Development of a flow cytometry method for detection of bovine multi-cytokines / 生物工程学报

This study aims to develop a method to detect bovine multi-cytokines based on flow cytometry. Previously we have prepared and screened monoclonal antibodies against bovine cytokines IFN-γ, IL-2, TNF-α, IP-10 and MCP-1. These bovine cytokine monoclonal antibodies were fluorescently labeled, and the combination of antibody and cell surface molecules were used to develop the method for detecting bovine multi-cytokines. Subsequently, the developed method was used to determine the cytokine expression profile of Mycobacterium bovis BCG infected bovine peripheral blood mononuclear cells in vitro, and evaluate the cytokine expression level of peripheral blood CD4+ T cells of tuberculosis-positive cattle. The bovine multi-cytokine flow cytometry detection method can effectively determine the cytokine expression of BCG-infected bovine peripheral blood T lymphocytes. Among them, the expression levels of IFN-γ, IL-2, and TNF-α continue to increase after 40 hours of infection, while the expression levels of IP-10 and MCP-1 decreased. The combined detection of IFN-γ, IL-2, and TNF-α on CD4+ T lymphocytes in peripheral blood of cattle can effectively distinguish tuberculosis-positive and tuberculosis-negative samples. This method may facilitate evaluating the level of cellular immune response after bovine pathogen infection and vaccine injection.

Preparation and immunogenicity evaluation of mRNA vaccine against porcine epidemic diarrhea / 生物工程学报

Porcine epidemic diarrhea (PED) is a highly contagious disease that causes high mortality in suckling piglets. Although several licensed inactivated and live attenuated vaccines were widely used, the infection rate remains high due to unsatisfactory protective efficacy. In this study, mRNA vaccine candidates against PED were prepared, and their immunogenicity was evaluated in mice and pregnant sows. The mRNA PED vaccine based on heterodimer of viral receptor binding region (RBD) showed good immunogenicity. It elicited robust humoral and cellular immune responses in mice, and the neutralizing antibody titer reached 1:300 after a single vaccination. Furthermore, it induced neutralizing antibody level similar to that of the inactivated vaccine in pregnant sows. This study developed a new design of PED vaccine based on the mRNA-RBD strategy and demonstrated the potential for clinical application.

Comparative efficacy of different Carbopol concentrations as a stabilizer of live attenuated sheep pox virus vaccine against lumpy skin disease

In Egypt, the lyophilized live attenuated sheep pox virus vaccine has been used for the vaccination of cattle against lumpy skin disease virus to control its economic impact on livestock industry. In this endeavor, we validate the efficacy of Carbopol® as a stabilizer and adjuvant to enhance immunogenicity of such a heterologous sheep pox virus vaccine against lumpy skin disease. Lyophilization of sheep pox virus vaccine stabilized with Carbopol® produced better physical and antigenic properties than freeze-drying with lactalbumin/sucrose stabilizer; this was manifested by superior disc uniformity, thermo-stability at 37oC, and less reduction in virus titer. Immunization of calves' groups with variable sheep pox vaccine doses containing different Carbopol® concentrations revealed that 103.5 TCID50 of sheep pox virus vaccine enclosing 0.5 percent Carbopol® is the field dose of choice. Moreover, it induced protective serum neutralizing index of 2.5 and a ELISA S/P ratio of 36, by the 4th week post vaccination. Besides, the inclusion of 0.5 percent Carbopol® in formulation of the sheep pox virus vaccine was safe in bovines and enhanced cellular immune response to lumpy skin disease virus, as evidenced by increased T cell proliferation. Hence, it is recommended to use Carbopol® as 0.5 percent in preparation of live attenuated sheep pox virus vaccine to confer better protection against lumpy skin disease virus infection(AU)

