Hepatic stellate cell activation and hepatic fibrosis in children with type 1 autoimmune hepatitis: an immunohistochemical study of paired liver biopsies before treatment and after clinical remission.

OBJECTIVES: The activation of hepatic stellate cells (HSC) is considered the most important event in hepatic fibrogenesis. The precise mechanism of this process is unknown in autoimmune hepatitis (AIH), and more evidence is needed on the evolution of fibrosis. The aim of this study was to assess these aspects in children with type 1 AIH. METHODS: We analyzed 16 liver biopsy samples from eight patients, paired before treatment and after clinical remission, performed an immunohistochemical study with anti-alpha actin smooth muscle antibody and graded fibrosis and inflammation on a scale of 0-4 (Batts and Ludwig scoring system). RESULTS: There was no significant reduction in fibrosis scores after 24+/-18 months (2.5+/-0.93 vs. 2.0+/-0.53, P=0.2012). There was an important decrease in inflammation: portal (2.6+/-0.74 vs. 1.3+/-0.89, P=0.0277), periportal/periseptal (3.0+/-0.76 vs. 1.4+/-1.06, P=0.0277), and lobular (2.8+/-1.04 vs. 0.9+/-0.99, P=0.0179). Anti-alpha actin smooth muscle antibodies were expressed in the HSC of the initial biopsies (3491.93+/-2051.48 mum), showing a significant reduction after remission (377.91+/-439.47 microm) (P=0.0117). CONCLUSION: HSC activation was demonstrated in the AIH of children. The reduction of this activation after clinical remission, which may precede a decrease in fibrosis, opens important perspectives in the follow-up of AIH.

Comparação de três protocolos de extração de DNA a partir de tecido fixado em formol e incluído em parafina / Comparison of three DNA extraction protocols from formaldehyde-fixed and paraffin-embedded tissues

OBJETIVO: Padronizar um método alternativo para extração de DNA a partir de tecido fixado em formol e conservado em arquivos de blocos de parafina, visando à realização de estudos retrospectivos. MÉTODOS: Comparou-se a eficiência de protocolos de extração de DNA a partir de tecido parafinado, para análise por reação em cadeia de polimerase (PCR), tomando-se como parâmetro um protocolo baseado em um kit comercial. Foram feitas extrações do DNA de 60 espécimes por três métodos: o protocolo A, baseado no kit GlassMAX; o B, utilizando-se o kit GFX TM; e o C, tendo como base o método de Banerjee et al.(2). A integridade e a suficiência do DNA presente na amostra foram avaliadas pela amplificação por PCR de um segmento de 110pb do gene da beta-globina humana, com visualização por meio de eletroforese em gel de poliacrilamida, corado pela prata. Resultados: Das 60 amostras analisadas, 45 apresentaram resultado positivo na PCR quando o DNA foi extraído por qualquer um dos três protocolos. Em seis amostras, a amplificação foi positiva apenas para o DNA extraído pelos protocolos A e C. Em três amostras, o resultado foi positivo apenas para o DNA extraído pelo protocolo A, e em duas, apenas para o DNA extraído pelo protocolo C. CONCLUSÕES: O protocolo C apresentou desempenho semelhante ao do protocolo A, com as vantagens de apresentar menor custo, dispensar o uso de kit comercial, além de não utilizar solventes orgânicos, revelando-se uma alternativa viável para a obtenção de DNA a partir de tecido fixado em formol e incluído em parafina.

Effect of topical application of fibronectin in duodenal wound healing in rats

Fibronectin (FN), a large family of plasma and extracellular matrix glycoproteins, plays an important role in wound healing. PURPOSE: To evaluate the effect of fibronectin on the healing of sutured duodenal wounds, correlating with the serum and tissue level of the substance. METHODS: An experimental study was done in 30 adult Wistar rats divided into two group. In the control group (n=15) a duodenal suture was treated with saline solution 0,9 percent and in the test group the duodenal wounds were treated with 1 percent FN. The duodenal wound healing process was studied in the 5th, 7tn and 10th postoperative days, by histological sections stained by hematoxylin-eosin, Masson trichromic and immunohistochemical reaction for FN. A digital histological grading system was used to obtain a score for each group and to observe the healing process. RESULTS: the FN was present in the several layers of the duodenum and the cellular and plasmatic FN increased with the evolution of healing. In the test group the FN enhanced the wound healing within 5, 7 and 10 days after injury, when compared with the control group. CONCLUSION: The topical use of FN in duodenal sutured wounds in rats enhances healing by stimulating the appearence of fibroblasts into the wound site and development of granulation tissue. This acceleration of the repair process may have an important application in the healing of duodenal wounds.