Lack of evidence for the pathogenic role of iron and HFE gene mutations in Brazilian patients with nonalcoholic steatohepatitis.

The hypothesis of the role of iron overload associated with HFE gene mutations in the pathogenesis of nonalcoholic steatohepatitis (NASH) has been raised in recent years. In the present study, biochemical and histopathological evidence of iron overload and HFE mutations was investigated in NASH patients. Thirty-two NASH patients, 19 females (59%), average 49.2 years, 72% Caucasians, 12% Mulattoes and 12% Asians, were submitted to serum aminotransferase and iron profile determinations. Liver biopsies were analyzed for necroinflammatory activity, architectural damage and iron deposition. In 31 of the patients, C282Y and H63D mutations were tested by PCR-RFLP. Alanine aminotransferase levels were increased in 30 patients, 2.42 1.12 times the upper normal limit on average. Serum iron concentration, transferrin saturation and ferritin averages were 99.4 31.3 g/dl, 33.1 12.7% and 219.8 163.8 g/dl, respectively, corresponding to normal values in 93.5, 68.7 and 78.1% of the patients. Hepatic siderosis was observed in three patients and was not associated with architectural damage (P = 0.53) or with necroinflammatory activity (P = 0.27). The allelic frequencies (N = 31) found were 1.6 and 14.1% for C282Y and H63D, respectively, which were compatible with those described for the local population. In conclusion, no evidence of an association of hepatic iron overload and HFE mutations with NASH was found. Brazilian NASH patients comprise a heterogeneous group with many associated conditions such as hyperinsulinism, environmental hepatotoxin exposure and drugs, but not hepatic iron overload, and their disease susceptibility could be related to genetic and environmental features other than HFE mutations.

Lack of evidence for the pathogenic role of iron and HFE gene mutations in Brazilian patients with nonalcoholic steatohepatitis

The hypothesis of the role of iron overload associated with HFE gene mutations in the pathogenesis of nonalcoholic steatohepatitis (NASH) has been raised in recent years. In the present study, biochemical and histopathological evidence of iron overload and HFE mutations was investigated in NASH patients. Thirty-two NASH patients, 19 females (59 percent), average 49.2 years, 72 percent Caucasians, 12 percent Mulattoes and 12 percent Asians, were submitted to serum aminotransferase and iron profile determinations. Liver biopsies were analyzed for necroinflammatory activity, architectural damage and iron deposition. In 31 of the patients, C282Y and H63D mutations were tested by PCR-RFLP. Alanine aminotransferase levels were increased in 30 patients, 2.42 + or - 1.12 times the upper normal limit on average. Serum iron concentration, transferrin saturation and ferritin averages were 99.4 + or - 31.3 g/dl, 33.1 + or - 12.7 percent and 219.8 + or - 163.8 æg/dl, respectively, corresponding to normal values in 93.5, 68.7 and 78.1 percent of the patients. Hepatic siderosis was observed in three patients and was not associated with architectural damage (P = 0.53) or with necroinflammatory activity (P = 0.27). The allelic frequencies (N = 31) found were 1.6 and 14.1 percent for C282Y and H63D, respectively, which were compatible with those described for the local population. In conclusion, no evidence of an association of hepatic iron overload and HFE mutations with NASH was found. Brazilian NASH patients comprise a heterogeneous group with many associated conditions such as hyperinsulinism, environmental hepatotoxin exposure and drugs, but not hepatic iron overload, and their disease susceptibility could be related to genetic and environmental features other than HFE mutations

Analysis of HLA-A antigens and C282Y and H63D mutations of the HFE gene in Brazilian patients with hemochromatosis.

