A corn straw-based diet increases release of inflammatory cytokines in peripheral blood mononuclear cells of dairy cows / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Recent studies have shown that diet can affect the body's immunity. Roughage of dairy cows consists of a variety of plant materials which make different contributions to health. This study investigated the effect of different roughages on the immunity of dairy cows. Serum, peripheral blood mononuclear cells (PBMCs), and milk samples were collected from 20 multiparous mid-lactation cows fed mixed forage (MF)- or corn straw (CS)-based diets. Expression profile analysis was used to detect the differentially expressed genes (DEGs) from PBMCs. The results showed that milk protein in the MF group increased to 3.22 g/100 ml, while that of the CS group milk was 2.96 g/100 ml; by RNA sequencing, it was found that 1615 genes were differentially expressed between the CS group and the MF group among the 24 027 analyzed probes. Gene ontology (GO) and pathway analysis of DEGs suggested that these genes (especially genes coding cytokines, chemokine and its receptors) are involved in the immune response. Results were confirmed at the protein level via detecting the levels of interleukin-2 (IL-2), IL-6, IL-10, IL-12, leptin (LEP), interferon-γ (IFN-γ), transforming growth factor-β1 (TGF-β1), and tumor necrosis factor-α (TNF-α) in peripheral blood by enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay analysis. Our data supported the conclusions that the protein content in milk of the MF group was higher than that of the CS group, the CS-based diets induced more release of cytokines than the MF-based diets in dairy cows' PBMCs, and milk protein content may be affected by cytokines.

Imbalance of Th17 and Treg in peripheral blood mononuclear cells of active tuberculosis patients

Abstract Objective: Maintaining a right balance between Th17 and Treg might be critical to the immunopathogenesis of active tuberculosis (TB). This study aimed to assess whether the Th17/Treg balance is altered in active TB patients. Methods: 250 study subjects (90 active TB patients, 80 latent TB subjects, and 80 healthy controls) were recruited for the study. The expression of Th17 and Treg in peripheral blood mononuclear cells (PBMCs) in the 250 subjects was investigated by flow cytometry. Plasma levels of cytokines IL-17 and IL-10, which are related to Th17 and Treg, respectively, were determined by ELISA. Results: The percentages of Th17 and Treg in PBMCs from active TB patients were significantly higher than those from latent TB or control groups (Th17: 4.31 ± 1.35% vs. 1.58 ± 0.71% or 1.15 ± 0.49%, p < 0.05; Treg: 11.44 ± 2.69% vs. 7.54 ± 1.56% or 4.10 ± 0.99%, p < 0.05). The expression of IL-17 and IL-10 was significantly increased in active TB patients in comparison to that in latent TB or control groups (IL-17: 16.85 ± 9.68 vs. 7.23 ± 5.19 or 8.21 ± 5.51 pg/mL, p < 0.05; IL-10: 28.70 ± 11.27 vs. 20.25 ± 8.57 or 13.94 ± 9.00 pg/mL, p < 0.05). Conclusions: Our study demonstrated an altered balance of Treg/Th17 in active TB patients, with higher percentages of Th17 and Treg in PBMCs. Further research on this imbalance may offer a new direction for TB treatment.

Identificaiton of Novel Immunogenic Human Papillomavirus Type 16 E7-Specific Epitopes Restricted to HLA-A*33;03 for Cervical Cancer Immunotherapy

PURPOSE: To identify new immunogenic HLA-A*33;03-restricted epitopes from the human papillomavirus (HPV) 16 E7 protein for immunotherapy against cervical cancer. MATERIALS AND METHODS: We synthesized fourteen overlapping 15-amino acid peptides and measured intracellular interferon-γ (IFN-γ) production in PBMC and CD8+ cytotoxic T lymphocytes (CTLs) after sensitization with these peptides using flow cytometry and ELISpot assay. The immunogenicity of epitopes was verified using a ⁵¹Cr release assay with SNU1299 cells. RESULTS: Among the fourteen 15-amino acid peptides, E7₄₉₋₆₃ (RAHYNIVTFCCKCDS) demonstrated the highest IFN-γ production from peripheral blood mononuclear cells (PBMCs), and CD8+ CTLs sensitized with E7₄₉₋₆₃ showed higher cytotoxic effect against SNU1299 cells than did CD8+ CTLs sensitized with other peptides or a negative control group. Thirteen 9- or 10-amino acid overlapping peptides spanning E7₄₉₋₆₃, E7₅₀₋₅₉ (AHYNIVTFCC), and E7₅₂₋₆₁ (YNIVTFCCKC) induced significantly higher IFN-γ production and cytotoxic effects against SNU1299 cells than the other peptides and negative controls, and the cytotoxicity of E7₅₀₋₅₉- and E7₅₂₋₆₁-sensitized PBMCs was induced via the cytolytic effect of CD8+ CTLs. CONCLUSION: We identified E7₅₀₋₅₉ and E7₅₂₋₆₁ as novel HPV 16 E7 epitopes for HLA-A*33;03. CD8+ CTL sensitized with these peptides result in an antitumor effect against cervical cancer cells. These epitopes could be useful for immune monitoring and immunotherapy for cervical cancer and HPV 16-related diseases including anal cancer and oropharyngeal cancer.

