Study on rAd5F35-SIVenvT vaccine in combination with rMVA-SIVenvT vaccine in mice / 中华微生物学和免疫学杂志

Objective:To analyze the immune response in mice after immunization with vaccine of rAd5F35-SIVenvT in combination with rMVA-SIVenvT to evaluate the efficacy of different immunization strategies.Methods:Two recombinant viruses were identified in vitro by PCR and Western blot. The BALB/c mice were immunized with homologous and heterologous immune strategies. The numbers of splenic lymphocytes secreting IFN-γ were measured by ELISPOT assay, meanwhile SIV gp120 antibody titer were measured by ELISA assay. Results:SIVenvT protein was expressed effectively by rAd5F35-SIVenvT and rMVA-SIVenvT in HEK293 cells. The specific immune response reached its peak at 4-week post first immunization, then decreased. SIV Env specific cellular immune response and SIV gp120 specific antibody could be detected at 4-16 weeks post first immunization. The specific cellular response was significant stronger in heterologous immunization group than homologous group at 4 week and 16 week. Furthermore, heterologous immunization induced significant higher titer of SIV gp120 antibody at 4 week than homologous group.Conclusions:Specific immune response induced by rAd5F35-SIVenvT in combination with rMVA-SIVenvT was stronger than homologous vector immunization. The results provided references for further study in nonhuman primates.

Study on T helper cells in the patients of pulmonary tuberculosis and the clinical significance of Th polarization / 中国免疫学杂志

Objective:This study was to analyze the changes of CD4~+ T lymphocytes and their subtypes,Th1 and Th2 cells,in the peripheral blood of patients with pulmonary tuberculosis disease and to investigate their clinical significance in the pathologic process of pulmonary tuberculosis.Methods:For polarization measurement of T-helper cells,1∶100 diluted Ionomycin and 1∶10 diluted Monensin were added in sequence into the equivalent volume mixture of heparin anti-coagulated whole blood and RPMI-1640 culture liquid.After being well mixed,the mixture was incubated at 5% CO_2,37℃ for 4 hours or overnight.To 100 μl of the mixture and in sequence,the antibodies of CD3-PerCP,CD8-APC,mIgG1-FITC,Rat IgG1-PE,IL-4-PE or IFN-γ-FITC were added.The stained CD4~+ IL-4~+ (Th2) and CD4~+ IFN-γ~+ (Th1) were analyzed by flow cytometry.Results:Compared with those from healthy controls,the peripheral blood of pulmonary tuberculosis patients contained significantly fewer Th1 (P＜0.01) but significantly more Th2 cells (P＜0.05).Th1 cells in the peripheral blood of the patients with miliary pulmonary tuberculosis were obviously fewer (P＜0.05) than in infiltrative pulmonary tuberculosis patients.The amount of Th2 cells in the peripheral blood of the patients with miliary pulmonary tuberculosis was significantly more (P＜0.05) than in either infiltrative pulmonary tuberculosis or tuberculous pleurisy patients.The ratio of CD4~+/CD3~+ tended to decrease in all these patients,and it was much lower (P＜0.05) in the patients of miliary pulmonary tuberculosis than in infiltrative pulmonary tuberculosis patients.Patients suffering from both pulmonary tuberculosis and diabetes had significantly lower levels of Th1 cells and CD4~+/CD3~+ (P＜0.05) and more Th2 cells,compared with those of pulmonary tuberculosis patients without diabetes.Levels of Th1 and Th2 cells restored significantly (P＜0.05) in 15 severe pulmonary tuberculosis patients after receiving tuberculosis chemotherapy and microcalorie theropy for three months.Patients with positive sputum examination tended to have decreased Th1 and CD4~+/CD3~+ (P＞0.05) and significantly increased Th2 (P＜0.05).Conclusion:Immunosuppression existed,in different extents,in patients of infiltrative pulmonary tuberculosis,tuberculous pleurisy,miliary pulmonary tuberculosis as well as patients with both pulmonary tuberculosis and diabetes.Analysis of Th1,Th2 and CD4~+/CD3~+ may be benefit for the judgments of disease conditions and therapeutic effects.

