Isolated specific IgA against respiratory viruses, Influenza or SARS-CoV-2, present in the saliva of a fraction of healthy and asymptomatic volunteers.

OBJECTIVES: Defense against respiratory viruses depends on an immune response present in the mucosa, as saliva IgA secretes antibodies. During the pandemic, such as influenza or SARS-CoV-2, most infected patients are asymptomatic but retain specific antibodies post-infection. The authors evaluated IgG and IgA antibodies against SARS-CoV-2 and influenza in the saliva of asymptomatic volunteers, validated with controls or vaccinated individuals. METHODS: The authors detected specific antibodies by validated conventional ELISA using natural SARS-CoV-2 antigens from infected Vero cells or capture-ELISA for influenza using natural antigens of the influenza vaccine. RESULTS: Saliva from influenza-vaccinated individuals had more IgA than paired serum, contrary to the findings for specific IgG. In COVID-19-vaccinated samples, specific IgA in saliva increased after vaccination, but IgG levels were high after the first dose. In saliva from the asymptomatic population (226), anti-Influenza IgG was found in 57.5% (130) of samples, higher than IgA, found in 35% (79) of samples. IgA results were similar for SARS-CoV-2, with IgA present in 30% (68) of samples, while IgG was less present, in 44.2% (100) of samples. The proportion of influenza IgG responders was higher than that for SARS-CoV-2 IgG, but both populations presented similar proportions of IgA responders, possibly due to variable memory B cell survival. For both viruses, the authors found an important proportion (> 10%) of IgA+IgG- samples, suggesting the occurrence of humoral immunity directed to the mucosa. CONCLUSION: Specific antibodies for respiratory viruses in saliva are found in either infection or vaccination and are a convenient and sensitive diagnostic tool for host immune response.

Virulence and DNA sequence analysis of Cronobacter spp. isolated from infant cereals.

Cronobacter spp. is an opportunistic pathogen that causes severe infections, affecting newborns and infants, and is also an emerging cause of hospital-acquired infection in elderly populations. These infections are mainly associated with the consumption of infant formulas, even though these bacteria have been isolated from other foods as well. Cronobacter spp. invades epithelial cells and escapes the immune response mechanisms, multiplying inside macrophages. However, the pathogenesis and virulence factors of these bacteria have not been fully elucidated and need to be further studied. Therefore, this study aimed to evaluate the ability of Cronobacter spp. strains isolated from infant cereals to invade and survive within macrophages, investigate the virulence phenotype using the Galleria mellonella model, and identify possible genes involved in bacterial pathogenesis through pan-genome analysis. All the isolates were able to invade macrophages and the survival of bacteria decreased over a 72 h period, with bacterial cell counts reaching up to 106 CFU/ml. Cronobacter sakazakii isolate 112 exhibited a similar mortality rate (40-70%) to the ATCC BAA 894 strain (Cronobacter sakazakii) in G. mellonella assay. In addition, some unique virulence genes (isolate 7, ada_2, tcmA_1, acrB_3; isolate 78, ampC_2, rihC_1 and isolate 112, fimH, ylpA, gtrA) were identified within isolates with the invasive profile in the in vivo and in vitro assays. Furthermore, isolates from different species were grouped into seven distinct clusters in the pan-genome analysis. The most virulent isolates (7, 78, and 112) were grouped in distinct subclusters in the cladogram. This work revealed potential Cronobacter spp. pathogenic strains recovered from infant cereals.

Isolated specific IgA against respiratory viruses, Influenza or SARS-CoV-2, present in the saliva of a fraction of healthy and asymptomatic volunteers

