In situ cellular immune response in non-ulcerated skin lesions due to Leishmania (L) infantum chagasi infection

Skin lesions of patients affected by non-ulcerated cutaneous leishmaniasis (NUCL) caused by L. (L.) infantum chagasi are characterized by lymphohistiocytic inflammatory infiltrate associated with epithelioid granuloma and scarce parasitism. However, the in situ cellular immune response of these patients is unclear. Therefore, the aim of the present study was to characterize the cellular immune response in the skin lesions of patients affected by NUCL. Methods Twenty biopsies were processed by immunohistochemistry using primary antibodies to T lymphocytes (CD4, CD8), NK cells, B lymphocytes, macrophages, nitric oxide synthase and interferon-gamma. Results Immunohistochemistry revealed higher expression of all cellular types and molecules (IFN-γ, iNOS) in the dermis of diseased skin compared to the skin of healthy individuals (p < 0.05). Morphometric analysis performed in the skin lesions sections showed the predominance of CD8+ T lymphocytes in the mononuclear infiltrate, followed by macrophages, mostly iNOS+, a response that could be mediated by IFN-γ. Conclusion Our study improves knowledge of the cellular immune response in non-ulcerated or atypical cutaneous leishmaniasis caused by L. (L.) infantum chagasi in Central America and pointed to the pivotal participation of CD8+ T lymphocytes in the host defense mechanisms against the parasite in patients with NUCL.(AU)

In situ cellular immune response in non-ulcerated skin lesions due to Leishmania (L) infantum chagasi infection

Skin lesions of patients affected by non-ulcerated cutaneous leishmaniasis (NUCL) caused by L. (L.) infantum chagasi are characterized by lymphohistiocytic inflammatory infiltrate associated with epithelioid granuloma and scarce parasitism. However, the in situ cellular immune response of these patients is unclear. Therefore, the aim of the present study was to characterize the cellular immune response in the skin lesions of patients affected by NUCL. Methods Twenty biopsies were processed by immunohistochemistry using primary antibodies to T lymphocytes (CD4, CD8), NK cells, B lymphocytes, macrophages, nitric oxide synthase and interferon-gamma. Results Immunohistochemistry revealed higher expression of all cellular types and molecules (IFN-γ, iNOS) in the dermis of diseased skin compared to the skin of healthy individuals (p < 0.05). Morphometric analysis performed in the skin lesions sections showed the predominance of CD8+ T lymphocytes in the mononuclear infiltrate, followed by macrophages, mostly iNOS+, a response that could be mediated by IFN-γ. Conclusion Our study improves knowledge of the cellular immune response in non-ulcerated or atypical cutaneous leishmaniasis caused by L. (L.) infantum chagasi in Central America and pointed to the pivotal participation of CD8+ T lymphocytes in the host defense mechanisms against the parasite in patients with NUCL.(AU)

Avaliação da resposta imune de gatos naturalmente infectados por Leishmania (Leishmania) infantum

