High SARS-CoV-2 tropism and activation of immune cells in the testes of non-vaccinated deceased COVID-19 patients.

BACKGROUND: Cellular entry of SARS-CoV-2 has been shown to rely on angiotensin-converting enzyme 2 (ACE2) receptors, whose expression in the testis is among the highest in the body. Additionally, the risk of mortality seems higher among male COVID-19 patients, and though much has been published since the first cases of COVID-19, there remain unanswered questions regarding SARS-CoV-2 impact on testes and potential consequences for reproductive health. We investigated testicular alterations in non-vaccinated deceased COVID-19-patients, the precise location of the virus, its replicative activity, and the immune, vascular, and molecular fluctuations involved in the pathogenesis. RESULTS: We found that SARS-CoV-2 testicular tropism is higher than previously thought and that reliable viral detection in the testis requires sensitive nanosensors or RT-qPCR using a specific methodology. Through an in vitro experiment exposing VERO cells to testicular macerates, we observed viral content in all samples, and the subgenomic RNA's presence reinforced the replicative activity of SARS-CoV-2 in testes of the severe COVID-19 patients. The cellular structures and viral particles, observed by transmission electron microscopy, indicated that macrophages and spermatogonial cells are the main SARS-CoV-2 lodging sites, where new virions form inside the endoplasmic reticulum Golgi intermediate complex. Moreover, we showed infiltrative infected monocytes migrating into the testicular parenchyma. SARS-CoV-2 maintains its replicative and infective abilities long after the patient's infection. Further, we demonstrated high levels of angiotensin II and activated immune cells in the testes of deceased patients. The infected testes show thickening of the tunica propria, germ cell apoptosis, Sertoli cell barrier loss, evident hemorrhage, angiogenesis, Leydig cell inhibition, inflammation, and fibrosis. CONCLUSIONS: Our findings indicate that high angiotensin II levels and activation of mast cells and macrophages may be critical for testicular pathogenesis. Importantly, our findings suggest that patients who become critically ill may exhibit severe alterations and harbor the active virus in the testes.

Schistosoma mansoni Tegument (Smteg) Induces IL-10 and Modulates Experimental Airway Inflammation.

BACKGROUND: Previous studies have demonstrated that S. mansoni infection and inoculation of the parasite eggs and antigens are able to modulate airways inflammation induced by OVA in mice. This modulation was associated to an enhanced production of interleukin-10 and to an increased number of regulatory T cells. The S. mansoni schistosomulum is the first stage to come into contact with the host immune system and its tegument represents the host-parasite interface. The schistosomula tegument (Smteg) has never been studied in the context of modulation of inflammatory disorders, although immune evasion mechanisms take place in this phase of infection to guarantee the persistence of the parasite in the host. METHODOLOGY AND PRINCIPAL FINDINGS: The aim of this study was to evaluate the Smteg ability to modulate inflammation in an experimental airway inflammation model induced by OVA and to characterize the immune factors involved in this modulation. To achieve the objective, BALB/c mice were sensitized with ovalbumin (OVA) and then challenged with OVA aerosol after Smteg intraperitoneal inoculation. Protein extravasation and inflammatory cells were assessed in bronchoalveolar lavage and IgE levels were measured in serum. Additionally, lungs were excised for histopathological analyses, cytokine measurement and characterization of the cell populations. Inoculation with Smteg led to a reduction in the protein levels in bronchoalveolar lavage (BAL) and eosinophils in both BAL and lung tissue. In the lung tissue there was a reduction in inflammatory cells and collagen deposition as well as in IL-5, IL-13, IL-25 and CCL11 levels. Additionally, a decrease in specific anti-OVA IgE levels was observed. The reduction observed in these inflammatory parameters was associated with increased levels of IL-10 in lung tissues. Furthermore, Smteg/asthma mice showed high percentage of CD11b+F4/80+IL-10+ and CD11c+CD11b+IL-10+ cells in lungs. CONCLUSION: Taken together, these findings demonstrate that S. mansoni schistosomula tegument can modulates experimental airway inflammation.

Unlipidated outer membrane protein Omp16 (U-Omp16) from Brucella spp. as nasal adjuvant induces a Th1 immune response and modulates the Th2 allergic response to cow's milk proteins.