En Egipto, la vacuna atenuada liofilizada contra el virus de la viruela ovina ha sido utilizado para la vacunación del ganado, contra el virus de la dermatosis nodular contagiosa, para controlar su impacto económico en la industria ganadera. En este trabajo, validamos la eficacia del Carbopol®, como estabilizador y adyuvante, para mejorar la inmunogenicidad de dicha vacuna heteróloga contra la dermatosis nodular contagiosa. La liofilización de la vacuna contra el virus de la viruela ovina estabilizada con Carbopol®, resultó en mejores propiedades físicas y antigénicas que la liofilización con el estabilizador de lactoalbúmina/sacarosa; lo anterior se manifestó en la uniformidad superior del disco, la termoestabilidad a 37°C y la menor reducción del título del virus. La inmunización de grupos de terneros con dosis variables de vacuna contra el virus de la viruela ovina, que contenían diferentes concentraciones de Carbopol®, reveló que la dosis de campo de elección fue 103,5 TCID50 de la vacuna contra el virus de la viruela ovina conteniendo 0,5 por ciento de Carbopol®, la que indujo un índice de neutralización sérica protectora de 2,5 y una relación S/P de ELISA de 36 a la cuarta semana después de la vacunación. Además, la inclusión de Carbopol® al 0,5 por ciento en la formulación de la vacuna contra el virus de la viruela ovina fue segura en los bovinos y potenció la respuesta inmunitaria celular contra el virus de la dermatosis nodular contagiosa, como lo demuestra el aumento de la proliferación de células T. Por lo tanto, se recomienda el uso de Carbopol® al 0,5 por ciento en la preparación de la vacuna viva atenuada contra el virus de la viruela ovina para conferir una mejor protección contra la infección por el virus de la dermatosis nodular contagiosa(AU)

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

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

Comparison of immunoadjuvant activities of four bursal peptides combined with H9N2 avian influenza virus vaccine

The bursa of Fabricius (BF) is a central humoral immune organ unique to birds. Four bursal peptides (BP-I, BP-II, BP-III, and BP-IV) have been isolated and identified from the BF. In this study, the immunoadjuvant activities of BPs I to IV were examined in mice immunized with H9N2 avian influenza virus (AIV) vaccine. The results suggested that BP-I effectively enhanced cell-mediated immune responses, increased the secretion of Th1 (interferon gamma)- and Th2 (interleukin-4)-type cytokines, and induced an improved cytotoxic T-lymphocyte (CTL) response to the H9N2 virus. BP-II mainly elevated specific antibody production, especially neutralizing antibodies, and increased Th1- and Th2-type cytokine secretion. BP-III had no significant effect on antibody production or cell-mediated immune responses compared to those in the control group. A strong immune response at both the humoral and cellular levels was induced by BP-IV. Furthermore, a virus challenge experiment followed by H&E staining revealed that BP-I and BP-II promoted removal of the virus and conferred protection in mouse lungs. BP-IV significantly reduced viral titers and histopathological changes and contributed to protection against H9N2 AIV challenge in mouse lungs. This study further elucidated the immunoadjuvant activities of BPs I to IV, providing a novel insight into immunoadjuvants for use in vaccine design.

Immune response elicited by graphene oxide-based nanovaccine / 国际生物医学工程杂志

Objective To study the antigen-specific immune response induced by the graphene oxide (GO) in mice.Methods OVA-loaded GO nano-immunocomplexes (GO-OVA) were prepared by co-incubation of nano GO with model antigen ovalbumin (OVA).Nano GO was characterized by atomic force microscopy and laser particle sizeanalyzer.The cytotoxicity of GO to mouse bone marrow dendritic cells (BMDCs) was detected by cell counting kit (CCK-8).The GO-OVA uptake of BMDCs were observed by fluorescent staining.C57BL/6 mice were divided into OVA group,aluminum adjuvant OVA (Al-OVA) group and GO-OVA group (6 mice in each group) by body weight for in vivo immunization.The levels of OVA-specific antibody IgG (total IgG,IgG1,and IgG2a) in serum of mice were detected by enzyme-linked immunosorbent assay (ELISA).The T lymphocyte subsets in spleen and inguinal lymph nodes of mice were detected by flow cytometry.Results The average particle size of the prepared nano GO was (294.34±4.68) nm,and the polydispersity coefficient was 0.208.Nano GO has less toxicity to mouse BMDCs.The results of in vitro experiments indicated that GO-OVA nanovaccine can be efficiently internalized by mouse BMDCs.The antigen-specific IgG antibodies induced by the GO-OVA was similar to that of aluminum adjuvant and the difference was not statistically significant (P＞0.05),and the Th1-type response was predominant.The proportions of CD4+ and CD8+ T lymphocytes in the spleen and inguinal lymph nodes in GO-OVA group were significantly higher than those in OVA and Al-OVA groups,and the differences were statistically significant (all P＜0.05).Conclusions GO-OVA nano-immunocomplexes can induce both humoral and cellular immune responses in mice,which provides basis for the development of novel vaccine vectors and adjuvants.

