Mantas não tecidas de nanofibras de três polímeros biodegradáveis poli(ácido láctico), PDLLA, poli(Ε-caprolactona), PCL, e poli(butileno adipato-co-tereftalato), PBAT e seus nanocompósitos com uma nanoargila montmorilonita (MMT) foram produzidas por eletrofiação. A morfologia, o comportamento térmico e a estrutura interna das nanofibras foram analisados por microscopia eletrônica de varredura e transmissão, calorimetria diferencial de varredura e difração de raios X, respectivamente. Observou-se que as nanofibras dos nanocompósitos possuíam diâmetros menores do que os correspondentes polímeros puros e que as nanofibras de PBAT puro e de PBAT/MMT apresentavam a menor cristalinidade de todas as mantas. A viabilidade celular de todas as nanofibras foi analisada pela técnica de redução do sal de tetrazolium pelo complexo enzimático piruvato desidrogenase presente na matriz de mitocôndrias (teste MTT). Os resultados mostraram que nenhuma manta nanofibrílica apresentou toxicidade às células e que as nanofibras de PBAT puro e seu nanocompósito propiciaram ainda um ambiente mais favorável ao desenvolvimento celular de fibroblastos de cardiomiócitos do que as condições oferecidas pelo controles, provavelmente por apresentarem menores diâmetros e baixa cristalinidade em relação às demais nanofibras. Estes resultados mostram o potencial de uso destas mantas nanofibrílicas como suportes de crescimento celular.

Non-woven mats of nanofibers of three biodegradable polymers, viz. poly(lactic acid), PDLLA, poly(Ε-caprolactone), PCL, and poly(butylene adipate-co-terephthalate), PBAT, and their nanocomposites with montmorillonite nanoclay (MMT) were produced by electrospinning. The morphology, thermal behavior and internal structure of the nanofibers were analyzed by scanning and transmission electron microscopy, differential scanning calorimetry and wide angle X-ray diffraction, respectively. The nanofibers of the nanocomposites had lower diameters than the nanofibers of the corresponding neat polymers, while the nanofibers from PBAT and PBAT/MMT were the least crystalline. The cell viability of all the nanofibers was analyzed by reduction of the tetrazolium salt by the pyruvate dehydrogenase enzymatic complex present in the mitochondria (MTT test). None of the nanofibers was toxic to the cells and the PBAT and PBAT/MMT nanofibers exhibited a more favorable environment for developing fibroblasts from cardiomyocytes than the control, probably due to their low crystallinity. These results demonstrated the potential use of nanofibers´ mats as scaffolds for cell growth.

OBJETIVO: O objetivo deste estudo foi avaliar o efeito da T3 na expressão da osteocalcina, osteopontina e colágeno I durante a diferenciação osteogênica das células-tronco mesenquimais (CTM). MATERIAIS E MÉTODOS: As células da medula óssea de ratas Wistar jovens foram extraídas, cultivadas e separadas em cinco grupos: controle (indiferenciado), diferenciado (estímulo osteogênico) e diferenciado com T3 (10-3 nM, 10-2 nM e 100 nM). Para cada grupo, foram cultivadas quatro amostras que foram analisadas por RT-PCR tempo real aos 7, 14 e 21 dias, para quantificação dos transcritos gênicos para osteocalcina, osteopontina e colágeno I. RESULTADOS: Todos os grupos diferenciados sem T3 ou com T3 independentemente da concentração apresentaram expressão de colágeno I significativamente menor e expressão de osteocalcina e osteopontina significativamente maior em comparação a das CTM indiferenciadas. Mas o grupo T3 100 nM apresentou concentração de osteocalcina mais elevada e semelhante à da cultura de osteoblastos. CONCLUSÃO: Conclui-se que a triiodotironina não altera a expressão de osteopontina e de colágeno pelas CTM, mas aumenta a expressão da osteocalcina durante a diferenciação osteogênica in vitro, sendo esse efeito dose-dependente.