Os gatos são animais que podem se infectar por parasitos do gênero Leishmania spp. No entanto, pouco se conhece sobre a relação das manifestações clínicas da leishmaniose felina (LF) com as respostas imunológicas desses animais. Neste trabalho, nós realizamos diagnóstico laboratorial, clínico, quantificação de IgA, IgG e IgM anti-L. (L.) infantum, quantificação de linfócitos T CD4+ e CD8+ nas células mononucleares do sangue periférico (PBMC) e análise do transcriptoma afim de identificar as diferenças das resposta imune humoral e celular de gatos naturalmente infectados por L. (L.) infantum. Foi avaliado um total de 166 gatos, onde, 15,06% (25/166) apresentaram anticorpos anti-L. (L.) infantum pelo ELISA e 53,61% (89/166) pela RIFI, enquanto que pela PCR convencional (cPCR), 3,61% (6/166) apresentaram DNA do protozoário Leishmania no sangue, com 100% de identidade à espécie L. (L.) infantum (GenBank: KY379078.1). Desses seis animais, três apresentaram amastigotas de L. (L.) infantum em esfregaços do aspirado de linfonodo e/ou medula óssea no exame parasitológico e um teve a Leishmania isolada em cultura de aspirado de linfonodo. Assim, os gatos com testes sorológicos, parasitológicos, moleculares e de sequenciamento positivo para L. (L.) infantum, compuseram nosso grupo infectado (G1), enquanto que o grupo controle (G2) foi composto por seis gatos saudáveis. De acordo com anamnese clínica observamos que a magreza, alopecias e lesões de pele estavam entre os sinais clínicos mais frequentes entre os felinos. A quantificação de IgG, IgA e IgM totais anti-Leishmania foram significativamente maiores no G1 em comparação ao G2, (p = 2,966x10-6; p = 0,0002348 e p = 2,945x10-5, respectivamente). Nos exames hematológicos dos gatos detectamos a redução das plaquetas (p = 0,0062, p < 0,01) nos gatos G1 em relação ao G2, enquanto os leucócitos foram aumentados para gatos G2 (p = 0,014, p <0,05). Em relação aos parâmetros bioquímicos, os gatos infectados (G1) apresentaram aumento na concentração de proteína total (p = 4,4832e-06, p <0,01) com baixa albumina (p = 0,0065, p <0,01) e baixa aspartato aminotransferase (p = 0,0025, p <0,01) fora do intervalo de referência para a espécie. Pela citometria de fluxo observamos diferença significativa entre os linfócitos T CD4+ do G1 em relação ao G2 (p = 0.0427), entretanto, o mesmo não aconteceu para as subpopulações de linfócitos T CD8+ (p = 0.06199). Pela análise do transcriptoma destacamos a down regulação das vias metabólicas que controlam a atividade da proteína arginina desaminase (PAD), o processo de hemopoiese, a resposta ao estresse e o desenvolvimento do sistema imunológico e a up regulação da via sinalizadora do fator de crescimento dos fibroblastos (FGF) e do gene CXCR6 que participa da via sinalizadora da inflamação mediada pela sinalização de quimiocinas e citocinas. Possivelmente, a regulação negativa ou positiva dessas vias pode indicar a imunorregulação, por parte do parasita, como mecanismo de evasão do sistema imune do hospedeiro. Em contrapartida, a down regulação das vias sinalizadoras de ativação das células B (gene MAP3k2) de ativação das células T (gene LCP2), dos receptores Toll (gene TLR4) associado a down regulação dos genes IFNAR1, IFNGR2, IL13RA1, IL10 e IL1B nos gatos infectados (G1) do nosso estudo, indicam imunorregulação por parte do sistema imune felino em controlar o parasitismo intracelular por L. (L.) infantum, mas que não foram suficientes para controlar a progressão da doença. Esses resultados trazem as primeiras informações para elucidação da resposta imunológica às infecções por L. (L.) infantum em felinos.