Biocompatibilidade da resina acrílica de prótese ocular: análise in vitro e in vivo / Biocompatibility of ocular prosthesis acrylic resin: in vitro and in vivo analysis

O objetivo desse estudo foi avaliar a influência de diferentes ciclos e métodos de polimerização da resina acrílica (RA) branca de próteses oculares sobre a biocompatibilidade de células da conjuntiva humana e resposta inflamatória do tecido subcutâneo de ratos. Para isso, foram confeccionados corpos de prova em RA termopolimerizados em água aquecida (RNAA), por energia de microondas (RNTM) e quimicamente ativados (RNQA). Para a análise in vivo, a resposta inflamatória desses 3 grupos (n=20/grupo) foi avaliada no tecido subcutâneo de 20 ratos Wistar por 7, 15, 30 e 60 dias (d). Células inflamatórias foram contadas no tecido adjacente ao corpo de prova após coloração com hematoxilina e eosina. A análise imunohistoquímica foi realizada para a detecção de IL-1ß, IL-6, TNFα, IL-17 e CCL20. Para a análise in vitro, diferentes ciclos de polimerização para cada método citado foram avaliados, totalizando 11 grupos (n=8/grupo). Foram realizadas análises de grau de conversão (GC), MTT, Alamar Blue, ELISA, RT-PCR em tempo real e dupla marcação de Anexina V e iodeto de propídio. Dados quantitativos foram submetidos à Análise de Variância e ao teste de Tukey com significância de 5%. Os resultados qualitativos foram comparados visualmente. Na análise in vivo, houve infiltrado inflamatório moderado para os grupos RNTM e RNQA e leve para o grupo RNAA após 7 d. O infiltrado inflamatório e a imunomarcação dos alvos testados diminuiu gradativamente ao longo dos 60 d. O grupo RNTM exibiu mais células inflamatórias, com exceção do grupo RNAA, que apresentou mais eosinófilos e linfócitos após 15 d, e do grupo RNQA, onde foi observado mais macrófagos em 15 d e neutrófilos em 60 d. Os grupos RNAA e RNQA apresentaram maior imunomarcação de IL-1ß após 7 d. O grupo RNQA apresentou maior imunomarcação de IL-1ß (15 e 30 d), IL-6 (30 e 60 d), IL-17 (15 e 30 d) e TNF-α (7 d). Os grupos RNAA e RNTM apresentaram maior imunomarcação de TNF-α nos períodos de 15 e 30 d, enquanto o grupo RNTM, aos 60 d. Na análise in vitro, todos os grupos apresentaram proliferação celular maior que 75%. O ciclo longo de polimerização em microondas apresentou menor GC e percentual de proliferação celular no MTT e resultou em grande liberação de IL-2. No ensaio de Alamar Blue, esse grupo apresentou baixo percentual de proliferação celular, assim como o grupo que recebeu ciclo longo de polimerização em água aquecida e grupos submetidos à ativação química. Maior liberação de IL-6 foi observada nos grupos submetidos à ativação química e de IL-23 para o ciclo curto de polimerização em microondas. Maior expressão gênica de TGF ß1 ocorreu para o grupo que recebeu ciclo longo de polimerização em água aquecida seguido de 30 min de armazenamento em água. Maior expressão gênica de CASP9 ocorreu para o grupo ativado quimicamente sobre a bancada. Pode-se concluir que os métodos de polimerização por meio de energia de microondas (ciclos longo e curto) e por ativação química desencadearam uma resposta inflamatória mais intensa. Dentre os métodos de polimerização recomendados pelo fabricante, a polimerização em água aquecida apresentou resultados mais satisfatórios(AU)

