Pulmonary influenza infection promotes the awakening of dormant metastatic breast cancer cells

Breast cancer is the most common form of cancer and the second cancer-causing death in females. Although remission rates are high if detected early, survival rates drop substantially when breast cancer becomes metastatic. The most common sites of metastatic breast cancer are bone, liver and lung. Respiratory viral infections inflict illnesses on countless people. The latest pandemic caused by the respiratory virus, SARS-CoV-2, has infected more than 600 million worldwide, with documented COVID-related death upward of 1 million in the United States alone. Respiratory viral infections result in increased inflammation with immune cell influx and expansion to facilitate viral clearance. Prior studies have shown that inflammation, including through neutrophils, can contribute to dormant cancer cells reawakening and outgrowth. Moreover, inhibition of IL6 has been shown to decrease breast cancer lung metastasis in mouse models. However, how respiratory viral infections contribute to breast cancer lung metastasis remains to be unraveled. Using MMTV/PyMT and MMTV/NEU mouse models of breast cancer lung metastasis and influenza A virus as a model respiratory virus, we demonstrated that acute influenza infection and the accompanying inflammation and immune cell influx awakens and dramatically increased proliferation and expansion of dormant disseminated cancer cells (DCC) in the lungs. Acute influenza infection leads to immune influx and expansion, including neutrophils and macrophages, with increased proportion of MHCII+ macrophages in early time points, and a sustained decrease in CD206+ macrophages starting 6 days post-infection until 28 days after the initial infection. Additionally, we observed a sustained accumulation of CD4+ T cells around expanding tumor cells for as long as 28 days after the infection. Notably, neutrophil depletion or IL6 knockout reversed the flu-induced dormant cell expansion in the lung. Finally, awakened DCC exhibited downregulation of vimentin immunoreactivity, suggesting a role for phenotypic plasticity in DCC outgrowth following viral infection. In conclusion, we show that respiratory viral infections awaken and increase proliferation of dormant breast cancer cells in the lung, and that depletion of neutrophils or blocking IL6 reverses influenza-induced dormant cell awakening and proliferation.

Embryology and Clinical Development of the Human Olfactory System

The olfactory system is unique as a special sensory system in its developmental neuroanatomy and function. Neonatal olfactory reflexes can be detected in the fetus from 30 weeks gestation and can be tested in term and preterm neonates and older children. Most efferent axons from the olfactory bulb terminate in the anterior olfactory nucleus within the olfactory tract, with secondary projections to the amygdala, hypothalamus, hippocampus, and entorhinal cortex (parahippocampal gyrus), with tertiary projections also to the insula and other cortical regions. The olfactory bulb and tract incorporate an intrinsic thalamic equivalent. The olfactory bulb may be primary in generating olfactory auras in some cases of temporal lobe epilepsy. Developmental malformations may involve the olfactory bulb and tract, isolated or as part of complex cerebral malformations and genetic syndromes. Primary neural tumors may arise in the olfactory bulb or nerve. Impaired olfaction occurs in neonatal hypoxic/ischemic and some metabolic encephalopathies. Loss of sense of smell are early symptoms in some neurodegenerative diseases and in some viral respiratory diseases including coronavirus disease 2019. Testing cranial nerve I is easy and reliable at all ages, and is recommended in selected neonates with suspected brain malformations or encephalopathy.

