, the preparation has immunomodulatory actions which may be beneficial. we decided to use hyperimmune anti-cmv globulins while the preparation contains immunoglobulins directed against the multiple viral pathogens (ebv, measles, parvovirus b19…) (2), and thus may imitate (at least partially) the convalescent plasma. convalescent plasma therapy, has been used in treatment of numerous infectious diseases including sars and mers pandemic (3) . based on the theory that it may neutralize viremia in patients with sars-cov-2 infection, one dose of 200 ml of convalescent plasma derived from recently recovered donors, was transfused to the patients along with the supportive care and antiviral drugs. the treatment was well tolerated, resulted with clinical and laboratory improvement, but with varying degrees of absorption of lung lesions (4, 5) . in conclusion, we suggest the use of hyperimmune anti-cmv immunoglobulins for treatment of covid-19 especially when occur as coinfection with cmv instead of the convalescent plasma which may be unavailable for majority of patient. the author declares no conflict of interests literature internal report. data on file the effectiveness of convalescent plasma and hyperimmune immunoglobulin for the treatment of severe acute respiratory infections of viral etiology: a systematic review and exploratory meta-analysis convalescent plasma as a potential therapy for covid-19 effectiveness of convalescent plasma therapy in severe covid-19 patients key: cord-306146-i4a74j3r authors: fitzgerald, paul j. title: noradrenergic and serotonergic drugs may have opposing effects on covid-19 cytokine storm and associated psychological effects date: 2020-06-09 journal: med hypotheses doi: 10.1016/j.mehy.2020.109985 sha: doc_id: 306146 cord_uid: i4a74j3r nan funding sources: none that directly supported this letter. in previous publications i have suggested that the neurotransmitters norepinephrine (ne) and serotonin (5ht), which are engaged in a wide range of physiological processes outside the brain as well, are largely functionally opposed to one another 1,2 . in addition to its role in various psychiatric, neurologic, and cardiovascular functions, there is increasing evidence that ne plays a role in the symptomatology associated with serious systemic infections such as influenza, including activation of cytokine signaling such as il-6 as well as the manifestation of psychological effects associated with infection 3 . it has also been suggested that catecholamines, including ne, play a role in producing the potentially lethal "cytokine storm" associated with coronavirus disease 2019 (covid-19) 4 . i suggest here that this cytokine storm may be counteracted by a range of clinically used drugs that reduce ne transmission (but that may not act through direct effects on viral replication or viral entry into cells) 5 : alpha2 agonists such as clonidine, guanfacine, dexmedetomidine; various beta blockers such as propranolol, nebivolol, carvedilol, atenolol; and various alpha1 antagonists such as prazosin 4 . since hdac inhibitors like valproic acid and vorinostat may also decrease ne transmission 6 , they may also be therapeutic in covid-19. since 5ht may be functionally opposed to ne, drugs that facilitate 5ht transmission may also dampen the cytokine storm associated with covid-19: ssris (fluoxetine, citalopram, sertraline, paroxetine, fluvoxamine; tca (clomipramine); maois (phenelzine, tranylcypromine); and the 5ht2c agonist lorcaserin. there are also theoretical underpinnings supporting functional opposition between ne and the neurotransmitter acetylcholine 7 , where the latter molecule participates in the cholinergic antiinflammatory pathway 8 . in this scenario, clinically available cholinesterase inhibitors (pyridostigmine, donepezil, rivastigamine, galantamine, physostigmine) may also counteract the cytokine storm associated with covid-19. there are already several ongoing clinical trials, in various countries, testing whether some of the above pharmacological agents reduce morbidity and mortality accompanying covid-19. i suggest that all of the above compounds are promising candidates for additional prospective clinical trials, and they can also be examined immediately through retrospective epidemiological investigation of the medical records of individuals who were taking these drugs for various indications and were hospitalized and had pneumonia or acute respiratory distress, where their clinical outcomes may be relevant to treating today's ongoing covid-19 pandemic 4 . if covid-19 is accompanied by systemically elevated ne signaling, the various drugs listed above may also counteract deleterious psychological effects associated with this infection 3 . the author declares that he has no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. gamma oscillations as a biomarker for major depression: an emerging topic in vivo electrophysiological recordings of the effects of antidepressant drugs serious infection may systemically increase noradrenergic signaling and produce psychological effects covid-19 using α-1 adrenergic receptor antagonists can beta-adrenergic blockers be used in the treatment of covid-19? med hypotheses vorinostat increases expression of functional norepinephrine transporter in neuroblastoma in vitro and in vivo model systems the neuro-immuno-senescence integrative model (nisim) on the negative association between parasympathetic activity and cellular senescence conflicts of interest the author reports no conflicts of interest, and no funding source directly supported this work key: cord-308279-gsk4qel5 authors: suzuki, yuichiro j. title: the viral protein fragment theory of covid-19 pathogenesis date: 2020-09-11 journal: med hypotheses doi: 10.1016/j.mehy.2020.110267 sha: doc_id: 308279 cord_uid: gsk4qel5 severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is causing the current pandemic of coronavirus disease 2019 (covid-19) that have killed nearly one million people so far. while this appears to be a respiratory virus, surprisingly, it has been recognized that patients with cardiovascular disease are likely to be affected severely and die of covid-19. this phenomenon cannot be explained by the generally accepted logic that the sars-cov-2 infection/replication is the sole determinant of the actions of the virus to define the fate of host cells. i herein propose the viral protein fragment theory of covid-19 pathogenesis based on my observations in cultured human vascular cells that sars-cov-2 spike protein can activate cell signaling events without the rest of the viral components. it is generally thought that sars-cov-2 and other single-stranded rna viruses attach to the host cells through the interactions between surface proteins of the viral capsid and the host cell receptors; the fusion and the entry of the viral components, resulting in the replication of the viruses; and the host cell responses are the consequence of these events. i hypothesize that, as humans are infected with sars-cov-2, the virus releases a fragment of the spike protein that can target host cells for eliciting cell signaling without the rest of the viral components. thus, covid-19 patients are subjected to the intact virus infecting the host cells for the replication and the amplification as well as the spike protein fragment that are capable of affecting the host cells. i propose that cell signaling elicited by the spike protein fragment that occurs on cardiovascular cells would predispose infected individuals to develop complications that are seen in severe and fatal covid-19 conditions. if this hypothesis is correct, then the strategies to treat covid-19 should include, in addition to giving agents that inhibit the viral replication, therapeutics that inhibit the virus fragment-mediated cell signaling on cardiovascular cells. severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is causing the current pandemic of coronavirus disease 2019 (covid-19) that have killed nearly one million people so far. while this appears to be a respiratory virus, surprisingly, it has been recognized that patients with cardiovascular disease are likely to be affected severely and die of covid-19. this viruses invade host cells, resulting in cell death. the major target cells for sars-cov-2 should be respiratory cells. however, surprisingly, patients with cardiovascular disease, rather than respiratory disease, are predisposed to severe covid-19 symptoms and death. to explain this phenomenon, i herein propose the viral protein fragment theory of covid-19 pathogenesis ( fig. 1) . this hypothesis is based on my experimental observations in cultured human vascular cells that the recombinant full length s1 subunit of sars-cov-2 spike protein can activate cell signaling events without the rest of the viral components. 11 thus, the pathology of covid-19 may not merely depend on the spike protein serving as a fusion protein to facilitate the viral entry and the infection. i propose a scenario that, as humans are infected with sars-cov-2, the virus releases a fragment of the spike protein that can target host cells for eliciting cell signaling. thus, infected patients would have at least two entities introduced in response to the sars-cov-2 infection. one is the intact virus that uses its spike protein to target ace2, resulting in the entry into the host cells for the viral replication and amplification. the other is the circulating fragment of the spike protein that independently elicits actions that ultimately results in severe pathological conditions. my experiments using cultured vascular cells also showed that, while the full length s1 subunit of sars-cov-2 spike protein elicits cell signaling, the shorter protein that only contains the receptor binding domain (rbd) does not. 11 these results suggest that other regions of the s1 subunit are responsible for eliciting cell signaling in host cells. thus, if the sars-cov-2 spike protein fragment that has been released into our blood is responsible for the pathogenesis of severe covid-19, therapies that target the viral replication such as inhibitors of rna-dependent rna polymerase alone would not work. also, if the regions of spike protein other than the rbd are responsible for eliciting pathological cell signaling and if the fragment is released quickly after the host is exposed to sars-cov-2, then vaccines that target rbd would not affect the viral fragment-mediated cell signaling. thus, therapeutic strategies to inhibit viral fragment-mediated cell signaling in cardiovascular cells, in addition to drugs that inhibits the viral replication, are necessary to reduce the covid-19-associated death. my specific hypothesis is that certain viruses shed fusion protein fragments that circulate in the blood that, in turn, elicit cell signal transduction that makes the infected individuals with cardiovascular diseases predisposed to severe covid-19 conditions and death. thus, infected patients would have at least two entities that may cause complications when they are infected with sars-cov-2: (i) virus itself that gets incorporated into the host cells where the viral replication and amplification occur; and (ii) components of viral fusion proteins (i.e. spike protein for sars-cov-2) that are circulated to elicit distinct processes that promote pathologic conditions. this is based on my observations that recombinant sars-cov-2 spike protein (without the rest of the viruses) is capable of eliciting cell signaling in cultured human vascular cells. 11 interestingly, my experiments also showed that rbd domain-only containing protein region of sars-cov-2 spike protein does not activate cell signaling, suggesting the role of other regions with in the s1 subunit of the sars-cov-2 spike protein to activate this cell signaling event. thus, the first experiments that are needed to test this hypothesis is to identify the regions of sars cov-2 spike protein that are responsible for activating cell signaling, perhaps by using cultured human cells. after identifying active protein regions of the s1 subunit of sars-cov-2 spike protein, the occurrence of protein fragments that possess such protein sequences should be detected in patients infected with sars-cov-2. experimental animals should also be employed to determine whether the administrations of such fragments promotes cardiovascular disease conditions or worsens the existing cardiovascular pathology. i herein propose the viral protein fragment theory of covid-19 pathogenesis, in which a fragment or fragments of sars-cov-2 spike protein is/are released into the patients' blood circulation, predisposing patients with cardiovascular disease conditions to severe covid-19 outcomes. thus, covid-19 deaths of patients with cardiovascular co-comorbidity are due to the death of host cells as a consequence of the viral infection and replication as well as the spike protein fragment-mediated cell signaling in human host cardiovascular cells. the current focus for developing therapeutic strategies against covid-19 focuses on the vaccine development and searching for agents that inhibit the viral replication. if my hypothesis is correct, it is critical to also inhibit the unexpected biological events that are elicited by sars-cov-2 such as the release of spike protein fragments that affect cardiovascular cells in addition to inhibiting the viral replication in order to successfully reduce the mortality and morbidity associated with covid-19 and to end the pandemic. epidemiology, genetic recombination, and pathogenesis of coronaviruses the molecular biology of sars coronavirus a new coronavirus associated with human respiratory disease in china clinical features of patients infected with 2019 novel coronavirus in wuhan structural basis for the recognition of sars-cov-2 by full-length human ace2 characterization of the receptor-binding domain (rbd) of 2019 novel coronavirus: implication for development of rbd protein as a viral attachment inhibitor and vaccine pathological findings of covid-19 associated with acute respiratory distress syndrome risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease prevalence and impact of cardiovascular metabolic diseases on covid-19 in china prevalence of comorbidities and its effects in patients infected with sars-cov-2: a systematic review and meta-analysis sars-cov-2 spike protein-mediated cell signaling in lung vascular cells this work was supported in part by nih (r21ai142649, r03ag059554, and r03aa026516) to y.j.s. the content is solely the responsibility of the authors and does not necessarily represent the official views of the nih. the author has no conflict of interest. key: cord-312912-i1yyz5lg authors: bou khalil, rami title: lithium chloride combination with rapamycin for the treatment of covid-19 pneumonia date: 2020-05-01 journal: med hypotheses doi: 10.1016/j.mehy.2020.109798 sha: doc_id: 312912 cord_uid: i1yyz5lg nan dear sir, the current rapid spread of the coronavirus disease (covid-19) originating from wuhan, china, has been recently declared a pandemic by the world health organization [1] . meanwhile the number of deceased individuals from covid-19 has been exponentially increasing. any live-attenuated vaccines may take time and give a low level of cross-strain immunity which renders the research for effective antiviral agents a high necessity. lithium chloride is a known efficacious treatment for bipolar disorder mainly because of its direct inhibition on glycogen synthase kinase 3β, a phosphorylating and inactivating agent of glycogen synthase involved in energy metabolism, neuronal cell development, and body pattern formation [2] . since lithium chloride has been demonstrated efficient in the treatment of human herpes simplex virus via its role in inhibiting viral dna synthesis, it has been essayed as an antiviral agent especially for coronaviruses infections in many studies. as a matter of fact, lithium chloride seems to be a protective agent against the infective effect of the avian coronavirus infectious bronchitis virus mostly through an inhibition of viral protein production at the level of genomic rna and subgenomic mrna synthesis without affecting host cell protein production [3, 4] . in addition, lithium chloride has been shown to inhibit the entry and replication of the porcine epidemic diarrhea virus into cells [5] . rapamycin and its analogs are clinically important macrolide compounds produced by streptomyces hygroscopicus. streptomyces are bacteria that live in symbiosis with plants and it is expected that rapamycin is one of the products that helps the plants fight against pathogens such as other bacteria, fungi and viruses [6] . the mammalian target of rapamycin (mtor) is a serine/threonine kinase that functions as a central regulator of cell growth and metabolism. rapamycin and its analogs are specific inhibitors of mtor kinase and, as a result, are wellestablished immunosuppressants and antitumorigenic agents that play a role in cell proliferation and survival as well as macroautophagy suppression [7] . when rapamycin forms a complex with its receptor and interacts with mtor, this blocks the cell-cycle progression of t cells thereby suppressing their induced proliferation by cross-linking of the t-cell receptors, antigenic peptides or cytokines such as inteleukin-2 (il-2) [8] . on another level, mtor complex and glycogen synthase kinase 3β have a direct mutual influence in order to regulate the immune response since mtor complex inhibition regulates pro-and antiinflammatory cytokine production via its capacity to inactivate glycogen synthase kinase 3β [9] . in the transmissible gastroenteritis virus, autophagy seems to play an important role in this coronavirus infection. treatment with rapamycin seems to induce autophagy and to protect cells against viral replication [10] . patients with the most important lung injury related to covid-19 are those who have a dysregulated response for the viral infection predominating in t cells function [11] . it might be hypothesized that covid-19 may delay the interferon response until the virus has sufficiently replicated which causes a sensitization of t cells towards apoptosis and macrophage stimulation. patients with the highest threshold for interferon-response such as the elderly seem to be most affected by covid-19 pneumonia. the rationale of combining lithium chloride and rapamycin consists on reducing the possibility of viral replication at its lowest by reducing viral entry to cells and viral rna synthesis (the effect of lithium due to protein synthesis inhibition via glycogen synthase kinase 3β deactivation) from one part, and regulating the immune response and stimulating autophagy without apoptosis from another part (the effect of rapamycin via mtor inhibition and t cell regulation). clinical studies are further needed in this domain. the-media-briefing-on-covid oldie but goodie: lithium in the treatment of bipolar disorder through neuroprotective and neurotrophic mechanisms lithium chloride inhibits the coronavirus infectious bronchitis virus in cell culture comparative analysis of the effect of glycyrrhizin diammonium and lithium chloride on infectious bronchitis virus infection in vitro antiviral effect of lithium chloride on porcine epidemic diarrhea virus in vitro antibiotics produced by streptomyces mtor inhibitors lower an intrinsic barrier to virus infection mediated by ifitm3 biosynthesis of rapamycin and its regulation: past achievements and recent progress convergence of the mammalian target of rapamycin complex 1-and glycogen synthase kinase 3-β-signaling pathways regulates the innate inflammatory response autophagy negatively regulates transmissible gastroenteritis virus replication dysregulation of immune response in patients with covid-19 in wuhan, china key: cord-318114-q29zax2j authors: bara, gregor a.; de ridder, dirk; maciaczyk, jaroslaw title: can neuromodulation support the fight against the covid19 pandemic? transcutaneous non-invasive vagal nerve stimulation as a potential targeted treatment of fulminant acute respiratory distress syndrome date: 2020-07-18 journal: med hypotheses doi: 10.1016/j.mehy.2020.110093 sha: doc_id: 318114 cord_uid: q29zax2j the covid-19 pandemic has rapidly spread all over the world and caused a major health care crisis. about 20% of patients develop severe disease and require hospitalisation, which is associated with a high mortality rate of up to 97% in those being ventilated and respiratory failure being the leading cause of death. despite many therapeutic agents being under current investigation there is yet no panacea available. with increasing rates of infection throughout the world, there is an urgent need for new therapeutic approaches to counteract the infection. as the nervous system has shown to be a strong modulator of respiratory function and the immune response, we want to highlight pathways involved in regulation of respiratory function, the neuro-immune axis as well as the rationale for a potential targeted treatment of fulminant acute respiratory distress syndrome via transcutaneous non-invasive vagal nerve stimulation in critically-ill covid-19 patients. can neuromodulation support the fight against the covid19 pandemic? transcutaneous non-invasive vagal nerve stimulation as a potential targeted treatment of fulminant acute respiratory distress syndrome gregor this is a pdf file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. this version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. dear dr. manku, i am writing to submit our manuscript entitled, "can neuromodulation support the fight against the covid19 pandemic? transcutaneous non-invasive vagal nerve stimulation as a potential targeted treatment of fulminant acute respiratory distress syndrome" for consideration as a clinical opinion in your very much respected journal. we discuss the current threat of the covid-19 pandemic and present an innovative approach for targeted treatment of the disease. we underscore the underlying physiology of the strong interconnection of the immune system and the nervous system. we present vagus nerve stimulation as a potential non invasive treatment and present clinical data to undermine the hypothesis. we draw the conclusion that vagus nerve stimulation may be a supporting treatment in the therapy continuum of covid-19 patients by combined modulation of respiratory function as well as the neuro-immune axis. each of the authors confirms that this manuscript has not been previously published and is not currently under consideration by any other journal. additionally, all of the authors have approved the contents of this paper and have agreed to the submission policies. i am happy to answer any further questions and can be contacted as follows: email: gregor.bara@ukbonn.de mobile: +49-172-5831998 i am very much looking forward to your response. yours sincerely, hypothesis: can neuromodulation support the fight against the covid19 pandemic bara 1 , dirk de ridder 2 , jaroslaw maciaczyk 1,2 1. stereotactic and functional neurosurgery none for all authors. key: cord-314679-lmfalzni authors: sangith, nikhil; diagnotek, xact title: unique fibrinogen-binding motifs in the nucleocapsid phosphoprotein of sars cov-2: potential implications in host-pathogen interactions date: 2020-06-24 journal: med hypotheses doi: 10.1016/j.mehy.2020.110030 sha: doc_id: 314679 cord_uid: lmfalzni novel coronavirus (sars cov-2), the etiological agent for the highly contagious corona virus disease-2019 (covid-19) pandemic has threatened global health and economy infecting around 5.8 million people and causing over 359,200 deaths (as of 28(th) may, 2020, https://www.worldometers.info/coronavirus/). the clinical manifestations of infected patients generally range from asymptomatic or mild to severe illness, or even death. the ability of the virus to evade the host immune response have been major reasons for high morbidity and mortality. one of the important clinical observations under conditions of critical illness show increased risk of developing disseminated intravascular coagulation. molecular mechanisms of how sars cov-2 induces such conditions still remain unclear. this report describes the presence of two unique motifs in the sars cov-2 nucleocapsid phosphoprotein (n-protein) that can potentially interact with fibrinogen and possibly prothrombin. this is based on an established function of secretory proteins in staphylococcus aureus (s. aureus)coagulase, efb (extracellular fibrinogen binding) and vwbp (von willebrand factor binding protein), which are known to regulate the blood clotting cascade and the functions of host immune response. it is hypothesized that having protein interaction motifs that are homologous to these s. aureus proteins, the n-protein of this virus can mimic their functions, which may in turn play a crucial role in formation of blood clots in the host and help the virus evade host immune response. however, this hypothesis needs to be tested in vitro. considering the overwhelming increase in the incidence of sars cov-2 infection globally, this information may be useful for further investigation and could help in deducing new therapeutic strategies to combat advanced stages of this disease. signalling pathways to escape from the host immune response. fibrinogen interaction with coagulase and efb of s. aureus is well established (5, 7) . the role of the fibrinogen-binding motif of n-protein in formation of blood clots and 142 mimicking functions of efb for pathogen survival in host will be investigated. the 143 affinity of the motif towards fibrinogen will play a major role in regulating the functions 144 coagulation disorders in coronavirus infected patients journal of clinical virology : the 168 official publication of the pan american society for abnormal coagulation parameters are associated with poor 170 prognosis in patients with novel coronavirus pneumonia immune responses in covid-19 and potential 173 vaccines: lessons learned from sars and mers epidemic the sars coronavirus 176 nucleocapsid protein--forms and functions staphylococcus aureus have a common fibrinogen binding motif multiple ligands of von willebrand factor-binding 181 protein (vwbp) promote staphylococcus aureus clot formation in human plasma binding of efb from staphylococcus 184 aureus to fibrinogen blocks neutrophil adherence staphylococcus aureus secretes coagulase 187 and von willebrand factor binding protein to modify the coagulation cascade and establish 188 host infections fibrin(ogen)-alpha m beta 2 interactions regulate leukocyte 190 function and innate immunity in vivo leukocyte engagement of fibrin(ogen) via the integrin 193 receptor alphambeta2/mac-1 is critical for host inflammatory response in vivo phagocytosis escape by a staphylococcus aureus 196 protein that connects complement and coagulation proteins at the bacterial surface coronaviruses hijack the complement system of fibrinogen and in turn the host immune system. at this juncture, the knowledge of 145 existence of such a motif will help investigators to further probe and establish its role key: cord-293860-6kz0iws6 authors: qutayba almerie, muhammad; daniel kerrigan, david title: the association between obesity and poor outcome after covid-19 indicates a potential therapeutic role for montelukast date: 2020-05-27 journal: med hypotheses doi: 10.1016/j.mehy.2020.109883 sha: doc_id: 293860 cord_uid: 6kz0iws6 it is widely believed that infection with the sars-cov2 virus triggers a disproportionate immune response which causes a devastating systemic injury, particularly in individuals with obesity, itself a chronic, multi-organ inflammatory disease. immune cells accumulate in visceral adipose tissue and together with paracrine adipocytes release a wide range of biologically active cytokines (including il-1β, il5, il6 and il8) that can result in both local, pulmonary and systemic inflammation. a more intense ‘cytokine storm’ is postulated as the mechanism behind the extreme immune response seen in severe covid-19. it is striking how dangerous the combination of obesity and covid-19 is, resulting in a greater risk of icu admission and a higher mortality. furthermore, patients from a bame background appear to have increased mortality after sars-cov2 infection; they also have a higher prevalence of central obesity and its metabolic complications. in the absence of an effective vaccine, the therapeutic potential of immune-modulating drugs is a priority, but the development of new drugs is expensive and time-consuming. a more pragmatic solution would be to seek to repurpose existing drugs, particularly those that might suppress the heightened cytokine activity seen in obesity, the major risk factor for a poor prognosis in covid-19. montelukast is a cysteinyl leukotriene receptor antagonist licensed to treat asthma and allergic rhinitis. it has been shown to diminish pulmonary response to antigen, tissue eosinophilia and il-5 expression in inflammatory cells. it has also been shown to decrease elevated levels of il-1β and il8 in humans with viral upper respiratory tract infections compared with placebo-treated patients. in addition, in silico studies have demonstrated a high binding affinity of the montelukast molecule to the terminal site of the virus’s main protease enzyme which is needed for virus rna synthesis and replication. montelukast, which is cheap, safe and widely available would appear to have the potential to be an ideal candidate drug for clinical trials, particularly in early stage disease before irreparable tissue damage has already occurred. hypothesis: through a direct anti-viral effect, or by suppression of heightened cytokine release in response to sars-cov2, montelukast will reduce the severity of immune-mediated multiorgan damage resulting from covid-19, particularly in patients with central obesity and metabolic syndrome. in the absence of an effective vaccine, the therapeutic potential of immune-modulating drugs is a priority, but the development of new drugs is expensive and time-consuming. a more pragmatic solution would be to seek to repurpose existing drugs, particularly those that might suppress the heightened cytokine activity seen in obesity, the major risk factor for a poor prognosis in covid-19. montelukast is a cysteinyl leukotriene receptor antagonist licensed to treat asthma and allergic rhinitis. it has been shown to diminish pulmonary response to antigen, tissue eosinophilia and il-5 expression in inflammatory cells. it has also been shown to decrease elevated levels of il-1 and il8 in humans with viral upper respiratory tract infections compared with placebo-treated patients. in addition, in silico studies have demonstrated a high binding affinity of the montelukast molecule to the terminal site of the virus's main protease enzyme which is needed for virus rna synthesis and replication. montelukast, which is cheap, safe and widely available would appear to have the potential to be an ideal candidate drug for clinical trials, particularly in early stage disease before irreparable tissue damage has already occurred. through a direct anti-viral effect, or by suppression of heightened cytokine release in response to sars-cov2, montelukast will reduce the severity of immune-mediated multiorgan damage resulting from covid-19, particularly in patients with central obesity and metabolic syndrome. it is widely believed that infection with the sars-cov2 virus triggers a disproportionate immune response which causes a devastating systemic injury, particularly in individuals with obesity, itself a chronic, multi-organ inflammatory disease. immune cells accumulate in visceral adipose tissue and together with paracrine adipocytes release a wide range of biologically active cytokines (including il-1, il5, il6 and il8) that can result in both local, pulmonary and systemic inflammation 1-3 . a more intense 'cytokine storm' is postulated as the mechanism behind the extreme immune response seen in severe covid-19. it is striking how dangerous the combination of obesity and covid-19 is, resulting in a greater risk of icu admission and a higher mortality 4 , suggesting that the already heightened background inflammatory process resulting from obesity might prime the immune system for a more catastrophic response to sar-cov2 infection. furthermore, patients from a bame background appear to have increased mortality after sars-cov2 infection 5 , an observation that has at least partly ben explained by the higher prevalence of central obesity and its metabolic complications in this group 6,7 . in the absence of an effective vaccine, exploring the therapeutic potential of immunemodulating drugs is a priority, but the development of new drugs is expensive and time-consuming. a more pragmatic solution would be to seek to repurpose existing drugs, particularly those that might suppress the heightened cytokine activity seen in obesity, the major risk factor for a poor prognosis in covid-19. montelukast is a cysteinyl leukotriene receptor antagonist licensed to treat asthma and allergic rhinitis. it has been shown to diminish pulmonary response to antigen, tissue eosinophilia and il-5 expression in inflammatory cells 8 . it has also been shown to decrease elevated levels of il-1 and il8 in humans with viral upper respiratory tract infections compared with placebo-treated patients 9 . in addition, in silico computer modelling studies have demonstrated a high binding affinity of the montelukast molecule to the sars-cov2 virus's main protease enzyme substrate-binding pocket that is involved in virus rna synthesis and replication 10, 11 . evidence supporting montelukast as a candidate covid-19 therapy in individuals with obesity: patients affected by severe obesity share a common physiological response with patients with covid-19 as both have raised concentrations of pro-inflammatory cytokines (e.g., tnf-a, il1 and il6) and t-helper-2 cytokines (il4, il10) [12] [13] [14] . whilst it is impossible to exclude increased viral burden as the driver for this inflammatory response in covid-19, there is evidence that some patients develop 'cytokine storm (release) syndrome', described as an uncontrolled and overwhelming release of proinflammatory mediators by an overly-activated immune system 12, 13, 15 . an overwhelming production of proinflammatory mediators in the lungs causes localised pulmonary injury characterised by diffuse alveolar damage with epithelial and endothelial apoptosis, and thrombosis due to dysregulated coagulation and fibrinolysis. these mediators may also leak into systemic circulation leading to cardiac, renal and hepatic inflammation and multiorgan failure 16 . the background chronic inflammatory state associated with obesity could act as a catalyst for the a massive inflammatory response to sars-cov2 by priming the immune system, triggered by the 'second hit' of covid-19, and explaining why those with obesity are more likely to become severely ill, require icu care and die. severe lymphopenia with hyperactivated proinflammatory t cells and decreased regulatory t cells are commonly seen in critically ill covid-19 patients supporting the likelihood of a dysregulated immune response to the viral infection 12 . leukotrienes (ltc4, ltd4, and lte4) are peptide-conjugated lipids that are prominent products of activated eosinophils, basophils, mast cells and macrophages 17, 18 . they are generated de novo from cell membrane phospholipid-associated arachidonic acid via the 5-lipoxygenase pathway. known to cause contraction of bronchial smooth muscle, leukotrienes are now also recognised as potent inflammatory mediators that initiate and propagate a diverse array of biologic responses including macrophage activation, mast cell cytokine secretion and dendritic cell maturation and migration 17, 18 . animal studies indicate a significant role for leukotrienes in adaptive immunity, particularly the induction of th2 response in the lung via their effect on dendritic cells and cytokine generation (mainly il4), the recruitment and/or activation of effector cells (especially eosinophils and mast cells), pulmonary inflammation, microvascular permeability and pulmonary fibrosis 17, 19 . montelukast 20,21 (montelukast sodium: c 35 h 36 clno 3 s) is a leukotriene receptor antagonist which demonstrates a marked affinity and selectivity to the cysteinyl leukotriene receptor type-1, thus it elicits substantial blockage of any ltd4 leukotriene-mediated effect, resulting in reduced inflammation and relaxation of smooth muscle. it has also been shown to diminish the pulmonary response to antigen, tissue eosinophilia and the number of inflammatory cells expressing il-5 8 . montelukast decreases elevated levels of il-1 and il8 in nasal washouts in humans with viral upper respiratory tract infections compared with placebo-treated patients 9 . montelukast was first approved for clinical use by the us fda in 1998. it is currently licensed to treat asthma, exercise-induced bronchospasm and allergic rhinitis. montelukast 10mg is given orally once daily (oral bioavailability 64%). it is highly metabolised in the liver by cytochrome p450. the drug and its metabolites are almost exclusively excreted in the bile and into the faeces. with its prominent effect in reducing leukotriene-mediated cytokine release montelukast would have the potential to moderate the background inflammation associated with obesity and the body's inflammatory response to sars-cov2. coronaviruses are enveloped viruses with a positive rna genome. the virus contains at least four structural proteins: spike (s) protein, envelop (e) protein, membrane (m) protein, and nucleocapsid (n) protein 22, 23 . sars-cov-2 entry into the host cells is mediated by an interaction between the spike protein and angiotensin-converting enzyme 2 (ace2) receptors 24, 25 , cell surface proteins highly expressed in the lung, heart and kidney. wu et al 10 performed in silico, target-based virtual ligand screening for sars-cov-2 main proteins including the main protease, spike protein, rna-dependent rna polymerase and papain-like protease. the authors screened approved drugs from a subset of the zinc database which contains 2924 compounds 26 . they also screened a natural product database of 1,066 chemicals separated from traditional chinese herbals and 78 known anti-viral drugs. their computer modelling study found that montelukast should demonstrate binding energy to the virus's main protease enzyme. it fitted well into the active pocket of the main protease, in which hydrophobic amino acids would create a relatively hydrophobic environment to contain the drug and stabilise its conformation 10 . similarly, a report by farag et al 11 predicted a high affinity of montelukast to the same binding domain in the main protease enzyme with hydrophobic-hydrophobic and hydrogen bond-interactions. farag et al 11 focussed on targeting the sars-cov-2 main protease enzyme at its two potential binding sites in the main substrate-binding pocket: the central site and terminal site. the authors screened >2,000 fda-approved drugs to identify potential hits based on drug molecules' binding energies, binding modes and the predicted interaction of amino acids with the enzyme's substrate binding pockets. whilst antiviral drugs unsurprisingly had the best binding to the central site of the main protease enzyme substrate-binding pocket, montelukast demonstrated a very high affinity for the terminal site, better than any other drug tested at this site whist drug binding in in silico modelling studies such as these may not translate into clinical effect, the protease enzyme is essential for the sars-cov-2 virus rna synthesis and replication and if montelukast was shown to confer benefit in clinical studies it may be, at least in part, due drug-induced disruption of the virus replication cycle. one of the main treatment strategies for covid-19 is to identify new targeted antiviral drugs based on the genomic information and pathological characteristics of sars-cov-2. these drugs are likely to be the most effective against the virus. however, anti-viral drug development and registration is time-consuming, the drug might not be available for the current outbreak and it is likely to be very expensive, an important factor particularly in poorer countries afflicted by covid-19. an alternative, faster strategy is 'drug repurposing'. this relies on identification of potential therapeutic characteristics of drugs currently licensed for other purposes. there are many clinical trials enrolling patients with covid-19 worldwide investigating the potential benefits of established antimicrobial, antiviral and anti-inflammatory drugs 27 . the strong association between the pro-inflammatory state found in metabolic syndrome and obesity and a more aggressive clinical course in covid-19 suggests a potential treatment role for drugs that inhibit cytokine release and macrophage activation. there is, however, so far no convincing clinical evidence to support the use of corticosteroids, one of the most widely utilised anti-inflammatory agents. if anything, there is concern about patients being harmed with such a treatment 28, 29 . anti-cytokines (interleukin 6 inhibitors, e.g., tocilizumab) have also attracted much interest recently 30 with a few ongoing clinical trials assessing their role 27 . however, these monoclonal antibodies are expensive and like corticosteroids have the potential for promoting secondary infections. cost and complications are likely to limit their application on a global scale, other than selective use in severely ill patients 30, 31 . we believe there is a good theoretical basis to consider montelukast as a therapeutic candidate with a hypothetical dual mode of action through suppression of cytokine activity and interference with viral replication. to date, the cochrane covid-19 study register does not identify any studies exploring the clinical benefits of montelukast. whilst it is unlikely that montelukast would be effective at curing covid-19 or reversing severe immune-modulated multiorgan damage, it could have a powerful role in preventing disease progression when used at an early stage in the disease, reducing the need for hospital admission, or the requirement for the care of hospitalised patients to be escalated up to level 3 critical care. it may shorten the course of mild to moderate covid-19 with the attendant social, economic and healthcare system benefits that would bring. furthermore, it has a good safety profile. but perhaps most importantly, if montelukast was shown to be of clinical benefit in well-conducted studies it is a widely applicable treatment, accessible to all patients, and at just over £4 per month 21 , not just available to those who live in nations with firstworld healthcare resources that can afford highly expensive new drugs. neither of the authors have any conflicting interests and neither receive funding from any third party or external source. professor kerrigan was the original creator of the hypothesis linking potential therapeutic benefits of montelukast in covid-19, with the inflammatory state associated with obesity. both authors were involved in background research and the writing of the manuscript. this paper did not require ethics approval. regulation of adipose tissue inflammation by interleukin 6 circulating levels of mcp-1 and il-8 are elevated in human obese subjects and associated with obesity-related parameters inflammatory cytokines in general and central obesity and modulating effects of physical activity features of 16,749 hospitalised uk patients with covid-19 using the isaric who clinical characterisation protocol are more black, asian and minority ethnic people dying with covid-19 than might be expected? southall and brent revisited: cohort profile of sabre, a uk population-based comparison of cardiovascular disease and diabetes in people of european, indian asian and african caribbean origins is ethnicity linked to incidence or outcomes of covid-19? montelukast, a leukotriene receptor antagonist, inhibits the late airway response to antigen, airway eosinophilia, and il-5-expressing cells in brown norway rats effect of montelukast on pro-inflammatory cytokine production during naturally acquired viral upper respiratory infections (vuris) in adults analysis of therapeutic targets for sars-cov-2 and discovery of potential drugs by computational methods identification of fda approved drugs targeting covid-19 virus by structure-based drug repositioning (pre-print) dysregulation of immune response in patients with covid-19 in wuhan, china. clin infect dis clinical features of patients infected with 2019 novel coronavirus in wuhan clinical features of 69 cases with coronavirus disease covid-19: consider cytokine storm syndromes and immunosuppression pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology cysteinyl leukotrienes and their receptors: cellular distribution and function in immune and inflammatory responses cysteinyl leukotrienes: multifunctional mediators in allergic rhinitis leukotriene e4 and granulocytic infiltration into asthmatic airways singulair (montelukast sodium) us fda label coronaviruses -drug discovery and therapeutic options structural insights into coronavirus entry a pneumonia outbreak associated with a new coronavirus of probable bat origin structure, function, and antigenicity of the sars-cov-2 spike glycoprotein zinc: a free tool to discover chemistry for biology a real-time dashboard of clinical trials for covid-19 clinical evidence does not support corticosteroid treatment for 2019-ncov lung injury immunosuppression for hyperinflammation in covid-19: a double-edged sword? can we use interleukin-6 (il-6) blockade for coronavirus disease 2019 (covid-19)-induced cytokine release syndrome (crs)? why tocilizumab could be an effective treatment for severe covid-19? key: cord-313382-prxc0lue authors: hossein norooznezhad, amir; hantoushzadeh, sedigheh; shamshirsaz, alireza a. title: empiric antibiotics in management of inpatient pregnant women infected with coronavirus disease 2019 (covid-19): focusing on inflammation and preterm labor date: 2020-09-12 journal: med hypotheses doi: 10.1016/j.mehy.2020.110269 sha: doc_id: 313382 cord_uid: prxc0lue nan the outbreak of novel coronavirus (srars-cov-2) caused coronavirus disease 2019 (covid-19) and led to emergency pandemic according to the world health organization (who) [1] . this disease with the main manifestation of pneumonia [2] may cause a wide range of morbidities which might lead to mortality [3] . not only the maternal outcomes but also fetal/neonatal outcomes might be affected by covid-19. data have shown that prevalence of preterm birth among inpatient pregnant women with covid-19 up to 47% [4] [5] [6] . herein, we would evaluate one of possible cause of non-viral induced preterm labor in pregnant women diagnosed with covid-19 which could be administration of empiric antibiotics with inflammatory properties in absence of bacterial infection in the inpatients. in a recent paper [7] , we have reviewed the role of inflammation as one of the most important key elements of preterm labor. although, in most cases the inflammation cascade for preterm labor has been started with a bacterial infection. however, we have highlighted that the use of antibiotics in the absence of bacterial infection might result in the release of pro-inflammatory cytokine and therefore, an inflammatory storm. as we have shown, antibiotics (in absence of infection) seem to cause gut bacterial decontamination and lipopolysaccharide (lps) release and therefore endotoxemia. during this endotoxemia, the activation of toll-like receptors (tlrs) cause the release/expression of pro-inflammatory cytokines (such as interleukin 1β (il-1β), il-6, and tumor necrosis factor α (tnf-α)), chemokines, prostaglandins, and proteases which are involved in preterm labor. we have called this pathway as the indirect pathway while some antibiotics have been shown to affect immune cells such as monocytes to release pro-inflammatory cytokines (direct pathway) [7] . interestingly, a study has evaluated the outcome of empiric antibiotic therapy on gestational duration in two groups of microbe positive and negative pregnant individuals using 3 rapid and high-sensitive polymerase chain reaction (pcr). among 104 evaluated pregnant cases, it was shown that the antibiotic-treated microbe negative group had a significantly shorter gestation duration (p<0.0001) [8] . also, at the beginning of the pandemic, we have suggested avoiding any non-indicated antibiotic consumption to prevent pro-inflammatory cytokine storm which might be one of the causes of septic shock in critically ill covid-19 patients who have been admitted in intensive care unit (icu) [2] . altogether, we have raised some concern regarding the increased risk of inflammation in patients received antibiotics without evidence of bacterial infection(s). after collecting data from critically ill pregnant patients with covid-19, we have observed that the most of them had used antibiotics at admission or just a few days following admission and while most cases reported no evidence of bacterial infection [3, 9] . another study which has compared covid-19 pregnant women with healthy controls showed the significant increase in the prevalence of preterm labor while all the covid-19 cases had received antibiotic from admission [10] . as we have shown, some antibiotics can induce inflammation [7] ; on the other hand, some others (such as macrolides) could modulate inflammation. [11] it is reasonable to start empiric antibiotics for some hospitalized pregnant patients (especially those in icu), however, we suggest to consider all aspects of the treatment first. if antibiotic(s) should be administrated, it is wiser to choose the one with anti-inflammatory activity such as macrolids which is able to reduce/modulate inflammation (especially in airways) as well as pro-inflammatory cytokines (such as il-1, il-6, tnf-α) [11] when there is no other significant difference between the choices. this issue may lead to a decrease in pro-inflammatory cytokines release and possibly preventing preterm labor in pregnant women with covid-19. 4 authors declare no actual or potential compacting interest related to this study. this study was not funded. primary symptoms, comorbidities, and outcomes of 431 hospitalized patients with confirmative rt-pcr results for covid-19 inappropriate antibiotic consumption as a possible cause of inflammatory storm and septic shock in patients diagnosed with coronavirus-19 disease (covid-19) maternal death due to covid-19 disease coronavirus disease 2019 (covid-19) pandemic and pregnancy coronavirus in pregnancy and delivery: rapid review severe coronavirus infections in pregnancy: a systematic review antibiotics, inflammation, and preterm labor: a missed conclusion antibiotic therapy increases the risk of preterm birth in preterm labor without intra-amniotic microbes gestation period in preterm labor with microbes, evaluated by rapid and high-sensitive pcr system a case of 2019 novel coronavirus in a pregnant woman with preterm delivery maternal and neonatal outcomes of pregnant women with covid-19 pneumonia: a case-control study antibiotics as immunomodulant agents in copd title: empiric antibiotics in management of inpatient pregnant women infected with coronavirus disease 2019 (covid-19): focusing on inflammation and preterm labor amir hossein norooznezhad declares no actual or potential conflict of interests related to this study sedigheh hantoushzadeh declares no actual or potential conflict of interests related to this study shamshirsaz declares no actual or potential conflict of interests related to this study key: cord-317238-uszhwugw authors: parodi, aurora; cozzani, emanuele title: coronavirus disease 2019 (covid 19) and malaria. have anti glycoprotein antibodies a role? date: 2020-06-25 journal: med hypotheses doi: 10.1016/j.mehy.2020.110036 sha: doc_id: 317238 cord_uid: uszhwugw nan to the dear editor, italian people are actually living a severe crisis due to a coronavirus disease 2019 . the north of italy, is particularly affected with a great proportion of severe acute respiratory syndrome. in the northern italian regions the presence of african people, usually employed in iron-steel factories and agricultural works, is high. now, among the people recovered in the hospitals with moderate or severe covid-19, the presence of black people is very low. according to the data of italian superior institute of health, in bergamo, one of the cities of lombardia mostly affect, the black people with moderate to severe covid-19 are about 1,6% of covid-19 patients [1] . these data are real because the italian health system is free and allows all people to access to treatments. so we think that are not underestimation of the number of asymptomatic people of black people affected by covid-19. we also noted that in central african countries the distribution of covd-19 is nil or very low as referred by napoli and nioi in their work [2] . why? because the black people who live in italy are young and it is known that covid-19 is more frequent in old people with comorbidities. or it can be due to a different genetic susceptibility of african people as occur in women and men the latter having higher range of severity and mortality [3] [4] . however it is known that in african-americans the severity of covid-19 is high so, we think that the theory of immune genetic factors does not completely explain the answer. in addition according to the data of who the dashboard of the pandemic in the most african countries is very low and in some nations the total cases of covid-19 are less than 10 units [5] and nowadays experts still do not know how so few cases of the new virus had been reported despite china, where the virus originated, being the continent's top trading partner. it can be due to a lack of detection or the monitoring efforts are weak [6] . in the african continent malaria is one of the diseases which cause significant morbidity and mortality. we speculate that anti-malarial immunity may also play a role. we know that individuals who have had malaria or asymptomatic malaria have antibodies (igg) to plasmodium specific antigens. these antibodies are directed to glycosylphosphatidylinositol (gpi), the anchor molecules of some membrane proteins of plasmodium species. although in malaria previous infection is not fully protective, as evidenced by repeated infections experienced by people living in endemic areas, clinical presentation among "such immune" people, is less severe than in non-immune people [7] . in addition children who, usually have antibodies to different viruses or bacteria are known to be protected towards covid-19 [8] . gpi is a phospholipid which can activate leukocytes, trigger the release of pro-inflammatory cytokines and induce the expression of adhesion molecules via toll like receptors 2 and 4. anti gpi antibodies may neutralize the toxic effect of plasmodium gpi. also coronavirus 19 presents different glycoproteins (gps):membrane gps, spike gps and gps that have acetyl esterase and hemagglutination properties .these gps could be recognized by the antibodies produced in malaria and could protect by virus infection or induced a milder disease [9] . in addition covid-19 and malaria present other 4 similarities. blood group 0 patients are associated with a low risk of malaria infection [10] probably due to mimicry of a or b antigens by infectious agents [10] . the same occurs in covid-19. the natural anti histo-blood group antibodies may play a favorable role in antiviral immunity since the viruses may carry abh structures in their envelope glycoproteins as previously demonstrated for other coronavirus [11] . the second similarity is that the 2 diseases respond to anti-malarials. for some authors these drugs could represent a sort of chemoprophylaxis against covid-19 in african countries because they promote also in covid-19 a virus negative conversion, shortening of the disease course and improving lung imaging findings as seen both in vitro and in vivo [2, 12] . superior institute of health global spread of coronavirus disease 2019 and malaria: an epidemiological paradox in the early stage of a pandemic the ocular surface and the coronavirus disease 2019: does a dual 'ocular route' exist? covid-19 and individual genetic susceptibility/receptivity: role of ace1/ace2 inflammation and coagulation. might the double xchromosome in females be protective against sars-cov-2 compared to the single x-chromosome in males? who coronavirus disease safety recommendations and medical liability in ocular surgery dur ing the covid-19 pandemic: an unsolved dilemma immunoregulation in human malaria: the challenge of understanding asymptomatic infection why are children less affected by covid-19 ? could there be an overlooked bacterial co-infection ? autoimmunity, phospholipid-reacting antibodies and malaria immunity relative susceptibilities of abo blood groups to plasmodium falciparum malaria in ghana inhibition of the interaction between the sars-cov spike protein and its cellular receptor by anti-histo-blood group antibodies of chloroquine and covid-19 key: cord-297178-moxhk2e0 authors: novaes rocha, vinicius title: viral replication of sars-cov-2 could be self-limitative the role of the renin-angiotensin system on covid-19 pathophysiology date: 2020-10-01 journal: med hypotheses doi: 10.1016/j.mehy.2020.110330 sha: doc_id: 297178 cord_uid: moxhk2e0 currently, the world is suffering with one of the biggest pandemics of recent history. caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2), the coronavirus disease 2019 (covid-19) is provoking devastating consequences on economic and social fields throughout all continents. therefore, pathophysiological knowledge about covid-19 is imperative for better planning of preventive measures, diagnosis, and therapeutics of the disease. based on previous studies, this work proposes new hypothesis related to the role of the renin-angiotensin system on the pathophysiology of covid-19, and its purpose is to enrich the discussion and to offer alternative ways for experimental and clinical studies aiming at the formulation of new diagnosis and / or treatment methods. the renin-angiotensin system (ras) is currently one of the focuses of worldwide research due to coronavirus 2019 (covid-19), a pandemic that has destabilized the world and created devastating consequences for economic and social areas [1] [2] [3] . amongst the components of rennin-angiotensin system (ras), the angiotensin-converting enzyme 2 (ace2) has gained great prominence for being directly associated with severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection, the coronavirus related to covid-19 [4, 5] . thus, the protagonism of ace2 is being debated amongst researchers with the aim of establishing a role of ras on the pathophysiologic context of covid-19 [6] [7] [8] . ras was initially described as a regulating component of blood pressure and hydric balance, being called "classic pathway". subsequently, with the discovery of new components and finding of a "local tissue ras", the existence of a more complex system was observed with endocrine, paracrine and intracrine characteristics [9] [10] [11] . ras elucidation continues to expand, new components are being discovered and also new ways of interaction between the local and systemic pathways, as well as interactions with the external environment [12] . nowadays, ras is known as a complex system, with systemic and local (tissue) activities which communicate between themselves and involve different sign pathways, leading to different results depending on the chosen pathway [9, 12] . this system's balance is imperative for homeostasis, in which the angiotensinconverting enzyme (ace) and ace2 are the main weights in this scale ( figure 1 ). when the scale plate tends to the ace side, the production of angiotensin ii (ang ii) and the activation of its respective receptors (at1) increases. the 3 activation of the detrimental pathway (ace -ang ii -at1r) stimulates actions such as cell proliferation, inflammation, fibrosis, and thrombosis. on the other hand, when the scale plate tends to the ace2 side, the production of angiotensin 1-7 (ang 1-7) increases, as well as the activation of its respective receptors (mas) [13] . the activation of the protective pathway (ace2 -ang 1-7 -mas) neutralizes the detrimental pathway, stimulating actions such as antiinflammatory, anti-fibrotic, and anti-thrombotic effects [9, 14] . coronaviruses are classified into four different genus, in which 3 species (α hcov-nl63, sars-cov, and sars-cov-2) have ace2 as a receptor [15] . the process of cell invasion occurs similarly amongst the different species. briefly, the virus-cell interaction occurs through the ace2 receptor coupling, and the internalization of the virus then occurs through fusion or endocytosis, with the participation of other components (clathrin, tmprss2, and others) [16] [17] [18] . although they belong to the same subfamily coronavirinae and share the same receptor, these viruses have significant differences, as presented in table 1 . amongst these differences, it is important to highlight in sars-cov-2 (the virus related to covid-19) the longer incubation period and the faster viral peak, observed a few days after the beginning of symptoms. such characteristics might be related to the fact that sars-cov-2 presents the most extreme deficiency of cpg amongst the known betacoronavirus genomes. the reduction of the cpg index may have allowed the virus to escape from immune response mediated by human zap (zinc finger antiviral protein), thus 4 becoming a severe human pathogen [24] . furthermore, sars-cov-2 has a much higher infection and replication capacity than sars-cov, however without inducing in a significant way the increase in interferons type i, ii or iii, and inflammatory mediators (pro-inflammatory cytokines / chemokines) [25] . such characteristics of sars-cov-2 are imperative for the understanding of covid-19 pathophysiology. the high replication rate associated with low inflammation leads to the viral peak even at the onset of symptoms, which makes early disease identification difficult, and as a consequence, makes treatment with antivirals ineffective because the effectiveness of their action occurs during the rise of the replication curve, the most recommended phase for such medicine [25] . although sars-cov and sars-cov-2 use ace2 as cell coupling receptor, the pathogenic characteristics of sars-cov-2 provide a great difference on disease progression, as shown in figure 2 . ace2 is a fundamental piece in the pathophysiology of covid-19, since the high replication capacity of sar-cov-2 is directly related to the coupling to ace2 and cell infection. studies show that the virus causes a reduction of ace2 in the infected organs [26] . therefore, the higher the amount of virus, the higher will be the use of ace2 by it, creating an inverse correlation between the amount of sars-cov-2 and ace2. at a certain time, the constant and gradual reduction of ace2 will cause a ras imbalance, increasing the action of ace in detriment of the decrease in the action of ace2, thus prevailing the harmful factors ( figure 3 ). the ace2 level reduction caused by sars-cov-2 infection may be directly related to the pathogenesis of covid-19 [26] . the ang 1-7 decrease and the lower activation of mas receptors and / or the ang ii production increase, with 5 subsequent activation of at1 receptor, is widely known for the triggering of inflammation and fibrosis [27] . for example, a study with mice has shown that acute lung lesion results in a marked decrease of ace2, and the use of recombinant ace2 has a protective effect against the lung lesion. the same study noted that ace2, ang ii, and the ang ii receptor 1 (at1) provoke the disease pathogenesis. thus, the important role of ras on acute lung lesion pathogenesis is demonstrated [28] . histologically, patients with covid-19 present a pattern of diffuse alveolar damage and perivascular lymphocyte infiltration, similar to what is observed in influenza cases. a surprising histopathological finding on covid-19 was the lung angiogenesis, which is 2.7 times greater than on the lungs of patients with influenza [29] . angiogenesis, a complex process by which new blood vessels are formed from existing, is induced by hypoxia. hypoxia is a condition in which tissues are not properly oxygenated, resulting in considerable cell stress and adaptive responses. the transcriptional responses to hypoxia are mostly mediated by the hypoxiainducible factor (hif), a transcription factor that acts as oxygen sensors and are related to the activation of vegf-a expression [30, 31] . under hypoxia, hif influences ace and ace2 in different ways, positively regulating the expression of ace and negatively of ace2 [32, 33] . in covid-19, such circumstances may worsen the clinical condition, for stimulating even more the inflammatory, fibrotic, and thrombogenic pathways of ras. although covid-19 is clinically classified as an acute respiratory syndrome, the increased angiogenesis suggests a chronic cell adaptation towards progressive hypoxia. the chronic adaptation to hypoxia could explain the higher resistance of some patients towards low o2 saturation, in which such patients do not present an involuntary increase of ventilation, even when great lung damage is present ("ground-glass opacity"). the replicative cycle of sars-cov-2 causes a local immune response as a result of cell death and tissue damage. in some cases, the immune response occurs in an unregulated manner, triggering a cytokine storm, and consequently, generalized pulmonary inflammation [34] . as with covid-19, other acute respiratory diseases (sars, mers, influenza) can also present the clinical picture of cytokine storm [35] . high virus titers and dysregulation of the cytokine / chemokine response cause an inflammatory cytokine storm, mainly related to the influx of inflammatory mononuclear macrophages. the activation of these macrophages by interferon-α / β produces more chemotactic factors for monocytes (ccl2, ccl7 and ccl12), resulting in the additional accumulation of mononuclear macrophages, and subsequently, an increase in the levels of pro-inflammatory cytokines (tnf, il-6, il1-β and inducible nitric oxide synthase), thus increasing the severity of the disease [35] . in severe cases of covid-19, patients demonstrate elevated plasma levels of il-2, il-7, il-10, granulocyte colony stimulating factor (g-csf), ip-10, mcp1, macrophage inflammatory protein 1α (mip1α) and tumor necrosis factor (tnf) [34] . viral infection and replication in airway epithelial cells can cause high levels of pyroptosis. pyroptosis is a highly inflammatory form of programmed cell death seen in cytopathic viruses such as sars-cov-2. il-1β, an important cytokine released during pyroptosis, is elevated in infection by sars-cov-2, thus being a possible trigger for the cytokine storm [34] . the excessive or uncontrolled release of pro-inflammatory cytokines, characteristic of the cytokine storm, is also associated with non-infectious 7 diseases, such as autoimmune diseases [36] . yiguo qiu et al. analyzed sars in mice with autoimmune uveitis. the study demonstrated that the administration of ang 1-7 antagonist reversed the protective effects of ace2 on inflammatory signals and on the production of inflammatory cytokines, as well as on the regulation of local immune responses. the inhibition of ang 1-7 increased the production of the pro-inflammatory cytokines il-6, il-1β, tnf-α, and mcp-1 [37] . this study demonstrates the importance of ang 1-7 in mediating the inflammatory process, as its inhibition can cause an increase in the production of pro-inflammatory cytokines, similar to that observed in the cytokine storm in covid-19. ace2 plays a crucial role in ras because it neutralizes ace activity by degrading ang i in ang (1-9), as well as hydrolyzing ang ii, producing ang (1-7) [38] . thus, ace2 is essential to stimulate the beneficial effects of the protective axis of ras, ace2 / ang (1-7) / mas, and to mitigate the deleterious effects of the harmful axis of ras, ace / ang ii / at1. maintaining normal levels of ace2 in the lung is beneficial for combating inflammatory lung disease. the reduction in ace2 expression may be related to pulmonary inflammation and subsequent cytokine storm seen in patients with severe covid-19. ace2 maintains the proper function of the heart and kidneys, and the negative regulation of ace2 by sars-cov-2 can compromise this protective characteristic and contribute to the damage caused by the infection of these organs [38] . renin-angiotensin system and the self-limitative replication of sars-cov-2 ras is classified as systemic (classic) and local, and the interaction between them is fundamental to homeostasis [9,10,39]. this interaction between local 8 and systemic ras probably occurs through the circulation of soluble ace and ace2. although they are transmembrane proteins, ace and ace2 may undergo a cleavage process by ace secretase and adam17, respectively, releasing an active extracellular domain. this soluble component is capable of converting angiotensin i into ang ii (ace) and ang ii into ang 1-7 (ace2) [39, 40] . the balance between the amount of transmembrane ace and soluble ace is imperative, because the excessive release of the soluble form to the extracellular environment could lead to local tissue injury, since it will cause misbalance on the production of local ang ii and ang 1-7. the local ras misbalance makes the harmful pathway effects prevail (ace -ang ii -at1r) on the referred organ or tissue [41] . in an ace2 receptor-dependent viral infection (coronavirus nl63), the reduction of ace2 cellular level occurs without changes in ace levels [42] . this finding, plus the fact that sars-cov-2 elicits a low immune and inflammatory response, even when compared to sars-cov [25] , is more evidence that covid-19 pathogenesis is more related to ras misbalance than to the viral action itself. mossel although the protease activity of adam17 over ace2 is observed, the same is 11 not observed in ace [55] [56] [57] . interestingly, ace can regulate the activity of adam17, since it can be triggered from