Comparative study of lymphocytes from individuals that were vaccinated and unvaccinated against the pandemic 2009-2011 H1N1 influenza virus in Southern Brazil

ABSTRACTINTRODUCTION:While no single factor is sufficient to guarantee the success of influenza vaccine programs, knowledge of the levels of immunity in local populations is critical. Here, we analyzed influenza immunity in a population from Southern Brazil, a region with weather conditions that are distinct from those in the rest of country, where influenza infections are endemic, and where greater than 50% of the population is vaccinated annually.METHODS:Peripheral blood mononuclear cells were isolated from 40 individuals. Of these, 20 had received the H1N1 vaccine, while the remaining 20 were unvaccinated against the disease. Cells were stimulated in vitro with the trivalent post-pandemic influenza vaccine or with conserved major histocompatibility complex I (MHC I) peptides derived from hemagglutinin and neuraminidase. Cell viability was then analyzed by [3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide)]-based colorimetric assay (MTT), and culture supernatants were assayed for helper T type 1 (Th1) and Th2-specific cytokine levels.RESULTS:Peripheral blood lymphocytes from vaccinated, but not unvaccinated, individuals exhibited significant proliferation in vitro in the presence of a cognate influenza antigen. After culturing with vaccine antigens, cells from vaccinated individuals produced similar levels of interleukin (IL)-10 and interferon (IFN)-γ, while those from unvaccinated individuals produced higher levels of IFN-γ than of IL-10.CONCLUSIONS:Our data indicate that peripheral blood lymphocytes from vaccinated individuals are stimulated upon encountering a cognate antigen, but did not support the hypothesis that cross-reactive responses related to previous infections can ameliorate the immune response. Moreover, monitoring IL-10 production in vaccinated individuals could comprise a valuable tool for predicting disease evolution.

High interleukin-4 expression and interleukin-4 gene polymorphisms are associated with susceptibility to human paracoccidioidomycosis

Paracoccidioidomycosis (PCM) is caused by dimorphic fungi from theParacoccidioides brasiliensis complex. Previous studies have demonstrated that the severity of disease is associated with a T-helper 2 immune response characterised by high interleukin (IL)-4 production. In the present study we analysed two polymorphisms in the IL-4gene (-590 C/T and intron-3 microsatellite) in 76 patients with PCM and 73 control subjects from an endemic area. The production of IL-4 by peripheral blood mononuclear cells after antigen or phytohaemagglutinin stimulation was determined by ELISA. A significant correlation was observed between the RP2/RP2 intron-3 genotype and infection with Paracoccidioides sp.(p = 0.011), whereas the RP1/RP1 genotype was correlated with resistance. No significant correlation was observed for the IL-4promoter polymorphism. Furthermore, the low IL-4 expression observed in the control group compared with patients was associated with the RP1/RP1 genotype. These results suggest that IL-4polymorphisms might be associated with the ability of the host to control Paracoccidioides sp.infection. The relevance of this polymorphism is supported by the observation that patients with disease produce high levels of IL-4 following mitogen or antigen stimulation. The IL-4gene is located in the cytokine cluster region of chromosome 5 where other polymorphisms have also been described.

