Immunological Characteristics between αß TDC and γδ TDC Cells in the Spleen of Breast Cancer-Induced Mice / Características imunológicas entre células TDC αß e TDC γδ no baço de camundongos com câncer de mama induzido

Abstract Objective To evaluate the antitumoral role of γδ TDC cells and αβ TDC cells in an experimental model of breast cancer. Methods Thirty female Balb/c mice were divided into 2 groups: control group (n=15) and induced-4T1 group (n=15), in which the mice received 2 x 105 4T1 mammary tumor cell line. Following the 28-day experimental period, immune cells were collected from the spleen and analyzed by flow cytometry for comparison of αβ TDC (TCRαβ+ CD11c+MHCII+) and γδ TDC (TCRγδ+CD11c+MHCII+) cells regarding surface markers (CD4+ and C8+) and cytokines (IFN-γ, TNF-α, IL-12 and IL-17). Results A total of 26.53% of γδ TDC- control group (p<0.0001) - the proportion of αβ TDC was lower in splenic cells than γδ TDC; however, these 2 cell types were reduced in tumor conditions (p<0.0001), and the proportion of IFN-γ, TNF-α, IL-12 and IL-17 cytokines produced by γδ TDC was higher than those produced by αβ TDC, but it decreased under conditions of tumor-related immune system response (p<0.0001). Conclusion Healthy mice engrafted with malignant cells 4T1 breast tumor presented TDC with γδ TCR repertoire. These cells express cytotoxic molecules of lymphocytes T, producing anti-tumor proinflammatory cytokines.

Resumo Objetivo Esclarecer o possível papel antitumoral das células TDC γδ e TDC αβ em um modelo experimental de câncer de mama. Métodos Trinta baços de camundongos Balb/c analisados por citometria de fluxo, separados entre grupo controle (n=15) e o grupo tumoral induzido por 4T1 (n=15). Resultados Presença de 26,53% de TDC γδ nos camundongos do grupo controle (p<0,0001), proporção de TDC αβ menor em células esplênicas do que TDC γδ; no entanto, estes dois tipos de células são reduzidos emcondições tumorais (p<0,0001), e a proporção de citocinas IFN-γ, TNF-α, IL-12 e IL-17 produzidas pelas célula TDC γδ foi maior do que as produzidas pelas células TDC αβ, mas foram diminuídas sob condições de resposta ao sistema imunológico relacionada ao tumor (p<0,0001). Conclusão Camundongos saudáveis induzidos ao tumor de mama 4T1 apresentaram TDC com repertório TCR γδ. Estas células expressam moléculas citotóxicas de linfócitos T, produzindo citocinas proinflamatórias anti-tumor.

CD4+CD69+ T cells and CD4+CD25+FoxP3+ Treg cells imbalance in peripheral blood, spleen and peritoneal lavage from pristane-induced systemic lupus erythematosus (SLE) mice

Abstract Background: Adaptive immune cells, including CD4+CD69+ and CD4+CD25+FoxP3+ regulatory T (Treg) cells, are important for maintaining immunological tolerance. In human systemic lupus erythematosus (SLE), CD4+CD25+FoxP3+ Treg cells are reduced, whereas CD69 expression is increased, resulting in a homeostatic immune imbalance that may intensify autoreactive T cell activity. To analyze the mechanisms implicated in autotolerance failure, we evaluated CD4+CD69+ and CD4+CD25+FoxP3+ T cells and interleukin profiles in a pristane-induced SLE experimental model. Methods: For lupus induction, 26 female Balb/c mice received a single intraperitoneal 0.5 ml dose of pristane, and 16 mice received the same dose of saline. Blood and spleen samples were collected from euthanized mice 90 and 120 days after pristane or saline inoculation. Mononuclear cells from peripheral blood (PBMC), peritoneal lavage (PL) and splenocytes were obtained by erythrocyte lysis and cryopreserved for further evaluation by flow cytometry using the GuavaEasyCyte TM HT. After thawing, cells were washed and stained with monoclonal antibodies against CD3, CD4, CD8, CD25, CD28, CD69, FoxP3, CD14 and Ly6C (BD Pharmingen TM). Interleukins were quantified using Multiplex® MAP. The Mann-Whitney test and the Pearson coefficient were used for statistical analysis, and p < 0.05 considered significant. Results: Compared with the controls, SLE-induced animals presented increased numbers of CD4+CD69+ T cells in the blood on T90 and T120 (p = 0.022 and p = 0.008) and in the spleen on T120 (p = 0.049), but there were decreased numbers in the PL (p = 0.049) on T120. The percentage of Treg was lower in blood (p < 0.005 and p < 0.012) on T90 and T120, in spleen (p = 0.043) on T120 and in PL (p = 0.001) on T90. Increased numbers of CD4+ CD69+ T cells in the PL were positively associated with high IL-2 (p = 0.486) and IFN-γ (p = 0.017) levels, whereas reduced Treg cells in the blood were negatively correlated with TNFα levels (p = 0.043) and positively correlated with TGFβ1 (p = 0.038). Conclusion: Increased numbers of CD4+CD69+ T cells and reduced numbers of CD4+CD25+FoxP3+ Treg cells with an altered interleukin profile suggests loss of autotolerance in pristane-induced lupus mice, which is similar to human lupus. Therefore, this model is useful in evaluating mechanisms of cellular activation, peripheral tolerance and homeostatic immune imbalance involved in human SLE.