Abstract Objectives: Defense against respiratory viruses depends on an immune response present in the mucosa, as saliva IgA secretes antibodies. During the pandemic, such as influenza or SARS-CoV-2, most infected patients are asymptomatic but retain specific antibodies post-infection. The authors evaluated IgG and IgA antibodies against SARS-CoV-2 and influenza in the saliva of asymptomatic volunteers, validated with controls or vaccinated individuals. Methods: The authors detected specific antibodies by validated conventional ELISA using natural SARS-CoV-2 antigens from infected Vero cells or capture-ELISA for influenza using natural antigens of the influenza vaccine. Results: Saliva from influenza-vaccinated individuals had more IgA than paired serum, contrary to the findings for specific IgG. In COVID-19-vaccinated samples, specific IgA in saliva increased after vaccination, but IgG levels were high after the first dose. In saliva from the asymptomatic population (226), anti-Influenza IgG was found in 57.5% (130) of samples, higher than IgA, found in 35% (79) of samples. IgA results were similar for SARS-CoV-2, with IgA present in 30% (68) of samples, while IgG was less present, in 44.2% (100) of samples. The proportion of influenza IgG responders was higher than that for SARS-CoV-2 IgG, but both populations presented similar proportions of IgA responders, possibly due to variable memory B cell survival. For both viruses, the authors found an important proportion (> 10%) of IgA+IgG- samples, suggesting the occurrence of humoral immunity directed to the mucosa. Conclusion: Specific antibodies for respiratory viruses in saliva are found in either infection or vaccination and are a convenient and sensitive diagnostic tool for host immune response.

Phenotype evaluation of human and canine isolates of Leishmania infantum.

Human visceral leishmaniasis (VL) and canine leishmaniasis (CanL) in countries of South and Central America are caused by Leishmania infantum and has been endemic in Brazil for several years. The parasite biodiversity as well as the pharmacologic properties of drugs and the host species, are involved in the efficacy or inefficacy of leishmaniasis treatments. Although there are substantial number of reports describing the genetic characterization of the clinical field isolates of L. infantum,the phenotypic parameters have been less studied. In this study isolates from human and canine leishmaniasis (Hum1 and Can1) obtained in Campinas, São Paulo state, Brazil were identified as L. infantum. The Hum1 and Can1 isolates exhibited typical promastigote growth pattern. Regarding morphological features Can1 isolate differed in cell size. The infectivity in vitro of both isolatesis lower compared to the reference strain of L. infantum. Moreover, the in vivo infectivity of the three parasites is similar in Balb/c mice. The Hum1 isolate is more sensitive to leishmanial drugs (amphotericin B, miltefosine and glucantime) than the Can1 isolate when inside human macrophages, but not when inside canine macrophages. These findings indicated that L. infantum isolates differs in some phenotypic characteristics.

Leishmania infantum transfected with toxic plasmid induces protection in mice infected with wild type L. infantum or L. amazonensis.

Leishmania infantum infection may cause visceral leishmaniasis (VL), a fatal disease having worldwide distribution, that may be silent or asymptomatic. The latter indicates that immunity is naturally developed in some individuals, and, therefore, a vaccine against VL would be possible. Molecular mechanisms of gene expression are being understood in Leishmania, and this knowledge may be useful for vaccine development. The aim of this study was developing an attenuated strain by regulating the expression of toxic proteins in a stage specific manner. For that purpose, the 3' UTR of an amastin gene, known by its increased expression in the amastigote phase, was selected for direct the expression of exogenous proteins. This construct (pFL-AMA), firstly, was proved effective for the expression of mCherry specifically in the intracellular form of L. infantum, as demonstrated by fluorescence microscopy, flow cytometry and Western blotting. Afterwards, mCherry coding sequence was replaced, in the pFL-AMA plasmid, by either egg avidin or the active form of bovine trypsin. Viability of transfected parasites was evaluated in promastigote axenic cultures and in in vitro infection of macrophages. Both lines of transfected parasites showed a limited capacity to multiply inside macrophages. BALB/c mice were inoculated intraperitoneally (i.p.) with a single dose consisting of 2 × 106L. infantum promastigotes transfected with plasmids bearing the toxic genes. After 10 weeks post-inoculation, no parasites were recovered by limiting dilution in either liver or spleen, but a specific immunological response was detected. The immunization with transfected parasites induced cellular and humoral immune responses with activation of TCD4+, TCD8+ and B cells, having a TH1-type response with increased levels of pro-inflammatory cytokines such as IFN-γ, TNF-α and IL-6. In parallel groups of mice, a challenge consisting on 1 × 106 virulent parasites of either L. infantum (inoculated i.p.) or L. amazonensis subcutaneously (s.c.) was performed. Vaccinated mice, challenged with L. infantum, showed lower parasite burdens in liver, spleen and bone marrow than infected mice with WT L. infantum (non-vaccinated); similarly, vaccinated mice developed smaller footpad inflammation than control group. These data support this strategy as an efficient immunization system aimed to the development of vaccines against different forms of leishmaniasis.