the activation of the at1 receptor by ang ii [58] . thus, ace directly regulates the formation of ang ii from ang i, and interferes via the action of adam17 in ace2, in the tissue formation of ang 1-7 from ang ii. therefore individuals with greater expression of tissue ace, and consequently, greater production of ang ii, will present a decrease in tissue ace2 and an increase in soluble ace2 (systemic), due to greater activation of adam17 and ace2 cleavage. this interaction between ace, adam17 and ace2 can explain the difference in covid-19 involvement between men and women. androgens increase renin levels and ace activity, while estrogen decreases renin levels, ace activity, and at1 receptor density, and is also related to increased levels of ang 1-7 [59] , an important actor in the protective axis of ras. this explains the fact that men have higher circulating (soluble) ace2 levels than women [60], due to the greater action of adam17 via androgens / eca / ang ii / at1. thus, due to higher levels of circulating ace2, men may present a lower level of tissue ace2, becoming more vulnerable to local sars imbalance after sars-cov-2 infection. this argument gains more strength when we observe that children [61] and the elderly [62] do not show a difference in the involvement of covid-19 between genders, probably due to the lack of hormonal interference before puberty and also due to the loss of protection from estrogen in women after menopause. show similar responses to the disease, presenting a great increase in the 13 production of ang 1-7 when compared to the control group [67, 71] . in sick adults, the ang 1-7 level is also high when compared to healthy subjects [68, 69] , but the response intensity is much lower when compared to dogs and covid-19 brings the world economy to its knees if the world fails to protect the economy, covid-19 will damage health not just now but also in the future developing a sustainable exit strategy for covid-19: health, economic and public policy implications system blockers and the risk of covid-19 renin-angiotensin-aldosterone system inhibitors in patients with covid-19 ace2: the key molecule for understanding the pathophysiology of severe and critical conditions of covid-19: demon or angel? viruses association between reninangiotensin system inhibitors and covid-19 complications covid-19: the renin-angiotensin system imbalance hypothesis renin-angiotensin system revisited a novel angiotensin-converting enzyme-related carboxypeptidase (ace2) converts angiotensin i to angiotensin 1-9 aceh/ace2 is a novel mammalian metallocarboxypeptidase and a homologue of angiotensinconverting enzyme insensitive to ace inhibitors ectodomain shedding of angiotensin converting enzyme 2 in human airway epithelia angiotensin-(1-7) is an endogenous ligand for the g protein-coupled receptor mas renin-angiotensin-aldosterone system inhibitors in covid-19. reply mechanisms of coronavirus cell entry mediated by the viral spike protein entry of human coronavirus nl63 into the cell efficient activation of the severe acute respiratory syndrome coronavirus spike 18 protein by the transmembrane protease tmprss2 coronavirus membrane fusion mechanism offers a potential target for antiviral development clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study insight into 2019 novel coronavirus -an updated interim review and lessons from sars-cov and mers-cov sars-cov-2 viral load in upper respiratory specimens of infected patients differential downregulation of ace2 by the spike proteins of severe acute respiratory syndrome coronavirus and human coronavirus nl63 extreme genomic cpg deficiency in sars-cov-2 and evasion of host antiviral defense comparative replication and immune activation profiles of sars-cov-2 and sars-cov in human lungs: an ex vivo study with implications for the pathogenesis of covid-19 the pivotal link between ace2 deficiency and sars-cov-2 infection targeting the renin-angiotensinaldosterone system in fibrosis angiotensin-converting enzyme 2 protects from severe acute lung failure pulmonary vascular thrombosis, and angiogenesis in covid-19 roles of runx in hypoxia-induced responses and angiogenesis hypoxia inducible factor pathway and physiological adaptation: a cell survival pathway? role of hif-1alpha in the regulation ace and ace2 expression in hypoxic human pulmonary artery smooth muscle cells mirna let-7b promotes the development of hypoxic pulmonary hypertension by targeting ace2 the trinity of covid-19: immunity, inflammation and intervention the pathogenesis and treatment of the `cytokine storm' in covid-19 into the eye of the cytokine storm aav8-mediated angiotensin-converting enzyme 2 gene delivery prevents experimental autoimmune uveitis by regulating mapk, nf-κb and stat3 sars-cov-2 pandemic and research gaps: understanding sars-cov-2 interaction with the ace2 receptor and implications for therapy signaling by the angiotensin-converting enzyme role of circulating angiotensin converting enzyme 2 in left ventricular remodeling following myocardial infarction: a prospective controlled study replication-dependent downregulation of cellular angiotensin-converting enzyme 2 protein expression by human coronavirus nl63 exogenous ace2 expression allows refractory cell lines to support severe acute respiratory syndrome coronavirus replication inhibition of sars-cov-2 infections in engineered human tissues using clinical-grade soluble human ace2 ace2 receptor expression and severe acute respiratory syndrome coronavirus infection depend on differentiation of human airway epithelia endotoxin induces rapid metalloproteinase-mediated shedding followed by up-regulation of the monocyte hemoglobin scavenger receptor cd163 epidemiological, comorbidity factors with severity and prognosis of covid-19: a systematic review and meta-analysis baseline chronic comorbidity and mortality in laboratory-confirmed covid-19 cases: results from the precovid study in spain polymorphism interacts with dietary n-6 polyunsaturated fatty acids to modulate obesity risk in the genetics of lipid lowering drugs and diet network study role of adam17 in kidney disease a score including adam17 substrates correlates to recurring cardiovascular event in subjects with atherosclerosis the egfr-adam17 axis in chronic obstructive pulmonary disease and cystic fibrosis lung pathology the shedding protease adam17: physiology and pathophysiology tumor necrosis factor-alpha convertase (adam17) mediates regulated ectodomain shedding of the severeacute respiratory syndrome-coronavirus (sars-cov) receptor, angiotensinconverting enzyme-2 (ace2) the secretases that cleave angiotensin converting enzyme and the amyloid precursor protein are distinct from tumour necrosis factor-alpha convertase receptor signaling pathways in the cardiovascular system gender and the reninangiotensin-aldosterone system circulating angiotensin-converting enzyme 2 activity in patients with chronic kidney disease without previous history of cardiovascular disease epidemiology of covid-19 among children in china patients with covid-19 aged 60 years and older in a university hospital in risks to children during the covid-19 pandemic: some essential epidemiology covid-19 in children: epidemiology, presentation, diagnosis and management spike protein recognition of mammalian ace2 predicts the host range and an optimized ace2 for sars-cov-2 infection infection of dogs with sars-cov-2 suessenbach fk, burckhardt bb. levels of angiotensin peptides in healthy and cardiovascular/renal-diseased paediatric population-an investigative review relationship between circulating levels of angiotensin-converting enzyme 2-angiotensin-(1-7)-mas axis and coronary heart disease. heart vessels roles of angiotensin peptides and recombinant human ace2 in heart failure circulating angiotensin converting enzyme 2 activity as a biomarker of silent atherosclerosis in patients with chronic kidney disease plasma and tissue angiotensin-converting enzyme 2 activity and plasma equilibrium concentrations of angiotensin peptides in dogs with heart disease the author has no conflict of interest or commercial sponsor for this work thanks to isabela tanuri bessa for her helpful comments. thanks also to the researchers from different knowledge areas for all their work undertaken as the basis for the construction of this article. the author has no conflict of interest or commercial sponsor for this work. 16 key: cord-331140-5b0y1xzb authors: cardona maya, walter d.; carvajal, alejandro title: sars-cov-2 and prostatitis: dangerous relationship for male sexual and reproductive health date: 2020-06-01 journal: med hypotheses doi: 10.1016/j.mehy.2020.109914 sha: doc_id: 331140 cord_uid: 5b0y1xzb nan . recently, sars-cov-2 was detected in semen samples (5) . therefore, it is not unreasonable to believe that the latest coronavirus could potentially be transmitted via semen (6) . it was also reported that angiotensin converting enzyme 2 (ace2) is a functional receptor that mediates the entry of sars-cov-1 (7) and 2 (8) , and this receptor is expressed in the prostate. thus, virus binding to these ace2 positive cells can potentially not only cause tissue alterations but also serve as a source of the virus secreted and actively pass to the ejaculate. thus, there is a significant group of patients with covid-19 who are susceptible to suffering from prostatitis. perhaps in the coming years, the real effect of sars-cov-2 on prostatitis cases will be evaluated and scope for researching factors that cause the clinical syndrome will be expanded. further, it is possible that viral typing tests will be routinely carried out, among which the coronavirus will be mandatory. the article has no funding source the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. epidemiology of prostatitis in finnish men: a population-based cross-sectional study the role of chronic prostatic inflammation in the pathogenesis and progression of benign prostatic hyperplasia (bph) male infertility: a public health issue caused by sexually transmitted pathogens orchitis: a complication of severe acute respiratory syndrome (sars) clinical characteristics and results of semen tests among men with coronavirus disease sars-cov-2 and the testis: similarity to other viruses and routes of infection angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus a pneumonia outbreak associated with a new coronavirus of probable bat origin key: cord-289905-dvl2pud2 authors: gan, rosemary; rosoman, nicholas p.; henshaw, david j.e.; noble, euan p.; georgius, peter; sommerfeld, nigel title: covid-19 as a viral functional ace2 deficiency disorder with ace2 related multi-organ disease date: 2020-06-23 journal: med hypotheses doi: 10.1016/j.mehy.2020.110024 sha: doc_id: 289905 cord_uid: dvl2pud2 sars-cov-2, the agent of covid-19, shares a lineage with sars-cov-1, and a common fatal pulmonary profile but with striking differences in presentation, clinical course, and response to treatment. in contrast to sars-cov-1 (sars), covid-19 has presented as an often bi-phasic, multi-organ pathology, with a proclivity for severe disease in the elderly and those with hypertension, diabetes and cardiovascular disease. whilst death is usually related to respiratory collapse, autopsy reveals multi-organ pathology. chronic pulmonary disease is underrepresented in the group with severe covid-19. a commonality of aberrant renin angiotensin system (ras) is suggested in the at-risk group. the identification of angiotensin-converting-enzyme 2 (ace2) as the receptor allowing viral entry to cells precipitated our interest in the role of ace2 in covid-19 pathogenesis. we propose that covid-19 is a viral multisystem disease, with dominant vascular pathology, mediated by global reduction in ace2 function, pronounced in disease conditions with ras bias toward angiotensin-converting-enzyme (ace) over ace2. it is further complicated by organ specific pathology related to loss of ace2 expressing cells particularly affecting the endothelium, alveolus, glomerulus and cardiac microvasculature. the possible upregulation in ace2 receptor expression may predispose individuals with aberrant ras status to higher viral load on infection and relatively more cell loss. relative ace2 deficiency leads to enhanced and protracted tissue, and vessel exposure to angiotensin ii, characterised by vasoconstriction, enhanced thrombosis, cell proliferation and recruitment, increased tissue permeability, and cytokine production (including il-6) resulting in inflammation. additionally, there is a profound loss of the “protective” angiotensin (1-7), a vasodilator with anti-inflammatory, anti-thrombotic, antiproliferative, antifibrotic, anti-arrhythmic, and antioxidant activity. our model predicts global vascular insult related to direct endothelial cell damage, vasoconstriction and thrombosis with a disease specific cytokine profile related to angiotensin ii rather than “cytokine storm”. our proposed mechanism of lung injury provides an explanation for early hypoxia without reduction in lung compliance and suggests a need for revision of treatment protocols to address vasoconstriction, thromboprophylaxis, and to minimize additional small airways and alveolar trauma via ventilation choice. our model predicts long term sequelae of scarring/fibrosis in vessels, lungs, renal and cardiac tissue with protracted illness in at-risk individuals. it is hoped that our model stimulates review of current diagnostic and therapeutic intervention protocols, particularly with respect to early anticoagulation, vasodilatation and revision of ventilatory support choices. disease in the elderly and those with hypertension, diabetes and cardiovascular disease. whilst death is usually related to respiratory collapse, autopsy reveals multiorgan pathology. chronic pulmonary disease is underrepresented in the group with severe covid-19. a commonality of aberrant renin angiotensin system (ras) is suggested in the at-risk group. the identification of angiotensin-converting-enzyme 2 (ace2) as the receptor allowing viral entry to cells precipitated our interest in the role of ace2 in covid-19 pathogenesis. we propose that covid-19 is a viral multisystem disease, with dominant vascular pathology, mediated by global reduction in ace2 function, pronounced in disease conditions with ras bias toward angiotensin-converting-enzyme (ace) over ace2. it is further complicated by organ specific pathology related to loss of ace2 expressing cells particularly affecting the endothelium, alveolus, glomerulus and cardiac microvasculature. the possible upregulation in ace2 receptor expression may predispose individuals with aberrant ras status to higher viral load on infection and relatively more cell loss. relative ace2 deficiency leads to enhanced and protracted tissue, and vessel exposure to angiotensin ii, characterised by vasoconstriction, enhanced thrombosis, cell proliferation and recruitment, increased tissue permeability, and cytokine production (including il-6) resulting in inflammation. additionally, there is a profound 3 loss of the "protective" angiotensin (1-7), a vasodilator with anti-inflammatory, antithrombotic, antiproliferative, antifibrotic, anti-arrhythmic, and antioxidant activity. our model predicts global vascular insult related to direct endothelial cell damage, vasoconstriction and thrombosis with a disease specific cytokine profile related to angiotensin ii rather than "cytokine storm". our proposed mechanism of lung injury provides an explanation for early hypoxia without reduction in lung compliance and suggests a need for revision of treatment protocols to address vasoconstriction, thromboprophylaxis, and to minimize additional small airways and alveolar trauma via ventilation choice. our model predicts long term sequelae of scarring/fibrosis in vessels, lungs, renal and cardiac tissue with protracted illness in at-risk individuals. it is hoped that our model stimulates review of current diagnostic and therapeutic intervention protocols, particularly with respect to early anticoagulation, vasodilatation and revision of ventilatory support choices. sars-cov-2, the agent of covid-19, shares a lineage with sars-cov-1 disease (sars), and a common fatal pulmonary profile but with striking differences in presentation, clinical course, and response to treatment [1] [2] [3] [4] . in contrast to sars, covid-19 has presented as an often bi-phasic, multi-organ pathology, with a proclivity for severe disease in the elderly and those with hypertension, diabetes and cardiovascular disease. whilst death is almost universally related to respiratory collapse with multiple systems revealed to be failing at autopsy, the co-morbidity of chronic pulmonary disease is surprisingly relatively underrepresented in the group with severe covid-19 [4] . covid-19 has not responded consistently to immunosuppressive therapy, antimicrobial agents or invasive ventilation [5] . there is evolving evidence for a mechanism of lung injury that is not always typical of acute respiratory distress syndrome (ards) [2] . there is a need to revise the disease model for covid-19 away from historical pulmonary-centric septic disease states typically characterised by lymphopaenia and cytokine storm with secondary bacterial sepsis. the diagnosis, assessment and management of covid-19 cannot be based entirely on previous sars, mers or influenza pandemics. our disease hypothesis suggests a revision of our use of diagnostic tools (to better capture atypical disease presentations) and consider early treatment and/or prevention of microvascular thrombosis and constriction in identified groups at high risk of severe or fatal disease. our hypothesis provides a mechanism of pulmonary insult, that warrants revision of indications for invasive ventilation particularly in view of poor outcomes [1, 6], with higher mortality for invasively ventilated patients than that recorded in sars [1, 4, 6]. 5 whilst the respiratory system is almost universally involved in clinical presentation and dominates current therapy in covid-19, those with more severe disease have been observed to have comorbidities in the vascular, renal, and cardiovascular systems [7, 8] . appreciating the clear differences between sars and covid-19 in presentation, poor prognostic indicators related to individuals' co-morbid status, and biochemical and radiologic profiles, a novel disease model may assist in: 1) the early recognition of atypical (non-respiratory) presentations of disease; 2) early prophylactic treatment intervention for individuals at risk of severe and critical disease which could take place 6 in the community; 3) revised management of pulmonary complications including those related to prone posturing and ventilation protocols; 4) allowing better utilisation of data collated at a global level in the absence of an evidence-based disease model at this time; 5) identification of different markers of disease progression in at-risk individuals. the hypothesis of covid-19 as a viral functional angiotensin-converting-enzyme 2 (ace2) deficiency disorder, with ace2 related multi-organ cell loss, comes some way to addressing this need. this model is supported by global data and observations, including that gathered phenotypically, biochemically, radiologically, and at autopsy. it evolved from the early observation of hypoxic pulmonary insult occurring without reduction in pulmonary compliance and often in the absence of fever. this is not consistent with ards, documented in historical pulmonary septic disease states with cytokine storm complicated by hypoxia. a pulmonary vascular pathology was sought in parallel with a possible disruption of surfactant function. thrombosis and vasoconstriction were implied, with the former supported by consistent, but not specific radiologic findings of ground glass change. thrombosis has now been confirmed in late disease and at autopsy [11] . the commonality of an aberrant renin angiotensin system (ras), was identified in the group at risk of severe disease, rather than the presence of underlying lung disease. the biochemical markers in early disease suggested coagulation, renal, cardiac, and respiratory pathology in evolution. with the identification of ace2 as the receptor for sars-cov-2 entry, the role of and distribution of ace2 in the human body was investigated. the hypothesis evolved, providing an explanation for pathophysiologic commonality in the at-risk group in covid-19, a mechanism for hypoxic pulmonary pathology and the unexpected degree of lung injury consequent to ventilation as well as vascular, renal and cardiac 7 complications. it also predicts that presentation may be atypical and that long term sequelae related to endothelial, renal, pulmonary and cardiac involvement may occur. the hypothesis also predicts a cytokine profile driven by ace2 deficiency in the ras, rather than viral-related immune modulation. the immunologic effects of the ras are well documented [12] , favouring il-6 production and affecting cd4+ and cd8+ cell populations. ace2 is a membrane-associated aminopeptidase expressed in the endothelium, renal and cardiovascular tissue, and epithelia of the small intestine and testes [13] . ace2 is also expressed in the lung, kidney, and gi tract according to pcr analysis -tissues shown to harbor sars-cov-1 [13, 14] . the entry receptor utilized by sars-cov-2 in covid-19 is ace2, and the enzyme utilized for s protein priming is transmembrane protease serine 2 (tmprss2) [15] or the controversial poly-basic furin cleavage site. we propose that covid-19 is a multiorgan pathology with dominant morbidity related to respiratory insult that is compounded by a more generalised disease driver that leads to a poorer prognosis and higher mortality in an identified at-risk group. we propose that the disease driver is relative ace2 deficiency related to viral destruction of ace2 expressing tissues, with more severe disease manifesting in a cohort with abnormality in the ras favouring the angiotensin-converting-enzyme (ace) arm. this is supported by the observed frequency of hypertension, cardiovascular disease and diabetes as co-morbidities in the group with severe and fatal disease [16, 17] . these disease entities have been well studied, with documentation of ras abnormality characterised by increased ace and angiotensin ii (atii) levels (fig. 1) . we hypothesise that in these conditions, there is a greater dependence on ace2 to 8 abrogate the effects of atii and increase angiotensin (1-7) (at (1-7) ). an upregulation of ace2 expressing cells related to chronic atii elevation [18] or treatment with aceinhibitors [19] , may increase the infective potential of sars-cov-2 in this group as a consequence of the duality of ace2 functioning as both a receptor for viral entry to cells and as an enzyme. with infection related cell damage and loss, enzymatic ace2 activity would be globally compromised. organ specific loss of ace2 expressing cells (endothelial, alveolus in lungs, proximal tubule and glomerulus in kidneys, pericytes in heart) is likely to also contribute to the pathophysiology of covid-19. these conditions are by no means the only ones that may confer escalated risk of severe or critical disease. we suggest that the pathogenesis is mediated by enhanced and protracted tissue, and vessel exposure to atii, characterised by vasoconstriction, enhanced thrombosis, cell proliferation and recruitment, increased tissue permeability, and cytokine production (including il-6) resulting in inflammation. a recent study noted serum levels of atii were elevated in covid-19 patients and correlated with viral load and lung injury [20] . atii is generated by both ace and non-ace pathways, but is the dominant substrate for ace2, being converted to at(1-7) [21] , a vasodilator with antiinflammatory, anti-thrombotic, antiproliferative, antifibrotic, anti-arrhythmic, and antioxidant activity [12, 22] . the viral destruction of ace2 expressing cells may lead to the profound loss of the "protective" at(1-7) effects in an environment of atii effect dominance (fig. 1) . enhanced thrombosis may occur via several atii driven pathways, including increased thrombin [23] , procoagulant endothelial cell activity via bradykinin [24, 25] , immune effects via t cells (cd4+, cd8+) [12, 26] , platelet aggregation [27] , and 9 reduced fibrinolysis via bradykinin and endothelin. the prothrombotic effects of atii appear to extend to the microvasculature in murine models [23] . platelet aggregation and endothelial damage in the septic setting may contribute further to thrombosis. we hypothesise that ace2 expressing cells are rapidly infected by sars-cov-2. initially, this is by ingestion and/or aspiration. early respiratory symptomatology is consistent with previously described sars-cov-1 infection via ace2 receptors in the ciliated respiratory epithelial cells, with apical release of replicated virus [28] rapidly infecting the downstream ace2 expressing cells -including type 2 alveolar cells. as opposed to sars-cov-1 infection, significant hypoxia in covid-19 is not accompanied in early disease by a reduction in pulmonary compliance [6, 29] . this and colleagues reported that abnormal coagulation parameters, particularly d-dimer and fibrin degradation products (fdps), were associated with poor prognosis in covid-19 [30] . an association with anticoagulation and improved survival after adjustment for ventilation was noted in a large united states cohort [31] . possible atii driven thrombosis and vasoconstriction are likely to be compounded by alveolar damage via the viral loss of ace2-rich type 2 alveolar cells. these cells in health are tasked with surfactant production, maintenance of alveolar homeostasis, and transdifferentiation into type 1 alveolar cells, which comprise 90% of the lining of the alveolus. the demise of these cells is likely to lead to hypoxia via surfactant loss, alveolar flooding, and apoptosis of type 1 alveolar cells without replacement via transdifferentiation from type 2 alveolar cells (fig. 2) . our hypothesis suggests that as disease progresses, the destruction of alveoli leads to dad, rather than ards. the destruction of alveoli is evident at autopsy, with dad [11] and radiologic appearances consistent with dad are documented in advanced disease [6, 32] . our hypothesis supports a possible mechanism for disappointing outcomes in ventilated patients, particularly related to those requiring or receiving high positive end expiratory pressure (peep) and the observed autopsy findings [6, 11, 33] . the advantage of prone posturing and peep application is well documented in ards [34] . advantage in ards was felt to be largely related to the distribution of oedema, where the mass of the diseased lung may be increased to 300% of the normal [35] . in normal subjects, prone posturing improved alveolar ventilation, however, the addition of 10cmh2o peep in ventilated patients reduced v/q matching via perfusion changes 11 in dependent lung [36] . it is possible that prior to the evolution of pulmonary oedema in covid-19 (when lung compliance is near normal), escalating peep in response to deteriorating hypoxia may be self-defeating. in addition to this, our disease model identifies that the at-risk group characteristic of aberrant ras status favouring ace activity may be more susceptible to volume induced lung injury related to peep. decreased ace2 activity with increased ace activity contributed to lung injury [7] in in vitro studies of cyclical stretch of human lung epithelial cells and to volume induced lung injury in animal models [6, 33] . viral spread to the vascular system may occur via pulmonary capillaries given the intimacy of the pulmonary capillary bed to the infected alveoli. we postulate the viraemia to lead to vascular, renal and myocardial infection at this time, supported in a remote manner by the somewhat unpredictable timing of fever, and the reports of biphasic clinical decline that involves shortness of breath, hypoxia (pao2/fio2 <300mmhg), transaminitis, low-normal procalcitonin, and abnormal chest imaging ( fig. 3) [32]. in hypertension and renal disease, particularly diabetic nephropathy, an aberrant ras is well documented, with a bias to higher ace/ace2 in renal biopsy [37] [38] [39] . we suggest that ace2 deficiency leads to increased and unabating atii activity resulting in vasoconstriction, water and sodium retention, cell proliferation, thrombosis, inflammation, oxidative stress, and fibrosis. in addition to the damaging effects of atii, the deficiency in ace2 leads to a reduction in at(1-7), with loss of its vasodilatory, 12 diuretic, antiproliferative, antioxidant, and antithrombotic effects (via bradykinin) [22] . in the acute setting, renal injury is likely due initially to the sustained effects of atii, including vasoconstriction that favours the efferent over afferent vessels, resulting in increased glomerular pressures, water and sodium retention, albuminuria, and possible microvascular thrombosis (fig. 4) . this is supported by documentation of albuminuria and elevated creatine kinase [16, 40] . the physical loss of ace2 expressing glomerular cells may further add to the renal insult. as disease progresses, a more diffuse renal impairment develops with the absence of overt septic inflammation in autopsy specimens [38] . an abnormal renal ace/ace2 ratio is also seen in diabetics without established nephropathy, iga nephropathy, and subtotal nephrectomy [37] . this group of patients may well carry higher risk for covid-19 renal disease. the hypothesis also predicts that renal scarring is likely to occur in survivors particularly related to vessel and glomerular damage. myocardial damage is suggested by the elevation in hypersensitive troponin i (tni) and dysrhythmia in a small series (7.2% and 16.7%, respectively) [16] . with the disease model proposed here, elevated atii activity with suboptimal at(1-7) activity may lead in the predisposed heart to microvascular thrombosis, vasoconstriction related hypoxia, and cell/fibroblast proliferation, with both acute and intermediate term sequelae of progressive heart failure, and adverse remodelling and fibrosis (fig. 5) . right ventricular dysfunction is likely to reflect the pulmonary vascular burden of thrombosis and vasoconstriction. ace2 is expressed by cardiac myocytes, fibroblasts, and endothelial cells. ace2 expression is increased in human heart failure and may reflect a predisposition to 13 cardiac disease susceptibility in the context of covid-19 [41, 42] . chen and colleagues identified that the pericyte, with very high ace2 expression, may be the entry point for myocardial infection [41] . in the autopsy series from new orleans, fox and colleagues acknowledged that the finding of scattered individual myocyte necrosis without evidence of viral myocarditis would be consistent with microvascular dysfunction related to pericyte infection [11] , as hypothesized by chen and colleagues [41] . chen's findings are consistent with our belief that cardiac infection follows viraemia, with a delayed declaration of pathological involvement. all subjects had gross right ventricular dilatation. none of the 4 autopsy subjects had known cardiac disease. more recently, peng and colleagues described echocardiographic features of severe covid-19 [43] . this included left ventricular segmental contraction abnormalities (takotsubo cardiomyopathy), right ventricular dilatation and systolic dysfunction, and, finally, global systolic and diastolic heart failure. these findings are not inconsistent with cardiac microvascular dysfunction and ischaemia with, as described previously, the right ventricular failure reflecting the pulmonary vascular burden of thrombosis and vasoconstriction. tni elevation and echocardiographic assessment remain useful tools for assessing disease progression or prognosis, particularly in subjects with existing cardiac failure [10, [43] [44] [45] . the hypothesis predicts adverse remodelling of the myocardium as a consequence of microvascular cardiac disease. our hypothesis supports the revision of use of diagnostic tools with reference to the possibility of atypical (non-respiratory) disease presentations. tissues with high ace2 14 expression may be targeted for sampling in early or protracted infection to minimise community spread and false negative results. faecal or urine pcr testing for viral infection may be reconsidered if respiratory sampling is unhelpful. we propose that identified high risk groups be considered for early treatment or prevention of microvascular thrombosis and constriction, given the likelihood of progression to multisystem failure and the possibility of vascular, pulmonary, myocardial, and renal scarring sequelae in survivors. [44, 45] . we suggest consideration of low molecular weight heparin (lmwh) or unfractionated heparin as these agents are likely to have both adequate anti-coagulant and additional antiinflammatory effects (via reduction in il-6). in a recent retrospective analysis of lmwh anticoagulation, shi and colleagues reported a reduction in levels of d-dimer, fdps, and il-6, with an improvement in lymphocyte percentages when compared to a control group [46] . sildenafil, with a number of studies in progress, may be considered as a vasodilator for efficacy of administration and cost in parallel with long term safety data and welldefined recommendations for monitoring of side effects. sildenafil may also have positive effects on cilial function in covid-19 [47] . reconsideration of parameters for invasive ventilation and multitargeted therapy may be in order given poor outcomes [1, 6], with higher mortality for invasively ventilated patients than that recorded in sars [1, 4, 6] , and the possible alternative mechanism of pulmonary insult proposed -thrombosis, vasoconstriction, surfactant loss, and alveolar destruction via viral loss of type 2 alveolar cells. peep <10cmh2o with prone posturing and supplemental oxygen should be considered in patients requiring respiratory support in the absence of ards. 15 with respect to individuals with only mild or moderate disease, we propose that future treatment study protocols consider prophylactic level anticoagulation in at-risk individuals with a low threshold for escalation to full anticoagulation and vasodilation if disease progression is supported by measures of clinical state (particularly hypoxia), and well documented pathology parameters (including d-dimer, fdps, crp, neutrophil, lymphocyte and platelet levels, tni, and liver and renal function profiles, including albuminuria and il-6). prevention of thrombosis may be commenced in the community in symptomatic infected patients and at-risk populations with mild disease or positive exposure, given their increased risk of venous thromboembolism and the widespread safety data for lmwh and heparin use to establish efficacy. whilst thrombosis may not be universal, the intervention is low risk and may abrogate one of the irreversible elements of this disease. in areas with high disease prevalence, prophylactic-level anti-coagulation could be administered and monitored by local medical officers in the outpatient setting and in residential care facilities. emerging results of single-target therapies will necessarily impact on the evolution of disease models like ours, with autopsy data unfortunately offering our best evidence for end stage disease description, but little guidance for early therapeutic options. in conclusion, we present a covid-19 disease hypothesis with the intention of stimulating discussion and further research on diagnostic and therapeutic intervention protocols, particularly with respect to early anticoagulation, vasodilatation and revision of ventilatory support choices. this may benefit a large group of people who are at risk 5 . cardiac effects of ace2 deficiency in an ace/ace2 biased renin-angiotensin system as a mechanism for thrombosis, adverse remodelling, and fibrosis. 24 audit/audits/cmp/reports. 2020 covid-19 does not lead to a typical acute respiratory distress syndrome management of critically ill patients with severe acute respiratory syndrome (sars) presenting characteristics, comorbidities, and outcomes among 5700 patients hospitalized with covid-19 in the clinical evidence does not support corticosteroid treatment for 2019-ncov lung injury covid-19 pneumonia: different respiratory treatments for different phenotypes? intensive care medicine a crucial role of angiotensin converting enzyme 2 (ace2) in sars coronavirus-induced lung injury characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study correction to: clinical predictors of mortality due to covid-19 based on an analysis of data of 150 patients from wuhan, china pulmonary and cardiac pathology in covid-19: the first autopsy series immunologic effects of the renin-angiotensin system high expression of ace2 receptor of 2019-ncov on the epithelial cells of oral mucosa tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis prostate-localized and androgen-regulated expression of the membrane-bound serine protease tmprss2 clinical characteristics of 138 hospitalized patients with novel coronavirus infected pneumonia in wuhan, china clinical characteristics of patients who died of coronavirus disease 2019 in china chapter 25 -ace2 cell biology, regulation, and physiological functions in: the protective arm of the renin angiotensin system (ras). t unger, um steckelings and ras dos santos are patients with hypertension and diabetes mellitus at increased risk for covid-19 infection? clinical and biochemical indexes from 2019-ncov infected patients linked to viral loads and lung injury a novel angiotensin-converting enzyme-related carboxypeptidase (ace2) converts angiotensin i to angiotensin 1-9 acute respiratory distress syndrome leads to reduced ratio of ace/ace2 activities and is prevented by angiotensin-(1-7) or an angiotensin ii receptor antagonist roles of coagulation and fibrinolysis in angiotensin ii-enhanced microvascular thrombosis angiotensin ii, tissue factor and the thrombotic paradox of hypertension bradykinin enhances in vitro procoagulant and antifibrinolytic properties of rat vascular endothelial cells role of t lymphocytes in angiotensin iiâmediated microvascular thrombosis physiological and pathophysiological functions of the at 2 subtype receptor of angiotensin ii ace2 receptor expression and severe acute respiratory syndrome coronavirus infection depend on differentiation of human airway epithelia covid-19 pneumonia: ards or not? abnormal coagulation parameters are associated with poor prognosis in patients with novel coronavirus pneumonia association of treatment dose anticoagulation with in-hospital survival among hospitalized patients with covid-19 covid-19 illness in native and immunosuppressed states: a clinical-therapeutic staging proposal mechanical stress and the induction of lung fibrosis via the prone positioning in severe acute respiratory distress syndrome prone positioning: beyond physiology positive end-expiratory pressure redistributes regional blood flow and ventilation differently in supine and prone humans ace and ace2 in kidney disease renal histopathological analysis of 26 postmortem findings of patients with covid-19 in china synergistic expression of angiotensin-converting enzyme (ace) and ace2 in human renal tissue and confounding effects of hypertension on the ace to ace2 ratio kidney disease is associated with in-hospital death of patients with covid-19 the ace2 expression in human heart indicates new potential mechanism of heart injury among patients infected with sars-cov-2 clinical characteristics of 113 deceased patients with coronavirus disease 2019: retrospective study using echocardiography to guide the treatment of novel coronavirus pneumonia covid-19 and the heart clinical management of severe acute respiratory infection when covid-19 is suspected: interim guidance the potential of low molecular weight heparin to mitigate cytokine storm in severe covid-19 patients: a retrospective clinical study sildenafil in the treatment of pulmonary hypertension marcus cremonese, medical illustration (http://www.medicalillustration.com.au/), and danielle santarelli, medical writing (genesis research services). no conflicts of interest to report. no sources of funding to report. key: cord-337493-8yhd697t authors: choquenaira-quispe, celia; saldaña-bobadilla, vanessa; kenedy ramirez, j. title: factors involved in low susceptibility to covid-19: an adaptation of high altitude inhabitants date: 2020-07-02 journal: med hypotheses doi: 10.1016/j.mehy.2020.110068 sha: doc_id: 337493 cord_uid: 8yhd697t nan master of science  hypoxia inducible factor regulation: high altitude inhabitants have the hypoxia-inducible factor activated, leading to a subsequent gene overexpression on erythropoietin-erythropoiesis production [6] . in addition, this signaling pathway decreases ace-2 expression [7] . consequently, populations from high altitude areas would be less susceptible to hypoxia during the infection.  pulmonary adaptation in high-altitude areas: low partial pressure of oxygen in the environment and lower inspired partial pressure of oxygen leads to pulmonary physiological and anatomical adaptations [8] , improving the pulmonary perfusion and capacity. consequently, sars-cov-2 infected populations from high altitude areas have greater lung adaptive capacity than those that live at low altitude. the three factors above would perform an important role in high altitude inhabitants, because they decrease the susceptibility to develop severe symptoms in sars-cov-2 infection. it is worth mentioning that it is possible to quantify the ace-2 receptor expression, interleukins and elucidate the molecular mechanisms involved. clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study clinical features of patients infected with 2019 novel coronavirus in wuhan, china immunology of covid-19: current state of the science hepcidin and anemia: a tight relationship does the pathogenesis of sars-cov-2 virus decrease at high-altitude? hif stabilizers in the management of renal anemia: from bench to bedside to pediatrics hypoxia inducible factor-1 protects against covid-19: a hypothesis high altitude genetic adaptation in tibetans: no role of increased hemoglobin-oxygen affinity the authors declare that there is no conflict of interest. the authors declare that there is no conflict of interest. key: cord-319822-h1zm94p4 authors: carelli, pasquale title: a physicist's approach to covid-19 transmission via expiratory droplets date: 2020-06-17 journal: med hypotheses doi: 10.1016/j.mehy.2020.109997 sha: doc_id: 319822 cord_uid: h1zm94p4 in this paper, a physicist's approach is given to support the necessity to wear surgical masks during the covid-19 pandemics; they have become compulsory in eastern countries, while in western countries they are still an optional. my thesis is supported and described on the basis of a physicist's model which studies the droplets behavior when emitted by the respiratory apparatus of an infected person, symptomatic or not. the intermediate dimensioned droplets are proved to be changed into aerosol, losing their water content and becoming seriously contagious, but in their initial phase they could be easily caught by a simple surgical mask. the actual efficiency of ffp3 masks has been examined and found to be lower than expected. while the covid-19 pandemic is spreading around all over the world in a dramatic way, i think it is necessary to state a reference date in order to make any reflections, i chose the 3rd april 2020. on that date in italy there were 14,681 dead people and 119,827 infected, the situation was dramatically changing with a number of deaths higher than 500 a day. that rate (mortality) caused by the virus is of 0·11 (deaths/population). at the same date in south korea there were 120 dead people and 9,037 infected, mortality was of 0·002% with a lethality rate (deaths/infected people) of 1·3%. as the population in south korea has a life expectation very similar to the italian one (even if koreans are younger than italians[1]), the korean data about lethality is more plausible. one of the reasons of such a serious pandemic in italy and afterwards in many other western countries, could be the following: in asian countries surgical masks have been widely worn for many years as a means to protect other people. this habit, there are non doubts about the fact that expiratory particles transmit the pandemic, but we must make a coarse distinction among droplets and their dynamic evolutions when emitted by infected people. this paper wants to afford the problem from a physical point of view. the airborne disease is caused by violent coughing [3] and sneezing [4] , but many infected people were minimally symptomatic or asymptomatic at all [5, 6, 7] and transmission of virus from asymptomatic carriers has been identified [8] . we always emit a large quantity of droplets[9,10] even just breathing and speaking. whatever is the possible expiratory particle source, i assume that the smallest droplets are more numerous than the bigger ones and i won't distinguish between speaking and any other more violent expiratory events. a single initial very large droplet (r0=1 cm radius, v0=4·2 cm 3 volume) containing a large viral load (10 8 virions for milliliters [8] ) splits in many smaller droplets (the largest droplets having a radius of r3=50 µm ). the model that i propose assumes that the number of fragments with a radius between r1 and r2 is given by: where a is a dimensionless constant. a is simply 3 times the cube of ratio between r0 and r3 because the volume of the initial droplet must be equal to that of all the smaller droplets; in this specific case a's value is 24000. , their viral load is absolutely negligible. those particles cannot be stopped by a surgical mask, but there is no reason to intercept them because they are just water or mucous. for the aerosol the gravity has a negligible effect, unless it is in an environment with brackish air. the second group (in green in fig.1) , is made of droplets with the biggest size, having a radius larger than 25 µm, they are a bit more than 15,000. they constitute almost the total volume of the initial large droplet and then they contain most of the viral load. they follow a parabolic trajectory and constitute what is called fomite [12] , which can contaminate surfaces where they settle. the third group (in black in fig.1) is made of droplets with an initial radius between 2·5 µm and 25 µm, they are about 55,000. their dynamic is strongly dependent from the stokes law, that states that a spherical droplet of radius r, moving in the air, undergoes a drag force given by: where µ[13] is the air viscosity. at first they don't behave like the aerosol, because the gravity action has an important role compared with the drag force; anyway at the end the two equal and opposite forces rapidly balance and the droplet reaches a descending speed limit. that movement is slow and it allows the evaporation of water, till the droplet is reduced to its particulate or virus and it has become aerosol. these droplets have a substantial viral load, they are quite numerous, some thousands of them can contain just one single virion each and they are probably the most contagious and dangerous elements[14]; if not stopped on time, they constitute the real element of airborne infection [15] . take note that at the starting phase, when they are larger, they could be easily caught by a common surgical mask. the evolution of those droplets has been roughly studied a long time ago by wells [16] ; more recently it has been deeply analyzed [17] . of course the distinction of the three different typologies of droplets is purely as an indication, they are not exact measurements. in the fluids dynamics we define fluidodynamical resistance the ratio between the gas flux (usually measured in liter/s) and the pressure difference. in a normal person breathing through the nose, the fluidodynamical resistance is about 250 pa liter/s [18]. ffp3 masks [19] have very specific technical characteristics, they should have a fluidodynamical resistance lower than 63 pa liter/s and filters at least 99% of airborne particles larger than 0·3 µm. we know that virions of sars-cov2 are about 0·1 µm. fig.2 shows a drawing on scale of a filter (simulating the ffp3 mask filters) with holes of 800 nm diameter and a particulate with a 300 nm diameter (down, smaller than the canal): i presume that in 99% of the cases, whatever is the coming direction, it will hit the wall and it will be stopped. the other particulate with a 100 nm diameter, moving on the same direction and angle, goes inside in the same position, but it passes through the canal without being stopped. therefore a filter able to stop particulates of 300 nm diameter in 99% of cases, can't have the same efficiency for smaller particles. shortage of ffp3 masks has been considered a very bad event, but i don't believe we need them so much: as a matter of fact, to be an absolutely safe protection they should cling to the face completely, which is impossible, especially when worn by bearded men. success in fighting against a virus depends on how deep is knowledge of its characteristics: to understand how the virus aerosol changes after being emitted from the otorhinolaryngological apparatus is important in order to recommend a widely spread use of surgical masks everywhere people speak, especially indoors. moreover i underline how ffp3 masks are poorly efficient in preventing infection, considering the nature of infectious aerosol. waiting for a new vaccine we could hope to extirpate the sars-cov-2 like it happened for smallpox [19] . a cough aerosol simulator for the study of disease transmission by human cough-generated aerosols violent expiratory events: on coughing and sneezing kin-hang kokn and others, a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster xingwang li and others, a novel coronavirus from patients with pneumonia in china guangfu jin, and others, clinical characteristics of 24 asymptomatic infections with covid-19 screened among close contacts in nanjing, china gisela bretzel and others, transmission of 2019-ncov infection from an asymptomatic contact in germany the size and the duration of air-carriage of respiratory droplets and droplets-nuclei the size distribution of droplets in the exhaled breath of healthy human subjects holbrook and others, aerosol and surface stability of sars-cov-2 as compared with sars-cov-1 on air-borne infection: study ii. droplets and droplet nuclei how far droplets can move in indoor environments--revisiting the wells evaporation-falling curve zdenek jezek, and others, smallpox and its eradication key: cord-323310-tq9muytr authors: ozdemir, burak; yazici, ahmet title: could the decrease in the endothelial nitric oxide (no) production and no bioavailability be the crucial cause of covid-19 related deaths? date: 2020-06-07 journal: med hypotheses doi: 10.1016/j.mehy.2020.109970 sha: doc_id: 323310 cord_uid: tq9muytr nan burak ozdemir 1 , ahmet yazici 2 1 md, consultant ophtalmologist, nurlu goz clinique d'ophtalmologie, dakar, senegal 2 md, associate professor of ophtalmology, smartlens inc. santa clara, ca, usa dear editor, there is a pandemic that suddenly appeared on the agenda of humanity for the past few months. when covid-19 related deaths are analyzed, higher mortality rates in older people and men are quite evident in all case series [1-3] (table1). hypertension is obviously the leading comorbidity in all studies followed by diabetes, obesity and other cardiovascular disorders. angiotensin-converting enzyme 2(ace2), which is the receptor for sars-cov-2 [4] , is a regulator of vascular function by modulating nitric oxide (no) release and oxidative stress [5] . we realized that the common prominent feature in older vs younger, hypertensive vs healthy, men vs women comparison is decreased endothelial no production and decreased no bioavailability which also links cardiovascular and metabolic diseases as their common deficiency. physiological no signaling is a key determinant of endothelial function, metabolic and vascular health. it is major regulator of vascular tone and has antioxidant, antiinflammatory and antithrombotic activities. [6] endothelial dysfunction leads to a shift of endothelial cell actions by various chemokines, cytokines and other factors and finally induces proinflammatory, proliferative and prothrombotic status [7, 8] . previous studies have shown that age is the most significant predictor of the endothelium dependent vasodilatation and no availability progressively declines with aging [9-12] ( figure 1 ). the vascular changes associated with essential hypertension, such as endothelial dysfunction, are generally considered to be an accelerated form of changes seen in aging [12] . estrogen increases expression and activity of endothelial nitric oxide synthase (enos) and no production is higher in the systemic vasculature of females [13] . females are known also to produce higher innate and adaptive immunity responses than males which can result in faster clearance of viruses [14] . covid-19 mortality has correlation with the decrease in endothelial no production and bioavailability in different age groups and/or comorbidities. whether this is a direct antiviral effect or associated with immune modulation needs to be further investigated. this suggests that increasing endothelial no availability should be directly effective in antiviral resistance against covid-19. diet rich in polyphenols, caloric restriction and regular exercise are non-pharmacological strategies to increase endothelial no production and bioavailability [6, 15] . in situations like aging, cardiovascular and metabolic disorders where endothelial no production is compromised, there are considerable published data suggesting that nitroso compounds can act as storage pools and donors of no and can compensate for insufficient enos activity. [15] [16] [17] [18] [19] [20] . ace2 expression was shown in vascular endothelium and arterial smooth muscle of all organs and on alveolar epithelial cells and enterocytes of small intestine [21] . from this aspect it is noteworthy, there are also studies reporting that there is a marked increase in exhaled no after dietary nitrate consumption [22, 23] and no produced from nitrite in the upper intestine is up to 10000 times the concentrations that occur in tissues from enzymatic synthesis [12] . another interesting point is that despite being an important risk factor in a large spectrum of diseases, cigarette smoke contains huge amounts of nitrogen oxides and smoking habits can increase the blood pool of nitroso compounds [24] . âkerström et al demonstrated that the inhibitory effect of no on sars-cov is happening inside the cells, where viral material is out of the capsid [25] . we speculate that if we divide the total no amount in this study to the cell count in the media, the results will be comparable to physiological no production levels in the vascular endothelium. from this aspect, in the endothelium, especially in early stages of viral invasions, when viral material is in low amounts and out of the capsid, any amount of no produced inside the cell should have effect on susceptible viruses and a healthy enos function may be very valuable as a first line defense. due to the urgent nature of the covid-19 pandemic, simultaneous learning and practice is inevitable. the relation between decreasing no production and bioavailability with covid-19 mortality has potential to open alternative preventive and therapeutic ways. characteristics of covid-19 patients dying in italy report based on available data on march 20 th , 2020. istitvto svperiore di sanita presenting characteristics, comorbidities, and outcomes among 5700 patients hospitalized with covid-19 in new york city area the novel coronavirus 2019(2019-ncov) uses the sars-coronavirus receptor ace2 and the cellular protease tmprs s2 for entry into target cells genetic deletion of ace2 induces vascular dysfunction in c57bl/6 mice: role of nitric oxide imbalance and oxidative stress the role of nitric oxide and its clinical applications nitric oxide and oxidative stress in vascular disease endothelial dysfunction and inflammation: immunity in rheumatoid arthritis aging progressively impairs endotheliumdependent vasodilatation in forearm resistance vessels of humans effects of age on endothelium-dependent vasodilatation of resistance coronary artery by acetylcholine in humans age related reduction of no availability and oxidative stress in humans nitric oxide and geriatrics: implications in diagnostics and treatment of the elderly estrogen induces nitric oxide production via nitric oxide synthase activation in endothelial cells sex influences immune responses to viruses, and efficacy of prophylaxis and therapeutic treatments for viral diseases targeting nitric oxide with naturally derived compounds as a therapeutic strategy in vascular diseases no generation from nitrite and its role in vascular control acute supplementation with nitrate rich beetroot juice causes a greater increase in plasma nitrite and reduction in blood pressure of older compared to younger adults acute effects of nitrate rich beetroot juice on blood pressure, hemostasis and vascular inflammation markers in healthy older adults: a randomized, placebo-controlled crossover study therapeutic value of stimulating the nitrate-nitrite-nitric oxide pathway to attenuate oxidative stress and restore nitric oxide bioavailability in cardiorenal disease dietary inorganic nitrate: from villain to hero in metabolic disease tissue distribution of ace 2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis acute ingestion of beetroot juice increases exhaled nitric oxide in healthy individuals dietary nitrate supplementation increases functional exhaled nitric oxide: implications for the assessment of airway health in athletes nitric oxide metabolites and cardiovascular disease, markers, mediators, or both dual effect of nitric oxide on s5rs-cov: viral rna production and palmitation of the s protein are affected aging is associated with endothelial dysfunction in healthy men years before the age-related decline in women figure 1: hypothetical representation of endothelial nitric oxide (no) production based on sex table 1 case fatality rate in covid-19 in china according key: cord-323749-lvtfv7ny authors: sai suresh chalichem, nehru; bethapudi, bharathi; mundkinajeddu, deepak title: aminoglycosides can be a better choice over macrolides in covid-19 regimen: plausible mechanism for repurposing strategy date: 2020-06-10 journal: med hypotheses doi: 10.1016/j.mehy.2020.109984 sha: doc_id: 323749 cord_uid: lvtfv7ny in the current covid-19 pandemic, prioritizing the immunity enhancers is equally important to anti-virals. defensins are the forgotten molecules that enhance the innate immunity against various microbes. although macrolides like azithromycin and clarithromycin etc., have been reported to act against respiratory infections but they lack the ability of immunity enhancement through defensins. the aminoglycosides were proved to have defensin mediated antiviral activity, that could enhance the immunity. so, consideration of aminoglycosides can be a double edge sword viz., against respiratory infection as well as immunity enhancer (along with anti-virals) for covid-19 regimen. although various nations throughout the globe making several strategies viz, different drug combinations, proposing lock downs and herd immunity etc., to control the wide spread of covid-19 and to tolerate its severity, the radical behaviour of virus is evident. understanding the virus behaviour, its complete genome, identifying suitable therapeutic targets, cost and ethical issues are the bars to come with new molecule, in the current (pandemic) situation. so, ddrug repurposing strategy i.e., application of widely and existing approved drugs for different focus, could be an ideal approach in the current pandemic situation. the current situation enlightened many people about importance of immune health which boosted up the usage of various traditional things like aashwagandha, eechinacea, llicorice and aandrographis etc., as immune supplements, out of which aandrographolides of aandrographis were proved as immune builders through defensins and inhibitor of virus immunity in the respiratory organs is one of the important aspects to consider as they are under continuous challenges with different particulates through inhalation and other contacts with nasal or oral surfaces. in the instances like current pandemic of covid-19, where infection control becomes a great challenge, immune mediators could be a vital link. so, strengthening respiratory epithelium is also an important aspect to control lung infection 4, 5 . defensins are part of innate immunity and it is the only group of antimicrobial peptides present in both animals (vertebrates and invertebrates) and plants 6, 7 . they present in cytoplasmic granules of immune cells like neutrophils and macrophages along with other antimicrobial factors and if from epithelial cells, released into extracellular environment 8 . defensins (mammalian) are classified as, the alpha, beta and theta defensins, which differ in their distribution and disulphide links (bonds) between conserved cysteine residues. based on their disulfide bonding pattern, divided into alpha, beta and theta defensins. the theta defensins exist as pseudo genes (cannot express) due to the presence of premature termination codon 9 . however, aminoglycosides (ags) were proved to produce functional peptides from theta defensins (called as retrocyclins) that are active against hiv 10 . so, herewith we hypothesize addition of ags into the therapeutic regimen of covid-19, could be a beneficial one. macrolides like clarithromycin and azithromycin etc., are well known for h. influenza as well as upper and lower respiratory infections. and moreover, certain clinical studies supported the usage of macrolides during panbronchiolitis and cystic fibrosis 11 and also due to their possible anti-inflammatory nature 12 . these might be the reasons for the inclusion aazithromycin in combination with hhydroxy cchloroquine. but we come with novel idea of inclusion of aaminoglycosides rather than mmacrolides with other combinations. as mentioned below, defensins, the natural immune enhancers, could play a role in enhancing the immunity. although widespread proved research is lacking to claim the ability of aminoglycosides (ags) in enhancing the defensins, it has been proved against hiv, which is also a deadly virus. as ags were proved against deadly immune suppressing virus 10 and moreover ags are currently in use, they can be used for current situation without any regulatory or ethical restrictions. direct iinstillation of retrocyclins into mouse lung was proved to reduce the mortality from sars corona virus infection. and moreover, various studies claimed the protective role of defensins against influenza viruses 13-16 .  they promote the viral aggregation which could reduce infectious titers and finally will be cleared from airway 17 .  defensins can block viral infection by directly acting on the virion or by affecting the target cell and thereby indirectly interfering with viral infection. so, that fusion of virion to target membrane is blocked results in the impairment of replication process 18, 19 . drug repurposing strategy is an ideal and rationale strategy to bring the therapeutic product in to market in quick times. although ags were proved only for their enhancing/modulating capability towards ttheta defensins (against hiv), their role in enhancing other defensins cannot be eliminated. since the safety profile of ags is already established, selection of these drugs for covid-19 therapeutic regimen could be an ideal choice. none to declare andrographolide prevents ev-d68 replication by inhibiting the acidification of virus-containing endocytic vesicles dehydroandrographolide enhances innate immunity of intestinal tract through up-regulation the expression of hbd-2 defensins and cathelicidins in lung immunity airway epithelial versus immune cell stat1 function for innate defense against respiratory viral infection plant defensins human defensins defensins: natural component of human innate immunity the chemistry and biology of theta defensins humanized θ-defensins (retrocyclins) enhance hapivirins and diprovirins: novel θ-defensin analogs with potent activity against influenza a virus βdefensin inhibits influenza virus replication by cell-mediated mechanism (s) human neutrophil defensins increase neutrophil uptake of influenza a virus and bacteria and modify virus-induced respiratory burst responses selsted me. θ-defensins: cyclic peptides with endless potential defensins in innate antiviral immunity key: cord-322880-zfc9w5y6 authors: rohit, anusha; rajasekaran, shankar; karunasagar, indrani; karunasagar, iddya title: respiratory droplets get suspended longer and spread wider in temperate environments compared to tropics and implications for sars-cov-2 transmission date: 2020-06-09 journal: med hypotheses doi: 10.1016/j.mehy.2020.109958 sha: doc_id: 322880 cord_uid: zfc9w5y6 the new pandemic of sars-cov-2 has shown stark differences in number of affected patients between countries in the tropics and those with temperate environments. though there have been many theories on reasons for these differences, we hypothesise that this could be due to differences in the fate of respiratory droplets in the two environments. a simple understanding of the mechanics of droplet size, dispersion and displacement could help infection control and public health measures to minimize spread and mitigate the risk of people getting infected especially in hotspots like hospital environments or other closed spaces. this paper discusses the possibility of differences in number of infections and spread between different countries based on the spread of droplets. the situation with respect of covid-19 globally is very grim with 3,939,119 cases and 274,917 deaths as on may 9 th (1). interestingly, resource rich countries like us, germany, uk, italy, spain seem to have been much more affected than many of the tropical countries in south and south east asia and africa. for example, vietnam, which shares a long border with china has only 288 cases and no deaths though 2 cases were detected as early as 23 rd january. laos, sharing border with china had 19 cases with no deaths, myanmar, sharing border with china had 177 cases with 6 deaths. thailand had cases detected in january, but in april, the case count is only 3,004 with 56 deaths. in contrast, in us, cases started appearing only early march, but in a span of a month, the numbers have increased dramatically to 1,322,154 and 78,616 deaths. in italy, the cases started showing up during the last week of february, but escalated to 217,185 with 30,201 deaths. in spain, cases started appearing early march, but soon increased to 260,117with 26,299 deaths, while in germany, cases began appearing late february, but increased to 170,588 with 7,510 deaths. the explosive increase in cases in resource rich countries with well-established public health system compared to a very slow increase in countries in south east asia, which are resource limited and having weak public health system is perplexing. though inadequate testing contributes to lower numbers in some countries, the difference is indeed stark and calls for other explanations based on transmission of this virus. the predominant mode of virus spread is said to be through droplets generated by infected individuals during coughing, talking or sneezing and contact transmission (2) . the droplets may range in size from sub-micrometers to hundred of micrometers. from "stoke's drag force equation", the terminal settling velocity of a particle of known diameter and density can be calculated. the settling velocity of the particle would be directly proportional to the density of the particle and to the square of the particle diameter. owing to their size, larger particles settle down at a much faster speed than smaller particles. for instance, the settling velocity of a 10 micron water droplet will be approximately 88 times greater than a 1 micron water droplet. thus smaller particles will stay airborne for a longer time, while larger particles will settle down on surfaces more quickly. according to law of brownian motion, which is applicable to smaller particles, the diffusive forces would transport the particles in random directions. the diffusion coefficient of a particle as given by "stokes-einstein equation" is inversely proportional to the diameter of the particle. so, smaller the particle, higher the diffusivity. higher diffusivity helps smaller particles to travel through the room at a faster speed than larger particles. for instance, a 1 micron particle will have a net diffusion displacement that is 3 times more than that for a 10 micron particle (figure 1 ). thus a smaller particle will spread throughout the room at a much faster rate than a large particle. the relative humidity of the environment influences the density of the particle as well as the size of the particle (figure 2 ). upon release, the droplets may be subject to partial or total evaporation, depending on environmental conditions and would reduce in size. the environmental relative humidity (rh) will influence the extent of evaporation and final equilibrium size of the droplet. thus lower rh reduces the size of the particle as the particle desiccates. furthermore, relative humidity could affect the particle density too, as the particle desiccates. the combined effect of change in size of the droplet as well as the density amplifies the influence that environmental relative humidity would have on the particle settling velocity. marr et al (3) studied the effect of rh on droplet equilibrium using droplets containing 9mg/ml nacl, 3 or 76 mg/ml protein and 0.5mg/l surfactant. they noted that a droplet of 10μm shrinks to 2.8μm at 90%rh and to 1.9μm at less than 64%rh. the droplet size would greatly affect settling times, with 10μm droplet remaining suspended for only 8min at 100% rh while the 1.9 μm droplet at 64% rh remaining suspended for more than 3h. all the above apply to the dynamics of particles in still air, with no air currents. however, in real life scenario, air currents and turbulences are unavoidable. these would further affect the settling patterns of particles. therefore a significant portion of the particles are likely to be airborne indefinitely, drifting around freely. the expelled aerosol by a person on account of exhaling, talking, coughing, sneezing, etc would have a poly dispersed particle size distribution. the mean, median, mode and standard deviation of the distribution would vary based on expulsion characteristics. a significant portion of this cocktail of particles expelled by a person could hence stay airborne, influenced by environmental conditions and perhaps even get transported out of the source room, when provided with suitable conditions. our hypothesis suggests that mechanistically, in tropical conditions of high temperature and high humidity, droplets settle quickly, while in temperate conditions of low temperature and low humidity, droplets tend to remain suspended in air. in the case of respiratory droplets, these would dramatically affect airborne transmission of viruses in the droplets. it is possible that droplets discharged from infected individuals in places like new york city could shrink in size quickly and remain airborne for considerable period of time. a droplet size of 1 μm could be airborne for 8 hrs. the humidity in new york city which is experiencing a serious covid situation is around 64%, and in berlin, the humidity is around 60%. on the other hand, in hanoi, vietnam, the humidity is above 80%. under conditions of temperature and humidity in cities like hanoi and hochi minn city in vietnam, the droplets would settle quickly. this might explain higher transmission rates observed in temperate environments compared to tropical ones. the same argument holds good for air conditioned spaces, where the temperatures and relative humidity levels could be low. this may influence the measures needed to prevent infections. hand hygiene and social distancing could be the most important step in tropical settings, since transmission could be predominantly through droplets settling on various surfaces, while, use of masks, hand hygiene and social distancing would be important in temperate places, since virus could remain airborne for considerably longer period of time. for air conditioned environments, suitable modifications and adjustments would be required to the air conditioning system to control the spread of infections. in addition to differences in mode of dispersion, there could be differences in infectivity of virus due to survival under conditions of temperature and sensitivity. chan et al (4) studied survival of sars coronavirus on various surfaces at different temperature and humidity conditions. virus viability was retained up to 5 days on plastic surface at temperature of 22-25°c and humidity of 40-50% with only one log reduction in titre. temperature range of 28-33°c and humidity of >95% did not make much difference, but temperature of 38°c at 80-90% rh led to up to 2 log reduction in titres in 24hrs. studies with porcine coronavirus (transmissible gastroenteritis virus, tgev) showed that they remain viable longer at low relative humidity (rh) than at high humidity (5). tgev is also an enveloped virus like sars-cov-2. the persistence of influenza virus in the environment has been studied by a number of investigators. using systematic review methodology, irwin et al (6) surmised that increasing temperature (2-12°c vs >27°c) resulted in shorter virus half life. published studies indicate that virus half life was 16.5 times longer at temperatures between 7-12°c as compared to >27°c. harper (7) studied influenza survival at three temperatures (7-8°c, 20.5-24°c and 32°c) and five rh ranges (20-25%, 34-36%, 49-51%, 64-65% and 81.82%) and noted that virus survival was highest at lowest temperature and lowest rh studied. using enveloped virus phi6 in droplets as surrogate for influenza and coronaviruses, prussin ii et al (8) noted significant decrease in infectivity at rh range 60-85%. infectivity decreased two orders of magnitude between 19-25°c at humidity level of 75%. it can be expected that sars-cov-2 virus behaves on similar lines as sars-cov-1 and influenza virus. infectivity could be reduced under environmental conditions prevailing in cities like hanoi in vietnam compared with new york city in us. therefore, reduced infectivity and shorter airborne transmission could explain lower number of cases observed in south east asia. virology, epidemiology, pathogenesis and control of covid-19 mechanistic insights into the effect of humidity on airborne influenza survival, transmission and incidence effect of temperature and relative humidity on the viability of sars coronavirus fate of respiratory droplets in tropical vs temperate environments and implications for sars-cov-2 transmission the research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors the new pandemic of sars-cov-2 has shown stark differences in number of affected patients between countries in the tropics and those with temperate environments. though there have been many theories on reasons for these differences, we hypothesise that this could be due to differences in the fate of respiratory droplets in the two environments. a simple understanding of the mechanics of droplet size, dispersion and displacement could help infection control and public health measures to minimize spread and mitigate the risk of people getting infected especially in hotspots like hospital environments or other closed spaces. this paper discusses the possibility of differences in number of infections and spread between different countries based on the spread of droplets. