Low frequency of regulatory B-cells and increased CD4+ and CD8+ interferon-γ-producing cells in patients with tropical spastic paraparesis associated with human T-cell lymphotropic virus type

Abstract INTRODUCTION: Tropical spastic paraparesis/HTLV-1 associated myelopathy (TSP/HAM) is a disease caused by human T-cell lymphotropic virus type 1 (HTLV-I) that mainly infects CD4 T cells-for example, those of the CD4+CD25hiFOXP3+ [Treg] phenotype-where it inhibits forkhead box protein P3 (FOXP3) expression and promotes interferon-γ (IFN-γ) expression. However, the role it exerts on regulatory B cells (CD19+CD24hiCD38hi; Breg) is unknown. METHODS: The frequencies of Treg and Breg cells was evaluated and the Th1 profiles were assessed in TSP/HAM patients and healthy control subjects. RESULTS: Low percentages of Breg cells and high production of IFN-γ were observed in patients compared to those in healthy control subjects. CONCLUSIONS: The low percentage of Breg cells in patients and the increase in the frequency of Th1 cells suggest an imbalance in the control of the inflammatory response that contributes to the immunopathogenesis of TSP/HAM.

Evaluation of the profile of inflammatory cytokines, through immunohistochemistry, in the skin of patients with allergic contact dermatitis to nickel in the acute and chronic phases

Abstract: Background: Allergic contact dermatitis to ion nickel (Ni+2) is an inflammatory dermatosis, common in industrialized countries. It involves the activation of nickel-specific T-cells, followed by proliferation and induction of a mixed profile of both proinflammatory and regulatory cytokines, suggesting that several T-cell subtypes (helper - Th and cytotoxic - Tc) are involved. A broader understanding of the cytokine profile may lead to new therapeutic approaches. Objectives: This study aimed to analyze the cytokines TNF-α, INF-γ, IL-2, IL-4, IL-10, IL-13, IL-17 and IL-23 using the immunohistochemistry technique in order to try to identify their prevalence in chronic and acute eczema of patients with allergic contact dermatitis to Ni+2. Methods: We performed an immunohistochemical study for eight cytokines in 20 patients with Ni+2 allergic contact dermatitis, biopsied at the site of chronic eczema, triggered by the patient's daily contact with Ni+2, and at the site of acute eczema caused by nickel sulfate, 48 hours after applying the contact test. Results: The stained samples showed positive results for the eight cytokines studied. TNF-α, IFN-γ, IL-4, IL-13 and IL-17 had a higher prevalence in chronic eczema, IL-2 and IL-23 in acute eczema, and IL-10 presented a similar prevalence in both acute and chronic eczema. However, these prevalences were statistically significant only for IL-4 and IL-13. Study Limitations: Small sample size. Conclusions: In chronic and acute eczema, we observed the presence of a mixed cytokine profile of the T cell subtypes (Th/Tc), suggesting that the responses are expressed at the same time.

Histopathologic aspects of experimental cysticercosis and in situ cytokines profile in C57BL/6 mice / Aspectos histopatológicos da cisticercose experimental e perfil de citocinas in situ em camundongos C57BL/6

ABSTRACT Neurocysticercosis (NCC) is one of the parasitic infections that most affects the central nervous system. The knowledge regarding its immunopathogenesis and pathophysiology needs broadening. Taenia crassiceps cysticerci are used as the NCC experimental model. The aim of this work was to describe the general pathological processes and the in situ cytokine profile in C57BL/6 mice inoculated intracranially with viable T. crassiceps cysticerci. The histopathology analysis showed cysticerci in the extraparenchymal and intraventricular region, mononuclear inflammatory infiltration surrounding the parasite, microgliosis and meningitis. The analysis of the in situ immune profiles showed a predominance of the Th2 response. The IL-4 and IL-10 dosages were significantly increased in the infected group. The decrease in the INF-gamma dosage reflects the immunomodulation from the cysticerci. In conclusion, a T. crassiceps NCC infection in C57BL/6 mice triggers an inflammatory response, a predominance of Th2 type in situ profile, with mononuclear inflammatory cell infiltration, meningitis and microgliosis.

RESUMO Neurocisticercose (NCC) é uma das doenças parasitárias que mais afeta o sistema nervoso central. É necessário aprofundar o conhecimento em relação à sua imunopatogênese e patofisiologia. Os cisticercos de Taenia crassiceps são utilizados como modelo experimental para estudos da NCC. O objetivo deste trabalho foi descrever os processos patológicos gerais e o perfil de citocinas in situ em camundongos C57BL/6 inoculados via intracerebral com cisticercos viáveis de T. crassiceps. A análise histopatológica demonstrou cisticercos nas regiões extra-parenquimatosa e intraventricular, infiltrado inflamatório de células mononucleares ao redor do parasita, microgliose e meningite. A análise in situ do perfil de citocinas mostrou uma predominância da resposta Th2. As dosagens de IL-4 e IL-10 foram significativamente maiores no grupo infectado. Conclui-se que a NCC por T. crassiceps em camundongos C57BL/6 induz uma resposta inflamatória com predominância in situ de citocinas do perfil Th2, com infiltrado inflamatório de células mononucleares, meningite e microgliose.