Estudo da resposta imune celular em pacientes com cromoblastomicose / Study of cellular immune response in patients with chromoblastomycosis

A cromoblastomicose é uma micose crônica que causa lesões granulomatosas e supurativas que atingem a pele e o tecido subcutâneo. Micose cosmopolita e freqüentemente observada no Brasil. As lesões aumentam progressivamente e posteriormente podem desenvolver um processo crônico e que geralmente não respondem a uma terapia convencional. Entretanto o mecanismo de defesa da resposta imune adaptativa, principalmente das células T na cromoblastomicose ainda não está definido. Em nosso estudo avaliamos a produção de citocinas e a resposta linfoproliferativa de diferentes amostras de sangue de pacientes com cromoblastomicose e indivíduos saudáveis in vitro após estimulação com antígenos do fungo. Além disso, nos acompanhamos esses pacientes sob terapia antifúngica em diferentes períodos de tratamento. Este estudo mostrou que a forma grave da cromoblastomicose é caracterizada pelo aumento na produção de IL-10 e deficiência na proliferação das células T após estimulação com antígenos do fungo. Ao contrario, pacientes com a forma leve da doença foram capazes de secretar predominantemente IFN-γ, que é uma citocina importante para defesa do hospedeiro. Em adição eles secretaram menores quantidades de IL-10 e suas células T proliferaram eficientemente in vitro após estimulação do fungo. Os pacientes avaliados após 6 meses de terapia antifúngica as células T proliferaram e secretaram altos níveis de IFN-γ eficientemente após estimulação. Ao contrário, pacientes com 12 meses de tratamento ocorreu um aumento na produção de IL-10 uma diminuição nos níveis de linfoproliferação. Interessantemente, os monócitos obtidos desses pacientes durante a doença foram capazes expressar moléculas co-estimulatorias (CD80 e CD86) e também aumento nos níveis de HLA-DR após estimulação com LPS. Além disso, monócitos desses pacientes secretam altos níveis de IL-12 e TNF-α, sugerindo que a suscetibilidade desses pacientes não apresentam uma deficiência na apresentação de antígeno por monócitos. Em suma, em nossos resultados mostraram que alta secreção de IFN-γ e eficiente proliferação de células T de pacientes com cromoblastomicose está diretamente relacionada com a forma leve da doença, enquanto que a produção de IL-10 e diminuição na proliferação de células T caracterizam a forma grave da doença

Chromoblastomycosis is a chronic granulomatous and suppurative disease that causes lesions mainly in skin and subcutaneous tissues. Although found worldwide, this mycosis is frequently observed in tropical countries such as Brazil. The skin lesions increase slowly and progressively in a chronic process that usually relapse even after canonical treatment. However, the mechanism of the host adaptive immune response, specially the role of T cells, in chromoblastomycosis is still unclear. In studies here, we evaluated the cytokine production and T cell response of peripheral blood mononuclear cells (PBMC) from different patients and healthy controls upon in vitro stimulation with fungal antigens. Moreover, we performed a follow-up study in patients undergoing long-term antifungal treatment. We collected PBMC samples from patients with an active form (either severe or mild skin lesions) of chromoblastomycosis and PBMC samples from healthy individuais. In PBMC from patients with a severe form of the disease we found a predominant production of IL-10 over IFN-gamma and a deficiency in T cell proliferation upon fungal antigen stimulation. In contrast, PBMC from patients in a mild form of the disease were able to secrete predominantly IFN-gamma, a cytokine important for host defense. In addition, they secreted low amounts of IL-10 and their T cells efficiently proliferated under in vitro stimulation with the fungal antigens. Surprisingly, the patients undergoing 6 months antifungal therapy PBMC from patients secreted higher amounts of IFN-gamma and their T cells proliferated efficiently upon stimulation. On the contrary, PBMC from patients after 12m of treatment showed an increase in IL-10 secretion followed by an inefficient T cell proliferation. Interestingly, monocytes obtained from patients during chronic phase of the disease were able to up-regulate their co-stimulatory molecules (CD80 and CD86) as well as their HLA-DR upon in vitro fungal stimulation. Moreover, monocytes from these patients secreted high amounts of pro-inflammatory cytokines IL-12 and TNF-alfa, suggesting that susceptibility of patients must be due to a immune deficiency other than a monocyte deactivation. Altogether, our data clearly show that a higher secretion of IFN-gamma and an efficient T cell proliferation of PBMC from infected individuals can distinguish the mild from the severe form of the chromoblastomycosis