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. all authors have no conflict of interest to declare. this work has not been published previously. key: cord-311673-z4hkw17g authors: uzzan, mathieu; corcos, olivier; martin, jerome; treton, xavier; bouhnik, yoram title: why is sars-cov-2 infection more severe in obese men? the gut lymphatics lung axis hypothesis date: 2020-06-23 journal: med hypotheses doi: 10.1016/j.mehy.2020.110023 sha: doc_id: 311673 cord_uid: z4hkw17g consistent observations report increased severity of sars-cov-2 infection in overweight men with cardiovascular factors. as the visceral fat possesses an intense immune activity, is involved in metabolic syndrome and is at the crossroad between the intestines, the systemic circulation and the lung, we hypothesized that it plays a major role in severe forms of sars-cov-2 infection. sars-cov2 presents the ability to infect epithelial cells of the respiratory tract as well as the intestinal tract. several factors may increase intestinal permeability including, direct enterocyte damage by sars-cov2, systemic inflammatory response syndrome (sirs) and epithelial ischemia secondary to sars-cov2associated endothelial dysfunction. this increase permeability further leads to translocation of microbial components such as mamps (microbial-associated molecular pattern), triggering an inflammatory immune response by tlr-expressing cells of the mesentery fat (mostly macrophages and adipocytes). the pro-inflammatory cytokines produced by the mesentery fat mediates systemic inflammation and aggravate acute respiratory distress syndrome (ards) through the mesenteric lymph drainage. the recent outbreak of the sars-cov-2 has led to a worldwide pandemic responsible for a major health crisis. coronavirus-induced disease can evolve to severe forms characterized by hyperinflammation, acute respiratory distress syndrome (ards) and death, which are more frequently observed in subsets of patients with comorbidities. observational studies in chinese, european and us cohorts consistently highlighted a significant association between intensive care unit (icu) admission and male gender, as well as hypertension, diabetes and cardiovascular diseases, as compared to non-icu patients [1, 2] . in a cohort of 138 patients hospitalized in wuhan, males accounted for 61% of icu patients (n=36) as compared to 52% (n=102) in conventional units; 58%, 25% and 22% of icu patients had hypertension, diabetes and cardiovascular history, respectively, as compared to 22%, 11% and 6% in non-icu patients (all p values below 0.05) [2] . higher risk of death was further reported for males with cardiovascular comorbidities within covid-19 icu patients [3, 4] . [5] . similar results were obtained in a large cohort from new york city (ny), in which obesity was an independent risk-factor for hospitalization in patients under 60 years old who were admitted to the emergency department [6] . in an italian cohort of more than 1,000 icu patients hospitalized in lombardia, 83% of patients were males; 50% had high blood pressure and about 20% had type 2 diabetes, hypercholesterolemia and/or a cardiovascular disease [7] . overall, these studies indicate that overweight males with cardiovascular risk factors are at significantly higher risk of developing severe forms of covid-19. as the increased volume of mesentery fat in overweight men play a key role in the occurrence of metabolic syndrome [8] , we hypothesized that the visceral adipose tissue plays a central role in severe forms of covid-19. the initial phase of the disease is considered as the viral phase. at that phase, covid-19 behaves as a classical lymphocytic pneumonia, evidenced by ct-scan and/or lung biopsies [9, 10] ,. at a later phase of the disease, lung tissue lesions are unusual, presenting notably with major intra-alveolar fibrin deposition and intraluminal loose connective tissue in alveolar ducts and bronchioles. additionally, vascular damage is significant with cytoplasmic vacuolization of the endothelium and cell detachment in small to medium-sized pulmonary arteries [10] . at this later phase, along with endothelial damage, clinical and biological characteristics are suggestive of a "cytokinic storm". in critically ill patients with covid-19 an intense inflammatory response during a secondary respiratory and systemic worsening at day 7 to day 15 from the onset of initial symptoms has been widely described [7, 11, 12] . hadjadj et al. performed an immunological extensive assessment (immune blood cell phenotyping, whole blood transcriptomic and cytokine quantification) in patients with mild form and severe form of covid-19 at day 8 to day 12 after onset of symptoms [13] . interestingly, they found that type 1 interferon response was profoundly impaired. in addition, innate immunity-associated cytokinic pathways were markedly activated. specifically, they found a strong increase in expression of il-6-induced genes, such as il6r, socs3 and stat3 as well as an up-regulation of tnf pathway-related genes, including tnfsf10. another key finding is the up-regulation of the ccl2-ccr2 pathway suggesting a key role of monocytes and macrophages in the lung and in the intense systemic inflammation state [13] . other studies described increased plasma concentrations of a series of inflammatory cytokines, such as granulocyte-macrophage colony-stimulating factor (gm-csf), il-6 [14, 15] , tnf-α, and granulocyte colony-stimulating factor (g-csf), in severe covid-19. further investigations demonstrated that peripheral inflammatory monocytes and t cells through their production of gm-csf may incite cytokine storm in severe covid-19 patients [15, 16] . this excess of inflammatory response is a critical cause of mortality from covid-19. a study of 548 patients showed that, high cytokine levels (il-2r, il-6, il-10, and tnf-α), and high ldh level were significantly associated with severe forms of covid-19 [17] . accordingly, anti-il-6r therapy is being investigated to treat severely ill patients [18, 19] with preliminary results reporting efficacy in a french multicenter randomized clinical trial. other anti-cytokines therapies are also under investigation. sars-cov-2 binds ace2 which is vastly expressed in cardiopulmonary tissues, including endothelial cells. some hematopoietic cells also express it, and particularly monocytes and macrophages [20] . from studies focusing on other coronaviruses, it is known that the infection of cells of the myeloid lineage (monocytes, macrophages and dendritic cells) results in their activation and production of inflammatory cytokines including il-6 [20] . il-6 can signal through 2 main pathways, the trans-and the cis-signaling pathways. the cispathway involves gp130 and mil-6r (membrane-bound il-6r) mainly restricted to immune cells and results in a pleiotropic effect on the cells of the adaptative and innate immunity. trans signaling only involves the soluble form of il-6r (sil-6r) forming a complex with gp130 which is virtually express on all hematopoietic and non-hematopoietic cell types. therefore, it activates cells that do not express mil-6r like endothelial cells, resulting in a cytokinic storm [20] . overall, the day 7 to day 12 clinical worsening is a typical feature of the course of covid-19 and involves a major release of cytokines and particularly il-6 and tnf-α with a key role of myeloid cells. this cytokine release syndrome highly correlates with severity and mortality. additionally, a key feature of covd-19 course, is lymphopenia -a hallmark of severe forms -, suggesting that most of the cytokines released during the cytokinic storm are produced by myeloid cells or non-hematopoietic cells such as adipocytes. visceral adipose tissue is more developed in men, as it represents 10 to 20% of total fat as compared to 5-10% in women [21] . visceral fat possesses multiple properties that make it a key site for intestinal and systemic immune response. in comparison with subcutaneous adipose tissue, visceral adipose tissue including mesenteric fat has a higher vascularization and contains greater number of immune cells. it also expresses higher level of pro-inflammatory cytokines such as il-6, tnf-α and il-8 [21] . visceral adipose tissue is enriched in macrophages that are skewed toward an m2 phenotype (the pro-inflammatory subtype) [22] . importantly, in case of visceral obesity, the pro-inflammatory state of the visceral fat is further enhanced [21] . macrophages of the mesentery fat from patients with nonalcoholic steatohepatitis (nash) present elevated levels of cytokines and chemokines compared with healthy subjects suggesting, that mesenteric inflammation may uncouples and mediates inflammation through the portal flow [23] . in addition to the immune response provided by mesentery fat-associated macrophages, adipocytes and stromal cells may also play their own role as immune enhancers [24] . for example, adipokine produced by adipocytes, can regulate metabolic homeostasis and affect immune functions [25] . in crohn's disease, it has been shown that mesenteric fat could be a major source of c-reactive protein as well as il-6. it is also enriched in microbial particles of the microbiota as an evidence of enhanced intestinal microbial translocation [26] . remarkably, antitnf-α and antiil-6 therapies have been shown to reduce intestinal inflammation during crohn's disease [27, 28] . therefore, the visceral adipose tissue is a site with an intense immune activity that is able to produce high level of innate immunity-associated cytokines such as il-6 and tnf-α. it haspro-inflammatory properties compared with subcutaneous adipose tissue. a systemic proinflammatory potential is further enhanced in case of android obesity. whether mesenteric fat can be responsible for an intense cytokine production raises the question of the role of the gut in this mesenteric activation. [29] . it has been demonstrated that sars-cov-2 is capable of binding to the angiotensin-converting enzyme 2 (ace2) which is express in many cell types including the epithelial cells of the respiratory and the gastrointestinal tract [31, 32] . an austrian team reported that patient with covd-19-associated diarrhea had an elevated fecal calprotectin with a mean of 123 µg/g in patients with active diarrhea. the fecal calprotectin could reach values above 200 µg/g in some patients without any preexisting digestive condition [33] . interestingly, the level of fecal calprotectin did not correlate with the level of viral rna in the stool but positively correlated with the level of il-6 in plasma [33] . cov-2. moreover, they showed that the virus can replicate in intestinal epithelial cell lines as well as in human colon organoid models, therefore participating in the spread and enhanced viremia of sars-cov-2 [34, 35] . overall, sars-cov-2 has an intestinal tropism and may induce acute intestinal inflammation which associates with systemic inflammation rather than with intestinal viral load. the ultimate consequence is an increase translocation of the intestinal luminal content (including bacteria of the microbiota) to the mesentery fat and the systemic blood circulation. therefore, the ability of sars-cov2 to directly bind and invade the intestinal epithelium and to induce intestinal mucosa inflammation could lead to enhance mesentery antigenic stimulation. this inflammation of the intestinal tract and subsequently of the mesentery fat could be aggravated by other covid-19-associated features such as blood coagulopathy and microthrombotic disorders [36] . another key digestive characteristic of covd-19 is the high prevalence of hepatic cytolysis that is associated with severe forms of the disease [37] . while the underlying mechanism is not well understood, multiple factors are likely involved. increased intestinal translocation to the portal circulation may be one of the participating factors of liver damage. overall, the gastrointestinal tract is a key organ with probable direct viral invasion evidenced by fecal shedding of sars-cov-2 rna and prevalent digestive symptoms during covid-19 course, which are associated with a pejorative outcome patients with systemic inflammatory response syndrome (sirs) have increase intestinal permeability even if they don't have any gastrointestinal condition [38] . therefore, there is an increase bacterial translocation of either live bacteria, or mamps (microbial-associated molecular patterns) that will trigger the innate immunity through toll-like receptors. increased intestinal permeability was found in icu patients by measuring urinary excretion of orally administered lactulose or mannitol [39] . interestingly, increased intestinal permeability was associated with clinical worsening including multiorgan failure in critically ill patients. portal venous and the lymph flows are the two main routes that could explain how this modified intestinal permeability as well as the bacterial translocation impact other organs such as the lungs. in a study published in 1991, moore and al. performed a venous catheterism in the portal vein of critically ill patients to do sequential blood cultures. while 60% of patients were in shock and 30% developed multiorgan failure, only 2% of portal venous blood cultures were positive suggesting that the portal circulation is not the main way for intestinal bacterial translocation [40] . an alternative route of translocation could be the intestinal lymphatics. intestinal lymph is drained from the mesenteric lymphatics to the thoracic duct, subsequently merging with the blood circulation in the left subclavian vein. therefore, products drained by the intestinal lymph are first in contact with the pulmonary arterial vasculature. in that regard, it is relevant to note that critically ill patients often develop ards even without any primary respiratory condition or infections such as patients with severe pancreatitis or trauma developing ards. interestingly, experiments performed in a hemorrhagic shock model in rats showed that lymph collected in the mesenteric lymphatics but not blood from the portal vein could have negative effect in vitro on the endothelium permeability as well as on neutrophils activation and several other relevant cell types. reinjection of lymph but not portal blood from shocked rat to other rats could worsen lung failure in vivo. strikingly, the ligation of the major intestinal lymph duct could prevent early lung damage [41] [42] [43] . therefore, multiple experimental in vivo and ex vivo data both in human and animal models support a gut lymph-lung axis. sars-cov-2 that has a direct effect on the intestine and that triggers a sirs may then critically increase intestinal permeability leading to the recirculation of pro-inflammatory mediators to the circulation and primarily to the lungs. additionally, extensively reported sars-cov-2-associated vascular injury and coagulopathy [10, 36] may lead to ischemia of the intestinal epithelium, further favoring microbial translocation. male sex, advanced age and metabolic syndrome are factors strongly associated with the severity of covid-19. they are associated with a high mortality rate. the daily expanding literature shows overwhelming data on the secondary worsening associated with high systemic inflammation including tnf-α and il-6 as two of the key cytokines. moreover, it is more and more evident that the intestine is targeted by the sars-cov-2 and that the intestinal phase may be critical in severe forms of covid-19. therefore, we propose a pathophysiogeny model of severe forms (figure 1) where the visceral fat, which is already in a pro-inflammatory state in patients with dysmetabolic syndrome, acts as an enhancer of inflammation, being an uncoupling link between the intestine and systemic and pulmonary inflammation through the mesenteric lymphatics. a better understanding of the clinical characteristics of coronavirus disease 2019 in china clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china clinical characteristics of 113 deceased patients with coronavirus disease 2019: retrospective study characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china high prevalence of obesity in severe acute respiratory syndrome coronavirus-2 (sars-cov-2) requiring invasive mechanical ventilation obesity in patients younger than 60 years is a risk factor for covid-19 hospital admission baseline characteristics and outcomes of 1591 patients infected with sars-cov-2 admitted to icus of the lombardy region mesenteric fat thickness is an independent determinant of metabolic syndrome and identifies subjects with increased carotid intima-media thickness covid-19 pneumonia: different respiratory treatments for different phenotypes? time to consider histologic pattern of lung injury to treat critically ill patients with covid-19 infection early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study impaired type i interferon activity and exacerbated inflammatory responses in severe covid-19 patients pathological inflammation in patients with covid-19: a key role for monocytes and macrophages pathogenic t cells and inflammatory monocytes incite inflammatory storm in severe covid-19 patients coronavirus infections and immune responses risk factors for severity and mortality in adult covid-19 inpatients in wuhan tocilizumab for the treatment of severe covid-19 pneumonia with hyperinflammatory syndrome and acute respiratory failure: a single center study of 100 patients in tocilizumab therapy reduced intensive care unit admissions and/or mortality in covid-19 patients cytokine release syndrome in severe covid-19 adipose-tissue and intestinal inflammation -visceral obesity and creeping fat adipose tissue macrophages association of adipose tissue inflammation with histologic severity of nonalcoholic fatty liver disease macrophage-secreted factors induce adipocyte inflammation and insulin resistance adipokines and their role in intestinal inflammation mesenteric fat as a source of c reactive protein and as a target for bacterial translocation in crohn's disease infliximab for the treatment of fistulas in patients with crohn's disease randomised trial and open-label extension study of an anti-interleukin-6 antibody in crohn's disease (andante i and ii) gastrointestinal manifestations of sars-cov-2 infection and virus load in fecal samples from the hong kong cohort and systematic review and meta-analysis real-time tracking of self-reported symptoms to predict potential covid-19 diarrhoea may be underestimated: a missing link in 2019 novel coronavirus receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus faecal calprotectin indicates intestinal inflammation in covid-19 critical role of type iii interferon in controlling sars-cov-2 infection, replication and spread in primary human intestinal epithelial cells sars-cov-2 productively infects human gut enterocytes coagulopathy and antiphospholipid antibodies in patients with covid-19 liver injury in covid-19: management and challenges gut-origin sepsis in the critically ill patient: pathophysiology and treatment increased intestinal permeability is associated with the development of multiple organ dysfunction syndrome in critically ill icu patients gut bacterial translocation via the portal vein: a clinical perspective with major torso trauma gut-derived mesenteric lymph but not portal blood increases endothelial cell permeability and promotes lung injury after hemorrhagic shock post-hemorrhagic shock mesenteric lymph is cytotoxic to endothelial cells and activates neutrophils role of the gut in the development of injury-and shock induced sirs and mods: the gut-lymph hypothesis, a review figure legends way that the mesentery fat aggravates the course of covid-19 may open new and innovative therapeutic avenues. the authors have no conflict of interest related to this work to declare. key: cord-320508-egw7bvzf authors: kennedy, james r. title: phosphatidylserine’s role in ebola’s inflammatory cytokine storm and hemorrhagic consumptive coagulopathy and the therapeutic potential of annexin v date: 2019-10-28 journal: med hypotheses doi: 10.1016/j.mehy.2019.109462 sha: doc_id: 320508 cord_uid: egw7bvzf the phosphatidylserine (ps) molecule is present in cell membranes where it is actively kept on their inner leaflets but when cells are damaged it moves to the surface and become a signal for their removal, the platform upon which the coagulation cascade takes place and a ligand that activates a feedback cycle of inflammatory cytokine secretion and initiates the wakeup call for the innate immune response. these are physiologic responses to ps but the ebola virus displays ps molecules on its membrane’s surface and the huge numbers of viruses cause a pathologic inflammatory cytokine storm and a hemorrhagic consumptive coagulopathy. annexin v is an innate molecule that can cloak membrane displayed ps and prevents its th1 cell’s inflammatory cytokine generation and cascade thrombin generation. the hypothesis presented is that its administration will cloak ps and prevent ebola’s consumptive coagulopathy and its cytokine storm. the phosphatidylserine (ps) molecule is present in cell membranes where it is actively kept on their inner leaflets but when cells are damaged it moves to the surface and become a signal for their removal, the platform upon which the coagulation cascade takes place and a ligand that activates a feedback cycle of inflammatory cytokine secretion and initiates the wakeup call for the innate immune response. these are physiologic responses to ps but the ebola virus displays ps molecules on its membrane's surface and the huge numbers of viruses cause a pathologic inflammatory cytokine storm and a hemorrhagic consumptive coagulopathy. annexin v is an innate molecule that can cloak membrane displayed ps and prevents its th1 cell's inflammatory cytokine generation and cascade thrombin generation. the hypothesis presented is that its administration will cloak ps and prevent ebola's consumptive coagulopathy and its cytokine storm. phosphatidylserine (ps) molecules are present in cell membranes where they are actively kept on their inner leaflets. in senescent and otherwise damaged cells ps moves to their surface where it becomes a signal for their phagocytic removal [1] , the platform upon which the coagulation cascade takes place [2] , a ligand for the tim-1 receptor on t helper one (th1) cells that activates its inflammatory cytokine secretion [3] and a ligand that binds to tim receptors on mononuclear immune cells to provide the wakeup call for the innate immune response [4] . billions of cells become apoptotic daily and display ps on their surface but they are rapidly removed by phagocytes that recognize it [5] and the th1inflammation [3] , cascade generated blood coagulation [2] and the innate immune activation do not take place [4] . in ebola ps on the surface of its virus activates th1 inflammatory cytokine secretion and the ps exposure is so great that a cytokine storm is produced [3] and all the thrombin generated on the cascade's ps platforms generates enough thrombin to consume coagulation components and produce a hemorrhagic consumptive coagulopathy [2] . macrophages and dendritic cells are phagocytes that secrete inflammatory cytokines when tlrs on their surface recognize foreign or damage associated molecular patterns but they also display tim-1 receptors and in ebola those receptors bind to and phagocytize the ps+ viruses and they become infected [6] . when infected these cells are prime viral replicators that generate viruses to infect other cells. in addition, the infected dendritic cells may no longer be able to mhc present antigenic pathogen peptides to immune cells in the adaptive immune response producing immunologic depression. in 2017 it was discovered that ps on the ebola virus could bind to the tim-1 receptor on th1 cells and independently produce a lethal cytokine storm in mice [3] . this was proven when an ebola infection in knockout tim-1 −/− negative did not produce a cytokine storm and the mice survived even though the viral load was only minimally affected [3] . the hypothesis presented here is that annexin v's therapeutic administration in ebola can prevent its th1 cell generated inflammatory cytokine storm, stop the cascade generated hemorrhagic consumptive coagulopathy and prevent macrophage and dendritic cell infection. unfortunately when annexin v cloaks ps the cascade can't function and hemostasis will not be possible so let us very briefly examine the components of inflammation, blood coagulation and annexin v with regard to the possibility that its administration can prevent ebola's cytokine storm and coagulopathy without causing bleeding. t continued in a late mediated inflammatory response when some of the cells damaged by those cytokines become necrotic causing high mobility group box one (hmgb1) molecules release by nuclear disruption. the hmgb1 molecules then bind to the tlr4 receptors on dendritic cells and cause their inflammatory cytokine secretion [7] . the ps exposed on the surface of cells damaged by the innate and late mediated inflammatory responses binds to the tim-1 receptor on th1 cells and they begin a feedback cycle of inflammatory cytokine secretion that expose more ps and amplifies the inflammation [3] . this amplified inflammation damages and kills both pathogens and healthy cells and the ps displayed on the latter activate the innate immune response when an infection is present and make hemostasis possible when a vascular wall is breached. this physiologic ps feedback generation contrast with the pathologic cytokine storm and hemorrhagic consumptive coagulopathy in ebola where membrane displayed ps is on every virus. for blood coagulation to begin thrombin must be present and its generation begins when cells are damaged and the procoagulant molecules tissue factor (tf) and ps are exposed. in ebola the inflammatory storm damages many cells and the large numbers of tf and ps molecules exposed cause intravascular blood coagulation that consumes coagulation components and leads to a hemorrhagic consumptive coagulopathy. tf initiates blood coagulation but ps is the platform upon which the coagulation cascade's thrombin generation produces the consumptive coagulopathy. in ebola ps is not only exposed by inflammatory cell damage it is also on every virus. tf begins blood coagulation by activating factors x and ix. activated factor xa changes prothrombin to thrombin and factor ixa is an essential component of the coagulation cascade. tf generated thrombin directly or indirectly activates all coagulation components including the coagulation cascade. once activated the cascade generates thrombin in a feedback cycle of factor x activation in its tenase complex and by a catalytic generation of thrombin in its prothrombinase complex [8] . in the tenase complex activated factor viiia and ixa bind to membrane displayed ps and are joined there by factor x to be activated [9] . the activated factor xa then joins factor va on ps in the prothrombinase complex and the factor xa, factor va and ps amalgam catalytically begins changing prothrombin to thrombin and exponentially increases its generation making hemostasis possible [10] . in hemophilia factors viii and/or ix are not available and so the cascade can't function and hemostasis is not possible even though tf thrombin generation is not affected [11] . factor ixa is required for cascade thrombin generation but tf can't supply enough of it for cascade function so cascade generated thrombin activates factor xi and it activates factor ix. this increases thrombin generation but not exponentially. this is a good thing because though fibrin generation is desirable during cellulitis intravascular blood coagulation is not. for the cascade's exponential thrombin generation to take place factor xii must activate factor xi so it can activate factor ix and to do this factor xii must be activated by binding to sulfatide on activated platelets [12] . this is not possible intravascularly because activated platelets secrete a factor xii activation inhibitor [13] . it is possible when a vascular breach is present and collagen is exposed because platelets displaying sulfatide and ps on their surface bind to the collagen and the blood flow washes away the inhibitor. in a vascular breach tf and ps are only exposed at the breach site and factor ix is activated there by factor xiia making the cascade's exponential increase in thrombin generation possible. the same thing happens when an atherosclerotic plaque ruptures and collagen is exposed. the physiologic feedback cycles of ps exposure that cause inflammation and thrombin generation are needed to assure a rapid innate immune response to an infection and to rapidly control bleeding when a vascular wall is breached but such cycles, like atomic chain reactions, are dangerous and require controls and it is proposed that annexin v, like factor ixa is one of them. the following 2016 study by park supports the hypothesis that annexin v can block cytokine storms and consumptive coagulopathies [14] . in this study gram negative septicemia was induced in mice where the lipopolysaccharide (lps) molecule on those bacteria generated a lethal cytokine storm and generated excess fibrin (not a coagulopathy). lps generates thrombin by binding to tlr4 on dendritic cells. it was found that a single injection of annexin v stopped the cytokine storm, reduced fibrin generation and enabled the mice survival [14] . the annexin v did this by cloaking ps on damaged cells and by blocking the binding of lps to tlr4. let us now take a brief look at annexin v. annexin v is an innate molecule found in cells and in nano molecular amounts in plasma where its levels increase during inflammation and blood coagulation [15] . what it does in cells and how it gets from there to plasma is uncertain [16] . it is a small protein molecule that is rapidly removed by kidneys causing it to have a half-life of minutes in plasma [17] . it has a great affinity for ps and because of this its labeled molecules are used in the lab and clinically for its identification but it has no therapeutic application. when annexin v binds to ps on a cell's membrane it crystalizes and this forms a two dimensional ps cloaking shield that can internalize it into the cells cytoplasm [16] . when annexin v cloaks ps the th1 cell's inflammatory cytokine secretion that causes ebola's cytokine storm [3] and the cascade thrombin generation that produces its hemorrhagic consumptive coagulopathy [2] can't take place. this will also prevent hemostasis. will this prevent its therapeutic use? using annexin v to treat the pathologic aspects of inflammation and blood coagulation will only be possible if its therapeutic benefits are rapid and long lasting, its pathologic ones transitory and the physiologic recovery from its ps cloaking is rapid. all seem possible. when annexin v cloaks exposed ps this will instantly and permanently prevent those ps's ability to activate the th1 cell's inflammatory cytokine secretion [16] and the blood coagulation that ps makes possible [2, 14] . annexin v's half-life of minutes [17] means it will be rapidly eliminated so that ps exposed by a subsequent vascular breach will be met by breach exposed tf generating thrombin and by ps and sulfatide on activated platelets adhering to the collagen. regarding annexin v's blocking of the innate immune cell activation [4] , annexin v will only be given during an infection so that the innate immune response will already have been initiated. the ability to monitor the effect of annexin v administration would be desirable and since radio and florescent labeled annexin v molecules are used in the lab and clinically to detect ps+ cells it is possible that they can be used to quantify the ps+ cells that produce the inflammation and blood coagulation. could conditions other than ebola be considered for annexin v treatment? the same inflammatory and coagulation pathology is present in severe septicemia as is present in ebola and as in ebola it has little effective treatment, annexin v may be effective there. coronavirus sars and severe influenza infections are other lethal conditions to consider for annexin v therapy. in them their lethal respiratory failure may be the result of the inflammation and blood coagulation caused by the ps exposure on infected alveolar and bronchiolar cells resulting in the consolidation of the air space by the massive infiltration of mixed mononuclear/neutrophilic cells, edema and fibrin deposits [18] . the administration of annexin v in ebola and in other critically ill patients should probably be by intravenous bolus so that its cloaking of ps will instantly stop further th1 cell feedback secretion of inflammatory cytokines and coagulation cascade thrombin generation that is causing the inflammatory organ damage and the thrombotic coagulopathy. in ebola its bolus administration may also prevent further macrophage and dendritic cell infection. in non-critical sars and influenza infections monitoring the levels of ps+ cells could be used to determine the necessity for annexin v use and when it is needed the ps + cell count can be used to monitor its continuous intravenous administration. annexin v's ability to cloak ps and block its roles in inflammation and blood coagulation is well documented but the therapeutic possibilities presented are speculative and require animal studies for their verification. at present there is little or no effective treatment for these lethal conditions so those studies would seem to be indicated. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. the role of phosphatidylserine in recognition of apoptotic cells by phagocytes the regulator of phosphatidylserine-catalyzed inflammation and coagulation during apoptosis ebola virus binding to tim-1 on t lymphocytes induces a cytokine storm tim genes: a family of cell surface phosphatidylserine receptors that regulate innate and adaptive immunity find-me and eat-me signals in apoptotic cell clearance: progress and conundrums phosphatidylserine receptors: enhancers of enveloped virus entry and infection cell death in the pathogenesis of immune-mediated diseases: the role of hmgb1 and damp-pamp complexes differential roles of tissue factor and phosphatidylserine in activation of coagulation kinetics of factor x activation by the membrane-bound complex of factor ixa and factor viiia membrane-dependent interaction of factor xa and prothrombin with factor va in the prothrombinase complex ratelimiting roles of the tenase complex of factors viii and ix in platelet procoagulant activity and formation of platelet-fibrin thrombi underflow human annexin v binds to sulfatide: contribution to regulation of blood coagulation platelet surface-associated activation and secretion-mediated inhibition of coagulation factor xii annexin a5 increases survival in murine sepsis model by inhibiting hmgb1-mediated pro-inflammation and coagulation clinical significance of measurement of plasma annexin v concentration of patients in the emergency room extracellular annexin a5: functions of phosphatidylserine-binding and two-dimensional crystallization diannexin, an annexin a5 homodimer, binds phosphatidylserine with high affinity and is a potent inhibitor of platelet-mediated events during thrombus formation animal models of acute lung injury not applicable. none. supplementary data to this article can be found online at https:// doi.org/10.1016/j.mehy.2019.109462. key: cord-310928-g553afo9 authors: murch, simon h title: common determinants of severe covid-19 infection are explicable by sars-cov-2 secreted glycoprotein interaction with the cd33-related siglecs, siglec-3 and siglec-5/14 date: 2020-08-07 journal: med hypotheses doi: 10.1016/j.mehy.2020.110168 sha: doc_id: 310928 cord_uid: g553afo9 sars-cov-2 interaction with the ace-2 receptor cannot alone explain the demography and remarkable variation in clinical progression of covid-19 infection. unlike sars-cov, the cause of sars, several sars-cov-2 spike glycans contain sialic acid residues. in contrast to the sars secreted glycoprotein (sgp), sars-cov-2 sgp are thus potential ligands for sialic acid-binding siglecs on host immune cells, known to regulate immune function. such sars-cov-2 glycoproteins would contribute to immune deviation. cd33-related siglecs are important immune regulators. siglec-5 and -14 are paired receptors with opposed actions on the nlrp3 inflammasome, which is critical in early viral clearance. sgp binding in persons of siglec-14 null genotype (30-70% in black, asian and minority ethnic (bame) persons, 10% in north europeans) would induce unopposed inhibitory signalling, causing viral persistence through inflammasome inhibition. siglec-3 (cd33) and siglec-5 are expressed on cd33 myeloid derived suppressor cells (cd33 mdsc). immunosuppressive cd33 mdsc populations are increased in all groups at risk of severe covid-19 infection. cd33 expression is increased in persons with the cd33 rs3865444 cc allele, associated with alzheimer’s disease, who would thus show enhanced susceptibility. viral sgp ligation of cd33, potentially in conjunction with siglec-5, would promote expansion of cd33 mdsc cells, as occurs in cancers but at much greater scale. cd33 is expressed on cns microglia, potentially activated by sgp penetration through the porous cribriform plate to cause anosmia. genotyping of severe or fatal covid-19 cases can confirm or refute this pathophysiological mechanism. early data have confirmed extremely high-level increase of cd33 mdsc numbers in severe covid-19 infection, consistent with the proposed mechanism. an additional sialylated carbohydrate motif, associated with shorter glycan chain and less microheterogeneity, is found in sialyl-tn glycans. both cd33 and siglec-5 are significantly bound by sialyl-tn 6, 9 . sialyl tn glycan binding is a functionally important modulator of cd33 signalling, as identified for hiv envelope glycoprotein gp120 3 . sars-cov-2 has 16 n-linked glycans between the s1 subunit, which binds the angiotensin-2 receptor, and the s2 subunit, which mediates membrane fusion 10 . glycosylation mapping has identified n-glycans terminating in 3'-slacnac structures at n-234 and n282 on spike-1 and n1098 on spike 2, while the o-glycan at t323 shows short sialic acid capped chains, including sialyl t antigen, and others trimmable to sialyl tn 11, 12 . this represents a fundamental difference to sars-cov, where glycans show negligible sialylation. the major sars-cov glycoprotein, mediating membrane fusion for viral entry in sars, is also released into the circulation in soluble form 13 . similar release through cleavage would allow sars-cov-2 sialylated secreted glycoproteins (sgp) to modulate immune response through cd33-r siglecs. in particular, such interaction with siglecs-3, -5 and -14 would explain specific features of the demography and pathogenesis of covid-19 infection. siglec-5 and siglec-14 are paired regulators of the nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing-3 (nlrp-3) inflammasome, which responds to danger signals in infection by production of inflammatory mediators (figure 1 ) 14 . while unregulated nlrp-3 inflammasome activation may contribute to damaging inflammatory responses in chronic infection, it is critical in the early response to infection 6 necessary for viral eradication: viruses including epstein-barr virus and zika virus have developed immune evasion strategies by inhibiting the nlrp3 inflammasome 14, 15 . siglec-5 and -14 are paired receptors, having essentially identical extracellular binding domains but differing intracellular signalling pathways 8 . siglec-5 associates with tyrosine phosphatase shp-1 to send an inhibitory signal upon ligation, while siglec-14 associates with the adapter protein dap-12 and the tyrosine kinase syk to send an activating signal. thus siglec-5 binding downregulates nlrp3 inflammasome activity, while siglec-14 binding upregulates it 8, 16 . importantly, there is a common polymorphism, in which siglec-14 is replaced by a siglec-5 like fusion protein (siglec 14/5) signalling via the inhibitory shp-1 pathway identically to siglec-5 17 . individuals can express both siglec-5 and -14 (siglec-14 +/+ ), can have one allele where siglec-14 is replaced by 14/5 (siglec-14 +/heterozygous) or have both alleles where siglec-14 is replaced by 14/5 (siglec-14 -/-, known as siglec-14 null allele) 18 . thus, persons with siglec-14 null allele receive an inhibitory input to the nlrp3 inflammasome upon siglec-5 binding while those without have a stimulatory input, greater in siglec-14 +/+ homozygotes than heterozygotes ( figure 1 ) 16, 18 . there are recognised functional consequences. adults with the siglec-14 null allele are at reduced risk for infective exacerbation of copd 18 and may be less susceptible to severe tuberculosis 19 . conversely siglec-14 null women are at increased risk of premature delivery if infected with gbs 17 . there is substantial ethnic variation in siglec-14 null allele, with a frequency of 50-70% in chinese, south east asians and middle eastern persons, 30-40% in indo-pakistanis and sub-saharan africans and only 10% in northern europeans 9 . the pathogen drive currently or previously underlying such major variability remains so far unknown. the nlrp3 inflammasome is a critical element in effective antiviral responses and its inhibition by pathogens an important cause of immune evasion, as seen in zika virus infection 14, 15 . binding of sars-cov-2 sgp to siglec-5 would allow such immune evasion in persons of siglec-14 null genotype through inflammasome inhibition. newly recruited monocytes would be susceptible to similar inflammasome inhibition. siglec-14 null women would also be more prone to preterm labour with covid-19, as occurs if gbs colonised 8 . this mechanism would put persons of origin from china or the far east, the middle east, the indian subcontinent and sub-saharan africa at increased risk of inability to clear sars-cov-2 virus upon initial exposure and thus of more severe and chronic disease. cd33 + mdsc populations release arginase-1 to cause arginine depletion, inducing decreased t cell receptor (tcr)- chain expression and impaired adaptive immune responses 20, 21 . they also release the immunosuppressive cytokines tgf- and il-10, as well as effector molecules such as nitric oxide and reactive oxygen metabolites. in addition b cell proliferation and antibody production are diminished by mdsc activation 23 . cd33 mdsc populations are not restricted to cancer, and are increased in a number of other conditions. indeed, in all other conditions known to be associated with increased covid-19 mortality, circulating cd33 + mdsc are increased. significant increase is seen in persons aged 61-76, compared to those aged 19-59, with further increase in frail elderly 22 . increased cd33+ mdsc numbers in cancer are associated with decreased tcr- expression, contributing to compromised anti-tumour responses 24 . cd33 + hla-dr low/-mdsc cells are significantly increased in obesity, associated with decreased tcr- expression 25 . type-2 diabetes mellitus and arterial hypertension cause increased circulating cd33 + mdsc cells producing immunosuppressive tgf- and il-10 26 . circulating cd33 + hla-dr -mdsc are upregulated in smokers with or without chronic obstructive pulmonary disease (copd) 27 . in copd this persists after stopping smoking, associated with significant downregulation of tcr- expression 27 . stopping smoking reduces cd33+ mdsc numbers. persons with dementia, likely to have high age-related cd33 mdsc, have increased frequency of the cd33 rs3865444 cc allele which upregulates cd33 expression on microglia and immune cells 7 . monocyte expression of cd33 is upregulated 7-fold in young persons carrying this allele, which explains over 70% of variance in cd33 cell expression 28 . while its putative role in dementia is that this cd33 variant impairs protective uptake of -amyloid, the relevance of this allele in cd33 rs3865444 cc carriers of whatever age in covid-19 infection would likely be of increased response by mdsc and monocytes to a cd33-binding viral sgp. this may therefore be a second genetic predisposition to severe covid-19 infection. the excess of cd33 mdsc in persons with older age or frailty, diabetes, hypertension, copd, cancer or previous malignancy or in those who smoke cigarettes would put them at there is evidence that persistent elevation of monocytic mdsc cells, identified as cd14 + hla-dr low , is strongly associated with worse outcome in septic shock 29 . cd14 + hla-dr low cells have been identified at high frequency in cases of severe covid-19 infection with immune dysregulation 30 . further characterisation would be required to determine how many of these cells are monocytic mdsc and how many are monocytes in which hla-dr has been downregulated as suggested by the authors. in addition, there is evidence that mdsc of proinflammatory potential can be released from the bone marrow in inflammation 31 . there is so far no clear consensus on their eventual phenotype but these new marrow derived cells are initially characterised as early stage mdsc (emdsc), which do not initially express lineage markers but do express cd33 and would thus be susceptible to sars-cov-2 sgp manipulation until the virus was cleared ( figure 3 ). proinflammatory mdsc are recognised in murine and human arthritis and in ibd, where they suppress t cell proliferation, as do mature mdsc, but also strongly promote th17 responses 31, 32, 33 . analysis of immune responses in severe covid-19 infection identified two distinct groups 30 . around a quarter developed a macrophage activation syndrome with hypercytokinaemia and hyperferritinaemia in association with reduced total neutrophils and monocytes compared to persons with milder disease. three quarters showed preservation of circulating cell numbers, with a state of chronic immune deviation but without macrophage activation syndrome. this association of reduced circulating cells with a hyperinflammatory response is consistent with bone marrow suppression, where release of new mdsc is prevented. by contrast, the prolonged state of immune suppression with immunopathology is explicable by viral sgp stimulation of the newly released proinflammatory emdsc ( figure 3 ). there is increasing evidence that sars-cov-2 may invade the central nervous system 34 . this would be most likely in severe disease with blood brain barrier disruption, and sars-cov-2 sgp would show tropism for cd33-expressing microglia. a common presentation of covid-19 disease is anosmia (loss of smell) with ageusia (loss of taste}. it has been postulated that the virus might gain access through the sieve-like barrier at the cribriform plate, in proximity to the olfactory bulb 34 . penetration of a cd33 binding sgp would potentially induce activation of cd33-expressing microglia within the olfactory bulb to cause this symptom. this would not require ingress of whole viral structures and could occur even if blood brain barrier function was intact elsewhere. in severe covid-19 infection there is a significant increase of venous and arterial thrombosis 2 . several mechanisms are likely to contribute to this. a cd33-r siglec binding viral sgp would provide a further and independent risk factor for vascular thrombosis. analysis of 274 circulating cytokines in diabetic patients with critical limb ischaemia identified soluble siglec-5 as the outstandingly increased marker, and confirmed this in a large second cohort, which excluded confounding risk factors 35 . soluble siglec-5 concentrations were considerably more than an order of magnitude greater than reported previously in healthy controls and patients with sle 36 . the source was identified as irregularly-shaped siglec-5+ cells that were present within atherosclerotic plaques but not normal vascular endothelium 35 . these cells were not further characterised, and thus may have been either siglec-5+ macrophages or siglec-5+ mdsc. the mechanism by which siglec-5 contributes to the progression of vascular ischaemia has yet to be determined, but there is clear potential for modulation of the siglec-5+ cells within existing vascular atherosclerotic plaques by such a siglec-5 binding viral sgp. there has been recent recognition of a covid-19 associated syndrome of multisystem inflammation in previously healthy paediatric patients, usually without the initial presentation with respiratory disease seen in adults. by contrast, gastrointestinal symptoms were noted in around 90% of cases in both the uk and usa multicentre cohorts 37, 38 . what is notable about paediatric cases of covid-19 infection is that sars-cov-2 is more readily detected in faeces than the respiratory tract 39 . there is now evidence that the virus can productively infect enterocytes, which strongly express the ace-2 receptor. 40 . i suggest that the lack of respiratory symptoms at presentation may point to preferential infection via the gastrointestinal tract in children. the subsequent mechanism of disease would then be as for complicated adult disease (figures 1-3) , but without the dominance of respiratory symptoms seen in adult disease. in support of this, there was evidence of ethnic predisposition in both cohorts. the uk patients showed significant overrepresentation of afro-caribbean and asian children (47% and 28% of the cohort respectively, compared to 8% and 7% of the overall uk population) 37 . the large usa cohort had ethnicity data on 147 children, of whom only 35 were white, non-hispanic 38 . while most children had no recognised comorbidities, 37% of the us children for whom data was available had diagnosed or bmi-based obesity while the uk group showed a significant increase in the observed to expected weight ratio. thus, a significant comorbidity in children may be an increase in mdsc cells due to overweight. it is also notable that mdsc populations are increased in children with atopic rhinitis 41 and asthma 42 . the relative susceptibility of males is not explained by this, and the mechanism is more likely to relate to a distinct antiviral immune response (figure 1 ). tlr7 responds to intracellular viral rna in innate immune cells, and plasmacytoid dendritic cells of females produce more antiviral type-1 interferons than do males 42 , related to the number of x chromosomes and serum testosterone production 44 . this may underpin sex-related symptomatic differences in rna viral infections more generally, but may have a fundamentally more important impact with a virus as virulent as sars-cov-2. this model of sars-cov-2 pathogenesis can rapidly be supported or excluded. such interaction could be confirmed or excluded by genetic analysis of persons with severe or fatal illness compared to the less affected. 1. persons with homozygous siglec-14 null allele would have higher incidence of severe disease than those siglec-5 + siglec 14 + , with heterozygotes intermediate. preterm delivery would be more common in siglec-14 null women, as in gbs infection. 2. persons of cd33 rs3865444 cc allele, when stratified by disease susceptibility, would have higher incidence of severe disease than those with cd33 rs3865444 tt allele. 3. increased numbers of cd33 mdsc at presentation in those who go on to develop severe disease. proceeding from the direction of assessing in vitro sgp binding to cell lines or tissues suffers from the disadvantage that glycan expression is known to be modulated by enzymes within the golgi apparatus of host cells during viral replication 12 . this may lead to impaired glycan maturation resulting in shorter glycan chains 12 . thus, predicted glycans may not be representative of those functioning in vivo to mediate pathogenesis. however, detection of the footprints of siglec-defined immunopathology would provide imperative for such extensive studies to be undertaken in order to characterise binding sites. prediction 4 appears to have been fulfilled, as mdsc populations (cd33+ but including both cd14+ monocytic and cd15+ granulocytic mdsc) were increased from 0.3% (iqr 0. 13-2.13) in healthy donors to 47.5% (iqr 28.4-65.6%) in covid-19 infection 45 47 . this is a strong pointer towards a specific mechanism of induction, unique to covid-19 infection. prediction 6 is supported by evidence that proinflammatory mdsc of immature phenotype occur in human and murine arthritis, and strongly promote th17 type responses 31, 32 . the cytokine storm in severe covid-19 infection has indeed been characterised to be of th17 type 48 , with over 30% of total cd4 cells being of th17 phenotype (cd4 + ccr6 + ) in one wellstudied case with fatal outcome 49 . if predictions based on this model are confirmed, inhibition of sars-cov-2 secreted glycoprotein interaction with host cd33-related siglecs should be of clinical benefit. if the testing predictions are confirmed, the next stage would be to characterise the binding, which may be mediated by a single sgp or by two. if the cd33-related siglec-binding sgp is of sialyl tn type, there may well be an existing sialyl tn monoclonal that serendipitously blocks sgp binding to siglecs. this would potentially be therapeutic. if not, characterisation of sgp-siglec binding would allow design of a small molecule that blocks interaction (possibly two), which would then be suitable for creating blocking immunoglobulin-(ig)-fusion protein(s) for clinical use. in due course, this might allow persons who fail to respond to immunisation to be pre-treated to block siglec binding sites, allowing them to be exposed to small infecting doses of sars-cov-2 in clinically controlled circumstances and thus generate a broad immune response, including to the pathogenic glycan determinants the clinical course of covid-19 infection is highly variable. the currently enigmatic determinants of disease severity can be fully explained on the basis of viral manipulation of host cd33-related siglecs. there is evidence that both cd33 and siglec-5 (thus also siglec the initial immune response to sars-cov-2 by innate immune cells, after its entry via the ace-2 receptor on lung or gut epithelium, determines whether or not infection is swiftly controlled and minimally symptomatic. such inflammasome activation is critical in an effective antiviral response. 5. nlrp3 activation is regulated by signals from cell surface siglecs, if these are stimulated by siglec ligands. an appropriately configured sialyl tn or 3'slacnac secreted glycoprotein would bind equally to siglec-5 or siglec-14 as their extracellular domain is almost identical. 6 . in a homozygous siglec-14 positive person such glycoprotein binding induces strong activation of the nlrp3 inflammasome, while in a siglec-14 null person this induces strong inhibition. in a person who is heterozygous, a more modest activation response occurs. thus, an infected person who is homozygous siglec-14 positive is more likely to mount a successful early antiviral response than one who is siglec-14 null, with heterozygotes making an intermediate response. release of arginase-1 by these cells, causing depletion of arginine, would synergise with release of the immunosuppressive cytokines il-10 and tgf- to impair t cell proliferation and memory cell generation, as well as prevent the effective t-b cell interaction required for antiviral antibody production. b cell proliferation and antibody production would also be impaired. release of nitric oxide and reactive oxygen species by these cells would contribute to ongoing inflammatory responses. the response by bone marrow as infection progresses may determine the clinical phenotype. persons suffering bone marrow suppression with severe disease will not release new mdsc into the circulation. these persons will show reduced numbers of circulating monocytes and neutrophils and will tend to progress towards macrophage activation syndrome as mdsc suppression wanes. those without bone marrow suppression maintain higher numbers of circulating cells. these include early phase mdsc, which can be stimulated through cd33 and siglec-5. these cells are immunosuppressive but also proinflammatory, particularly promoting th17 responses. in both groups, a separate prothrombotic pathway will be driven by sgp stimulation of siglec-5+ cells within atherosclerotic plaques. role of angiotensinconverting enzyme 2 (ace2) in covid-19 risk factors of critical & mortal covid-19 cases: a systematic literature review and meta-analysis siglecs at the host-pathogen interface siglecs--the major subfamily of i-type lectins siglecs: a journey through the evolution of sialic acid-binding immunoglobulin-type lectins rapid evolution of binding specificities and expression patterns of inhibitory cd33-related siglecs in primates alzheimer's risk-altering polymorphism, cd33 expression, and exon 2 splicing siglec-5 and siglec-14 are polymorphic paired receptors that modulate neutrophil and amnion signaling responses to group b streptococcus new aspects of siglec binding specificities, including the significance of fucosylation and of the sialyl-tn epitope structure, function, and antigenicity of the sars-cov-2 spike glycoprotein deducing the n-and o-glycosylation profile of the spike protein of novel coronavirus sars-cov-2 site-specific glycan analysis of the sars-cov-2 spike the sars-cov s glycoprotein nlrp3 inflammasome-a key player in antiviral responses inflammasome inhibition as a pathogenic stealth mechanism siglec-14 enhances nlrp3-inflammasome activation in macrophages deletion polymorphism of siglec14 and its functional implications loss of siglec-14 reduces the risk of chronic obstructive pulmonary disease exacerbation the siglec14 null allele is associated with mycobacterium tuberculosis-and bcg-induced clinical and immunologic outcomes mdsc; the most important cell you have never heard of on the armament and appearances of human myeloid-derived suppressor cells +)hla-dr(-) myeloid-derived suppressor cells are increased with age and a history of cancer myeloidderived suppressor cells modulate b-cell responses myeloid-derived suppressor cells impede t cell functionality and promote th17 differentiation in oral squamous cell carcinoma increased monocytic cd14⁺hladr low/-myeloid-derived suppressor cells in obesity myeloid-derived suppressor cells show different frequencies in diabetics and subjects with arterial hypertension differential effects of smoking and copd upon circulating myeloid derived suppressor cells alzheimer's disease locus: altered monocyte function and amyloid biology delayed persistence of elevated monocytic mdsc associates with deleterious outcomes in septic shock: a retrospective cohort study complex immune dysregulation in covid-19 patients with severe respiratory failure myeloidderived suppressor cells are proinflammatory and regulate collageninduced arthritis through manipulating th17 cell differentiation myeloid-derived suppressor cells in inflammatory bowel disease neurological insights of covid-19 pandemic siglec-5 is a novel marker of critical limb ischemia in patients with diabetes soluble siglec-5 is a novel salivary biomarker for primary sjogren's syndrome intensive care admissions of children with paediatric inflammatory multisystem syndrome temporally associated with sars-cov-2 (pims-ts) in the uk: a multicentre observational study characteristics of pediatric sars-cov-2 infection and potential evidence for persistent fecal viral shedding sars-cov-2 productively infects human gut enterocytes myeloid-derived suppressor cells and costimulatory molecules in children with allergic rhinitis peripheral blood mdscs, il-10 and il-12 in children with asthma and their importance in asthma development tlr7 ligands induce higher ifn-alpha production in females sex and pubertal differences in the type 1 interferon pathway associate with both x chromosome number and serum sex hormone concentration an inflammatory profile correlates with decreased frequency of cytotoxic cells in covid-19 expansion of myeloidderived suppressor cells in patients with severe coronavirus disease the clinical significance of myeloid-derived suppressor cells in dengue fever patients th17 responses in cytokine storm of covid-19: an emerging target of jak2 inhibitor fedratinib pathological findings of covid-19 associated with acute respiratory distress syndrome key: cord-334709-z70oevy2 authors: niv, yaron title: defensin 5 for prevention of sars-cov-2 invasion and covid-19 disease date: 2020-09-04 journal: med hypotheses doi: 10.1016/j.mehy.2020.110244 sha: doc_id: 334709 cord_uid: z70oevy2 corona virus disease 2019 (covid-19), a pandemia emerged recently, caused by severe acute respiratory syndrome corona virus 2 (sars-cov-2). the receptor for corona virus and influenza a is the mucosal cell membrane protein angiotensin converting enzyme 2 (ace2), which is abundant on the membrane of alveolar cells and enterocytes. viral spike protein 1 (s1) is the ligand, with an affinity of 14.7 nm to the receptor. the main port of entry for the virus is the upper respiratory tract, and the diagnosis is usually by pcr of the viral rna with nasal and pharyngeal swab test. human defensin 5 (hdef5) is a protein encoded by the defa gene, secreted by paneth cells in the small intestine and by granules of neutrophils. it has an affinity of 39.3 nm to ace2, much higher than that of the corona s1. hdef5 may also attach to glycosylated corona s1 protein, make its efficiency even better. the issues to be investigated are the affinity of hdef5 to s1 protein, the ability of recombinant hdef5 function in attaching both ace2 and s1, and the feasibility to perform aerosol spray of this protein. in addition, safety and efficiency should be studied in phases i, ii and ii clinical protocols. thus, an aerosol spray of hdef5 given through the nose and throat, once to several times a day, may be a very efficient approach to prevent infection with sara-cov-2 as well as influenza a. corona virus disease 2019 (covid-19), a pandemia emerged recently, caused by severe acute respiratory syndrome corona virus 2 (sars-cov-2). the receptor for corona virus and influenza a is the mucosal cell membrane protein angiotensin converting enzyme 2 (ace2), which is abundant on the membrane of alveolar cells and enterocytes. viral spike protein 1 (s1) is the ligand, with an affinity of 14.7 nm to the receptor. the main port of entry for the virus is the upper respiratory tract, and the diagnosis is usually by pcr of the viral rna with nasal and pharyngeal swab test. human defensin 5 (hdef5) is a protein encoded by the defa gene, secreted by paneth cells in the small intestine and by granules of neutrophils. it has an affinity of 39.3 nm to ace2, much higher than that of the corona s1. hdef5 may also attach to glycosylated corona s1 protein, make its efficiency even better. the issues to be investigated are the affinity of hdef5 to s1 protein, the ability of recombinant hdef5 function in attaching both ace2 and s1, and the feasibility to perform aerosol spray of this protein. in addition, safety and efficiency should be studied in phases i, ii and ii clinical protocols. thus, an aerosol spray of hdef5 given through the nose and throat, once to several times a day, may be a very efficient approach to prevent infection with sara-cov-2 as well as influenza a. similarly, to other corona viruses and influenza a, the receptor for adherence of sars-cov-2 is ace2 which is abundant on respiratory and gastrointestinal mucosal cells, especially on alveolar type 2 pneumocytes [2] . the spike protein of coronavirus is divided into the s1 and s2 domains, in which s1 is the ligand for the receptor binding, and s2 is responsible for cell membrane fusion [3, 4] . the affinity of s1 to ace2 is 14.7 nm. some patients, especially older with background diseases or immunocompromised, progressed rapidly with acute respiratory distress syndrome (ards), septic shock, and multiple organ failure. 