Social conditions and immune response in human immunodeficiency virus-seropositive pregnant women: a cross-sectional study in Brazil

Abstract INTRODUCTION The functioning of the immune system during pregnancy is altered in both human immunodeficiency virus (HIV)-infected and uninfected women. Unfavorable socioeconomic conditions have been indicative of higher morbidity and mortality and worsening of the immune system. The aim of this study was to correlate social status with levels of interleukin (IL)-10 (non-inflammatory) and interferon-gamma (IFN-γ; inflammatory) cytokines. METHODS A cross-sectional study was conducted with three groups of women: 33 pregnant HIV-infected (G1); 40 non-pregnant, HIV-infected (G2); and 35 pregnant, HIV-uninfected. To measure the social status, a compound indicator called the social status index (SSI), was established using sociodemographic variables (i.e., education level, housing conditions, per capita income, and habitation and sanitary conditions). RESULTS The HIV-infected women had a higher proportion of unfavorable SSI (73% and 75% of G1 and G2, respectively). There were significantly lower IL-10 levels in the G1 group with both unfavorable and favorable SSI than in the other groups. No significant difference in IFN-γ levels was observed among groups. However, the G1 group had higher IFN-γ values among both favorable and unfavorable SSI groups. CONCLUSIONS Higher rates of unfavorable conditions, including lower education levels, IL-10 levels, and a trend for higher IFN-γ levels, were identified among HIV-infected women, pregnant and non-pregnant. These factors may interfere in health care and lead to poor outcomes during pregnancy. Therefore, we suggest that health policies could be created to specifically address these factors in this population.

Humoral and cellular immune response of mice challenged with Yersinia pestis antigenic preparations

ABSTRACT Objectives: The plague, which is an infectious disease caused by Yersinia pestis, still threatens many populations in several countries. The worldwide increase in human plague cases and the potential use of the bacteria as a biological weapon reinforce the need to study the immunity that is induced by potential vaccine candidates. To determine the immunogenicity of antigenic preparations based on the F1 protein and the total extract from Y. pestis, we assessed the role of these antigens in inducing an immune response. Methods: The immunogenicity of antigenic preparations based on the Y. pestis (YP) total extract and the Y. pestis fraction 1 capsular antigen protein (F1) was determined in Swiss-Webster mice immunized with 40 µg or 20 µg for each preparation. Immunophenotyping was performed by flow cytometry. Results: Animals immunized with the YP total extract did not elicit detectable anti-F1 antibodies (Ab) in the hemaglutination/inhibition (HA/HI) test. Animals immunized with 40 µg or 20 µg of the F1 protein produced anti-F1 Abs, with titres ranging from 1/16 to 1/8132. The average of CD3+-CD4+ and CD3+-CD8+ T cells did not differ significantly between the groups. Neither YP total extract nor F1 protein induced a significant expression of IFN-γ and IL-10 in CD4+ T lymphocytes. In addition, F1 failed to induce IFN-γ expression in CD8+ T cells, unlike the YP total extract. Conclusion: The results showed that F1 protein is not an immunogenic T cell antigen, although the YP total extract (40 µg dose) favoured CD8+ T cell-mediated cellular immunity.

Lactobacillus rhamnosus could inhibit Porphyromonas gingivalis derived CXCL8 attenuation

ABSTRACT An increasing body of evidence suggests that the use of probiotic bacteria is a promising intervention approach for the treatment of inflammatory diseases with a polymicrobial etiology. P. gingivalis has been noted to have a different way of interacting with the innate immune response of the host compared to other pathogenic bacteria, which is a recognized feature that inhibits CXCL8 expression. Objective The aim of the study was to determine if P. gingivalis infection modulates the inflammatory response of gingival stromal stem cells (G-MSSCs), including the release of CXCL8, and the expression of TLRs and if immunomodulatory L. rhamnosus ATCC9595 could prevent CXCL8 inhibition in experimental inflammation. Material and Methods G-MSSCs were pretreated with L. rhamnosus ATCC9595 and then stimulated with P. gingivalis ATCC33277. CXCL8 and IL-10 levels were investigated with ELISA and the TLR-4 and 2 were determined through flow cytometer analysis. Results CXCL8 was suppressed by P. gingivalis and L. rhamnosus ATCC9595, whereas incubation with both strains did not abolish CXCL8. L. rhamnosus ATCC9595 scaled down the expression of TLR4 and induced TLR2 expression when exposed to P. gingivalis stimulation (p<0.01). Conclusions These findings provide evidence that L. rhamnosus ATCC9595 can modulate the inflammatory signals and could introduce P. gingivalis to immune systems by inducing CXCL8 secretion.