The hemochromatosis gene, HFE, is located on chromosome 6 in close proximity to the HLA-A locus. Most Caucasian patients with hereditary hemochromatosis (HH) are homozygous for HLA-A3 and for the C282Y mutation of the HFE gene, while a minority are compound heterozygotes for C282Y and H63D. The prevalence of these mutations in non-Caucasian patients with HH is lower than expected. The objective of the present study was to evaluate the frequencies of HLA-A antigens and the C282Y and H63D mutations of the HFE gene in Brazilian patients with HH and to compare clinical and laboratory profiles of C282Y-positive and -negative patients with HH. The frequencies of HLA-A and C282Y and H63D mutations were determined by PCR-based methods in 15 male patients (median age 44 (20-72) years) with HH. Eight patients (53%) were homozygous and one (7%) was heterozygous for the C282Y mutation. None had compound heterozygosity for C282Y and H63D mutations. All but three C282Y homozygotes were positive for HLA-A3 and three other patients without C282Y were shown to be either heterozygous (N = 2) or homozygous (N = 1) for HLA-A3. Patients homozygous for the C282Y mutation had higher ferritin levels and lower age at onset, but the difference was not significant. The presence of C282Y homozygosity in roughly half of the Brazilian patients with HH, together with the findings of HLA-A homozygosity in C282Y-negative subjects, suggest that other mutations in the HFE gene or in other genes involved in iron homeostasis might also be linked to HH in Brazil.

Analysis of HLA-A antigens and C282Y and H63D mutations of the HFE gene in Brazilian patients with hemochromatosis

The hemochromatosis gene, HFE, is located on chromosome 6 in close proximity to the HLA-A locus. Most Caucasian patients with hereditary hemochromatosis (HH) are homozygous for HLA-A3 and for the C282Y mutation of the HFE gene, while a minority are compound heterozygotes for C282Y and H63D. The prevalence of these mutations in non-Caucasian patients with HH is lower than expected. The objective of the present study was to evaluate the frequencies of HLA-A antigens and the C282Y and H63D mutations of the HFE gene in Brazilian patients with HH and to compare clinical and laboratory profiles of C282Y-positive and -negative patients with HH. The frequencies of HLA-A and C282Y and H63D mutations were determined by PCR-based methods in 15 male patients (median age 44 (20-72) years) with HH. Eight patients (53 percent) were homozygous and one (7 percent) was heterozygous for the C282Y mutation. None had compound heterozygosity for C282Y and H63D mutations. All but three C282Y homozygotes were positive for HLA-A3 and three other patients without C282Y were shown to be either heterozygous (N = 2) or homozygous (N = 1) for HLA-A3. Patients homozygous for the C282Y mutation had higher ferritin levels and lower age at onset, but the difference was not significant. The presence of C282Y homozygosity in roughly half of the Brazilian patients with HH, together with the findings of HLA-A homozygosity in C282Y-negative subjects, suggest that other mutations in the HFE gene or in other genes involved in iron homeostasis might also be linked to HH in Brazil

Size of gastroesophageal varices: its behavior after the surgical treatment of portal hypertension.

The size of gastroesophageal varices is one of the most important factors leading to hemorrhage related to portal hypertension. An endoscopic evaluation of the size of gastroesophageal varices before and after different operations for portal hypertension was performed in 73 patients with schistosomiasis, as part of a randomized trial: proximal splenorenal shunt (PSS n=24), distal splenorenal shunt (DSS n=24), and esophagogastric devascularization with splenectomy (EGDS n=25). The endoscopic evaluation was performed before and up to 10 years after the operations. Variceal size was graded according to Palmer's classification: grade 1 - up to 3 mm, grade 2 - from 3 to 6 mm, grade 3 - greater than 6 mm, and were analyzed in four anatomical locations: inferior, middle or superior third of the esophagus, and proximal stomach. The total number of points in the pre-operative grading minus the number of points in the post-operative grading gave a differential grading, allowing statistical comparison among the surgical groups. Good results, in terms of disappearance or decrease of variceal size, were observed more frequently after PSS than after DSS or EGDS - 95.8%, 83.3%, and 72%, respectively. When differential grading was analyzed, a statistically significant difference was observed between PSS and EGDS, but not between proximal and distal splenorenal shunts. In conclusion, shunt surgeries were more efficient than devascularization in diminishing variceal size.

Liver biopsy in neonatal cholestasis: a review on statistical grounds.