Clinical risk factors for predicting anti-Sars-CoV-2 antibody immunoreactivity duration after mild COVID-19 infection

Introdução: a doença causada pelo coronavírus (COVID-19) é complexa e multissistêmica. Ainda não se sabe se os sintomas da fase aguda estão correlacionados com a duração da resposta imune e com a persistência dos sintomas crônicos. Objetivo: o presente estudo visa acessar e monitorar os sintomas clínicos do COVID-19, correlacionando-os com a produção de anticorpos neutralizantes. Método: uma coorte de 69 profissionais da saúde da Universidade Federal do Espírito Santo (HUCAM-UFES/EBSERH) diagnosticados com infecção por SARS-CoV-2 confirmada via RT-PCR (Real-Time Reverse Transcription-Polymerase Chain Reaction) foram avaliados do início dos sintomas até seis meses depois. Exames laboratoriais de IgG e IgM foram utilizados para detectar a presença de IgG e IgM contra a proteína do nucleocapsídeo do vírus SARS-CoV-2 nas amostras de plasma sanguíneo. Sorologia de anticorpos IgG e IgM, função pulmonar via espirometria e avaliação clínica dos pacientes foram realizadas nos dias 15, 30, 45, 60, 90 e 180 após o início dos sintomas da doença. Resultados: sessenta e nove profissionais da saúde (idade, 40 ± 10 anos;74% mulheres) foram avaliados por seis meses. Todos apresentaram a forma leve a moderada do COVID-19. O número médio de sintomas foi 5.1 (± 2.3). O sintoma inicial mais comum foi dor muscular (77%), cefaleia (75%), anosmia (70%), ageusia (64%), coriza (59%), febre (52%), e tosse (52%). Após 30 dias, os pacientes mantiveram anosmia (18%), astenia (18%), adinamia (14%), dor muscular (7%), e ageusia (7%). Em relação à função pulmonar, 9.25% apresentaram padrão obstrutivo e todos recuperaram ao final dos seis meses. Dentre todos os participantes analisados, 18/69 (26%) não obtiveram nenhum valor de IgG e IgM considerados reagentes nos exames realizados. A curva sorológica de IgG mostrou um pico enquanto a de IgM apresentou seu maior valor médio no 15º dia. Houve um declínio progressivo e níveis similares aos basais aos 90. 15/53 (28%) permaneceram com IgG reagente após seis meses. Dor de garganta e dispneia foram considerados fatores de risco independentes, e os pacientes com esses sintomas tiveram 5,9 vezes mais chances de apresentar IgG reativa no 180º dia. Pacientes com diarreia tiveram quatro vezes mais chances de apresentar IgM reagente. Conclusão: nossos achados mostraram que 26% dos pacientes não produziram uma resposta humoral pós-COVID-19 leve. Seus títulos de anticorpos caíram significativamente após 90 dias e apenas 28% mantiveram anticorpos IgG reativos após seis meses. Dor de garganta e dispneia foram preditores de maior duração da resposta imune humoral.Alternate abstract: Introduction: coronavirus disease 2019 (COVID-19) is a complex multisystem disorder. It is not yet well known whether symptoms in the acute phase correlate with the duration of the immune response and the persistence of chronic symptoms. Objective: this study aimed to assess and monitor the clinical symptoms of COVID-19 and correlate them with the production of neutralizing antibodies. Methods: a cohort of 69 health workers at the University Hospital of the Federal University of Espírito Santo (HUCAM-UFES/EBSERH) diagnosed with SARS-CoV-2 infection confirmed via RT-PCR (Real-Time Reverse Transcription–Polymerase Chain Reaction) were evaluated from the onset of symptoms up to six months. SARS-CoV-2 IgG and IgM assays were used to detect the presence of IgG and IgM against the nucleocapsid protein of SARS-CoV-2 in serum samples. IgG and IgM antibody serology, pulmonary function via spirometry, and the clinical evolution of patients were performed at 15, 30, 45, 60, 90, and 180 days after the onset of COVID-19 symptoms. Results: sixty-nine health workers (age, 40 ± 10 years;74% women) were evaluated for six months. All subjects showed mild to moderate COVID-19. The mean number of symptoms was 5.1 (± 2.3). The most common initial symptoms were muscle pain (77%), headache (75%), anosmia (70%), ageusia (64%), runny nose (59%), fever (52%), and coughing (52%). After 30 days, the patients had anosmia (18%), asthenia (18%), adynamia (14%), muscle pain (7%), and ageusia (7%). Regarding lung function, 9.25% presented with an obstructive pattern, and all recovered after six months. Of all analyzed participants, 18/69 (26%) did not have any reactive IgG or IgM values in any of the assessments. The IgG serology curve showed a peak, whereas IgM had the highest mean value on the 15th day. There was a progressive decrease and levels similar to those at baseline after 90 days, and 15/53 (28%) remained with reactive IgG after six months. Sore throat and shortness of breath were found to be independent risk factors, and patients with these symptoms were 5.9 times more likely to have reactive IgG on the 180th day. Patients with diarrhea were four times more likely to have reactive IgM. Conclusion: our findings showed that 26% of patients did not produce a humoral response post-mild COVID-19. Their antibody titers dropped significantly after 90 days, and only 28% maintained reactive IgG antibodies after six months. Sore throat and shortness of breath are predictors of a longer duration of the humoral immune response.