Increased circulating macrophage migration inhibitory factor levels are associated with coronary artery disease

BACKGROUND: To evaluate the macrophage migration inhibitory factor and E-selectin levels in patients with acute coronary syndrome. MATERIALS/METHODS: We examined the plasma migration inhibitory factor and E-selectin levels in 87 patients who presented with chest pain at our hospital. The patients were classified into two groups according to their cardiac status. Sixty-five patients had acute myocardial infarction, and 22 patients had non-cardiac chest pain (non-coronary disease). We designated the latter group of patients as the control group. The patients who presented with acute myocardial infarction were further divided into two subgroups: ST-elevated myocardial infarction (n = 30) and non-ST elevated myocardial infarction (n = 35). RESULTS: We found higher plasma migration inhibitory factor levels in both acute myocardial infarction subgroups than in the control group. However, the E-selectin levels were similar between the acute myocardial infarction and control patients. In addition, we did not find a significant difference in the plasma migration inhibitory factor levels between the ST elevated myocardial infarction and NST-elevated myocardial infarction subgroups. DISCUSSION: The circulating concentrations of migration inhibitory factor were significantly increased in acute myocardial infarction patients, whereas the soluble E-selectin levels were similar between acute myocardial infarction patients and control subjects. Our results suggest that migration inhibitory factor may play a role in the atherosclerotic process. .

Análisis molecular del cáncer de colon esporádico / Molecular analysis of sporadic colon cancer

Background: In Chile, colorectal cancer (CRC) is often diagnosed in late stages. Thus, surgical treatment must be complemented with chemotherapy. KRAS mutations and microsatellite instability have been detected in these tumors. However, the response to treatment in patients without KRAS mutations varies and requires a better understanding. Aim: To determine the frequency and distribution of somatic point mutations in KRAS, BRAF and PIK3CA genes and microsatellite instability status (MSI) in patients with colon cancer (CC). Material and Methods: A prospective observational study of patients undergoing surgery for colon cancer. Tumor-derived DNA was analyzed by polymerase chain reaction (PCR) for the most frequent mutations of KRAS, BRAF and PIK3CA. PCR was also used to analyze MSI. Results: Fifty-eight patients with sporadic CC were analyzed, 16 showed KRAS mutations (G12R, G12D, G12V, G13D) and out of the 42 patients that did not show any mutation, 10 had mutations in BRAF (V600E) and PIK3CA (E542K, E545D, E545K, Q546E, H1047R). BRAF mutations alone or in combination with PIK3CA mutations were observed in 27% of high MSI tumors and in 2% of tumors without instability (p < 0.049). A higher percentage of high MSI tumors were located in the right colon (p < 0.001), and showed BRAF mutation (p < 0.020). Conclusions: The highest percentage of high MSI and BRAF mutations was observed in the right colon. Therefore, this study suggests the presence of different molecular features between right and left colon tumors that should be considered when defining the therapeutic management.

Vaccination with Klebsiella pneumoniae-derived extracellular vesicles protects against bacteria-induced lethality via both humoral and cellular immunity

The emergence of multidrug-resistant Klebsiella pneumoniae highlights the need to develop preventive measures to ameliorate Klebsiella infections. Bacteria-derived extracellular vesicles (EVs) are spherical nanometer-sized proteolipids enriched with outer membrane proteins. Gram-negative bacteria-derived EVs have gained interest for use as nonliving complex vaccines. In the present study, we evaluated whether K. pneumoniae-derived EVs confer protection against bacteria-induced lethality. K. pneumoniae-derived EVs isolated from in vitro bacterial culture supernatants induced innate immunity, including the upregulation of co-stimulatory molecule expression and proinflammatory mediator production. EV vaccination via the intraperitoneal route elicited EV-reactive antibodies and interferon-gamma-producing T-cell responses. Three vaccinations with the EVs prevented bacteria-induced lethality. As verified by sera and splenocytes adoptive transfer, the protective effect of EV vaccination was dependent on both humoral and cellular immunity. Taken together, these findings suggest that K. pneumoniae-derived EVs are a novel vaccine candidate against K. pneumoniae infections.

Oral coinfection can stress peripheral lymphocyte to inflammatory activity in leprosy