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.

BJ-1108, a 6-Amino-2,4,5-trimethylpyridin-3-ol analogue, regulates differentiation of Th1 and Th17 cells to ameliorate experimental autoimmune encephalomyelitis

BACKGROUND: CD4+ T cells play an important role in the initiation of an immune response by providing help to other cells. Among the helper T subsets, interferon-γ (IFN-γ)-secreting T helper 1 (Th1) and IL-17-secreting T helper 17 (Th17) cells are indispensable for clearance of intracellular as well as extracellular pathogens. However, Th1 and Th17 cells are also associated with pathogenesis and contribute to the progression of multiple inflammatory conditions and autoimmune diseases. RESULTS: In the current study, we found that BJ-1108, a 6-aminopyridin-3-ol analogue, significantly inhibited Th1 and Th17 differentiation in vitro in a concentration-dependent manner, with no effect on proliferation or apoptosis of activated T cells. Moreover, BJ-1108 inhibited differentiation of Th1 and Th17 cells in ovalbumin (OVA)-specific OT II mice. A complete Freund's adjuvant (CFA)/OVA-induced inflammatory model revealed that BJ-1108 can reduce generation of proinflammatory Th1 and Th17 cells. Furthermore, in vivo studies showed that BJ-1108 delayed onset of disease and suppressed experimental autoimmune encephalomyelitis (EAE) disease progression by inhibiting differentiation of Th1 and Th17 cells. CONCLUSIONS: BJ-1108 treatment ameliorates inflammation and EAE by inhibiting Th1 and Th17 cells differentiation. Our findings suggest that BJ-1108 is a promising novel therapeutic agent for the treatment of inflammation and autoimmune disease.

Gestation and breastfeeding in schistosomotic mothers differently modulate the immune response of adult offspring to postnatal Schistosoma mansoni infection

Schistosoma mansoni antigens in the early life alter homologous and heterologous immunity during postnatal infections. We evaluate the immunity to parasite antigens and ovalbumin (OA) in adult mice born/suckled by schistosomotic mothers. Newborns were divided into: born (BIM), suckled (SIM) or born/suckled (BSIM) in schistosomotic mothers, and animals from noninfected mothers (control). When adults, the mice were infected and compared the hepatic granuloma size and cellularity. Some animals were OA + adjuvant immunised. We evaluated hypersensitivity reactions (HR), antibodies levels (IgG1/IgG2a) anti-soluble egg antigen and anti-soluble worm antigen preparation, and anti-OA, cytokine production, and CD4+FoxP3+T-cells by splenocytes. Compared to control group, BIM mice showed a greater quantity of granulomas and collagen deposition, whereas SIM and BSIM presented smaller granulomas. BSIM group exhibited the lowest levels of anti-parasite antibodies. For anti-OA immunity, immediate HR was suppressed in all groups, with greater intensity in SIM mice accompanied of the remarkable level of basal CD4+FoxP3+T-cells. BIM and SIM groups produced less interleukin (IL)-4 and interferon (IFN)-g. In BSIM, there was higher production of IL-10 and IFN-g, but lower levels of IL-4 and CD4+FoxP3+T-cells. Thus, pregnancy in schistosomotic mothers intensified hepatic fibrosis, whereas breastfeeding diminished granulomas in descendants. Separately, pregnancy and breastfeeding could suppress heterologous immunity; however, when combined, the responses could be partially restored in infected descendants.

B cells expressing IL-10 mRNA modulate memory T cells after DNA-Hsp65 immunization

In DNA vaccines, the gene of interest is cloned into a bacterial plasmid that is engineered to induce protein production for long periods in eukaryotic cells. Previous research has shown that the intramuscular immunization of BALB/c mice with a naked plasmid DNA fragment encoding the Mycobacterium leprae 65-kDa heat-shock protein (pcDNA3-Hsp65) induces protection against M. tuberculosis challenge. A key stage in the protective immune response after immunization is the generation of memory T cells. Previously, we have shown that B cells capture plasmid DNA-Hsp65 and thereby modulate the formation of CD8+ memory T cells after M. tuberculosis challenge in mice. Therefore, clarifying how B cells act as part of the protective immune response after DNA immunization is important for the development of more-effective vaccines. The aim of this study was to investigate the mechanisms by which B cells modulate memory T cells after DNA-Hsp65 immunization. C57BL/6 and BKO mice were injected three times, at 15-day intervals, with 100 µg naked pcDNA-Hsp65 per mouse. Thirty days after immunization, the percentages of effector memory T (TEM) cells (CD4+ and CD8+/CD44high/CD62Llow) and memory CD8+ T cells (CD8+/CD44high/CD62Llow/CD127+) were measured with flow cytometry. Interferon γ, interleukin 12 (IL-12), and IL-10 mRNAs were also quantified in whole spleen cells and purified B cells (CD43−) with real-time qPCR. Our data suggest that a B-cell subpopulation expressing IL-10 downregulated proinflammatory cytokine expression in the spleen, increasing the survival of CD4+ TEM cells and CD8+ TEM/CD127+ cells.