Structural and Immunoreactivity Properties of the SARS-CoV-2 Spike Protein upon the Development of an Inactivated Vaccine.

Inactivated vaccines are promising tools for tackling the COVID-19 pandemic. We applied several protocols for SARS-CoV-2 inactivation (by ß-propiolactone, formaldehyde, and UV radiation) and examined the morphology of viral spikes, protein composition of the preparations, and their immunoreactivity in ELISA using two panels of sera collected from convalescents and people vaccinated by Sputnik V. Transmission electron microscopy (TEM) allowed us to distinguish wider flail-like spikes (supposedly the S-protein's pre-fusion conformation) from narrower needle-like ones (the post-fusion state). While the flails were present in all preparations studied, the needles were highly abundant in the ß-propiolactone-inactivated samples only. Structural proteins S, N, and M of SARS-CoV-2 were detected via mass spectrometry. Formaldehyde and UV-inactivated samples demonstrated the highest affinity/immunoreactivity against the convalescent sera, while ß-propiolactone (1:2000, 36 h) and UV-inactivated ones were more active against the sera of people vaccinated with Sputnik V. A higher concentration of ß-propiolactone (1:1000, 2 h) led to a loss of antigenic affinity for both serum panels. Thus, although we did not analyze native SARS-CoV-2 for biosafety reasons, our comparative approach helped to exclude some destructive inactivation conditions and select suitable variants for future animal research. We believe that TEM is a valuable tool for inactivated COVID-19 vaccine quality control during the downstream manufacturing process.

SARS-CoV-2 specific T Cell: clinical validation of a rapid and easy to use method based on direct realtime PCR amplification

Introduction: SARS-CoV-2 immune-response is mediated by both humoral and cellular immunity. However, since Ab levels wane faster than SARS-CoV-2 specific T cells immunity, cellular immunity represents an important factor for COVID-19 immune defence. Determining immunoreactivity of SARS-CoV-2 specific T cells is of clinical relevance in transplant recipients or patients treated with immunomodulant therapy. SARS-CoV-2 specific T cells assays are currently based on ELISA, whilst rapid tests are pivotal for real-time patients' evaluation. In this study, a novel direct real-time PCR (dRT-PCR) targeting mRNA of CXCL10 for measuring SARS-CoV-2 specific T cells, was tested and evaluated. Method(s): A total of 104 healthcare workers, with two or three doses of homologous (Pfizer/BioNTech, n = 82) or heterologous (Pfizer/BioNTech and Vaxzevria or Moderna, n = 22) vaccinations were asked to collect a blood (Li-He) sample. Blood was stimulated overnight with SARS-CoV-2 spike peptides (S-peptide) or treated with non-stimulating substance. Stimulated/treated samples were diluted in Buffer A, mixed with dqTACT MS then loaded into the cartridge. The analysis was performed using SCV2 T Activation kit, bCube and bApp (Hyris srl, Lodi, Italy), equipped by an automatic result interpretation based on artificial intelligence. For a subgroup of 49 samples, IFN-y releases to SARS-CoV-2 spike peptides were tested by Quant-T-Cell SARS-CoV-2 and ELISA (Euroimmune, Lubeck, Germany). Result(s): Seventy-nine (75.9%) and 25 (24.1%) were females and males, respectively. Twenty-nine subjects were previously infected by SARS-CoV-2. Overall mean age (+/- SD) was 45.9+/-13.3 years. At qualitative analyses, 97 subjects (93.2%) resulted reactive to S-peptides, 3 (2.8%) were borderline and 4 were negative (3.8%). These negatives had their third vaccinal dose in December/November 2021. Previous infected individuals presented reactivity to S-peptides, with the exception of one subject with resulted reactive also in the untreated sample. Samples tested with both dRT-PCR and ELISA perfectly agreed (100%) with both methods. At quantitative analyses, between-assay correlation was 0.32 (p<0.001). Conclusion(s): Hyris dRT-PCR appeared accurate for determining presence or absence of immunoreactivity of SARS-CoV-2 specific T cell, especially when rapid analyses are required, such as for organ transplantation.