5% to 10% of patients have died [5] . theoretically using of recombinant s1 protein, which will attach to the ace2 receptors, may actively compete with the sars-cov-19 and prevent infection. a protein with higher affinity may be even better in this regard. defensins are a family of cytotoxic peptides, involved in host defense. human defensin 5 (hdef5) is a 94 amino acids protein encoded by the defa gene on chromosome 8, secreted by paneth cells in the small intestine and by granules of neutrophils. it has an affinity of 39.3 nm to ace2, much higher than that of the corona s1. wang et al looked at the efficiency of hdef5 attachment to ace2 and the inhibition of hdef5 against sars-cov-2 in vitro [6] . they found affinity of hdef5 binding to ace2 of 76.2 nm, efficient blocking of sars-cov-2 receptor binding domain (rbd), and significant inhibition of invasion into caco-2 cells. hdef5 protein has also the capability to attach s1 protein through the glycosylated residue of the protein [7] . thus, theoretically, enhances its ability to prevent the virus invasion acting dually on the ligand (s1) and the receptor (ace2). the next step, after preparing recombinant hdef5, active and functioning, with high affinity to both receptor and ligand, is to make the protein available in powder, with small enough particles to be aerosolized and sprayed. spray-drying is an effective, efficient means of producing peptide-loaded powders suitable for pulmonary delivery. if the correct formulation and spray-drying conditions can be identified, then a product can be obtained with a high yield and having a large fine-particle dose [8] . thus, can be applied to the upper airways for prophylaxis and prevention of covid-19 disease. the spray will be applied to high risk populations and medical teams, on a regular basis, being protective together with face masks and shields [9] . the quality of a spray dried powder is determined by the reconstitution behavior, flowability, and morphology of the powder particles. the size, shape, weight, bulk density, and porosity of the particles influence the flow of a powder as was demonstrated for lactose, suspensions of glass spheres, calcium carbonate crystals, plate-shaped talc, whey protein and maltodextrin [10] . sensitive proteins should be embedded in stabilizing excipients ("carriers") such as disaccharides or amino acids. the spray-drying of protein aqueous solution without carriers can lead to inactivation (due to unfolding or aggregation). several basic science and clinical steps are essential for advancing my hypothesis: preparing recombinant hdef5 protein, protein lyophilization, preparing aerosol spray, performing clinical study of three phases for studying safety and efficiency and then randomized controlled studies of several doses and frequency of application. blocking the receptor for sars-cov-2 virus, ace2, on the respiratory tract epithelium, especially the alveolar type 2 pneumocytes, will probably prevent invasion of the virus, at least for a short while. using a spray of defensin 5 protein before potential exposure to covid-19 active, infective patient, in addition to face mask and shield, will enhance the ability of high risk population such as the elderly, immunocompromised patients or those with background diseases, (such as chronic pulmonary diseases, diabetes mellitus, arterial hypertension, congestive heart failure, chronic renal failure, chronic liver diseases), to avoid this devastating infection. medical teams, especially those that take care of covid-19 patients, may add this line of protection to their personal protective equipment (ppe) on a routine basis. this approach, together with immunization against influenza, will minimize the danger of both, corona, and influenza in the upcoming winter. coronavirus disease 2019 (covid-19): a literature review angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus genomic characterization and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding chain a. spike glycoprotein -protein -ncbi epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study human intestinal defensin 5 inhibits sars-cov-19 invasion by cloaking ace2. gastroenterology -in press cryo-em structure of the 2019-ncov spike in the prefusion conformation the surface composition of spray-dried trehalose/bovine serum albumin/surfactant particle moving personal protective equipment into the community. face shields and containment of covid-19 particle morphology and powder properties during spray drying of maltodextrin and whey protein mixtures professor yaron niv defensin 5 for prevention of sars-cov-2 invasion and covid-19 disease yaron niv, md, facg, agaf ministry of health and faculty of medicine, ariel university, israel declaration of competing interest: the author declares that he has no known competing financial interests or personal relationships that could have appeared to influence the work key: cord-344170-qrupbtem authors: biswas, subrata k; mudi, sonchita r title: genetic variation in sars-cov-2 may explain variable severity of covid-19 date: 2020-05-24 journal: med hypotheses doi: 10.1016/j.mehy.2020.109877 sha: doc_id: 344170 cord_uid: qrupbtem nan to the editor: the coronavirus disease 2019 (covid-19) is highly contagious and is rapidly spreading all over the world. although the majority of the covid-19 patients are either asymptomatic or mildly symptomatic, many symptomatic patients who require hospitalization and intensive care are dying. disease severity and fatality have been found to be associated with many host factors, including age, gender, race, ethnicity and presence of co-morbidities. disease severity and fatality have also been noted to differ between countries and between geographical locations within the same country. many hypotheses have so far been proposed to explain the underlying basis of the variation in the severity of covid-19 in the population but none of the proposed hypotheses has been proved or appeared to be convincing. the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) was discovered as the viral pathogen that causes covid-19. the reference sequence of the full-genome of the sars-cov-2 was explored soon after the manifestation of the disease in wuhan, china in december 2019. when covid-19 quickly spread outside china, sequencing of the viral genome was conducted from infected patients in other countries to compare the sequence with the reference sequence. intriguingly, genomic sequence of sars-cov-2 of the first two covid-19 patients diagnosed in italy during january and february 2020 already showed mutations compared to the reference sequence established in china [1] . variations in the genomic sequences were also observed when sequencing data from viral isolates of patients from other european and north american countries were compared with each other and with reference sequence [1] . several studies have so far been conducted utilizing sequencing data of sars-cov-2 obtained from publicly available repositories. one such study, analyzed 95 sars-cov-2 complete genome sequences obtained from genbank, found 156 variants in total and 116 unique variants [2] . however, this recent study [2] did not explore the association between genetic variation in sars-cov-2 and the severity of covid-19. another analysis of 86 genomic sequences obtained from gisaid database (https://www.gisaid.org/) identified 3 deletions in the genomes of sars-cov-2 from japan, usa and australia, and 93 mutations over the entire sars-cov-2 genomes [3] . several of these mutations were located in the receptor binding domain (rbd) of the spike surface glycoprotein [3] . since the rbd of the spike surface glycoprotein plays essential role in binding with receptors on the host cell, mutation in spike protein may alter the virulence of the virus. pachetti et al. [4] analyzed 220 sars-cov-2 genomic sequences from the gisaid database and found 8 novel mutation hot spots: 5 of them were predominantly observed in europe and 3 were exclusively present in north america but none of them was detected in asia. they also identified a novel mutation in the viral gene for rna dependent rna polymerase (rdrp), an enzyme that is related to proofreading functionality [4] . mutation in the rdrp gene in european viral genome was associated with higher numbers of point mutations compared to viral genomes from asia [4] . these findings suggest that the sars-cov-2 is evolving, and european, north american and asian variants with different mutation pattern may coexist. therefore, it is an urgent need to explore the association of the mutation pattern of sars-cov-2 genome with the severity and fatality of covid-19. based on the above discussion, we hypothesize that the genetic variation in sars-cov-2 may explain variable severity of covid-19 in the population. analysis of sequencing data of sars-cov-2 genome showed evidence of mutation at the beginning of the worldwide spread of covid-19 [1] . existence of multiple genetic variants of the virus was also identified in the same population, same country or same geographical location. on the other hand, some unique variants have been found in a particular region or country [4] . furthermore, mutations have been identified in the rdrp gene that determines frequency of mutation that will be acquired in the future generation of the virus [4] . mutations have also been detected in the rbd of the spike surface glycoprotein that determines viral virulence [3] . thus, among the various variants of sars-cov-2 circulating in the population, some may be highly virulent and lethal whereas others may be less virulent. in conclusion, it is plausible that genetic variation in sars-cov-2 may at least partly explain variable severity of covid-19. it is now very important to test this hypothesis. this hypothesis can be tested by obtaining genetic sequences of sars-cov-2 from two groups of covid-19 patients. one group will include patients who have died due to severe covid-19 and another group will include patients who had only mild symptoms of covid-19. genetic sequences of sars-cov-2 will be compared between the groups to identify unique variation in the genomic sequence, and will be compared within group to identify similarities in the genomic sequence. this hypothesis, if accepted, will help identify virulent variant of sars-cov-2 and will thus help in drug design and vaccine development. no conflict of interest to be declared. whole genome and phylogenetic analysis of two sars-cov-2 strains isolated in italy in genomic characterization of a novel sars-cov-2 genetic diversity and evolution of sars-cov-2 emerging sars-cov-2 mutation hot spots include a novel rna-dependent-rna polymerase variant key: cord-326849-vqpwdlj7 authors: rao us, vishal; thakur, shalini; rao, jyothsna; arakeri, gururaj; brennan, peter a; jadhav, sachin; suhail sayeed, mufti; rao, gururaj title: mesenchymal stem cells -bridge catalyst between innte and adaptive immunity in covid 19 date: 2020-05-18 journal: med hypotheses doi: 10.1016/j.mehy.2020.109845 sha: doc_id: 326849 cord_uid: vqpwdlj7 majority of patients infected with the covid 19 virus display a mild to moderate course of disease and spontaneously recover at 14 20 days,. however, about 15 % of patients progress to severe stages and 2.5% of these patients succumb to this illness. most patients with severe disease belong to the elderly age group (< 65 years of age) and have multiple associated co-morbidities. the immune responses induced by the covid 19 virus, during the incubation and non-severe stages, requires the early initiation of a specific adaptive immune response to eliminate the virus and prevent the progress to severe stages. in patients with a dysfunctional bridge adaptive immunity, the innate immune response becomes exaggerated due to the lack of feedback from the adaptive immune cells. the resultant cytokine storm is responsible for the severe lung injury leading to acute respiratory distress syndrome seen in covid 19 patients. mesenchymal stem cells are known to suppress overactive immune responses as well as bring about tissue regeneration and repair. this immuno-modulatory effect of mscs could hold potential to manage a patient with severe symptoms of covid 19 infection due to a dysfunctional adaptive immune system. elderly age group (< 65 years of age) and have multiple associated co-morbidities. the immune responses induced by the covid 19 virus, during the incubation and non-severe stages, requires the early initiation of a specific adaptive immune response to eliminate the virus and prevent the progress to severe stages. in patients with a dysfunctional bridge adaptive immunity, the innate immune response becomes exaggerated due to the lack of feedback from the adaptive immune cells. the resultant cytokine storm is responsible for the severe lung injury leading to acute respiratory distress syndrome seen in covid 19 patients. mesenchymal stem cells are known to suppress overactive immune responses as well as bring about tissue regeneration and repair. this immuno-modulatory effect of mscs could hold potential to manage a patient with severe symptoms of covid 19 infection due to a dysfunctional adaptive immune system. introduction individuals infected by the covid 19 virus display a clinical course ranging from asymptomatic carriers, mildly symptomatic individuals to those with severe respiratory distress and death, based on age and other co-morbid features. 1, 2, 3 about 80-85% of patients infected with the covid 19 virus display a mild to moderate course of the disease and spontaneously recover at 14 -20 days from the point of the first contact. however, about 15 % of patients progress to severe stages of the disease, often requiring icu admission and mechanical ventilation with around 2.5% succumbing to this illness. 1, 2 research has shown that most of the patients who progress to severe stages of disease belong to the elderly age group < 65 years of age and have multiple associated co-morbidities. 1 this paper serves to bring out the immunological responses of patients with severe disease, in order to design pertinent interventions to decrease morbidity and mortality of covid 19. the immune responses induced by the covid 19 virus, during the incubation and non-severe stages, requires the early initiation of a specific adaptive immune response to eliminate the virus and prevent the progression to severe stages. 4 this is carried out by neutralization of free virus particles and termination of viral replication by antiviral cytotoxic t cells (ctls). the innate cytokine responses, like interferon(ifn) alpha/beta or ifn-gamma have roles in controlling and initiating downstream adaptive immune responses. 4 interferon-alpha and beta are synthesized by most virally infected cells whereas ifn-γ is synthesized only by certain cells of the immune system like the natural killer (nk) cells, cd4 th1 cells, and cd8 cytotoxic suppressor cells. a decrease in lymphocytic count is a deterrent to the adaptive immune response. mild to moderate lymphocytopenia is a common haematological finding (about 44% of all patients ) in individuals with covid 19 infections. studies have shown an absolute lymphocytic count of about 1-1.2x 10 9 / l . 5, 6 early recruitment of cd4+and cd8+ cells is crucial in the recovery of patients with covid 19 infections. studies have shown that although cd4+ and cd8+ expression is lesser in patients with covid 9 than healthy individuals, there is a steady increase in expression upto 4-5 days. 6, 7, 8 individuals that recovered from the disease with only mild to moderate symptoms show activated cd4+ and cd8+ t cells, (co-expression of cd38 and hla-dr), along with igm and igg viralspecific antibodies. 4, 7, 8 the emergence and rapid increase in activated t cells, especially cd8+ t cells, at days 7-9 has been shown to precede the resolution of symptoms. following this, there is a rapid decline cd8+ cells and minor decline in cd4+ cell population upto day 20 . 4 this coincides with complete recovery of the patient, thus establishing the importance of a functional bridge adaptive immunity. in cases that progress to severe or critical stages, counts of peripheral cd4+ and cd8+ t cells were this implies an over-activation of t cells, and high cytotoxicity of cd8 t cells leading to an exaggerated immune response. this is substantiated by the increased expression of il6, il10, il2 and ifn-γ in the sera of severe cases when compared with that of the mild cases. 6 the levels of inflammatory cytokines (il1, tnf alpha, il6 ) are high in the lungs of covid-19 patients and these cytokines are strong inducers of hyaline production in lung alveolar epithelial cells, and fibroblasts. 8 lung inflammation is the main cause of life-threatening respiratory disorders and the severity of pulmonary immune injury correlates with extensive infiltration of neutrophils and macrophages in the lungs. 10 more pertinently, it has been demonstrated that when t cell counts drop to their lowest levels at day 4-6, the serum il10, il2, il4, tnf-α and ifn-γ levels reach their peaks thereby initiating a cytokine storm. 6 this excessive, ineffective host immune response may lead to severe lung injury leading to acute respiratory distress syndrome (ards). 7 as the infection progresses and becomes severe in intensity , the remaining t cells are unable to sustain long term activation, leading to immune exhaustion and decline in effector activity and proliferation. 11 . the overstimulated innate immune response , without feedback from adaptive immune system clearly demonstrates the dysfunctional bridge adaptive immunity . mesenchymal cells derived from umbilical cord, dental pulp , bone marrow etc , have been used for immune modulation in various autoimmune disorders . 12 mesenchymal stem cells are known to suppress overactive immune responses as well as maintain a pro-inflammatory phenotype when inflammatory responses are absent. mscs also bring about tissue regeneration and repair . 12, 13 the plasticity of these mesenchymal cells according to the presence or absence of pro inflammatory cytokines has been described and is being intensively studied. 14 this immunomodulatory effect of mscs could hold the potential to manage a patient at risk of manifesting the severe symptoms of covid 19 infection due to a dysfunctional adaptive immune system. a few reports of use of mesenchymal stromal / stem cell in the treatment of covid 19 has emerged in recent times with promising results. 15 wilson et al conducted a study to investigate the effectiveness and safety of the use of mesenchymal stem cells for ards with favourable results . 16 mscs are non-immunogenic due to their low expression of major histocompatibility complex (mhc). this makes it well suited for therapeutic interventions using allogenic transfers without hla matching. 17 in the presence of an exaggerated immune response, as seen in covid 19 patients with cytokine storm and ards, the pro-inflammatory cytokines bring out an anti-inflammatory effect on the mscs. the mscs secrete several soluble factors such as nitric oxide (no) , transforming growth factor (tgf) beta , prostaglandin e2 (pge2), indoleamine 2,3-dioxygenase (ido) , hla -g5 and soluble interleukin 6 . these molecules then inhibit the proliferation and activation of th1 and th17 cell lines, thereby decreasing the production of interferon-gamma and interleukin 17. they also inhibit the activation of cytotoxic cd8 +cells , thereby reducing direct injury to lung parenchyma. the activity and maturity of dendritic cells are also reduced via pge2 pathways, hence, decreasing tnf alpha and increasing il10, which is anti-inflammatory . this then activates t reg cells that regulate the t cell activity and further increase the production of il10. the nk cells are suppressed both by the secretion of these soluble molecules as well as contact-mediated communication with mscs. mscs also bring about increased mobilization and decreased chemotaxis of neutrophils. 12, 13, 14 . mesenchymal stem cells may be lysed or inactivated by the already activated nk cells . this phenomenon can be controlled by priming the mscs with interferon-gamma before transfusion . 8, 12, 13 to explore the use of mesenchymal stem cells in the treatment of covid positive patients with severe disease, we have initiated a clinical trial (imd/cov/001/2020) to transfuse allogeneic mesenchymal stem cells derived from donated bone marrow or umbilical cord administered via intrapulmonary implantation where possible or administered via the intravenous route. in order to offset the neutralization of mscs, we have proposed priming of msc with a cytokine cocktail derived from th1 cells of healthy donors to suppress the hyperactive immune response and promote tissue repair. no conflict of interest between the authors no financial affiliations/ external funding agency involved irb ethical clearance has been obtained for the study *the study mentioned has been registered under ctri and clinical trials. clinical characteristics of coronavirus disease 2019 in china clinical features of patients infected with 2019 novel coronavirus in wuhan pathological findings of covid-19 associated with acute respiratory distress syndrome breadth of concomitant immune responses prior to patient recovery: a case report of non-severe covid-19 jing liu et al longitudinal characteristics of lymphocyte responses and cytokine profiles in the peripheral blood of sars-cov-2 infected patients , lancet infectious diseases clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china covid-19 infection: the perspectives on immune responses covid-19: consider cytokine storm syndromes and immunosuppression. the lancet infectious diseases doi sars and mers: recent insights into emerging coronaviruses covid-19 immunopathology & immunotherapy multipotent mesenchymal stromal cells and the innate immune system mesenchymal stem cell therapy for the treatment of inflammatory diseases: challenges, opportunities, and future perspectives plasticity of mesenchymal stem cells in immunomodulation: pathological and therapeutic implications msc transplantation improves covid-19 patient outcome mesenchymal stem (stromal) cells for treatment of ards: a phase 1 the lancet respiratory medicine mesenchymal stem cells: mechanisms of potential therapeutic benefit in ards and sepsis. the lancet respiratory medicine key: cord-338585-ep9r1n8t authors: perrin, david m. title: a hypothesis for examining dihydroxyacetone, the active component in self-tanning products, as a topical prophylactic against sars-cov-2 transmission date: 2020-09-16 journal: med hypotheses doi: 10.1016/j.mehy.2020.110280 sha: doc_id: 338585 cord_uid: ep9r1n8t this hypothesis raises the interesting prospect that dihydroxyacetone (dha), the key ingredient in self-tanning creams, when applied daily to the face and hands may have prophylactic action against sars-cov-2 transmission and infection. the scientific and mechanistic basis for this hypothesis is elaborated based on our understanding of the chemical reactivity of dha with proteins to afford advanced glycation products. this piece ends with a proposal for doing key experiments that can be run to test this hypothesis. as more than 20 million people have been infected with this disease world-wide, a safe method for stopping spread is worthy of consideration. publication of this hypothesis would enable the scientific community at large to test this in a clinically meaningful setting to address the potential for dha-based prophylaxis. given the calamity of this crisis, it is anticipated that the publication of this hypothesis, which is supported by key studies on protein and nucleoside glycation, can be disseminated to as many researchers as possible. david m. perrin, ph.d., chemistry department, 2036 main mall -ubc, vancouver, bc, v6t1z1, dperrin@chem.ubc.ca as sars-cov-2 ravages the world, over 28 million people and counting have now tested positive for the disease while mortality rates are of great concern. the virus is transmitted by: 1) respiration of aerosolized droplets containing the virus; 2) contaminated hands passing the virus directly into the eyes or mouth; and 3) transferring the virus that lands on the face to the eyes, nose, or mouth by face touching. as the hands and face is known to be a major point of transmission, there has been an urgent need to identify safe chemicals that could be applied topically to the skin to reduce the risk of facial transmission. dihydroxyacetone (dha, figure 1 ), the active ingredient in sunless tanning creams, which gives a characteristic orange color, may be of significant and immediate interest. dha reacts chemically with proteins to form glycation products and thus would be expected to react analogously with viral components such that it might provide prophylactic action against anti-sars-cov-2. if so, this cheap, nontoxic molecule may be worthy of consideration for limiting facial transmission, notwithstanding the cosmetic value. here, i will review the paraclinical history of dha and provide sound biochemical evidence of its chemical reactivity towards either protein e.g. the viral coat and nucleocapsid proteins, or rna e.g. the viral genome to support measured exploration of its topical use to prevent facial transmission. dihydroxyacetone is the simplest keto-sugar and its monophosphate ester (dihydroxyacetone phosphate -dhap) is an intermediate in glycolysis. nearly a century ago, dha was first administered orally in a failed attempt to treat type-i diabetic children with a "keto" diet(1, 2) designed to bypass glycolysis. while ineffective for treating diabetes, dha was found to be nontoxic. nevertheless, this treatment resulted in apparent superficial discoloration in the gums and mouth(3) while regurgitation of dha on the skin resulted in notable discoloration that led to the serendipitous discovery that dha could be used for sunless tanning. subsequently, dha was investigated for topical applications to treat vitiligo(4). considered to be safe, dha is now used as the key ingredient in sunless tanning products that are widely available without prescription(5, ). while such products may be used with varied frequency, the more frequent the use, the greater the effect, with twice-daily use providing maximal effect (7) often marked by a distinctive orange hue. dha's action is limited to the outer dermal layer of the stratum corneum where it colors dead skin cells. there, it condenses dehydratively with the amine and guanidine nucleophiles of lysine and arginine found in keratin and other proteins(8-10) via the famous maillard reaction(11) (or browning reaction) to afford chromophores accounting for the characteristic pumpkin color. the maillard reaction, comprises a manifold of reactions including schiff-base formation, aldol-type condensations, amadori rearrangements, and dehydrative and/or oxidative aromatization events that modify lysine amines and give rise to crosslinks, (12) which are consistent with other advanced glycation events seen in diabetic patients. a recent report characterized certain products of this cascade in the context of several reducing sugars, most notably dha (10) , which led to several substituted lysine-modified pyridinium salts, exemplified by compound 5a, figure 2 (and detailed therein for those who might not be familiar with this process), which is further capable of crosslinking the nitrogen atoms of lysine and arginine. figure 2 : maillard reaction of "3-carbon" sugars with lysine to give glycation products 5a-c by a series of reactions including aldol, amadori rearrangements and oxidation events. compound 5b is one of several conceivable adducts to arginine resulting from oxidation. the formation of 5c, currently unknown, is proposed here as an alternative product resulting from a hantzsch-type pyrrole synthesis. the formation of 5a and related congeners has been extensively characterized by hrms analysis as well as by 1 h-and 13 c-nmr, and uv-vis spectroscopy, based on the reaction of various three-carbon sugars with lysine. as these aromatic amadori products are capable of acting as photosensitizers that generate free radicals (13) , the formation of heterocycles e.g. 5c, which might serve as important reductants in radical processes that may contribute to a much more extensive array of chemical reactions. besides the chromogenic reaction that occurs between dha and proteins, dha has also been found to glycate the nucleic acids: under in vitro conditions, reducing sugars are known to glycate the exocyclic amines of nucleosides, in particular guanine, as seen for dna (14) (15) (16) preventing infectivity of bacteriophages. in addition, there is evidence that reducing sugars including fructose (a related keto sugar) rapidly crosslink lysine to dna (17) in vitro. while the evidence for amino-acid (protein) glycation at ambient ph is much stronger than that for nucleoside modification, the potential relevance to sars-cov-2 topical prophylaxis becomes apparent. as the outer dermal lay of skin is expected to be much more dehydrating that the aqueous conditions that are generally used to assay glycation in vitro, it reasonable to suspect that dha may be capable of modifying one or more of the 61 lysines and/or 48 arginines on the spike protein (genebank qhd43416.1), which interacts with ace2 receptor enabling viral entry. additionally, the n-terminal domain of the nucleocapsid protein, with 7 lysines and 5 arginines, which provide electrostatic complementarity to the polyanionic rna genome, may also be a target for glycation, does the rna genome itself, which might undergo crosslinking to the nucleocapsid protein. given the need to stanch viral transmission, it would be especially attractive to identify a molecule that is cheap, safe, fda-approved, and already found in commercial formulations for topical application. dha necessarily meets all of these characteristics. taken together, a solid body of scientific evidence provides a reasonable chemical basis to support the hypothesis that dha may dehydratively glycate viral proteins and/or viral genomic rna, which would in turn be expected to inactivate the virus, thus prophylactically protecting the wearer from facial transmission. finally, independent support for dha application is found in an isolated report that describes the antifungal activity of dha to treat dermatomycosis(18) further highlighting potential biocidal activity. in the absence of a clinical study, i can only provide sound biochemical evidence to support this hypothesis to highlight the chemical rationale for exploring the potential of studying whether dha could protect against sars-cov-2 transition. herein, several studies are proposed. to start, studies could be undertaken to score viral transfectivity following application of live virus to non-living surfaces (19) that have been coated with dha compared to those that have not. it may be possible to explore a human trial, which would involve little concern for side effects. for example, in assisted living facilities where there is the highest risk to life, residents could be asked to apply a commercially available dha-containing cream to face and hands. likewise, health workers, who are typically at higher risk of exposure, could volunteer to apply self-tanning cream to hands and face on semi-daily basis. incidence of infection could then be scored in relation to the dha application or lack thereof. together, such studies would systematically advance this hypothesis from well-established in vitro results to an easily monitored clinical trial, that would lead to a medically justified recommendation for daily topical prophylactic application of dha to the face and hands for reducing sars-cov-2 infection. as a chemist, i have based this proposal on sound biochemical logic and wellestablished in vitro evidence of dha-based glycation with citations of peer-reviewed scientific publications. with limited funding and no access to viral strains in my own lab, i submit this hypothesis in the hope that it will inspire medical researchers who are equipped with the proper biosafety containment level-3 and clinicians who are certified to treat high-risk individuals in clinical settings/care facilities to consider the proposed experiments. in no way do i advocate ingestion of dha. nor do i advocate use of self-tanning products as a prophylactic until these experiments are properly conducted. furthermore, in no way do i suggest dha applications should obviate wearing a mask, social distancing or hand washing. the influence of dihydroxyacetone upon the blood sugar and glycosuria investigative studies with the skin coloring agents dihydroxyacetone and glyoxal -preliminary report staining of skin with dihydroxyacetone dihydroxyacetone in a new formulation -a powerful therapeutic option in vitiligo dihydroxyacetone and sunless tanning: knowledge, myths, and current understanding dihydroxyacetone: an updated insight into an important bioproduct dihydroxyacetone -a suntan simulating agent dihydroxyacetone (dha) -a keratin coloring agent reaction of dihydroxyacetone (dha) with human skin callus and amino compounds triosidines: novel maillard reaction products and cross-links from the reaction of triose sugars with lysine and arginine residues a synthesis of humic matter by effect of amine acids on sugar reducing agents pyridinium-carbaldehyde: active maillard reaction product from the reaction of hexoses with lysine residues 3-hydroxypyridine chromophores are endogenous sensitizers of photooxidative stress in human skin cells modificaiton of dna by reducing sugars -a possible mechanism for nucleic acid aging and age-related dysfunction in geneexpression modification of dna by glucose-6-phosphate induces dna rearrangements in an escherichia-coli plasmide reaction of guanosine with glucose under oxidative conditions the formaiton of reactive intermediate(s) of gluocose-6-phosphate and lysine capable of rapidly reacting with dna vitro antifungal activity of dihydroxyacetone against causative agents of dermatomycosis stability of human metapneumovirus and human coronavirus nl63 on medical instruments and in the patient environment i attest that i have no conflicts in submitting this work. key: cord-334543-gavnscor authors: lim, rachel k.; wambier, carlos g.; goren, andy title: are night shift workers at an increased risk for covid-19? date: 2020-07-29 journal: med hypotheses doi: 10.1016/j.mehy.2020.110147 sha: doc_id: 334543 cord_uid: gavnscor recent data has revealed an association between coronavirus disease-19 (covid-19) incidence and seasonally regulated androgen sensitivity. this potential relationship between sars-cov-2 infection and clock genes, coupled with previously reported effects of night shift work on health leads us to hypothesize that night shift workers may be at an increased physiological risk of coronavirus disease-19 (covid-19). shift work, especially night shift work, has long been associated with several chronic health conditions. the mechanisms that drive these associations are not well understood; however, current literature suggests that the disruption of circadian rhythms may cause downstream hormonal and immune effects that render night shift workers more susceptible to disease. first, circadian rhythms may play a role in the mechanism of viral infection, as viral vaccines administered in the morning elicit greater immune responses than those administered in the afternoon. next, increased exposure to light at night may inhibit melatonin production, which has been observed to enhance dna repair and shown to upregulate expression of bmal1, an established as an inhibitor of herpes simplex virus and influenza. finally, abnormal immune cell and cytokine levels have been observed following night-shift work. these data suggest that further research is warranted and that high-risk occupations should be considered as public health policies are introduced and evolve. (covid19) incidence and seasonally regulated androgen sensitivity. this potential relationship between sars-cov-2 infection and clock genes, coupled with previously reported effects of night shift work on health leads us to hypothesize that night shift workers may be at an increased physiological risk of coronavirus disease-19 (covid19) . shift work, especially night shift work, has long been associated with several chronic health conditions. the mechanisms that drive these associations are not well understood; however, current literature suggests that the disruption of circadian rhythms may cause downstream hormonal and immune effects that render night shift workers more susceptible to disease. first, circadian rhythms may play a role in the mechanism of viral infection, as viral vaccines administered in the morning elicit greater immune responses than those administered in the afternoon. next, increased exposure to light at night may inhibit melatonin production, which has been observed to enhance dna repair and shown to upregulate expression of bmal1, an established as an inhibitor of herpes simplex virus and influenza. finally, abnormal immune cell and cytokine levels have been observed following night-shift work. these data suggest that further research is warranted and that high-risk occupations should be considered as public health policies are introduced and evolve. although many occupations have transitioned to remote settings during the pandemic, certain "essential" workers such as health care workers, food industry workers, and police officers have continued to work in-person. it would be interesting to test this hypothesis through a large-scale survey of essential shift workers who have continued to work night shifts as the covid-19 pandemic began and progressed. comparing the incidence of covid-19 infections among night shift and day-shift workers will shed light upon this topic. perspectives and conclusions: 7 in conclusion, because several viral infections, including influenza, are closely linked to circadian rhythms, and night shift workers regularly disrupt their circadian rhythms, it is likely that night shift workers are at a greater risk of covid-19 infection and further research is warranted. a novel coronavirus from patients with pneumonia in china a four year seasonal survey of the relationship between outdoor climate and epidemiology of viral respiratory tract infections in a temperate climate androgenetic alopecia present in the majority of hospitalized covid-19 patients -the "gabrin sign what does androgenetic alopecia have to do with covid-19? an insight into a potential new therapy clock genes may drive seasonal variation in sars-cov-2 infectivity: are we due for a second wave of covid-19 in the fall? genetics of circadian rhythms health consequences of shift work and insufficient sleep night shift work and risk of breast cancer night-shift work and risk of prostate cancer: results from a canadian case-control study, the prostate cancer and environment study metabolic syndrome in shift healthcare workers rotating night shift work and adherence to unhealthy lifestyle in predicting risk of type 2 diabetes: results from two large us cohorts of female nurses is shift work associated with a higher risk of overweight or obesity? a systematic review of observational studies with metaanalysis the effects of nocturnal life on endocrine circadian patterns in healthy adults interplay between circadian clock and viral infection preliminary evidence that morning vaccination is associated with an enhanced antibody response in men morning vaccination enhances antibody response over afternoon vaccination: a cluster-randomised trial cell autonomous regulation of herpes and influenza virus infection by the circadian clock genome-wide effect of pulmonary airway epithelial cell-specific bmal1 deletion epidemiology of urinary melatonin in women and its relation to other hormones and night work melatonin for the prevention and treatment of cancer a versatile protector against oxidative dna damage simulated night shift disrupts circadian rhythms of immune functions in humans conflicts of interest: the authors do not have any conflicts of interest to report none 9 the authors do not have any conflicts of interest to report. 10 key: cord-349718-x5i460bc authors: chandrasekaran, baskaran title: dr. chandrasekaran’s reply to “exercising and face masks: an important hypothesis buried in a selective review” date: 2020-09-23 journal: med hypotheses doi: 10.1016/j.mehy.2020.110302 sha: doc_id: 349718 cord_uid: x5i460bc nan dr. chandrasekaran's reply to "exercising and face masks: an important hypothesis buried in a selective review" this is a pdf file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. this version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. dr. chandrasekaran's reply to "exercising and face masks: an important hypothesis buried in a selective review" thank you very much for your interest and critical feedback on our recent hypothesis article published in "medical hypotheses" journal. we are happy to address the concerns posed by prof greenhalgh and colleagues with respect to our recent paper. please find below clarifications with our viewpoint, to the concerns directed to us. firstly, we would like to clarify to the authors and readers that our article focuses only on the possible physiological mechanisms that could be associated with wearing the tight custom made cloth masks during moderate to heavy exercise (60 -80% vo2 max or above anaerobic threshold). our article does not try to demote the theory of mask usage in the community. we second the directives given by the reputed health institutions regarding the usage of mask during community visits/ interaction/ use of community resources ( while using public transportation, grocery store visits, meetings etc) ) as this measure will help reduce the airborne transmission of the deadly virus. second, being a hypothetical proposition our literature search was directected specifically towards the possible potential physiological mechanisms that the non-respiratory masks could induce in the exercising population during moderate to heavy exercise, rather than the effects of n95 or surgical masks used by health care professionals. the systematic review by bahl et al. 2020 investigated droplet transmission during social distancing, but they have not investigated the effect of face masks [1] . the systematic review by bakhit et al. investigated the effects of facemasks on the difficulty in breathing, as well as face irritation, and our hypothesis solely tries to address the physiological mechanisms underpinning the use of facemasks during exercise [2] . though the systematic review by bakhit investigated downsides of the use of facemasks, majority (34 of 37) of the studies that were included, mainly reported comparison of surgical and facemasks and only a few studies reported discomfort claims. our hypothesis pertains to the use of tight custom made masks during moderate to vigorous exercise, not general household work nor in healthcare professionals. we agree with the authors that facemasks should be used in public places and can help curb covid transmission to almost 40% (mitzie 2020), however, this was published after the acceptance of our manuscript http://ftp.iza.org/dp13319.pdf. we would once again like to reiterate that our hypothesis was framed on the premise to link potential physiological mechanisms associated with strenuous exercise rather than general public gathering or household or healthcare work. fourth, we respect the work of the authors on facemask usage during covid 19 [3] , and by no means meant to offend the sentiment of the authors or their paper. we feel the message from our hypothesis has been gravely misconstrued. we are aware of the limited evidence available, with respect to facemasks curbing virus transmission (from the references the authors quoted), and we only meant to convey that the general population who do not generally exercise are exploiting the use of masks and are getting out to exercise going against the government regulations of home quarantine (regulations that have urged individuals to step outdoors only for essential services and supplies, using adequate precautionary measures). we meant no false interpretation or disregard for the author's work. fifth, we would like to clarify the authors that the concept of our hypothesis is more relatable with low-middle income countries that would potentially practise use of facemasks during exercise, where sports medicine practises are still at the stage of infancy, compared to the established western world of sports medicine. the authors would agree with us, when we say that social exercisers engaging in strenuous exercise in public places during the pandemic, would mainly be using tight-fitting masks (not made for therapeutic purposes) that are easily available, worldwide. the knowledge, availability and access of aerodynamically designed masks as mentioned by the authors is highly doubtful, especially in developing countries. further, the authors seem to agree with our stand that masks are necessary unless the people are exercising alone or at home. sixth, in our opinion the results from roberge rj et al study that reported "2 out of 10 health care workers, going into hypercapnia with the use of masks [4] ", does contribute to a significant risk (20%) in patients with chronic diseases during strenuous exercise. prof greenhalgh and colleagues quoted a study by person et al., which showed a significant variation in dyspnea associated even with surgical masks [5] . we hypothesised ill-fitting tight facemasks may aggravate the underlying dyspnea in persons with chronic diseases while exercising. further person et al. 2017 demonstrated the efficacy of surgical mask on the six minute walk test, which is a submaximal test [5] . however, our hypothesisrevolves around th anticipated physiological alterations with facemasks during maximal exercise (moderate to vigorous intensity -60 -80% vo2max) we agree with the authors that our arguments regarding the risks of respiratory alkalosis, increased lactate levels and early fatigue, downregulation of immune response, increased cardiac overload, compromised renal function and reduced cerebral perfusion are speculative. there remains a dearth in literature investigating facemask effects on strenuous exercises (not low intensity activities such as, household or office work, activities of daily living etc). we propose future studies that could concentrate on the molecular level changes induced by even slight hypercapnia in healthy adults with facemasks not asymptomatic covid 19 patients, as mentioned by the authors in their third query. seventh, we too agree with the authors that only newspaper articles and blogs are available to claim that masks can increase the spread covid19 and thus warrants further investigations at the primary care level. lastly, the author's statement is in line with our hypothesis that during prolonged exercise, there may be increased hypercapnia and hypoxia. the available evidence is not strong enough to emphasize the physiological alterations even during low-moderate exercise. we again stress our point that our hypothesis focuses only on the speculated physiological changes associated with tight custom made masks during moderate to vigorous or prolonged exercise and not with basic household, occupational activities or public gathering. we commend the authors for proposing a study that could objectively measure the effects of face masks on strenuous exercise. as exercise and physical activity professionals, we eagerly await these results, which could be further advised to the general healthy population. authors declare no conflict of interests airborne or droplet precautions for health workers treating coronavirus disease downsides of face masks and possible mitigation strategies: a systematic review and meta-analysis face masks for the public during the covid-19 crisis physiological impact of the n95 filtering facepiece respirator on healthcare workers. respir care assistant professor in physical activity, behavioral science and health promotion key: cord-331428-6pvr2vew authors: heffernan, kevin s.; ranadive, sushant; jae, sae young title: exercise as medicine for covid-19: on ppar with emerging pharmacotherapy date: 2020-08-17 journal: med hypotheses doi: 10.1016/j.mehy.2020.110197 sha: doc_id: 331428 cord_uid: 6pvr2vew coronavirus disease 2019 (covid-19) may have a metabolic origin given strong links with risk factors such as lipids and glucose and co-morbidities such as obesity and type 2 diabetes mellitus. the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) spike protein mediates viral cellular entry via the ace2 receptor. the cytoplasmic tail of this spike protein is heavily palmitoylated. emerging studies suggest that sars-cov-2 alters lipid metabolism in the lung epithelial cells by modulating peroxisome proliferator-activated receptor alpha (pparα), possibly contributing to lipotoxicity, inflammation and untoward respiratory effects. disruption of this process may affect palmitoylation of sars-cov spike protein and thus infectivity and viral assembly. covid-19 is also increasingly being recognized as a vascular disease, with several studies noting prominent systemic endothelial dysfunction. the pathogenesis of endothelial dysfunction may also be linked to covid-19-mediated metabolic and inflammatory effects. herein, exercise will be compared to fenofibrate as a possible therapeutic strategy to bolster resilience against (and help manage recovery from) covid-19. this paper will explore the hypothesis that exercise may be a useful adjuvant in a setting of covid-19 management/rehabilitation due to its effects on pparα and vascular endothelial function. severe acute respiratory syndrome coronavirus 2 (sars-cov-2) currently shows no sign of disappearing on its own. globally, coronavirus disease 2019 (covid-19) cases have surpassed 19 million, contributing to over 700,000 deaths. in the united states, the cdc projects that covid-19 will be a top 10 leading cause of death for the year 2020. while we all eagerly await the development of a vaccine, scientists and clinicians have begun exploring "off-label" use of various drugs with that hope that strategic repurposing may help manage and treat covid-19.(1) fenofibrate (a peroxisome proliferator-activated receptor alpha agonist) is one such medication that holds promise given its favorable effects on inflammation and endothelial function. (1) herein, exercise will be compared to fenofibrate as a possible therapeutic strategy to bolster resilience against (and help manage recovery from) covid19 . this paper will explore the hypothesis that exercise may be a useful adjuvant in a setting of covid-19 management/rehabilitation due to its effects on ppar and vascular endothelial function. covid-19 progression has been suggested to have a metabolic origin given that elevated glucose and lipid levels are risk factors. the sars-cov-2 spike protein mediates viral cellular entry via the ace2 receptor (please see our previous paper on the possible role of exercise as a mediator of ace2). (2) the cytoplasmic tail of this spike protein is heavily palmitoylated (i.e. a 16 carbon fatty acid chain is added to palmitate), a common posttranslational modification that increases the hydrophobic nature of a protein. (3) emerging studies suggest that sars-cov-2 alters lipid metabolism in the lung epithelial cells by modulating ppar, possibly contributing to lipotoxicity and untoward respiratory effects. (4) ppar belongs to the nuclear receptor (nr) family and is considered a key transcriptional factor that regulates lipid metabolism. pparα is constitutively expressed in the lung. not surprisingly, alveolar epithelial cells have been shown to conduct fatty acid oxidation, a function that serves a critical role in maintaining optimal lung function.(5) disruption of this process may affect palmitoylation of sars-cov spike protein and thus infectivity and viral assembly. (3, 4) in response to pulmonary inflammation induced by lipopolysaccharide (lps) or tnf, ppar mrna in the lung can be reduced by 50-60%. likely leads to inflammation and cytokine production (i.e. cytokine storm syndrome), reduction of nitric oxide and impaired vascular reactivity. alterations in pulmonary vascular reactivity may affect gas exchange (i.e. alveolar-capillary barrier disruption) and be partially responsible for ventilation-perfusion mismatches and hypoxemia seen with covid-19. (11) as alluded to previously, ppar-activation has anti-inflammatory effects mainly achieved via transrepression, a process whereby pro-inflammatory genes are downregulated. as such, use of the ppar agonists may serve a useful therapeutic role by helping to reverse the inflammatory and metabolic changes induces by sars-cov-2. a recent study by ehrlich et much of the research to date on ppars and exercise has focused on modulation of other isoforms (namely ppar but also pparδ/β) or key co-regulator/co-activators (e.g. peroxisome proliferator-activated receptor γ 1α, pgc-1α) in skeletal muscle. ppar is expressed in cardiac myocytes, hepatocytes, enterocytes, lymphocytes, monocytes, adipocytes, smooth muscle cells, and as alluded to previously endothelial cells and epithelial cells. as such, ppar plays an important role for systemic metabolic processes (heart, kidney, central nervous system, bone, intestines, pancreas, liver, lung). while ppar is responsible for synthesis and storage although studies have yet to explore the effect of exercise on ppar in the lung, it is reasonable to speculate that mechanisms responsible for transcriptional changes in the heart and skeletal muscle would be similar in the lung. that is, the lung as a target organ is essential for mounting an optimal exercise response and delivering oxygen rich blood to the working skeletal muscle (i.e. cardio-respiratory fitness). the classic karlman wasserman "gear wheel model" describes the integrated exercise response as linking mitochondria, skeletal muscle, heart-blood (circulatory system) and lungs as inter-connected cogs. increases in mechanical and metabolic factors that govern changes in ppar in the heart and skeletal muscle may spill over to the respiratory system to ensure a concerted effort to match metabolic demand with cardio-respiratory supply. that habitual exercise training can modulate pparα in the lung remains, at this time, a hypothesis. conversely, there may be some redundancy between pparα and pparδ/β such that pparδ/β can compensate for reductions pparα and this may be target organ specific and differentially affected by exercise. (42) empirical data will be needed to support (or refute) our hypothesis. given the known effect of covid-19 on the heart as an incendiary for cardiac damage, (43, 44) we have no conflicts of interest to disclose. delivering benefits at speed through real-world repurposing of off-patent drugs: the covid-19 pandemic as a case in point exercise as medicine for covid-19: an ace in the hole? palmitoylation of sars-cov s protein is necessary for partitioning into detergent-resistant membranes and cell-cell fusion but not interaction with m protein the sars-cov-2 transcriptional metabolic signature in lung epithelium impairment of fatty acid oxidation in alveolar epithelial cells mediates acute lung injury regulation of peroxisome proliferatoractivated receptor-α expression during lung inflammation pparalpha downregulates airway inflammation induced by lipopolysaccharide in the mouse endotheliopathy in covid-19-associated coagulopathy: evidence from a single-centre, cross-sectional study endothelial cell infection and endotheliitis in covid-19 pulmonary vascular endothelialitis, thrombosis, and angiogenesis in covid-19 endothelial cell dysfunction: a major player in sars-cov-2 infection (covid-19)? expression of peroxisome proliferatoractivated receptor alpha (ppar alpha) in primary cultures of human vascular endothelial cells fatty acid carbon is essential for dntp synthesis in endothelial cells a protective effect of pparα in endothelial progenitor cells through regulating metabolism pparalpha and gr differentially down-regulate the expression of nuclear factor-kappab-responsive genes in vascular endothelial cells nad(p)h oxidase/nitric oxide interactions in peroxisome proliferator activated receptor (ppar)alpha-mediated cardiovascular effects fenofibrate suppresses microvascular inflammation and apoptosis through adenosine monophosphate-activated protein kinase activation fenofibrate activates ampk and increases enos phosphorylation in huvec the peroxisome proliferator-activated receptor α agonist fenofibrate decreases airway reactivity to methacholine and increases endothelial nitric oxide synthase phosphorylation in mouse lung pparalpha activators upregulate enos activity and inhibit cytokine-induced nf-kappab activation through amp-activated protein kinase activation peroxisome proliferator-activated receptor α agonists increase nitric oxide synthase expression in vascular endothelial cells ppar-α agonist fenofibrate upregulates tetrahydrobiopterin level through increasing the expression of guanosine 5'-triphosphate cyclohydrolase-i in human umbilical vein endothelial cells effect of fenofibrate on serum nitric oxide levels in patients with hypertriglyceridemia, advances in clinical and experimental medicine : official organ fenofibrate improves vascular endothelial function by reducing oxidative stress while increasing endothelial nitric oxide synthase in healthy normolipidemic older adults fibrate therapy and flow-mediated dilation: a systematic review and meta-analysis of randomized placebocontrolled trials strong relationship between vascular function in the coronary and brachial arteries peripheral vascular endothelial function and the pulmonary blood pressure response to exercise in pulmonary hypertension coronary flow velocity reserve is improved by ppar-α agonist fenofibrate in patients with hypertriglyceridemia fenofibrate improves cerebral blood flow after middle cerebral artery occlusion in mice pparα activation improves endothelial dysfunction and reduces fibrosis and portal pressure in cirrhotic rats effects of the ppar-α agonist fenofibrate on acute and short-term consequences of brain ischemia effect of endurance training on lipid metabolism in women: a potential role for pparalpha in the metabolic response to training transcriptional adaptations of lipid metabolism in tibialis anterior muscle of endurance-trained athletes vascular effects of exercise: endothelial adaptations beyond active muscle beds exercise modalities and endothelial function: a systematic review and dose-response meta-analysis of randomized controlled trials, sports medicine the antiinflammatory effects of exercise: mechanisms and implications for the prevention and treatment of disease physical exercise as a tool to help the immune system against covid-19: an integrative review of the current literature prevention of age-related endothelial dysfunction by habitual aerobic exercise in healthy humans: possible role of nuclear factor κb adolph distinguished lecture: the remarkable anti-aging effects of aerobic exercise on systemic arteries effects of exercise training on endothelial progenitor cells in cardiovascular disease: a systematic review pparδ is required for exercise to attenuate endoplasmic reticulum stress and endothelial dysfunction in diabetic mice fatty acid homeostasis and induction of lipid regulatory genes in skeletal muscles of peroxisome proliferator-activated receptor (ppar) alpha knock-out mice. evidence for compensatory regulation by ppar delta coronavirus disease 2019 (covid-19) and cardiac injury extrapulmonary manifestations of covid-19 ppars: regulators of metabolism and as therapeutic targets in cardiovascular disease. part i: ppar-α the peroxisome proliferator-activated receptor alpha regulates amino acid metabolism long-term clinical outcomes in survivors of severe acute respiratory syndrome and middle east respiratory syndrome coronavirus outbreaks after hospitalisation or icu admission: a systematic review and meta-analysis obesity and outcomes in covid-19: when an epidemic and pandemic collide loss of hepatic pparα promotes inflammation and serum hyperlipidemia in diet-induced obesity ppar-α agonist elicits metabolically active brown adipocytes and weight loss in diet-induced obese mice ppars, obesity, and inflammation role of increasing the aerobic capacity on improving the function of immune and respiratory systems in patients with coronavirus (covid-19): a review outcomes of cardiovascular magnetic resonance imaging in patients recently recovered from coronavirus disease follow-up study of the pulmonary function and related physiological characteristics of covid-19 survivors three months after recovery respiratory rehabilitation in elderly patients with covid-19: a randomized controlled study acknowledgements. we wish to thank sara mascone for her assistance creating figure 1 .conflicts of interest: none to disclose key: cord-328587-vctvcyim authors: jun, sun title: the hypothesis that sars-cov-2 affects male reproductive ability by regulating autophagy date: 2020-07-10 journal: med hypotheses doi: 10.1016/j.mehy.2020.110083 sha: doc_id: 328587 cord_uid: vctvcyim the outbreak of coronavirus disease19 (covid19) in december 2019 posed a serious threat to public safety, and its rapid spread caused a global health emergency. clinical data show that in addition to respiratory system damage, some male patients with covid-19 are also accompanied by abnormal renal function and even renal damage. as the main receptor of syndrome coronavirus 2 (sars-cov-2), angiotensin converting enzyme 2 (ace2) is also found to be highly expressed not only in respiratory mucosa and alveolar epithelial cells, but also in renal tubule cells, testicular leydig cells and seminiferous tubule cells. this suggests that sars-cov-2 has the possibility of infecting the male reproductive system, and the recent detection of sars-cov-2 in the patient's semen further confirms this theory. in previous studies, it has been found that ace2 has the ability to regulate autophagy. not only that, recent studies have also found that sars-cov-2 infection can also lead to a reduction in autophagy. all of these associate sars-cov-2 with autophagy. furthermore, autophagy has been shown to have an effect on male reproduction in many studies. based on these, we propose the hypothesis that sars-cov-2 affects male reproductive function by regulating autophagy. this hypothesis may provide a new idea for future treatment of covid-19 male patients with reproductive function injury, and it can also prompt medical staff and patients to consciously check their reproductive function. the hypothesis that sars-cov-2 affects male reproductive ability by regulating autophagy. 【abstract】：the outbreak of coronavirus disease19 (covid19) in december 2019 posed a serious threat to public safety, and its rapid spread caused a global health emergency. clinical data show that in addition to respiratory system damage, some male patients with covid-19 are also accompanied by abnormal renal function and even renal damage. as the main receptor of syndrome coronavirus 2 (sars-cov-2), angiotensin converting enzyme 2 (ace2) is also found to be highly expressed not only in respiratory mucosa and alveolar epithelial cells, but also in renal tubule cells, testicular leydig cells and seminiferous tubule cells. this suggests that sars-cov-2 has the possibility of infecting the male reproductive system, and the recent detection of sars-cov-2 in the patient's semen further confirms this theory. in previous studies, it has been found that ace2 has the ability to regulate autophagy. not only that, recent studies have also found that sars-cov-2 infection can also lead to a reduction in autophagy. all of these associate sars-cov-2 with autophagy. furthermore, autophagy has been shown to have an effect on male reproduction in many studies. based on these, we propose the hypothesis that sars-cov-2 affects male reproductive function by regulating autophagy. this hypothesis may provide a new idea for future treatment of covid-19 male patients with reproductive function injury, and it can also prompt medical staff and patients to consciously check their reproductive function. 【key words】sars-cov-2 , ace2 , autophagy , reproduction coronavirus disease19 (covid-19) is a new kind of infectious respiratory disease caused by syndrome coronavirus 2 (sars-cov-2). like severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov) , sars-cov-2 is highly pathogenic and lethal [1] . according to the existing clinical data, some patients not only suffer from respiratory diseases, but also have complications such as acute renal injury and even renal necrosis [2] [3] [4] , in addition, sars-cov-2 was also found in recent semen analysis of male patients [5] . these show that sars-cov-2 has a certain infection to the male reproductive system and may cause damage to male fertility. as a receptor protein of sars-cov-2, ace2 not only mediates sars-cov-2 infection in host cells, but also is associated with the decrease of autophagy [6] . moreover, recent studies have found that sars-cov-2 infection limits autophagy by interfering with multiple metabolic pathways [7] . the majority of studies have further found that autophagy is often associated with male reproductive ability [8] [9] . therefore, we associate sars-cov-2 infection with male reproduction and autophagy, and put forward the hypothesis that sars-cov-2 affects male reproductive function by regulating autophagy. the outbreak of covid-19 in late december 2019 has attracted much attention because of its high infectivity. with the in-depth study of sars-cov-2, we found that the virus is more contagious than sars-cov and mers-cov [10] . in addition, according to survey data, of the 44672 confirmed cases in mainland china, there were 1023 deaths, with a crude case fatality rate of 2.3 % [11] , which was lower than that of mers-cov (37 %) and sars-cov (10 %) [12] . it can be seen that compared with mers-cov and sars-cov, sars-cov-2 is more prominent in that it is highly contagious and the case fatality rate is relatively low. 1918-1919 influenza pandemic has similar characteristics to covid-19. the case fatality rate of 1918-1919 influenza pandemic is less than 5%, but due to its wide spread, it has a great impact on society [13] . besides, 1918-1919 influenza pandemic is accompanied by many complications and sequelae, which eventually cause a lot of irreversible damage to patients. thus, in the face of this kind of highly contagious but relatively low mortality infectious diseases, we should not only pay attention to the treatment of the disease itself, but also pay attention to its complications and sequelae so as to avoid serious complications or sequelae in the course of clinical treatment or after cure. lung injury is considered to be the main damage of sars-cov-2 infection. the common symptoms of covid-19 are fever and dry cough, followed by the most typical symptoms-respiratory distress, and severe patients can deteriorate rapidly and die of respiratory failure in a short period of time [14] . nevertheless, in addition to respiratory diseases, liver biopsies from patients with severe covid-19 death showed moderate microvascular steatosis, mild lobular and portal vein activity and other liver injury [15] . moreover, complications such as acute renal injury, acute heart injury and myocarditis were also found in dead patients [2] [3] [4] , suggesting that sars-cov-2 may cause damage to the non-respiratory system. sars-cov-2 is similar to sars-cov in that it also uses ace2 as its receptor [16] . ace2 was found to be highly expressed not only in alveolar epithelial cells, but also in hepatic endothelial cells, making the liver a target for sars-cov. in addition, sars-cov may also induce hepatocyte apoptosis, resulting in liver injury [17] [18] . in the study of sars-cov-2, it may lead to liver injury through direct cytopathic effect induced by virus or immunopathological effect caused by excessive inflammatory response [19] . not only that, when using drugs to treat covid-19, some drugs will also cause liver, kidney and other organ damage. all these further prove that sars-cov-2 has the ability to infect the non-respiratory system and can cause non-respiratory system damage. based on this, we can realize that from a clinical point of view, in addition to actively dealing with the primary disease caused by sars-cov-2 infection, we should also pay attention to monitoring the damage of other organs, such as liver, kidney and other non-respiratory organs. at the same time, it is also a kind of education for doctors and patients, reminding them to consciously check the organs of the non-respiratory system from time to time, so as to prevent other functional disorders of the body. covid-19 patients are not limited to respiratory injuries, but may also be accompanied by non-respiratory diseases. previous clinical data have shown that in addition to respiratory diseases, some male patients with covid-19 are accompanied by kidney damage and even renal failure [2] [4], which suggest that sars-cov-2 may affect male fertility. at the same time, orchitis has been found to cause male reproductive dysfunction as a complication of sars [20] , which also supports this view. similar to sars-cov, sars-cov-2, it can also infect host cells through ace2. it is worth emphasizing that ace2 exists not only in respiratory system cells such as alveoli and trachea, but also in testicular cells, seminiferous tubule cells, testicular leydig cells, kidney cells and so on [4] [21] , indicating that sars-cov-2 is likely to invade the reproductive system. coronavirus spike protein (s protein) plays an important role in virus infection and pathogenesis。as the key surface localization glycoprotein of the virus, it mediates the invasion of the virus by binding to the host cell surface receptor [1] . the s protein of sars-cov-2 has the typical characteristics of coronavirus s protein, and its two subunits s1 and s2 are responsible for receptor recognition and membrane fusion, respectively. s1 subunit can be further divided into n-terminal domain (ntd) and c-terminal domain (ctd), both of which can play a role as ligands binding to receptors [22] . from this we can know that the signal recognition of sars-cov-2 in the process of entering host cells may be related to the ntd or ctd of s1 subunit. wang q et al. [22] found that for sars-cov-2, its s1 subunit ctd and ace2 were co-located on the surface of host cells, and its ntd had no ability to bind to ace2. these results suggest that the entry of sars-cov-2 into the host cell may be through the binding of its s1 subunit ctd to ace2, thus invading the host cell. to sum up, we can know that the existence of ace2 on the surface of testicular cells provides the possibility for sars-cov-2 to infect the reproductive system, and it may induce sars-cov-2 to infect a series of reproduction-related cells by combining with the ctd sequence of s protein s1 subunit of sars-cov-2, and finally affect male fertility. in mers-cov-infected cells, autophagy is limited by a virus-induced akt1-dependent activation of the e3-ligase s-phase kinase-associated protein 2 (skp2) [23] . similarly, recent studies have found an increase in the level of autophagy receptor sqstm1/p62 in sars-cov-2-infected cells, suggesting a decrease in autophagy flux. ars-cov-2 infection also promotes akt1/skp2-dependent autophagy to initiate beclin-1 (becn1) degradation [7] . these results suggest that sars-cov-2 infection can limit the level of autophagy. as the main receptor of sars-cov-2, angiotensin converting enzyme 2 (ace2) not only mediates the entry of sars-cov-2 into host cells, but also has some relationship with autophagy. in previous studies, it was found that the level of autophagy decreased under the action of ace2 [6] [24] , while the overexpression of ace2 appeared in autophagy deficient cells [25] . the regulation of autophagy by ace2 also confirms the effect of sars-cov-2 on autophagy from the side。 