INTRODUCTION: This study evaluated the intracellular profile of interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-10 (IL-10) and interferon-γ (IFN-γ) in peripheral blood mononuclear cells (PBMCs) from leprosy patients based on oral infections presence to determine whether these coinfections could be associated with pro-inflammatory activity in leprosy. METHODS:Leprosy patients regardless of clinical form and specific leprosy treatment (n=38) were divided into two groups: Group I - leprosy patients with oral infections (n=19), and Group II - leprosy patients without oral infections (n=19). Non-leprosy patients presenting oral infections were assigned to the control Group (n=10). Intracellular IL-2, IL-4, IL-10 and IFN-γ production was evaluated by flow cytometry (FACS) before and 7 days after controlling the oral infection in the Group I, before and 7 days after dental prophylaxis in the Group II, and during oral infection process in control Group. RESULTS: Low percentages of CD3+ lymphocytes bearing IL-2, IL-10 and IFN-γ were observed in the Group I and Group II at baseline and 7 days after therapy or prophylaxis compared to controls. Group I showed reduced percentages of IL-4 at baseline and 7 days after therapy compared to controls, or at baseline of Group II, and the Group II showed reduced percentages of CD3+ cells bearing IL-4 compared to control. An increase of the percentages of CD3+cells bearing IL-4 was observed in the Group I after the oral infections treatment. CONCLUSIONS: The occurrence of oral infections favors the intracellular cytokines expression and, probably, the inflammatory reaction operating as a stimulatory signal triggering the leprosy reactions.

Estado funcional dos linfócitos T CD4+ e CD8+ e seu papel na progressão lenta da infecção por HIV em pacientes pediátricos / Functional state of CD4+ and CD8+ T lymphocytes and their role in the slow progression of HIV infection in pediatric patients

OBJETIVO: Avaliar o estado funcional dos linfócitos T CD4+ e CD8+ de pacientes pediátricos venezuelanos infectados pelo HIV-1. MÉTODOS: As crianças foram agrupadas como progressoras rápidas (PRs) ou progressoras lentas (PLs), com base no quadro clínico. Para determinar a funcionalidade dos linfócitos T CD4+ e CD8+, foram utilizadas técnicas de citometria de fluxo e caracterizados parâmetros de funcionalidade dessas células por meio de testes ex vivo como expressão de CD95/Fas e de CD127 e frequência de apoptose. Além disso, determinamos, em células mononucleares de sangue periférico, a proliferação do HIV e a produção de interleucina-10 (IL-10), do fator de necrose tumoral alfa (TNF-α) e de interferon gama (IFN-γ), e também estimamos o IFN-γ intracelular em células T CD4+. RESULTADOS: Nossos resultados indicam que vários mecanismos moleculares e celulares dos linfócitos T CD4+ e CD8+ tiveram piora nos PRs em comparação com PLs e controles. Ambos os tipos de linfócitos T dos PRs apresentaram aumento na expressão de CD95/Fas (p < 0,01), redução na expressão de CD127 (p < 0,01) e elevação na frequência de apoptose (p < 0,01). Além disso, as células T desses pacientes apresentaram diminuição na capacidade de proliferação mitogênica (p < 0,05), redução na porcentagem de linfócitos T CD4+ produtores de IFN-γ (p < 0,05) e menor capacidade de produção de IL-10, TNF-α e IFN-γ (p < 0,01) em comparação com PLs e controles. CONCLUSÃO: Nossos resultados indicam que o declínio das respostas moleculares e celulares dos linfócitos T está relacionado a uma rápida progressão e à diminuição na resistência à infecção pelo HIV-1 em crianças.

OBJECTIVE: To evaluate simultaneously the functional state of CD4+ and CD8+ T lymphocytes from Venezuelan HIV-1-infected pediatric patients. METHODS: Children were assigned to subgroups of rapid progressors (RPs) and slow progressors (SPs), based on clinical features. To determine the degree of CD4+ and CD8+ T-lymphocyte functionality, flow cytometry techniques were used, and diverse parameters of the functionality of these cells were characterized by ex vivo tests, such as expression of CD95/Fas and CD127, and frequency of apoptosis. In addition, we determined, in cultured peripheral blood mononuclear cells, HIV-specific proliferation and the production of interleukin-10 (IL-10), tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ), besides measuring intracellular IFN-γ in CD4+ T cells. RESULTS: Our results indicate that several molecular and cellular mechanisms of CD4+ and CD8+ T lymphocytes are deteriorated in RPs in comparison with SPs and controls. Indeed, both types of T lymphocytes from RPs exhibited an increased expression of CD95/Fas (p < 0.01), a significantly reduced expression of CD127 (p < 0.01), and an augmented frequency of apoptosis (p < 0.01). Furthermore, T cells from these patients displayed a diminished capacity of mitogen proliferation (p < 0.05), a reduced percentage of IFN-γ producing CD4+ T lymphocytes (p < 0.05) and a smaller capacity of IL-10, TNF-α and IFN-γ production (p < 0.01) in comparison with SP and control patients. CONCLUSION: Our findings indicate that the decline of the normal T lymphocyte molecular and cellular responses is related to a rapid progression and a decreased resistance to HIV-1 infection in children.