Abnormal phenotypic distribution of regulatory and effector T cells in octogenarian and nonagenarian women / Distribuição fenotípica anormal das células T reguladoras e efetoras em mulheres octogenárias e nonagenárias

SummaryIntroduction:aging is associated with several immunologic changes. Regulatory (Treg) and effector T cells are involved in the pathogenesis of infectious, neoplastic, and autoimmune diseases. Little is known about the effects of aging on the frequency and function of these T cell subpopulations.Methods:peripheral blood mononuclear cells (PBMC) were obtained from 26 young (under 44 years old) and 18 elderly (above 80 years old) healthy women. T cell subpopulations were analyzed by flow cytometry.Results:elderly individuals had lower frequency of several activated effector T cell phenotypes as compared with young individuals: CD3+CD4+CD25+ (3.82±1.93 versus 9.53±4.49; p<0.0001); CD3+CD4+CD25+CD127+(2.39±1.19 versus 7.26±3.84; p<0.0001); CD3+CD4+CD25+ (0.41±0.22 versus 1.86±0.85, p<0.0001); and CD3+CD4+CD25highCD127+(0.06±0.038 versus 0.94±0.64, p<0.0001). Treg (CD3+CD4+CD25+CD127øFoxp3+) presented lower frequency in elderly individuals as compared to young adults (0.34±0.18 versus 0.76±0.48; p=0.0004) and its frequency was inversely correlated with age in the whole group (r=-0.439; p=0.013). The elderly group showed higher frequency of two undefined CD25øFoxp3+ phenotypes: CD3+CD4+CD25øFoxp3+(15.05±7.34 versus 1.65±1.71; p<0.0001) and CD3+CD4+CD25øCD127øFoxp3+(13.0±5.52 versus 3.51±2.87; p<0.0001).Conclusions:the altered proportion of different T cell subsets herein documented in healthy elderly women may be relevant to the understanding of the immunologic behavior and disease susceptibility patterns observed in geriatric patients.

ResumoIntrodução:o envelhecimento está associado a diversas alterações imunológicas. Células T reguladoras e efetoras estão envolvidas na patogênese de enfermidades infecciosas, neoplásicas e autoimunes. Pouco se sabe acerca dos efeitos da idade sobre a frequência e a função dessas populações celulares.Métodos:células mononucleares do sangue periférico foram obtidas de participantes saudáveis (26 com idade inferior a 44 anos e 18 acima de 80 anos). As subpopulações celulares foram analisadas por citometria de fluxo.Resultados:o grupo constituído por idosas apresentou menor frequência de vários fenótipos de células T efetoras ativadas em comparação com jovens: CD3+CD4+CD25+ (3,82±1,93 versus 9,53±4,49, p<0,0001); CD3+CD4+ CD25+CD127+ (2,39±1,19 versus7,26±3,84, p<0,0001); CD3+CD4+CD25high(0,41±0,22 versus 1,86±0,85, p<0,0001); CD3+CD4+CD25highCD127+(0,06±0,038 versus 0,94±0,64, p<0,0001). As células T reguladoras CD3+CD4+CD25highCD127øFoxP3+ apresentaram menor frequência em indivíduos idosos em comparação com adultos jovens (0,34±0,18 versus0,76±0,48, p=0,0004) e sua frequência foi inversamente correlacionada com a idade em todo o grupo (r=-0,439; p=0,013). O grupo de idosas apresentou maior frequência de dois fenótipos indefinidos (CD25øFoxP3+), células CD3+CD4+CD25øFoxP3+ (15,05±7,34 versus 1,65±1,71, p<0,0001) e células CD3+CD4+CD25øCD127øFoxP3+(13,0±5,52 versus 3,51±2,87, p<0,0001).Conclusão:as proporções alteradas de diferentes subpopulações de células T em idosas saudáveis contribuem para a compreensão dos padrões de comportamento e suscetibilidade a doenças imunológicas evidenciadas em pacientes geriátricos.

Instrument to assess the nursing care product: development and content validation / Instrumento para avaliação do produto do cuidar em enfermagem: desenvolvimento e validação de conteúdo / Instrumento para evaluación del producto del cuidar en enfermería: desarrollo y validación de contenido

AbstractObjectives: develop and validate the content of a tool about nursing care production.Method: the data were collected between 2011 and 2013, based on focus groups, the application of semistructured questionnaires (prototype test) and the Delphi technique. The focus groups were used to produce the instrument items and held at three hospitals in the interior of the State of São Paulo, involving 20 nurses. A panel of 10 experts evaluated the instrument.Results: after two phases of the Delphi technique, the tool consisted of eight items. The content validity index of the scale corresponded to ≥0.9 and the content validity of the items ranged between 0.8 and 1.0, indicating the maintenance of the structure and content. The assertion on the applicability in daily nursing practice showed a content validity index of the scale equal to 0.8.Conclusion: this study permitted the development and content validation of scale on nursing care production, equipping the nurses in their management practice.