autophagy is a degradation system in eukaryotic cells, which can not only degrade intracellular aging-damaged organelles and unneeded metabolites to provide cell energy and nutrients, but also play an important role in the elimination of intracellular pathogenic microorganisms. moreover, more and more studies have proved that autophagy is involved in a wide range of cellular events in the male reproductive system, affecting male reproductive ability. in the process of spermatogenesis, autophagy is very important to ensure the formation of specific structures and the degradation of some components in spermatogenesis [26] . in autophagy-deficientt mouse testis, due to the accumulation of a negative cytoskeleton organization regulator, pdlim1, the cytoskeleton structure is disordered, the assembly of extracellular specialized (es) is destroyed, and finally lead to sperm head deformity [26] . besides, autophagy has also been shown to synthesize testosterone by promoting cholesterol uptake and degradation of intracellular low density lipoprotein, which may be involved in the metabolism and elimination of testosterone, as well as the production of other cholesterol-based hormones [8] . it can be seen that autophagy plays an active role in male spermatogenesis and endocrine process. we know that sars-cov-2 may infect host cells by binding to ace2 on the surface of reproduction-related cells such as leydig cells. sars-cov-2 itself or ace2 can cause cell dysfunction by regulating the level of autophagy, and some viral proteins can also directly induce or inhibit the autophagy pathway to achieve virus survival [27] . for example, the dual action of hiv and autophagy pathway increases virus production by using the early stage of autophagy and inhibiting the late stage of autophagy [28] [29] . this information links sars-cov-2, autophagy and male fertility, leading us to speculate whether sars-cov-2 may eventually cause male reproductive disorders by regulating the level of autophagy in male germ cells. in the recent treatment of covi-19, it was found that male patients had renal dysfunction and even renal injury [2] [4] . furthermore, clinical data also show that the receptor protein ace2 that mediates the entry of sars-cov-2 into host cells is not only expressed in alveolar cells, but also highly expressed in male renal tubular cells [4] [21] [30] .all these suggest that sars-cov-2 not only causes damage to the respiratory system of patients, but also has a certain impact on the reproductive system of male patients. therefore, in the process of treating covid-19 patients, clinicians should not only focus on respiratory diseases, but also pay attention to whether the reproductive function of male patients is normal, especially the fertility evaluation and appropriate intervention of young patients. in addition, when a male patient comes to see a doctor because of symptoms such as fever and reproductive system disorders, doctors need to consider whether the patient is likely to be infected by novel coronavirus and take timely protective measures. through education to make medical staff and patients understand the impact of sars-cov-2 on male reproduction, doctors can not only use drugs to protect male reproductive system in the process of clinical treatment, so as to reduce the impact of such complications on male reproduction, but also enable patients with fertility needs to consciously carry out fertility physical examination after rehabilitation, so as to avoid irreversible damage to their fertility because of sequelae. the hypothetical mechanism that sars-cov-2 affects male reproductive ability by regulating autophagy can provide a new idea for the treatment of male covid-19 patients with impaired reproductive ability in the future. clinicians can treat reproductive function damage in male covid-19 patients , or sequelae such as reproductive decline after recovery by regulating autophagy. through analysis, we systematically linked sars-cov-2, autophagy and male fertility, and realized that sars-cov-2 has the possibility of infecting male germ cells. sars-cov-2 can also cause some damage to male germ cells by regulating the level of autophagy, and finally affect male fertility. although the specific mechanism of injury is not clear, our hypothesis provides a new idea, suggesting that we can explain the effect of sars-cov-2 on male reproduction from the aspect of autophagy. for male patients with reproductive dysfunction, regulating autophagy may be a good treatment. not only that, it is also a kind of education for medical staff and male patients, so that they can understand the impact of sars-cov-2 on male reproductive function, and enable them to consciously carry out semen and sex hormone tests, so as to prevent such complications or sequelae from damage to the reproductive function of male patients. the authors declare that there is no conflict of interests regarding the publication of this article from sars to mers thrusting coronaviruses into the spotlight clinical characteristics of 113 deceased patients with coronavirus disease 2019: retrospective study surgical infection society guidance for operative and peri-operative care of adult patients infected by the severe acute respiratory syndrome coronavirus-2 (sars-cov-2) ace2 expression in kidney and testis may cause kidney and testis damage after 2019-ncov infection clinical characteristics and results of semen tests among men with coronavirus disease spironolactone alleviates diabetic nephropathy through promoting autophagy in podocytes analysis of sars-cov-2-controlled autophagy reveals spermidine, mk-2206, and niclosamide as putative antiviral therapeutics autophagy in male reproduction diet-induced obesity impairs spermatogenesis: a potential role for autophagy a novel coronavirus emerging in china -key questions for impact assessment the novel coronavirus pneumonia emergency response epidemiology team. the epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (covid-19) -china a novel coronavirus outbreak of global health concern age-and sex-specific mortality associated with the 1918-1919 influenza pandemic in kentucky clinical features of patients infected with 2019 novel coronavirus in wuhan forner alejandro et al. covid-19 and liver disease the novel coronavirus 2019 (2019-ncov) uses the sars-1 coronavirus receptor2 ace2 and the cellular protease tmprss2 for entry into target cells sars-associated viral hepatitis caused by a novel coronavirus: report of three cases overexpression of 7a, a protein specifically encoded by the severe acute respiratory syndrome coronavirus, induces apoptosis via a caspase-dependent pathway liver injury during highly pathogenic human coronavirus infections orchitis: a complication of severe acute respiratory syndrome (sars) the novel angiotensin-converting enzyme (ace) homolog, ace2, is selectively expressed by adult leydig cells of the testis structural and functional basis of sars-cov-2 entry by using human ace2 skp2 attenuates autophagy through beclin1-ubiquitination and its inhibition reduces mers-coronavirus infection mirna-30e mediated cardioprotection of ace2 in rats with doxorubicin-induced heart failure through inhibiting cardiomyocytes autophagy severe acute respiratory syndrome coronavirus replication is severely impaired by mg132 due to proteasome-independent inhibition of m-calpain autophagy is required for ectoplasmic specialization assembly in sertoli cells matrix protein 2 of influenza a virus blocks autophagosome fusion with lysosomes autophagy pathway intersects with hiv-1 biosynthesis and regulates viral yields in macrophages hepatitis b virus x protein sensitizes cells to starvation-induced autophagy via up-regulation of beclin 1 expression scrna-seq profiling of human testes reveals the presence of the ace2 receptor, a target for sars-cov-2 infection in spermatogonia, leydig and sertoli cells key: cord-324949-sqy03dks authors: poe, francis l.; corn, joshua title: n-acetylcysteine: a potential therapeutic agent for sars-cov-2 date: 2020-05-30 journal: med hypotheses doi: 10.1016/j.mehy.2020.109862 sha: doc_id: 324949 cord_uid: sqy03dks covid-19, a respiratory disease caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2), continues to spread across the globe. predisposing factors such as age, diabetes, cardiovascular disease, and lowered immune function increase the risk of disease severity. t cell exhaustion, high viral load, and high levels of tnf-ɑ, il1β, il6, il10 have been associated with severe sars-cov-2. cytokine and antigen overstimulation are potentially responsible for poor humoral response to the virus. lower cellular redox status, which leads to pro-inflammatory states mediated by tnf-ɑ is also potentially implicated. in vivo, in vitro, and human clinical trials have demonstrated n-acetylcysteine (nac) as an effective method of improving redox status, especially when under oxidative stress. in human clinical trials, nac can be used to replenish glutathione stores and increase the proliferative response of t cells. nac has also been shown to inhibit the nlrp3 inflammasome pathway (il1β and il18) in vitro, and decrease plasma tnf-ɑ in human clinical trials. mediation of the viral load could occur through nac’s ability to increase cellular redox status via maximizing the rate limiting step of glutathione synthesis, and thereby potentially decreasing the effects of virally induced oxidative stress and cell death. we hypothesize that nac could act as a potential therapeutic agent in the treatment of covid-19 through a variety of potential mechanisms, including increasing glutathione, improving t cell response, and modulating inflammation. in this article, we present evidence to support the use of nac as a potential therapeutic agent in the treatment of covid-19. covid-19, a respiratory disease caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2), continues to spread across the globe. sars-cov-2 is an enveloped positivesense, single stranded-rna virus in the betacoronavirus genus [1, 2] . sars-cov-2 is genomically similar to severe acute respiratory coronavirus 1 (sars-cov-1), which led to an outbreak of severe acute respiratory syndrome (sars) in the early 2000s. no cases of sars have been reported globally since 2004 [1] . similar to sars-cov-1, sars-cov-2 gains entry into cells through spike protein affinity for angiotensin converting enzyme 2 receptors (ace2) and uses host cell serine protease tmprss2 to mediate entry [3] [4] [5] . ace2 receptors are expressed in lung, heart, kidney, and intestinal tissue and primarily function physiologically in the maturation of angiotensin [6] . the pathogenicity of the sars-cov-1 stemmed from nod like receptor, pyrin domain containing 3 (nlrp3) inflammasome activation in monocytes and macrophages [7, 8] . this led to high levels of cytokines: interleukin-1β (ilβ), interleukin-18 (il18), tumor necrosis factor alpha (tnfɑ); and an immunopathological response associated with acute respiratory distress syndrome (ards), cytokine storms, organ damage, and death [7, [9] [10] [11] . although sars-cov-2 is 78% genomically similar to sars-cov-1, there are variations in the open reading frames (orfs) involved in the inflammatory monocyte and macrophage response [3, 12] . research is underway to elucidate these mechanisms further. in severe covid-19, there are increased levels of cytokines interleukin-6 (il6), interleukin-10 (il10), and tnf-ɑ [13] . in some cases, these increased cytokine levels create a "cytokine storm" and cause significant damage to lung tissue [14] . clinically, covid-19 is mild in most cases, with severe cases characterized by pneumonia and critical cases characterized by ards, sepsis, and multiple organ failure [14] . older adults, aged 65 years and older, appear to be most susceptible to covid-19 [14, 15] . the most susceptible persons to sars-cov-2 are the elderly: the typical profile of the critically ill patient is 65 years and older, presents with comorbidities, and ards [9] . these patients had a mortality rate of 67% from the time of admittance to 28 days later [9] . n-acetylcysteine (nac) is a precursor of glutathione and acts as a powerful antioxidant and free radical scavenger in the body [16] . nac has been used for paracetamol toxicity and conditions with viscous mucous secretions [17] . nac may be administered orally, intravenously, or nebulized. we hypothesize that nac could act as a potential therapeutic agent in the treatment of covid-19 through a variety of potential mechanisms, including increasing glutathione [18] , improving t cell response [19] [20] [21] , and modulating inflammation [22, 23] . in this article, we present evidence to support the use of nac as a treatment for covid-19. sars-cov-2 and the elderly sars-cov-2 is especially virulent in elderly populations. the virus gains entry primarily via ace2 receptors on alveolar type 2 epithelium [24] . hypertension, a known risk factor for developing more severe covid-19 disease, is more common in older adults [25, 26] . in adults with a history of taking angiotensin converting enzyme inhibitors and angiotensin receptor blockers to treat hypertension, there is a possible increase in ace2 receptors [27] . although the relationship has yet to be clearly defined, viral infectivity may be increased due to increased expression of ace2 secondary to treatment with acei and arbs in the elderly [28] . in sars-cov-1, the immune response was dominated by a th1 response, but in sars-cov-2 there are both th1 and th2 responses [29] . in sars-cov-1, an inflammatory monocyte and macrophage cascade activated via the nlrp3 inflammasome pathway [7, 8] . this resulted in pro-inflammatory cytokines il1β and il18, and an immunopathological response causing injury to lung tissues [7] . in sars-cov-2, there is a remarkable increase of cytokines il6, il10, and tnf-ɑ [15] , however, similar to sars-cov-1, there appears to be elevated levels of il1β, which suggests possible pro-inflammatory macrophage and monocyte activity activated by the nlrp3 inflammasome pathway [15, 30] . t cell exhaustion has been noted in severe sars-cov-2, illustrated by high levels of programmed cell death protein 1 (pd1) and low cd4+ and cd8+ counts [15] . t cell exhaustion leads to poor cd4+ and cd8+ responses, which limits viral clearance via humoral immunity [31] . the likely cause of t cell exhaustion in sars-cov-2 is hypothesized to be from excessive stimulation of t cells by viral antigen and cytokines (il6, il10, tnf-ɑ [15] . notwithstanding sars-cov-2 infection, the elderly have an increased risk of mortality due to age related proinflammatory changes [32] . one mechanism suggested is increased tnf-ɑ induced apoptosis that occurs in the elderly [33] . in severe sars-cov-2, diao et al. suggest that age related proinflammatory changes to tnf-ɑ expression may be a causative factor in t cell exhaustion [15] . additionally, elderly adults may also have decreased redox potential via lower glutathione levels [34] [35] [36] . lowered redox status of a cell increases susceptibility to oxidative stress that may lead to cell death and viral release [37] . glutathione conjugates oxidative species through phase 2 detoxification. it is a central oxidative species mediator that impacts cell cycle and apoptosis [38, 39] . l-cysteine is the rate-limiting substrate in the intracellular synthesis of glutathione [39] . nac directly affects the amino acid pool of extracellular cystine and intracellular cysteine through a series of redox reactions in the plasma [40] . nac increases extracellular cysteine, and through transport channels increases intracellular cysteine levels [18, 38, 41] . during oxidative stress, nac will increase glutathione synthesis [18, 42] . without oxidative stress, the pool of cysteine and cystine appears to primarily mediate cellular stress through thiols other than glutathione [18, 38] . individuals 60 years and older demonstrate lower plasma glutathione levels and increased oxidative stress [43] . those with diabetes have lower glutathione levels compared to control subjects [35, 43] . dietary supplementation of cysteine and glycine can increase glutathione levels and reduce oxidative stress in the elderly [36] and persons with diabetes [35] . glutathione concentrations affect the proliferative capacity of lymphocytes: low concentrations lead to less lymphocyte proliferation and high concentrations lead to more lymphocyte proliferation [44] . t cell exhaustion occurs when lymphocytes (cd4+ and cd8+) counts are low, and can be measured through program cell death protein 1 (pd1) [31] . it is commonly seen in chronic viral infections [31] . pd1 was elevated in severe covid-19 cases [15] . diao et al. referred to this state, in conjunction with significantly lowered cd4+ and cd8+ counts, as functional t cell exhaustion [15] . the investigators posited that functional t cell exhaustion is an etiological factor in severe sars-cov-2 infection. they determined the observed t cell exhaustion is associated with and likely caused by overstimulation of cytokines (il6, il10, tnf-ɑ). lymphocyte proliferation has been noted with the administration of nac. in patients with hiv, oral nac increased both whole blood glutathione levels and lymphocyte count (cd4+ and cd8+) [20, 21] . nac has also shown to decrease pd-1 levels and increase the longevity of cd8+ cells in vitro [19] . data from sars-cov-1 indicates that this coronavirus mediates the nlrp3 inflammasome pathway in infected cells by orfs 3a and 8b [7, 45, 46] . this pathway has been indicated in inflammatory cell death (necroptosis) and is likely responsible for the pathological findings found in lung biopsy of sars-1 patients [45, 47] . likewise in sars-cov-2, there is an elevated level of il1β suggesting nlrp3 activation in macrophages and monocytes [29, 30, 48] . tnf-ɑ is usually increased early in the inflammatory response [49, 50] , and can be secreted by monocytes, macrophages, and alveolar epithelium [50] . tnf-ɑ attaches to receptor sites and depending on the redox potential of the cell, may induce an apoptotic or a proliferative pathway [51] . in vitro, nac interrupts the nlrp3 inflammasome pathway in a dose dependent manner through effects on mrna expression of nlrp3 and caspase-1 activation [22] . nac lowers il1β, il18 [22] . nac has been shown to lower mucin production, and il6 and tnf-ɑ in vitro [52] . nac inhibits the downstream activities post tnf-ɑ receptor activation [52] , and while under oxidative stress nac inhibits gene expression of tnf-ɑ and il-6 [23] . there have been several clinical trials investigating the use of nac in respiratory illness in humans. intravenous nac has been used clinically for the treatment of ards. in a metaanalysis of randomized clinical trials investigating the use of nac as a treatment for ards, administration of nac resulted in a decreased length of stay in intensive care units, however, there was no change in overall short term mortality. the meta-analysis found no adverse reactions with doses similar to those used in drug-induced liver injury [53] . in both in vivo and human trials, nebulized nac may improve arterial oxygen tension [54, 55] ; and attenuate pulmonary fibrosis [56] , and ards [57] . in a randomized clinical trial, oral nac demonstrated decreases in tnf-ɑ and no adverse reactions at 1200 mg daily, however there were no changes in computed tomography scores between those treated with nac and the control group [58] . in another trial involving multiple elder care facilities, oral nac was investigated as a prophylactic and therapeutic agent for influenza. participants who were given 1200 mg daily for six months experienced fewer influenza and influenza-like episodes, decreased severity of illness, and fewer days confined to bed [59] . in this trial, nac did not alter viral seroconversion but participants taking nac were less likely to develop a symptomatic infection [59] . these findings are especially impactful given the higher-risk study population. ventilator use is common in severe cases of covid-19, with approximately 3% of all cases requiring intubation and invasive ventilation at some point during the course of illness [60] . nac has been studied as a prophylactic intervention for ventilator associated pneumonia (vap), a complication of the use of mechanical ventilation [61] . in a randomized, double-blind, placebocontrolled trial of 60 participants receiving nasogastric administration of 1200 mg nac daily, participants in the treatment group were less likely to develop vap and had a shorter duration of hospital and icu stay. additionally, the incidence of complete recovery from vap was higher in the nac group. nac has an excellent safety record in clinical trials. oral nac can cause stomatitis, nausea, vomiting, gastroesophageal reflux [17, 62] . oral nac may be used if there has been an anaphylactoid reaction to iv nac [17] . nebulized nac may cause bronchoconstriction and prolonged coughing and may worsen asthma [55, 63] . serious adverse reactions with iv nac are rare, loading dose dependent, and primarily occur in drug-induced liver injury [17, 64] . in drug-induced liver injury from paracetamol, 10 grams of nac is typically loaded in 15 minutes. this can result in upwards of 31% risk of anaphylactoid reactions ranging from pruritus, rash, angioedema, bronchospasm, and hypotension [65] . with an alternate loading schedule the overall risk of anaphylactic reaction can be lowered to 5% [65] .anaphylactoid reactions occur with iv nac and can be managed with attentive care [17] . but as noted in the ards meta-analysis no adverse reactions were recorded with a similar loading dosage used in drug-induced liver injury [53] . discussion nac has shown activity in a variety of potential therapeutic target pathways involved in the pathophysiology of sars-cov-2 infection. the pathogenic factors of sars-cov-2 that could possibly be mediated by nac are (1) t cell exhaustion, which manifests as lower counts and decreased functional capacity of cd4+ and cd8+ cells; (2) pro-inflammatory state via increase in tnf-ɑ, il1β, il18; and (3) modulation of viral activity through increased glutathione. the virus is especially virulent in the elderly population. certain physiological conditions, including diabetes, cardiovascular disease, and lowered immune function that may affect the severity of sars-cov-2 are more common in older adults. lowered redox status, common in high-risk groups including older adults and those with uncontrolled diabetes, causes alterations in the tnf-ɑ receptor activity towards a pro-inflammatory state [33, 51] . nac has been shown to replenish glutathione stores and increase the proliferative response of t cells. nac has also been shown to inhibit the nlrp3 inflammasome pathway (il1β and il18) in vitro, and decrease plasma tnf-ɑ. mediation of the viral load could occur through the ability of nac to increase cellular redox status by maximizing the rate limiting step of glutathione synthesis, and thereby decreasing the effects of virally induced oxidative stress and cell death. to test these hypotheses further, we recommend both in vitro and in vivo studies investigating nac. in vitro studies may include investigations into the action of nac on certain cell types such alveolar type 2 epithelial cells, monoctyes, or macrophages, as well changes in receptor and cytokine expression in these cell types. other studies could investigate the action of nac in human cell lines with low redox potential. human clinical trials would build on current evidence for the use of nac in other infectious respiratory illnesses. hospital-based clinical trials could administer nebulized, oral, or iv nac during various stages of illness, perhaps utilizing grouping identified by diao et al. similar to clinical trials in influenza, ards, and vap, outcome measures could include change in sars-cov-2 associated cytokines, change in lymphocyte count and activation, change in icu status, and overall mortality. while no clinical trials investigating the use of nac in covid-19, trials completed in using nac for influenza, ards, and vap have shown promising results in reducing disease severity and duration of hospital stay. currently, a protocol for using both nac and heparin has been developed by a seattle-based biotherapeutics researcher. the protocol is available for use by clinicians but at the date of this publication, data from studies using this protocol have not been published [66] . epidemiology, virology, and clinical features of severe acute respiratory syndrome -coronavirus-2 (sars-cov-2 coronavirus disease-19) severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and coronavirus disease-2019 (covid-19): the epidemic and the challenges genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding severe acute respiratory syndrome-related coronavirus: the species and its viruses -a statement of the coronavirus study group sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor structural basis for the recognition of sars-cov-2 by full-length human ace2 severe acute respiratory syndrome coronavirus viroporin 3a activates the nlrp3 inflammasome host factors in coronavirus replication clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study covid-19: consider cytokine storm syndromes and immunosuppression the origin, transmission and clinical therapies on coronavirus disease 2019 (covid-19) outbreak -an update on the status the establishment of reference sequence for sars-cov-2 and variation analysis sars-cov-2: a storm is raging evaluation and treatment coronavirus (covid-19) reduction and functional exhaustion of t cells in patients with coronavirus disease 2019 (covid-19) a review on various uses of n-acetyl cysteine interaction of nacetylcysteine and cysteine in human plasma nacetyl cysteine protects anti-melanoma cytotoxic t cells from exhaustion induced by rapid expansion via the downmodulation of foxo1 in an akt-dependent manner the role of oxidative imbalance in progression to aids: effect of the thiol supplier n-acetylcysteine nacetylcysteine replenishes glutathione in hiv infection the anti-inflammatory effects of acetaminophen and n-acetylcysteine through suppression of the nlrp3 inflammasome pathway in lps-challenged piglet mononuclear phagocytes n-acetylcysteine modulates lipopolysaccharide-induced intestinal dysfunction a crucial role of angiotensin converting enzyme 2 (ace2) in sars coronavirus-induced lung injury hypertension and its severity or mortality in coronavirus disease 2019 (covid-19): a pooled analysis hypertension and aging covid-19 and angiotensin-converting enzyme inhibitors and angiotensin receptor blockers: what is the evidence organ-protective effect of angiotensin-converting enzyme 2 and its effect on the prognosis of covid-19 clinical features of patients infected with 2019 novel coronavirus in wuhan, china spotlight on the nlrp3 inflammasome pathway molecular and cellular insights into t cell exhaustion inflammageing: chronic inflammation in ageing, cardiovascular disease, and frailty increased tnf-alpha-induced apoptosis in lymphocytes from aged humans: changes in tnf-alpha receptor expression and activation of caspases r)-alpha-lipoic acid reverses the age-related loss in gsh redox status in post-mitotic tissues: evidence for increased cysteine requirement for gsh synthesis glutathione synthesis is diminished in patients with uncontrolled diabetes and restored by dietary supplementation with cysteine and glycine deficient synthesis of glutathione underlies oxidative stress in aging and can be corrected by dietary cysteine and glycine supplementation redox biology of respiratory viral infections the cystine/cysteine cycle: a redox cycle regulating susceptibility versus resistance to cell death glutathione and apoptosis role of n-acetylcysteine and cystine in glutathione synthesis in human erythrocytes the utilization of n-acetylcysteine and 2-oxothiazolidine-4-carboxylate by rat hepatocytes is limited by their rate of uptake and conversion to cysteine pharmacokinetics of n-acetylcysteine following repeated intravenous infusion in haemodialysed patients glutathione in human plasma: decline in association with aging, age-related macular degeneration, and diabetes proliferative capacity of human peripheral blood lymphocytes sorted on the basis of glutathione content sars-coronavirus open reading frame-3a drives multimodal necrotic cell death sars-coronavirus open reading frame-8b triggers intracellular stress pathways and activates nlrp3 inflammasomes lung pathology of severe acute respiratory syndrome (sars): a study of 8 autopsy cases from singapore tnf regulates transcription of nlrp3 inflammasome components and inflammatory molecules in cryopyrinopathies tnf regulates chemokine induction essential for cell recruitment, granuloma formation, and clearance of mycobacterial infection tumor necrosis factoralpha mediates lipopolysaccharide-induced macrophage inflammatory protein-2 release from alveolar epithelial cells. autoregulation in host defense redox regulation of tumor necrosis factor signaling n-acetyl-l-cysteine (nac) inhibit mucin synthesis and pro-inflammatory mediators in alveolar type ii epithelial cells infected with influenza virus a and b and with respiratory syncytial virus (rsv) effects of n-acetylcysteine treatment in acute respiratory distress syndrome: a meta-analysis effect of n-acetylcysteine on gas exchange after methacholine challenge and isoprenaline inhalation in the dog evaluation of the effect of nebulized n-acetylcysteine on respiratory secretions in mechanically ventilated patients: randomized clinical trial aerosolized administration of n-acetylcysteine attenuates lung fibrosis induced by bleomycin in mice influence of nebulized unfractionated heparin and n-acetylcysteine in acute lung injury after smoke inhalation injury n-acetylcysteine improves oxidative stress and inflammatory response in patients with community acquired pneumonia: a randomized controlled trial attenuation of influenza-like symptomatology and improvement of cell-mediated immunity with long-term n-acetylcysteine treatment intubation and ventilation amid the covid-19 outbreak: wuhan's experience safety and efficacy of n-acetyl-cysteine for prophylaxis of ventilatorassociated pneumonia: a randomized, double blind, placebo-controlled clinical trial acetaminophen-induced hepatotoxicity: a comprehensive update endotracheal nebulization of n-acetylcysteine increases airway resistance in cats with experimental asthma evidence for the changing regimens of acetylcysteine reduction of adverse effects from intravenous acetylcysteine treatment for paracetamol poisoning: a randomised controlled trial new covid-19 hope clinical trial recommendations introduced today may reduce or eliminate mechanical ventilation for coronavirus patients | biospace heather zwickey, phd hzwickey@nunm.edu james dumbauld, do pdumbauld@live.com bracey dangerfield, phd bdangerfield@nunm.edu key: cord-299911-v95pf3eg authors: el-ghiaty, mahmoud a.; shoieb, sherif m.; el-kadi, ayman o.s. title: cytochrome p450-mediated drug interactions in covid-19 patients: current findings and possible mechanisms date: 2020-06-26 journal: med hypotheses doi: 10.1016/j.mehy.2020.110033 sha: doc_id: 299911 cord_uid: v95pf3eg at the end of 2019, the entire world has witnessed the birth of a new member of coronavirus family in wuhan, china. ever since, the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has swiftly invaded every corner on the planet. by the end of april 2020, almost 3.5 million cases have been reported worldwide, with a death toll of about 250000 deaths. it is currently well-recognized that patient’s immune response plays a pivotal role in the pathogenesis of coronavirus disease 2019 (covid-19). this inflammatory element was evidenced by its elevated mediators that, in severe cases, reach their peak in a cytokine storm. together with the reported markers of liver injury, such hyperinflammatory state may trigger significant derangements in hepatic cytochrome p450 metabolic machinery, and subsequent modulation of drug clearance that may result in unexpected therapeutic/toxic response. we hypothesize that covid-19 patients are potentially vulnerable to a significant disease-drug interaction, and therefore, suitable dosing guidelines with therapeutic drug monitoring should be implemented to assure optimal clinical outcomes. coronavirus disease 2019 (covid-19) is an infectious disease that was first reported as pneumonia of ambiguous etiology in a cluster of patients in the chinese city of wuhan by the end of december 2019 [1] . the causative organism was identified several days later as a novel coronavirus (2019-ncov). later, its name was changed to severe acute respiratory syndrome coronavirus 2 (sars-cov-2) as it was found to be genetically related to the coronavirus responsible for the sars outbreak of 2003 [2] . the infection broke out of china to spread to every continent around the world and was declared as an emerging pandemic by who in march 2020 [3] . full-length genome sequences have revealed that sars-cov-2 is 96% identical to a bat coronavirus, thus providing a clue about its original reservoir host [4] . human-to-human transmission of sars-cov-2 is evident and, as a respiratory infectious disease, covid-19 primarily spreads with close contact through respiratory droplets and secretions. controlling the disease is based mainly on directing the public towards reducing the transmission [5] . the disease is contagious, but people are reacting differently upon exposure to the virus. the virus may get eliminated by immune system and the infection can pass unnoticed. however, after an asymptomatic incubation period of up to 14 days, infected persons may develop mild flu-like symptoms, including fever, dry cough, fatigue, and shortness of breath [6] . other reported manifestations include upper respiratory symptoms as sneezing, runny nose and sore throat, in addition to gastrointestinal symptoms as nausea, vomiting and diarrhea [7] . anosmia or ageusia have been also reported as characteristic signs of infection [8] . while most cases develop mild symptoms, some may progress rapidly to a more sever stage that necessitates admission to an intensive care unit and probably mechanical ventilation [9] . the complications of this critical stage include severe 4 pneumonia, acute respiratory distress syndrome (ards), respiratory failure, multiple organ failure, and ultimately death [10] . in the absence of a proven therapy for covid-19, scientists are currently endeavoring to find an effective drug capable of eradicating this infection. several agents are currently under extensive laboratory and clinical investigations. some of these agents are investigational new drugs while the others are repurposed drugs which are already approved for other ailments [11] . when it comes to medications, pharmacokinetics should be strongly considered especially when dealing with such critical illnesses. drug metabolism is a highly important aspect of its pharmacokinetics that may significantly influence its clearance and, eventually, its efficacy and/or toxicity. cytochromes p450 (cyps) are a superfamily of heme-containing monooxygenase enzymes that have been identified in all kingdoms of life [12] . they represent the major enzyme family involved in the oxidative biotransformation of most drugs and other lipophilic xenobiotics [13] . in mammals, these enzymes are found primarily in microsomes of the liver, the dominant metabolizing organ, in addition to other extrahepatic tissues [14] . in humans, there are 57 functional members in cyps families, most of which have specific endogenous functions including the metabolism of arachidonic acid, cholesterol, bile-acids, steroid hormones, vitamin d, and others [15] . the biotransformation of the majority of hepatically cleared drugs and other foreign chemicals involves members belonging to the cyp1, cyp2, and cyp3 families. pathways involving cyp3a4/5, cyp2c9, cyp2c19 and cyp2d6 are the most common, and responsible for about 80% of the phase i oxidation system reactions [16, 17] . through their profound contribution to xenobiotic biotransformation, cyps can significantly modulate the overall body exposure to a drug. the metabolic activity of the cyps may result in decreasing efficacy and/or toxicity of a drug by enhancing the 5 clearance of its active form. for another drug, such metabolic activity may lead to increased efficacy or toxicity by activating its inert prodrug or generating toxic metabolites, respectively. understanding cyps activity in relation to the target drug is crucial in predicting its behavior inside the body and the consequences of its exposure [18] . we hypothesize a pharmacokinetic disease-drug interaction in which hepatic cyps metabolizing capacity, and eventually drug response, is altered in covid-19 patients. based on the conclusions drawn from the currently rapidly evolving knowledge about covid-19, our hypothesis is built on the potential modulation of cyps activity by the inflammatory environment provoked by sars-cov-2 infection, as well as the pathologic involvement of the liver which harbors the majority of the drug metabolizing enzymes (dmes). patient characteristics are also believed to increase the likelihood of the incidence of such interaction. systemic inflammation and immune response represent a substantial element in many acute and chronic diseases which is strongly implicated in altering drug pharmacokinetics through, mainly, modulating the expression and activity of dmes. as a main contributor to the metabolic biotransformation of most drugs, cyps are widely involved in such disease-drug interactions [19] . regulation of cyps has been linked to inflammation in several disease states such as infectious diseases (including viral infections), cancer, type 1 diabetes, rheumatoid arthritis, and inflammatory bowel 6 disease, in addition to age-related disorders such as normal aging, metabolic disorders, and neurodegenerative diseases [20] . alterations in hepatic cyps expression and activity are caused by the mediators produced during the inflammation process which are mainly cytokines [21] . cytokines are a broad category of small cell signaling proteins responsible for keeping homeostasis of the immune system and its components. the immune response is driven by a complex interplay between pro-and anti-inflammatory signals mediated by different cytokines [22] . it has been reported that different cyps are regulated differently, that is, multiple cytokines may participate in regulating single enzymes, while a subset of enzymes can be regulated by individual cytokines. this fact about specificity of regulation is highly important when considering drug interactions because drug pharmacokinetics will ultimately vary depending on disease type and its released cytokines, as well as the administered drug and its involved metabolizing enzymes [23, 24] . since the 1990s, cytokine-induced changes in hepatic cyps activity have been described using hepatocyte cultures from different mammals including rats [25], rabbits [26], pigs [27] , and humans [28, 29] . a strong evidence has been also provided through in vivo studies in mice [30] , rats [31] , and humans [32] . some studies have also demonstrated the suppression of extrahepatic cyps by inflammatory mediators [33] [34] [35] [36] . interleukin 1 (il-1) [37] [38] [39] , interleukin 6 (il-6), tumor necrosis factor-α (tnf-α), and interferon gamma (ifn-γ) [40] [41] [42] are the most prominent proinflammatory cytokines that have exhibited suppression of cyps expression and activity, in addition to other cytokines such as interleukin 2 (il-2) [43, 44] and interleukin 10 (il-10) [45] . theophylline is metabolized by several cyps isoforms (including cyp1a1, cyp1a2, and cyp3a) yielding different metabolites. serum treatment resulted in reducing the formation of all theophylline metabolites as well as the amount of total cyps, indicating a significant down-regulation of these metabolizing enzymes [46] . in a later study using the same experimental model, the down-regulatory effect was mainly attributed to il-6, interleukin 1β (il-1β), and ifn-γ [42] . cyps down-regulation was also proven to be subsequent to the reduction of their activity. additionally, the inflammatory mediators in human serum provoked varying degrees of suppression to different cyps with the cyp3a being more vulnerable than cyp1a to such effect [47] . for decades, il-6 has been recognized as the major inflammatory element that provokes a significant repressive effect on the expression and activity of different cyps. human recombinant interleukin 6 (rhil-6) has shown concentration-dependent blocking of phenobarbital-mediated induction of cyp2b1/2 mrna and activity in rat hepatocytes [48] . in fischer 344 rats, rhil-6 resulted in reducing different cyps activities with variable degrees [49] . the mrna levels of cyp1a1, cyp1a2, and cyp3a3 were markedly suppressed in three human hepatoma cell lines (hepg2, hepg2f, and hep3f3) because of rhil-6 treatment [50] . acute-phase inflammatory reaction provoked by turpentine or purified bacterial lipopolysaccharide (lps) administration to male rats resulted in significant suppression of hepatic cyp2c11 [51] , an effect that was later confirmed to be transcriptional, involving cyp2c11 promoter sequences, using rhil-6 treatment [52, 53] . il-6-knockout (il-6 -/-) mouse was adopted in several studies as a model to assess the extent of il-6 contribution to the suppression of different cyp isoenzymes during inflammatory response generated by various stimuli. in turpentine-induced inflammation, the inhibitory effect observed in wild-type (wt) mice on cyp1a2, cyp2a5, and cyp3a11 mrnas was revoked in il-6-deficient mice [54] . induction of immune response by bacillus calmette-guérin (bcg) has led to down-regulation of transcriptional expression of both cyp3a11 and cyp2c29, but only cyp3a11 in il-1α/β-knockout mice and tnf-α-knockout mice, respectively. however, tuberculosis vaccine had non-significant effect on these enzymes in il-6-knockout mice [55] . in a disease model, citrobacter rodentium infection was used to elicit inflammatory response in il-6 -/mice, where cyp3a11 mrna suppression seen in wt mice was abolished [56] . the mentioned studies have shown that the role of il-6 is usually critical and can't be played by another cytokine or mediator, however, sometimes il-6 -/mice may react similarly as wt mice, i.e. show down-regulation too, for certain cyps in certain inflammation models. this can be explained by the functional redundancy existing among the released cytokines which, sometimes, may replace il-6 [54] [55] [56] . interestingly, sometimes the suppression of cyps may happen only in il-6 -/mice, but not wt mice, thus indicating that il-6 may have an opposing signal with inductive effect [56, 57] . several studies have evaluated the impact of il-6 triggered by malignancies. in an interesting study by charles et al, the hepatic levels of both human cyp3a4 as well as its murine orthologue cyp3a11 were simultaneously assessed in a tumor-derived inflammation model using transgenic mice loaded with genetic constructs containing the upstream regulatory elements of the human cyp3a4 gene linked to the lacz reporter gene. the tumor generated a systemic inflammatory response with high levels 9 of circulating il-6 resulting in down-regulation of cyp3a11 orthologue at mrna, protein, and activity levels as well as cyp3a4 transgene product which was assessed through measuring β-galactosidase enzyme activity [58, 59] . different tumor types have also demonstrated a significant suppressive effect on hepatic cyp3a11 with an accompanying increase in il-6, and such effect was progressively promoted by tumor growth [60] . the pivotal role of il-6 in cancer-mediated repression of hepatic cyp3a has been further demonstrated by attenuation of such effect via anti-il-6 monoclonal antibody treatment [61] , or interleukin-6 receptor blocking [62] . anti-il-6 antibody intervention was also tested in il-6-treated primary human hepatocytes. the inhibitory effect of il-6 affected cyp1a1, cyp1a2, cyp2b6, and cyp3a4; and it was also capable of overriding cyp1a2 and cyp3a4 induction mediated by omeprazole and rifampicin, respectively. anti-il-6 antibody partially abrogated enzyme activity suppression [63] . the basis of cytokine-induced down-regulation of cyps activity is not fully elucidated; however, the associated decrease in their respective mrnas strongly suggests a transcriptional mechanism involving a number of transcriptional factors [64, 65] . nuclear factor kappa b (nf-κb), a pivotal regulatory transcription factor in the inflammatory and immune response, has been shown to regulate gene expression of many hepatic cyp enzymes in humans, rats, and mice [66, 67] . the aryl hydrocarbon receptor (ahr) is a gene battery that regulates a group of dmes including cyp1a1, cyp1a2, and cyp1b1. both nf-κb and ahr have a mutual inhibitory effect thus repressing each other's functions [68, 69] . for instance, pyrrolidine dithiocarbamate (pdtc), an inhibitor of nf-κb signal, has demonstrated partial blocking of the inflammatory reduction in cyp1a2 activity [70, 71] . the reported inhibitory effect of nf-κb activators on the pregnane x receptor (pxr) and its target genes (most notably of which is cyp3a4) on one hand, and enhanced pxr activity by inhibiting nf-κb on the other hand, indicates how inflammatory stimuli can manipulate hepatic cyps expression [72] [73] [74] . activation of nf-κb results in repressing glucocorticoid receptor (gr), thus down-regulating constitutive androstane receptor (car) expression and its associated genes such as cyp2b, cyp2c, and cyp3a [75] . other studies have shown that nf-κb can interfere with cyps expression, such as cyp2c11 and cyp2e1, by binding directly to their respective genes [38, 76] . 1.2. immunopathological aspects of covid-19: the immune system plays a crucial role in resolving covid-19 infection, but it also can go out of control and contribute to its progression. after invading the alveolar cells, the host body starts mounting an immune response during the incubation period and the mild stage to exterminate the virus and hamper disease progression to the severe stage. however, if body defenses are impaired, the suboptimal antiviral immune reaction will allow viral proliferation resulting in lung damage and dissemination to other angiotensin-converting enzyme 2 (ace2)-expressing cells such as enterocytes [77] [78] [79] . the initial containment of the infection by innate immune response generates a strong local inflammatory reaction with a myriad of inflammatory cytokines. upon failure of this attempt, the highly specific adaptive immune cells are summoned, by systemically disseminated cytokines, to augment the immune response and clear the increasing viral load [80] . if coordinated recruitment of innate and adaptive immunity fails to effectively control the pathogen, more immune cells will get activated, with subsequent release of more cytokines, in a positive feedback loop of pathogenic inflammation. this state of systemic inflammation is called cytokine storm syndrome (css) and it has been reported in severe cases as an important cause of ards and multiple organ failure [81] . at this point, the immune system is doing more harm than good to the body and 11 becoming a major cause of lung damage and subsequent mortality, thus suppressing such hyperinflammation is strongly recommended together with symptomatic treatment [82, 83] . it is noteworthy that the immune system starts its response upon viral exposure and continues to reinforce it till, in most cases, it effectively diminishes viral burden. premature halting of immune response may delay virus clearance and perpetuate the infection, therefore the use of immunosuppressants should be the last resort and only for severe cases with profound inflammatory lung injury [84] . it has been reported that a wide range of proinflammatory cytokines are elevated in peripheral blood of covid-19 patients such as interferon gamma (ifn-γ), tumor necrosis factor-α (tnf-α), granulocyte-macrophage colony stimulating factor (gm-csf), macrophage colony-stimulating factor (mcsf), and interleukins il-1, il-6, and il-12; and many more [7, 85] . interfering with these inflammatory mediators can modulate the amplified immune response that ends up with lung damage. interleukin-6 is one of the important targets for anti-cytokine therapy as it is believed to be a key player in the process and its elevation is associated with poor prognosis [86] [87] [88] . tocilizumab is a monoclonal antibody against the interleukin-6 receptor (il-6r) approved for the treatment of rheumatoid arthritis. it has been reported that a single dose of tocilizumab significantly improved the clinical outcomes in covid-19 patients [89, 90] . sarilumab is another il-6r antagonist which is also approved for treating rheumatoid arthritis and is currently assessed for its efficacy against covid-19 [11]. different organs, such as lung, kidney, and intestine, contribute to drug biotransformation in the body; however, the liver is generally regarded as the major metabolizing organ responsible for drug clearance [91] . through several enzyme systems, the most prominent of which is cyps, the liver can drive numerous metabolic reactions. acute as well as chronic insults to the liver will eventually hamper its metabolic machinery resulting ultimately in inefficient drug clearance [92, 93] . regardless of the cause, it is believed that the severity of liver disease is correlated to the extent of metabolism alteration [94, 95] . lungs represent the hot spot for the pathogenesis of covid-19, however, potential involvement of other organs, such as the liver, has been also reported [96, 97] . the hepatic involvement can be tied to the incidence of other extrapulmonary, or more specifically gastrointestinal, manifestations [98, 99] , and it has been reported in covid-19 patient with or without underlying liver disease [100] . reported findings of hepatic dysfunction in covid-19 cases include elevated aminotransferases [101] and bilirubin [102] , hypoalbuminemia [6] , in addition to microvesicular steatosis with mild lobular activity [77] . it is worth mentioning that hepatic impairment has been also associated with severe acute respiratory syndrome coronavirus (sars-cov) [103] , which is highly homologous to sars-cov-2 [104] , as well as middle east respiratory syndrome coronavirus (mers-cov) [105] . the exact mechanism behind liver function deterioration is not fully understood, however, it has been proposed that sars-cov-2 causes direct liver injury. viral hepato-tropism can be attributed to the relatively high expression of its cellular entry gate, i.e. ace2, in cholangiocytes, rather than hepatocytes, rendering them vulnerable to viral attack [106, 107] . this assumption is supported by the increase in circulating levels of serum gamma-glutamyl transferase (ggt) in covid-19 patients [108] . hepatic abnormalities can be also a secondary effect induced after initiating disease management by consumption of hepatotoxic antipyretic agents (e.g. acetaminophen), antiviral medications (e.g. oseltamivir and lopinavir), antibiotics, and steroids [109, 13 110] . despite the absence of viral antigens in the liver, the overwhelming systemic inflammatory reaction elicited by sars-cov-2 can be also a major cause of multiple organ dysfunction including liver damage as seen in other respiratory viral infections [111] [112] [113] . 3. drug outcome alteration in a vulnerable patient population: cyps have been implicated in mediating clinically relevant drug interactions in several disease states [114] . such metabolic interactions have been regarded as a major reason behind revising and updating safety profiles of pharmaceutical products [115] . diseasedrug interactions are commonly observed in inflammatory conditions with drugs whose clearance is predominantly cyps-mediated [116] . examples of such conditions include rheumatoid arthritis [117] , hepatitis [118] , crohn's disease [119] , acquired immunodeficiency syndrome (aids) [120] , influenza [121] , congestive heart failure [122] , and cancer [123] . as a central inflammatory regulator, agents interfering with il-6 actions have led to regaining the inhibited cyps activity, thus normalizing drug disposition pattern. blocking il-6 receptor by the monoclonal antibodies; tocilizumab [124] and sarilumab [125] in rheumatoid arthritis patients co-administering simvastatin has reversed cyp3a4 activity suppression as demonstrated by a clinically significant decrease in simvastatin exposure. halting il-6 signaling by direct binding with the monoclonal antibody sirukumab has also proven effective in rheumatoid arthritis patients who received a cyps probe cocktail consisting of midazolam (cyp3a), omeprazole (cyp2c19), and warfarin (cyp2c9). area under the plasma concentration-time curve (auc) for probe substrates was reduced after sirukumab administration indicating that the activity of their respective metabolizing enzymes was recovered [126] . as an 14 attempt to predict the risk of such interactions, physiologically based pharmacokinetic (pbpk) models were developed to assess the impact of anti-il-6 agents on the metabolism of concomitant medications [127] [128] [129] . the immune response, with il-6 in the core of its network of mediators, is a hallmark of covid-19 pathogenesis. based on the aforementioned examples, it is strongly postulated that cyps metabolic activity will be inevitably altered, mostly downregulated, during the course of sars-cov-2 infection in a similar manner, resulting in a clearance-related pharmacokinetic interaction with the administered drugs. additionally, liver involvement in covid-19 may further complicate the picture. in may 2020, the investigational antiviral agent, remdesivir, has received u.s. food and drug administration (fda) emergency use authorization for the treatment of covid-19. according to its manufacturer, remdesivir undergoes extensive metabolism by cyps especially cyp3a4 [130] . moreover, other potential candidates for treating covid-19 such as chloroquine [131] and colchicine [132] are also hepatically metabolized, so understanding the nature of such interaction is highly essential as it can influence the therapeutic/toxic response of patients to the agents intended for managing the disease [133, 134] . full attention should be paid when anti-cytokine therapy comes into play. in this case, partial or complete regain of normal metabolic status can be achieved as a result of the immunomodulatory effect. in case of co-administration of multiple drugs, the risk of drug interactions increases, however, a more complex disease-drug-drug interaction is expected in covid-19. sars-cov-2 infection has resulted in high rates of hospitalization and intensive care unit (icu) admission [135, 136] . this can be regarded as a major clinical concern especially when considering that critically ill patients are more predisposed to drug interactions, and that cyps are involved in the metabolism of commonly prescribed 15 drugs in the icus [137] . moreover, demographic analysis of covid-19 patients has revealed that older people with comorbidities are the most affected group [98, 138] who also represent the potential candidates for advancing to severe stages and inevitable icu admission [85] . medical comorbidities in elderly patients warrant concomitant drug therapies, thus rendering them the most vulnerable group for both drug-drug and disease-drug interactions [139, 140] . therefore, the utmost level of caution is required to deliver the optimal dose for these patients. clinical characteristics of coronavirus disease 2019 in china sars-cov-2 is an appropriate name for the new coronavirus who declares covid-19 a pandemic a pneumonia outbreak associated with a new coronavirus of probable bat origin down-regulation of multiple cytochrome p450 gene products by inflammatory mediators in vivo. independence from the hypothalamo-pituitary axis plasma levels of tnf-alpha and il-6 are inversely related to cytochrome p450-dependent drug metabolism in patients with congestive heart failure modulation of cytochrome p450 by inflammation in astrocytes cytokines influence mrna expression of cytochrome p450 3a4 and mdri in intestinal cells role of cytokines in the lipopolysaccharide-evoked depression of cytochrome p450 in the brain and liver the impact of cytokines on the expression of drug transporters, cytochrome p450 enzymes and chemokine receptors in human pbmc repression of cytochrome p450 by cytokines: il-1 beta counteracts clofibric acid induction of cyp4a in cultured fetal rat hepatocytes suppression of cyp2c11 gene transcription by interleukin-1 mediated by nf-kappab binding at the transcription start site effects of interleukin 1beta (il-1beta) and il-1beta/interleukin 6 (il-6) combinations on drug metabolizing enzymes in human hepatocyte culture pretranslational down-regulation of cytochromes p450 2c11 and 3a2 in male rat liver by tumor necrosis factor alpha nitric oxidemediated inhibition of cytochrome p450 by interferon-gamma in human hepatocytes inactivation by serum from humans with a viral infection and serum from rabbits with a turpentine-induced inflammation: the role of cytokines the interleukin-2 receptor downregulates the expression of cytochrome p450 in cultured rat hepatocytes decrease in hepatic cytochrome p450 after interleukin-2 immunotherapy in vivo effects of interleukin-10 on human cytochrome p450 activity down-regulation of the hepatic cytochrome p450 by an acute inflammatory reaction: implication of mediators in human and animal serum and in the liver cytochrome p450 down-regulation by serum from humans with a viral infection and from rabbits with an inflammatory reaction effect of interleukin 6 on phenobarbital induction of cytochrome p-450iib in cultured rat hepatocytes chauvelot-moachon l. effects of interleukin-6 on cytochrome p450-dependent mixed-function oxidases in the rat interleukin-6 down regulates the expression of transcripts encoding cytochrome p450 ia1, ia2 and iiia3 in human hepatoma cells suppression of constitutive cytochrome p-450 gene expression in livers of rats undergoing an acute phase response to endotoxin selective suppression of cytochrome p-450 gene expression by interleukins 1 and 6 in rat liver suppression of the constitutive expression of cytochrome p-450 2c11 by cytokines and interferons in primary cultures of rat hepatocytes: comparison with induction of acute-phase genes and demonstration that cyp2c11 promoter sequences are involved in the suppressive response to interleukins 1 and 6 hepatic cytochrome p450 down-regulation during aseptic inflammation in the mouse is interleukin 6 dependent involvement of interleukin-6 and tumor necrosis factor alpha in cyp3a11 and 2c29 down-regulation by bacillus calmette-guerin and lipopolysaccharide in mouse liver modulation of hepatic cytochrome p450s by citrobacter rodentium infection in interleukin-6-and interferon-{gamma}-null mice cytochrome p450 and antioxidant activity in interleukin-6 knockout mice after induction of the acute-phase response transgenic mouse models of human cyp3a4 gene regulation transcriptional repression of hepatic cytochrome p450 3a4 gene in the presence of cancer downregulation of drug transport and metabolism in mice bearing extra-hepatic malignancies extrahepatic cancer represses hepatic drug metabolism via interleukin (il)-6 signalling effects of cytokines on cyp3a4 expression and reversal of the effects by anti-cytokine agents in the three-dimensionally cultured human hepatoma cell line flc-4 effects of interleukin-6 (il-6) and an anti-il-6 monoclonal antibody on drug-metabolizing enzymes in human hepatocyte culture gene transcription in hepatocytes during the acute phase of a systemic inflammation: from transcription factors to target genes regulation of cytochrome p450 by inflammatory mediators: why and how? nf-kappab and the immune response role of nf-kappab in the regulation of cytochrome p450 enzymes mechanism of suppression of cytochrome p-450 1a1 expression by tumor necrosis factor-alpha and lipopolysaccharide ah receptor and nf-kappab interactions: mechanisms and physiological implications modulation of cyp1a2 and cyp3a6 catalytic activities by serum from rabbits with a turpentine-induced inflammatory reaction and interleukin 6 inhibition of nuclear factor-kappab signal by pyrrolidine dithiocarbamate alleviates lipopolysaccharide-induced acute lung injury mutual repression between steroid and xenobiotic receptor and nf-kappab signaling pathways links xenobiotic metabolism and inflammation role of nf-kappab in regulation of pxr-mediated gene expression: a mechanism for the suppression of cytochrome p-450 3a4 by proinflammatory agents pregnane x receptor is required for interleukin-6-mediated down-regulation of cytochrome p450 3a4 in human hepatocytes is nuclear factor kappa-b the missing link between inflammation, cancer and alteration in hepatic drug metabolism in patients with cancer? a novel lipopolysaccharide-modulated jun binding repressor in intron 2 of cyp2e1 pathological findings of covid-19 associated with acute respiratory distress syndrome genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding the digestive system is a potential route of 2019-ncov infection: a bioinformatics analysis based on single-cell transcriptomes covid-19 infection: the perspectives on immune responses covid-19: consider cytokine storm syndromes and immunosuppression induction of pro-inflammatory cytokines (il-1 and il-6) and lung inflammation by coronavirus-19 (covi-19 or sars-cov-2): anti-inflammatory strategies covid-19: combining antiviral and antiinflammatory treatments immunosuppression for hyperinflammation in covid-19: a double-edged sword? the origin, transmission and clinical therapies on coronavirus disease 2019 (covid-19) outbreak -an update on the status clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study the cytokine release syndrome (crs) of severe covid-19 and interleukin-6 receptor (il-6r) antagonist tocilizumab may be the key to reduce the mortality cytokine release syndrome in severe covid-19 effective treatment of severe covid-19 patients with tocilizumab tocilizumab treatment in severe covid-19 patients attenuates the inflammatory storm incited by monocyte centric immune interactions revealed by single-cell analysis drug-metabolizing activity of human and rat liver, lung, kidney and intestine slices the effect of liver cirrhosis on the regulation and expression of drug metabolizing enzymes cytochrome p450 and liver diseases selective effect of liver disease on the activities of specific metabolizing enzymes: investigation of cytochromes p450 2c19 and 2d6 liver disease selectively modulates cytochrome p450--mediated metabolism covid-19 and the liver: little cause for concern liver and kidney injuries in covid-19 and their effects on drug therapy; a letter to editor. archives of academic emergency medicine clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china first case of 2019 novel coronavirus in the united states coronavirus disease 2019 (covid-19) and prevalence of chronic liver disease: a meta-analysis. liver international evaluation of hepatic enzymes changes and association with prognosis in covid-19 patients bilirubin levels in patients with mild and severe covid-19: a pooled analysis sars-associated viral hepatitis caused by a novel coronavirus: report of three cases covid-19: gastrointestinal manifestations and potential fecal-oral transmission histopathology of middle east respiratory syndrome coronovirus (mers-cov) infection -clinicopathological and ultrastructural study exploring the mechanism of liver enzyme abnormalities in patients with novel coronavirus-infected pneumonia. zhonghua gan zang bing za zhi = zhonghua ganzangbing zazhi = chinese journal of hepatology specific ace2 expression in cholangiocytes may cause liver damage after 2019-ncov infection liver injury in covid-19: management and challenges covid-19 and liver dysfunction: current insights and emergent therapeutic strategies covid-19 and liver disease systemic viral infections and collateral damage in the liver kupffer cell-dependent hepatitis occurs during influenza infection liver involvement during influenza infection: perspective on the 2009 influenza pandemic complex drug-drug interactions involving cytochromes p450: systematic review of published case reports and clinical perspectives trends in new drug interactions for pharmaceutical products in japan infection and inflammation leading to clozapine toxicity and intensive care: a case series decreased dromotropic response to verapamil despite pronounced increased drug concentration in rheumatoid arthritis influence of chronic hepatitis c infection on cytochrome p450 3a4 activity using midazolam as an in vivo probe substrate drug-disease interaction: crohn's disease elevates verapamil plasma concentrations but reduces response to the drug proportional to disease activity variability in drug metabolizing enzyme activity in hiv-infected patients altered theophylline clearance during an influenza b outbreak the use of imipramine in depressed patients with congestive heart failure understanding disease-drug interactions in cancer patients: implications for dosing within the therapeutic window disease-drug-drug interaction involving tocilizumab and simvastatin in patients with rheumatoid arthritis disease-drug interaction of sarilumab and simvastatin in patients with rheumatoid arthritis evaluation of disease-mediated therapeutic protein-drug interactions between an anti-lnterleukin-6 monoclonal antibody (sirukumab) and cytochrome p450 activities in a phase i study in patients with rheumatoid arthritis using a cocktail approach a physiologically based pharmacokinetic modeling approach to predict disease-drug interactions: suppression of cyp3a by il-6 development of a physiologically based pharmacokinetic model to predict disease-mediated therapeutic protein-drug interactions: modulation of multiple cytochrome physiologically based pharmacokinetic model to assess the influence of blinatumomab-mediated cytokine elevations on cytochrome p450 enzyme activity summary on compassionate use of remdesivir gilead breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid-19 associated pneumonia in clinical studies the greek study in the effects of colchicine in covid-19 complications prevention (grecco-19 study): rationale and study design in vitro metabolism of chloroquine: identification of cyp2c8, cyp3a4, and cyp2d6 as the main isoforms catalyzing ndesethylchloroquine formation colchicine--update on mechanisms of action and therapeutic uses critical care utilization for the covid-19 early experience and forecast during an emergency response forecasting covid-19 impact on hospital bed-days, icu-days, ventilator-days and deaths by us state in the next 4 months drug-associated disease: cytochrome p450 interactions baseline characteristics and outcomes of 1591 patients infected with sars-cov-2 admitted to icus of the lombardy region clinical implications from drug-drug and drug-disease interactions in older people an update on the clinical consequences of polypharmacy in older adults: a narrative review key: cord-327809-9uhhqasl authors: dimitriu, mihai c.t.; pantea-stoian, anca; smaranda, alexandru c.; nica, anca a.; carap, alexandru c.; constantin, vlad d.; davitoiu, ana m.; cirstoveanu, catalin; bacalbasa, nicolae; bratu, ovidiu g.; jacota-alexe, florentina; badiu, cristinel d.; smarandache, catalin g.; socea, bogdan title: burnout syndrome in romanian medical residents in time of the covid-19 pandemic date: 2020-06-07 journal: med hypotheses doi: 10.1016/j.mehy.2020.109972 sha: doc_id: 327809 cord_uid: 9uhhqasl burnout is a state of physical or mental collapse caused by overwork or stress. burnout during residency training has gained significant attention secondary to concerns regarding job performance and patient care. the new covid-19 pandemic has raised public health problems around the world and required a reorganization of health services. in this context, burnout syndrome and physical exhaustion have become even more pronounced. resident doctors, and especially those in certain specialties, seem even more exposed due to the higher workload, prolonged exposure and first contact with patients. this article is a short review of the literature and a presentation of some considerations regarding the activity of the medical residents in a non-covid emergency hospital in romania, based on the responses obtained via a questionnaire. burnout prevalence is not equal in different specialties. we studied its impact and imagine the potential steps that can be taken in order to reduce the increasing rate of burnout syndrome in the pandemics. the burnout syndrome in health care professionals has gained significant attention over the last several years. given the intense emotional demands of the work environment, clinicians are particularly susceptible to developing this syndrome more than in other jobs. residency can cause a significant