Cats are animals that can be infected by parasites of the genus Leishmania spp. However, little is known about the relationship of the clinical manifestations of feline leishmaniasis (FL) with the immunological responses of these animals. In this work, we performed laboratory, clinical, quantification of IgA, IgG and IgM anti-L. (L.) infantum, quantification of CD4+ and CD8+ T lymphocytes in peripheral blood mononuclear cells (PBMC) and transcriptome analysis in order to identify differences in the humoral and cellular immune responses of cats naturally infected by L. (L.) infantum. A total of 166 cats were evaluated, where 15.06% (25/166) presented antibodies anti-L. (L.) infantum by ELISA and 53.61% (89/166) by RIFI, whereas by conventional PCR (cPCR), 3.61% (6/166) presented Leishmania protozoal DNA in the blood, with 100% identity to the species L. (L.) infantum (GenBank: KY379078.1). Of these six animals, three presented amastigotes of L. (L.) infantum in smears of the lymph node aspirate and/or bone marrow in parasitological examination and one had Leishmania isolated in lymph node aspirate culture. Thus, cats with serological, parasitological, molecular and positive sequencing tests for L. (L.) infantum, composed our infected group (G1), while the control group (G2) was composed of six healthy cats. According to clinical anamnesis we observed that thinness, alopecias and skin lesions were among the most frequent clinical signs among felines. Quantification of total anti-L. (L.) infantum IgG, IgA and IgM were significantly higher in G1 than in G2, (p = 2,966x10-6, p = 0,0002348 and p = 2,945x10-5, respectively). In the hematological examinations of cats, we detected the reduction of platelets (p = 0.0062, p <0.01) in G1 cats compared to G2, whereas leukocytes were increased for G2 cats (p = 0.014, p <0.05). In relation to the biochemical parameters, infected cats (G1) showed an increase in the total protein concentration (p = 4.4832e-06, p <0.01) with low albumin (p = 0.0065, p <0.01) and low aspartate aminotransferase (p = 0.0025, p <0.01) all outside the reference range for the species. By flow cytometry we observed a significant difference between G1 CD4+ T lymphocytes compared to G2 (p = 0.0427); however, the same did not occur for the CD8+ T lymphocyte subpopulations (p = 0.06199). By the analysis of the transcriptome we highlight the down regulation of the metabolic pathways that control the activity of the protein arginine deaminase (PAD), the hemopoiesis process, the response to stress and the development of the immune system and the up regulation of the signaling pathway fibroblast growth factor (FGF) and the CXCR6 gene that participate in the signaling pathway of inflammation mediated by chemokine and cytokine signaling. Possibly, the negative or positive regulation of these pathways may indicate the immunoregulation, by the parasite, as a mechanism to evade the immune system of the host. In contrast, down regulation of B cell activation (MAP3k2 gene) activation cells of T cells (LCP2 gene), Toll receptors (TLR4 gene) associated with down regulation of the genes IFNAR1, IFNGR2, IL13RA1 , IL10 and IL1B in the infected (G1) cats of our study indicate immunoregulation by the feline immune system to control intracellular parasitism by L. (L.) infantum but not sufficient to control the progression of the disease. These results provide the first information to elucidate the immunological response to L. (L.) infantum infections in felines.

Fontes de óleo na dieta e sua influência no desempenho e na imunidade de frangos de corte / Sources of oil in the diet and its influence on the performance and the immunity of broilers

[...] O objetivo deste estudo foi avaliar o efeito do consumo de óleos ricos em ácidos graxos poli-insaturados ômega-6 (PUFAs n-6) e ômega-3 (PUFAs n-3) sobre o desempenho e a resposta imunológica de frangos de corte frente a um desafio antigênico. Foram comparadas dietas formuladas com 7% de óleo de soja (OS), linhaça (OL) ou sardinha (OP), fornecidas a 240 frangos da linhagem Cobb, divididos em 24 grupos de 10 aves cada, num arranjo experimental 3x2 (3 tipos de óleo e aves vacinadas ou não vacinadas) e 4 repetições. O óleo de soja é rico em ácido linoleico, um PUFA n-6, o óleo de linhaça é fonte de ácido alfa-linolênico, um PUFA n-3, e o óleo de sardinha, de outros PUFAs n-3, como os ácidos eicosapentaenoico e docosahexaenoico. O consumo de ração, o ganho de peso e a conversão alimentar foram avaliados aos 21, 35 e 42 dias. Aos 7 e aos 21 dias de idade, metade das aves recebeu vacina contra doença de Newcastle. Quinze dias após a imunização, avaliou-se a produção de anticorpos pelo método de ELISA, expressa pela densidade óptica a 450 nm (D.O. 450nm). Apenas as aves alimentadas com ração contendo OS apresentaram maior imunidade humoral (P<0,05) após a vacinação. A resposta linfoproliferativa das aves, que expressa a imunidade celular, foi maior entre as aves vacinadas, em comparação às aves não vacinadas (P<0,05), independentemente do óleo utilizado. A fonte de óleo da ração ou a vacinação não influenciaram o ganho de peso das aves (P>0,05). Entre as aves que receberam dieta com OS, as aves vacinadas apresentaram pior conversão alimentar (P<0,05). Nos grupos que consumiram ração com OL ou OP, a vacinação não influenciou a conversão alimentar (P>0,05), considerando todo o período experimental. A utilização de óleo rico em PUFA n-6 na dieta de frangos de corte aumentou a resposta humoral, mas não influenciou a resposta celular frente a um desafio antigênico.(AU)