Presence of Toll Like Receptor-2 in spleen, lymph node and thymus of Swiss albino mice and its modulation by Staphylococcus aureus and bacterial lipopolysaccharide.

Toll-like receptors (TLR) are a family of pattern recognition receptors identifying pathogen associated molecular patterns (PAMPs). They play a critical role in the innate immune response during the initial interaction between the infecting microorganism and phagocytic cells. Here, we verified the presence of TLR-2 in spleen, lymph node and thymus of Swiss albino mice and their modulation after infection with Staphylococcus aureus and Lipopolysaccharide (LPS) challenge. It was seen that TLR-2 gene transcribed to its respective mRNA on S. aureus infection, in thymus, spleen and lymph node of mice but their levels and mode of expression varied. When challenged with LPS no prominent changes in the expression of TLR-2 receptor was observed but its expression increased gradually with time in the thymus, spleen and lymph node of S. aureus infected mice. TLR-2 expression was also found enhanced in infected splenic macrophages. By studying the serum cytokine profile the functionality of the receptor was measured. The results indicate the presence of TLR-2 in thymus, spleen and lymph node of Swiss albino strain of mice and that they are modulated by S. aureus.

Detección de antígenos del virus del dengue en tejidos post mórtem / Detection of dengue virus antigen in post-mortem tissues

El panorama epidemiológico del dengue ha empeorado durante la última década. Las dificultades para prevenir su transmisión, así como la ausencia de una vacuna o tratamiento específico, lo convierten en un riesgo que desafía las medidas de salud pública y desborda la capacidad de los centros de salud y los sistemas de investigación a muchos niveles. Actualmente, la mayoría de los estudios sobre la patogenia de la infección centran su atención en la respuesta inmunitaria de las células T casi exclusivamente en infecciones secundarias y están dirigidos a identificar los mecanismos implicados en el desarrollo de la permeabilidad vascular y de los eventos hemorrágicos que lo acompañan. En este reporte se describe el caso de una menor de 45 días de edad con signos clínicos de dengue grave, cuyo diagnóstico se confirmó por reacción en cadena de la polimerasa de transcripción inversa en muestras de tejido post mórtem y por herramientas de apoyo diagnóstico de inmunohistoquímica, las cuales detectaron antígenos virales en todos los órganos obtenidos en la necropsia. Este caso subraya la importancia del estudio de las infecciones primarias asociadas a dengue grave, particularmente en niños, en quienes es más probable el desarrollo de la forma grave de la enfermedad sin una infección previa, y, además, pone de relieve la importancia de un diagnóstico que no se limite a las muestras de tejido hepático en el estudio de la patogenia de la infección viral.

The epidemiological situation of dengue has worsened over the last decade. The difficulties in preventing its transmission and the absence of a vaccine or specific treatment have made dengue a serious risk to public health, health centers and research systems at different levels. Currently, most studies on the pathogenesis of dengue infection focus on the T-cell immune response almost exclusively in secondary infections and are aimed at identifying the mechanisms involved in the development of vascular permeability and bleeding events that accompany the infection. This report describes the case of a baby girl less than 45 days of age with clinical signs of severe dengue, whose diagnosis was confirmed by reverse transcription polymerase chain reaction in post-mortem tissue samples and by the ancillary diagnostic use of immunohistochemistry, which detected viral antigens in all organs obtained at autopsy. This case highlights the importance of studying primary infections associated with severe dengue, particularly in children, who are more likely to develop the severe form of the disease without previous infection, and it further stresses the importance of a diagnosis that should not be based solely on the examination of liver tissue samples when studying the pathogenesis of the viral infection.

Distress do paciente oncológico: prevalência e fatores associados na opinião de familiares / Distress of cancer patients: prevalence and associated factors in the opinion of family / Angustia de los pacientes con cáncer: prevalencia y factores asociados en la opinión de la familia

Este estudo verificou a prevalência e fatores associados ao distress de pacientes oncológicos, na opinião de familiares. Foram entrevistados 140 familiares responsáveis pelo cuidado de pacientes com câncer. O Termômetro de Distress foi adaptado para uso em familiares. Estes consideraram que 72,9% dos pacientes estavam com distress relacionado a preocupações (80,4%), nervosismo (78,4%), tristeza (74,5%), dor (67,6%), fadiga (67,6%) e problemas com alimentação (57,8%). Modelos de regressão logística hierárquica mostraram que, enquanto familiares do sexo masculino (OR=0,025) e idades mais avançadas (OR=0,006 a 0,059) tiveram menor risco de perceber o distress, indivíduos protestantes, comparados a católicos, tiveram chance 12,77 vezes maior de percebê-lo. Quanto aos fatores associados, nervosismo (OR=10,8) contribuiu significativamente mais com a percepção de distress pelos familiares quando comparado a fadiga (OR=3,38) ou ter plano de saúde privado (OR=2,55). Familiares podem ser grandes aliados na avaliação e acompanhamento do distress de pacientes com câncer.