In vitro immunotoxicological assessment of a potent microbicidal nanocomposite based on graphene oxide and silver nanoparticles.

Graphene oxide (GO) and silver nanoparticles (AgNPs) can be formed into a hybrid nanomaterial, known as GOAg nanocomposite, which presents high antibacterial activity. The successful translation of this nanomaterial into medical use depends on critical information about its toxicological profile. In keeping with a Safe-by-design approach, we evaluated the immunotoxicity of GOAg using J774 and primary murine macrophages. The interaction between GOAg and macrophages was investigated with a scanning electron microscope (SEM). High-throughput technologies were employed to evaluate cell viability, apoptosis/necrosis, mitochondrial depolarization and lipid peroxidation. The inflammogenicity of nanomaterials was predicted after quantification of the cytokines IL-1ß, TNF-α and IL-10 before and after stimulation with interferon-γ (IFN-γ). The ratio between CD80 and CD206 macrophage populations were also estimated. In addition, the production of nitric oxide (NO) was investigated. SEM surveys revealed the potential of GOAg to induce frustrated phagocytosis. GOAg induced a dose-dependent mitochondrial depolarization, apoptosis and lipid peroxidation to J774 macrophages. GOAg toxicity was not modified in an inflammatory microenvironment, but its toxicity was within the range of concentrations used in bacterial inactivation. GOAg did not induce primary macrophages to significantly produce inflammatory cytokines, and previous macrophage stimulation did not enhance GOAg inflammogenicity. Additionally, the pristine nanomaterials and GOAg do not shift macrophages polarization towards M1. Sublethal concentrations of GOAg did not impair macrophages NO production. Finally, we suggest options for improvement of GOAg nanocomposite in ways that may help minimize its possible adverse outcomes to human health.

Gamma irradiation of Toxoplasma gondii protein extract improve immune response and protection in mice models.

Gamma radiation induces protein changes that enhance immunogenicity for venoms, used in antivenin production. Coccidian parasites exposed to gamma radiation elicit immune response with protection in mice and man, but without studies on the effect of gamma radiation in soluble acellular extracts or isolated proteins. Toxoplasmosis is a highly prevalent coccidian disease with only one vaccine for veterinary use but with remaining tissue cysts. Total parasite extracts or recombinant proteins used as immunogen induce usually low protection. Here, we study gamma radiation effect on T. gondii extracts proteins (STAG) and its induced immunity in experimental mice models. By SDS-PAGE, protein degradation is seen at high radiation doses, but at ideal dose (1500 Gy), there are preservation of the antigenicity and immunogenicity, detected by specific antibody recognition or production after mice immunization. Immunization with STAG irradiated at 1500 Gy induced significant protection in mice immunized and challenged with distinct T. gondii strains. In their blood, higher levels of specific CD19+, CD3+CD4+ and CD3+CD8+ activated cells were found when compared to mice immunized with STAG. Irradiated T. gondii tachyzoites extracts induce immune response and protection in mice in addition, could be a feasible alternative for Toxoplasma vaccine.

CD19 LYMPHOCYTE PROLIFERATION INDUCED BY Bifidobacterium animalis subsp. lactis IN C57BL/6 MICE EXPERIMENTALLY INFECTED WITH Toxoplasma gondii.

Toxoplasmosis is frequently acquired through the oral route by the ingestion of cysts or oocysts of Toxoplasma gondii. Once ingested, the parasites penetrate the intestinal epithelial cells and rapidly disseminate to all organs in the host. During T. gondii infection, the intestinal microbiota plays an important role in stimulating a protective immune response against the parasite. In this sense the use of probiotics is worthy of note since they are live microorganisms that have beneficial effects on the host through stimulation of the immune response that can be important in the control of T. gondii proliferation and dissemination in the host. In the present study, the action of the probiotic Bifidobacterium animalis subsp. lactis was investigated in C57BL/6 mice infected with oocysts of ME49 strain of T. gondii. The probiotic had an immunomodulatory action, inducing CD19 lymphocyte proliferation and consequently increasing anti-T. gondii antibody level. Bifidobacterium animalis subsp. lactis provided protection in supplemented mice, compared to the control group. In addition, supplemented animals had milder inflammatory process in the small intestine, indicating that the probiotic protects the intestinal mucosa during infection with T. gondii. It was concluded that the probiotic B. animalis subsp. lactis induces humoral immune response capable of providing protection against T. gondii infection.