OBJECTIVE: The aim of this study was to evaluate the effect of T3 on the expression of osteocalcin, osteopontin and collagen I during osteogenic differentiation of mesenchymal stem cells (MSC). MATERIALS AND METHODS: The bone marrow cells of Wistar rats with 30 days of age were extracted, cultured and separated into five groups: control (undifferentiated), differentiated (osteogenic stimulus) and differentiated with T3 (10-3 nM, 10-2 nM and 100 nM). For each group, four samples were cultured and were analyzed by real time RT-PCR at 7, 14 and 21 days for quantification of gene transcripts for osteocalcin, osteopontin and collagen I. RESULTS: All the different groups without T3 or with T3 regardless of the concentration, showed the collagen I expression significantly lower expression, and osteocalcin and osteopontin expression significantly greater than that of undifferentiated MSC. Nevertheless, the group T3 100 nM showed higher expression of osteocalcin and a similar expression of the osteoblast culture. CONCLUSION: In conclusion, the triiodothyronine does not affect the expression of osteopontin and collagen I, but increases ostecalcin expression during osteogenic differentiation in vitro of the MSC, and this effect is dose-dependent.

The CTLA-4 protein is expressed in activated T cells and plays an essential role in the immune response through its regulatory effect on T cell activation. Polymorphisms of the CTLA-4 gene have been correlated with autoimmune, neoplastic and infectious illnesses. This work aimed to verify possible associations between single nucleotide polymorphisms (SNPs) in CTLA-4, -318C/T in the promoter and +49A/G in exon 1 and paracoccidioidomycosis (PCM) caused by Paracoccidioides brasiliensis. For this purpose, 66 chronic form PCM patients and 76 healthy controls had their allele, genotype and haplotype frequencies determined. The genetic admixture structure of the patients and controls was evaluated to eliminate ancestral bias. The comparison of frequencies indicated no significant differences between patients and controls that could link the SNPs to PCM. Groups were admixture matched with no difference observed in population ancestry inference, indicating that the absence of association between CTLA-4 polymorphisms and PCM could not be attributed to ancestral bias. This study showed that there was no association between the CTLA-4 SNPs -318 and +49 and the resistance or susceptibility to PCM.

A citocompatibilidade de compósitos sintéticos hidroxiapatita/colágeno dopados ou não com Zn+2 foi avaliada, usando cultura primária de osteoblastos. A hidroxiapatita (HAP) foi sintetizada tendo como precursores o hidróxido de cálcio e o ácido ortofosfórico. Um novo compósito de HAP foi desenvolvido adicionando 1,05% em peso de Zn(NO3)2.6H2O, obtendo-se HAPZn. Colágeno tipo I puro (COL) foi obtido de pericárdio bovino pelo método da digestão enzimática. Os compósitos HAP/COL e HAPZn/COL foram desenvolvidos e caracterizados através de MEV/EDS. A viabilidade celular e a produção de fosfatase alcalina na presença dos compósitos foram avaliadas pelos ensaios de MTT e NBT-BCIP, respectivamente, e comparadas às células osteoblásticas do controle. Três experimentos individuais foram realizados em triplicatas e submetidos à análise de variância e pós-teste de Bonferroni com significância estatística p<0,05. Os compósitos HAPZn/COL não estimularam a proliferação e o aumento da produção de fosfatase alcalina das células osteoblásticas. Os compósitos testados não alteraram a viabilidade celular nem causaram alterações na morfologia celular em 72 h, mostrando propriedades adequadas para aplicações biológicas.

The cytocompatibility of synthetic hydroxyapatite/collagen composites alone or doped with Zn+2 was tested by using primary culture of osteoblasts. The hydroxyapatite (HAP) was synthesized having calcium hydroxide and orthophosphoric acid as precursors. A new HAP composite was developed adding 1.05 w% of Zn(NO3)2.6H2O forming HAPZn. The pure type I collagen (COL) was obtained from bovine pericardium by enzymatic digestion method. The HAP/COL and HAPZn/COL composites were developed and characterized by SEM/EDS. The cell viability and alkaline phosphatase activity in the presence of composites were evaluated by MTT assay and NBT-BCIP assay, respectively, and compared to osteoblastic cells of the control. Three individual experiments were accomplished in triplicates and submitted to the variance analysis and Bonferroni’s post-test with statistically significant at p<0.05. The HAPZn/COL composite did not stimulate the proliferation and increasing of alkaline phosphatase activity of the osteoblastic cells. The tested composites did not alter the cellular viability neither caused alterations in the cellular morphology in 72 h showing adequate properties for biological applications.