The study aimed to verify the opinion of family members about distress on cancer patients and the factors associated with it. Interviews with 140 family members of cancer patients were conducted. The Distress Thermometer was adapted to be used with family members. Approximately 72.9% of patients were considered in distress, related to concern (80.4%), nervousness (78.4%), sadness (74.5%), pain (67.6%), fatigue (67.6%) and problems with eating (57.8%). The hierarchical logistic regression models showed that while male (OR=0.025) and older ages (OR=0.006 to 0.059) had lower risk of perceiving the distress, individuals Protestants, compared to Catholics, were 12.77 times more likely to perceive it. About the associated factors, nervousness (OR=10.8) contributed significantly more to the perception of distress for family members when compared to fatigue (OR=3.38) or have private health insurance (OR=2.55). Family can be great allies in the evaluation and monitoring of distress in patients with cancer.

Este estudio examinó la prevalencia y los factores asociados con el distress de los pacientes de cáncer, de acuerdo con los familiares. Fueran entrevistados 140 cuidadores familiares de pacientes con cáncer. El Termómetro de Distress fue adaptado para el uso en los familiares. Ellos encontraron que el 72,9% de los pacientes estaban con distress relacionado con preocupaciones (80,4%), nerviosismo (78,4%), tristeza (74,5%), dolor (67,6%), fatiga (67, 6%) y problemas con la alimentación (57,8%). Modelos de regresión logística jerárquica mostraran que, mientras los familiares de sexo masculino (OR=0,025) y de edades más avanzadas (OR=0,006 a 0,059) tuvieron un menor riesgo de percibir el distress, los individuos protestantes, comparados a los católicos, tuvieron oportunidad 12,77 veces mayor para percibirlo. En cuanto a los factores asociados, el nerviosismo (OR=10,8) contribuyó significativamente más a la percepción del distress de los familiares, en comparación con la fatiga (OR=3,38) o tener un seguro de salud privado (OR=2,55). Familiares pueden ser grandes aliados en la evaluación y el seguimiento de sufrimiento en los pacientes con cáncer.

Síndrome de falla multiorgánica secundario a hematoma esplénico por Plasmodium vivax / Mulitorgan dysfunction syndrome due to splenic hematoma secondary to Plasmodium vivax infection

Los cuadros de falla orgánica múltiple y mortalidad secundarios a Plasmodium vivax son escasos. Generalmente esta infección es considerada un enfermedad de curso benigno. El compromiso esplénico con la formación de hematoma es una complicación poco frecuente por este parásito pero, al presentarse, puede asociarse a un compromiso mayor de los sistemas cardiovascular, respiratorio, hematológico, renal y nervioso central; en algunos casos puede ser fatal. Para el diagnóstico de esta complicación se debe tener una alta sospecha clínica y descartar otras enfermedades, por lo cual presentamos el caso de un paciente quien cursó con un cuadro de hematoma esplénico con falla orgánica múltiple con desenlace fatal por Plasmodium vivax .

Cases of multiple organ failure and mortality secondary to Plasmodium vivax are scarce, and the infection produced by this parasite is generally considered of a benign nature. Splenic compromise with formation of a haematoma is a complication not usually seen; however, when present, it can be associated with a greater compromise of the cardiovascular, respiratory, hematologic, renal and central nervous systems, leading to death in some reports. To diagnose this type of complication, physicians must have a high clinical suspicion and exclude other associated pathologies. We present the case of a patient who developed splenic haematoma associated with multiple organ failure and death secondary to infection with Plasmodium vivax .

Effects of splenic allograft in lipid profile of non-splenectomized rats: the immune and metabolic role of the "double spleen" / Efeitos do aloenxerto esplênico no lipidograma de ratos não esplenectomizados: papel imunológico e metabólico do "baço duplo"

OBJECTIVE: To elucidate the role of the spleen and splenic allograft in lipid control and evaluate its effect on the lipid profile of rats. METHOD: 32 male Wistar rats were randomly assigned into four groups: control group (1), total splenectomy group (2), splenectomy and implantation of allograft group (3) and double spleen group (4). Each group was subdivided into two subgroups: A and B, based on the death of the animals after 30 or 120 days of monitoring. The procedures in groups 2, 3 and 4 were made simultaneously, and splenectomized animals, groups 2 and 3 were donors, respectively, for the animals of groups 3 and 4. In group 4 the spleen was preserved and the animals received implants from the spleens of rats from group 3. The regeneration of splenic tissue was evaluated by macroscopic and microscopic analyzes of the grafts and own spleens, as well as with measurements of VLDL, HDL, LDL, total cholesterol and triglycerides. RESULTS: after 120 days, Group 4 showed levels of total cholesterol and LDL lower than the other groups. Group 1 had higher levels of lipids. CONCLUSION: The technique of double spleen was effective in the control of lipid metabolism, corroborating the function of the spleen as a reserve of lipids. .