ResumoObjetivos:desenvolver e validar o conteúdo de um instrumento sobre produção do cuidado de enfermagem.Método:a coleta de dados ocorreu entre 2011 e 2013, a partir de grupos focais, aplicação de questionários semiestruturados (teste do protótipo) e técnica Delphi. Os grupos focais foram utilizados para geração de itens do instrumento e realizados em três hospitais do interior do Estado de São Paulo, com a participação de 20 enfermeiros. A apreciação do instrumento foi conduzida por um painel de 10 especialistas.Resultados:após duas fases da técnica Delphi, o instrumento passou a ser constituído por oito itens. O índice de validade do conteúdo da escala foi de ≥0,9 e a validade dos conteúdos dos itens apresentou variação de 0,8 a 1,0, indicando a manutenção da estrutura e do conteúdo. A afirmativa referente à aplicabilidade na prática diária do enfermeiro apresentou índice de validade do conteúdo da escala de 0,8.Conclusão:este estudo possibilitou desenvolver e validar o conteúdo de uma escala sobre produção do cuidado de enfermagem, instrumentalizando os enfermeiros em sua prática gerencial.

ResumenObjetivos:desarrollar y validar el contenido de un instrumento sobre producción del cuidado de enfermería.Método:los datos fueron recolectados entre 2011 y 2013, a partir de grupos focales, aplicación de cuestionarios semiestructurados (prueba del prototipo) y técnica Delphi. Los grupos focales fueron utilizados para generar ítems del instrumento y organizados en tres hospitales del interior del Estado de São Paulo, con la participación de 20 enfermeros. La apreciación del instrumento fue conducida por un panel de 10 especialistas.Resultados:tras dos fases de la técnica Delphi, el instrumento pasó a ser constituido por ocho ítems. El índice de validez de contenido de la escala fue ≥0,9 y la validez de los contenidos mostró variación de 0,8 a 1,0, indicando la manutención de la estructura y del contenido. La afirmativa respecto a la aplicabilidad en la práctica diaria del enfermero mostró índice de validez del contenido de la escala de 0,8.Conclusión:este estudio permitió desarrollar y validar el contenido de una escala sobre producción del cuidado de enfermería, instrumentalizando los enfermeros en su práctica gerencial.

The TLR7 agonist Imiquimod promote the immunogenicity of msenchymal stem cells

BACKGROUND: Mesenchymal stem cells (MSCs) are considered the best candidate in stem cells therapy due to their multipotent differentiation ability, low expression of co-stimulatory molecules (CD80, CD86, CD34 and HLA-II) and immunosuppression effects on in vivo immune responses. MSCs were now widely used in clinical trials but received no encourage results. The major problem was the fate of engrafted MSCs in vivo could not be defined. Some studies indicated that MSCs could induce immune response and result in the damage and rejection of MSCs. As toll like receptors (TLRs) are important in inducing of immune responses, in this study we study the role of TLR7 in mediating the immune status of MSCs isolated from umbilical cord. RESULTS: Our results indicated that TLR7 agonist Imiquimod could increase the proliferation of PBMC isolated from healthy human volunteers and release of lactate dehydrogenase (LDH) in supernatant from PBMC-UCMSCs co-culture system. Flow cytometry and quantitative PCR also confirmed the regulated expression of surface co-stimulatory molecules and pro-inflammatory genes (IL-6, IL-8, IL-12, TGF-β and TNF-α). And the down-regulation expression of stem cell markers also confirmed the loss of stemness of UCMSCs. We also found that the osteo-differentiation ability of UCMSCs was enhanced in the presence of Imiquimod. CONCLUSION: To our knowledge, this is the first report that activation of TLR7 pathway increases the immunogenicity of UCMSCs. Extensive researches have now been conducted to study whether the change of immune status will be help in tumor rejection based on the tumor-tropism of MSCs.