Immunomodulatory effects of human amniotic membrane-derived mesenchymal stem cells

Human amniotic membrane-derived mesenchymal stem cells (hAM-MSCs) are capable of differentiating into several lineages and possess immunomodulatory properties. In this study, we investigated the soluble factor-mediated immunomodulatory effects of hAM-MSCs. Mitogen-induced peripheral blood mononuclear cell (PBMC) proliferation was suppressed by hAM-MSCs in a dose-dependent manner as well as hAM-MSC culture supernatant. Moreover, interferon-gamma and interleukin (IL)-17 production significantly decreased from PBMC, whereas IL-10 from PBMCs and transforming growth factor beta (TGF-beta) production from hAM-MSCs significantly increased in co-cultures of hAM-MSCs and PBMCs. Production of several MSC factors, including hepatocyte growth factor (HGF), TGF-beta, prostaglandin E2 (PGE2), and indoleamine 2, 3 dioxygenase (IDO), increased significantly in hAM-MSCs co-cultured with PBMCs. These results indicate that the immunomodulatory effects of hAM-MSCs may be associated with soluble factors (TGF-beta, HGF, PGE2, and IDO), suggesting that hAM-MSCs may have potential clinical use in regenerative medicine.

Human dendritic cells sequentially matured with CD4+ T cells as a secondary signal favor CTL and long-term T memory cell responses

Dendritic cells (DCs) are professional antigen-presenting cells involved in the control and initiation of immune responses. In vivo, DCs exposed at the periphery to maturation stimuli migrate to lymph nodes, where they receive secondary signals from CD4+ T helper cells. These DCs become able to initiate CD8+ cytotoxic T lymphocyte (CTL) responses. However, in vitro investigations concerning human monocyte-derived DCs have never focused on their functional properties after such sequential maturation. Here, we studied human DC phenotypes and functions according to this sequential exposure to maturation stimuli. As first signals, we used TNF-α/polyI:C mimicking inflammatory and pathogen stimuli and, as second signals, we compared activated CD4+ T helper cells to a combination of CD40-L/ IFN-γ. Our results show that a sequential activation with activated CD4+ T cells dramatically increased the maturation of DCs in terms of their phenotype and cytokine secretion compared to DCs activated with maturation stimuli delivered simultaneously. Furthermore, this sequential maturation led to the induction of CTL with a long-term effector and central memory phenotypes. Thus, sequential delivery of maturation stimuli, which includes CD4+ T cells, should be considered in the future to improve the induction of long-term CTL memory in DC-based immunotherapy.

Expresión de IL-10, IL-4 e IFN- γ en lesiones activas de piel en niños con urticaria papular por picadura de pulga / Expression of IL-10, IL-4 and IFN-γ in active skin lesions of children with papular urticaria

Introducción. La urticaria papular por picadura de pulga se conoce como una enfermedad alérgica. Sin embargo, las investigaciones no muestran una clara relación con las enfermedades alérgicas. Objetivo. Estudiar la expresión de IL-10, IL-4 e IFN-γ, como marcadores de la respuesta efectora de células T en lesiones de piel de pacientes con urticaria papular por picadura de pulga. Materiales y métodos. Se incluyeron 14 biopsias de lesiones de piel de niños con diagnóstico de urticaria papular por picadura de pulga y 5 biopsias de piel sana obtenidas de niños sometidos a cirugía por enfermedades no inflamatorias. Todas las muestras se obtuvieron de niños menores de 12 años. Se extrajo ARN con trizol y se cuantificaron los niveles de expresión de las citocinas con la técnica de reacción en cadena de la polimerasa en tiempo real. Resultados. En los pacientes con urticaria papular por picadura de pulga, se encontró amplia diversidad en los niveles de expresión de IFN-γ e IL-10, y valores bajos constantes para IL-4. Se observaron tres perfiles que no corresponden a un patrón común en los pacientes. Las muestras obtenidas de tejidos sanos no presentaron expresión de las citocinas. Conclusiones. Los datos corresponden a la primera descripción de citocinas que median la respuesta inmunitaria en el sitio de la lesión cutánea en niños con con urticaria papular por picadura de pulga, lo cual indica que la respuesta local es mixta ya que no se encuentra predominio de un fenotipo específico en ninguno de los pacientes.

Introduction: Papular urticaria caused by the bites of fleas traditionally has been defined as a chronic allergic disease. However, currently no clear relationship has been described between this pathology and common allergic diseases. Objective: The expression of IL-10, IL-4 and IFN-γ as markers of effector T cell responses was examined in skin lesions of patients with papular urticaria by flea bite. Materials and methods: Fourteen skin lesion biopsies were sampled from children with a clinical diagnosis of papular urticaria by flea bite and were compared with 5 healthy skin biopsies of children with no history of the disease. All children were under 12 years old. RNA was extracted with trizol and the expression levels of cytokines were analyzed by real time PCR technique. Results: A wide range in the expression levels of IFN-γ and IL-10 was noted as well as constant low values of IL-4. Three distinct profiles were observed, but which did not correspond to a recognizable pattern among the patients. The samples obtained from healthy tissues showed no expression of any of the cytokines. Conclusions: This is the first characterization of cytokines that mediate the immune response at the site of the skin lesion in children with papular urticaria by flea bite. The data indicated that the local response was mixed and that a single phenotype is not predominant among the patients.