The aim of this study was to improve the accuracy of the histopathologic diagnosis in the differential diagnosis between obstructive and nonobstructive forms of neonatal cholestasis, using this clinical situation as a model for a mathematical approach. The study was blind, and we performed it in two steps. In the first step, 49 histologic parameters were visually estimated and were scored on a scale of 0 to 4+ in 100 liver biopsy specimens obtained between 1980 and 1985 from 78 patients with neonatal cholestasis. Forty-eight of these 100 specimens were from patients with final diagnosis of obstructive cholestasis (Group I), and 52 were from patients with nonobstructive cholestasis (Group II). The age range was 3 to 24 weeks (median, 12.5 wk). Twelve histologic variables were selected by chi 2 and Fisher's exact test (P < .05). Next, a series of combinations among these variables were submitted to statistical analysis by logistic regression method, defining a six-variable model that had the most powerful predictive value to classify the type of cholestasis. The variables were portal ductal proliferation, bile plugs in portal bile ductules, portoportal bridges, neutrophils, hepatocyte swelling, and multinucleated giant hepatocytes. The score obtained by this model correspond to the probability of a case belonging to Group I. The accuracy, sensitivity, and specificity rates were 94.0%. In the second step, the model was applied to a new sample of 74 needle-liver biopsy specimens obtained between 1990 and 1995, 45 from patients in Group I and 29 from patients in Group II. The age range was 3 to 15 weeks (median, 8 wk). The accuracy, sensitivity, and specificity rates were 90.5%, 100%, and 75.9%, respectively. In our diagnostic routine, this score has been systematically reported and has been helpful in orienting the therapeutic decision in this group of patients.

Hepatocellular carcinoma in Brazil: report of a national survey (Florianópolis, SC, 1995).

In order to investigate epidemiological aspects of hepatocellular carcinoma (HCC) in Brazil, basic informations about cases diagnosed from January 1992 to December 1994 were requested to several medical centers of different Brazilian States. A simple questionnaire included age, sex, alcohol abuse (over 80g/day), associated liver cirrhosis, persistent HBV infection (HBsAg), HCV infection (anti-HCV) and serum levels of alpha fetoprotein. 287 cases, over 16 years old, from 19 medical centers of 8 States (Pará, Bahia, Minas Gerais, Espirito Santo, Rio de Janeiro, São Paulo, Paraná and Rio Grande do Sul) were analysed. The results showed: (a) Mean age was 56.3 +/- 14.4 for men and 54.7 +/- 16.8 yr for women and the male/female ratio was 3.4:1. (b) 69.6% were caucasians, 21.8% mullatoes, 4.8% orientals and 3.7% blacks. (c) HBsAg (+) in 77/236 cases (41.6%) without differences between males and females. (d) Anti-HCV (+) in 52/193 cases (26.9%). (e) 7/180 cases were positive both for HBsAg and anti-HCV (3.8%). (f) There was chronic alcoholism in 88/235 cases (37%). (g) HCC was found in cirrhotic livers in 71.2% of 202 cases in which the presence or absence of cirrhosis was reported. (h) Alpha-fetoprotein above 20 ng/ml was found in 124/172 cases (72%) and above 500 ng/ml only in 40 cases (23.2%). These results showed that the HCC in Brazil has an intermediate epidemiological pattern as compared to those from areas of low and high incidence of the tumor. In spite of the high frequency of the association of HCC with the HBV and/or HCV infections, 42% of 180 cases were negative both for HBsAg and anti-HCV, indicating the possible role of other etiological factors. The comparison of data from different States showed some regional differences: higher frequency of associated HBsAg in Pará, Bahia, Minas Gerais and Espírito Santo, higher frequency of associated HCV infection in Rio de Janeiro, São Paulo and States of the Southern region and low frequency of associated liver cirrhosis in Salvador and Rio de Janeiro (55.5 and 50% respectively). Further investigation will be necessary to study the presence of other possible etiological factors as aflatoxins, suggested by the favourable climatic conditions for food contamination by fungi in the majority Brazilian regions.