Mycobacterium tuberculosis epitope-specific interferon-g production in healthy Brazilians reactive and non-reactive to tuberculin skin test

The interferon (IFN)-γ response to peptides can be a useful diagnostic marker of Mycobacterium tuberculosis (MTB) latent infection. We identified promiscuous and potentially protective CD4+ T-cell epitopes from the most conserved regions of MTB antigenic proteins by scanning the MTB antigenic proteins GroEL2, phosphate-binding protein 1 precursor and 19 kDa antigen with the TEPITOPE algorithm. Seven peptide sequences predicted to bind to multiple human leukocyte antigen (HLA)-DR molecules were synthesised and tested with IFN-γ enzyme-linked immunospot (ELISPOT) assays using peripheral blood mononuclear cells (PBMCs) from 16 Mantoux tuberculin skin test (TST)-positive and 16 TST-negative healthy donors. Eighty-eight percent of TST-positive donors responded to at least one of the peptides, compared to 25% of TST-negative donors. Each individual peptide induced IFN-γ production by PBMCs from at least 31% of the TST-positive donors. The magnitude of the response against all peptides was 182 ± 230 x 106 IFN-γ spot forming cells (SFC) among TST-positive donors and 36 ± 62 x 106 SFC among TST-negative donors (p = 0.007). The response to GroEL2 (463-477) was only observed in the TST-positive group. This combination of novel MTB CD4 T-cell epitopes should be tested in a larger cohort of individuals with latent tuberculosis (TB) to evaluate its potential to diagnose latent TB and it may be included in ELISPOT-based IFN-γ assays to identify individuals with this condition.

The use of Mycobacterium tuberculosis HspX and GlcB proteins to identify latent tuberculosis in rheumatoid arthritis patients

Rheumatoid arthritis (RA) is an autoimmune disease characterised by the destruction of articular cartilage and bone damage. The chronic treatment of RA patients causes a higher susceptibility to infectious diseases such as tuberculosis (TB); one-third of the world’s population is latently infected (LTBI) with Mycobacterium tuberculosis (Mtb). The tuberculin skin test is used to identify individuals LTBI, but many studies have shown that this test is not suitable for RA patients. The goal of this work was to test the specific cellular immune responses to the Mtb malate synthase (GlcB) and heat shock protein X (HspX) antigens of RA patients and to correlate those responses with LTBI status. The T-helper (Th)1, Th17 and Treg-specific immune responses to the GlcB and HspX Mtb antigens were analysed in RA patients candidates for tumour necrosis factor-α blocker treatment. Our results demonstrated that LTBI RA patients had Th1-specific immune responses to GlcB and HspX. Patients were followed up over two years and 14.3% developed active TB. After the development of active TB, RA patients had increased numbers of Th17 and Treg cells, similar to TB patients. These results demonstrate that a GlcB and HspX antigen assay can be used as a diagnostic test to identify LTBI RA patients.

effect of HLA-DRB1 sharing between the couples with recurrent pregnancy loss on the pregnancy outcome after leukocyte therapy

Miscarriage is a common phenomenon complicating more than half of pregnancies. Recurrent Pregnancy Loss [RPL] is defined as three or more pregnancies lost before the twentieth week of gestation. It is believed that abnormality in maternal immune reaction to fetus and sharing of HLA antigens might be associated with RPL. To investigate the effect of HLA-DRB1 sharing between the couples with recurrent pregnancy loss on the pregnancy outcome after leukocyte therapy. Sixty primary RPL women who were immunized and followed after therapy [30 successful and 30 unsuccessful] and their husbands formed the cases of this study. In addition, one hundred healthy women were considered as the controls. HLA-DRB1 genotypes of all the cases and controls were checked by PCR-SSP method. HLA typing indicated that the prevalence of HLA-DRB1 sharing [defined as at least one allele sharing] between the couples with unsuccessful outcomes was significantly higher compared to those with successful outcomes [63.3% vs. 23.3%, p<0.004]. Moreover, HLA DRB1*07:01 allelic group was significantly more frequent in the patients with unsuccessful outcome compared to the controls [18.3% vs. 8%, p<0.04]. Our results confirmed the role of HLA sharing in RPL and revealed that HLA-DRB1 typing may be a valuable prognostic factor for the leukocyte therapy outcome