The aim of this study was to evaluate the influence of different cycles and methods of white color acrylic resin (AR) for ocular prosthesis on the biocompatibility of human conjunctival cells and on the inflammatory response of rat subcutaneous tissue. For this, AR specimens were prepared in water bath (NRWB), by microwave energy (NRME), and chemically activated (ANR). For in vivo analysis, the inflammatory response of these 3 groups (n=20/group) was assessed in subcutaneous tissue of 20 Wistar rats at 7, 15, 30 and 60 days (d). Inflammatory cells were counted in the tissue adjacent to specimen after staining with hematoxylin and eosin. The immunohistochemical analysis was performed for the detection of IL-1ß, IL-6, TNFα, IL-17, and CCL20. For in vitro analysis, different cycles of polymerization for each method were evaluated, with a total of 11 groups (n=8/group). The degree of conversion (DC), MTT, ELISA, real-time RT-PCR and Annexin V and propidium iodide assays were performed. Quantitative data were submitted to Analysis of Variance and Tukey test with a 5% significance. Qualitative data were submitted to visual comparison. In in vivo analysis, there was a moderate inflammatory infiltrate for groups NRME and ANR, and a light infiltrate for the group NRWB after 7 d. The inflammatory infiltrate and the immunolabeling of tested targets decreased gradually during the 60 d. The group NRME exhibited the highest number of inflammatory cells, except for the group NRWB, which presented a higher number of eosinophils and lymphocytes after 15 d, and for the group ANR, where a higher number of macrophages and neutrophils were observed at 15 d and at 60 d, respectively. Groups NRWB and ANR showed higher IL-1ß immunolabeling after 7 d. The group ANR had the highest immunolabeling of IL-1ß (15 and 30 d), IL-6 (30 and 60 d), IL-17 (15 and 30 d), and TNF-α (7 d). Groups NRWB and NRME showed greater immunolabeling in the periods of 15 and 30 d, while the group NRME had also high results at 60 d. In in vitro analysis, all groups showed cell proliferation higher than 75%. The long cycle of polymerization using microwave energy resulted in lower DC and lower percentage of cell proliferation in the MTT assay and in large release of IL-2. In the Alamar Blue assay, this group had a low percentage of cell proliferation, as well as the group that received a long cycle of polymerization in water bath and groups submitted to chemical activation. A higher release of IL-6 was observed in groups submitted to chemical activation and of IL-23, for the short cycle of polymerization in microwave. Higher TGF ß1 gene expression occurred for the group that received long cycle of polymerization in water bath followed by 30 min of storage in water. Higher CASP 9 gene expression occurred for the chemically activated group on bench. It can be concluded that the polymerization by microwave energy (long and short cycles) and by chemical activation resulted in higher inflammatory response. Among methods recommended by the manufacturer, the water bath polymerization showed more satisfactory results(AU)

Kinesis of cellular immune response to persistent infection with BK virus after renal transplantation / 中华器官移植杂志

Objective To analyze and discuss the dynamics of cellular immune response to persistent infection with BK virus after renal transplantation.Methods The recipients of renal transplantation in our center were selected and BK virus load in urine and blood was regularly observed.The victims of persistent infection with BK virus (defined as two successive positive results of BK virus load in urine or blood) were followed up and peripheral blood mononuclear cells (PBMCs) were collected for mixed cultivation with overlapping peptide pool,which contained peptide fragments (VP1,VP2,VP3,LT-Ag and st-Ag) extracted from BK virus.Flow cytometry was used to examine the in vitro proliferation of IFN-γ/IL-2/TNF-ininduced T cells and analyze the dynamics of cellular immune response to BK virus.Results A total of 46 recipients of renal transplantation were enrolled and 6 victims of persistent viruria were identified.Of the 6 victims,3 were complicated with persistent viremia,and 2 were diagnosed as BK virus nephropathy by biopsy,presenting with persistent viruria and viremia.The victims of persistent BK viremia after renal transplantation showed a significantly decreasing trend in cellular immune response to 5 BKV-specific proteins,according to the proliferation of TNF-γ/IL-2/TNF-α-induced T cells.However,this trend was not observed in the victims of persistent BK viruria.Conclusion At the stage of viremia,the victims of BKV infection after renal transplantation have seriously inhibited specific immune response to BKV.Thus,if the antiviral mechanisms are not restored in time,these recipients suffering persistent viremia are prone to virus nephropathy (BKVN),delayed graft function,and even graft loss.

The cellular immune response and protective efficacy against tuberculosis infection of a novel mycobacterium tuberculosis DNA vaccine p846 / 实用医学杂志

Objective To construct a novel M.tb DNA vaccine (p846) co-expressing mycobacterial triple antigens including Rv3615c, Mtb10.4 and Rv2660c, and evaluate its cellular immune response and protective efficacy against tuberculosis infection in BALB/c mice. Methods We constructed the p846 by using the cloning technology. The 6- to -8-week old female BALB/c mice were randomly divided into 4 groups: p846, pcDNA3.1, PBS and the BCG group. All mice were administrated intramuscularly with 50 μg recombinant plasmids at 0, 2, 4, 6 week. A single dose of BCG was injected subcutaneously in the BCG group. Two weeks after the final immunization, 10 mice in each group were used for cell proliferation, ELISPOT and FCM assay, BCG challenge experiment and HE staining of lung were performed at 4, 6 weeks later, respectively. Results The p846 vaccine could effectively induce the specific T cell proliferation(P < 0.001) and increase the numbers of IFN-γ+T cells(P <0.001), compared with those in the PBS group and the vector conreol group. The mouse lung tissue presented very mild lung inflammation in the p846 group, compared with other groups. Conclusion Vaccine p846 could not only induce strong cellular immune response, but also efficiently protect BALB/c mice against M.tb infection.