Immunity in the spleen and blood of mice immunized with irradiated Toxoplasma gondii tachyzoites.

Toxoplasma gondii infection induces a strong and long-lasting immune response that is able to prevent most reinfections but allows tissue cysts. Irradiated, sterilized T. gondii tachyzoites are an interesting vaccine, and they induce immunity that is similar to infection, but without cysts. In this study, we evaluated the cellular immune response in the blood and spleen of mice immunized with this preparation by mouth (v.o.) or intraperitoneally (i.p.) and analyzed the protection after challenge with viable parasites. BALB/c mice were immunized with three i.p. or v.o. doses of irradiated T. gondii tachyzoites. Oral challenge with ten cysts of the ME-49 or VEG strain at 90 days after the last dose resulted in high levels of protection with low parasite burden in the immunized animals. There were higher levels of specific IgG, IgA and IgM antibodies in the serum, and the i.p. immunized mice had higher levels of the high-affinity IgG and IgM antibodies than the orally immunized mice, which had more high-affinity IgA antibodies. B cells (CD19(+)), plasma cells (CD138(+)) and the CD4(+) and CD8(+) T cell populations were increased in both the blood and spleen. Cells from the spleen of the i.p. immunized mice also showed antigen-induced production of interleukin-10 (IL-10), interferon gamma (IFN-γ) and interleukin 4 (IL-4). The CD4(+) T cells, B cells and likely CD8(+) T cells from the spleens of the i.p. immunized mice proliferated with a specific antigen. The protection was correlated with the spleen and blood CD8(+) T cell, high-affinity IgG and IgM and antigen-induced IL-10 and IL-4 production. Immunization with irradiated T. gondii tachyzoites induces an immune response that is mediated by B cells and CD4(+) and CD8(+) T cells, with increased humoral and cellular immune responses that are necessary for host protection after infection. The vaccine is similar to natural infection, but free of tissue cysts; this immunity restrains infection at challenge and can be an attractive and efficient model for vaccine development in toxoplasmosis.

Humoral responses and immune protection in mice immunized with irradiated T. gondii tachyzoites and challenged with three genetically distinct strains of T. gondii.

Toxoplasma gondii is an obligate intracellular parasite that infects a variety of mammals and birds. T. gondii also causes human toxoplasmosis; although toxoplasmosis is generally a benign disease, ocular, congenital or reactivated disease is associated with high numbers of disabled people. Infection occurs orally through the ingestion of meat containing cysts or by the intake of food or water contaminated with oocysts. Although the immune system responds to acute infection and mediates the clearance of tachyzoites, parasite cysts persist for the lifetime of the host in tissues such as the eye, muscle, and CNS. However, T. gondii RH strain tachyzoites irradiated with 255Gy do not cause residual infection and induce the same immunity as a natural infection. To assess the humoral response in BALB/c and C57BL/6J mice immunized with irradiated tachyzoites either by oral gavage (p.o.) or intraperitoneal (i.p.) injection, we analyzed total and high-affinity IgG and IgA antibodies in the serum. High levels of antigen-specific IgG were detected in the serum of parenterally immunized mice, with lower levels in mice immunized via the oral route. However, most serum antibodies exhibited low affinity for antigen in both mice strain. We also found antigen specific IgA antibodies in the stools of the mice, especially in orally immunized BALB/c mice. Examination of bone marrow and spleen cells demonstrated that both groups of immunized mice clearly produced specific IgG, at levels comparable to chronic infection, suggesting the generation of IgG specific memory. Next, we challenged i.p. or p.o. immunized mice with cysts from ME49, VEG or P strains of T. gondii. Oral immunization resulted in partial protection as compared to challenged naive mice; these findings were more evident in highly pathogenic ME49 strain challenge. Additionally, we found that while mucosal IgA was important for protection against infection, antigen-specific IgG antibodies were involved with protection against disease and disease pathogenesis. Most antigen responsive cells in culture produced specific high-affinity IgG after immunization, diverse of the findings in serum IgG or from cells after infection, which produced low proportion of high-avidity IgG.