Potential biomarkers for the clinical prognosis of severe dengue

Currently, several assays can confirm acute dengue infection at the point-of-care. However, none of these assays can predict the severity of the disease symptoms. A prognosis test that predicts the likelihood of a dengue patient to develop a severe form of the disease could permit more efficient patient triage and treatment. We hypothesise that mRNA expression of apoptosis and innate immune response-related genes will be differentially regulated during the early stages of dengue and might predict the clinical outcome. Aiming to identify biomarkers for dengue prognosis, we extracted mRNA from the peripheral blood mononuclear cells of mild and severe dengue patients during the febrile stage of the disease to measure the expression levels of selected genes by quantitative polymerase chain reaction. The selected candidate biomarkers were previously identified by our group as differentially expressed in microarray studies. We verified that the mRNA coding for CFD, MAGED1, PSMB9, PRDX4 and FCGR3B were differentially expressed between patients who developed clinical symptoms associated with the mild type of dengue and patients who showed clinical symptoms associated with severe dengue. We suggest that this gene expression panel could putatively serve as biomarkers for the clinical prognosis of dengue haemorrhagic fever.

Distinct cytokine profiles of circulating mononuclear cells stimulated with Staphylococcus aureus enterotoxin A in vitro during early and late episodes of chronic osteomyelitis

We investigated the cytokine profile of peripheral mononuclear cells from chronic osteomyelitis (OST) patients following in vitro stimulation with staphylococcal enterotoxin A (SEA). We demonstrate that stimulation with SEA induced prominent lymphocyte proliferation and high levels of tumour necrosis factor (TNF)-α, interleukin (IL)-4 and IL-10 secretion in both OST and non-infected individuals (NI). Even though stimulation with SEA had no impact on IL-6 production in either patient group, the baseline level of IL-6 production by cells from OST patients was always significantly less than that produced by cells from NI. After classifying the osteomyelitic episodes based on the time after the last reactivation event as "early" (1-4 months) or "late" osteomyelitis (5-12 months), we found that increased levels of TNF-α and IL-4 in combination with decreased levels of IL-6 were observed in the early episodes. By contrast, increased levels of IL-10, IL-2 and IL-6 were hallmarks of late episodes. Our data demonstrate that early osteomyelitic episodes are accompanied by an increased frequency of "high producers" of TNF-α and IL-4, whereas late events are characterised by increased frequencies of "high producers" of IL-10, IL-6 and IL-2. These findings demonstrate the distinct cytokine profiles in chronic osteomyelitis, with a distinct regulation of IL-6 production during early and late episodes.

Production of cytokine and chemokines by human mononuclear cells and whole blood cells after infection with Trypanosoma cruzi / Produção de citocinas e quimiocinas por células mononucleares e células do sangue total humano após infecção com Trypanosoma cruzi

INTRODUCTION: The innate immune response is the first mechanism of protection against Trypanosoma cruzi, and the interaction of inflammatory cells with parasite molecules may activate this response and modulate the adaptive immune system. This study aimed to analyze the levels of cytokines and chemokines synthesized by the whole blood cells (WBC) and peripheral blood mononuclear cells (PBMC) of individuals seronegative for Chagas disease after interaction with live T. cruzi trypomastigotes. METHODS: IL-12, IL-10, TNF-α, TGF-β, CCL-5, CCL-2, CCL-3, and CXCL-9 were measured by ELISA. Nitrite was determined by the Griess method. RESULTS: IL-10 was produced at high levels by WBC compared with PBMC, even after incubation with live trypomastigotes. Production of TNF-α by both PBMC and WBC was significantly higher after stimulation with trypomastigotes. Only PBMC produced significantly higher levels of IL-12 after parasite stimulation. Stimulation of cultures with trypomastigotes induced an increase of CXCL-9 levels produced by WBC. Nitrite levels produced by PBMC increased after the addition of parasites to the culture. CONCLUSIONS: Surface molecules of T. cruzi may induce the production of cytokines and chemokines by cells of the innate immune system through the activation of specific receptors not evaluated in this experiment. The ability to induce IL-12 and TNF-α contributes to shift the adaptive response towards a Th1 profile.