Análise in vitro da proliferação celular e produção de mediadores inflamatórios por células da conjuntiva estimuladas por materiais utilizados na confecção de próteses oculares / In vitro analysis of cell proliferation and the production of inflammatory mediators by conjunctiva cells stimulated by ocular prosthesis materials

A prótese ocular é utilizada para a reabilitação estética e funcional da ausência ocular. O conhecimento da sua biocompatibilidade é importante para a utilização sem reações danosas aos usuários. O objetivo neste estudo foi avaliar a citotoxicidade de materiais utilizados na confecção de próteses oculares, por meio da análise da proliferação celular e da produção de citocinas pró-inflamatórias e de proteínas de matriz extracelular por células da conjuntiva humana. Inicialmente, foi analisada a influência de diferentes períodos de formação e de exposição dos extratos de resina acrílica branca (N1), termopolimerizada em água aquecida, sobre culturas de células da conjuntiva. Foram confeccionados 24 corpos de prova em resina, sendo 12 para cada período de exposição de células da conjuntiva aos extratos da resina testada (24 e 72 horas). Após a formação dos extratos por 24, 48 e 72 horas de imersão em meio de cultura e, 24 horas em água seguido de 24 horas de imersão em meio, os ensaios propostos foram realizados (n=3). Em seguida, foi avaliado o efeito citotóxico de diferentes métodos de polimerização de resina acrílica N1 em células da conjuntiva. Foram confeccionados 9 corpos de prova em resina (n=3), termopolimerizados em água aquecida, por energia de microondas ou ativados quimicamente, utilizados para a formação dos extratos dessas resinas. Os extratos foram obtidos após 72 horas de imersão dos corpos de prova em meio de cultura e, então, expostos às células da conjuntiva por 72 horas para a realização dos ensaios propostos. Adicionalmente, foi analisada a influência da presença do pigmento acrílico na confecção da prótese de resina acrílica N1, termopolimerizada em água aquecida. Foram confeccionados 9 corpos de prova (n=3): resina N1, resina N1 + pigmento e, pigmento, utilizados para a formação dos extratos desses materiais. Os extratos formaram-se por 72 horas de imersão dos corpos de prova em meio de cultura e, então, foram expostos às células da...

Ocular prosthesis is a treatment option for esthetical and functional rehabilitation of ocular absence. The knowledge of ocular prosthesis material’s biocompatibility is important to ensure a safe use in patients. The aim of this study was to evaluate the cytotoxic effect of ocular prosthesis materials, through the analysis of the cell proliferation, and the production of proinflammatory cytokines and extracellular matrix proteins by a human conjunctival cell line. Initially, the influence of different preparation and exposition periods of eluates from heat-polymerized ocular prosthesis N1 color acrylic resin in human conjunctival cell line was evaluated. A total of 24 acrylic resin samples were manufactured and divided into 2 groups, according to the eluate exposition period to conjunctival cell line (24 and 72 hours). Eluates corresponding to 24, 48 and 72 hours of resin sample immersion in medium and, 24 hours of resin sample immersion in water followed by 24 hours of immersion in medium, were prepared (n=3) for the proposed tests. Then, the cytotoxic effect of different polymerization methods of ocular prosthesis N1 color acrylic resin was analyzed. A total of 9 acrylic resin samples were manufactured (n=3), according to the polymerization method: heat-polymerization in water bath, polymerization by microwave energy and auto-polymerization. Eluates corresponding to 72 hours of resin sample immersion in medium were prepared for proposed tests and exposed to conjunctival cell line for 72 hours. Additionally, the influence of pigment incorporation on the cytotoxicity of heat-polymerized ocular prosthesis N1 color acrylic resin was evaluated. A total of 9 samples were manufactured (n=3): N1 color acrylic resin without pigment incorporation, N1 color acrylic resin with pigment incorporation, and acrylic pigment. Eluates corresponding to 72 hours of sample immersion in medium were prepared and exposed to conjunctival cell line for 72 hours. The cytotoxic effect...