An experimental protocol for the establishment of dogs with long-term cellular immune reactions to Leishmania antigens

Domestic dogs are considered to be the main reservoirs of zoonotic visceral leishmaniasis. In this work, we evaluated a protocol to induce Leishmania infantum/Leishmania chagasi-specific cellular and humoral immune responses in dogs, which consisted of two injections of Leishmania promastigote lysate followed by a subcutaneous inoculation of viable promastigotes. The primary objective was to establish a canine experimental model to provide positive controls for testing immune responses to Leishmania in laboratory conditions. After inoculation of viable promastigotes, specific proliferative responses of peripheral blood mononuclear cells (PBMCs) to either Leishmania lysate or recombinant proteins, the in vitro production of interferon-γ by antigen-stimulated PBMCs and a significant increase in circulating levels of anti-Leishmania antibodies were observed. The immunized dogs also displayed positive delayed-type hypersensitivity reactions to Leishmania crude antigens and to purified recombinant proteins. An important finding that supports the suitability of the dogs as positive controls is that they remained healthy for the entire observation period, i.e., more than seven years after infection. Following the Leishmania antigen lysate injections, the infection of dogs by the subcutaneous route appears to induce a sustained cellular immune response, leading to an asymptomatic infection. This provides a useful model for both the selection of immunogenic Leishmania antigens and for immunobiological studies on their possible immunoprotective activities.

Avaliação do interferon-gama no diagnóstico e no monitoramento do tratamento da tuberculose latente / Evaluation of interferon-gamma in the diagnosis and monitoring of treatment of latent tuberculosis

A identificação e o tratamento dos indivíduos com tuberculose latente (TBL), ou seja, infectados, são medidas essenciais para o controle e eliminação da TB. Entretanto, os recursos disponíveis para o diagnóstico da TBL no Brasil são falhos, o que dificulta a identificação e o tratamento precoce. A fim de eliminar as atuais limitações dos testes empregados, novos testes têm sido desenvolvidos, a exemplos do ensaios de liberação de interferon-gama (IGRAs). A introdução dos IGRAs para uso de rotina em países de alta prevalência tem sido questionada. Principalmente, devido ao grande número de resultados discordantes gerado entre os testes diagnósticos: IGRA e o teste tuberculínico (TT). O Objetivo do estudo foi analisar os fatores associados com a discordância entre o resultados dos testes IGRA e o TT; avaliar a prática e a aderência do tratamento da TBL em uma população de alto risco; e avaliar o IGRA comercial para monitorar a resposta ao tratamento. Comunicantes domiciliares de pacientes com TB pulmonar recém diagnosticados foram submetidos a radiografia do tórax, coleta de sangue para realização do IGRA, e a um questionário específico. Os comunicantes que tiveram resultado do TT positivo, ≥ 10 mm, foram convidados a retornar ao hospital a cada mês para reposição dos comprimidos de isoniazida (H). Após um período de seis meses eles foram novamente submetidos a uma segunda coleta de sangue para a realização do IGRA. Dos 261 comunicantes satisfatoriamente testados pelo TT, 145 (55,6%) tiveram resultados positivos; dos 298 satisfatoriamente testados pelo IGRA, 127 (43,1%) tiveram resultados positivos. A concordância entre os testes foi de 0,76 (κ = 0,53, IC 95% 0,43-0,63). Sessenta e um (24%) tiveram resultados discordantes: 44 (72%) com o TT-positivo e IGRA-negativo e 17 (28%), com TT-negativo e IGRA-positivo. Comparado com o grupo TT-negativo e IGRA-negativo, os grupos TT-positivo e IGRA-negativo e o grupo TT-positivo e IGRA-positivo foram significativamente mais propensos a ter uma radiografia de tórax apresentando nódulo calcificado (OR = 6,8, IC 95% 1,3-35,0; OR = 7,4, IC 95% 2,2-24,4, respectivamente). O grupo TT-negativo e IGRA-positivo foi exposto ao paciente índice por mais tempo do que o grupo TT-negativo e IGRA-negativo (OR = 7,2, IC 95% 1,7-29,3). Dos 101 comunicantes que iniciaram o tratamento para TBL, 54 (53,5%)completaram o esquema de 6 meses. O risco de não conclusão do tratamento foi significativamente maior nos comunicantes que relataram efeitos colaterais para H (RR 2,69, IC 95% 1,3-5,8, P = 0,01), e os que necessitavam ingressar em duas rotas de ônibus para uma viagem de ida ao hospital (RR 1,8, IC 95% 1,01-3,3, P = 0,04). Dos 26 comunicantes que retornaram para uma segunda coleta de sangue, 25 (96%) apresentaram nível de IFN-γ superior ao seu nível antes do tratamento (P ≤ 0,001). Apenas uma pessoa teve uma diminuição do nível de IFN-γ após o tratamento, mas manteve-se positivo para TBL. Em um cenário de alta transmissão, o nível de IFN-γ aumentou após o tratamento da TBL. Os grupos TT-positivo e IGRA-negativo e TT-positivo e IGRA-positivo compartilham características mais semelhantes entre si do que com o grupo TT-negativo e IGRA-negativo. Em uma configuração endêmica para a TB, os resultados do TT parecem ser mais adequado na decisão de tratar a TBL. Quase 50% dos comunicantes com alto risco de desenvolver TB, concluíram um curso de 6 meses de tratamento para TBL. O tratamento foi mais afetado por intolerância à medicação e pelas dificuldades para visitas de acompanhamento. Além disso, mais estudos devem ser realizados para entender se a elevação dos níveis de IFN-γ é passageira.