Spike antibody seroconversion and breadth following SARS-CoV-2 vaccination in Australian people with multiple sclerosis

Background: COVID-19 vaccination induces protective Spike antibodies. Some responses are attenuated in people with multiple sclerosis (MS) on high efficacy disease-modifying therapies (DMT).Whether antibodies afford immunity against emerging SARS-CoV-2 Variants of Concern (VoC) such as Delta and Omicron is unknown. Aim(s): To assess the longevity and breadth of Spike antibody in MS patients after COVID-19 vaccination. Objective(s): To determine seroconversion and antibody binding toVoC Spike. Method(s): Spike antibodies to Clade A SARS-CoV-2 were assessed in 535 MS sera at baseline (n=292), 1 (n=141) and 6 month (n=67) post-second dose, and 1 month post-third dose (n=35), and 489 health worker controls. When known, COVID- 19 vaccines were BNT162b2 (n= 489 controls, n=108 MS patients) and ChAdOx1-S (n=37).Spike antibody binding to VoC Delta and Omicron BA1 was assessed in 68 sera 1 month post-second dose. Demographic and DMT information was available in 269 patients. Result(s): 123/141 sera at 1 month post-second dose, 66/67 at 6 months post-second dose, and 26/35 at 1 month post-third dose were positive for Spike antibodies.Patients who did not seroconvert at 1 and 6 month post-second and 1 month post-third dose (n=28) were treated with ocrelizumab (n=22), cladribine (n=1), fingolimod (n=4), and siponimod (n=1). At 1 month post-second dose, the median and IQR Spike antibody levels were 67,224+/- 101,251 in the seroconverted MS group compared to 145,510+/- 99,669 in controls (n=489). When patient sera were assessed for binding to Clade A Spike, and VoC Delta and Omicron BA1 Spikes, most sera were able to bind the three different Spike antigens (n=61). However, Spike antibody immunoreactivity was decreased by 70% against Delta Spike and 90% for Omicron BA1 Spike compared to the original clade A Spike.As observed for Clade A Spike antibody, DMTs, such as ocrelizumab, fingolimod, and ofatumumab, decreased the antibody binding to Delta and Omicron Spike. Still, the pattern of antibody recognition was similar between the three Spikes and all DMTs analysed, i.e. alemtuzumab, natalizumab, teriflunomide, and interferons. Our data suggest that, irrespectively of DMTs, antibodies generated after vaccination did not bind Spike from recent VoCs to the same extent as the original Spike used in COVID-19 vaccines. Conclusion(s): Some DMTs reduce Spike antibody titres or prevent seroconversion. The sequence of Spike used in the first generation of vaccines may need to be updated for emerging VoC.