Perfil da resposta imune celular em pacientes infectados pelo HIV com leishmaniose ou tuberculose / profile of the cellular immune response in HIV-infected patients with tuberculosis or leishmaniasis

A infecção pelo HIV promove a redução do número de linfócitos T CD4+ e, consequentemente, o surgimento de doenças oportunistas. A leishmaniose visceral e a tuberculose são comumente reconhecidas como doenças oportunistas importantes e associadas ao óbito de indivíduos infectados por HIV. Ambos os patógenos, Leishmania e Mycobacterium tuberculosis (Mtb) infectam cronicamente macrófagos. A imunidade protetora associada a estas infecções envolve linfócitos Th1 produtores de IFN-g. O prejuízo na resposta imune celular causado pelo HIV perturba a resposta imune contra estes patógenos. Não são bem determinadas quais alterações imunológicas causadas pelo HIV promovem o prejuízo na resposta imune específica contra a Leishmania spp. e Mtb, induzindo o desenvolvimento de formas atípicas e graves destas infecções. Deste modo, esta tese teve como objetivo descrever o perfil da resposta imune celular aos antígenos de Leishmania spp. ou Mtb em pacientes infectados com HIV. Para tal., foram recrutados pacientes infectados por HIV e com diagnóstico de leishmaniose (HIV/LV) e tuberculose (HIV/TB). Indivíduos não infectados por HIV e diagnóstico de leishmaniose (LV) ou tuberculose (TB) forma incluídos como controles. Foram avaliadas a linfoproliferação e a frequência das subpopulações de memória dos linfócitos T CD4+...

The HIV-infection promotes reduced number of CD4+ T-lymphocytes and manifestation of opportunistic diseases. Visceral leishmaniasis and tuberculosis are commonly known as main opportunistic infections and are associated with mortality in HIV-infected individuals. Both pathogens, Leishmania and Mycobacterium tuberculosis (Mtb), infect macrophages. The protect immune response involve T-lymphocytes help 1 (Th1) and producing of IFN-g. The impairment of cellular immune response caused by HIV disrupts the immune response against these pathogens. It is unclear which immunological alterations caused by HIV infection promote the damage in specific cellular immune response against Leishmania and Mtb and induces the development of atypical and severe forms. Thus, this thesis aimed to describe the profile of the cellular immune response to Leishmania antigens or Mtb in HIV infected patients. To this end, were recruited HIV infected patients with visceral leishmaniasis (HIV/VL) and HIV infected patients with active tuberculosis (HIV/TB). Moreover, HIV uninfected individuals with VL or TB were also included as controls. Lymphoproliferation and frequency of memory CD4+...

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

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

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

Caracterización de las respuestas inmunocelular y humoral en pacientes con virus del papiloma humano / Characterization of humoral and cellular immune responses in patients with human papilloma virus

Se realizó un estudio descriptivo y transversal de 30 féminas con infección por el virus del papiloma humano, atendidas en la consulta de Inmunología del Policlínico de Especialidades perteneciente al Hospital Provincial Docente Clinicoquirúrgico "Saturnino Lora Torres" de Santiago de Cuba, desde junio del 2009 hasta igual mes del 2010, a fin de caracterizarlas según la respuesta inmunitaria. Para evaluar la respuesta inmune celular y humoral se empleó el test de rosetas y la cuantificación de inmunoglobulinas, respectivamente. En la serie prevaleció la infección por el virus antes mencionado en las mujeres de 25-35 años (40,0 %), en especial las procedentes de la zona urbana y se evidenció una disminución de la respuesta celular significativa con relación a la humoral.

A descriptive and cross-sectional study was carried out in 30 females infected with the human papilloma virus, attended in the office of Immunology of the Specialty Polyclinic belonging to "Saturnino Lora" Provincial Clinical Surgical Teaching Hospital in Santiago de Cuba, from June 2009 to June 2010, in order to characterize them according to immune response. To evaluate the humoral and cellular immune response rosetting assay and quantification of immunoglobulines were used respectively. Women between 25-36 years of age (40 %) infected with this virus, especially those coming from urban areas, prevailed in the series, and a significant decrease of the cellular response as compared to the humoral response was evidenced.