degree of burnout, leading to individuals' ability to establish correct communication with the patient, solve diagnostic dilemmas, and have a good overview of the correct treatment. burnout is associated with a variety of negative consequences including depression, risk of medical errors, and negative effects on patient safety. the goal of this review is to provide an overview of the prevalence in different specialties of the burnout syndrome, even more pronounced in covid context, provide team leaders with options to minimize the risks and prevalence and recognize its potential hazards towards the medical act and its medical caregiver [1] . although reported and discussed long before the pandemic, after its outbreak, burnout syndrome became even more pregnant [2] . the coronavirus epidemic broke out in wuhan, china, in a metropolis of 11 million people in december 2020 [3] . free movement of people led to the spread of the virus in europe (italy), the united kingdom and the united states in january-february 2020, becoming soon a pandemic that affected almost all countries [4] . resident physicians often represent the communication interface between the attending physician and the patient, so that the time spent with the patient is often longer. in the context of the pandemic, physical fatigue is added to the mental stress associated with a possible infection. resident physicians have the most prolonged contact with patients, including in the time window from the testing moment to the arrival of the result for sars-cov-2, therefore the stress is even more pronounced. some physical factors are added to the equation. prolonged wearing of personal protective equipment, excessive heat provided by them, lack of hydration, alimentation, sleep deprivation, all together accentuate fatigue and the burnout syndrome. from this point of view, the most affected doctors are from the emergency units, radiology, intensive care units, but also from the specialties that ensure surgical, obstetrical, orthopedic and neurosurgery emergencies. the modification of the shifts and of the work schedule, of the type of the current activity, the time and the technique of dressing and undressing the protective equipment, all constitute the novelty that adds an additional stress factor. last, but not the least, wearing facial protection equipment leads to a depersonalization of the activity, both in contact with patients and medical staff. the impossibility of reading facial expressions, the lack of interpersonal interactions and facial expressions decreases the possibility of socialization and mutual encouragement. in this sense, the idea related to wearing a photo of the holder on the protective suit appeared. there are studies that paradoxically indicate a higher level of stress in regular, non-covid wards of hospitals, compared to front-line ones [5] . a possible explanation would be that better organization and a sense of control reduce the stress level of those in the front line, while the fear of being exposed when the protocols do not seem perfect is higher in non-covid wards. here, however, there is a permanent threat of a positive patient, so no one seems to be safe. some studies in china and uk have also revealed depression and anxiety due to the covid-19 outbreak that medical staff experienced [6, 7, 8] . the pandemic has led to numerous cases of depression and anxiety, as well as worsening preexisting mental illnesses [9] . the term "burnout" was described by psychologist herbert freudenberger [10] in 1974 in an article entitled "staff burnout" in which he discussed job dissatisfaction precipitated by workrelated stress. a broadly applicable description defines burnout as a state of mental and physical exhaustion related to work or care-giving activities. a long-standing conceptual and operational definition characterized burnout as a triad of emotional exhaustion (emotional overextension and exhaustion), depersonalization (negative, callous, and detached responses to others), and reduced personal accomplishment (feelings of competence and achievement in one's work) [11] . the maslach burnout inventory (mbi) [12] is the most used questionnaire to measure burnout in research studies. the mbi human services survey is a self-administered, 22 item questionnaire that was developed to measure burnout in human services workers and is the "gold standard" of measuring burnout [13] . the mbi items are rated on a likert scale from 0 to 6 (0 = never, 1 = a few times per year, 2 = once a month, 3 = a few times per month, 4 = once a week, 5 = a few times per week, and 6 = every day) and score sample items such as: "i feel emotionally drained from my work.". it is designed to assess the 3 primary dimensions of burnout: emotional exhaustion, depersonalization, and personal accomplishment. burnout is detected using cutoff scores of high emotional exhaustion (≥27), high depersonalization (≥10), and low personal accomplishment (≤33), based on normative data from 1104 medical professionals [14] . a study in china, comparing burnout of front line workers with that of normal ward staff, in covid context, used maslach questionnaire for medical workers and the results were interesting and quite unexpected [3] . in 2004, martini et al [15] did a unique study that compared burnout rates among the different specialties using the mbi. the overall burnout rate was 50% and ranged from 27% to 75% among different specialties. this variation among specialties was not statistically significant; however, burnout rates were as follows: 75% in obstetrics-gynecology followed by 63% in internal medicine, 63% in neurology, 60% in ophthalmology, 50% in dermatology, 40% in general surgery, 40% in psychiatry, and 27% in family medicine. however, this variation among specialties was not statistically significant. being in one's first year in residency, mood fluctuation, dissatisfaction with clinical faculty, recent family stress, and being unmarried were all associated with increased likelihood to meet burnout criteria [15] . psychiatry residents were noted to have additional stressors including fear and exposure to patient violence and suicide [16] [17] [18] [19] [20] in a study by fahrenkopf et al [21] , no actual correlation was found between burnout and the number of medical errors seen in collected data. one plausible explanation may be that residents reporting symptoms of burnout may be more likely to over-report their errors. in this pandemics, there is a need for practical methods to assess medical stuff burnout. some researchers have even proposed the continuous wearing of sensors to quantify fatigue [22] . the aim of our study was to compare the frequency of burnout syndrome between medical residents considered to work on the front line (emergency unit, radiology and intensive care unit) and those working in normal hospital wards (surgery, obstetrics and gynecology, obstetrics). our hypothesis that we wanted to prove is that there is higher prevalence of burnout syndrome in regular, non-covid wards of hospitals, compared to front-line ones. the study was conducted in a hospital with an emergency clinical profile, which is not in the frontline in the fight against coronavirus, ensuring non-covid emergencies or suspects until the result of the real time polymerase chain reaction test is obtained. during the pandemic, the teaching processes in the hospital were stopped, both for medical students and residents, opting for online teaching. students' access to the hospital was stopped by a university decision to limit the spread of the infection. the medical residents continued to carry out medical activity, restricted to the activity in this only hospital where they have an employment contract. we distributed a survey of 30 questions to 100 medical residents, 50 for resident doctors in the emergency department, radiology and intensive care unit (considered first-contact with the 50 questionnaires for first-line medical resident were distributed as follows: 30 for residents in emergency unit, 10 for residents in intensive care unit and 10 for residents in radiology department (lot a). the other 50 questionnaires were distributed in that we considered normal wards. they were allocated for residents in general surgery (25 questionnaires), gynecology (15) and orthopedics (10) -lot b in our study. all the invited participants anonymously completed the survey and the responses were valid to be analyzed. demographics characteristics can be found in table 1. demographic characteristics showed that the two groups were relatively homogeneous in terms of distribution by age and gender. the extreme ages were 24 and 35 years, respectively, given that the target was represented by resident doctors. burnout was defined as a high level of emotional exhaustion (≥27), and/or high level of depersonalization (≥10), and/or low personal accomplishment (≤33). according to our results, we obtained an average burnout for medical residents of 76%, about two months after the outbreak of the pandemics in our country, which is superior to studies conducted in normal periods. the global prevalence of burnout syndrome among medical residents is high, proving that the threat posed by sars-cov-2 is a major stressor for medical staff. the results are all the more worrying as the target group was represented by resident doctors, of young age (maximum 35 years), who, at least theoretically, should have a better adaptability to the new condition represented by this pandemics, compared to senior doctors. the burnout was significantly more frequent in normal wards workers (lot b) (prevalence 86%) compared to medical residents working in places that we considered front-line departments: emergency unit, radiology, including ct/mri department and intensive care unit -lot a in our study, that showed a prevalence of burnout of only 66% (p<0.05, from chi-square statistic test) (table 2). we considered emergency unit, radiology, including ct/mri department and intensive care unit as front-line departments as all the patients, at presentation time, are now considered potentially infected ones till invalidation by a negative real time polymerase chain reaction test that usually takes 24 hours in our hospital. the prolonged time is due to the fact that this test is analyzed in an external laboratory. there can be an array of methods that can be used to fight against the burnout sydrome. in a sample of 200 professionals, maslach [23] showed that venting, laughing, and discussing care with colleagues decreased personal anxiety. in conditions of social distancing, even between colleagues, all these mechanisms are annulled. the place of direct socialization is partially replaced by social media groups and net socialization. there are no studies on suggested interventions for reducing the prevalence or how it works on each individual. each person is different genetically, racially, different sex and culture, different family environment and so on. with these many factors comes the problem of pinpointing the trigger factor for each individual. that is why with these many stress factors comes a multitude of anti-stress factors which can be used: from physical activity to meditation. mentoring programs in residency training can also be helpful in this regard [24, 25] . the negative impact of burnout on patient care includes risk of medical errors, patient safety risks, and potential compromise of quality of care. burnout and fatigue can affect the caution of medical staff, lead to negligence on self-protection measures and increase the risk of infection. negative consequences of burnout on physicians in training include depression, suicidal tendencies, and medical illnesses. the problems of medical errors related to fatigue and burn-out syndrome seem to be more serious for the surgical specialties [26] . effective interventions to address burnout should be developed at both the individual and institutional levels [1] . in a previous study conducted in our hospital, in non-pandemic conditions, the conclusion was that surgeons' fatigue seems to be a more subjective self-perception of surgeons than an objective fact and that surgeons tend to attribute their mistakes to burn-out syndrome, this being more acceptable for their conscience. in normal times, the rates of complications were not statistically higher on call-days and the days immediately after, when exhaustion should be maximum [27] . maslach [28] summarized effective working through burnout by stating: "if all of the knowledge and advice about how to beat burnout could be summed up in 1 word, that word would be balance-balance between giving and getting, balance between stress and calm, balance between work and home." pandemics of this magnitude have appeared in humanity about once every 100 years [29] . so you cannot talk about personal experience in managing a crisis like this. it is also difficult to assume that the competent centralized structures, such as governments, public health organizations, could fully manage the situation at individual level. in certain critical situations, it even turned out that local authorities, along with the population, had more competent organization and involvement (the example of hurricanes). in our opinion, the local organization at the hospital level is much more important for decreasing the stress level and the prevalence of the burnout syndrome. the existence of clear protocols for any possible situation, the practical trainings with the personnel regarding the protection measures, the adequate use of the protection equipment are all measures that ensure a state of confidence and control, which obviously decreases the stress level. this could explain the higher prevalence of burnout syndrome in staff in regular wards, compared to employees in the emergency department. medical residents in emergency unit had more training hours about the protective equipment and the wearing of the personal protection equipment was continuous, throughout the working time, that gave them the feeling of being safe, reducing the stress. all the successes of medical teams must be promoted by all means, being a source of positive emotions. the shift program must be organized in such a way as to respect the epidemiological timing (incubation period or quarantine time). periods of rest and relaxation are important and must be observed to prevent burnout, even if, often, they cannot take place in the privacy of families. the burnout-syndrome is a real phenomenon and may manifest in many forms. each resident and other higher level caregiver is susceptible to it. being aware of this, the new physician generations are shown to have an attraction towards balancing activities. all in all medical leaders and mentors should be aware of their colleagues and residents, thus allowing themselves to partake in the well-being of the team and making the work environment less stressful. the research in individual stress-factors and its many ways in which to actively fight them it is a gateway to making a whole medical environment better by concentrating upon the individual and giving a successful education to the next generation of physicians. in the context of covid-19, the best way to combat burnout seems to be, in our opinion, the precise local organization within the hospital and practical training sessions on the use of personal protective equipment, source of a mental comfort feeling. mean age (standard deviation) 27 low personal accomplishment 13 11 total (burnout frequency) 33 (66%) 43 (86%) p=0.019208 table 2 . characteristics of burnout syndrome elements of the two lots burnout during residency training: a literature review coronavirus disease 2019 (covid-19) and beyond: micropractices for burnout prevention and emotional wellness emergence of a novel coronavirus causing respiratory illness from wuhan the outbreak of coronavirus disease 2019 (covid-19)-an emerging global health threat a comparison of burnout frequency among oncology physicians and nurses working on the frontline and usual wards during the covid-19 mental health care for medical staff in china during the covid-19 outbreak occupational risks for covid-19 infection covid-19: give nhs staff rest spaces and free parking not thank yous, says doctor factors associated with mental health outcomes among health care workers exposed to coronavirus disease staff burnout burnout: a multidimensional perspective professional burnout: recent developments in theory and research maslach burnout inventory manual stress in health professionals: psychological and organizational causes and interventions evaluating stress: a book of resources burnout comparison among residents in different medical specialties general psychiatry in no-man's land hidden ethical dilemmas in psychiatric residency training: the psychiatry resident as a dual agent observations on burnout in family medicine and psychiatry residents burnout among dutch medical residents burnout and internal medicine resident work-hour restrictions rates of medication errors among depressed and burnt out residents: prospective cohort study continuous monitoring and detection of post-traumatic stress disorder (ptsd) triggers among veterans: a supervised machine learning approach burned out an exploratory study of resident burnout and wellness mentoring matters: mentoring and career preparation in internal medicine residency training burnout and medical errors among american surgeons surgical malpractice in relation to long calls burnout: the cost of caring the mother of all pandemics is 100 years old (and going strong)! maria sklodowska curie carol davila sos. pantelimon, 021659, bogdan.socea@umfcd.ro, +40788491091, kindly ask you to take in consideration our submission for your journal, having the title all authors have been read and approved the final manuscript. the study was performed according to the world medical association declaration of helsinki and according to national legislation, using a protocol approved by the local bioethics committee. all subjects have previously signed an informed written consent about future publication of data all authors have significant scientific contribution to the manuscript. all authors have been read and approved the final manuscript. the study had no funding. there are no conflicts of interest. key: cord-337627-1a2gpqdl authors: alvarado-vasquez, noé title: could a family history of type 2 diabetes be a risk factor to the endothelial damage in the patient with covid-19? date: 2020-11-06 journal: med hypotheses doi: 10.1016/j.mehy.2020.110378 sha: doc_id: 337627 cord_uid: 1a2gpqdl in december 2019, in china, a disease derived from a new beta coronavirus (sars-cov-2) was reported, which was termed coronavirus disease 2019 (covid-19). currently, it is known that endothelial cell dysfunction is a critical event in the infection by this virus. however, in a representative percentage of patients with covid-19, neither cardiovascular disease nor diabetes mellitus, which could be linked with endothelial dysfunction, has been reported. previous evidence has shown the presence of early endothelial dysfunction in healthy subjects but with a family history of type 2 diabetes (fh-dm2), where glucose metabolism, the synthesis of nitric oxide (no), reactive oxygen species (ros), as well as expression of genes involved with their synthesis are impaired. besides, in subjects with an fh-dm2, the presence of hyperinsulinemia and high glucose levels are common events that could favor the infection of endothelial cells by the coronavirus. interestingly, both events have been reported in patients with covid-19, in whom hyperinsulinemia increases the surface expression of ace2 through a diminution of adamts17 activity; whereas hyperglycemia induces higher expression of ace2 in different tissues, including microvascular endothelial cells from the pancreatic islets, favoring chronic hyperglycemia and affecting the release of insulin. therefore, we hypothesized that an fh-dm2 should be considered an important risk factor, since the individuals with this background develop an early endothelial dysfunction, which would increase the susceptibility and severity of infection and damage to the endothelium, in the patient infected with the sars-cov-2. in december of 2019, a disease associated with a new beta coronavirus (sarswas reported in china, and which was termed coronavirus disease 2019 (covid-19) [1] . at the same time, some reports showed that cardiovascular disease or hypertension were important risk factors for developing covid-19, especially in people above 65 years old [1] . recent works have also shown that patients with diabetes mellitus have a worse prognosis if they get infected by the sars-cov-2 [2] . type 2 diabetes mellitus (dm2) per se courses with high morbidity and mortality worldwide [3] , and, although, it affects especially low-and middle-income countries of latin america [4] , the rest of the world is not free of this disease. in europe, in 2019, about 59 million individuals were diagnosed with diabetes mellitus, a number that is anticipated to rise to about 68 million people in 2045 [5, 6] . this higher susceptibility of patients with diabetes to infection by different infectious agents (including the sars-cov-2) has been associated with the numerous alterations of innate immunity observed in these patients [7] . current reports show that the virus affects the lower respiratory tract, as well as the heart, kidney, gastrointestinal tract, and distal vasculature, using the angiotensin-converting enzyme 2 (ace2) as its receptor [8] . the ace2 is expressed higher in arterial and venous endothelial cells, as well as in smooth muscle cells of the heart; it is also present in kidneys, pancreas, adipose tissue, and lung epithelium [9] . nowadays, it is accepted that the endothelial cell is one important entryway used by this coronavirus due to its high expression of ace2 [9] . recently, in patients with covid-19, signals of inflammation and viral infection were observed, as well as evidence of endothelial cells death [10] . furthermore, severe endothelial damage associated with the intracellular presence of the virus has been described in the lungs of patients who died due to covid-19, as well as an increase in the growth of new vessels by intussusceptive angiogenesis [11] . the infection of endothelial cells by the sars-cov-2 induces their dysfunction [12] . the latter is of great relevance since the endothelium plays a central role in maintaining cell homeostasis, by regulating the synthesis of different molecules with an important biological activity [13] . for example, numerous evidences have shown the association between endothelial damage and pulmonary microvascular thrombosis, which is associated with a worse prognosis in patients with covid-19 [14] . likewise, the presence of sars-cov-2 induces the release of different pro-inflammatory molecules that favor the hypercoagulation state reported in the patient with covid-19 [8] . moreover, the presence of sars-cov-2 has negative effects in the synthesis of nitric oxide (no), and in the anticoagulant factors synthesized by the endothelial cell. in addition, an increase in the expression of the von willebrand factor (vwf), in leukocyte adhesion molecules (icam-1, p-selectin) has been reported, as well as in the synthesis of reactive oxygen species and of pro-inflammatory cytokines (il-1α, -β, -6, -10, tnf-α, tgf-β), by the endothelium of the covid-19 patient [14] [15] [16] [17] . however, and in spite of all the new knowledge generated, there are still doubts about all the elements that intervene in the infection and evolution of the disease. for example, in a representative percentage of patients with covid-19, pathologies such as cardiovascular disease or diabetes mellitus, which could be associated with an early endothelial dysfunction, have not been reported. huang et al. [18] showed that only 27.2% of patients with covid-19 had an underlying pathology, such as hypertension, diabetes mellitus, lung, liver, or cardiovascular diseases. results shown by li et al. [12] indicate that 33% of patients with covid-19 had hypertension, while 5% had a diagnosis of coronary heart disease. on the other hand, niu et al. [19] , after evaluating the clinical characteristics of elderly patients with covid-19, found that 15% had hypertension and 5% coronary heart disease, and only 3% of all patients had diabetes. on the other side, ebinger et al. [20] reported in their study that of a total of 442 patients diagnosed with covid-19, 16.1% had obesity and 36.4% hypertension, whereas 11.1% mentioned prior myocardial infarction, and 19% reported to have diabetes mellitus. in comparison, goel et al. [21] found that diabetes mellitus was present in 30.9% of patients in america, in 16% in europe, whereas it was present in 12.3% of asian patients with covid-19. also, the authors showed that obesity was present in 37.6, 29.6, and 17.9% of patients positive to the virus in america, europe, and asia, respectively. currently, it is known that the patient with covid-19 shows different severity levels and variations in the immune response, facts that are associated with the age or gender of the patient [20, 22, 23] . interestingly, and in spite of all still existing doubts, endothelial dysfunction is considered now a critical event for patients with covid-19 [10] [11] [12] . therefore, early endothelial dysfunction could be the reason why some apparently healthy (or asymptomatic) individuals are more susceptible to infection. based on the above, we hypothesized that a family history of type 2 diabetes (fh-dm2) could be a risk factor for the severe endothelial damage observed in the patient infected with sars-cov-2. this hypothesis is supported by evidences of early endothelial dysfunction in subjects with fh-dm2 or in asymptomatic patients with type 2 diabetes [24, 25] , which could explain the greater susceptibility of these patients to infection by this virus. in relation to this, mcsorley et al. [26] report a minor response to inducers of nitric oxide synthesis in young healthy adults with fh-dm2. likewise, fh-dm2 has been associated with an impaired vascular function in healthy offspring, as well as with the development of dm2 in adult life [27] . also, other authors have shown that endothelial cells from the umbilical cord from newborns of diabetic mothers had alterations in the cell membrane structure, a deficient glucose metabolism, and were less resistant to shear stress [28] . additionally, the endothelial cells from newborns with an fh-dm2 had a diminished synthesis of reactive oxygen species (ros), nitric oxide (no), as well as an impaired expression of genes (enos, glut1, and p53) associated with the synthesis and metabolism of no and glucose [29, 30] . in comparison, the infection by the sars-cov-2 induced endothelial dysfunction, which was associated with pulmonary microvascular thrombosis and with impaired no synthesis [12, 14, 16] . this has led to suggest the importance of increasing the synthesis and levels of no in the patient with covid-19, who has reduced levels of this mediator [31] . in young, healthy, insulin-sensitive women but with an fh-dm2, a reduced body metabolic rate and an impaired vascular endothelial function have been reported [32] . the above is similar to that observed in the covid-19 patient, where both an impaired glucose metabolism and apoptosis have been described in the endothelial cells. in their work, varga et al. [10] report the presence of apoptosis, viral elements within endothelial cells, and endotheliitis. in turn, ackermann et al. [11] observed severe endothelial damage, intracellular presence of the virus, disturbance in the cell membrane, and variation in the expression of genes linked to the inflammatory process and cell metabolism. these negative effects can be associated too with an impaired expression of molecules involved in the inflammatory response and with both glucose metabolism and no synthesis, as has been reported in the subjects with fh-dm2; paralleling the recent findings in covid-19 patients, in whom a total of 70 inflammation-related genes that influence cell metabolism has been reported [11, 29, 30, 31, 35] . furthermore, here it is relevant to comment that the mitochondrion, which is associated with the origin of dm2, apoptosis, glucose metabolism, and no synthesis in the endothelial cell, is affected in the patient with covid-19 too [33] ; similarly to the observations made in the endothelial cell from subjects with hf-dm2 [29] . it is also important to mention that several clinical features reported in patients infected with sars-cov-2 are similar to those observed in patients with metabolic syndrome or diabetes, such as hyperinsulinemia and hyperglycemia [34] . in patient [35, 37] . moreover, the high glucose observed in the covid-19 patient, consequence of impaired glucose metabolism, is associated too with the presence of "cytokines storm" [35, 38] . in comparison, hyperinsulinemia and hyperglycemia have also been found in subjects with fh-dm2 [27, 39] . due to the aforementioned, the early treatment of endothelial cells as a new target has been suggested, aimed at reducing the vascular damage caused by the virus in the patient with covid-19 [40] . therefore, an fh-dm2 should be considered a relevant factor for the endothelial damage in the patient infected with the coronavirus because healthy individuals, but with a family history of diabetes, develop an early endothelial dysfunction. nowadays, the presence of endothelial dysfunction is considered a critical event for the infection and severity of vascular damage in the patient infected with the sars-cov-2. the author reports having no relation that could be considered a conflict of interest. a close-up on covid-19 and cardiovascular diseases diabetes as a risk factor for greater covid-19 severity and in-hospital death: a meta-analysis of observational studies risk scores for type 2 diabetes mellitus in latin america: a systematic review of population-based studies the risk of mortality among people with type 2 diabetes in latin america: a systematic review and meta-analysis of population-based cohort studies the burden of type 2 diabetes in europe: current and future aspects of insulin treatment from patient and healthcare spending perspectives international diabetes federation. idf diabetes atlas the diabetes pandemic and associated infections: suggestions for clinical microbiology covid-19: towards understanding of pathogenesis the ace-2 in covid-19: foe or friend? endothelial cell infection and endotheliitis in covid-19 pulmonary vascular endothelialitis, thrombosis, and angiogenesis in covid-19 cardiovascular disease potentially contributes to the progression and poor prognosis of covid-19 overcoming barriers: the endothelium as a linchpin of coronavirus disease the contribution of diabetic micro-angiopathy to adverse outcomes in covid-19 the assessment of endothelial function: from research into clinical practice the vascular endothelium: the cornerstone of organ dysfunction in severe sars-cov-2 infection. version 2 an evolving new paradigm: endothelial cells--conditional innate immune cells clinical findings of patients with coronavirus disease 2019 in jiangsu province, china: a retrospective, multi-center study clinical characteristics of older patients infected with covid-19: a descriptive study pre-existing traits associated with covid-19 illness severity clinical characteristics and in-hospital mortality for covid-19 across the globe the war against the sars-cov2 infection: is it better to fight or mitigate it? endothelial dysfunction in coronavirus disease 2019 (covid-19): gender and age influences family history of diabetes is a major determinant of endothelial function endothelial dysfunction and cardiovascular outcome in asymptomatic patients with type 2 diabetes: a pilot study endothelial function, insulin action and cardiovascular risk factors in young healthy adult offspring of parents with type 2 diabetes: effect of vitamin e in a randomized double-blind, controlled clinical trial microvascular and macrovascular reactivity is reduced in subjects at risk for type 2 diabetes human endothelial cells are defective in diabetic vascular disease huvecs from newborns with a strong family history of diabetes show diminished ros synthesis in the presence of high glucose concentrations reduced no synthesis and enos mrna expression in endothelial cells from newborns with a strong family history of type 2 diabetes could the decrease in the endothelial nitric oxide (no) production and no bioavailability be the crucial cause of covid-19 related deaths? metabolic rate and vascular function are reduced in women with a family history of type 2 diabetes mellitus the role of genetic sex and mitochondria in response to covid-19 infection coronavirus and obesity: could insulin resistance mediate the severity of covid-19 infection? endocrine conditions and covid-19 covid-19 pandemic, coronaviruses, and diabetes mellitus factors leading to high morbidity and mortality of covid-19 in patients with type 2 diabetes clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study maternal and paternal histories differentially influence risks for diabetes, insulin secretion and insulin resistance in a chinese population systemic endothelial dysfunction: a common pathway for covid-19, cardiovascular and metabolic diseases key: cord-332365-20u06444 authors: raciti, loredana; calabrò, rocco salvatore title: can volcanic trace elements facilitate covid-19 diffusion? a hypothesis stemming from the mount etna area, sicily date: 2020-06-27 journal: med hypotheses doi: 10.1016/j.mehy.2020.110058 sha: doc_id: 332365 cord_uid: 20u06444 in december 2019, severe cases of pneumonia of unknown aetiology were reported in wuhan city, in china. lately, the pneumonia was related to the severe acute respiratory syndrome coronavirus-2 (sars-cov-2), and the diseases was termed coronavirus disease-2019 (covid-19). at the end of january 2020, the infection spread all over italy, but with high infection rates and mortality in the northern part, especially in lombardy, the most industrialized and polluted region of the country. it is noteworthy that a strong association between severe viral respiratory disease and air pollution has been described. air pollutant could be solid particles, liquid droplets, or gases and can be of natural origin (such as ash from a volcanic eruption) or released from motor vehicle depletes (carbon monoxide gas) or factories (sulfur dioxide). volcanic eruptions release large amounts of sulphuric acid, hydrogen sulfide, and hydrochloric acid into the atmosphere. pulmunary diseases spread by means of small droplets in the breath, also called aerosols, and air pollution may facilitate the outside survival of viruses. we suppose that ash and gases emitted from the mount etna contributed to air pollution, potentially favouring the major contagion of covid-19 in the eastern flank of the mountain, as in catania city. in fact, ash and gases (with regard to radon) are usually particularly intense in winter, with a reduction of emission of specific metals with warmer weather. this is the first paper that elaborates the hypothesis of a potential role of volcanic gases and heavy metals-related air pollution, combined to specific climatic conditions and regional topography, in favouring severe covid-19 diffusion in sicily. clinical and epidemiological studies are needed to support the hypothesis and plan the due prevention and awareness-raising campaigns. mount etna is the highest active volcano in europe, and it is located on the east coast of sicily, between the cities of messina and catania. the etna covers an area of 1,190 km 2 with a basal circumference of 140 km ( figure 1 ) [1] . during the uprising of the volcanic eruptive plume, soluble ash fraction and solid particles are emitted [2] . these latter include gases trapped in the volcanic rocks, dissolved or dissociated gases in magma and lava or ashes, or gases emanating directly from lava or indirectly through ground water heated by the volcanic action. volcanoes may discharge flows of ash and gas up to 50 kilometres away, with the generation of cloud columns 1-5 km high and blasting about 5 km-wide chunk off the tip of the volcano [3, 4] . in volcanic areas, the emissions and deposits of volcanogenic elements are key factors for geochemical mobility of trace elements (tes), and their distribution in the environment might impair animals and human health. among all the volcano elements, metals represent the main natural source [3, 4] . fine particulate matter with an aerodynamic diameter of 2.5m or less (pm2.5), 10m or less (pm10) are sulfur dioxide (so2), nitrogen dioxide (no2), carbon monoxide (co) and ozone (o3) that affect airways through inhalation and exacerbate the susceptibility to and severity of respiratory virus infections [5] [6] [7] . moreover, trace elements deposit in soil and plants representing a high risk of ingestion of metals by the population with serious toxic effects for public health [8] . when added to other environmental toxicants, such as herbicides, pesticides and industrial emissions, heavy metals are associated with an increased risk of neurodegenerative diseases [9] [10] [11] [12] . a recent study evaluating the level of tes in scalp hair of school-children living around the mount etna area, showed a high level of metals and nickels [12] . moreover, areas of different flank present different susceptibility to neurodegenerative diseases, being eastern to southern flank more exposed than western one [13] . heavy metals have been dosed in the groundwater of the etna (used for water plants or to drink), especially in the eastern and southern sectors of the volcano, and they are believed to contribute to intoxication of public health and to pulmonary or neurodegenerative diseases [12, 14, 15] . 4 moreover, food, especially, fishes are highly concentrate of several metals [16, 17] , and this is likely related to gases emitted by the etna mountain [18] [19] [20] , that blow from the eastern to the southern of the volcano, most of the time westerly to north-westerly trade winds, emitting about 16% of the global volcanic heavy metals, including radon [21] . radon (222rn) is a noble gas, invisible, odorless, tasteless, and short-lived decay product of uranium (238u), whose high activity concentration in radon emissions at the topographic surface is produced by convective flow of gases that facilitate the transport of radon from greater depth within soils (22) (23) . the collapsing flanks are bordered by numerous active faults [24] that cross urban areas, and are likely the locations of high soil degassing and elevated radon activities [25] . in the areas of strong degassing, particularly in the east and south-west flanks of the volcano, due to continuous tectonic deformations and gravitational collapses [26] [27] , radon activity was up to 60,000 bq/m3 [28] . in sicily radon has been highly related with lung cancer (29) (30) , and could favour pulmonary infections, as well as other elements do [31] . in december 2019, severe cases of pneumonia of unknown aetiology were reported in wuhan city, in china. lately, the pneumonia was related to the severe acute respiratory syndrome coronavirus-2 (sars-cov-2), previously named 2019 novel coronavirus (2019-ncov), and the diseases was termed coronavirus disease-2019 (covid-19) [32] [33] . at the end of january 2020, after the first case in codogno, the infection spread all over italy, but with high infection rates and mortality in the northern part, especially in lombardy, the most industrialized and polluted region of the country. it is noteworthy that a strong association between severe viral respiratory disease and air pollution has been described [34] . air pollutant could be solid particles, liquid droplets, or gases. a pollutant can be of natural origin or man-made and classified as primary or secondary, based on natural origin (such as ash from a volcanic eruption: primary) or released from motor vehicle depletes (carbon monoxide gas) or factories (sulfur dioxide) [35] [36] . 5 the inhalable fraction of particles in the air that can enter the nose or mouth depends on external wind speed and direction, as well as on the particle-size distribution by aerodynamic diameter [37] [38] [39] . two alternative size-selective criteria, often used in atmospheric monitoring, are pm10 and pm2.5. pm10 is defined by iso as "particles which pass through a size-selective inlet with a 50% efficiency cut-off at 10 μm aerodynamic diameter (defined as "thoracic convention"), whereas pm2.5 as "particles which pass through a size-selective inlet with a 50% efficiency cut-off at 2.5 μm aerodynamic diameter"; these latter correspond to the "high-risk respirable convention" [40] . then, particles with a diameter smaller than 10 μm can enter the bronchi, while the ones with an effective diameter smaller than 2.5 μm can enter as far as the gas exchange region in the lungs [41] , because of prolonged permanence in the air than heavier particles, bypassing the nose and throat. high concentrations of pm 2.5 particles characterize the air of hubei region in china, as well as the po valley (italy), revealing the possible correlation between the major distribution of covid-19 and the concentration of pollutants [42] . in fact, pollution insult decreases airway ciliary activity, and increases excessive mucus production, exposing to progressive and chronic inflammation of the respiratory airways with severe respiratory diseases after viral infections. several factors contribute to individual reactions to air pollutants: the type of pollutant a person is exposed to, the degree of exposure, and the individual's health status and genetics [43] . to this end, air pollution is a significant risk factor for viral respiratory infections, heart disease, chronic obstructive pulmonary disease, stroke and lung cancer [44] [45] . origin of pollutant could be volcanoes emissions. volcanic eruptions release large amounts of sulphuric acid, hydrogen sulfide, and hydrochloric acid into the atmosphere. these gases react with other atmospheric particles to form aerosols and eventually return to earth as acid rain, having a number of adverse effects on the environment and human life [37] . pulmunary diseases spread by means of small droplets in the breath, also called aerosols [38] , and air pollution may facilitate the outside survival of viruses. 6 on the other hand, heavy metals origin from geologic cycle (e.g. erosion, volcanic activity, windblown dust, etc.) and other industrial or artificial activities (industrialization, fuel combustion, roadway traffic, etc.). indeed, metals accumulate in aquatic and terrestrial systems with biomagnification in food chain, after being conveyed by air further contributing to air pollution. [47] . another active and very dangerous asian volcano is the taal, on the island of luzon, philippines. in december 2019, an alert level to four due to an explosive eruption was raised and a "total evacuation" order to population living within 17-kilometer around the volcano was done for the high risk of toxicity of volcanic ash (that could travel hundreds of kilometres an hour), toxic gases emitted from the eruption, and mud flows caused by ash mixing with water vapour in the atmosphere. a column of ash raised up as it erupts on january 2020 shower down on the south-west sector of the volcano [49] . then, volcanoes gases and heavy metals could contribute to air pollution of the china. as well as in china, we suppose that ash and gases emitted from the mount etna contributed to air pollution, potentially favouring the major contagion in the eastern flank of the mountain, as in catania. moreover, the reduction in number of contagion and virulence in the last month would be justified by the interaction of several factors, as social distances and lockdown, and environmental issues, including a warmer climatic season and concentration of gases emitted. in fact, ash and gases are usually particularly intense in winter, with a reduction of emission of specific metals with warmer weather [50] . in particular, radon concentration is generally lower in summer, when air temperature is higher. a negative correlation between radon concentration and air temperature has 7 been found with consequent lowering of indoor radon concentration. on the other hand, no seasonal cyclicity due to meteorological parameters was found. then, active faults (40-150m) and volcanic substrates could be an alternative explanation of radon and other pollution elements variation [50] . based on all this information, our hypothesis is that the volcano ash/gas together with climatic conditions may have promoted a longer persistence of the viral particles in the air, mleading to a higher prevalence of covid-19 in the eastern to southern part of sicily. air pollution has been suggested as cofactor of the spread of covid-19 in industrial cities, in an attempt to explain the major incidence of the virus infection in the north of italy, especially in the po valley. the main involved italian cities are, indeed, lodi, cremona and bergamo, defined as the "industrial triangle" which is characterized by a high density of factories, traffic and intensive agriculture with the highest pollution levels [51] . in sicily, the highest incidence of infection was in catania, followed by messina and palermo, although this latter city is the biggest sicilian one (see fig 3 and 3) . because of its altitude and geographical position of mount etna, the volcano displays a role in the exposure of the flanks to the dominant winds: most of the rainfall is on the eastern flank, as the volcano itself induces condensation of wet air masses coming from the ionian sea, where catania is [52] . gases blow from eastern to the southern due to the westerly to north-westerly trade winds [52] . in fact, quantitative of heavy metals is more representative in the ground waters in the eastern and southern sectors of the volcano. the presence of tes in the water is the results of degassing of magmatic volatiles, such as radon, by the known faults or faults that are not clearly visible at the surface (26, 53) . due to direction of gas emissions, we should expect (how it is actually) a lower incidence of infectious in the western flank of the volcano (palermo, trapani, etc) than the eastern or southern (messina and catania), as demonstrated by prevalence data in figure 2 and 3. 8 on the other hand, the reduction of specific and dangerous gases emitted with warmer weather, reveals a potential correlation between the distribution of severe covid-19 in sicily and the metals diffusion resulting from a combination of volcano emissions, locally climatic conditions, population genetic predispositions and regional topography. this is the first paper that elaborates the hypothesis of a potential role of volcanic gases and heavy metals-related air pollution, combined to specific climatic conditions and regional topography, in favouring severe covid-19 diffusion in sicily. the idea stems from the fact that tes likely lead to a major susceptibility of respiratory system to infection. based on the frontera hypothesis, heavy metal air pollutants combined to climatic conditions prolonged the permanence of the virus in the air, as well as the susceptibility to pulmonary virus infection. in biancavilla, town located in in eastern sicily, a relation between a higher risk of mesothelioma, as well as chronic obstructive pulmonary disease, and asbestiform fiber used in the local building industry (fluoro-edenite) was found. then, preventive manoeuvres were done, such as covering with asphalt of roads previously paved with local soil materials, and removal of sources of dust in the urban area [54] [55] . moreover, a recent environmental survey showed the presence of both c. neoformans and c. gattii species complexes in the environment. in particular, these species were previously recovered from messina (northeast sicily) in several samples of bird excreta, as well as in eucalyptus camaldulensis, prunus dulcis (almond), and ceratonia siliqua (carob) [56] [57] [58] . in 2019, trovato et al found that c. neoformans and c. gattii species colonize olive trees, samples from 124 olive trees collected from 14 different sites in eastern sicily around mount etna, as well as carob trees [59, 60] . assays showed that volcanic soil is a suitable substrate for the growth of c. neoformans and c. gattii species for the characteristics of the soil rich in iron and copper, potassium, phosphorus and magnesium, but poor of nitrogen and calcium [61] . therefore, the blastospores and basidiospores producted by cryptococcal yeasts represent a potential source of infection since soil aerosols could transfer small cells and spores (and potentially virus particles) in pulmonary alveoli of humans and animals causing the onset of the infection [59] . pulmunary diseases caused by tes were also demonstrated by censi et al. [62] . it is noteworthy that two rare pulmonary diseases, i.e. dendriform pulmonary ossification and pulmonary microlithiasis were related to inhalation of atmospheric particles released by industrial practices or hydrocarbon combustion [63] . one possible cause of these pathologies is the disposal of metal dust with large affinity to phosphate precipitation, such as lanthanides (yln), produced during the manufacture of mirrors, optical lenses, and certain electronic [64, 65] . lanthanides can crystallize in interstitial lung spaces and it could be diagnosed by broncho-alveolar lavage fluid dosing yln content. pulmunary characteristics are phosphatic microcrysts in intraaveolar areas of the lungs [66] . the microcrysts may precipitate with yln-phosphates and progress to pulmonary fibrosis due to dissolution of atmospheric particles [67] . due to the increasing utilization of yln for agricultural and industrial applications, the measurement of yln fractionation in lung fluid has the potential to be a viable tracer of human exposure to heavy fluxes of fine particulates enriched in heavy metals pollutants. based on these information, we could hypothesize that volcanic trace elements might play an important role in the predisposition and development of viral infection, as covid-19, and thus prevention of respiratory diseases due to pollutions is fundamental. because of the underestimation of volcanic gases emitted, we would like to encourage an accurate surveillance of the level of heavy metals and air pollution in the predisposed areas. our hypothesis may imply a higher level of attention to the risk of infection spread in sicily. it would be very important to carry out a clinical and epidemiological survey to identify heavy metals in soils and water, especially in the exposed flank areas of the volcanoes, reducing the usage and consumption of products with the higher levels of tes. further epidemiological and ecological surveys to confirm our hypothesis should be carried out. the authors state neither conflict of interest nor financial support. lava flow hazards at mount etna: constraints imposed by eruptive history and numerical simulations tephra fallout of 2001 etna flank eruption: analysis of the deposit and plume dispersion endogenous magma degassing and storage at mount etna eruptive and diffuse emissions of carbon dioxide from etna volcano air pollution and respiratory viral infection so2 emissions at mt. etna with particular reference to the period 1993-1995 so2 flux measurements at mount etna (sicily) human exposure to heavy metals. rare earth pneumoconiosis in occupational workers the role of environmental exposures in neurodegeneration and neurodegenerative diseases genetics meets environment: evaluating gene-environment interactions in neurologic diseases heavy metals and neurodegenerative diseases: an observational study the epidemiology of amyotrophic lateral sclerosis in the mount etna region: a possible pathogenic role of volcanogenic metals multiple sclerosis in the mount etna region: possible role of volcanogenic trace elements trace elements in cerebrospinal fluid and blood from patients with a rare progressive central and peripheral demyelinating disease cytokine profiles in human exposure to metals (iupac technical report) heavy metals content by icp-oes in sarda sarda atmospheric aerosols: characterization, chemistry, modeling, and climate. 1005 the interaction between volcanic gases and tephra: fluorine adhering to tephra of the 1970 hekla eruption fertilizing potential of volcanic ash in ocean surface water gas/aerosol-ash interaction in volcanic plumes: new insights from surface analyses of fine ash particles preliminary indoor radon measurements near faults crossing urban areas of mt. etna volcano (italy). public health modelling the 3-dimensional microfissure network in quartz in a thin section of granite soil 222rn pulse during the initial phase of the june-august 1995 eruption of cerro negro a pilot gis database of active faults of mt. etna (sicily): a tool for integrated hazard evaluation reconnaissance soil-gas radon survey over faulted crystalline area of ile-ife spatial distribution of soil radon as a tool to recognize active faulting on an active volcano: the example of mt. etna (italy) gravitational collapse of mount etna's southeastern flank high spatial resolution radon measurements reveal hidden active faults on mt. etna lung cancer in radonexposed miners and estimation of risk from indoor exposure risk assessment of radon in drinking water cryptococcus neoformans and cryptococcus gattii species complex isolates on the slopes of mount clinical features of patients infected with 2019 novel coronavirus in wuhan, china a pneumonia outbreak associated with a new coronavirus of probable bat origin global seasonal influenza-associated mortality collaborator network history of air pollution selections from the history of environmental pollution, with special attention to air pollution atmospheric aerosols: what are they, and why are they so important? surgical technology -e-book: principles and practice aerosol technology particulate pollution -pm10 and pm2.5". recognition, evaluation, control. news and views from diamond environmental limited earth observation data group arpa lombardia: air pollutants monitoring fundamentals of air pollution global seasonal influenza-associated mortality collaborator network three active volcanoes in china and their hazards three active volcanoes in china and their hazards three active volcanoes in china and their hazards preliminary indoor radon measurements near faults crossing urban areas of mt. etna volcano (italy). front public health regional air pollution persistence links to covid-19 infection zoning atmospheric sources ad sinks of volcanogenic elements in a basaltic volcano natural and anthropogenicfactors affecting groundwater quality of an active volcano adverse health effects of fluoro-edenitic fibers: epidemiological evidence and public health priorities mortality from chronic obstructive pulmonary disease and pleural mesothelioma in an area contaminated by natural fiber (fluoro-edenite) serotyping of cryptococcus neoformans isolates from environmental and clinical sources in extreme southern italy (calabria and sicily, central mediterranean area) molecular subtyping of clinical and environmental strains of cryptococcus neoformans variety neoformans serotype a isolated from southern italy cryptococcus neoformans/ cryptococcus gattii species complex in southern italy: an overview on the environmental di?usion of serotypes, genotypes and mating-types cryptococcus neoformans and cryptococcus gattii species complex isolates on the slopes of mount etna environmental distribution of cryptococcus neoformans and c. gattii around the mediterranean basin rare earth elements distribution in grapevine varieties grown on volcanic soils: an example from mount etna yttrium and lanthanides in human lung fluids, probing the exposure to atmospheric fallout inhalation of hydrocarbon combustion products as a cause of dendriform pulmonary ossification long-term occupational risk of rare-earth pneumoconiosis. a case report as investigated by neutron activation analysis dissolution of functional materials and rare earth oxides into pseudo alveolar fluid dendriform pulmonary ossification in patient with rare earth pneumoconiosis report of two cases with unique findings fig. 1. shows the location of mount etna, with images of its eruptions valle bove 2013; c: etna view landing to catania airport; d: etna view from giarre, a city in the eastern flank of the mountain; e: strombolian etna eruption with high km of ash emission weekly reported numbers of positive cases for covid-19 in sicily, evaluated per single province *legend: decreto "zone rosse": national decree where "red zones" where closed without any possibility to go to or to travel from all these individualized areas (lumbardy); decreto #iorestoacasa": national decree with any limitations to go out, unless strictly necessary and to keep social distances (quarantine effort); decreto "chiudi italia": all commercial activities were suspended key: cord-353716-gxgvhhv1 authors: kumar, ashutosh; narayan, ravi k.; kumari, chiman; faiq, muneeb a.; kulandhasamy, maheswari; kant, kamla; pareek, vikas title: sars-cov-2 cell entry receptor ace2 mediated endothelial dysfunction leads to vascular thrombosis in covid-19 patients date: 2020-09-30 journal: med hypotheses doi: 10.1016/j.mehy.2020.110320 sha: doc_id: 353716 cord_uid: gxgvhhv1 several studies have described unusually high incidence of vascular thrombosis in coronavirus disease-2019 (covid-19) patients. pathogenesis of the vascular thrombosis in covid-19 is least understood for now and presents a challenge to the treating physicians. severe acute respiratory syndrome coronavirus-2 (sars-cov-2), the causative pathogen for covid-19, has been shown to bind to angiotensin converting enzyme 2 (ace2) protein in human epithelial cells which facilitates its entry in the organ and mediate tissue specific pathogenesis. for ace2 mediated cell entry of the sars-cov-2, co-expression of one more protein—transmembrane protease serine 2 (tmprss2) is essential. existing studies suggested significant expression of ace2 and tmprss2 in human vascular endothelium. vascular endothelial dysfunction can potentially activate coagulation cascade eventually resulting in thrombosis. ace2 has proven role in the maintenance of endothelial integrity inside the vessels. existing in situ evidence for sars-cov-1 (the causative agent for sars pandemic of 2002, which shared ace2 as cell entry receptor) suggested that virus binding can downregulate ace2, thus can induce endothelial dysfunction. recently, in situ evidence has been presented that sars-cov-2 can infect cells in engineered human vascular endothelium, which can be effectively blocked by using clinical-grade recombinant human ace2. based on the circumstantial evidence present in the literature, we propose a sars-cov-2 cell entry receptor ace2 based mechanism for vascular thrombosis in covid-19 patients. several studies have described high incidence of vascular thrombosis in coronavirus disease-2019 (covid19) patients (1) (2) (3) . elevated levels of prothrombotic markers including d-dimer and fibrin degradation products (fdps), thrombocytopenia, abnormalities in prothrombin time and partial thromboplastin time are among the frequent findings in covid-19 patients (2) ( table 1) , especially those with severe symptoms and in icu admission (1) (2) (3) . though, probability of vascular thrombosis remains relatively high in icu admitted patients, in covid19 it has been found to be unusually high (1, 3) . pathogenesis of the vascular thrombosis in covid-19 is rather obscure and hence management of such cases presents a considerable challenge to the treating physicians (1) . severe acute respiratory syndrome coronavirus-2 (sars-cov-2), the causative pathogen for covid-19 has been shown to bind to angiotensin converting enzyme 2 (ace2) protein in human epithelial cells, which facilitates its entry in the organ and mediate tissue specific pathogenesis (4,5). ace2 is also known as a receptor for sars-cov-1 (causative agent for sars epidemic of 2002) and human coronavirus nl63 (hcov-nl63) (6). for ace2 mediated cell entry of the sars viruses, co-expression of one more protein-transmembrane protease serine 2 (tmprss2) is essential (4). in this article, we propose a sars-cov-2 cell entry receptor ace2 based mechanism for vascular thrombosis in covid-19 patients. sars-cov-2 binding to the cell entry receptor ace2 downregulates receptor expression that in turn induces vascular endothelial dysfunction, which activates prothrombotic cascade and eventually leads to vascular thrombosis observed in covid-19 patients. evaluation of the hypothesis sars-cov-2 cell entry receptor ace2 and entry associated receptor tmprss2 are coexpressed across various tissue and cell types in humans (4). ace2 expression is known in endothelial cells of human blood vessels and microvasculature (7,8). though, studies are limited, tmprss2 expression has been noted in human vascular endothelium (9,10). ace2 is an analogue of angiotensin converting enzyme (ace)-a key regulator of renin angiotensin system regulating hemodynamic homeostasis in body (11) . an ace/ace2 balance appears to be crucial in maintaining the ras and a dysregulation of this ratio is associated with vascular thrombosis (12, 13) . an ace/ace2 balance is also critical for maintenance of endothelial integrity in vessels, a breach of which can potentially induce thrombosis (14, 15) . although, any sars-cov-2 specific evidence is lacking for now, existing in vitro studies for sars-cov1 and hcov-nl63 binding on ace2 in lung tissue indicate that virus binding downregulates ace2 (but not ace) creating ace/ace2 imbalance and thereby promotes tissue injury which potentially can activate prothrombotic cascades inside the vessels (6,16). a lower ace/ace2 ratio in the vascular endothelium prevents prothrombotic cascade from activation by catalyzing degradation of angiotensin ii to angiotensin 1-7. angiotensin 1-7 also induces antithrombotic effects through binding to g-protein coupled mas receptors (17) . conversely, a higher ace/ace2 ratio will allow binding of angiotensin ii to at1 receptors which will induce vasoconstriction, inflammation, and eventually thrombosis (14, 17) . sars-cov-2 mediated ace2 downregulation in vascular endothelium also can activate the kallikrein-bradykinin pathway inducing platelet aggregation and leaking of the vessels which can further contribute to thrombosis (14, 18) . ( fig. 1 gives a schematic description of proposed mechanism for ace2 mediated dysfunction of vascular endothelium leading to thrombotic incidences in covid-19 patients.) studies describing the effect of sars-cov-2 binding on ace2 signaling in vessels are lacking. knock-out of ace2 gene in mice was found to induce vascular endothelial dysfunction leading to release of chemokines and pro-inflammatory markers (19) . ace2 knock-out mediated release of chemokine like ccl2 and other pro-inflammatory cytokines and interleukins are known to induce platelet aggregation and adhesion to the vascular endothelium, which consequently leads to increased propensity for thrombosis (19, 20) . conversely, activated ace2 has been found to protect against vascular thrombosis (13) . sars-cov-2 infection of vascular endothelium is mediated by ace2 was proven by a recent study. monteil et al provided in situ evidence that sars-cov-2 can infect engineered human vessels which can be prevented by application of clinical grade recombinant human ace2 (21) . (apart from sharing of the virus entry receptor). cases of vascular thrombosis were also reported in sars patients (22) . sars-cov-1:ace2 binding analogy to covid-19 can explain the pathogenesis of vascular thrombosis with articulate coherence. ace2 is secreted in blood and expresses in the endothelium of the small vessels (8,23,24). the histopathological examination of autopsy tissues in sars, and now in covid-19, showed presence of the virus and cellular damage in ace2 expressing tissues (22, 25, 26) . damaged tissue also displays presence of pro-inflammatory markers and cytokines, (e.g. c-reactive protein, d-dimer, ferritin and interleukin-6), which can induce thrombosis (1, 22, 27) . viral inclusion structures, an accumulation of inflammatory cells associated with endothelium, as well as apoptotic bodies were evidenced in vascular endothelial cells of multiple organs in 6 autopsied patients (28) , indicating for the widespread endothelial dysfunction as the etiology for the vascular thrombosis in covid-19 (29) . widespread thrombosis, more specifically in microvasculature, was found present in majority of the autopsied patients (30) . in some cases, thrombosis was observed despite full anticoagulation and regardless of timing of the disease course of the duration of onset of the disease, which indicated that it could be a key pathology in covid-19 (30) . thrombus rich in megacaryocytes and platelets was detected in lung, and many other viscera like liver, heart, kidney, and brain, and also in skeletal tissue like bone (30) . pulmonary vessels were found most severely affected where distinctive vascular features, consisting of severe endothelial injury associated with the presence of intracellular virus and disrupted cell membranes, and perivascular t-cell infiltration were observed (31) . presence of vascular thrombosis was associated with multi organ injury and higher fatality. formations of clots were unusually high in patients who were critical and admitted in icu. clinically, early appearances of thrombotic symptoms predicted a more severe course of disease (32) . uniquely, purple or red lesions in toes and skin rashes have been reported in some patients (27) , which are supposedly caused by inflammation of the dermal micro-vessels and are complement system mediated (25) . the optimum conditions leading to thrombosis like presence of proinflammatory markers, reduced clotting time, activated platelets and fibrin degradation products, and complement cascades were observed in the blood samples of covid-19 patients (table 1) . existing studies for sars and mers, and now emerging evidence from autopsy studies in covid-19 provide clear indications that increased thrombosis is propagated by virus mediated tissue injury (28, 31) . an early presence of vascular thrombosis at systemic scale can be appropriately explained by ace2 mediated dysfunction of vascular endothelium and consequent activation 7 of prothrombosis cascade (14, 17, 29) . sars-cov-2 binding mediated downregulation of ace2 signaling in vascular endothelium and consequent imbalance of ace/ace2 ratio and dysregulation of ras, can potentially activate prothrombotic cascade at a scale matched to that observed in covid-19 (14, 17, 29) (fig. 1) . other than sars-cov-2 induced vascular endothelial dysfunction ace2 mediated injury of liver tissue can also contribute to vascular thrombosis. liver cell injury and functional impairment are usual features in covid-19 patients (33) . owing to low expression of ace2 in hepatocytes, it is not clear if liver cell injury is mediated through this molecule; howbeit, high expression of ace2 (and tmprss2) is known in common bile duct and gallbladder epithelium (4). literature suggests that acute liver injury should be considered as an independent risk factor for developing vascular thrombosis (34) . clinical outcomes in covid-19 were found depending on multiple factors specific to the virus and host (35) , that may apply to thrombotic incidences as well. apart from direct sars-cov-2 inflicted tissue injury, vascular thrombosis can also be a resultant of cell toxicity by viral proteins, pro-inflammatory markers released after viral tissue injury, sepsis, or cellular stress caused by critical illness (36) . additionally, host specific factors like old age and associated comorbid conditions which induce endothelial dysfunction like diabetes, hypertension, obesity, coronary artery diseases, atherosclerosis, chronic liver, renal or lung diseases, cancers, activator (43) , is currently being used as an anti-protozoan veterinary drug (an active ingredient of 'berenil'), however, in clinically effective doses it has serious toxicity risk for vital organs such as liver and kidney, and more particularly for the central nervous system (cns) and heart. adequate safety testing, and determination of safe dose, in human subjects, will be necessary, before it can be repurposed in covid-19 patients.) based on the circumstantial evidence present in the literature, we propose that thrombosis in organ vasculature and dermal micro-vessels in covid-19 are primarily caused by virus binding mediated dysregulation of ace2 signaling in vascular endothelium and consequent cellular injury and activation of hemostatic factors. however, contributory role of other prothrombotic factors (related to both virus and host) cannot be denied. 