DIFFERENT HUMORAL but SIMILAR CELLULAR RESPONSES of PATIENTS with AUTOIMMUNE INFLAMMATORY RHEUMATIC DISEASES under DISEASE-MODIFYING ANTI-RHEUMATIC DRUGS after COVID-19 VACCINATION

Background: The interplay between humoral and cellular response after vaccination against SARS-CoV-2 in patients (pts.) with autoimmune infammatory rheumatic diseases (AIRD) remains unknown. Objectives: To investigate the impact of different immunosuppressive therapies on the development of humoral and cellular immune responses to full 2-dose SARS-CoV-2 vaccination in AIRD pts. with stable low disease activity. Methods: The immune reactivity to COVID-19 vaccination was investigated in a prospectively recruited AIRD cohort with rheumatoid arthritis, axial spondy-loarthritis or psoriatic arthritis which received a therapy with IL-17i, TNFi, JAKi or MTX (alone or in combination). Almost all patients received mRNA-based vaccine, only 4 patients had a heterologous scheme. Anti-spike(S) antibodies(ab.) and sera neutralizing capacity (neutralization dilution 50;ND50) were measured 4 weeks after the frst (prime+4w) and 4 weeks after the second vaccination (boost+4w). Vaccine-specifc cellular immunity was evaluated by quantifying expression of activation markers on T cells as well as their production of key cytokines, at prime+4w and boost+4w. Results: Overall, a total of 92 pts. were included in the fnal cohort. 31 (33.7%) pts. were on TNFi, 24 (26.1%) on IL-17i, 24 (26.1%) on JAKi, each group encompassing pts. receiving drug inhibitors alone or in combination with MTX.13 (14.1%) were treated with MTX alone. The median time between the vaccination and blood sampling was 31 [IQR: 28-34] days after prime+4w and 28 [IRQ: 28-28] days after boost+4w. Although at prime+4w only 34/90 (37.8%) of pts. presented neutralizing ab., the majority (86/91, 94.5%), developed them at boost+4w. The highest neutralization titer developed the pts. on IL-17i both at prime+4w (74 [IQR: 13-91]) and boost+4w (798 [IQR: 511-1344]), while no statistically signifcant differences were found in the neutralization titer at boost+4w for the TNFi, JAKi, and MTX groups: 207 ND50 [IQR: 120-576], 319 [IQR: 133-461] and 749 [IQR: 264-1920], respectively. 81/90 (90.0%) pts. developed IgG ab. against SARS-CoV-2 S-protein at prime+4w and 91/92 (98.9%) at boost+4w. Pts. receiving IL-17i developed higher ab. titers (8295 U/mL [IQR: 4586-11,237]) compared to the other three groups: JAKi (4405 U/mL [IQR: 1436-7265], TNFi (2313 [IQR: 1156-3630] U/mL) and MTX (2010 U/mL [IQR: 693-9254]). Neutralization capacity correlated well with the titer of anti-S ab. at both timepoints. Co-administration of biologic/tsDMARDs and MTX led to lower titers compared to biologic/tsDMARDs mon-otherapy. All therapies left frequencies of CD154+CD137+ CD4+ T cells and CD137+ CD8+ T cells at prime+4w and boost+4w unchanged. Polyfunction-ality and T cell cytokine profiles across therapies did not signifcantly vary at boost+4w. Conclusion: Even after insufficient seroconversion for neutralizing capacity and ab. response against SARS-CoV-2 S-proteins between pts. of different mod of action agents, particularly for MTX and JAKi after frst vaccination, a second vaccination covered almost all pts. regardless of DMARDs therapy, with better outcomes in those on IL-17I. T cell immunity revealed similar frequencies of activated T cells in all modes of action after the second vaccination.