[...] The objective of this study was to evaluate the effect of the consumption of oils rich in omega-6 (n-6 PUFA) and omega-3 (n-3 PUFA) polyunsaturated fatty acids on the performance and the immune response of broilers submitted to an antigenic challenge. Diets were formulated with either 7% soybean oil (SO), linseed oil (LO) or sardine oil (PO) and provided to 240 Cobb broilers which were divided into 24 groups of 10 birds each, following a 3x2 experimental arrangement (3 types of oil and vaccinated or non-vaccinated birds) and four replications. Soybean oil is rich in linoleic acid (n-6 PUFA), linseed oil a source of alfa-linolenic acid (n-3 PUFA) and the sardine oil is a source of eicosapentaenoic and docosahexaenoic acids (other n-3 PUFA). Feed intake, weight gain and feed conversion were evaluated at 21, 35 and 42 days. Half of the birds were vaccinated against Newcastle disease at 7 and 21 days. Fifteen days after the immunization, the production of antibodies was evaluated by ELISA and expressed by optical density at 450 nm (O.D. 450 nm). Only the birds fed ration containing SO presented higher humoral immune response (p<0.05) after vaccination. The lymphoproliferative response, which expresses the cellular immunity, was higher in vaccinated than in the unvaccinated birds (P<0.05), regardless of the oil used. Neither the oil source in the ration nor the vaccination influenced birds' weight gain (P>0.05). The vaccination impaired the feed conversion of the birds fed diet containing SO (P<0.05) but did not influence feed conversion of the birds fed rations with LO or PO (P>0.05). The use of oil rich in n-6 PUFA in broilers' diet increased humoral response, but did not influence the cellular response against an antigenic challenge.(AU)

EFEITO DO MANANOLIGOSSACARÍDEO (MOS) SOBRE DESEMPENHO, SAÚDE INTESTINAL E RESPOSTA IMUNE EM LEITÕES PÓS-DESMAME DESAFIADOS POR Escherichia coli

Leitões no pós-desmame estão susceptíveis a quedas de desempenho, saúde intestinal e imunidade devido a alterações fisiológicas que ocorrem nesta fase de criação, concomitante com a ocorrência de infecções patogênicas por Escherichia coli. A fim de minimizar as perdas diretas e indiretas causadas por esta soma de fatores, o mananoligossacarídeo (MOS), prebiótico advindo de parede celular de levedura, é utilizado. Este trabalho teve por objetivo analisar o desempenho, saúde intestinal e resposta imunológica de leitões no pós-desmame em delineamento experimental inteiramente casualizado, com e sem inclusão de 0,1% MOS comercial (HyperGen® , Biorigin) na dieta e com e sem inoculação via oral de Escherichia coli K88+ enterotoxigênica. No cultivo microbiológico houve crescimento de E. coli em todos os tratamentos, o tratamento controle negativo (DC-INO) apresentou o menor crescimento e os tratamentos com desafio (DC+INO e MOS+INO) apresentaram os maiores crescimentos, mostrando que a inoculação foi eficaz devido a maior proliferação de E. coli no intestino dos animais com inoculação, além de maior formação de ácidos graxos voláteis (AGV) no ceco destes animais, devido a possível disbiose provocada pela ação do patógeno. Os animais que receberam MOS, independente de desafio sanitário (MOS+INO e MOS-INO), tiveram maior quantidade de células em proliferação nos tecidos linfoides de íleo e linfonodo mesentérico, com vilosidades intestinais maiores, além de melhores índices de desempenho, comparado ao tratamento controle negativo DC-INO. Isso comprova que a inclusão de MOS na concentração de 0,1% foi capaz de modular o sistema imune, principalmente frente a desafio sanitário, de forma controlada e eficaz, contribuindo com os resultados finais de desempenho. A ação da E. coli causou um desequilíbrio da microbiota intestinal, mas não prejudicou no desempenho de animais tratados com MOS. O tratamento com MOS+INO obteve mais ganhos e melhor desempenho, pela ação do MOS sob a saúde intestinal e a contribuição dos AGV, produzidos em maior quantidade devido a disbiose, que mesmo sendo uma alteração maléfica, aumentou a fonte de energia dos enterócitos, que aumentou o tamanho das vilosidades, o que contribuiu com os ganhos finais.