9 further in vivo/in vitro studies investigating sars-cov-2 binding influence on ace2 signaling and consequently on molecular cascade involved in clotting in vascular endothelium will be necessary to establish ace2 based mechanisms in thrombotic incidences in covida.k. designed research. a.k., r.k.n., and c.k. analyzed data. a.k., m.a.f., m.k., k.k., and v.p. wrote the paper. authors have no financial disclosure to report. covid-19 and thrombotic or thromboembolic disease: implications for prevention, antithrombotic therapy, and follow-up: jacc state-of-the-art review the procoagulant pattern of patients with covid-19 acute respiratory distress syndrome incidence of thrombotic complications in critically ill icu patients with covid-19 angiotensin-converting enzyme 2, angiotensin-(1-7) and mas: new players of the renin-angiotensin system the potential actions of angiotensin-converting enzyme ii (ace2) activator diminazene aceturate (dize) in various diseases ace2 activation promotes antithrombotic activity. molecular medicine covid-19: the vasculature unleashed evolving functions of endothelial cells in inflammation a crucial role of angiotensin converting enzyme 2 (ace2) in sars coronavirus-induced lung injury the pivotal link between ace2 12 deficiency and sars-cov-2 infection plasma kallikrein enhances platelet aggregation response by subthreshold doses of adp genetic ace2 deficiency accentuates vascular inflammation and atherosclerosis in the apoe knockout mouse. circulation research upregulation of the chemokine (cc motif) ligand 2 via a severe acute respiratory syndrome coronavirus spike-ace2 signaling pathway inhibition of sars-cov-2 infections in engineered human tissues using clinical-grade soluble human ace2 the clinical pathology of severe acute respiratory syndrome (sars): a report from china expression of severe acute respiratory syndrome coronavirus receptors, ace2 and cd209l in different organ derived microvascular endothelial cells. zhonghua yi xue za zhi complement associated microvascular injury and thrombosis in the pathogenesis of severe covid-19 infection: a report of five cases histopathologic changes and sars-cov-2 immunostaining in the lung of a patient with covid-19 clinical and coagulation characteristics of 7 patients with critical covid-2019 pneumonia and acro-ischemia. zhonghua xue ye xue za zhi endothelial cell infection and endotheliitis in covid-19 endothelial cell dysfunction: a major player in sars-cov-2 infection (covid-19)? megakaryocytes and platelet-fibrin thrombi characterize multi-organ thrombosis at autopsy in covid-19: a case series pulmonary vascular endothelialitis, thrombosis, and angiogenesis in covid-19 abnormal coagulation parameters are associated with 14 poor prognosis in patients with novel coronavirus pneumonia liver injury in covid-19: management and challenges effects of acute liver injury on blood coagulation viral and host factors related to the clinical outcome of covid-19 review: viral infections and mechanisms of thrombosis and bleeding risk factors for venous and arterial thrombosis covid-19, induced activation of hemostasis, and immune reactions: can an auto-immune reaction contribute to the delayed severe complications observed in some patients? the emerging threat of (micro)thrombosis in covid-19 and its therapeutic implications inhibition of sars pseudovirus cell entry by lactoferrin binding to heparan sulfate proteoglycans human coronavirus nl63 utilizes heparan sulfate proteoglycans for attachment to target cells covid-19 and the endothelium the ace 2 activator diminazene aceturate (dize) improves left ventricular diastolic dysfunction following myocardial infarction in rats authors declare there are no conflicts of interest. cell atlases reveal age, gender, and smoking status associations with cell type-specific expression of mediators of sars-cov-2 viral entry and highlights inflammatory programs in putative target cells. biorxiv. 2020 jan 1. key: cord-352911-9wbq9qo2 authors: de oliveira, pedro gonçalves; termini, lara; durigon, edison luiz; lepique, ana paula; sposito, andrei c; pierulivo, enrique mario boccardo title: diacerein: a potential multi-target therapeutic drug for covid-19 date: 2020-06-01 journal: med hypotheses doi: 10.1016/j.mehy.2020.109920 sha: doc_id: 352911 cord_uid: 9wbq9qo2 severe acute respiratory syndrome coronavirus 2 (sars-cov-2), which causes coronavirus disease 19 (covid-19), was declared pandemic by the world health organization in march 2020. sars-cov-2 binds its host cell receptor, angiotensin-converting enzyme 2 (ace2), through the viral spike (s) protein. the mortality related to severe acute respiratory distress syndrome (ards) and multi-organ failure in covid-19 patients has been suggested to be connected with cytokine storm syndrome (css), an excessive immune response that severely damages healthy lung tissue. in addition, cardiac symptoms, including fulminant myocarditis, are frequent in patients in a severe state of illness. diacerein (dar) is an anthraquinone derivative drug whose active metabolite is rhein. different studies have shown that this compound inhibits the il-1, il-2, il-6, il-8, il-12, il-18, tnf-α, nf-κb and nalp3 inflammasome pathways. the antiviral activity of rhein has also been documented. this metabolite prevents hepatitis b virus (hbv) replication and influenza a virus (iav) adsorption and replication through mechanisms involving regulation of oxidative stress and alterations of the tlr4, akt, mapk, and nf-κb signalling pathways. importantly, rhein inhibits the interaction between the sars-cov s protein and ace2 in a dose-dependent manner, suggesting rhein as a potential therapeutic agent for the treatment of sars-cov infection. based on these findings, we hypothesize that dar is a multi-target drug useful for covid-19 treatment. this anthraquinone may control hyperinflammatory conditions by multi-faceted cytokine inhibition and by reducing viral infection. the first case of coronavirus disease 19 (covid-19), a condition caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2), emerged in the city of wuhan in hubei province, china, on december 2019. due to its incubation time (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) days) and because it is highly contagious over a short time, infection with sars-cov-2 has traversed global borders and caused thousands of deaths, mainly in elderly patients. on march 11th, 2020, the world health organization declared covid-19 a pandemic disease. even though some infected persons are asymptomatic, sars-cov-2 infection can cause severe symptoms, including high fever, severe cough and shortness of breath, which often indicates pneumonia. severe acute respiratory distress syndrome (ards) has been identified as the leading cause of covid-19-associated mortality. in addition, sepsis, acute cardiac injury, and fulminant myocarditis are common critical complications (1, 2) . the mortality related to ards as well as multi-organ failure in covid-19infected patients may be connected with cytokine storm syndrome (css), an excessive immune response that severely damages healthy lung tissue (3). this response may lead to macrophage activation syndrome (mas) (4,5) or secondary haemophagocytic lymphohistiocytosis (shlh) with fulminant and fatal hypercytokinaemia (3,6). in this scenario, a dilemma emerges due to the potential deleterious effects of immunosuppressive agents used to treat hyperinflammation, such as corticosteroids and janus kinase (jak) inhibitors (3), on antimicrobial immunity (7). diacerein (dar), also called diacetylrhein, is an anthraquinone derivative used as a symptomatic slow-acting drug for the management of osteoarthritis (sysadoa) (8-12) licensed in countries of the european union, latin america and asia for up to 20 years. the drug is administered orally and entirely converted to its active metabolite, rhein, before reaching the systemic circulation (8,12). the main mechanism of action of dar is inhibition of the interleukin-1 (il-1) signalling pathway (8-10,12-20). in addition, several studies have described the inhibitory effect of this compound on il-6 (16, (20) (21) (22) (23) (24) and tnf-α (16, 21, 25, 26) . however, no reported studies have addressed the potential therapeutic use of dar in patients with covid-19. considering the current global health crisis caused by covid-19, the search for therapeutic alternatives is mandatory. the use of drugs with established mechanisms of action has emerged as a valid tool to identify compounds that can contribute to managing this disease. sars-cov-2 recognizes and binds its host receptor, angiotensin-converting enzyme 2 (ace2), through the viral spike (s) protein. this binding occurs through the s1 domain of the viral protein, while the s2 domain is responsible for fusion of the viral envelope to the cell membrane (27) (28) (29) . therefore, the level and pattern of human ace2 expression in different tissues might be critical for differences in susceptibility, symptoms and outcome between different individuals (30) . in addition, it is important to consider that the expression of ace2, including its related polymorphisms, can differ in the population (30) (31) (32) . this enzyme is mainly expressed in endothelial cells of the blood vessels but is also present in other organs, including the heart, lungs and kidneys. this may explain the extra-pulmonary manifestations of the disease as well as some focus on specific targets. on the other hand, other approaches, such as the use of corticoids, present a more nonspecific mechanism of action. it is clear from the data described above that several therapeutic alternatives are under examination. however, at present, no drug for covid-19 management has been explored from a multi-target perspective. the active metabolite of dar, an anthraquinone, is rhein (8, 12) . different studies have demonstrated that the main mechanism of action of dar is the inhibition of il-1β production (15, 17) . in addition, this drug reduced the number il-1 receptor (il-1r) in chondrocytes (15) , induced the upregulation of il-1 receptor agonist (il-1ra) in cartilage culture (17), and prevented il-1-induced nuclear factor-b (nf-b) activation by inhibiting the degradation of its main inhibitor, iκbα (18). moreover, rhein was shown to reduce the production of il-1 converting enzyme (ice) in cartilage, leading to a reduction in activation of il-1β from its inactive (pro-il-1) form (19). recently, chang et al. (60) demonstrated that rhein suppressed caspase-1 protease activity and il-1β production by interfering with formation of the nlrp3 multi-protein complex. in addition, it may exert its anti-inflammatory action through inhibition of the nf-b and nalp3 inflammasome pathways (61, 62) . based on these mechanisms of action, rhein could suppress lung inflammatory injury induced by human respiratory syncytial virus in mice (63) . finally, the inhibitory effect of this molecule on il-6 (20-24,64,65) and tnf-α monolayer cultures of lung, cardiac, endothelial and epidermal cells will be maintained under standard cell culture conditions. the different cell lines will be seeded in 24-well plates and infected with sars-cov-2 at different multiplicity of infection (moi) values. after 48 hours, cell cultures will be treated with rhein at different concentrations (1 m to 400 m) for 3, 6, 12, 24, 48 and 72 hours. production of the pro-inflammatory and anti-inflammatory cytokines il-1b, il-2, il-6, il-7, il-8, il-10, il-12, il-18, il-37, il-38 and tnf-α in cell extracts and the supernatants and whole-cell extracts of the cell cultures described will be determined by western blotting, flow cytometry, luminex assay and enzyme-linked immunosorbent assay (elisa). additionally, release of the damage-associated molecules apt and hmgb-1 in the cell culture supernatants will be measured by elisa. the expression and function of the nacht, lrr and pyd domains-containing protein (nalp3) inflammasome in the cell lines treated with rhein will be analysed by western blotting and rt-polymerase chain reaction (pcr). all the experiments will be performed in triplicate. moreover, the levels of activated caspase-1 and hmgb1 will be tested. the effect of rhein-mediated inhibition of the interaction between the s protein and ace2 on sars-cov-2 will be evaluated as described by ho et al. (73) . the method will consist of purification and biotinylation of recombinant sars-cov-2 s protein, biotinylated elisa, immunofluorescence assay (ifa), and the infection of vero cells and lung-, cardiac-, endothelial-and epidermal-derived cell lines with s proteinpseudotyped retrovirus. cell viability will be evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (mtt) assay. the study will be performed in the presence of rhein at varying concentrations (1 m-400 m). c57 black/6 (7 to 9 weeks old) mice will be infected with the mouse-adapted sars-cov-2 ma15 strain (85). for infection, mice will be intranasally inoculated with 1×10 4 pfu mouse-adapted sars-cov-2 in 50 l of pbs. treatment of 3 experimental groups and equivalent control and placebo-treated groups with rhein (1 m to 400 m) will start at 2, 6 or 12 hours after viral inoculation. body weight and pulmonary function (using a single-chamber, whole-body plethysmograph (buxco eletronics inc., nc, usa)) will be assessed daily until the 5 th day post-inoculation. at day 5, the blood, lungs, heart, kidneys and peripheral lymph nodes will be harvested after euthanasia. the lungs will be scored for haemorrhage, and the inferior right lobe will be used for viral load determination by vero e6 cell infection according to the protocol described by sheahan et al. (86) . for lung damage measurements, the lungs will be fixed and embedded in paraffin. scores will be given according to the american thoracic society scoring tool, which takes into consideration neutrophil infiltration in the alveolar space, neutrophils in the interstitial spaces, hyaline membranes, proteinaceous debris in air spaces, and alveolar septal thickening. moreover, necrosis, haemorrhagic areas and cellular sloughing will be examined. the hearts and kidneys will also be fixed and embedded for histology to search for necrosis and haemorrhagic areas. the lymph nodes will be mechanically dissociated into single-cell suspensions. cells will be labelled with proliferation cell dye (bd biosciences, carlsbad, ca) and stimulated with 10 mg/ml tpa and 1 g/ml ionomycin for 4 days. after that period, cell culture supernatants will be harvested for cytokine secretion determination (th1/th2/th17 cba kit, bd biosciences, carlsbad, ca), and cells will be harvested and labelled with anti-cd4, anti-cd8, anti-cd19 and anti-cd25 to determine the proliferation of specific populations. serum isolated from the blood will be used to determine the cytokine concentration by luminex and the lactate concentration to evaluate acidosis using a colorimetric assay (merck/sigma). the effect of rhein on sars-cov-2 replication will be evaluated as described by sun et al. (71) with modifications. briefly, the different cell lines described above will be infected with sars-cov-2 virions and cultured in the presence of rhein at different concentrations. after 72 hours, the effect of rhein on viral replication will be determined by measuring viral load by quantitative real-time pcr. the expression levels of sars-cov-2 surface antigens s and e will be determined by elisa and a virus neutralization test (vnt) after rhein treatment. rhein will be tested under the conditions described above. the half-maximal inhibitory concentration (ic50) will be calculated. total extracts from monolayer cell cultures infected with sars-cov-2 and treated with rhein under the conditions described above will be analysed using commercially available protein arrays to determine the levels and activation state of proteins involved in the tlr-, akt-, mapk-, and nf-b-regulated signalling pathways. data will be analysed using imagej software. the levels of reactive oxygen species (ros)/reactive nitrogen species (rns) will be determined in the total cell extracts and supernatants from monolayer cell cultures infected with sars-cov-2 and treated with rhein under the conditions described above. important tool to test our hypothesis in humans and, based on their results, accelerate the translational development of this potential therapeutic alternative for covid-19. considering the data presented above, we hypothesize that dar is a multi-target drug useful for covid-19 treatment. the mechanisms of action involved include the control of hyperinflammatory conditions by multi-faceted cytokine inhibition of il-1, il-2, il-6, il-8, il-12, il-18 and tnf-α; anti-platelet aggregation activity; and potential effects on viral infection and replication. pedro gonçalves de oliveira is responsible for r&d activities at trb pharma indústria química e farmacêutica ltda. trb pharma is the owner of the product artrodar ® , a diacerein-based product for osteoarthritis treatment. the other authors declare no conflict of interest. clinical predictors of mortality due to covid-19 based on an analysis of data of 150 patients from wuhan, china clinical characteristics of 113 deceased patients with coronavirus disease 2019: retrospective study symptomatic efficacy and safety of diacerein in the treatment of osteoarthritis: a meta-analysis of randomized placebo-controlled trials effects of diacerein at the molecular level in the osteoarthritis disease process benefit-risk assessment of diacerein in the treatment of osteoarthritis therapeutics in osteoarthritis based on an understanding of its molecular pathogenesis in vitro effects of diacerein and rhein on il-1 and tnf-α systems in human osteoarthritic synovium and chondrocytes effects of diacerhein on granuloma induced cartilage breakdown in the mouse. osteoarthr cartil analysis with dexamethasone, methotrexate and anti-tnf protocols characterization of the receptor-binding domain (rbd) of 2019 novel coronavirus: implication for development of rbd protein as a viral attachment inhibitor and vaccine genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding structural basis for the recognition of sars-cov-2 by full-length human ace2 comparative genetic analysis of the novel coronavirus (2019-ncov/sars-cov-2) receptor ace2 in different populations polymorphisms of angiotensin-converting a rush to judgment? rapid reporting and dissemination of results and its consequences regarding the use of hydroxychloroquine for covid-19 chloroquine and hydroxychloroquine as available weapons to fight covid-19 the cytokine storm of severe influenza and development of immunomodulatory therapy rhein, an anthraquinone drug, suppresses the nlrp3 inflammasome and macrophage activation in urate crystal-induced gouty inflammation rhein attenuates inflammation through inhibition of nf-κb and nalp3 inflammasome in vivo and in vitro rhein protects against barrier disruption and inhibits inflammation in intestinal epithelial cells rhein suppresses lung inflammatory injury induced by human respiratory syncytial virus through inhibiting nlrp3 inflammasome activation via nf-κb pathway in mice response of young, aged and osteoarthritic human articular chondrocytes to inflammatory cytokines: molecular and cellular aspects rhein inhibits lipopolysaccharide-induced intestinal injury during sepsis by blocking the toll-like receptor 4 nuclear factor-κb pathway efficacy and safety of diacerein and diclofenac in knee osteoarthritis in indian patients -a prospective randomized open label study rhein prevents endotoxin-induced acute kidney injury by inhibiting nf-κb activities anti-inflammatory effects of rhein and crude extracts from cassia alata l. in hacat cells diacerhein downregulate proinflammatory cytokines expression and decrease the autoimmune diabetes frequency in nonobese diabetic (nod) mice chrysophanol, an anthraquinone from ast2017-01, possesses the anti-proliferative effect through increasing p53 protein levels in human mast cells identification of natural compounds with anti-hepatitis b virus activity from rheum palmatum l. ethanol extract anti-influenza a virus activity of rhein through regulating oxidative stress, tlr4, akt, mapk, and nf-κb signal pathways emodin blocks the sars coronavirus spike protein and angiotensin-converting enzyme 2 interaction inhibitors of cathepsin l prevent severe acute respiratory syndrome coronavirus entry quinazoline derivatives as cathepsins b, h and l inhibitors and cell proliferating agents amino acid sequences of the human kidney cathepsins h and l anthraquinone-2,6-disulfonic acid as a disease-modifying osteoarthritis drug disseminated intravascular coagulation in patients with 2019-ncov pneumonia antithrombotic and antiplatelet activities of 2-chloro-3-[4-(ethylcarboxy)-phenyl]-amino-1,4-naphthoquinone (nq12), a newly synthesized 1,4-naphthoquinone derivative key: cord-315576-bgcqkz0p authors: yamamoto, naoki; bauer, georg title: apparent difference in fatalities between central europe and east asia due to sars-cov-2 and covid-19: four hypotheses for possible explanation date: 2020-08-05 journal: med hypotheses doi: 10.1016/j.mehy.2020.110160 sha: doc_id: 315576 cord_uid: bgcqkz0p the comparison of the numbers of cases and deaths due to sars-cov-2 / covid-19 shows that people in central europe are much more affected than people in east asia where the disease originally occurred. trying to explain this difference, this communication presents four hypotheses that propose the following reasons for the observed findings: 1) differences in social behaviors and cultures of people in the two regions; 2) possible outbreak of virulent viruses in central europe due to multiple viral infection, and the involvement of immuno-virological factors associated with it, 3) possibility of corona resistance gene mutation occurring among east asians as a result of long-term co-evolution of virus and host, and 4) possible involvement of hygienic factors. direct or indirect supportive evidences for each one of our hypotheses are presented and experimental approaches for their evaluation are discussed. finally, we suggest that the dynamics of the pandemic also shows that the problems of the new coronavirus can be overcome due to people's awareness of the epidemics, rational viral diagnostics and a high level of medical care. the new coronavirus sars-cov-2, causing an infectious disease named covid-19 has caused a major pandemic with more than 13 million people infected and more than 570,000 already dead [1, 2] . its momentum is disrupting the economy, society, and healthcare systems, filling hospitals with patients, emptying public spaces, and drawing people away from work and friends. due to the test strategy used in nearly all countries, "people infected with sars-cov-2" or simply named "covid-19 cases" are usually people with clinical symptoms or contacts to infected people. the number of infected people without clinical symptoms is therefore unknown. further testing, including antibody tests, will most likely reveal that much more people around the world have been infected than the number of defined cases of covid-19. this may upset modern societies on a scale never seen before. on the other side, it may be assumed that these infected people might contribute to herd immunity and slow down further spread of the virus, provided their antibody response is longer lasting and has neutralizing potential. these aspects need further experimentation and proof. in the 21st century, information (including scientific and technological knowhow) circulates around the world instantly. the world has entered an era where the traffic of people, economic goods, money, but also viruses and other microbes seems to be nearly unlimited. therefore, after the outbreak of the new coronavirus epidemics in china, the transport of the virus to other parts of the world, particularly to europe, 4 was fast and efficient. of particular note is the striking difference in the extent of medical impact caused by sars-cov-2 in eastern asia versus central europe [3, 4] . this difference is prominent, when i) the number of infected persons with clinical symptoms, ii) the ratio between infected people with clinical symptoms and the total population of the countries, iii) the number of deaths, or iv) the death ratios of infected people with symptoms are compared between eastern asia and central europe. in this communication, we compared selected countries from central europe and east asia with respect to the medical impact of sars-cov-2 and covid-19. based on this analysis, we propose four hypotheses that might explain the observed differences. for each one of these hypotheses, we present direct or indirect evidences to support them. none of these hypotheses by itself can explain the observed differences. rather, the mechanisms defined in individual hypotheses seem to interact and thus cause the overall effect observed. we are aware that additional mechanisms, not covered by our analysis, may also contribute to the pandemics. for the analysis of the difference regarding the number of infected people and the death tolls due to covid-19 between central european and east asian 5 countries, we have chosen italy, spain, france, germany and uk from central europe and china, south korea, japan, and taiwan from south east asia. mongolia and north korea were excluded, since not sufficient information was available from both countries. for comparison, the united states, which have the highest number of infected and dead people due to covid-19, were included into our analysis. table 1 first shows the populations and gdps, in order to help estimating the scale, medical level, and affluence scale of the selected countries. it is obvious that east asia is one of the most densely populated areas in the world. it can also be seen that there is not much difference with respect of the gdps, except for china. however, china's gdp has grown rather rapidly in recent years, and it is certain that it will reach the economic level of the other countries sooner or later. european countries ranged from 1993 to 5359 per million people, whereas those of east asian countries were in the range of 18~211. the ratio between the two was about 32. these numbers depend on the number of tests performed in individual countries, as well as on differences in the test strategy, resulting in a certain degree of uncertainty. to get a more precise picture of comparison, covid-19-related 6 deaths per nation's total population (per million people) were calculated based on the total population of each country. as seen in table 1 , the numbers of central european countries were in the range of 391-548 deaths/million people, with the only exception being germany's lower number of 83. in contrast, deaths/million people in east asian countries were in the range of 0.3-5, i. e. they were nearly 150 times lower than the values found for central european countries. importantly, also the death rate of infected people seems to be much lower in east asian countries, compared to the central european countries (except germany). the relatively lower number of infected people as well as covid-19-associated deaths in germany seems to be due to strong differences in the percentage of infected people with clinical symptoms between the south and north of germany (table 2) . it seems that measures taken after the first cases in bavaria (a large state in the south east of germany, which was first hit by the pandemics) were successful to prevent a strong spreading on the long run. germany had the advantage to be affected by covid-19 later than china and italy. getting valuable information from other countries was essential for slowing down and partially restricting the pandemics in germany. furthermore, the relatively high availability of equipment for intensive care reduced the death rate of infected people. importantly, the picture of inhomogenous distribution of the disease and associated deaths as seen for the whole of germany is also seen when the spreading of infections and 7 deaths is analyzed within bavaria, the state being affected the most ( table 2 ). the detailed analysis of cases and deaths in districts points to hotspots of infection and death, from which the neighbouring areas seem to be reached out. this results in an overall picture with a relatively high number of cases and deaths in the hotspot (tirschenreuth) that far exceeds the average number of cases / population and deaths / population of total germany. the immediate neighbouring districts in the north and south show about half the cases / population and less than half the deaths / population than the hotspot, whereas districts distant of the hotspot are characterized much lower numbers. this picture points to strong initial effects from local infection events, but, importantly, it also indicates that further spreading had obviously been prevented through adequate measures. since the data on the number of covid-19-caused deaths in each country gave reliable data that showed strong differences between east asian and central european countries, we tried to explain these differences by four intercalated hypotheses. given the host-virus relationship, it is necessary to consider factors affecting both virulence of the pathogens and the resistance of the hosts to virus 8 infection. several aspects might be involved in the difference. for example, lippi et al. investigated potential reasons for the noticeable difference in covid-19-associated mortality rates between asian and european populations from clinical and demographic aspects in italy versus china [5] . these authors confirmed that a higher burden of comorbidities, male sex, and older age may be considered substantial determinants of enhanced risk of death in italy compared to china. similarly, in new york city where the highest number of deaths is recorded compared to other states in the united states, factors such as high population density, the possible super spreaders of virus, the degree of poverty between races, and the lack of insurance have become important issues [6] . hypothesis #1 is based on the difference in social behavior and customs of people in each area, i.e. in europe and south east asia. this might explain the difference to some extent. when comparing the lifestyles of european countries with that of asia, the first noticeable difference is the closeness of direct human contact. in asia, bowing is the mainstream greeting, and even with the spread of western culture, shaking hands is still not very common. of course, besides handshaking, there can be no hugs or kisses that are popular in western culture (if any, those are performed only after building a very close relationship). it is easy to imagine that these common actions in western societies help to spread respiratory viruses like sars-cov-2 very efficiently. these differences between europeans and asians, considered from a perspective of the social science might be very important in the progress of the pandemic. there is also a marked difference in obesity between westerners and asians, mainly due to differences in food culture. in addition, people wear shoes indoors in western societies, while it is strictly forbidden to do so in east asia. although the weight of this habit in preventing virus infection is not clear at this moment, this difference indicates that asians very clearly distinguish between outdoor and indoor in daily life. the habit of wearing a mask in case of a cold or fear of it also seems quite asian-specific. thus, it is very likely that differences with respect to these actions play an important role in the spread of infection. social distancing has been proven to avoiding new corona infections, and is a drug-free means of controlling de novo infections. shaking hands, kissing, and hugging are not allowed during social distancing. in favour of our hypothesis #1, the switch from european style of close contact to the asian style during social distancing is regarded as an essential part of successful slowing down and preventing further infections with sars-cov-2. potential effects of repeated infection on the severity of the clinical outcome, contributions of antibody-mediated enhancement of infection to the severity of disease, as well as genetic changes of the virus due to mutation of its rna genome might be underlying the observed effects. in italy, the death toll from the new coronavirus was 25,085 by the april 22, the third highest in the world after the united states and spain. of these, more than 10,000 people were confirmed to be infected among medical staff. this finding shows that inadequate protection of medical staff from infection is a major issue. many of the doctors who died were practitioners. they were fighting the new coronavirus without adequate protective equipment (sometimes due to unavailability of material) or underestimation of the contagiousity of the new virus, driven by the strong wish to help their patients. thus, it is highly likely, especially during the medical collapse, that these doctors will have got infections repeatedly from the infected patients one after another. so the question is what would be severe disease [7] . mice developed higher viral loads, more severe lung pathology, and greater inflammatory responses and generated only limited influenza a virus-specific b and t cell responses. although several pathways could contribute to higher viral loads in mice that received influenza a virus repeatedly, the authors suggested the possibility that the inflammatory response elicited by the first dose of influenza a virus damaged the mucosal barrier, thus allowing the virus given as a second or third dose to penetrate more deeply into the lungs. higher viral loads will lead to the induction of stronger cytokine storms. these data are in agreement with clinical findings in humans, where high-risk individuals were exposed to multiple small doses of hbv, as well as in the woodchuck model, in which animals were repeatedly exposed to small quantities of infectious hbv [8] . in both cases, viral infection was molecularly evident and there were detectable virus-specific cd8-t cell responses but undetectable virus-specific antibody responses. as to repeated infections, another possibility is also considered that is involvement of the antibody-dependent enhancement (ade), which is a well-known phenomenon in the field of viral immunology. first, let's assume the situation of the individual who received a coronavirus infection followed by a second one. one week or so after initial infection, antibodies against the virus are elicited, and the second virus reacts with this antibody causing more serious effects. this phenomenon to increase viral infectivity and virulence has been observed with many viruses including several coronaviruses, hiv, zika virus and mosquito-borne flaviviruses (dengue virus and yellow fever virus). indeed, olsen and colleagues have shown antibody enhancement of infection with one of the animal coronaviruses, feline infectious peritonitis virus by a subset of specific monoclonal antibodies, especially those directed against specific sites on the spike protein [9] . similarly, wang et al. generated monoclonal antibodies against sars-cov spike proteins and found that these antibodies promoted sars-cov infection [10] . ade these aspects may be particulary relevant for a limited number of people such as medical staff. therefore, in addition to that, the emergence of more virulent virus due to mutation of the virus during the course of superinfection might also be assumed. rna viruses are, in general, highly susceptible to mutations, and the base substitution rate, which indicates the degree of change, is estimated to be 7 + 2 x10 -4 nucleotide substitutions per site and per year on average in case of transmissible gastroenteritis virus (tgev), an enteropathogenic coronavirus. this rate falls in the range reported for other rna viruses [11] . in addition, it is also known that coronavirus changes considerably easily due to gene recombination or part of gene loss (deletion). for example, the new strain of canine respiratory coronavirus identified in 2017 is reported to be a recombinant of the existing canine and bovine coronaviruses [12] . in addition, it has been known that the deletion of part of the spike gene has caused the emergence of a prcv from the ancestral porcine gastroenteritis virus [13] . it is possible that the doctor who actually examined many infected patients could be exposed to the coronavirus repeatedly, and among the multiple virus strains infected, more virulent virus strains suitable for 14 spread may be selected and prevailed, at least initially. according to the classical models of virulence evolution of the virus, multiple infections select for raised virulence. to explain why so many people are dying of covid-19 in new york city, sequence analysis of the viral genome has been performed. according to zimmer, the virus mainly came from asia through a small number of infected individuals in california, while in new york, more than 100 people initially brought the virus mainly from europe, suggesting as if the european type viruses are more virulent in its pathogenicity and infectivity than the asian type [14] . a number of studies have supported that through collective actions leading to common good production and immune system impairment, viral cooperation can lead to increased levels of virulence. researchers around the world have already infecting vero-e6 cells [15] . based on more molecular virological studies, tan et al. proposed that sars-cov-2 can be divided into two major lineages (l and s). intriguingly, the s and l lineages can be clearly defined by just two tightly linked 15 snps at positions 8,782 (orf1ab: t8517c, synonymous) and 28,144 (orf8: c251t, s84l). orf1ab encoding replicase/transcriptase is essential for viral genome replication and might also be important for viral pathogenesis [16] . however, there was no evidence so far that this mutation produced a more virulent form of the virus. moreover, the data examined up to now are still very limited, and follow-up analyses of a larger set of data are needed to have a better understanding of the evolution and epidemiology of sars-cov-2. thus, further virological studies must focus on the relationship between differences in nucleotide sequences and infectivity/ pathogenicity of viruses since there is no firm evidence, so far, of the existence of european strains of the coronavirus or its pathogenicity being more virulent than the asian strains. human hosts and their virus have co-evolved for millions of years, during which viruses have adapted to defense system of its host by regulating pathogenic mechanisms. therefore, the possible genetic change and resulted selection of people living in east asia should also be considered from an evolutional perspective. thus, the difference in viral susceptibility and mortality of east asian people to sars-cov-2 could also be explained if people living in east asia may have evolved to be more resistant to viral infections, including those of novel corona 16 viruses. in east asia, especially in china, agriculture started about 13,000 years ago, maybe 3,000 years ahead of europe. this led to an explosive increase in population, urbanization, and population density with the supply of abundant food. as a matter of course, acute viral infections such as measles, rubella, mumps, which could not be established until then, are believed to have taken roots in the human population (in the case of measles, it requires a population more than 250,000 to settle). unlike today, asia had long been much richer than europe before the industrial revolution. under the over-crowded and chaotic conditions, east asians must have experienced overwhelmingly with many plagues including several zoonoses due to the encounter with strange animal species. it is natural to consider that such epidemics are related to the change, choice, and evolution of the people who live there. east asians may have evolved to become more resistant against infectious agents in general including coronavirus. it is possible that difference of the past plagues could contribute to a difference in the susceptibility (and thus, pathogenicity) between europeans and asians against present new corona. present covid-19 is apparently derived from bats directly or via vector animals, and its appearance is closely related to chinese food culture. given this, it is not strange to consider the possibility that this area had been hit by coronavirus infections similar to this time before long ago. in fact, the country experienced similar endemics, sars and mers only 18 and 8 years ago, respectively. this suggests that coronavirus infection itself is one of the most likely candidates for east asian selection and evolution among the past plagues. although humans are a fairly homogeneous group of species as viewed from the genome, the diversity of the genome is well maintained. it avoids all human species from suffering the same disease and is a means of survival as a species, even if some disease prevails. although plague and people have been closely linked, one of the causes of human diversity is infectious disease. many genetic diseases are unfavorable to survival, but in some cases they are also advantageous for survival, and in some cases mutations have given the power to survive from the diseases that have hit the ground in the past. in east asia, where agriculture was established early on and urbanization has been achieved, plagues have been rushing to people in a messy environment since ancient times. we believe that it should be worth considering that individuals with advantageous gene mutations have been selected in relation to various epidemics, and have reached the present day. several genes may be involved in the genetic predisposition to covid-19, and 18 the combination of multiple genes may be important for the severity of the infection. among them, human leukocyte antigen (hla) polymorphisms are associated with susceptibility to various diseases such as autoimmune diseases and infectious diseases. the composition ratio of hla types varies greatly depending on the country and ethnic group. since hla is a protein of the immune system that is responsible for antigen presentation, hla has been attracting attention in relation to disease susceptibility. ellinghaus and colleagues performed gwas on patients in italy and spain [17] . but, it was found that certain hla types are unlikely to be associated with exacerbation of the novel coronavirus at least in italy and spain. nevertheless, since hla types, which are present only in japan and other asian countries, may show resistance to the novel coronavirus, further analysis is necessary. the human angiotensin-converting enzyme 1 (ace1) gene on chromosome 17 has an insertion (i) or deletion (d) of a 287 base pair (bp) alu repeat sequence in intron 16 [18] . therefore, in the i/d polymorphism, there are three different genotypes, ii, id and dd. ace1 is a metalloproteinase, which is a type i transmembrane glycoprotein. this protein plays an important regulatory function in the renin-angiotensin-aldosterone system (raas) and can convert angiotensin i (angi) vasoconstrictor, which is inactive, to angiotensin ii (angii). angii is the core product of the raas system, and causes various biological reactions through the angiotensin receptors (at1 and at2) while ace2 (angiotensin invertase 2) is a homologue of ace and is well known as a receptor for sars-cov-2. however, the original role of ace2 is to digest angii into ang1-7 polypeptides, and protect the heart, vasodilate, resist growth, and resist proliferation. also, the activity of bradykinin can be enhanced by ace2. very recently, we showed a strong negative correlation that the numbers of sars-cov-2 infected cases and deaths due to viral infection decreased with increasing ace1 ii genotype frequency [19] . the serum ace1 level was significantly higher in those with the dd genotype compared with those with either the id or the ii genotype [20] , and viral infection may lead to suppression of ace2 function and causes ace1/ace2 imbalance responsible for ras over-activation and pulmonary shut-down [21] . this can further reduce the effects of ace2, which counteracts the pathophysiological effects of ang ii produced by ace1, and may worsen the pathology. in patients with the d allele, especially those with the dd genotype, higher risk of morbidity and mortality from sepsis, acute respiratory distress syndrome (ards) and certain heart, lung and kidney conditions probably due to inflammation, vasculopathy and coagulopathy induced by angii [22] is reported. thus, the ace1/ace2 imbalance predicts that covid-19 patients with the d allele of ace1, especially the dd genotype, have a higher severity and prevalence of covid-19. further evidence for our hypothesis is provided by results obtained through the 20 study of other viral systems. there are a few lucky people who have escaped infection while being exposed to hiv-1. individuals homozygous for ccr5-î�32 show perfect resistance to hiv-1 [23] . infection with yersinia pestis or smallpox virus were suggested as potential selective pressures favoring ccr5-î�32 [24] . only european or central asians have this characteristic and in norway, ccr5-î�32 allele frequencies reaches nearly 20%. since aids, smallpox and plague are, by nature, independent infectious diseases that should be completely unrelated, it is very intriguing if these infectious diseases are linked by this mutant gene. indeed, in 1999, lalani et al. showed that poxviruses, can exploit chemokine receptors, especially ccr5 to infect some cell types, notably migratory leukocytes [25] . this further suggests a need searching for polymorphism and virological studies to see if there is a difference in the function of the ace2 gene as a sars-cov-2 entry receptor between asians and europeans. besides, several other examples are available in the resistance to certain type infections and genetic mutations. relationship between sickle cell anemia and malaria is well known [26] . although less dramatic, it is speculated that the relationship between typhoid fever and cystic fibrosis, which is a recessive genetic disease commonly found in europeans, is similar. there is also information regarding the relationship between human abo blood type and disease, while ab type is strong against cholera and o type is resistant to tuberculosis [27] . however, 21 our hypothesis suffers from the lack of analogous data available in other viral infections. in other words, if this theory is relevant, we should realize earlier that there is a difference in susceptibility and pathogenicity between western and asian people even in the case of other viral infections before this corona pandemic. still, it is not impossible to explain with a claim that there was no report from the past because there was no appropriate viral pandemic involving europe and east asia in the past. if there is, it may be the spanish flu occurred at the beginning of the last century or some influenzas after that. however, in the case of spanish flu, it was before the identification of the influenza virus, and it was a special event taking place towards the end of world war i in europe, so it is quite difficult to compare the difference in the pathogenicity of the influenza virus in both areas. or is the new coronavirus indeed the first case to show such ethnic differences in terms of pathogenicity? it is hoped that this point will be answered when data in the comparative studies on new coronavirus infections in the united states, where many european and asian immigrants live, will be accumulated in the very near future. in relation to this natural selection of human traits about viral resistance, there 22 are also some records on the relationship between hygienic condition and resistance to infectious diseases. it is generally accepted that asia used to have a denser human population and to have lower hygienic conditions than europe. is corona's low pathogenicity for east asians not related to the situation of asia being less-hygienic than in europe? a similar paradoxical seeming suggestion has been raised for childhood leukemia: the more often a child is infected during the first year of life, the less likely it seems to develop leukemia. epidemiological studies performed by kinlen [28] , and greaves and wiemels [29] separately showed that high socioeconomic status and lack of contact with multiple infections early in life could be a risk factor. based on these observations, zur hausen and de villiers made the following proposal by assuming the existence of a fairly universal lesser-known tumor virus such as ttv. many viral infections during pregnancy and perinatal have a negative effect on the persistence and increase of this oncovirus. intermittent viral infections, by inducing interferon, significantly reduce tumor virus load and reduce the risk of viral chromosomal alterations, prerequisites for malignant transformation [30] . very recently, katoh et al. reported a possible association between lower rate of fatality induced by coronavirus and immunization rate against encephalitis [31] . 23 artificial vaccines against japanese encephalitis (je) may result in relatively lower mortality rate in some countries. je immunization is widespread or included in national programs in many asian countries including china, south korea, japan, and taiwan. in all of these countries, the fatality rate due to covid-19 is very low when compared with countries that don't immunize against je. also, a similar hypothesis has been proposed that the inoculation of bcg vaccine may be effective against infectious diseases caused by the new coronavirus [32] . it is thus very intriguing to find out the reason why recipients are cross-protected against covid-19 conferring lower fatality rate in the countries where immunization is performed against some infectious diseases such as encephalitis or tuberculosis. it will be important to explore the underlying mechanism, such as whether this can be explained by mere nonspecific activation of immunity. as the aspect of nonspecific activation of immunity is considered as one possible mechanism that counteracts sars-cov-2 infection as well as the death rate of infected patients, it might seem worthwhile to ask the question whether the lower rate of infection and mortality due to sars-cov-2 in germany, compared to other european countries, might be connected to differences in vaccination coverages. unfortunately, such an analysis is difficult and has severe inherent limitations, as there is a very high degree of variability of vaccination coverage in germany with respect to age, sex and geography [33] . this does not allow to consider germany as one entity, but rather would require to focus on many different regions and groups, for which it seems difficult to obtain the required complete set of data. however, when the influence vaccination coverage for risk groups is compared between 11 european countries [34] germany is only on place 10 among 11 -certainly not in favour for a more advanced immunization profile of the country. furthermore, the analysis of the national vaccination calendars of france, united kingdom, italy, spain, sweden, portugal and germany [35] shows a large overlap of essential immunizations in these countries -a finding that is not in favour of assuming a specific aspect of immunization in germany that might have a particular negative impact on sars-cov-2 infections and disease. there is no doubt that central europe is much more affected by sars-cov-2 and covid-19 than east asia. the strong difference between east asia and central europe cannot be explained by eventual differences in the frequency of testing, as this would only affect the number of detected cases per inhabitants, but not the number of deaths. we are convinced that the behavioral difference in human contact in both areas of the world can be considered to have a very important influence on the spread of the sars-cov-2, as seen by the impressive positive effect of social distancing on the control of covid-19 in europe. this shows that hypothesis # 1 seems to be relevant to a significant degree for the differences between east asia and central europe. however, hypothesis # 1 cannot explain the complete picture observed, as it would only have an impact on the number of cases in relation to the population, but not on the death rate of cases. as the death rates per cases are also lower in east asia compared to central europe, mechanisms suggested in hypothesis # 2-4 might also contribute to the overall effect. in addition, mechanisms not included into our hypothesis might play essential roles and await to be defined in the future. essential parts of our hypotheses for which we have no direct supportive information so far can be experimentally verified or falsified in the future. these so far unresolved aspects are i) the possible existence of more virulent strains of 26 sars-cov-2 in europe, ii) the effects of repeated infections, possibly in combination with iii) ade, iv) polymorphism of ace2 or some other genes such as tmprss2 [36] and ace1 [19, 37] , and their relation to the function of viral receptor. covid-19 positive cases are already over 5.3 million even at this point in total while number of people infected with sars coronavirus-1 was only about 10,000. considering its near-future expansion in developing areas such as africa and south america, the new coronavirus may reach an even stronger impact than sars-cov-1. moreover, this coronavirus is very easy to mutate due to its original properties. taking these facts into account, this sars-cov-2 has the potential to persist in every corner of the world, has a great possibility of finding and adapting to the best environment in various climates and people's lives, and becoming established in human society. however, the pandemics have taught us some essentials for counteracting in the future. at the beginning of the outbreak of covid-19 in europe, the initial response, especially the delay in response to outbreaks (clusters), demographics, social behavior and lower testing capacity, etc. were sometimes very problematic in response to covid-19. these experiences allowed states that were hit later by the pandemics, like germany, to adjust countermeasures. in germany, the federal and local governments have been involved in the fight against covid-19 from an early stage, and especially with an emphasis on looking for signs of early onset, pcr 27 testing of very large numbers of samples for free, and isolation of defined cases. the medical system had time to be prepared and intensive care beds equipped with artificial respirators were reserved for covid-19 and increased in number. the needed specialized staff was trained. social distancing guidelines were introduced and widely followed. this resulted in slow-down of the pandemic. therefore, we can be confident, that even if european corona strains were more virulent than asian strains, or if europeans were more susceptible to coronaviruses, the authors declare no conflict of interest. world health organization. naming the coronavirus disease (covid-19) and the virus that causes it covid-19: what is next for public health? who scientific and technical advisory group for infectious hazards clinical and demographic characteristics of patients dying from covid-19 in italy versus china why are so many people dying of covid-19 repeated low-dose influenza virus infection causes severe disease in mice: a model for vaccine evaluation repeated exposure to trace amounts of woodchuck hepadnavirus induces molecularly evident infection and virus-specific t cell response in the absence of serological infection markers and hepatitis monoclonal antibodies to the spike protein of feline infectious peritonitis virus mediate antibody-dependent enhancement of infection of feline macrophages antibody-dependent sars coronavirus infection is mediated by antibodies against spike proteins genetic evolution and tropism of transmissible gastroenteritis coronaviruses discovery of a novel canine respiratory coronavirus support genetic recombination among betacoronavirus1 complete genomic sequence of human coronavirus oc43: molecular clock analysis suggests a relatively recent zoonotic coronavirus transmission event most new york coronavirus cases came from europe, genomes show. the new york times patient-derived mutations impact pathogenicity of sars-cov-2 on the origin and continuing evolution of sars-cov-2. national science review genomewide association study of severe covid-19 with respiratory failure online ahead of print sequence variation in the human angiotensin converting enzyme sars-cov-2 infections and covid-19 mortalities strongly correlate with ace1 i/d genotype an insertion/deletion polymorphism in the angiotensin i-converting enzyme gene accounting for half the variance of serum enzyme levels covid-19 and individual genetic susceptibility/receptivity: role of ace1/ace2 might the double x-chromosome in females be protective against sars-cov-2 compared to the single x-chromosome in male? implications of the angiotensin converting enzyme gene insertion/deletion polymorphism in health and disease: a snapshot review homozygous defect in hiv-1 coreceptor accounts for resistance of some multiply-exposed individuals to hiv-1 infection evaluating plague and smallpox as historical selective pressures for the ccr5-delta32 hiv resistance allele use of chemokine receptors by poxviruses sickle cell disease and malaria morbidity: a tale with two tails blood type biochemistry and human disease epidemiological evidence of an infectious basis for childhood leukaemia origins of chromosome translocations in childhood leukaemias virus target cell conditioning model to explain some epidemiologic characteristics of childhood leukemias and lymphomas cross-protection induced by encephalitis vaccines against covid-19 might be a reason for relatively lower mortality rate in some countries is bcg vaccination causally related to reduced covid-19 mortality? online ahead of print vaccination coverage in german adults influenza vaccination coverage among high risk groups of 11 european countries how much money is spent on vaccination across western european countries sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven potease inhibitor covid-19 infections are also affected by human ace1 d/i polymorphism the data show that the two most southern states, bavaria and baden-wã¼rttemberg have much more cases of covid-19 and covid-19-related deaths (both in absolute numbers and per 100 000 people) than the two northern states within bavaria, there are very strong differences between tirschenreuth (one of the hotspots of covid-19 in germany, located in the north east of bavaria on the border to the czech republic) and its directly neighbouring districts wunsiedel compared to districts that are more distant to the hotspot (oberallgã¤u in the north west corner, main-spessart in the south west corner and regensburg in the center of bavaria). taken together, these data show the high efficiency of sars-cov-2 for spreading from a site of initial infection, but also the efficiency of adequate countermeasures taken the authors state no conflict of interests. key: cord-320959-sgdqhtns authors: lee, hanjun title: vitamin e acetate as linactant in the pathophysiology of evali date: 2020-08-12 journal: med hypotheses doi: 10.1016/j.mehy.2020.110182 sha: doc_id: 320959 cord_uid: sgdqhtns the recent identification of vitamin e acetate as one of the causal agents for the e-cigarette, or vaping, product use associated lung injury (evali) is a major milestone. in membrane biophysics, vitamin e is a linactant and a potent modulator of lateral phase separation that effectively reduces the line tension at the two-dimensional phase boundaries and thereby exponentially increases the surface viscosity of the pulmonary surfactant. disrupted dynamics of respiratory compression-expansion cycling may result in an extensive hypoxemia, leading to an acute respiratory distress entailing the formation of intraalveolar lipid-laden macrophages. supplementation of pulmonary surfactants which retain moderate level of cholesterol and controlled hypothermia for patients are recommended when the hypothesis that the line-active property of the vitamin derivative drives the pathogenesis of evali holds. effectively reduces the line tension at the two-dimensional phase boundaries and thereby exponentially increases the surface viscosity of the pulmonary surfactant. disrupted dynamics of respiratory compression-expansion cycling may result in an extensive hypoxemia, leading to an acute respiratory distress entailing the formation of intraalveolar lipid-laden macrophages. supplementation of pulmonary surfactants which retain moderate level of cholesterol and 10 controlled hypothermia for patients are recommended when the hypothesis that the line-active property of the vitamin derivative drives the pathogenesis of evali holds. as of january 14, 2020, there were 2,668 reported hospitalized cases of e-cigarette, or vaping, product use associated lung injury (evali) in the united states [1] . patients with evali typically exhibit respiratory symptoms, such as dyspnea and tachypnea, and often require the 5 receipt of supplemental oxygen due to progressive hypoxemia [2, 3] . computed tomography (ct) scans of the chest of these patients mostly display diffuse ground-glass opacity, a non-specific sign characteristic of lung injury that is often indistinguishable from those induced by viral infections [4] . extensive investigation into evali has revealed an emerging link between the disease and tetrahydrocannabinol (thc). reportedly, as many as 82% of patients with evali 10 used thc-containing e-cigarettes, while those who solely used nicotine-containing products accounted for a mere 14% of all cases [1] . correspondingly, on october 4, 2019, the food and drug administration issued a public warning against thc-containing vaping products, and the rate of emergency department (ed) visits due to evali has significantly declined since then [5] . the pathophysiology and the definitive cause of evali are yet to be established. initial efforts 15 to elucidate the pathophysiology of the disease have mainly focused on its association with thc. 4 providers still mostly rely on a diagnosis of exclusion when a patient with a history of thccontaining vaping product usage exhibits severe respiratory symptoms indicative of evali [3] . since the epidemic of evali has already reached its post-peak period and is expected to enter a post-epidemic one in a near future [5] , thanks to global efforts to strengthen regulations against 5 e-cigarettes, it is likely that we might not be able to fully characterize the disease before the epidemic is finally over. nonetheless, for thousands of hospitalized patients who still suffer from the disorder and for the deceased who died without knowing what they were exactly dying from, the pathophysiological characterization of the disease should be continued and recent breakthroughs in the field are tremendously supporting us. 10 recently, investigators at the centers for disease control and prevention (cdc) identified vitamin e acetate, a chemical widely utilized as a diluent for thc-containing vaping fluids, as one of the potential causal agents of evali [12] . among 51 submitted samples of bronchoalveolar lavage (bal) fluid from patients, 48 samples contained high content of vitamin e acetate exceeding 2.32 nm, while none was observed in those of healthy e-cigarette users 15 without evali. primary toxicants other than vitamin e acetate were nearly absent in bal fluid samples from patients, indicating a strong association of vitamin e acetate in the pathology. in a field predominated by confusion, this discovery sets an important milestone. vitamin e has long been thought of as an antioxidant dwelling in the lipid bilayer. in fact, the discovery of the vitamin itself has come from reports of fetal resorption in murine models which were depleted of the vitamin [13] . the lethality of vitamin e deficiency was later attributed to 5 the lack of its antioxidative capacities, and it is now well-established that the vitamin is required for the protection of polyunsaturated fatty acids from being unwantedly oxidized [14] . however, vitamin e acetate, given the additional ester moiety, is deprived of the antioxidative property and is markedly more thermostable. furthermore, its affinity to α-tocopherol transfer protein (αttp) is ~50-fold lower than that of the natural stereoisomer of vitamin e [15] , indicating the 10 significance of an extensive water bridge between the hydroxyl group and residues tyr117, ser136, and ser140 in the binding of the vitamin to αttp [16] . despite αttp often being recognized as a hepatocyte-specific protein which presents vitamin e from endosomal compartments to the plasma membrane for secretion [17] , it is also expressed in the lung where it non-canonically increases the level of vitamin e upon hypercapnia [18] . due to these significant 15 differences between vitamin e and vitamin e acetate, it is important to clarify whether the lung injury observed in patients with evali is induced solely by the esterified derivative of the vitamin. unfortunately, the distinguishment between the two compounds has largely been neglected in the field of evali, and the scarcity of information on the esterified derivative of the vitamin has forced many investigators to rely on reported biochemical properties of 20 unmodified vitamin e in elucidating the pathophysiology of evali. unlike those administered via dietary uptake [19] , wherein the compound is readily hydrolyzed by cellular esterases, including pancreatic carboxyl ester hydrolase and cholesteryl ester hydrolase [20, 21] , inhaled vitamin e acetate does not undergo esterase-mediated hydrolysis in the time scale of several hours [22, 23] . this indicates that most of the inhaled vitamin e acetate would remain unhydrolyzed in the lung of e-cigarette users even until the next vaping session in a typical setting. this is analogous to those observations made in skin, wherein the hydrolysis rate of the esterified derivative was measured as mere 5% [24] . the mechanistic detail on why 5 inhaled vitamin e acetate is resistant to esterase-mediated hydrolysis, however, is poorly understood. despite the remaining controversy on which of these compounds is responsible for the pathogenesis of evali, there is ample amount of evidence to rule out the antioxidative property of vitamin e as its driving force. antioxidative activities have long been recognized as a 10 protective mechanism against injury in respiratory disorders. indeed, pulmonary surfactants contain significant amounts of superoxide dismutase and catalase activities, which act to scavenge extracellular reactive oxygen species, such as hydrogen peroxide, within the pulmonary system [25] . similarly, type ii alveolar pneumocytes secrete vitamin e alongside pulmonary surfactants to protect the respiratory system against inhaled oxidants [26, 27] . however, it is 15 worth to note that the protective effect of vitamin e in the pulmonary system is not solely due to its antioxidative property. indeed, the transcriptional activation of αttp, which increases the level of vitamin e in the pulmonary system, protects against ventilator-induced lung injury in murine models without affecting antioxidant response signaling, such as those dependent on nuclear factor erythroid 2-related factor 2 (nrf2) [18] . 20 recent investigations on the biological action of the vitamin have raised an emerging appreciation of its non-antioxidative properties [28, 29] . although it remains a matter of controversy whether non-antioxidative properties of the vitamin can be strictly discriminated from its antioxidative property [30] , thorough examination of the effects of vitamin e other than antioxidation in this poorly characterized respiratory disorder remains valuable. in this regard, herein i discuss several possible mechanisms by which vitamin e acetate in vaping fluids may drive the pathogenesis of evali. as the biochemical properties of the vitamin derivative have been insufficiently characterized, i focus on those of an unmodified vitamin. currently, there are 5 five established non-antioxidative properties of vitamin e in the biological system: i) its ability to induce gel-liquid crystalline phase transition, ii) its active deposition in the lipid droplet of macrophages, iii) its modulation of the antidiabetic cascade involving diacylglycerol kinase (dgk) and protein kinase c (pkc), iv) its activation of the xenobiotic-sensing pregnane x receptor (pxr) signaling, and v) its ability to modulate lateral phase separation. in each section, 10 i discuss possible ways by which these properties may contribute to the pathophysiology of evali and whether these properties are expected to be shared by vitamin e acetate. at last, i argue that the line-active property of the vitamin deserves academic attention. although toxic byproducts of heating vitamin e acetate have recently garnered considerable interest [31] , i instead focus on the biological action of the vitamin derivative itself, as there is yet lacking 15 amount of evidence that pinpoints toxic byproducts as the pathological driving force for evali. indeed, lanzarotta and colleagues have recently shown that the majority of vaporized vitamin e acetate exist either as an equimolar complex with thc or as a dimer [32] . 20 among the five non-antioxidative properties of vitamin e, investigators from the lung injury response laboratory working group have focused on its peculiar ability to induce gel (l β ) to liquid crystalline (l c ) phase transition in model saturated phosphatidylcholine bilayers [12] (l in the notation stands for lamellar). this property of the vitamin is believed to be the consequence 8 of its structural deviance from phospholipids. as the structure of vitamin e greatly differs from a typical phospholipid, especially in its rigid chromane double ring, the vitamin greatly perturbs the packing of phospholipids within the l β phase, resulting in a facilitated gel-liquid crystalline phase transition ( fig. 1 ) [33] . for instance, the addition of either vitamin e or vitamin e acetate 5 in molar ratios as high as 16 mol% in cholesterol-free model dimyristoylphosphatidylcholine (dmpc) membranes lowers the critical temperature required for gel-liquid crystalline phase transition for ~1.2 k [33, 34] . however, since cholesterol, a major constituent of biological membranes, also exhibits profound structural deviance from a typical phospholipid, it also facilitates gel-liquid crystalline phase transition in a similar manner. compared to free 10 cholesterol, vitamin e acetate is 1.5 times more potent in modulating the phase behavior of the lipid bilayer. as a consequence, one possible mechanism by which vitamin e acetate intoxication may lead to a fatal lung injury is its induction of liquid crystalline phase in pulmonary surfactants, which would likely influence the respiratory compression-expansion cycling. 