RESUMO
The current COVID-19 vaccines protect against severe disease, but are not effective in controlling replication of the Variants of Concern (VOCs). Here, we used the existing pre-clinical models of severe and moderate COVID-19 to evaluate the efficacy of a Spike-based DNA vaccine (pCTV-WS) for protection against different VOCs. Immunization of transgenic (K18-hACE2) mice and hamsters induced significant levels of neutralizing antibodies (nAbs) to Wuhan and Delta isolates, but not to the Gamma and Omicron variants. Nevertheless, the pCTV-WS vaccine offered significant protection to all VOCs. Consistently, protection against lung pathology and viral load to Wuhan or Delta was mediated by nAbs, whereas in the absence of nAbs, T cells controlled viral replication, disease and lethality in mice infected with either the Gamma or Omicron variants. Hence, considering the conserved nature of CD4 and CD8 T cell epitopes, we corroborate the hypothesis that induction of effector T-cells should be a main goal for new vaccines against the emergent SARS-CoV-2 VOCs.
RESUMO
Accurate testing to detect SARS-CoV-2 RNA is key to counteract the virus spread. Nonetheless, the number of diagnostic laboratories able to perform qPCR tests is limited, particularly in developing countries. We describe the use of a virus-inactivating, denaturing solution (DS) to decrease virus infectivity in clinical specimens without affecting RNA integrity. Swab samples were collected from infected patients and from laboratory personnel using a commercially available viral transport solution and the in-house DS. Samples were tested by RT-qPCR, and exposure to infective viruses was also accessed by ELISA. The DS used did not interfere with viral genome detection and was able to maintain RNA integrity for up to 16 days at room temperature. Furthermore, virus loaded onto DS were inactivated, as attested by attempts to grow SARS-CoV-2 in cell monolayers after DS desalt filtration to remove toxic residues. The DS described here provides a strategy to maintain diagnostic accuracy and protects diagnostic laboratory personnel from accidental infection, as it has helped to protect our lab crew.
Assuntos
Teste para COVID-19/métodos , COVID-19/diagnóstico , Estabilidade de RNA/efeitos dos fármacos , RNA Viral/análise , SARS-CoV-2/genética , Manejo de Espécimes/métodos , Testes Diagnósticos de Rotina , Genoma Viral/genética , Humanos , Desnaturação Proteica/efeitos dos fármacos , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real , SARS-CoV-2/efeitos dos fármacosRESUMO
The balance between pro- and antiinflammatory mechanisms is essential to limit immune-mediated pathology, and CD4+ forkhead box P3 (Foxp3+) regulatory T cells (Treg) play an important role in this process. The expression of inhibitory receptors regulates cytokine production by Plasmodium vivax-specific T cells. Our goal was to assess the induction of programmed death-1 (PD-1) and cytotoxic T-lymphocyte antigen (CTLA-4) on Treg during malaria and to evaluate their function. We found that P. vivax infection triggered an increase in circulating Treg and their expression of CTLA-4 and PD-1. Functional analysis demonstrated that Treg from malaria patients had impaired suppressive ability and PD-1+Treg displayed lower levels of Foxp3 and Helios, but had higher frequencies of T-box transcription factor+ and interferon-gamma+ cells than PD-1-Treg. Thus malaria infection alters the function of circulating Treg by triggering increased expression of PD-1 on Treg that is associated with decreased regulatory function and increased proinflammatory characteristics.