Subcutaneous immunization against Leishmania major - infection in mice: efficacy of formalin-killed promastigotes combined with adjuvants / Imunização subcutânea na infecção do camundongo pela Leishmania major: eficácia dos promastigotos mortos pela formalina combinada com adjuvantes

Formalin-killed promastigotes (FKP) of Leishmania major, in combination with Montanide ISA 720 (MISA), BCG or alum were used in vaccination of an inbred murine model against cutaneous leishmaniasis (CL). Significant and specific increases in anti-FKP IgG responses were detected for both alum-FKP and BCG-FKP compared to MISA-FKP (p < 0.001). Significant increases in splenic lymphocyte recall proliferation was obtained in the MISA-FKP vaccinated mice compared to alum-FKP or BCG-FKP vaccinated groups (p < 0.01). The highest interferon-ã responses were observed in the BCG-FKP group followed by the MISA-FKP while the alum-FKP gave the least responses. Significantly reduced lesion sizes were obtained in the MISA-FKP group compared to the BCG/alum adjuvants-FKP vaccinated groups. Although the BCG-FKP group showed the highest IFN-ã responses, it failed to control cutaneous lesions. Significant reductions in parasite numbers were observed in the MISA-FKP and BCG-FKP vaccinated groups (p < 0.001). There was a good correlation between parasite burden and IFN-ã level indicating IFN-ã response as a sensitive parameter of the immune status. In conclusion, MISA-FKP is the most efficacious vaccine formulation against murine cutaneous leishmaniasis.

Promastigotos mortos pela formalina (FKP) de Leishmania major combinados com Montanide ISA 720 (MISA), BCG ou alumen foram usados na vacinação de modelo murino cutâneo de leishmaniose (CL). Aumento significante e específico de resposta IgG anti FKP foram detectados tanto no FKP com alumen como naquele com BCG comparados ao MISA-FKP (p < 0,001). Aumento significante da proliferação esplênica de linfócitos de memória foi obtida nos camundongos vacinados com MISA-FKP quando comparados aos grupos vacinados com alumen-FKP ou BCG-FKP (p < 0,01). As maiores respostas por interferon-gama foram observadas no grupo BCG-FKP seguido pelo MISA-FKP enquanto que o alumen-FKP deu a menor resposta. No grupo MISA-FKP foram obtidas reduções significantes do tamanho das lesões quando comparado aos grupos vacinados com BCG/adjuvante de alumen-FKP. Embora o grupo BCG-FKP tenha mostrado a maior resposta por interferon-gama, não houve controle das lesões cutâneas. Redução significante no número de parasitas foi observada tanto no grupo vacinado com MISA-FKP como no BCG-FKP (p < 0,001). Houve boa correlação entre a carga parasitária e o nível de interferon-gama indicando que a resposta do interferon-gama é parâmetro sensível do estado imunológico. Em conclusão, MISA-FKP é a forma mais eficaz de vacina contra a leishmaniose cutânea murina.

Leishmania spp. parasite isolation through inoculation of patient biopsy macerates in interferon gamma knockout mice / Leishmania spp.: isolamento de parasitos pela inoculação de macerados de biopsias de pacientes em camundongos deficientes em interferon gama

Isolation of Leishmania parasite and species identification are important for confirmation and to help define the epidemiology of the leishmaniasis. Mice are often used to isolate pathogens, but the most common mouse strains are resistant to infection with parasites from the Leishmania (Viannia) subgenus. In this study we tested the inoculation of interferon gamma knockout (IFNγ KO) mice with biopsy macerates from Leishmania-infected patients to increase the possibility of isolating parasites. Biopsies from twenty five patients with clinical signs of leishmaniasis were taken and tested for the presence of parasites. Immunohistochemical assay (IHC) and conventional histopathology detected the parasite in 88 percent and 83 percent of the patients, respectively. Leishmania sp. were isolated in biopsy macerates from 52 percent of the patients by culture in Grace's insect medium, but 13 percent of isolates were lost due to contamination. Inoculation of macerates in IFNγ KO mice provides isolation of parasites in 31.8 percent of the biopsies. Most isolates belong to L. (Viannia) subgenus, as confirmed by PCR, except one that belongs to L. (Leishmania) subgenus. Our preliminary results support the use of IFNγ KO mice to improve the possibility to isolate New World Leishmania species.