OBJETIVO: Este estudo objetiva elucidar o papel do baço e do aloenxerto esplênico no controle lipídico e avaliar seu efeito no lipidograma de ratos. MÉTODO: Foram distribuídos aleatoriamente 32 ratos machos da linhagem Wistar em quatro grupos: grupo controle (1), grupo esplenectomia total (2), grupo esplenectomia e implante de aloenxerto (3) e grupo baço duplo (4). Cada grupo foi subdividido em dois subgrupos: A e B, com base na morte dos animais após 30 ou 120 dias de acompanhamento. Os procedimentos nos animais dos grupos 2, 3 e 4 foram feitos simultaneamente, sendo que os animais esplenectomizados, grupos 2 e 3, foram doadores, respectivamente, para os animais dos grupos 3 e 4. No grupo 4 preservou-se o baço dos animais e implantou-se outro baço oriundo dos ratos do grupo 3. A regeneração do tecido esplênico foi avaliada por análises macro e microscópicas dos enxertos e dos baço próprios, bem como dosagens de VLDL, HDL, LDL, colesterol total e triglicérides. RESULTADOS: O Grupo 4 apresentou, após 120 dias, níveis de LDL e colesterol total inferiores aos demais grupos. O Grupo 1 apresentou os níveis de lipidograma mais elevados. CONCLUSÃO: A técnica do baço duplo foi eficaz no controle do metabolismo lipídico, comprovando a função do baço como reserva de lipídios. .

A Novel Recombinant BCG Vaccine Encoding Eimeria tenella Rhomboid and Chicken IL-2 Induces Protective Immunity Against Coccidiosis

A novel recombinant Bacille Calmette-Guerin (rBCG) vaccine co-expressed Eimeria tenella rhomboid and cytokine chicken IL-2 (chIL-2) was constructed, and its efficacy against E. tenella challenge was observed. The rhomboid gene of E. tenella and chIL-2 gene were subcloned into integrative expression vector pMV361, producing vaccines rBCG pMV361-rho and pMV361-rho-IL2. Animal experiment via intranasal and subcutaneous route in chickens was carried out to evaluate the immune efficacy of the vaccines. The results indicated that these rBCG vaccines could obviously alleviate cacal lesions and oocyst output. Intranasal immunization with pMV361-rho and pMV361-rho-IL2 elicited better protective immunity against E. tenella than subcutaneous immunization. Splenocytes from chickens immunized with either rBCG pMV361-rho and pMV361-rho-IL2 had increased CD4+ and CD8+ cell production. Our data indicate recombinant BCG is able to impart partial protection against E. tenella challenge and co-expression of cytokine with antigen was an effective strategy to improve vaccine immunity.

Immunoexpression of proliferating cell nuclear antigen (PCNA) in spleen of splenectomized rats with preservation of inferior pole, submitted to hyperbaric oxygenation

PURPOSE: To analyze PCNA immunoexpression on the inferior pole of the spleen of splenectomized rats submitted to hyperbaric oxygenation (HBO). METHODS: Were analyzed fragments of the inferior pole of the spleen of 20 male Wistar rats submitted to splenectomy with preservation of the inferior pole. The rats were divided in two groups: group A (n=10) without HBO and group B (n=10) submitted to HBO at 2, 5 atmospheres per 120 minutes, twice a day for three days and once a day for seven days. The groups were then subdivided in four subgroups: A15 (n=5), with euthanasia on the 15th day; A45 (n=5), with euthanasia on the 45th day; B15 (n=5) with euthanasia on the 15th day and B45 with euthanasia on the 45th day. Respectively on these days, fragments of the inferior pole of the spleen of all animals were collected and analyzed with the immunohistochemistry technique in order to evaluate PCNA expression. RESULTS: There was an expressive increase in PCNA immunoreactivity in the group B. The 45 day postoperative period resulted in a higher level of positivity than the 15 day postoperative period (p<0.01). CONCLUSION: The quantitative analysis of proliferating cell nuclear antigen positive suggests that hyperbaric oxygenation increases cellular proliferation, contributing to splenic regeneration.