Assuntos
Malária Vivax/imunologia , Malária Vivax/parasitologia , Linfócitos T Reguladores/fisiologia , Adulto , Proliferação de Células , Citocinas/genética , Citocinas/metabolismo , Feminino , Regulação da Expressão Gênica/imunologia , Humanos , Imunofenotipagem , Masculino , Pessoa de Meia-Idade , Plasmodium vivax , Reticulócitos/parasitologia , Reticulócitos/fisiologia , Adulto JovemRESUMO
Using flow cytometry, we evaluated the frequencies of CD4(+) and CD8(+) T cells and Foxp3(+) regulatory T cells (Tregs) in mononuclear cells in the jejunum, colon, and cervical and mesenteric lymph nodes of dogs naturally infected with Leishmania infantum and in uninfected controls. All infected dogs showed chronic lymphadenitis and enteritis. Despite persistent parasite loads, no erosion or ulcers were evident in the epithelial mucosa. The colon harbored more parasites than the jejunum. Frequencies of total CD4(+), total Foxp3, and CD4(+) Foxp3(+) cells were higher in the jejunum than in the colon. Despite negative enzyme-linked immunosorbent assay (ELISA) serum results for cytokines, levels of interleukin-10 (IL-10), gamma interferon (IFN-γ), transforming growth factor beta (TGF-ß), and tumor necrosis factor alpha (TNF-α) were higher in the jejunum than in the colon for infected dogs. However, IL-4 levels were higher in the colon than in the jejunum for infected dogs. There was no observed correlation between clinical signs and histopathological changes or immunological and parasitological findings in the gastrointestinal tract (GIT) of canines with visceral leishmaniasis. However, distinct segments of the GIT presented different immunological and parasitological responses. The jejunum showed a lower parasite load, with increased frequencies and expression of CD4, Foxp3, and CD8 receptors and IL-10, TGF-ß, IFN-γ, and TNF-α cytokines. The colon showed a higher parasite load, with increasing expression of IL-4. Leishmania infantum infection increased expression of CD4, Foxp3, IL-10, TGF-ß, IFN-γ, and TNF-α and reduced CD8 and IL-4 expression in both the jejunum and the colon.
Assuntos
Colo do Útero/imunologia , Colo/imunologia , Jejuno/imunologia , Leishmania infantum/imunologia , Leishmaniose Visceral/imunologia , Linfonodos/imunologia , Linfócitos T Reguladores/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/microbiologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/microbiologia , Colo do Útero/microbiologia , Colo/microbiologia , Doenças do Cão/imunologia , Doenças do Cão/microbiologia , Cães , Enterite/imunologia , Enterite/microbiologia , Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Feminino , Fatores de Transcrição Forkhead/imunologia , Interferon gama/imunologia , Interleucina-10/imunologia , Interleucina-4/imunologia , Jejuno/microbiologia , Leishmaniose Visceral/microbiologia , Linfonodos/microbiologia , Linfadenite/imunologia , Linfadenite/microbiologia , Masculino , Mucosa/imunologia , Mucosa/microbiologia , Carga Parasitária , Fator de Crescimento Transformador beta/imunologia , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Canine visceral leishmaniasis (CVL) is a severe and fatal systemic chronic inflammatory disease. We investigated the alterations in, and potential associations among, antioxidant enzymes, trace elements and histopathology in CVL. Blood and tissue levels of Cu-Zn superoxide dismutase, catalase and glutathione peroxidase were measured in mixed-breed dogs naturally infected with Leishmania infantum chagasi, symptomatic (n = 19) and asymptomatic (n = 11). Serum levels of copper, iron, zinc, selenium and nitric oxide, and plasma lipid peroxidation were measured. Histological and morphometric analyses were conducted of lesions in liver, spleen and lymph nodes. We found lower blood catalase and glutathione peroxidase activity to be correlated with lower iron and selenium respectively. However, higher activity of Cu-Zn superoxide dismutase was not correlated with the increase in copper and decreased in zinc observed in infected animals compared to controls. Organ tissue was characterized by lower enzyme activity in infected dogs than in controls, but this was not correlated with trace elements. Lipid peroxidation was higher in symptomatic than in asymptomatic and control dogs and was associated with lesions such as chronic inflammatory reaction, congestion, haemosiderin and fibrosis. Systemic iron deposition was observed primarily in the symptomatic dogs showing a higher tissue parasite load. Dogs with symptomatic CVL displayed enhanced LPO and Fe tissue deposition associated with decreased levels of antioxidant enzymes. These results showed new points in the pathology of CVL and might open new treatment perspectives associated with antioxidants and the role of iron in the pathogenesis of CVL.
