Mitochondrial cytochrome C and ultrastructural changes in hepatic steatosis induced by hepatitis C virus

Hepatic steatosis is an important hallmark of hepatitis C virus [HCV] infection. There is substantial body of evidence to implicate steatosis in the development of hepatic fibrosis. The underlying mechanisms of HCV-related steatosis however are not yet clarified. This study was performed to evaluate ultrastructural mitochondrial changes in patients with HCV-induced hepatic steatosis and correlate these findings with serum cytochrome c and apolipoproteins. Thirty-seven HCV-positive patients admitted to Theodore-Bilharz Research Institute were selected. They did not have other confounding prosteatogenic variables: diabetes, overweight, alcohol consumption and prosteatogenic drugs as amiodarone; corticosteroids. In addition, 10 apparently age- matched subjects were selected as a reference group. All subjects were initially subjected to full history, thorough clinical examination, liver function tests; lipid profile, HCV-IgG antibody and hepatitis-B surface antigen. All patients were subjected to ultrasound-guided liver biopsy. Biopsy specimen was processed for light and electron microscopic histopathological examination. According to histopathological findings, patients were divided into 4 groups according to the stage of fibrosis[I, II, III and IV]into :-Group 1 [n=7]; Group 2 [n=9]; Group:3 [n=9] and Group 4 [n=12]or cirrhotic group respectively. Measurement of apolipoproteins A, B and II and specific estimation of serum cytochrome-C was performed. Interpretation of the results revealed accumulated fat droplets by ultrastructure identification in the hepatocytes together with hypobetalipoproteinemia and hypotriglyceridemia. This was accompanied with mitochondrial ultrastructural alterations in all the studied groups ranging from complete dissolution to loss of outer mitochondria] membrane. In addition, it is noteworthy that ultrastructural changes of the rough endoplasmic reticulum [RER] detected in this study may be a contributing factor to abnormal fat metabolism in HCV. Concomittantly, serum cytochrome c was significantly lowered in all the studied groups as compared to the reference mean value. Depletion of mitochondria] cytochrme c might result in accumulation of reactive oxygen species and further accentuation of steatosis. There was significant correlation between serum cytochrome c, apolipoprotein B and serum triglycerides in the patients' group, ushering that it might have its role in HCV-induced lipid changes. In the cirrhotic group, ultrastructural elucidation of homogenous unlocalized intracytoplasmic fat and evident intracytoplasmic collagen fibrils was reported in this study. This was associated with significantly lowered serum ApoAl, ApoB and triglycerides as compared to all other groups. The afore-mentioned data solidifies the evidence that hepatic steatosis and fibrosis are important sequence of HCV infection and confirm the ability of hepatocyte to synthesize collagen. Moreover, the discerned intracytoplasmic unsaturated fat droplets might mirror the inability of the HCV-dysfunctioning hepatocyte to cope with the excessive dietary intake of fat and hence accentuation of steatosis results. In conclusion, ultrastructural. mitochondrial and RER changes seems to confirm their relation- to the associated intracytoplasmic fatty acids accumulation, the decreased serum cytochrome c and apolipoproteins Al and B Moreover it confirms the cytopathic effect of HCV

Agarose cell block: innovated technique for the processing of urine cytology for electron microscopy examination

Easy manipulation and preservation of cells in suspension through the different steps of sample processing for electron microscopy examination is essential for proper diagnosis. The present study used agarose gel as an embedding media for the processing of cell in suspension for electron microscopic examination. The study also modified the agarcyto cell block procedure for multiple molecular diagnostic analyses on a single scraping from uterine cervix of Kerstens et al.[2000] to convene with electron microscopic processing of exfoliated urothelial cell in voided urine or cell in suspension. The applied protocol depends on the embedding or coating of urothelial cells sediment with melted and then solidified 2% agarose in distilled water. This step is preceded by fixation of urine sediment in 4% buffered glutaraldehyde in sodium cacodylate. The solidified agarose cell block [ACB] is then divided longitudinally into two portion. One portion is fixed in formalin and processed for paraffin block preparation and the other half is divided into tiny pieces and refixed in 4% buffered glutaraldehyde then processed for EM examination. The advantage of this previously discussed maneuver is getting from the same sample, paraffin section stained with haematoxylin and eosin for light microscopic examination and utrathin section for EM examination. Moreover, different histo and immunohistostains can be applied on the paraffin prepared sections. In this work, twenty urine samples were processed using the conventional EM techniques and another twenty urine samples were processed using the innovated ACB technique. The later one allowed an optimal condition for cell processing. It minimized the loss of the harvested cells during the running procedure. It provided good support to the cells. Moreover, the damage imposed on cells by centrifugal forces during the conventional technique was avoided

Role of pentoxifylline and anti-transforming growth factor-B in controlling hepatic fibrosis in murine Schistosomiasis

On the basis of fibroblast and myofibroblast-like cells, the inhibitory effect of pentoxifylline [PTX] and anti-transforming growth factor-beta [anti-TGF-beta] and their role in controlling hepatic fibrosis were investigated. Sixty albino mice were infected with schistosomiasis by subcutaneous [s.c.] injection of 60 cercariae/mouse. They were divided into two groups: The first group [30 mice] was sacrificed at seventh week and the other [30 mice] at fifteenth week after infection. Groups of infected mice were treated with PTX or anti-TGF-beta for three weeks before sacrifice at the acute [7 weeks] and chronic [15 weeks] phases of infection. Three weeks before sacrifice, ten mice from each group were treated by PTX. In parallel, another ten mice from each group received s.c. Injection with anti-TGF-beta three weeks before sacrifice. The remaining ten mice of each group served as controls. Human fibroblasts were incubated with supernatants derived from splenic or hepatic cell cultures and the cell proliferation was measured by the XTT assay

Cellular constituent and intercellular adhesion in schistosoma mansoni granuloma: an ultrastructural study

The present work dealt with the structural analysis of Schistosoma mansoni granuloma and the visualization of cellular interaction at an ultrastructural level in the acute [8 weeks] and chronic [20 weeks] stages of infection, for more detailed understanding of pathophysiology of the disease. Although S. mansoni granuloma is mediated by T-lymphocytes, yet in this work, the macrophage cells and not the lymphocytes represented the main cell type in cellular and fibrocellular granulomas. The cellular and fibrocellular granulomas detected in the acute stage of infection elicited no difference in cellular constituent to those of the chronic stage, respectively. Macrophage cells and fibrocytes were the only cell type detected in fibrotic granuloma. The monocytes may be considered the first cell, reaching the site of the trapped egg as they formed the first row of cells around the egg. The cellular infiltrate forming the granuloma, monocytes, macrophages, lymphocytes, eosinophils, fibroblasts and plasma cells, revealed direct contact or adherence between them and even between the individual cell type, through extending protrusion from the cell membrane of adjacent cells. They constituted an integrated network, which encircled the egg. Similar adhesion between inflammatory cells in the blood vessels and between the inflammatory cells and the endothelial cells were displayed