INTRODUÇÃO: A resposta imune inata é o primeiro mecanismo de proteção contra o Trypanosoma cruzi e a interação de células inflamatórias com moléculas do parasita pode ativar esta resposta e modular a resposta adaptativa. O objetivo deste trabalho foi analisar os níveis de citocinas e quimiocinas sintetizados por células do sangue total (WBC) e células mononucleares do sangue periférico (PBMC) de voluntários soronegativos para doença de Chagas depois da interação com Trypanosoma cruzi. MÉTODOS: IL-12, IL-10, TNF-α, TGF-β, CCL5, CCL2, CCL3, CXC-9 foram avaliados por ELISA. Níveis de nitrito foram determinados pelo método de Griess. RESULTADOS: Foram produzidos altos níveis de IL-10 por WBC quando comparado aos sintetizados por PBMC, inclusive após incubação com tripomastigotas. A produção de TNF-α foi significativamente maior nas culturas de PBMC e WBC após estímulo com o parasita. O aumento significativo dos níveis de IL-12 foi observado apenas em PBMC depois do estímulo com tripomastigotas. A adição de tripomastigotas nas culturas induziu aumento dos níveis de CXCL9 produzidos por WBC. Os níveis de nitrito produzidos pelos PBMCs de todos os voluntários após a adição de parasito nas culturas aumentaram. CONCLUSÕES: Moléculas de superfície do parasito podem induzir a produção de citocinas e quimiocinas pelas células da resposta imune inata através da ativação dos receptores específicos não avaliados neste experimento. A habilidade de induzir IL-12 e TNF-α contribui para direcionar uma resposta imune adaptativa de perfil Th1.

Schistosoma mansoni antigens alter the cytokine response in vitro during cutaneous leishmaniasis

Schistosoma mansoni infection or associated products are able to down-modulate the type 1 CD4+ T cell inflammatory response characteristic of autoimmune diseases. In this study, we evaluated how S. mansoni antigens altered the immune response that was induced by the soluble Leishmania antigen (SLA) from cutaneous leishmaniasis (CL) patients. Cytokines were measured from the supernatants of peripheral blood mononuclear cell cultures stimulated with SLA. This was performed using the sandwich enzyme linked immunosorbent assay technique in the presence or absence of S. mansoni recombinant antigens Sm29, SmTSP-2 and PIII. The addition of S. mansoni antigens to the cultures resulted in the reduction of interferon gamma (IFN-γ) levels in 37-50 percent of patients. Although to a lesser extent, the antigens were also able to decrease the production of tumour necrosis factor-alpha (TNF-α). We compared patients that either had or did not have reduction in IFN-γ and TNF-α production in cultures stimulated with SLA in the presence of S. mansoni antigens. We found that there was no significant difference in the levels of interleukin (IL)-10 and IL-5 in response to S. mansoni antigens between the groups. The antigens used in this study down-modulated the in vitro proinflammatory response induced by SLA in a group of CL patients through a currently undefined mechanism.

Response of peripheral blood mononuclear cells to conditioned medium from cultured oral squamous cell carcinomas

The current study investigated the capacity for tumor factors secreted by head and neck squamous cell carcinoma (HNSCC) cell lines, KB, KB16, and HEP, to induce the secretion of various cytokines from peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from blood samples collected from six healthy volunteers and these cells were incubated for 6, 24, 48, or 72 hours in the presence of 50 percent conditioned medium collected from cultured cell lines pretreated with, or without, stimulants such as phytohemagglutinin (PHA) or lipopolysaccharides (LPS). Aliquots of each supernatant were then assayed for levels of IFN-Γ, vascular endothelial growth factor (VEGF), TNF-α, and IL-4 using enzyme linked immunosorbent assays (ELISAs). Data collected were analyzed using Student's t-test, an ANOVA test followed by Tukey's test, and tests of Pearson's Correlation. PBMCs cultured with KB16-conditioned medium produced the highest levels of IFN-Γ. VEGF was also detected in conditioned media collected from all of the squamous cell carcinoma (SCC) cell lines used, and a significant difference in VEGF levels between control and KB- or KB16-conditioned media was observed. TNF-α was secreted by all PBMC groups within 6 hours of receiving conditioned media, and these levels increased up to the 24 hour timepoint, after which levels of TNF-α stabilized. In contrast, none of the supernatant samples contained detectable levels of IL-4. In combination, these data suggest that direct contact between fresh human PBMCs and conditioned media from tumor cells induces the secretion of TNF-α and VEGF by PBMCs, and this represents an initial angiogenic response.