The discovery of novel mucosal adjuvants will help to develop new formulations to control infectious and allergic diseases. In this work we demonstrate that U-Omp16 from Brucella spp. delivered by the nasal route (i.n.) induced an inflammatory immune response in bronchoalveolar lavage (BAL) and lung tissues. Nasal co-administration of U-Omp16 with the model antigen (Ag) ovalbumin (OVA) increased the amount of Ag in lung tissues and induced OVA-specific systemic IgG and T helper (Th) 1 immune responses. The usefulness of U-Omp16 was also assessed in a mouse model of food allergy. U-Omp16 i.n. administration during sensitization ameliorated the hypersensitivity responses of sensitized mice upon oral exposure to Cow's Milk Protein (CMP), decreased clinical signs, reduced anti-CMP IgE serum antibodies and modulated the Th2 response in favor of Th1 immunity. Thus, U-Omp16 could be used as a broad Th1 mucosal adjuvant for different Ag formulations.

Schistosoma mansoni antigens modulate experimental allergic asthma in a murine model: a major role for CD4+ CD25+ Foxp3+ T cells independent of interleukin-10.

In areas where schistosomiasis is endemic, a negative correlation is observed between atopy and helminth infection, associated with a low prevalence of asthma. We investigated whether Schistosoma mansoni infection or injection of parasite eggs can modulate airway allergic inflammation in mice, examining the mechanisms of such regulation. We infected BALB/c mice with 30 S. mansoni cercariae or intraperitoneally injected 2,500 schistosome eggs, and experimental asthma was induced by ovalbumin (OVA). The number of eosinophils in bronchoalveolar lavage fluid was higher in the asthmatic group than in asthmatic mice infected with S. mansoni or treated with parasite eggs. Reduced Th2 cytokine production, characterized by lower levels of interleukin-4 (IL-4), IL-5, and immunoglobulin E, was observed in both S. mansoni-treated groups compared to the asthmatic group. There was a reduction in the number of inflammatory cells in lungs of S. mansoni-infected and egg-treated mice, demonstrating that both S. mansoni infection and the egg treatment modulated the lung inflammatory response to OVA. Only allergic animals that were treated with parasite eggs had increased numbers of CD4(+) CD25(+) Foxp3(+) T cells and increased levels of IL-10 and decreased production of CCL2, CCL3, and CCL5 in the lungs compared to the asthmatic group. Neutralization of IL-10 receptor or depletion of CD25(+) T cells in vivo confirmed the critical role of CD4(+) CD25(+) Foxp3(+) regulatory T cells in experimental asthma modulation independent of IL-10.

Peptides containing T cell epitopes, derived from Sm14, but not from paramyosin, induce a Th1 type of immune response, reduction in liver pathology and partial protection against Schistosoma mansoni infection in mice.

Sm14 and paramyosin are two major Schistosoma mansoni vaccine candidate antigens. Recently, we have identified Sm14 and paramyosin epitopes that are recognized by T cells of resistant individuals living in endemic areas for schistosomiasis. Herein, mice were immunized with these peptides separately or in association in order to evaluate their vaccine potential. Immunization of mice with Sm14 peptides alone or mixed with paramyosin peptides was able to induce 26%-36.7% or 28%-29.2% of worm burden reduction, 67% or 46% of intestinal eggs reduction and also 54%-61% or 43%-52% of liver pathology reduction, respectively. Protection was associated with a Th1 type of immune response induced by Sm14 peptide immunization. In contrast, paramyosin peptide vaccination did not engender protective immunity or liver pathology reduction and immunization was associated with a Th2 type of immune response.

Schistosoma mansoni antigen-driven interleukin-10 production in infected asthmatic individuals

Asthmatics infected with Schistosoma mansoni have a less severe course of asthma and an inhibition of the Th2 inflammatory response that seems to be mediated by interleukin (IL-10). The objective of this study was to evaluate the capacity of some S. mansoni antigens to stimulate IL-10 production in vitro by cells of asthmatic infected individuals. Peripheral bloods mononuclear cells were stimulated with the S. mansoni recombinant antigens Sm22.6, Sm14, P24, and PIII antigen. IL-10 was measured in the supernatants of cultures. As the recombinant antigens were cloned in Escherichia coli, we blocked contaminant endotoxin with polymyxin B added to the cultures. We demonstrated that all antigens used drove high production of IL-10 in S. mansoni infected individuals (n = 13, 408 ± 514 and 401 ± 383 pg/ml, 484 ± 245 pg/ml, 579 ± 468 pg/ml, respectively). In asthmatics infected with S. mansoni (n = 21) rP24 induced higher levels of IL-10 (565 ± 377 pg/ml) when compared to PIII, rSm14 and rSm22.6 (184 ± 209 pg/ml; 292 ± 243 pg/ml; 156 ± 247 pg/ml, respectively). Conclusion: the S. mansoni antigens evaluated in this study stimulated IL-10 production by cells from infected individuals and therefore they have the potential to be used as a modulator of the inflammatory response in asthma.