15 a major downfall of blount and his colleagues' hypothesis is the abundance of free cholesterol in human pulmonary surfactants. indeed, free cholesterol level in bal fluids were measured as ~2.6 mm in murine models of pulmonary alveolar proteinosis (pap) [35] , while the level of vitamin e acetate observed in patients with evali, who occasionally feature signs of pap [36] , was in nanomolar range [12] . in contrast, the decrease in the critical temperature induced by the 20 addition of vitamin e acetate was of roughly the same orders of magnitude compared to that of free cholesterol. this indicates that the overall contribution of vitamin e acetate in the modulation of phase behavior is at least three orders of magnitude smaller compared to that of endogenously available free cholesterol. furthermore, the existence of cholesterol in human pulmonary surfactants is known to completely abrogate gel-liquid crystalline phase transition [37] . in explanation, the driving force for gel-liquid crystalline phase transition induced by vitamin e acetate in model saturated phosphatidylcholine bilayers has been its perturbation of phospholipid packing by structural deviance. the abundance of free cholesterol, of which 5 structure is as rigid as the head portion of the vitamin, in biological membranes offsets the structural deviance of vitamin e in lipid bilayers, resulting in an inability to induce gel-liquid crystalline phase transition (fig. 1) . indeed, the observation of gel-liquid crystalline phase transition has been limited to the cholesterol-free model saturated phosphatidylcholine bilayers [33, 34] and the membrane of mycoplasma laidlawii, which is depleted of cholesterol and is 10 enriched with saturated fatty acids [38] . accounting for the rationale behind the selection of mycoplasma laidlawii as the model system for investigating gel-liquid crystalline phase transition, steim and colleagues concisely stated, "because cholesterol interferes, to detect a phase change above the ice point in a membrane, an organism containing rather saturated fatty acids but little or no cholesterol must be chosen" [38] . 15 another important characteristic of vitamin e acetate in the biological system is its deposition in lipid droplets [39, 40] , and many investigators have especially focused on the observation of intraalveolar lipid-laden macrophages as it has been proven to be one of the most prominent 20 features of lung biopsies from patients with evali [41] . indeed, intraalveolar lipid-laden macrophages were also evident in a murine model of vitamin e acetate inhalation (daily inhalation of 77.3-167.5 μg vitamin e acetate per gram of body weight, two weeks of exposure), of which effect was largely absent in mice inhaled a mixture of propylene glycol and vegetable glycerin [42] . intriguingly, when a single dose of vitamin e acetate (inhalation of 0.1 μg vitamin e acetate per gram of body weight) was inhaled into the lipopolysaccharide-treated lungs of murine models, the substance greatly attenuated the inflammatory response, despite being 3-fold less potent than vitamin e [23]. this indicates that it is the excessive accumulation 5 of vitamin e acetate, rather than its transient exposure, which is responsible for the pathogenesis of evali. although it is tempting to speculate that intraalveolar lipid-laden macrophages observed are merely the consequence of excessive deposition of vitamin e acetate within the pulmonary system, increasing amount of reports suggest that such assumptions are erroneous. importantly, 10 there is a lacking histological evidence of exogenous lipoid pneumonia (elp) in patients suffering from evali, despite the prevalence of intraalveolar lipid-laden macrophages in bal fluids. for instance, when a 63-year-old woman had regularly smoked cod-liver oil, she acquired a non-oncologic perihilar mass in the right middle lobe of her lung, indicative of elp [43] , but these elp-like histological signs were mostly absent in patients with evali [11] . as guerrini 15 and colleagues importantly mention, intraalveolar lipid-laden macrophages do not always originate from an excessive uptake of exogenous lipids, but may often emerge as a common immunopathological response following sustained inflammation [44] . however, unmodified vitamin e is indicated in the downregulation of cd36 [45] , which functions to promote the formation of lipid-laden macrophages (foam cells) [46] . furthermore, vitamin e also ameliorates this hypothesis also does not provide us with an exact mechanism by which vitamin e acetate may initiate inflammation. since vitamin e acetate is a viscous liquid at physiological temperature, activation of the nlrp3 inflammasome through crystal-induced lysosomal rupture as in models of silicosis [48] seems unlikely, despite robust activation of the inflammasome in e5 cigarette users [49] . vitamin e is also indicated in an active inhibition of 5-lipoxygenase (5-lo), which converts fatty acids to leukotrienes, promoting inflammation [50, 51] . importantly, vitamin e acetate is also reported to inhibit 5-lo activity [50], despite being twice less potent than the unmodified vitamin, supporting the view that vitamin e acetate does not induce elp in patients with evali via the inflammatory response. 10 in the field of diabetes mellitus, vitamin e is a potent agonist of dgkα, which catalyzes the conversion of diacylglycerol (dag) to phosphatidic acid (pa) and thereby inhibits the catalytic activity of pkcα [52] [53] [54] . agonism of dgkα by vitamin e derivatives are known to be 15 dependent on the chromane double ring of the molecule, as both vitamin e analogs containing the moiety, such as troglitazone and trolox, and non-antioxidative vitamin e derivatives, such as vitamin e succinate, exhibited robust activation and subsequent plasmalemmal translocation of the kinase [55] . vitamin e acetate may also antagonize the catalytic activity of pkcα in a dgkindependent manner, as there are studies indicating that the substance is capable of competing 20 with dag for its binding site in pkcα [56]. intriguingly, d-α-tocopherol, but not d-β-tocopherol nor d-γ-tocopherol exhibited robust inactivation of pkcα, indicating a pivotal role of methylation at c5 and c7 of the chromanol structure [57] . supplementation of vitamin e acetate in murine models of diabetic cardiomyopathy showed largely protective role for the chemical [58] , but it is worth to note that most of the substances supplemented via dietary uptake undergo hydrolysis to yield vitamin e. the protective effect of vitamin e derivatives in diabetes mellitus was completely ameliorated in dgkα knockout mice, indicating a pivotal role of the kinase in this antidiabetic signaling cascade [54] . 5 since the observed level of vitamin e acetate in bal fluids is of same orders of magnitude with that of dag, a potent second messenger with which vitamin e acetate competes for binding [59] , the activity of the substance as a modulator of dgk-pkc pathway is physiologically relevant. however, the reported anti-inflammatory effects of d-α-tocopherol in the pulmonary system through post-translational inactivation of pkcα significantly undermines this hypothesis 10 [60, 61] . since many observations suggest that the antagonism of pkcα by d-α-tocopherol is independent of its antioxidative property [57], it is anticipated that vitamin e acetate might play an analogous anti-inflammatory role in the pulmonary system [62, 63] . however, whether the esterified derivative antagonizes pkcα still remains to be investigated. overall, the modulation of dgk-pkc pathway by vitamin e acetate seems yet incapable of accounting for the 15 observation of pro-inflammatory intraalveolar lipid-laden macrophages in patients with evali, although there is yet lacking amount of evidence to completely rule out this hypothesis. when vitamin e is administered into the biological system, it transforms into a plethora of 20 bioactive metabolites [64]. this includes several of the pxr agonists, such as α-tocopherol-13'-cooh, γ-tocotrienol, and garcinoic acid [65, 66] . since pxr forms a heterodimer with 9cis retinoic acid receptor (nr2b) to activate the transcription of cytochrome p450 monooxygenase genes indicated in the clearance and detoxification of xenobiotic substances 13 [67] , pro-agonist role of vitamin e acetate may be relevant in the pathophysiology of evali. indeed, polymorphisms in the cytochrome p450 genes have been associated with drug-induced interstitial lung diseases (diild), characterized by coarse reticular opacity and intraalveolar lipid-laden macrophages [68] . this pathology is consistent with that of evali, wherein patients 5 often reported patterns of giant-cell interstitial pneumonia [4] . in this picture, evali can be seen as a variant of diild caused by an excessive accumulation of vitamin e acetate in the pulmonary system. despite its pathophysiology being not fully characterized, diild often involves the generation of reactive oxygen species [69, 70] . vitamin e acetate, however, is known to protect the biological system against reactive oxygen species even in the absence of 10 apparent hydrolysis [24] , possibly through its inhibition of 5-lo [15] or through infinitesimal hydrolysis to vitamin e as previously speculated [24] . furthermore, diild is a highly heterogenous disorder, that is often dependent on genetic polymorphisms rare enough to avert federal regulations during drug approval [71, 72] . for instance, the demonstration of respiratory symptom after oral administration of fenfluramine or dexfenfluramine was on a patient with a 15 rare cyp2d6*1/*3 haplodeficient variant of the cytochrome p450 2d6 (cyp2d6) gene [68] . how haplodeficiency of the cyp2d6 gene contributes to the pathogenesis of diild remains unclear. if vitamin e acetate behaves as a pro-agonist of pxr, a transcriptional activator of cytochrome p450 genes, and thereby contributes to the pathogenesis of evali, the symptoms of evali 20 should be replicated by inhalation of other well-established pxr agonists, such as corticosteroids [73] [74] [75] . nebulized budesonide or fluticasone, however, did not induce respiratory distress when inhaled repeatedly (inhalation of 125-200 μg corticosteroids each session, 2-4 times daily, 8 days) in young children with a severe onset of asthma [76] . furthermore, even in the absence of 14 vitamin e acetate, vaping induced significant increase in the activity of cytochrome p450 enzymes, including those hydroxylates vitamin e, such as cyp2b1, cyp2c11, and cyp3a1, but without apparent respiratory distress [77] . however, it remains to be studied whether an excessive accumulation of vitamin e acetate produces pxr-agonizing metabolites above a 5 certain threshold such that it may induce an acute respiratory distress. overall, the pro-agonist role of vitamin e acetate on pxr seems relevant in the pathophysiology of evali, but there is yet lacking amount of evidence which indicates that the pxr agonism drives the pathogenesis of evali. 10 recent developments in membrane biophysics indicate that an unmodified vitamin e has an active role in lateral phase separation. due to the rigid chromane double ring, which significantly reduces the entropic cost required for its association with cholesterol [78] , unmodified vitamin e efficiently releases the line tension (i.e. two-dimensional counterpart of a surface tension) 15 between the liquid-disordered l α and the liquid-ordered l o phase of lipid polymorphs [79] . indeed, oral supplementation of vitamin e acetate in hiv/aids patients, which is readily converted to vitamin e by cellular esterases, has been successful in reducing the viral load [80] , as the molecule disrupts the tensile two-dimensional l α /l o phase boundary required for viral entry [81, 82] . in membrane biophysics, this vitamin is classified as a linactant [83] , which is a 20 two-dimensional counterpart of a surfactant, as it reduces the line tension at the two-dimensional phase boundaries (fig. 2a) . in explanation, when one puts a drop of oil into the water, gravity and surface tension compete to generate the lobular shape of the droplet. the addition of sodium dodecyl sulfate (sds), a well-known surfactant, reduces the surface tension such that the oil droplet loses its ability to maintain its structure against gravitational force. as a consequence, oil emulsifies into the water, if not become completely miscible [84] . similarly, when one adds vitamin e onto the lipid bilayer exhibiting l α /l o phase separation, it reduces the line tension, which competes with entropy to 5 form an enlarged cholesterol-enriched microdomain. as it has been shown that the molar ratio of vitamin e within a typical lipid bilayer is insufficient to completely abolish lateral phase separation [85] , vitamin e acts to reduce the size of the microdomain and thereby emulsifies cholesterol-enriched l o into cholesterol-depleted l α (fig. 2c) . despite the fact that the line-active property of vitamin e acetate has not yet been explicitly tested, the small size and the abundance 10 of ideally localized nonbonding electron donors of the ester moiety provide ample reason to hypothesize that the substance is a linactant. the content of vitamin e within the lipid mixture that is sufficient to function as a potent linactant is typically below 10 mol% [79, 85] , which is comparable to that of vitamin e acetate, estimated based on its concentration in bal fluid samples from patients with evali. 15 an important consequence of the dissemination of the cholesterol-enriched microdomain is a profound increment in the surface viscosity of pulmonary surfactants [86] . lateral phase separation is an important source of free area, defined as the difference between observed and minimum area per molecule [87] . this is analogous to the observation of reduction in the total volume after mixing ethanol with water. in such a case, the phenomenon can be interpreted as if 20 the free volume of each molecule has decreased following mixing. similarly, mixing of l o with l α results in a substantial decrease in the free area, while demixing induces its subsequent increase. according to the free area theory of surface viscosity [88] , surface viscosity exponentially increases following the reduction of free area, driven by reduced tensile force along the two-dimensional phase boundaries. indeed, a mere 3% decrease in the free area resulted in a striking 12-fold increase in the surface viscosity of pulmonary surfactants [87] . in accordance, upon hyperpnea, the biological system increases free cholesterol level in pulmonary surfactants, to reduce surface viscosity, which is required for facilitated respiration [89] . 5 similarly, the reduced free area following the inhalation of vitamin e acetate may considerably increase the surface viscosity of the pulmonary surfactant, resulting in a disrupted respiratory compression-expansion cycling and a subsequent hypoxemia (fig. 2b) . in this picture, intraalveolar lipid-laden macrophages are consequences of hypoxemia-induced excessive intracellular accumulation of triglycerides [90] , and extensive vacuolization, 10 multinucleation, and reactive hyperplasia observed in type ii alveolar pneumocytes are products of an extensive tissue hypoxia [91, 92] . taking into account that the pulmonary-associated surfactant protein b (sp-b) often cooperates with cholesterol in reducing the surface viscosity of pulmonary surfactants during respiratory compression-expansion cycling [86], other than its known role in the reduction of surface tension in the steady state, it is worth to note that the 15 partial deficiency of the protein results in a respiratory distress analogous to those observed in patients with evali [93] . in sum, vaporized vitamin e acetate, a thermostable derivative of the well-established linactant, may substantially increase the surface viscosity of pulmonary surfactants and thereby confer the pathogenesis of evali. 20 evaluating the hypothesis the above-described biological actions of vitamin e derivatives are closely intertwined to each other [83, 94] , and the effect of vitamin e acetate on the pulmonary system in patients with evali is likely to be the sum of all these actions, rather than originating from a single action of the vitamin derivative. for instance, cholesterol-enriched microdomains, or lipid rafts [95] , are involved in a plethora of cellular activities, including pkcα [96] and 5-lo signaling [97] . as a consequence, it is worth to note that the evaluation of these hypotheses should focus on identifying the major driving force for the pathogenesis of evali, rather than a sole cause, 5 which most likely does not exist. to evaluate whether the line-active property of vitamin e acetate is driving the pathogenesis of evali, we must answer the following questions. first, is vitamin e acetate, like d-α-tocopherol, a potent linactant? if it is, to what extent does the substance reduce the line tension at the twodimensional phase boundaries and increase the surface viscosity? molecular dynamics 10 simulations, such as those previously introduced by rosetti and colleagues [98] , would particularly be useful, as they enable us to roughly estimate the fold change in the surface viscosity of pulmonary surfactants and thereby to evaluate the validity of the hypothesis. second, are linactants, in general, capable of inducing respiratory distress when inhaled through ecigarettes? one obvious experiment that must be done is the repeated inhalation of hybrid lipids 15 in murine models. hybrid lipids, which are phospholipids that contain both saturated and unsaturated acyl chains, are one of the most widely accepted class of linactants [98] [99] [100] [101] . as a consequence, repeated inhalation of hybrid lipids, analogous to those attempted by bhat and colleagues, should induce similar histological signs, for the hypothesis to hold. to rule out the potential role of vitamin e acetate as an inducer of gel-liquid crystalline phase transition, rescue 20 experiments using an inhalation setup for dimethyl sulfoxide (dmso), which abrogates gelliquid crystalline phase transition [102] , can be attempted. to rule out the possibility that vitamin e acetate drives the pathogenesis of evali through modulating dgk-pkc or pxr signaling, vitamin e acetate inhalation experiment using either pkcα [103] or pxr knockout mice [104] can be pursued. characterization of the lipid composition of the intraalveolar lipidladen macrophages is also crucial, as it may assist us to rule out the hypothesis that an acute respiratory distress associated with evali is indeed a form of elp. lastly, as we still suffer from the lack of characterization of the type of injury in evali, i propose an rna sequencing 5 (rna-seq) experiment for lung tissues obtained from murine models of evali [42] . analyses on the level of transcripts associated with the biological action of the vitamin, such as cytochrome p450 monooxygenase genes, hypoxia-inducible factor 1 (hif-1), and nrf2, as well as gene ontology analyses would greatly assist us to better define the pathology of the new respiratory disorder. rna-seq experiment would also allow us to develop reliable biomarkers for 10 evali to better assist health care providers in the differential diagnosis of the vaping-induced respiratory disorder against viral infections. living in the era of the pandemic outbreak of the coronavirus disease 2019 (covid-19), wherein tens of millions of people worldwide are being hospitalized with symptoms often indistinguishable from those of evali [105, 106] , establishing diagnostic biomarkers for evali would particularly be valuable. in summary, i discussed several possible mechanisms by which vitamin e acetate may drive the pathogenesis of evali and specifically advocated the role of vitamin e acetate as a linactant (table 1) . based on recent discoveries from the field of membrane biophysics, i proposed that 5 the alleged linactivity of vitamin e acetate, a thermostable derivative of a well-established linactant, significantly increases the surface viscosity of pulmonary surfactants in the alveoli to confer acute respiratory distress associated with evali. this hypothesis leads to new possible medication approaches for the new pulmonary disease. if it is true that the line-active property of the vitamin derivative is what is causing the disease, supplementation of pulmonary surfactants 10 as well as corticosteroid treatments should be valid. to reduce the elevated viscosity of pulmonary surfactants, pulmonary surfactants which retain a moderate level of cholesterol, such as infasurf [107] , may be recommended for use. in addition, in a biophysical perspective, since the entropy component of the free energy, which is proportional to the temperature of the system, works against lateral phase separation in regular solutions [108, 109] , one can reverse the effects 15 of vitamin e acetate by slightly lowering the temperature of the system. controlled hypothermia to rescue lateral phase separation and thereby reduce surface viscosity, as introduced by autilio and colleagues [110] , might be a useful way to manage acute respiratory distress syndrome associated with evali. the current outbreak of evali is an emerging crisis affecting a great portion of the global 20 population. as pathophysiologists, we have a duty to decipher the pathophysiology of evali for the good of all patients suffering from the disease, especially after the recent identification of the widely utilized derivative of the vitamin as one of the causal agents for the new catastrophic pulmonary disease. that vitamin e acetate may facilitate gel-liquid crystalline phase transition to confer acute 5 respiratory distress [12] . in this picture, vitamin e acetate perturbs the packing of phospholipids within the lipid bilayer such that the system thermodynamically prefers the liquid crystalline phase over the gel phase. however, cholesterol, which exists in large excess to vitamin e acetate in the biological membrane, not only exerts similar effect in model saturated phospholipid bilayers, but also reportedly abolishes gel-liquid crystalline phase transition [38] . therefore, gel-10 liquid crystalline phase transition may be irrelevant in the pathogenesis of evali. reduced line tension within the two-dimensional phase boundary confers smaller microdomains 5 and decreases the free area of lipids [87] . as a result, the surface viscosity of pulmonary surfactants exponentially increases to perturb the dynamics of respiratory compressionexpansion cycling, causing acute respiratory distress analogous to those seen in patients with evali. update: characteristics of a nationwide outbreak of e-cigarette, or vaping, product use-associated lung injury -united states pulmonary illness related to e-cigarette use in illinois and wisconsin -final report update: interim guidance for health care providers evaluating and caring for patients with suspected e-cigarette, or vaping, product use associated lung injury -united states imaging of vaping-associated lung disease syndromic surveillance for e-cigarette, or vaping, product use-associated lung injury tolerance to hypothermic and antinoceptive effects of ∆9-tetrahydrocannabinol (thc) vapor inhalation in rats lasting effects of repeated ∆9-tetrahydrocannabinol vapour inhalation during adolescence in male and female rats tree-in-bloom": severe acute lung injury induced by vaping cannabis oil pathology of vaping-associated lung injury more on the pathology of vaping-associated lung injury pulmonary lipid-laden macrophages and 5 vaping vitamin e acetate in bronchoalveolarlavage fluid associated with evali a history of vitamin e vitamin e, antioxidant and nothing more affinity for α-tocopherol transfer protein as a determinant of the biological activities of vitamin e analogs the molecular basis of vitamin e retention: structure of human α-tocopherol transfer protein α-tocopherol transfer protein stimulates the secretion of α-tocopherol from a cultured liver cell line through a brefeldin a-insensitive pathway α-tocopherol transfer protein mediates 20 protective hypercapnia in murine ventilator-induced lung injury safety assessment of the substance α-tocopherol acetate for use in food contact materials all-rac-α-tocopherol acetate is a better vitamin e source than all-rac-α-tocopherol succinate for broilers the distribution and relative hydrolysis of tocopheryl acetate in the different matrices coexisting in the lumen of the 5 small intestine during digestion could explain its low bioavailability supercritical fluid-aerosolized vitamin e pretreatment decreases leak in isolated oxidant-perfused rat lungs aerosol-administered α-tocopherol attenuates lung inflammation in rats given lipopolysaccharide intratracheally hydrolysis of rrr-αtocopheryl acetate (vitamin e acetate) in the skin and its uv protecting activity (an in vivo 15 study with the rat) characterization of antioxidant activities of pulmonary surfactant mixtures type ii pneumocytes secrete vitamin e 20 together with surfactant lipids oxidative stress and antioxidants at biosurfaces: plants, skin, and respiratory tract surfaces vitamin e: non-antioxidant roles the rise, the fall and the renaissance of vitamin e vitamin e: emerging aspects and new directions potential for release of pulmonary toxic ketene from vaping pyrolysis of vitamin e acetate hydrogen bonding between tetrahydrocannabinol and vitamin e acetate in unvaped, aerosolized, and condensed 10 aerosol e-liquids interaction of vitamin e with saturated phospholipid bilayers modulated phases of phospholipid bilayers induced by tocopherols targeting cholesterol homeostasis in lung diseases a unique case of secondary pulmonary alveolar proteinosis following e-cigarette, or vaping, product use-associated lung injury (evali) studies on lecithin-cholesterol-water interactions by differential scanning calorimetry and x-ray diffraction calorimetric evidence for the liquid-crystalline state of lipids in a biomembrane lipid droplet functions beyond energy storage nonalcoholic fatty liver disease impairs the cytochrome p-450-dependent metabolism of α-tocopherol (vitamin e) vaping-associated acute 10 respiratory failure due to acute lipoid pneumonia an animal model of inhaled vitamin e acetate and evali-like lung injury a woman who took cod-liver oil and 15 smoked lipid-laden macrophages as biomarkers of vaping-associated lung injury antagonistic effects of oxidized low density lipoprotein and α-tocopherol on cd36 scavenger receptor 20 expression in monocytes: involvement of protein kinase b and peroxisome proliferatoractivated receptor-γ a cd36-dependent signaling cascade is necessary for macrophage foam cell formation vitamin e ameliorates ox-ldl-induced foam cells formation through modulating the activities of oxidative stress-induced nf-κb pathway silica crystals and aluminum salts activate the nalp3 inflammasome through phagosomal destabilization electronic versus combustible cigarette effects on inflammasome component release into human lung inhibition of 5-lipoxygenase by vitamin e endogenous metabolites of vitamin e limit inflammation by targeting 5-lipoxygenase diacylglycerol-protein kinase c activation by vitamin e in aorta of diabetic rats and cultured rat smooth muscle cells exposed to elevated glucose levels α-tocopherol specifically inactivates cellular protein kinase c α by changing its phosphorylation state diacylglycerol kinase alpha is involved in the vitamin e-induced amelioration of diabetic nephropathy in mice importance of chroman ring and tyrosine endothelial relaxation is disturbed by oxidative stress in the diabetic rat heart: influence of tocopherol as antioxidant intracellular diacylglycerol: a mitogenic second messenger proposable as marker of transformation in 15 squamous cell carcinoma of the lung α-tocopherol inhibits the respiratory burst in human monocytes: attenuation of p47(phox) membrane translocation and phosphorylation opposing immunoregulatory functions during inflammation by regulating leukocyte recruitment the α-tocopherol form of vitamin e reverses age-associated susceptibility to streptococcus pneumoniae lung infection by modulating pulmonary neutrophil recruitment the alpha-tocopherol form of vitamin e boosts elastase activity of human pmns and their ability to kill streptococcus pneumoniae vitamin e biotransformation in humans the long chain α-tocopherol metabolite α-13'-cooh and γ-tocotrienol induce p-glycoprotein expression and activity by activation of the pregnane x receptor in the intestinal cell line ls 180 garcinoic acid is a natural and selective agonist of pregnane x receptor the nuclear pregnane x receptor: a key regulator of xenobiotic metabolism relationship between drug-induced interstitial lung diseases and cytochrome p450 polymorphisms drug induced interstitial lung disease drug-induced interstitial lung disease: mechanisms and best diagnostic 20 approaches role of cytochrome p450 polymorphisms in the development of pulmonary drug toxicity: a case-control study in the netherlands whole genome sequencing to identify predictive markers for the risk of drug-induced interstitial lung disease pregnane x receptor and retinoid x receptor-α expression in human hepatocytes: synergistic increase of cyp3a4 induction by pregnane x receptor activators pxr-mediated induction of human cyp3a4 and mouse cyp3a11 by the glucocorticoid 10 budesonide identification and validation of novel human pregnane x receptor activators among prescribed drugs via ligand-based virtual screening effectiveness of high repeated doses of 15 inhaled budesonide or fluticasone in controlling acute asthma exacerbations in young children e-cigarettes induce toxicological effects that can raise the cancer risk cholesterol as a co-solvent and a ligand for 20 membrane proteins tuning membrane phase separation using nonlipid amphiphiles effects of vitamin e and c supplementation on oxidative stress and viral load in hiv-infected subjects line tension at lipid phase boundaries as driving force for hiv fusion peptide-mediated fusion hiv virions sense plasma membrane heterogeneity for cell entry line active molecules promote inhomogeneous structures in membranes: theory, simulations and experiments capillarity and wetting phenomena the antioxidant vitamin e as a membrane raft modulator: tocopherols do not abolish lipid domains effect of cholesterol and surfactant protein b on the viscosity of phospholipid mixtures effect of cholesterol nanodomains on monolayer morphology and dynamics in-plane steady shear viscosity of monolayers at the air/water interface and its dependence on free area effect of hyperpnea on the cholesterol to disaturated phospholipid ratio in alveolar surfactant of rats hypoxia converts human macrophages into triglyceride-loaded foam cells lysosomal alterations in hypoxic and reoxygenated hearts. i ultrastructural and cytochemical changes hif and the lung: role of hypoxia-inducible factors in pulmonary development and disease partial deficiency of surfactant protein b in an infant with chronic lung disease vitamin e: regulatory role on signal transduction functional rafts in cell membranes the nk1 receptor localizes to the plasma membrane microdomains, and its activation is dependent on lipid raft integrity protein profiling of plasma membranes defines aberrant signaling pathways in mantle cell lymphoma molecular insight into the line tension of bilayer 20 membranes containing hybrid polyunsaturated lipids partitioning of lipids at domain boundaries in model membranes modulation of a small two-domain lipid vesicle by linactants physical properties of the hybrid lipid popc on micrometer-sized domains in mixed lipid membranes insights into the dynamics of dmso in phosphatidylcholine bilayers enac activity and expression is decreased in the lungs of protein kinase c-α knockout mice pregnane x receptor knockout mice display aging-dependent wearing of articular cartilage clinical features of patients infected with 2019 novel coronavirus in wuhan, china notes from the field : e-cigarette, or vaping, product use-associated lung injury cases during the covid-19 response -california commercial versus native surfactants: surface activity, molecular components, and the effect of 20 calcium themodynamics of high polymer solutions solutions of long chain compounds controlled hypothermia may improve kim at the republic of korea army for their administrative assistance. key: cord-346281-sma6e891 authors: maldonado, valente; loza-mejía; chávez alderete, jaime title: repositioning of pentoxifylline as an immunomodulator and regulator of the renin-angiotensin system in the treatment of covid-19 date: 2020-06-09 journal: med hypotheses doi: 10.1016/j.mehy.2020.109988 sha: doc_id: 346281 cord_uid: sma6e891 pentoxifylline (ptx) is a phosphodiesterase inhibitor that increases cyclic adenosine monophosphate levels, which in turn activate protein kinase, leading to a reduction in the synthesis of proinflammatory cytokines to ultimately influence the renin-angiotensin system (ras) in vitro by inhibiting angiotensin 1 receptor (at1r) expression. the rheological, anti-inflammatory, and renin-angiotensin axis properties of ptx highlight this drug as a therapeutic treatment alternative for patients with covid-19 by helping reduce the production of the inflammatory cytokines without deleterious effects on the immune system to delay viral clearance. moreover, ptx can restore the balance of the immune response, reduce damage to the endothelium and alveolar epithelial cells, improve circulation, and prevent microvascular thrombosis. there is further evidence that ptx can improve ventilatory parameters. therefore, we propose repositioning ptx in the treatment of covid-19. the main advantage of repositioning ptx is that it is an affordable drug that is already available worldwide with an established safety profile, further offering the possibility of immediately analysing the result of its use and associated success rates. another advantage is that ptx selectively reduces the concentration of tnf-α mrna in cells, which, in the case of an acute infectious state such as covid-19, would seem to offer a more strategic approach. the immune dysregulation observed in covid-19 clinically translates into a secondary hemophagocytic syndrome or lymphohistiocytosis, acute respiratory distress syndrome (ards), and multiple organ failure. however, multiple studies have indicated that immunosuppression with corticosteroids impairs the immune response to various respiratory viruses as it affects the induction of antiviral interferon (ifn) type i. therefore, the decision to immunosuppress a hospitalized patient with covid-19 due to the possible benefits of reducing inflammation must be carefully weighed against the potential deleterious effect of delaying virus clearance or promoting bacterial superinfection. in addition to the adverse effects of immunosuppression therapy that further complicates the management of a hospitalized patient, the use of systemic steroids may worsen lung injury, which is particularly relevant in the case of treating patients with covid-19. similar risks of perpetuating viral infection and bacterial or fungal superinfection must be considered when administering a drug that influences ifn-mediated signalling, including jak inhibitors and interleukin (il)-6 blockers, because the il-6r-jak-stat3 signalling pathway is an essential component of the type i ifn pathway. moreover, il-6 can play an essential role in initiating the response against virus infections by promoting viral clearance by neutrophils; this was demonstrated in a study where deficiency of il-6 or il-6r in mice favoured the persistence of influenza infection leading to death in the experimental group. therefore, in the face of this global emergency that is witnessing rapid degeneration in patients' clinical conditions toward more serious situations that can be overwhelming to primary physicians (despite the best standards of care), the opportunity to redirect 4 medications offers an ethical and legal solution as medications outside of their usual indications can be prescribed as long as there is sufficient justification to do so. we here propose the repositioning of pentoxifylline (ptx) in the treatment of covid-19. ptx is a phosphodiesterase (pde) inhibitor that increases cyclic adenosine monophosphate (camp) levels, which in turn activate protein kinase (pka), leading to a reduction in the synthesis of the proinflammatory cytokines il-1, il-6, and tumour necrosis factor-alpha (tnf-α). ptx has also been shown to influence the renin-angiotensin system (ras) in vitro by inhibiting angiotensin 1 receptor (at1r) expression. the effects of ptx on restoration of glutathione levels, maintenance of mitochondrial viability, inhibition of tnf-α production, and preservation of microvascular blood flow, along with reports of improvement in endothelial function and coagulation have encouraged its use in the treatment of neonatal sepsis, leading to a reduction in hospital stay and mortality. moreover, in the context of covid-19, ptx has shown evidence of improvement in experimental ards models. moreover, since it is a short-lived drug, the effect of ptx can be rapidly suppressed if severe adverse reactions arise due to excessive suppression of tnf-. furthermore, the rheological properties of ptx could be useful when faced with the atypical presentation of ards associated with covid-19, characterized by marked hypoxemia with preservation of the ventilatory mechanics. this suggests that the loss of regulation of pulmonary perfusion and hypoxic vasoconstriction (vasoplegia) can be associated with microvascular obstructive inflammatory thrombus syndrome of the lungs (i.e., microclots). 5 overall, the rheological, anti-inflammatory, and renin-angiotensin axis properties of ptx highlight this drug as a therapeutic treatment alternative for patients with covid-19, which can help reduce the production of the inflammatory cytokines tnf-α, il-6, ifn-γ, and il-17 and increase the anti-inflammatory cytokine il-10. ptx also favours restoring the balance of the immune response, reducing damage to the endothelium and alveolar epithelial cells, improving circulation, and avoiding microvascular thrombosis. moreover, ptx decreases the neutrophil/lymphocyte ratio and restores the ratio of treg/th17 lymphocyte subpopulations. ptx decreases c-reactive protein (crp), ferritin, lactic dehydrogenase, and d-dimer, with consequent improvement in the ventilatory parameters pao 2 /fio 2 and sato 2 . in view of this evidence, it seems reasonable to reposition a drug with a proven safety profile and that is widely available as an alternative treatment of covid-19 to reduce inflammation without the trade-off of conventional immunosuppression. moreover, such repositioning avoids ethical or legal barriers allowing for immediate application in an acute setting. the sars-cov-2 spike protein (spike s) binds only to human angiotensin-converting enzyme type 2 (ace2) as a cell entry receptor [1] . this interaction partially explains why the lung is the organ most vulnerable to infection with sars-cov-2. zhao et al. [2] 6 demonstrated that 83% of the cells in the healthy lung tissue expressing ace2 are type ii alveolar epithelial cells (aecii cells), suggesting that these cells can serve as a reservoir for viral infection. moreover, genetic analysis revealed that ace2-expressing aecii cells have high levels of regulatory genes related to multiple viral processes, including the viral life cycle, assembly, and genome replication, further supporting that ace2 expressed in aecii cells facilitates the replication of coronavirus in the lungs [2] . similarly, other extrapulmonary tissues, including the heart, kidney, endothelium, and intestine, express ace2 [3] [4] [5] [6] [7] . this broad distribution ace2 tissue expression could partially explain the multiorgan dysfunction seen in patients with severe sars-cov infection [8] [9] [10] . compounding these effects, the coupling of s protein to its cell-binding site of ace2 leads to the downregulation of ace2, resulting in excess production of angiotensin (ang) by angiotensin-converting enzyme (ace). the reduction in ace2 concentrations in turn decreases the production of the heptapeptide vasodilator or ang1-7, which contributes to lung injury, since the stimulation of angiotensin type 1 receptors (at1rs) by ang ii increases the permeability of the pulmonary vasculature [11, 12] . the ras plays an essential role in maintaining blood pressure homeostasis, as well as in fluid and electrolyte balance [13] [14] [15] , whereas ace2 functions as a negative regulator of the ras [3, 16, 17] . specifically, ace cleaves the ang i decapeptide into the ang ii octapeptide [13, 14] , ace2 cleaves a single ang i residue to generate ang1-9 [16, 17] and a single ang ii residue to produce ang 1-7 [17] , thereby negatively regulating ras by inactivating ang ii [16] . 7 imai et al. [11] demonstrated that ang ii receptor subtypes are responsible for ace/ace2regulated acute lung injury (ali) based on experiments of a mouse model with genetic loss of at1a receptor (rat1a) expression, which showed markedly improved lung function and reduced oedema formation. conversely, inactivation of the at2 receptor (rat2) was shown to aggravate ali [15] . moreover, they attempted to prevent ali in the ace2-knockout mice using rat1-and rat2-specific antagonists. pharmacological inhibition of rat1 attenuated the severity of acid-induced lung injury, whereas rat2 inhibition had no apparent effect on the phenotypes of ali in ace2 knockout mice [11, 15] . collectively, these data show that ang ii-rat1a interactions play a role in the development and severity of ali [15] . ace contributes to an increase in vascular permeability and a decrease in the survival of pneumocytes [18] . at the same time, ang ii is considered to function as a growth factor that regulates cell proliferation/apoptosis and fibrosis, as well as a mediator that attracts inflammatory cells to sites of tissue injury [19] . all of the evidence accumulated to date has led to the widely accepted belief that ang ii is the primary factor responsible for the exacerbation of lung tissue lesions through the activation of rat1 [20] [21] [22] . similarly, ang ii exerts its fibrotic effects by inducing the production of transforming growth factor-beta 1 (tgf-β1), thereby triggering fibroblast proliferation and differentiation in collagen-secreting cells [23] . accordingly, it would be reasonable to consider that dysregulation of the ras could partially explain the observations made by a group of doctors in italy that patients with covid-19 pneumonia who meet the berlin criteria of ards present an atypical form of ards. the main characteristic of these patients was a dissociation between their relatively wellpreserved lung mechanics and the severity of hypoxemia. these doctors suggested that this 8 disconnect may be explained by the loss of regulation of pulmonary perfusion and hypoxic vasoconstriction (i.e., vasoplegia) [24] . the obstructive inflammatory thrombus syndrome of the lungs that occurs in patients with covid-19 could offer another explanation for this atypical presentation of ards involving inflammation of the endothelial vascular lesions, which may involve dysregulation of the ras (i.e., microclots) [25] . that is, the high concentration of ace2 in the endothelial cells of the arteries, veins, and arterial smooth muscle cells favour viral replication along with secondary cell damage, consequently releasing alarmins that are generated during cell death, thereby triggering the host's innate immune response through various mechanisms, including activation of complement via lectin and alveolar macrophages through tnf-α and other mediators [25] . complement activation would not only cause direct endothelial damage but also participates in leukocyte recruitment through c3a and c5a formation, leading to massive local production of the cytokines il-1, il-6, il-8, and ifn-γ. along with this massive host immune response, lymphocytes, resident macrophages, monocytes, and neutrophils perform their inflammatory functions, causing increased endothelial and alveolar epithelial cell damage and microvascular thrombosis, as reflected in elevations of lactic dehydrogenase and dimer d [25] . this endothelial damage could be progressive and potentially involve the microvascular beds of the kidney, brain, and other organs [1] . an attractive alternative to deal with the covid-19 pandemic, which may even be able to handle the emergence of new strains due to acquired mutations of sars-cov-2, is to search for agents that could block the activity of the at1rs [26] while simultaneously reducing the 9 vascular injury mediated by ang ii characterized by inflammation and oxidative stress. since nuclear factor-kappa b (nf-kb) is stimulated after the coupling of rat1 and ang ii, it would be beneficial to activate the expression of various cytokines such as tnf-α, il-6, il-8, mcp-1, and vcam-1 by generating oxidative stress, altering endothelial nitric oxide, and inducing the activity of nadph oxidase that increases the production of superoxide and peroxynitrite [27] [28] [29] [30] . therefore, increasing ace2 expression through at1r blockade [26] may affect viral replication and the release of useful viral materials. this would confer protection against the ali generated by sars-cov-2 due to the counterbalance of the excess production of ang ii and its potentially harmful effects [18, 26] . however, a limitation in the use of available at1r blockers is the risk or potential of deleterious hemodynamic effects of arterial hypotension [26] . although there is currently a lack of detailed information on hypotension rates among hospitalized patients with sars-cov-2 during this pandemic [26] , yu and colleagues [31] reported that half of the patients with sars-cov infection developed hypotension during their hospitalization. in this regard, ptx, as the drug we are considering repositioning for covid-19 treatment, has shown atr blocking effects in experimental studies, along with clinical evidence in humans showing no secondary effect on blood pressure [32] [33] [34] . all available reports and anecdotal information indicate that the pathological process contributing to severe cases of covid-19 is largely related to dysregulation of the immune system owing to a storm of cytokine production, characterized by increased expression levels of inflammatory cytokines and chemokines with a concomitant decrease in naive cd4+ t cells, cd8+ t cells, and regulatory t cells (tregs) [35, 36] . these findings may be related to previous descriptions of an association of higher levels of serum inflammatory cytokines with extensive lung damage in patients with other coronavirus infections in addition to sars-cov-2, including sars-cov and mers-cov [36] . this is due to induction of the production of large amounts of chemokines and cytokines (il-1, il-6, il-8, il-21, tnf-α, and mcp-1) by cov-infected cells, followed by the recruitment of lymphocytes [36] . coronaviruses also infects macrophages, which present the antigens of the virus to the t cells, thereby generating their activation and differentiation into the different subtypes of t lymphocytes. this results in the massive release of cytokines to amplify the immune response (39) . the continuous production of these mediators due to viral persistence ultimately harms cd8+ t cells and natural killer cells [36, 37] . the cd8+ t cells in sars-cov infection play a vital role in the elimination of infected cells and in generating inflammatory immune damage, accounting for 80% of the total inflammatory cells in the pulmonary interstitium in patients infected with sars-cov [36, 37] . in experimental models, t-cell-deficient balb/c mice (transduced with ad5-hdp4) were compared with controls and b-cell-deficient mice, demonstrating that t cells could survive in the infected lungs and destroy infected cells [37] . using these same models, the depletion of cd8+ t cells did not affect viral replication during sars-cov infection [38, 39] , whereas the depletion of cd4+ t cells was associated with reduced numbers of lung lymphocytes, decreased production of neutralizing antibodies and cytokines, and strong immune-mediated interstitial pneumonitis with delayed clearance of sars-cov from the lungs [40] . moreover, t-helper cells (th) produce inflammatory cytokines through the nf-b signalling pathway [41] , and il-17 cytokines recruit monocytes and neutrophils to the site of infection with inflammation to activate other downstream signalling pathways for cytokines and chemokines, such as il-1, il-6, il-8, il-21, tnf-, and mcp-1 [9, 42] . a study of 452 patients with covid-19 in wuhan, china demonstrated an increase in the neutrophil/lymphocyte ratio, with a high frequency of lymphopenia of t cells and cd4+ t cells [35] . this study further showed that patients with severe cases of covid-19 had higher serum levels of inflammatory cytokines (tnf-α, il-1, and il-6) compared to those of individuals of the same age suffering from a milder form of the disease [35] . host immune responses may play an essential role in the disease severity of covid-19 since subsets of naive cd4+ t cells were found to be elevated with a smaller percentage of memory cells and tregs [36, 43] . in particular, cd4+ t cells and cd8+ t cells play an essential role in attenuating innate immune responses during viral infection [36] . by contrast, tregs attenuate the activation, proliferation, and effector functions of a wide range of immune cells for the maintenance of auto-tolerance and immune homeostasis [37] [38] [39] . moreover, a histopathological study of biopsy specimens from patients that died of covid19 in china revealed that the lungs were full of a gelatinous substance [39] that was suspected to contain hyaluronic acid (ha) given its reported dysfunction and deregulation in the case of sars-cov infection [40] . the levels of inflammatory cytokines (e.g., il-1 and tnf-α) are elevated in patients with covid-19, and these cytokines are important inducers of hasynthase 2 (has2) in cd31+ endothelial cells, epcam+ alveolar epithelial cells, and fibroblasts [41] . 12 the chinese national health commission reported that patients infected with sars-cov-2 showed high leukocyte numbers and plasma levels of the inflammatory markers crp, globular sedimentation rate, and d-dimer [42] . another research group [9] found a correlation between viral rna in the blood with the incidence of ground-glass opacities and acute cardiac injury in patients with covid-19. they further reported high blood levels of in another study [44] , the viral load of sars-cov-2 in serum (rnaemia) was positive only in critically ill patients with more severe lymphopenia and a higher number of neutrophils, and the degree of rnaemia was positively correlated with both il-6 levels and the mortality rate. notably, the authors acknowledged that they did not measure other cytokines due to the lack of available reagents; however, this association of rnaemia with disease severity suggests a correlation with other inflammatory markers [44] . this possibility is particularly relevant given our suggestion of not limiting immunomodulatory strategies in the treatment of covid-19 to the current trend of focusing on the il-6r jak-stat3 pathway, whose cost-benefit analysis would also be questionable. xu et al. [39] documented the characteristics of a patient in china who died of severe sars-cov-2 infection, indicating substantial reduction in cd4+ and cd8+ t-cell counts. they also found that these cells were overactive, showing high proportions of hla−dr (cd4 3-47%) and cd38 (cd8 39.4%) double-positive fractions and a high concentration of proinflammatory cd4+ccr6+ th17 cells [39] . furthermore, this patient had high 13 concentrations of cytotoxic granules, which is another marker of hyperactive cd8+ t cells, 31.6% of which were perforin-positive, 64.2% were granulysin-positive, and 30.5% were both perforin-and granulysin-positive. this dysregulation of the immune system clinically manifests as respiratory failure with extensive lung damage, which is histopathologically related to the massive infiltration of neutrophils and macrophages, diffuse alveolar damage with the formation of hyaline membranes, and diffuse thickening of the alveolar wall [39, 42] . in addition to the pathological findings of patients who died from sars-cov-2 infection, splenic atrophy and necrosis of the lymph nodes have been observed, further suggesting the progression to shock and multiple organ failure due to tissue damage to the heart, liver, and kidneys [35, 39] . dysregulation of the immune response in covid-19 involves an immunosuppression stage with a subsequent inflammatory state characterized by a substantial reduction in peripheral lymphocyte counts that is correlated with disease severity [45] . as cd4+ and cd8+ t cells are the most affected subpopulations in this process, the immune response to the virus is consequently compromised, thereby increasing the risk of bacterial superinfection [46, 47] . although the detailed mechanism underlying the pathogenesis of lymphopenia is not fully understood, one of the pathways involved in lymphopenia induced by respiratory viral infections is cell death triggered by activation of the fas-fas-ligand interaction, as well as induction of the apoptosis axis from tnf-α ligands [47] [48] [49] . overall, these compounding effects of sars-cov-2 infection on the immune response and inflammation lead to the conclusion that it is necessary to search for appropriate immunomodulatory strategies to dampen inflammatory responses without simultaneously suppressing the immune response to avoid any deleterious effects. in this regard, in 2003, bermejo et al. [50] suggested the use of ptx for the treatment of sars-cov infection due to its anti-inflammatory effect by reducing tnf-α and ifn-γ. this proposal was further based on the ability of ptx to regulate activation of the transcription factors nf-b and nfat and to inhibit various viruses such as herpes simplex virus, human immunodeficiency virus, tick ptx is a drug with multifactorial action, including pde inhibition [52] and increasing camp levels, which in turn activate pka [53] , leading to a reduction in the synthesis of the inflammatory cytokines il-1, il-6, and tnf-α [52] [53] [54] . these inhibitory effects on pdes result in an overall reduction of inflammation [54, 55] . similarly, ptx modulates ifn-γ [56] [57] [58] and other molecules such as intracellular adhesion molecule type 1 (icam-1), vascular cellular adhesion molecule type 1 (vcam-1), and crp [59, 60] . based on these foundations, several models of kidney disease and clinical studies have shown that ptx is capable of attenuating proteinuria through modulating inflammatory cytokines [61] [62] [63] [64] [65] [66] [67] [68] [69] . in addition, clinical trials with diabetic nephropathy patients demonstrated that ptx enhanced the antiproteinuric effect of angiotensin receptor blockade [70, 71] . the effect of ptx on the ras appears to be primarily related to its inhibition of at1r expression. this speculation was supported by studies using a rat model of heart failure induced by coronary artery ligation, in which ptx infusion to the paraventricular hypothalamic nucleus attenuated the increase in at1r expression [32] and ptx treatment could prevent the overexpression of at1r mrna [33] . in addition, azhar and el-bassossy [72] showed that ptx significantly decreased at1r expression in the aortic tissue in a rat model of metabolic syndrome, and counteracted the induced hypertension by reducing the degree of inflammation, based on significantly lower levels of tnf-α and higher levels of the anti-inflammatory cytokine adiponectin. further, ptx shows promise as a useful therapeutic tool for covid-19 because the ras is one of the most critical systems activated during oxidative stress. upon binding of ang ii with at1rs, the secondary messengers inositol triphosphate and diacylglycerol are produced, resulting in the production of reactive oxygen and vasoconstriction [73] . a meta-analysis showed that ptx had an anti-inflammatory effect in adults with a variety of disorders, including coronary artery disease, type 2 diabetes mellitus, idiopathic or ischemic cardiomyopathy, and chronic kidney disease. the statistically significant differences were corroborated by a reduction in plasma concentrations of tnf- and crp and no deleterious effects on blood pressure [34] . from a practical perspective to reposition ptx for covid-19, li et al. [74] proposed using ptx as a treatment strategy for ards in 2016 based on its ability to specifically interfere with camp signalling [24] . considering that an increase in both tregs and th lymphocytes 16 producing interleukin il-17 (th17 cells) along with an imbalance of the treg/th17 ratio are among the main immune alterations observed in ards [75] [76] [77] [78] [79] [80] , targeting camp signalling is a reasonable strategy to induce immune tolerance. in particular, camp signalling plays a critical role give in the proliferation and function of tregs and effector t cells; that is, camp suppresses the adaptive differentiation of tregs mediated by tgf-β1 to reduce the treg content [81, 82] . using a mouse model of cephalic ligation and puncture-induced ards, li et al. [74] demonstrated that ptx pre-treatment attenuated lung injury and reduced the mortality rate. the authors further observed an increase in the camp levels of the spleens of the ptx-pretreated mice, whereas the number of tregs and th17 cells decreased. more interestingly, overexpression of stat3, which is required for th17 differentiation, restored the treg/th17 ratio, accompanied by a decrease in il-2, il-6, il-10, and il-17 levels, and significant inhibition of the expression of foxp3 and rorγt [74] . considering that these transcription factors are essential regulators of the differentiation and function of tregs/th17 cells [82, 83] , li and colleagues concluded that ptx-induced increases in camp might have partially restored the treg/th17 balance by modulating foxp3 and rorγt transcription through the stat3 pathway. therefore, regulating the treg/th17 balance and the subsequent immune response through camp signalling was proposed as a feasible treatment strategy for ards [74] . moreover, ptx was shown to inhibit tnf- production by alveolar macrophages [84, 85] . because pulmonary sarcoidosis is a chronic inflammatory disease, interactions between an antigen-presenting cell and an unknown antigen are perceived by naive cd4+ lymphocytes (th0 cells) and alveolar macrophages, leading to the activation and proliferation of both cell types and the consequent release of il-2, tnf-, and ifn-γ [86] . in support of these effects, adding ptx to a systemic steroid regimen allowed for a steroid dose reduction [87] . park et al. [88] confirmed this steroid-sparing effect of ptx in a randomized controlled clinical trial in patients with pulmonary sarcoidosis, in which ptx improved the pulmonary diffusion of carbon monoxide and arterial blood oxygen pressure during exercise, especially in patients who were naive to steroid treatment. in addition to these anti-inflammatory properties, ptx has also been reported to suppress tissue fibrosis by blocking tgf-β1 and preventing the deposition of type i collagen [89, 90] . several in vitro studies have shown that ptx inhibits fibroblast proliferation and extracellular matrix production [91] [92] [93] , and a clinical study demonstrated that administration of ptx to obese patients decreased the plasma levels of plasminogen activator inhibitor-1 (pai-1) [94] . these findings motivated lee and colleagues [95] to test the effect of ptx administration in an experimental model of radiation-induced pulmonary fibrosis in rats, and found a decrease in the expression levels of both fibronectin and pai-1. this was considered to be particularly relevant in light of evidence that pai-1 expression is elevated in fibrotic pathological conditions. indeed, pai-1 contributes to a reduction in fibrinolysis rates and a subsequent decrease in the degradation of components of the extracellular matrix, including fibronectin, leading to tissue fibrosis [96] . since there are no clinical guidelines for the management of pulmonary fibrosis, ptx is currently recommended for the prevention and treatment of this condition [97] . moreover, ptx was shown to prevent the development of pneumonitis in patients with breast and lung cancers [98, 99] . taken together, the evidence highlighted herein strongly points to the benefits of redirecting ptx as an ethical and legal attempt in the treatment of covid-19, which can help support patients in critical care and overwhelmed hospital resources in the face of this pandemic. the authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers' bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript. a pneumonia outbreak associated with a new coronavirus of probable bat origin single-cell rna expression profiling of ace2, the putative receptor of wuhan covid-19 angiotensin-converting enzyme 2 is an essential regulator of heart function essential role for collectrin in renal amino acid transport multiple organ infection and the pathogenesis of sars organ distribution of severe acute respiratory syndrome (sars) associated coronavirus (sars-cov) in sars patients: implications for pathogenesis and virus transmission pathways tissue distribution of ace2 protein, the functional receptor for sars coronavirus: a first step in understanding sars pathogenesis clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china clinical features of patients infected with 2019 novel coronavirus in wuhan, china clinical characteristics of coronavirus disease 2019 in china angiotensin-converting enzyme 2 protects from severe acute lung failure a crucial role of angiotensin converting enzyme 2 (ace2) in sars coronavirus-induced lung injury the biochemistry of the reninangiotensin system peptidyl dipeptidase a: angiotensin i-converting enzyme angiotensin-converting enzyme 2-a new cardiac regulator a novel angiotensin-converting enzymerelated carboxypeptidase (ace2) converts angiotensin i to angiotensin 1-9 a human homolog of angiotensin-converting enzyme. cloning and functional expression as a captoprilinsensitive carboxypeptidase good ace, bad ace do battle in lung injury, sars role of the renin-angiotensin system in vascular diseases: expanding the field angiotensin converting enzyme-2 is protective but downregulated in human and experimental lung fibrosis angiotensin-tgf-b1 crosstalk in human idiopathic pulmonary fibrosis: autocrine mechanisms in myofibroblasts and macrophages fibrosis, a common pathway to organ failure: angiotensin ii and tissue repair cellular and molecular mechanisms of fibrosis covid-19 does not lead to a "typical" acute respiratory distress syndrome microvascular covid-19 lung vessels obstructive thromboinflammatory syndrome (microclots): an atypical acute respiratory distress syndrome working hypothesis crit care resusc angiotensin receptor blockers as tentative sars-cov-2 therapeutics drug ace2 deficiency enhances angiotensin ii-mediated aortic profilin-1 expression, inflammation and peroxynitrite production angiotensin ii activates the proinflammatory transcription factor nuclear factor kappa b in human monocytes nuclear factor kappa b-molecular biomedicine: the next generation angiotensin ii and vascular injury cardiovascular complications of severe acute respiratory syndrome tnf-α in hypothalamic paraventricular nucleus contributes to sympathoexcitation in heart failure by modulating at1 receptor and neurotransmitters cytokine blockade attenuates sympathoexcitation in heart failure: cross-talk between nnos, at-1r and cytokines in the hypothalamic paraventricular nucleus effects of pentoxifylline on inflammatory markers and blood pressure: a systematic review and meta-analysis of randomized controlled trials dysregulation of immune response in patients with covid-19 in wuhan, china pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology foxp3+ regulatory t cells in the human immune system regulatory t cells: key controllers of immunologic self-tolerance pathological findings of covid-19 associated with acute respiratory distress syndrome accumulation of hyaluronan (hyaluronic acid) in the lung in adult respiratory distress syndrome defective lung function following influenza virus is due to prolonged, reversible hyaluronan synthesis ct imaging of the 2019 novel coronavirus coronavirus infections and immune responses detectable serum sars-cov-2 viral load (rnaaemia) is closely correlated with drastically elevated interleukin 6 (il-6) level in critically ill covid-19 patients longitudinal characteristics of lymphocyte responses and cytokine profiles in the peripheral blood of sars-cov-2 infected patients clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study sars-cov-2 and viral sepsis: observations and hypotheses lancet the impact of the interferon/tnf-related apoptosis-inducing ligand signaling axis on disease progression in respiratory viral infection and beyond apoptosis regulators from dna viruses pentoxifylline and severe acute respiratory syndrome (sars): a drug to be considered evaluation of immunomodulators, interferons and known in vitro sars-cov inhibitors for inhibition of sars-cov replication in balb/c mice cyclic nucleotide phosphodiesterase (pde) superfamily: a new target for the development of specific therapeutic agents cyclic nucleotide phosphodiesterase (pde) inhibitors: novel therapeutic agents for progressive renal disease pentoxifylline: a review of its pharmacodynamics and pharmacokinetic properties and its therapeutic efficacy of chloroquine and covid-19 pentoxifylline improves hemoglobin levels in patients with erythropoietin-resistant anemia in renal failure pentoxifylline reduces proinflammatory and increases anti-inflammatory activity in patients with coronary artery disease-a randomized placebo controlled study pentoxifylline decreases serum level of adhesion molecules in atherosclerosis patients acute-phase markers of inflammation and glomerular structure in patients with type 2 diabetes tnf-alpha increases albumin permeability of isolated rat glomeruli through the generation of superoxide renal pro-inflammatory cytokine gene expression in diabetic nephropathy: effect of angiotensin-converting enzyme inhibition and pentoxifylline administration cytokine modulation by px differently affects specific acute phase proteins during sepsis in rats effects of pentoxifylline, pentifylline and gamma interferon on proliferation, differentiation, and matrix synthesis of human renal fibroblasts the anti-inflammatory effects of the phosphodiesterase inhibitor pentoxifylline in the rat pentoxifylline diminishes the oxidative damage to renal tissue induced by streptozotocin in the rat the role of inflammatory cytokines in diabetic nephropathy inflammatory cytokines in diabetic nephropathy dexamethasone and pentoxifylline inhibit endotoxininduced cachectin/tumor necrosis factor synthesis at separate points in the signaling pathway urinary tumour necrosis factor-alpha excretion independently correlates with clinical markers of glomerular and tubulointerstitial injury in type 2 diabetic patients additive antiproteinuric effect of pentoxifylline in patients with type 2 diabetes under angiotensin ii receptor blockade: a short-term, randomized, controlled trial the effect of angiotensin system: a double blind and randomized clinical trial pentoxifylline alleviates hypertension in metabolic syndrome: effect on low-grade inflammation and angiotensin system the renin-angiotensin system in adipose tissue and its metabolic consequences during obesity resolution acute respiratory distress syndrome through reversing the imbalance of treg/th17 by targeting the camp signaling pathway acute respiratory distress syndrome: pathophysiology and therapeutic options the ratio of treg/th17 cells correlates with the disease activity of primary immune thrombocytopenia the imbalance of th17 cells and cd4(+) foxp3(+) treg cells in patients with atopic dermatitis an increased alveolar cd4+cd25+foxp3+t regulatory cell ratio in acute respiratory distress syndrome is associated with increased 30-day mortality cd4+cd25+foxp3+ tregs resolve experimental lung injury in mice and are present in humans with acute lung injury the ratio of th17/treg cells as a risk indicator in early acute respiratory distress syndrome cyclic amp concentrations in dendritic cells induce and regulate th2 immunity and allergic asthma transcriptional regulation of treg and th17 the expression of foxp3 and ror gamma t in lung tissues from normal smokers and chronic obstructive pulmonary disease patients pentoxifylline inhibits tnfalpha production from human alveolar macrophages inhibition of cytokine release from alveolar macrophages in pulmonary sarcoidosis by pentoxifylline: comparison with dexamethasone anti-tnf therapeutics for the treatment of sarcoidosis pentoxifylline in treatment of sarcoidosis steroid-sparing effects of pentoxifylline in pulmonary sarcoidosis l-arginine and phosphodiesterase (pde) inhibitors counteract fibrosis in the peyronie's fibrotic plaque and related fibroblast cultures pentoxifylline downregulates profibrogenic cytokines and procollagen i expression in rat secondary biliary fibrosis pentoxifylline attenuates tubulointerstitial fibrosis by blocking smad3/4-activated transcription and profibrogenic effects of connective tissue growth factor pentoxifylline inhibits normal human dermal fibroblast in vitro proliferation, collagen, glycosaminoglycan, and fibronectin production, and increases collagenase activity pentoxifylline, pentifylline, and interferons decrease type i and iii procollagen mrna levels in dermal fibroblasts: evidence for mediation by nuclear factor 1 down-regulation pentoxifylline lowers plasminogen activator inhibitor 1 levels in obese individuals: a pilot study pentoxifylline regulates plasminogen activator inhibitor-1 expression and protein kinase a phosphorylation in radiation-induced lung fibrosis pai-1, fibrosis, and the elusive provisional fibrin matrix radiation-induced lung injury: assessment and management pentoxifylline in prevention of radiationinduced lung toxicity in patients with breast and lung cancer: a double-blind randomized trial randomized, placebo-controlled trial of combined pentoxifylline and tocopherol for regression of superficial radiation-induced fibrosis we would like to thank ms. alejandra maldonado for guidance and support. 