Resistance to Toxoplasma gondii Infection in Mice Treated with Silk Protein by Enhanced Immune Responses

This study investigated whether elevated host immune capacity can inhibit T. gondii infection. For this purpose, we used silk protein extracted from Bombyx mori cocoons as a natural supplement to augment immune capacity. After silk protein administration to BALB/c mice for 6 weeks, ratios of T lymphocytes (CD4+ and CD8+ T-cells) and splenocyte proliferative capacities in response to Con A or T. gondii lysate antigen (TLA) were increased. Of various cytokines, which regulate immune systems, Th1 cytokines, such as IFN-gamma, IL-2, and IL-12, were obviously increased in splenocyte primary cell cultures. Furthermore, the survival of T. gondii (RH strain)-infected mice increased from 2 days to 5 or more days. In a state of immunosuppression induced by methylprednisolone acetate, silk protein-administered mice were resistant to reduction in T-lymphocyte (CD4+ and CD8+ T-cells) numbers and the splenocyte proliferative capacity induced by Con A or TLA with a statistical significance. Taken together, our results suggest that silk protein augments immune capacity in mice and the increased cellular immunity by silk protein administration increases host protection against acute T. gondii infection.

Clinical and immunological features of patients with atopy and concomitant HTLV-1 infection

Human T-cell lymphotropic virus type 1 (HTLV-1) induces an exacerbated type 1 immune response characterized by high spontaneous IFN-γ and TNF-α production. Allergic rhinitis and asthma are associated with the type 2 immune response, with elevated secretion of IL-4 and IL-5. The aim of this study was to characterize the immune response in atopic HTLV-1 carriers. The cytokine profile of atopic HTLV-1 carriers (N = 10; all females) was compared with that of non-atopic HTLV-1 carriers (N = 14; 9 females and 5 males). Mean patient age of atopic and non-atopic groups was 45 ± 8 and 38 ± 11 years, respectively. All atopic HTLV-1 carriers had rhinitis with or without asthma and a skin prick test positive for Dermatophagoides pteronyssinus antigen 1 (Derp-1). There was no difference in cytokine levels between the two groups in unstimulated peripheral blood mononuclear cell cultures. In cultures stimulated with Derp-1, IFN-γ levels tended to be higher (P = 0.06) and IL-5 levels were higher (P = 0.02) in atopic HTLV-1 patients than in non-atopic subjects. In contrast, IL-10 was lower (P = 0.004) in atopic than in non-atopic HTLV-1-infected subjects. This study shows that HTLV-1 infection with an exaggerated type 1 immune response does not prevent atopy. In this case, the exacerbated type 1 and type 2 immune responses were due to a lack of IL-10 production, a cytokine that plays an important role in down-modulating type 1 and type 2 immune responses and in preventing the development of chronic inflammatory diseases.

In vitro initial immune response against Leishmania amazonensis infection is characterized by an increased production of IL-10 and IL-13

The initial encounter of Leishmania with its host's immune system is important in the outcome of infection. Previous studies have shown that PBMCs from healthy volunteers (HV) exposed to Leishmania differ in IFN-γ production. We have expanded such observations evaluating the profile and kinetics of cytokines (IFN-γ, IL-12p70, IL-10, IL-13), chemokines (CCL5, CCL3, CCL4, CXCL10), and chemokine receptors (CCR1,CCR5, CXCR3, CCR4) in vitro L. amazonensis-stimulated of HV's PBMCs. HVs were divided in groups of high (HR) or low (LR) IFN-γ responders. In both groups, HR and LR, after L. amazonensis infection there was a predominance of IL-10 and IL-13 over IFN-γ production, while IL-12 was produced in similar amount. Regarding chemokines, a more striking difference was observed for CCL3 expression that was lower at 12 hours and 48 hours post infection in LR than in HR. Interestingly, a downregulation of CCR5 and a greater expression of CCR4 were found in low IFN-γ responders. These data suggest that early after L. amazonensis infection there is a cytokine milieu dominated by IL-13 and IL-10, and despite of this environment, IFN-γ is produced, supporting the complexity of the response. It is noteworthy that the pattern of immune response is mounted in first hours after Leishmania stimulation, with the definition of the differentiation of Th1 versus Th2 cells. It remains to be determined if such an in vitro difference has an in vivo counterpart in terms of susceptibility to infection.