Aluminum hydroxide associated to Schistosoma mansoni 22.6 kDa protein abrogates partial protection against experimental infection but not alter interleukin-10 production

The need to develop a vaccine against schistosomiasis led several researches and our group to investigate proteins from Schistosoma mansoni as vaccine candidates. Sm22.6 is a protein from S. mansoni that shows high identity with Sj22.6 and Sh22.6 (79 and 91 percent, respectively). These proteins are associated with high levels of IgE and protection to reinfection. Previously, we have shown that Sm22.6 induced a partial protection of 34.5 percent when used together with Freund's adjuvant and produced a Th0 type of immune response with interferon-g and interleukin-4. In this work, mice were immunized with Sm22.6 alone or with aluminum hydroxide adjuvant and high levels of IgG, IgG1, and IgG2a were measured. Unfortunately, no protection was detected. Since IL-10 is a modulating cytokine in schistosomiasis, we also observed a high level of this molecule in splenocytes of vaccinated mice. In conclusion, we did not observe the adjuvant effect of aluminum hydroxide associated with rSm22.6 in protective immunity.

Co-administration of plasmid expressing IL-12 with 14-kDa Schistosoma mansoni fatty acid-binding protein cDNA alters immune response profiles and fails to enhance protection induced by Sm14 DNA vaccine alone.

Schistosomiasis is an endemic disease that affects 200 million people worldwide. DNA-based vaccine is a promising strategy to induce protective immunity against schistosomiasis, since both humoral and cellular immune responses are involved in parasite elimination. In this study, we evaluated the ability of Sm14 cDNA alone or in association with a plasmid expressing murine interleukin (IL)-12 to induce protection against challenge infection. Mice were immunized with four doses of the DNA vaccine and the levels of protection were determined by worm burden recovery after challenge infection. Specific antibody production to rSm14 was determined by ELISA, and cytokine production was measured in splenocyte culture supernatants stimulated with rSm14 and in bronchoalveolar lavage of vaccinated mice after challenge infection. DNA immunization with pCI/Sm14 alone induced 40.5% of worm reduction. However, the use of pCI/IL-12 as adjuvant to pCI/Sm14 immunization failed to enhance protection against challenge infection. Protection induced by pCI/Sm14 immunization correlates with specific IgG antibody production against Sm14, Th1 type of immune response with high levels of interferon (IFN)-gamma and low levels of IL-4 in splenocyte culture supernatants and in bronchoalveolar lavage after challenge infection. IL-12 co-administration with pCI/Sm14 induced a significant production of nitric oxide in splenocyte culture supernatants and also lymphocyte suppression, with reduced percentage of T cells producing IFN-gamma and tumor necrosis factor-alpha.

Immunization with Schistosoma mansoni 22.6 kDa antigen induces partial protection against experimental infection in a recombinant protein form but not as DNA vaccine.

Schistosomiasis is a major public health problem that affects mainly developing countries. There are 200 million people worldwide infected with schistosomes resulting in more than 250,000 deaths per year. Although schistosomicidal drugs exist, the advent of an efficacious vaccine remains the most potentially powerful means for controlling this disease. In this study we isolated a cDNA clone encoding the Schistosoma mansoni lung-stage Sm22.6 protein, which is 100% and 79% identical with the 22.6 kDa adult worm tegument antigen of S. mansoni and S. japonicum, respectively. Further, we produced recombinant (r) Sm22.6 and constructed an Sm22.6 DNA vaccine. Western blot analysis confirmed the identity of purified MBP-Sm22.6 fusion protein using anti-MBP (maltose binding protein) and anti-rSm22.6 antibodies. Additionally, C57BL/6 mice were immunized and specific anti-Sm22.6 IgG responses were produced when both vaccination strategies were used. Importantly, only rSm22.6 vaccine provided levels of protection against challenge infection (34.5%). Mice immunized with rSm22.6 induced production of IgG1 and IgG2a and synthesis of IFN-gamma and IL-4 in cultured mouse splenocytes. Finally, rSm22.6 vaccination induced a Th0 type of immune response and protective immunity that suggests Sm22.6 as a potential candidate to compose an anti-schistosome vaccine.