La infección por Trypanosoma cruzi en ratones gestantes induce una respuesta inmune celular con producción de citocinas en sus fetos / Trypanosoma cruzi infection in pregnant mice induces a cellular immune response with citokines production in their fetuses

El objetivo del presente estudio fue detectar las citocinas IFN-g, IL-4 e IL-10 expresadas por células T CD4+ en tejidos de fetos de ratones con infección chagásica aguda. Para ello, se examinaron fetos de ratones NMRI cuyas madres fueron infectadas con 22×10³ tripomastigotes metacíclicos de la cepa M/HOM/BRA/53/Y de T. cruzi y preñadas durante la fase aguda de la infección. Para la detección y localización de infiltrados inflamatorios, nidos de parásitos, antígenos de T. cruzi y citocinas se emplearon las técnicas de hematoxilina-eosina, peroxidasa-anti-peroxidasa e inmunofluorescencia indirecta. Se detectaron infiltrados inflamatorios y antígenos con nidos de amastigotes en el músculo esquelético fetal. Células T CD4+ productoras de IFN-g así como depósitos de IFN-g e IL-10 fueron detectados en las secciones de placenta, corazón y músculo esquelético de fetos de ratones infectadas, mientras que células CD4+/IL-10+ se encontraron sólo en músculo esquelético, adicionalmente se detectaron depósitos de IL-4 sólo en placentas de ratones sanas. Estos resultados indican que el feto es capaz de generar una respuesta inmune propia frente a antígenos transmitidos por su madre, lo cual induce la secreción de citocinas que actuando en sinergia con los anticuerpos maternos le confieren un estado de protección contra la infección, y que la transmisión del parásito depende de factores específicos de cada madre, la cual puede modificar su capacidad de controlar tal transmisión ya sea a nivel placentario o sistémico.

The objective of this study was to detect the cytokines IFN-g, IL-4 and IL-10 expressed by CD4+ T cells in tissues of fetal mice with acute chagasic infection. For this, we examined the fetuses of NMRI mice whose mothers were infected with 22×10³ metacyclic trypomastigotes of the M/HOM/BRA/53/Y strain of T. cruzi and made pregnant during the acute phase of infection. For the detection and localization of inflammatory infiltrates, nest parasites, antigens of T. cruzi and cytokines we used hematoxylin-eosin techniques, peroxidase-anti-peroxidase and immunofluorescence. The immunohistochemical study revealed the presence of inflammatory infiltrates and antigens with amastigote nests in fetal skeletal muscle. CD4 + T cells producing IFN-g, as well as deposits of IFN-g and IL-10, were detected in sections of placenta, heart and skeletal muscle of fetuses of mice infected, while CD4+/IL-10+ was found only in skeletal muscle; in addition, deposits of IL-4 were detected only in placentas of healthy mice. These results indicate that fetuses are capable of generating their own immune response to antigens transmitted by their mother, which induces the secretion of cytokines and that, acting in synergy with the maternal antibodies, confer them a state of protection against infection; and that the transmission of the parasite depends on factors specific to each mother, which may modify its ability to control such transmission at the placental or systemic levels.

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.

Comparison of the early immune responses in mice vaccinated by different kinds of hepatitis B surface antigen / 中华微生物学和免疫学杂志

ObjectiveTo evaluate the early cellular immune responses to three kinds of hepatitis B surface antigen (HBsAg) in the immunized mice.MethodsAt day 4,the levels of IFN-γand IL-2 secreted by CD4+ and CD8+T cells which selected from splenic mononuclear cells (MNC) of the vaccinated mice were detected by enzyme-linked immunospot methods (ELISPOT) after stimulation in vitro with HBsAg MHC class Ⅰ peptide S28-39 of HBsAg or recombinant hepatitis B surface antigen(rHBsAg).ResultsAfter selected by MACs,the purity of CD3+/CD4+ and CD3+/CD8+T cell was more than 90％.The positive rate of IFN-γsecreted by CD4+T cells induced by HBsAg derived from Hansenula polymorpha(rHP) was higher than that of HBsAg derived from CHO cell (rCHO).Levels of IFN-γ secreted by CD8+T cells and IL-2 secreted by CD4+T cells induced by rHP antigen were significantly higher than those of rCHO( P＜0.05 ).Meanwhile,levels of IFN-γsecreted by CD4+T cells and CD8+T cells induced by rHP were also significantly higher than those of plasma HBsAg(pHB) (P＜0.05).ConclusionAt day 4,the cellular immune responses induced by HBsAg could be detected.But the immune responses induced by the three kinds of HBsAg are different in levels.According to early cellular immune response intensity,the rHP HBsAg are superior to the rCHO and pHB,in accordance with the high protection rate interrupting the mother-infant transmission immunized by rHP vaccine in clinical trial.It provides scientific basis for necessity of timely birth dose of HB vaccine and kind of HB vaccine for high risk newborn infants vaccinated.