IMMUNOMODULATORY EFFECTS of SARS-COV-2 VACCINATION: INCREASE of REGULATORY T CELLS after MRNA VACCINE

Background: Besides the ability to induce antigen-specifc responses, vaccines can be endowed with immunomodulatory properties including the capacity to induce or downregulate regulatory T cells (Treg) that suppress adaptative and autoreactive immune responses (1). Objectives: We asked if an anti-SARS-CoV-2 mRNA vaccine could also induce an accumulation of Treg cells in patients with mixed cryoglobulinemia vasculitis (MCV), who have a defciency of Treg cells (2) and in healthy individuals. We also investigated immunologic variables possibly associated with a low immunogenic-ity of SARS-CoV-2 mRNA vaccine in patients with MCV (3). Methods: We analyzed peripheral blood lymphocyte subpopulations and anti-SARS-CoV-2 serological response in 24 patients with MCV and 9 Healthy donors (HD) before and after 2 weeks after the second dose of the Pfzer/BioNTech vaccine. Results: Among MCV patients we found 15 serological responders and 9 non-responders. All 5 seronegative patients treated recently with rituximab had <5 B cells/μ L, whereas the absolute B cell count was increased in 2 of 4 untreated patients due to monoclonal B cell lymphocytosis, with monoclonal cells representing more than 90% of B cells, associated with non-Hodgkin lymphoma. The percentage of pathologic CD21low B cells was signifcantly increased in seronegative patients. Before receiving the Pfzer/BioNTech vaccine, patients with MCV had a signifcantly reduced frequency of Treg cells among CD4+ T cells compared to HD. After the second dose of the vaccine, there was in MCV patients a signifcant increase in the percent and absolute count of Treg among CD4+ T cells Concerning the pre-vaccination distribution of T cells subpopulations, including the percentages and absolute counts of total CD3+, CD4+, CD8+, HLA-DR+ activated, Treg or CD56+ natural killer T cells, we could not reveal any pattern signifcantly associated with lack of serological response to vaccine. Conclusion: Our fndings show that lack of immunoreactivity in patients with MCV may be associated with expansion of pathologic B cells and that anti-SARS-CoV2 mRNA vaccine may induce an increase of Treg cells.

Immune response to COVID-19 vaccination in patients with B-cell malignancies after CD19 CART cell therapy

Introduction: Patients with hematologic malignancies are at an increased risk of morbid/mortality from COVID-19. Our prospective clinical study evaluated immune responses to COVID-19 mRNA vaccines in patients with B-cell lymphoma who had received CD19-directed chimeric antigen receptor (CAR) T cell therapy. Methods: We measured SARS-CoV-2 neutralizing activity of plasma from 18 patients and 4 healthy controls (HC) and antibody titers against viral spike proteins (S1, S2, RBD) including their delta variants using an enzyme-linked immunoassay (ELISA). We measured B cell subpopulations in the patients' blood using flow cytometry. Results: We found that the peripheral blood plasma from 3/4 HCs showed substantial SARS-CoV-2 neutralizing activity already at 4 weeks after the first dose of COVID-19 mRNA vaccine while none of the CD19 CART patients evidenced any antibody-mediated neutralizing activity at the same point in time. At 4 weeks after receiving the second dose of the vaccine, all 4 HCs showed complete neutralizing activity. In marked contrast, only 1 of 14 CART patients evidenced any relevant antibody-mediated SARS-CoV-2 neutralizing activity. Assessing whether a globally insufficient antibody-mediated immunity was the underlying cause of the lack of a response to the COVID-19 vaccine in our CART patients, we found that IgG antibody levels against common microbial and viral antigens like influenza, Epstein-Barr virus (EBV), Cytomegalovirus (CMV), and tetanus toxoid, were comparable to those observed in HCs. However, while at 4 weeks post second dose of the vaccine the HCs showed high levels of vaccine-induced IgG antibody titers against all the viral spike proteins (S1, S2, RBD), including the delta variants of the S1 and RBD proteins, the vast majority of our CART patients did not evidence any SARS-CoV-2-specific antibodies. Importantly, a third booster vaccination did not lead to an improvement in the antiviral immunity in the 4 CART patients analyzed. When we assessed B cell subpopulations in the blood of patients and HCs, we found that prior treatments had completely eradicated all CD19+/CD20+ B cells in the patients while numbers of long-lived memory plasma cells were comparable to those of HCs. Conclusions: In this study, 17 of 18 patients with lymphoma who received CD19 CART therapy had very poor immunoreactivity to 1-3 doses of mRNA-based COVID-19 vaccines. Importantly, antibody responses to common recall antigens were preserved, suggesting impaired immune response primarily against novel antigens like SARS-COV-2. This lack of immunoreactivity against novel antigens was probably based on the eradication of earlier-stage B cell subpopulations after treatment with anti-CD19 and anti-CD20 immunotherapies.