T cell recognition of antigens displayed on the surface of antigen presenting cell results in rapid activation of protein tyrosine kinases and kinase C. This process leads to second messengers, such as inositol phosphates and diacylgycerol, and phosphorylation of multiple proteins. The role of different protein kinases in the activation of peripheral blood mononuclear cells (PBMC) from Schistosoma mansoni infected individuals was evaluated using genistein and H-7, specific inhibitors of protein tyrosine kinase and kinase C, respectively. Our results showed that proliferation in response to soluble egg antigen or adult worm antigen preparation of S. mansoni was reduced when PBMC were cultured in presence of protein kinase inhibitors. Using these inhibitors on in vitro granuloma reaction, we also observed a marked reduction of granuloma index. Taken together, our results suggest that S. mansoni antigen activation of PBMC involves protein kinases activity

A Schistosoma mansoni adult worm anionic fraction (PIII) has previously been shown to protect mice against challenge infection and to reduce pulmonary and hepatic granulomatous hypersensitivity. Serum from PIII-immunized rabbit was used to screen a lgt11 cDNA library from S. mansoni adult worm in order to identify antigens capable of modulating granulomatous hypersensitivity. We obtained four clones with 400 (Sm-III.11), 900 (Sm-III.16), 1100 (Sm-III.10) and 1300 (Sm-III.12) bp of length. All clone-specific antibodies were able to recognize most of the PIII components. The sequence analysis showed that these clones presented high homology with S. mansoni paramyosin (Sm-97). These findings ascribe a new function to this antigen with an important role in modulation of granulomatous hypersensitivity to S. mansoni eggs

Schistosomiasis is a disease whose pathology is strongly related to the granulomatous reaction formed around parasite eggs trapped in host tissues. Studies have shown that the chronic intestinal form (INT) of this infection is associated with a variety of immunoregulatory mechanisms which lead to a diminished granulomatous reaction. Using an in vitro model of granuloma reaction, we show that immune complexes (IC) isolated from sera of INT patients are able to reduce granulomatous reaction developed by peripheral blood mononuclear cells (PBMC) from acute (AC), INT and hepatosplenic (HE) patients to soluble egg antigen (SEA)-conjugated polyacrylamide beads (PB-SEA). This inhibitory activity is also observed in cell proliferation assay of PBMC from INT and HE patients stimulated with SEA and adult worm antigen (SWAP). Furthermore, IC isolated from sera of patients with different clinical forms of the disease are also able to suppress INT patients PBMC reactivity. Therefore, our results show that circulating IC present in sera of patients with different clinical forms of schistosomiasis may down-regulate PBMC reactivity to parasite antigens resulting in a diminished granuloma reaction to parasite eggs

We have developed an in vitro model of granuloma formation for the purpose of studying the immunological components of delayed type hypersensitivity granuloma formation in patients infected with Schistosoma mansoni. Our data show that 1) granulomatous hypersensitivity can be studied by examining the cellular reactivity manifested as multiple cell layers surrounding the antigen conjugated beads; 2) this reactivity is a CD4 cell dependent, macrophage dependent, B cell independent response and 3) the in vitro granuloma response is antigenically specific for parasite egg antigens. Studies designed to investigate the immune regulation of granulomatous hypersensitivity using purified populations of either CD4 or CD8 T cells have demonstrated the complexity of cellular interactions in the suppression of granulomatous hypersensitivity. The anti-S. mansoni egg immune responses of individual patients with chronic intestinal schistosomiasis can be classified either as soluble egg antigen (SEA) hypersensitive with maximal granulomatous hypersensitivity or SEA suppressive with activation of the T cell suppressor pathway with effective SEA granuloma modulation. Our data suggest that T cell network interactions are active in the generation of effective granuloma modulation in chronic intestinal schistosomiasis patients.