Piglets in postweaning are susceptible to performance declines, intestinal health and immunity due to physiological changes occurring during this breeding phase, concomitant with the occurrence of pathogenic Escherichia coli infections. In order to minimize the direct and indirect losses caused by this sum of factors, the prebiotic mananoligosaccharide (MOS), from yeast cell wall, is used. The objective of this study was to analyze the performance, intestinal health and immune response of piglets in postweaning in a completely randomized experimental design, with and without inclusion of 0.1% commercial MOS (HyperGen®, Biorigin) in the diet and with and without inoculation via oral Escherichia coli K88 + enterotoxigenic. In the microbiological culture there was growth of E. coli in all treatments, the negative control treatment (DC-INO) showed the lowest growth and the treatments with challenge (DC + INO and MOS + INO) showed the highest growth, showing that inoculation was effective due to the greater proliferation of E. coli in the gut of the inoculated animals, as well as a higher formation of volatile fatty acids (VFA) in these animals due to possible dysbiosis caused by the pathogen. Animals that received MOS, regardless of health challenge (MOS + INO and MOS-INO), had higher numbers of proliferating cells in the lymphoid tissues of ileum and mesenteric lymph node, with larger intestinal villi, and better performance indices compared to treatment negative control DC-INO. This proves that the inclusion of MOS in the concentration of 0.1% was able to modulate the immune system, mainly against sanitary challenge, in a controlled and effective way, contributing to the final results of performance. The action of E. coli caused an imbalance of the intestinal microbiota, but did not impair the performance of animals treated with MOS. The treatment with MOS + INO obtained more gains and better performance, due to the action of MOS under intestinal health and the contribution of the VFA, produced in greater quantity due to dysbiosis, that even being a malefic alteration, it increased the energy source of enterocytes, which increased the size of the villi, which contributed to the final gains.

Immunophenotypic characterization of patients with American cutaneous leishmaniasis prior to and after treatment in Pernambuco, Brazil

Leishmania infections induce a specific activation of host immunological response, particularly characterized by T cell expansion. Studies indicate the importance of the balance between CD4+ and CD8+ T cells, in which the first ones would have their number reduced during the healing process. Meanwhile, CD25+ T cells have been associated with the suppression of the immune response. Since the immune response has an essential role in both healing and progression of diseases, this study aimed to identify the percentage of CD3+, CD4+, CD8+, CD16+ and CD25+ T cells in the peripheral blood of patients afflicted with American cutaneous leishmaniasis (ACL) - before and after treatment - and healthy controls. Peripheral blood was collected and transferred to cytometry tubes containing monoclonal antibodies specific for cell surface markers CD3, CD4, CD8, CD16 e CD25. The immunophenotypic and morphometric parameters of cells were determined by flow cytometry and the results demonstrated a significant increase in the number of T CD8+ cells after treatment, suggesting a cytotoxic T cell response. An increase in CD25+ T cells in patients with active ACL and after treatment was also observed, suggesting the participation of these cells in the modulation of the exacerbated effector response.(AU)

Cytokine and antibody production during murine leptospirosis

The aim of the present study was to investigate the kinetics of humoral and cellular responses during leptospirosis. We observed that the presence of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) was associated with antibody production and bacterial recovery, and the compromising of both TNF-alpha and IL-6 in the immunopathogenesis of leptospirosis during an experimental infection of BALB/c mice inoculated with Leptospira interrogans serovar Canicola was verified. Results showed higher levels of TNF-alpha and IL-6 in the initial phase of infection, in which the greatest bacterial clearance was observed. However, when the bacterial recovery was compared with the kinetics of the production of antibodies, the results revealed a kinetics proportionally inverted to antibody production. This fact may be related to some inhibitory factor which could be responsible for the selective suppression of the cellular immune response. We concluded that during leptospirosis there was a greater mobilization of the cellular immune response activity, mainly in the initial phase of the infectious process, for posterior involvement of the humoral response, and that both TNF-alpha and IL-6 could be associated with the immunopathogenesis of the disease.