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

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

Immunogenicity of the Spike Glycoprotein of Bat SARS-like Coronavirus / 中国病毒学·英文版

A group of SARS-Iike coronaviruses(SL-CoV)have been identified in horseshoe bats.Despite SL-CoVs and SARS-CoV share identical genome structure and high-level sequence similarity,SL-CoV does not bind to the same cellular receptor as for SARS-CoV and the N-terminus of the S proteins only share 64% amino acid identity,suggesting there are fundamental differences between these two groups of coronaviruses.To gain insight into the basis of this difference,we established a recombinant adenovirus system expressing the S protein from SL-CoV(rAd-Rp3-S)to investigate its immune characterization.Our results showed that immunized mice generated strong humoral immune responses against the SL-CoV S protein.Moreover,a strong cellular immune response demonstrated by elevated IFN-γ and IL-6 levels was also observed in these mice.However,the induced antibody from these mice had weaker cross-reaction with the SARS-CoV S protein,and did not neutralize HIV pseudotyped with SARS-CoV S protein.These results demonstrated that the immunogenicity of the SL-CoV S protein is distinct from that of SARS-CoV,which may cause the immunological differences between human SARS-CoV and bat SL-CoV.Furthermore,the recombinant virus could serve as a potential vaccine candidate against bat SL-CoV infection.

Immune responses specific for ESAT-6 expressed by recombinant Salmonella typhimurium / 中华微生物学和免疫学杂志

Objective To determine the immune responses induced by recombinant Salmonella ty-phimurium expressing the secreting antigen ESAT-6 of Mycobacterium tuberculosis. Methods ESAT-6 cod-ing gene was cloned and identified by PCR and sequencing. Prokaryotic expression plasmid pYA33-esat car-rying the ESAT-6 coding sequence was constructed firstly and electro-transformed into an attenuated strain X4550 of Salmonella typhimurium, the recombinant bacteria was named as X4550(33-esat). C57BL/6 mice were immunized intranasally (I. N) with 108 CFU recombinant bacteria at day 0 and 18. Cells from spleen, lung, mesenteric lymph node (MLN) and Peyer's patch (PP) were collected from mice after second immu-nization, and the specific IFN-γ-secreting cells and IL-4-secreting cells were detected by ELISPOT assay u-sing ESAT-6 peptide as stimulus. Furthermore, CTL effects were in vivo evaluated by CFSE assay. Results The results showed that cellular immune responses specific for ESAT-6 could be detected by ELISPOT assay. In lung and PP cells, immune responses against ESAT-6 were biased toward Th1 type, the frequency of IFN-γ-secreting cells was much higher than that of IL-4-secreting cells. In splenocytes and MLN cells, the anti-gen specific immune responses acted as Thl and Th2 balance, the frequency of IFN-γ-secreting cells was close to that of IL-4-secreting cells. CFSE assay indicated that recombinant bacteria could induce the high level of CTL effects specific for ESAT-6 peptide. Conclusion These results suggested that recombinant Sal-monella typhimurium X4550(33-esat) not only can induce cellular immune responses, but also can elicit specific CTL responses after I. N immunization. It also provided the useful information for the control of infec-tious disease of tuberculosis.

Mechanism of Humoral and Cellular Immune Modulation Provided by Porcine Sertoli Cells

The understanding of main mechanisms that determine the ability of immune privilege related to Sertoli cells (SCs) will provide clues for promoting a local tolerogenic environment. In this study, we evaluated the property of humoral and cellular immune response modulation provided by porcine SCs. Porcine SCs were resistant to human antibody and complement-mediated formation of the membrane attack complex (38.41+/-2.77% vs. 55.02+/-5.44%, p=0.027) and cell lysis (42.95+/-1.75% vs. 87.99 +/-2.25%, p<0.001) compared to immortalized aortic endothelial cells, suggesting that porcine SCs are able to escape cellular lysis associated with complement activation by producing one or more immunoprotective factors that may be capable of inhibiting membrane attack complex formation. On the other hand, porcine SCs and their culture supernatant suppressed the up-regulation of CD40 expression (p<0.05) on DCs in the presence of LPS stimulation. These novel findings, as we know, suggest that immune modulatory effects of porcine SCs in the presence of other antigen can be obtained from the first step of antigen presentation. These might open optimistic perspectives for the use of porcine SCs in tolerance induction eliminating the need for chronic immunosuppressive drugs.