Corona, higher risk for testicular cancer development?

Introduction & Objectives: In November 2019 the first case of Sars-Cov2 was noticed in Wuhan, China. This virus was finally spreading all over the world and we have a pandemic situation with an unclear outcome. Several different groups have already published that the virus can be present in the testis after infection, however with unknown consequences. Materials & Methods: FPPE tissue samples from patients died with or of Corona (n=6) compared with healthy donors (n=5), seminoma without metastasis (n=5) and seminoma with metastasis (n=5) were analyzed and compared via qRT-PCR for the expression of microRNAs (miRs) which are predominantly overexpressed in Seminoma and in metastazing cells, miR-199-3p, miR-498 and miR-371a-3p. Beyond this, an IHC for Androgenreceptor (AR) and ACE2 was performed. The median age of the corona patients was 70 years. Results: In 50% of all corona FFPE samples, a significant upregulation of the seminoma specific miR-371a-3p was detectable, whereas all other tumor specific miRs were negative. In H&E staining of the FFPE samples in 50% of all patients the spermatogenesis was reduced/absent. IHC for AR in COVID positive and negative testes showed loss of immunoreactivity in Sertoli cells of Covid-positive cases vs controls. Conclusions: Our group presented here for the first time a possible late onset complication after Sars-Cov2 infection, namely the increased risk for developing seminoma due to the upregulation of the seminoma specific miR-371a-3p.

Effects of levamisole on sexual behavior, reproductive hormones and testicular histopathology in male rats

AIM: Levamisole, an antiparasitic drug, was reported to have positive effects in various clinical trials in the treatment of COVID-19. However, the number of studies on the effects of levamisole on the reproductive system and sexual behavior in male rats is limited. The present study aimed to investigate the possible effects of levamisole on sexual behavior, testicular histopathology, serum gonadotropin, and testosterone levels in male rats. METHODS: Twenty male Sprague-Dawley rats were divided into two groups as control and levamisole were used. Rats were given levamisole (2 mg/kg) dissolved in distilled water for 30 days, while only distilled water was administered to the control group by oral gavage. Finally, sexual behavior tests (SBT) were performed for 30 min. Then, the animals were decapitated, blood samples and testis tissues were taken. The Bax, Hsp70 and cytochrome c immunohistochemistry staining were performed in testis tissues, and gene expression levels were measured by real-time PCR. The luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone levels were measured by ELISA in serum samples. RESULTS: In SBT parameters, mount latency (ML, p<0.001), intromission latency (IL, p<0.01), and the postejaculatory interval (PEI, p<0.01) were significantly prolonged. Also, the copulatory rate (CR, p<0.05) was significantly reduced. Serum LH, FSH, and testosterone levels did not change. In the histopathological stainings, irregularities in the seminiferous tubule germinal epithelium, congestion, edema in the interstitial area, and metaphase arrest in some spermatocytes were detected in the levamisole group (p<0.001). Levamisole treatment also significantly increased cytochrome c, Bax, and Hsp 70 immunoreactivities and Bax (p=0.05) and Hsp 70 (p<0.01) gene expression levels in testicular tissue. CONCLUSION: Levamisole may decrease sexual motivation and copulation efficiency. Also, it may adversely affect testicular histopathology in male rats.