Fibroblast growth factor 9: cloning and immunolocalisation during tooth development in Didelphis albiventris.

There are no reports in literature about functional roles of fibroblast growth factor 9 (FGF-9) in tooth development in animals with complete tooth pattern. The classical model for studying tooth development is the mouse, which has small number of teeth and distinctive incisor and molar patterns. The opossum Didelphis albiventris with five upper and four lower incisors, one canine, three premolars, and four molars, on each side of the jaw, seems to be a convenient model to test results obtained in the mouse. Molecular expression studies indicate that FGF-9 participates in murine tooth initiation and regulation of morphogenesis. Searching for similarities and differences in FGF-9 expression between the opossum and the mouse, amino acid sequence and expression pattern of FGF-9 in the developing first molars of D. albiventris were characterised. FGF-9 cDNA sequence was obtained using RT-PCR and expressed in bacterial system for recombinant protein production and analysis of immunoreactivity. FGF-9 expression during tooth development was investigated by immunoperoxidase method. FGF-9 protein consists in a 209-residue polypeptide with a predicted molecular mass of 23.5 kDa. FGF-9 amino acid sequence has 98% of sequence identity to human and 97% to rodents. During tooth development, epithelial FGF-9 expression was seen at the dental lamina stage. Mesenchymal expression was seen at the bud stage and at the cap stage. No significant expression was found in the enamel knot. While in rodents FGF-9 is involved in initiation and regulation of tooth shape, it is suggested that it is only involved in tooth initiation in D. albiventris.

Schistosoma mansoni antigen-driven interleukin-10 production in infected asthmatic individuals.

UNLABELLED: Asthmatics infected with Schistosoma mansoni have a less severe course of asthma and an inhibition of the Th2 inflammatory response that seems to be mediated by interleukin (IL-10). The objective of this study was to evaluate the capacity of some S. mansoni antigens to stimulate IL-10 production in vitro by cells of asthmatic infected individuals. Peripheral bloods mononuclear cells were stimulated with the S. mansoni recombinant antigens Sm22.6, Sm14, P24, and PIII antigen. IL-10 was measured in the supernatants of cultures. As the recombinant antigens were cloned in Escherichia coli, we blocked contaminant endotoxin with polymyxin B added to the cultures. We demonstrated that all antigens used drove high production of IL-10 in S. mansoni infected individuals (n = 13, 408 +/- 514 and 401 +/- 383 pg/ml, 484 +/- 245 pg/ml, 579 +/- 468 pg/ml, respectively). In asthmatics infected with S. mansoni (n = 21) rP24 induced higher levels of IL-10 (565 +/- 377 pg/ml) when compared to PIII, rSm14 and rSm22.6 (184 +/- 209 pg/ml; 292 +/- 243 pg/ml; 156 +/- 247 pg/ml, respectively). CONCLUSION: the S. mansoni antigens evaluated in this study stimulated IL-10 production by cells from infected individuals and therefore they have the potential to be used as a modulator of the inflammatory response in asthma.

Aluminum hydroxide associated to Schistosoma mansoni 22.6 kDa protein abrogates partial protection against experimental infection but not alter interleukin-10 production.

The need to develop a vaccine against schistosomiasis led several researches and our group to investigate proteins from Schistosoma mansoni as vaccine candidates. Sm22.6 is a protein from S. mansoni that shows high identity with Sj22.6 and Sh22.6 (79 and 91%, respectively). These proteins are associated with high levels of IgE and protection to reinfection. Previously, we have shown that Sm22.6 induced a partial protection of 34.5% when used together with Freund's adjuvant and produced a Th0 type of immune response with interferon-g and interleukin-4. In this work, mice were immunized with Sm22.6 alone or with aluminum hydroxide adjuvant and high levels of IgG, IgG1, and IgG2a were measured. Unfortunately, no protection was detected. Since IL-10 is a modulating cytokine in schistosomiasis, we also observed a high level of this molecule in splenocytes of vaccinated mice. In conclusion, we did not observe the adjuvant effect of aluminum hydroxide associated with rSm22.6 in protective immunity.