O isolamento e a identificação da espécie de parasito do gênero Leishmania são importantes para a confirmação e auxiliam na epidemiologia da leishmaniose. Os camundongos são freqüentemente utilizados para isolar patógenos, porém, as linhagens mais comuns de camundongos são resistentes à infecção por parasitos do subgênero Leishmania (Viannia). Neste estudo, avaliamos a inoculação de macerados de biópsias de pacientes infectados em camundongos deficientes do gene do interferon gama (IFNγ KO) como um método para aumentar a possibilidade de isolar Leishmania spp. Biópsias de 25 pacientes infectados com Leishmania sp. foram avaliadas para a presença de parasitos pelos métodos de imunohistoquímica (IHC) e histopatologia convencional. Os parasitos foram observados, respectivamente, em 88 por cento e 83 por cento das biópsias. Leishmania sp. foi isolada de macerados de biópsia de 52 por cento dos pacientes infectados, quando cultivados em meio Grace, porém, 13 por cento destes isolados foram perdidos devido a contaminações. Inoculação dos macerados em camundongos IFNγ KO proporcionou o isolamento de parasitos oriundos de 31,8 por cento dos pacientes. A maioria dos isolados pertence ao subgênero L. (Viannia), exceto um que pertence ao subgênero L. (Leishmania), como confirmado pela reação da polimerase em cadeia. Nossos resultados preliminares sugerem que o uso de camundongos IFNγ KO pode ser útil para aumentar a possibilidade de isolamento de leishmânias encontradas nas Américas.

15d-PGJ2 modulates acute immune responses to Trypanosoma cruzi infection

The acute phase of Trypanosoma cruzi infection is associated with a strong inflammatory reaction in the heart characterised by a massive infiltration of immune cells that is dependent on the T. cruzi strain and the host response. 15d-PGJ2 belongs to a new class of anti-inflammatory compounds with possible clinical applications. We evaluated the effects of 15d-PGJ2 administered during the acute phase of T. cruzi infection in mice. Mice were infected with the Colombian strain of T. cruzi and subsequently treated with 15d-PGJ2 repeatedly for seven days. The inflammatory infiltrate was examined by histologic analysis. Slides were immunohistochemically stained to count the number and the relative size of parasite nests. Infection-induced changes in serum cytokine levels were measured by ELISA. The results demonstrated that treatment with 15d-PGJ2 reduced the inflammatory infiltrate in the skeletal muscle at the site of infection and decreased the number of lymphocytes and neutrophils in the blood. In addition, we found that 15d-PGJ2 led to a decrease in the relative volume density of amastigote nests in cardiac muscle. T. cruzi-infected animals treated with 15d-PGJ2 displayed a statistically significant increase in IL-10 levels with no change in IFN-ã levels. Taken together, we demonstrate that treatment with 15d-PGJ2 in the acute phase of Chagas disease led to a controlled immune response with decreased numbers of amastigote nests, as measured by the volume density.

IL-12-STAT4-IFN-gamma axis is a key downstream pathway in the development of IL-13-mediated asthma phenotypes in a Th2 type asthma model

IL-4 and IL-13 are closely related cytokines that are produced by Th2 cells. However, IL-4 and IL-13 have different effects on the development of asthma phenotypes. Here, we evaluated downstream molecular mechanisms involved in the development of Th2 type asthma phenotypes. A murine model of Th2 asthma was used that involved intraperitoneal sensitization with an allergen (ovalbumin) plus alum and then challenge with ovalbumin alone. Asthma phenotypes, including airway-hyperresponsiveness (AHR), lung inflammation, and immunologic parameters were evaluated after allergen challenge in mice deficient in candidate genes. The present study showed that methacholine AHR and lung inflammation developed in allergen-challenged IL-4-deficient mice but not in allergen-challenged IL-13-deficient mice. In addition, the production of OVA-specific IgG2a and IFN-gamma-inducible protein (IP)-10 was also impaired in the absence of IL-13, but not of IL-4. Lung-targeted IFN-gamma over-expression in the airways enhanced methacholine AHR and non-eosinophilic inflammation; in addition, these asthma phenotypes were impaired in allergen-challenged IFN-gamma-deficient mice. Moreover, AHR, non-eosinophilic inflammation, and IFN-gamma expression were impaired in allergen-challenged IL-12Rbeta2- and STAT4-deficient mice; however, AHR and non-eosinophilic inflammation were not impaired in allergen-challenged IL-4Ralpha-deficient mice, and these phenomena were accompanied by the enhanced expression of IL-12 and IFN-gamma. The present data suggest that IL-13-mediated asthma phenotypes, such as AHR and non-eosinophilic inflammation, in the Th2 type asthma are dependent on the IL-12-STAT4-IFN-gamma axis, and that these asthma phenotypes are independent of IL-4Ralpha-mediated signaling.