Assuntos
Catalase/metabolismo , Doenças do Cão/metabolismo , Doenças do Cão/patologia , Glutationa Peroxidase/metabolismo , Leishmaniose Visceral/veterinária , Superóxido Dismutase/metabolismo , Oligoelementos/metabolismo , Animais , Modelos Animais de Doenças , Cães , Ferro/metabolismo , Leishmaniose Visceral/metabolismo , Leishmaniose Visceral/patologia , Peroxidação de Lipídeos , Fígado/metabolismo , Fígado/patologia , Linfonodos/metabolismo , Linfonodos/patologia , Óxido Nítrico/metabolismo , Selênio/metabolismo , Baço/metabolismo , Baço/patologiaRESUMO
BACKGROUND: Infection with parasite protozoa is a long-term health issue in tropical and subtropical regions throughout the world. The Toll-like receptor (TLR) signaling pathway is one of the first-responding defense systems against Leishmania. The aim of this study was to investigate the expression of TLR2 and TLR9 in jejunum and colon and its correlation with CD11c, CD11b, and CD14 receptors used as markers for dendritic cells and macrophages. METHODS: Twenty four dogs infected with Leishmania infantum were used in this study. Cytometry was carried out in lamina propria cells from jejunum and colon using markers for TLR2, TLR9, CD11b, CD11c and CD14. RESULTS: Cellular inflammatory exudate was diffuse in the mucosa and submucosa, predominately comprising mononuclear cells: plasma cells, macrophages, and lymphocytes. Despite the parasite load, microscopy showed no erosion was evident in the epithelial mucosa layers. The colon harbored more parasites than the jejunum. Flow cytometry revealed higher frequency of TLR2+ and CD11c+ dendritic cells in the colon than in the jejunum. Conversely, TLR9-expressing cells were more frequent in jejunum. Moreover, frequency of macrophages (CD11b+ and CD14+) expressing simultaneity TLR9 were lower in the colon than in jejunum, while CD11c+ cells predominated in the colon. Despite of the negative ELISA serum results, IL-10 and TNF-α were higher in jejunum than colon of infected animals. However, IL-4 was higher in colon than jejunum of infected animals. A higher expression these cytokines were demonstrated in infected dogs compared to uninfected dogs. CONCLUSIONS: There was no correlation between clinical signs and pathological changes and immunological and parasitological findings in the gastrointestinal tract in canine visceral leishmaniasis. However, jejunum showed a lower parasite load with increased frequency and expression of CD11b, TLR9, CD14/CD11b/TLR9 receptors and IL-10 and TNF-α cytokines. Conversely, the colon showed a higher parasite load along with increased frequency and expression of TLR2, CD11c receptors, and IL-4 cytokine. Thus, Leishmania infantum is able to interfere in jejunum increased expression of TLR2, TLR9, CD11b, CD14, CD14/CD11b/TLR9 receptors, IL-10, and TNF-α; and in colon increased expression of CD11c, TLR2, TLR9, CD11b, CD14 e, CD14/CD11b/TLR9 receptors, IL-10, and TNF-α.
Assuntos
Colo/metabolismo , Doenças do Cão/imunologia , Doenças do Cão/parasitologia , Jejuno/metabolismo , Leishmania infantum/imunologia , Leishmaniose Visceral/veterinária , Receptor 2 Toll-Like/metabolismo , Receptor Toll-Like 9/metabolismo , Animais , Antígenos CD/metabolismo , Brasil , Colo/imunologia , Colo/parasitologia , Colo/patologia , Citocinas/metabolismo , Doenças do Cão/metabolismo , Doenças do Cão/patologia , Cães , Fluorescência , Jejuno/imunologia , Jejuno/parasitologia , Jejuno/patologia , Leishmania infantum/fisiologia , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/patologia , Células Mieloides/metabolismo , ParasitosRESUMO
We propose that canine visceral leishmaniasis (CVL) is a systemic fibrotic disease, as evidenced by the wide distribution of fibrosis that we have found in the dogs suffering from chronic condition. The inflammatory cells apparently direct fibrosis formation. Twenty-four cases (symptomatic dogs) were identified from a total of one hundred and five cases that had been naturally infected with Leishmania chagasi and had been documented during an epidemiological survey of CVL carried out by the metropolitan area of the municipality of Belo Horizonte, MG, Brazil. The histological criterion was intralobular liver fibrosis, as has been described previously in dogs with visceral leishmaniasis. In addition to the findings in the liver, here we describe and quantify conspicuous and systemic deposition of collagen in other organs, including spleen, cervical lymph nodes, lung and kidney of all the infected symptomatic dogs. Thus we report that there is a systematic fibrotic picture in these animals, where inflammatory cells appear to direct fibrosis in all organs that have been studied. Therefore we propose that CVL is a systemic fibrotic disease.