Sex differences in mortality from covid-19 pandemic: Are men vulnerable and women protected. Why?

The epidemiological studies shows that compared to women, men infected with the novel COVID-19 have more severe and threatening disease and have a higher mortality globally. The population which is under real panic comprises older men. The reason could be linked to sex chromosome genes and sex hormones with differential regulation of immune responses between the sexes. Objectives of Study: We need to recognize the phenotypical differences in severe case manifestations of COVID-19 in men and women as a fundamental step to understand the effects of this health emergency. Study Design/Methodological/Approach: The study was designed upon factors that are the major cause of gender inequality of infection. The statistical data was collected from the official record of COVID-19 Emergency Centre established in OJHA Centre Dow University Karachi. Results: It was found that, the mortality rate is very high in men. Less deaths of patients younger than 40 years even in past coronavirus leading to severe acute respiratory SARS-CoV, were found to infect more men than women. This may be due to not required enhanced response to infections on reproductive function in young men population, enhanced immune reactivity along with changes in immune cells during aging and pleiotropic nature of many genes. Conclusion: Evidence from this study tells us that sex is an important driver of risk of mortality and response to the COVID-19 pandemic. Age distribution is related with increased mortality rate among men, especially the older age men. Further research is warranted to investigate hormonal, inflammatory, immunologic, and phenotypical differences in severe COVID-19 disease presentations.

The Flu Vaccination May Have a Protective Effect on the Course of COVID-19 in the Pediatric Population: When Does Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Meet Influenza?

Background In the midst of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, a lot more chaos could be anticipated in the flu season due to the coexistence of SARS-CoV-2 and influenza with almost similar epidemiologic and clinical features. Could this become a "twindemic" or "syndemic" if there is any viral interference occurs? We investigated the effect of influenza and pneumococcal vaccines on the disease course of SARS-CoV-2 in the pediatric population and the possibility of viral interference. Material and methods After approval from Institutional Review Board, a retrospective electronic chart review on 20 years and younger SARS-CoV-2 polymerase chain reaction (PCR) positive patients who visited Arkansas Children's Hospital System between February 1 to August 30, 2020, was performed. The clinical data was collected along with influenza and pneumococcal vaccination status of these patients. Results The results showed that viral interference may have played a role in the current flu and coronavirus disease 2019 (COVID-19) twindemic. SARS-CoV-2 and influenza may have significantly affected each other's epidemiological features. Conclusion Understanding the relationship and co-existence of other viruses alongside SARS-CoV-2 and knowing the vaccination status of the host population may help in deploying the right strategies to get the best outcomes.

Expression and immunoreactivity of a recombinant multi-epitope antigen designed based on four major structural proteins of avian infectious bronchitis virus.

The development of rapid, simple, and sensitive diagnostic methods for identification of avian infectious bronchitis virus (IBV) is crucial for the effective control of avian infectious bronchitis. In the present study, a tandemly arranged multiepitope peptide (named SEMN) was designed with four antigenic regions derived from four major structural proteins of IBV. Then, we performed codon optimization of SEMN gene by changing the codon-adaptation index from 0.45 to 0.94 and expressed the optimized gene in codon bias-adjusted Escherichia coli Rosetta (DE3), followed by determination of the immunoreactivity of the purified protein. Bioinformatics analysis of SEMN showed a high antigenicity, surface probability and hydrophilicity. The recombinant protein rSEMN was expressed both in soluble forms and as inclusion bodies, and the molecular weight of rSEMN was about 39 kDa. The preliminary diagnostic performance of rSEMN was confirmed by Western blotting analysis using chicken anti-IBV polyclonal antibodies. Further studies are needed to evaluate the immunogenicity in animal models and to give a final assessment of the diagnostic utility of this recombinant multi-epitope antigen.