Desorganização da polpa branca esplênica está associada com a apresentação clínica mais grave da leishmaniose visceral em cães naturalmente infectados / Disorganization of the splenic white pulp is associated with more severe clinical presentation of visceral leishmaniasis in naturally infected dogs

O baço é o maior órgão linfoide secundário em seres humanos e em cães. Em ambos, a ausência do baço está associada com um risco aumentado de ocorrência de infecções localizadas e disseminadas, incluindo sepse generalizada. A leishmaniose visceral e outras infecções podem alterar a estrutura histológica do baço, o que leva a uma destruição dos microambientes da polpa branca. Trabalhos anteriores mostraram que a uptura da estrutura de polpa branca é mais frequente em cães com marcadores laboratoriais de suscetibilidade à leishmaniose visceral, como a cultura esplênica positiva e LST negativo, do que nos animais em que estes marcadores de susceptibilidade estavam ausentes. Neste estudo, o nosso objetivo é examinar a relação entre a desorganização histológica da polpa branca esplênica e a gravidade da leishmaniose visceral. As amostras e os dados utilizados neste estudo foram coletados de 206 cães de rua provenientes de uma área endêmica para leishmaniose visceral, a cidade de Jequié (Bahia, Brasil). Os animais foram examinados clinicamente e foram realizados os testes ELISA e LST. Aspirados de baço foram coletados para a cultura, e fragmentos de baço foram coletadas para estudos de biologia molecular e de estudos morfológicos. Os animais foram classificados de acordo com o grau de organização estrutural da polpa branca esplênica em grupos com baço (a), bem organizado, (b) ligeiramente desorganizado, (c) a moderadamente a extensivamente desorganizado. Em relação à positividade no ELISA juntamente com a desorganização do baço, conjuntivite ( P= 0,0116), hiperproteinemia (p= 0,021) foram mais frequentes no grupo de animais com ELISA positivo e baço desorganizado, quando comparado com os outros grupos. Os animais polissintomáticos são mais frequentes no grupo com ELISA positivo e baço do tipo 3 (p= 0,004). Os scores clínicos atribuídos à intensidade da conjuntivite (P <0,05), dermatite (P <0,05) e linfadenopatia (P <0,01), alopecia (P <0,01), onicogrifose (P <0,05) foram mais elevados nos animais com ELISA positivo e baço desorganizado do que outros grupos, bem como o número de sinais clínicos atribuíveis à leishmaniose visceral canina (p= 0,0014).Em relação à análise da positividade na cultura esplênica juntamente com a desorganização do baço, a frequência de cães polissintomáticos foi maior em animais com baço ligeiramente desorganizado (P <0,05) e baço desorganizado (P <0,005), do que em animais com baço organizado. Alopecia (P <0,01), conjuntivite (P <0,05), desidratação (P <0,001), dermatite (P <0,05), onicogrifose (p <0,01), anemia (P <0,05), úlcera (P <0,001) e alto escore clínico (P <0,001) foram mais frequentes em animais com cultura esplênica positiva e baço desorganizado, do que nos animais com cultura negativa e baço organizado. Em conclusão, os cães com desorganização do baço associada com leishmaniose visceral têm mais sinais clínicos e pior estado clínico do que os animais com leishmaniose visceral, mas sem desorganização do baço.

STAT6 Expression and IL-13 Production in Association with Goblet Cell Hyperplasia and Worm Expulsion of Gymnophalloides seoi from C57BL/6 Mice

In intestinal helminth infections, Th2 immune respones are generally associated with mucin secretion for worm expulsion from the host intestine. In particular, IL-4 and IL-13 are the important cytokines related with intestinal mucus production via STAT6 signalling in nematode infections. However, this perspective has never been studied in Gymnophalloides seoi infection. The present study aimed to observe the STAT6 signalling and cytokine responses in C57BL/6 mice, a mouse strain resistant to infection with this trematode. The results showed that worm expulsion occurred actively during days 1-2 post-infection (PI), when goblet cells began to proliferate in the small intestine. The STAT6 gene expression in the mouse spleen became remarkable from day 2 PI. Moreover, G. seoi infection induced a significant increase of IL-13 from day 4 PI in the spleen of infected mice. Our results suggested that goblet cell hyperplasia and worm expulsion in G. seoi-infected mice should be induced by STAT6 signalling, in which IL-13 may be involved as a dominant triggering cytokine.

Antigen-presenting cells transfected with Hsp65 messenger RNA fail to treat experimental tuberculosis

In the last several years, the use of dendritic cells has been studied as a therapeutic strategy against tumors. Dendritic cells can be pulsed with peptides or full-length protein, or they can be transfected with DNA or RNA. However, comparative studies suggest that transfecting dendritic cells with messenger RNA (mRNA) is superior to other antigen-loading techniques in generating immunocompetent dendritic cells. In the present study, we evaluated a new therapeutic strategy to fight tuberculosis using dendritic cells and macrophages transfected with Hsp65 mRNA. First, we demonstrated that antigen-presenting cells transfected with Hsp65 mRNA exhibit a higher level of expression of co-stimulatory molecules, suggesting that Hsp65 mRNA has immunostimulatory properties. We also demonstrated that spleen cells obtained from animals immunized with mock and Hsp65 mRNA-transfected dendritic cells were able to generate a mixed Th1/Th2 response with production not only of IFN-γ but also of IL-5 and IL-10. In contrast, cells recovered from mice immunized with Hsp65 mRNA-transfected macrophages were able to produce only IL-5. When mice were infected with Mycobacterium tuberculosis and treated with antigen-presenting cells transfected with Hsp65 mRNA (therapeutic immunization), we did not detect any decrease in the lung bacterial load or any preservation of the lung parenchyma, indicating the inability of transfected cells to confer curative effects against tuberculosis. In spite of the lack of therapeutic efficacy, this study reports for the first time the use of antigen-presenting cells transfected with mRNA in experimental tuberculosis.