Assuntos
Doenças do Cão/patologia , Leishmania infantum/fisiologia , Leishmaniose Visceral/veterinária , Animais , Brasil , Colágeno/metabolismo , Doenças do Cão/parasitologia , Cães , Feminino , Fibrose/veterinária , Rim/patologia , Leishmaniose Visceral/patologia , Fígado/patologia , Pulmão/patologia , Linfonodos/patologia , Baço/patologiaRESUMO
The aim of this study is to describe a case of an asymptomatic dog naturally infected with L. infantum chagasi with a surprising number of parasites in the duodenum. A mixed breed dog of unknown age was referred to the Center for Zoonoses Control of the Municipality of Ribeirão das Neves, Belo Horizonte Metropolitan area, Minas Gerais State, Brazil. The dog was diagnosed for Leishmania using an enzyme-linked immunosorbent assay (ELISA), direct parasitological examination of bone marrow aspiration, and immunohistochemistry of ear biopsy. After euthanasia samples of spleen, liver, lung, kidney, heart, cervical and mesenteric lymph nodes; ear, snout, abdominal skin and GIT segments (esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, and rectum) were evaluated histologically and immunohistochemically for the presence of parasite amastigotes. Gross and microscopic examination of necropsy samples showed no severe alterations of the mucosa in any gastrointestinal segment. A conspicuous parasite load was observed in the lamina propria of the duodenum, jejunum, ileum, cecum, colon, and rectum. Parasite distribution in the small intestine was diffuse through the lamina propria, whereas in the large intestine it was concentrated close to the muscularis mucosa and distant from the intestinal lumen. The parasite load in the duodenum, mainly in the subepithelial region, was higher...
Assuntos
Animais , Cães , Doenças do Cão , Ferimentos e Lesões/veterinária , Intestino Delgado/lesões , Leishmaniose Visceral/veterináriaRESUMO
The aim of this study is to describe a case of an asymptomatic dog naturally infected with L. infantum chagasi with a surprising number of parasites in the duodenum. A mixed breed dog of unknown age was referred to the Center for Zoonoses Control of the Municipality of Ribeirão das Neves, Belo Horizonte Metropolitan area, Minas Gerais State, Brazil. The dog was diagnosed for Leishmania using an enzyme-linked immunosorbent assay (ELISA), direct parasitological examination of bone marrow aspiration, and immunohistochemistry of ear biopsy. After euthanasia samples of spleen, liver, lung, kidney, heart, cervical and mesenteric lymph nodes; ear, snout, abdominal skin and GIT segments (esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, and rectum) were evaluated histologically and immunohistochemically for the presence of parasite amastigotes. Gross and microscopic examination of necropsy samples showed no severe alterations of the mucosa in any gastrointestinal segment. A conspicuous parasite load was observed in the lamina propria of the duodenum, jejunum, ileum, cecum, colon, and rectum. Parasite distribution in the small intestine was diffuse through the lamina propria, whereas in the large intestine it was concentrated close to the muscularis mucosa and distant from the intestinal lumen. The parasite load in the duodenum, mainly in the subepithelial region, was higher...(AU)
Assuntos
Animais , Cães , Doenças do Cão , Leishmaniose Visceral/veterinária , Ferimentos e Lesões/veterinária , Intestino Delgado/lesõesRESUMO
BACKGROUND: The aim of this study was to provide a systematic pathological and parasitological overview of the gastrointestinal tract (GIT), including the stomach, duodenum, jejunum, ileum, caecum and colon, of dogs naturally infected with Leishmania. METHODS: Twenty mongrel dogs naturally infected with Leishmania (Leishmania) infantum and obtained from the Control Zoonosis Center of the Municipality of Ribeirão das Neves, Belo Horizonte Metropolitan area, Minas Gerais (MG) state, Brazil, were analyzed. The dogs were divided into two groups: Group 1 comprised nine clinically normal dogs and group 2 comprised 11 clinically affected dogs. After necropsy, one sample was collected from each GIT segment, namely the stomach, duodenum, jejunum, ileum, caecum and colon. Furthermore, paraffin-embedded samples were used for histological and parasitological (immunohistochemistry) evaluation and a morphometrical study were carried out to determine the parasite load (immunolabeled amastigote forms of Leishmania). The Friedman and the Mann Whitney tests were used for statistical analysis. The Friedman test was used to analyze each segment of the GIT within each group of dogs and the Mann Whitney test was used to compare the GIT segments between clinically unaffected and affected dogs. RESULTS: The infected dogs had an increased number of macrophages, plasma cells and lymphocytes, but lesions were generally mild. Parasite distribution in the GIT was evident in all intestinal segments and layers of the intestinal wall (mucosal, muscular and submucosal) irrespective of the clinical status of the dogs. However, the parasite load was statistically higher in the caecum and colon than in other segments of the GIT. CONCLUSION: The high parasite burden evident throughout the GIT mucosa with only mild pathological alterations led us to consider whether Leishmania gains an advantage from the intestinal immunoregulatory response (immunological tolerance).