Expression of non-TLR pattern recognition receptors in the spleen of BALB/c mice infected with Plasmodium yoelii and Plasmodium chabaudi chabaudi AS

The spleen plays a crucial role in the development of immunity to malaria, but the role of pattern recognition receptors (PRRs) in splenic effector cells during malaria infection is poorly understood. In the present study, we analysed the expression of selected PRRs in splenic effector cells from BALB/c mice infected with the lethal and non-lethal Plasmodium yoelii strains 17XL and 17X, respectively, and the non-lethal Plasmodium chabaudi chabaudi AS strain. The results of these experiments showed fewer significant changes in the expression of PRRs in AS-infected mice than in 17X and 17XL-infected mice. Mannose receptor C type 2 (MRC2) expression increased with parasitemia, whereas Toll-like receptors and sialoadhesin (Sn) decreased in mice infected with P. chabaudi AS. In contrast, MRC type 1 (MRC1), MRC2 and EGF-like module containing mucin-like hormone receptor-like sequence 1 (F4/80) expression decreased with parasitemia in mice infected with 17X, whereas MRC1 an MRC2 increased and F4/80 decreased in mice infected with 17XL. Furthermore, macrophage receptor with collagenous structure and CD68 declined rapidly after initial parasitemia. SIGNR1 and Sn expression demonstrated minor variations in the spleens of mice infected with either strain. Notably, macrophage scavenger receptor (Msr1) and dendritic cell-associated C-type lectin 2 expression increased at both the transcript and protein levels in 17XL-infected mice with 50% parasitemia. Furthermore, the increased lethality of 17X infection in Msr1 -/- mice demonstrated a protective role for Msr1. Our results suggest a dual role for these receptors in parasite clearance and protection in 17X infection and lethality in 17XL infection.

Construction of a recombinant plasmid harbouring the glyceraldenyde-3-phosphate dehydrogenase gene of periodic Brugia malayi and observation on DNA immunity.

Purpose: Controlling and eliminating lymphatic filariasis will require further research of preventative measures and implementation. Parasite is dependent on glycolysis for ATP production. The glycolytic enzyme glyceraldenyde-3-phosphate dehydrogenase (GAPDH) plays an important role in glycolysis and therefore is either a potential target for anti-parasite drug development or a vaccine candidate. Therefore, we tried to investigate the DNA vaccine-elicited immune responses in BALB/c mice. Materials and Methods: We cloned a gene encoding the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from periodic Brugia malayi into vector pcDNA3.1. Mice were injected at a dosage of 100 μg recombinant plasmid DNA with CpG intramuscular injection and immunized three times at 2-week intervals. pcDNA3.1 and normal saline were used as control. The tissue of muscles at the 4 weeks after the third injection was collected and target genes were detected using RT-PCR. The humoral responses elicited in mice by inoculation with the recombinant plasmid pcDNA3.1-BmGAPDH were detected using a standard ELISA. Two weeks after the third immunization, stimulation index (SI) was measured using the MTT method and the level of secreted IL-4 and INF-g were detected using ELISA. Results: Specific gene fragment coding GAPDH was amplified and the recombinant plasmid pcDNA3.1-BmGAPDH was constructed. Post-challenge sera from the mice immunized with the DNA vaccine had specific antibody titres of 1:1600 to 1:6400, and the highest titre was observed in the mice that were inoculated by pcDNA3.1-BmGAPDH/CpG at 6 weeks. At 4 weeks after immunization, the spleens of the mice were obviously enlarged. The proliferation of spleen T lymphocytes seen on the MTT assay was higher in the pcDNA3.1-BmGAPDH group than in the control group (P value <0.05). The levels of IL-4 and INF-g in serums from the immunized mice were significantly higher than that of the control (P value <0.05). Conclusions: We conclude that the recombinant eukaryotic plasmid pcDNA3.1-BmGAPDH could elicit humoral and cellular immune responses in mice.

Consumo de óleo de peixe na resposta inflamatória em ratos submetidos a esplenectomia total isolada ou combinada com autoimplante esplênico e à indução de sepse abdominal / Consumption of fish oil on inflammatory response in rats submitted to total splenectomy or combined with autoimplantation spleen and induction of abdominal sepsis

Em órgãos potencialmente importantes na resposta imune, como o baço, alternativas como o autoimplante de segmentos esplênicos, quando a esplenectomia total torna-se necessária, e a utilização de nutrientes com funcionalidade imunomoduladora vêm sendo estudadas, objetivando minimizar o efeito pró-inflamatório persistente da sepse abdominal. O objetivo deste trabalho foi avaliar o efeito do consumo de óleo de peixe na modulação da resposta inflamatória em animais submetidos a esplenectomia total isolada ou combinada com autoimplante esplênico e à indução de sepse abdominal, verificando a possível otimização na resposta pró-inflamatória e a regeneração funcional do autoimplante. Utilizamos 64 ratos machos da linhagem Wistar, com peso variando entre 140-200 g, aleatoriamente distribuídos em oito grupos: quatro grupos-controle (100% óleo de soja) e quatro grupos-intervenção (35% de óleo de peixe), cada um com oito animais. Os dos grupos-controle (animais alimentados com ração purificada, segundo AIN-93, com conteúdo lipídico constituído por 100% óleo de soja) foram: I – sem intervenção cirúrgica e, 16 semanas após, submetidos à indução de sepse abdominal; II – esplenectomia total isolada e, 16 semanas após, submetidos à indução de sepse abdominal; III – esplenectomia total combinada com autoimplante esplênico e, 16 semanas após, submetidos à indução de sepse abdominal; e IV – esplenectomia total combinada com autoimplante esplênico e, oito semanas após, submetidos à indução de sepse abdominal. Os dos grupos-intervenção (V a VIII) foram submetidos a procedimentos similares aos executados nos grupos I a IV, respectivamente, sendo a única modificação fundamentada na substituição de 35% do conteúdo lipídico da alimentação dos animais por óleo de peixe. Todos os animais foram submetidos a sepse induzida por ligadura e perfuração cecal (CLP). Coletamos amostras sanguíneas de todos os animais antes da indução da sepse (período 1) e 2 e 4 horas (períodos 2 e 3...

In organs potentially important in the immune response, like the spleen, alternatives such as autotransplantation of splenic segments, when the total splenectomy becomes necessary, and the use of nutrients with immunomodulatory function have been studied, trying to minimize the effect of pro-inflammatory persistent abdominal sepsis. The aim of this study was to evaluate the effect of consumption of fish oil in modulating the inflammatory response in animals submitted to total splenectomy alone or combined with spleen autotransplantation and the induction of sepsis, verifying the possible optimization in the proinflammatory response and the functional regeneration of the autotransplant. We used 64 male Wistar rats, weighing between 140-200 g, were randomly distributed into eight groups: four control-groups (100% soybean oil) and four intervention-groups (35% fish oil), each one with ten animals. The rats in control groups (animals fed with purified according to the AIN-93 with lipid content consisting of 100% soybean oil) : I – without surgical intervention, and 16 weeks after, submitted to the induction of abdominal sepsis II – total spleenectomy alone, and 16 weeks after, submitted to the induction of abdominal sepsis; III – total splenectomy combined with spleen autotransplantation, and 16 weeks after, submitted to the induction of abdominal sepsis; and IV – total splenectomy combined with spleen autotransplantation, and 8 weeks after, submitted to the induction of abdominal sepsis. The rats in intervention groups (V to VIII) were subjected to similar procedures performed in groups I to IV, respectively, being the only modification based on the substitution of 35% of the lipid content of animal feed for fish oil. All animals were subjected to sepsis induced by cecal ligation and puncture (CLP). We collected blood samples from all animals before the induction of sepsis (period 1) and 2 and 4 hours (periods 2 and 3) after the induction of abdominal...

T Regulatory Cell Responses to Immunization with a Soluble Egg Antigen in Schistosoma mansoni-Infected Mice

The aim of the study is to characterize the phenotypes of CD4+ CD25+ T regulatory cells within the liver granulomas and association with both Foxp-3 gene expression and splenic cytokines. Naive C57BL/6 mice were intravenously injected with multiple doses of the soluble egg antigen (SEA) 7 days before cercarial infection. The immunized and infected control groups were sacrificed 8 and 16 weeks post-infection (PI). Histopathology, parasitological parameters, splenic phenotypes for T regulatory cells, the FOXP-3 expression in hepatic granuloma using real-time PCR, and the associated splenic cytokines were studied. Histopathological examination of the liver revealed remarkable increase in degenerated ova within hepatic granuloma which decreased in diameter at weeks 8 and 16 PI (P<0.01). The percentage of T regulatory cells (CD4+ CD25+) increased significantly (P<0.01) in the immunized group compared to the infected control at weeks 8 and 16 PI. The FOXP-3 expression in hepatic granulomas increased from 10 at week 8 to 30 fold at week 16 PI in the infected control group. However, its expression in the immunized group showed an increase from 30 at week 8 to 70 fold at week 16 PI. The splenic cytokine levels of pro-inflammatory cytokines, IFN-gamma, IL-4, and TNF-alpha, showed significant decreases (P<0.05) compared to the infected control group. In conclusion, the magnitude and phenotype of the egg-induced effects on T helper responses were found to be controlled by a parallel response within the T regulatory population which provides protection in worm parasite-induced immunopathology.