Spontaneous hepatitis B surface antigen clearance in a long-term follow-up study of patients with chronic type B hepatitis. Lack of correlation with hepatitis C and D virus superinfection.

We investigated the frequency of HBsAg clearance and the possible role of viral superinfection in a long-term follow-up of 184 patients with chronic hepatitis B (CHB). Our subjects were 184 patients with chronic hepatitis B and the follow-up was 12-216 months (mean 66.2 +/- 53.7 months). The investigative methods used were: immunoenzymatic assays for HBV, HCV, HDV, and HIV markers; polymerase chain reaction (PCR) for HBV DNA; and liver biopsy and immunoperoxidase. During the follow-up, 20 of the 184 patients cleared serum HBsAg. A comparison of patients with persistent HBsAg(group I) and of those who cleared this marker (group II) showed a significant difference in mortality (P = 0.002) between the two groups and a tendency to a more severe exacerbation (flare) in group II (P = 0.07). Antibodies to hepatitis C and D virus as well as antibodies to HIV were equally distributed in both groups. Thirteen patients (7.9%) from group I, but none from group II, subsequently developed hepatocellular carcinoma. These results suggest that the frequency of spontaneous clearance of HBsAg during chronic HBV infection is low. No determinant factor for the clearance was found, including the presence of liver cirrhosis. Serum HBV DNA was undetectable by PCR after clearance in 16 out of 17 patients.

Portal hypertension in schistosomiasis: a long-term follow-up of a randomized trial comparing three types of surgery.

The long-term follow-up of patients with the severe form of Manson's schistosomiasis who had had elective surgical treatment for portal hypertension, in a randomized trial, was clinically evaluated. Of 94 patients, proximal splenorenal shunting was performed in 32, esophagogastric devascularization with splenectomy in 32 and distal splenorenal shunting in 30. Patients were observed during a mean of 85.7 +/- 33.1 mo, excluding nine patients (9.6%) who were lost to follow-up. Recurrence of upper gastrointestinal tract bleeding occurred in 24.1% of the patients, without statistical differences among the three groups, but rebleeding because of varices was more frequent after esophagogastric devascularization with splenectomy. Hepatic encephalopathy was significantly higher after proximal splenorenal shunting (39.3%) when compared with distal splenorenal shunting (14.8%) and with esophagogastric devascularization with splenectomy (0%). Lethality was also significantly higher after proximal splenorenal shunting (42.9%) when compared with distal splenorenal shunting (14.8%) and with esophagogastric devascularization with splenectomy (7.1%). Indirect hyperbilirubinemia was absent after esophagogastric devascularization with splenectomy and more frequent after distal splenorenal shunting (52%) although also present after proximal splenorenal shunting (29.6%). Esophagogastric devascularization with splenectomy was demonstrated to be the best option because of the absence of encephalopathy and because of low mortality rates. Hepatic encephalopathy occurred after distal splenorenal shunting but in a lesser percentage than after proximal splenorenal shunting. The higher incidence of encephalopathy and lethality proscribes proximal splenorenal shunting in Manson'schistosomiasis.

Leptospiral antigens in the liver of experimentally infected guinea pig and their relation to the morphogenesis of liver damage.

In order to investigate the morphogenes of experimental leptospirosis by morphologic and immunohistologic methods, 24 guinea-pigs were inoculated intraperitoneally with L. interrogans serogroup Icterohaemorrhagiae. They were divided in 6 groups, sacrificed from the 1st to the 6th day of infection. Semiquantitative analyses of histopathological liver lesions were performed in 1 micron sections of tissue embedded in glycol-methacrylate. The distribution of leptospiral antigen (L. Ag) and its glycolipoprotein (GLP) was demonstrated by peroxidase-antiperoxidase on paraffin embedded tissue. Significant lesions appeared at the 4th day of infection, progressing to a peak on the 6th day. Inflammation was associated with injury of the portal triad. Liver cells showed either swelling or acidophilic degeneration and necrosis, together with loss of cell cohesion, leading to disarray of liver cell plates. Mitochondria were found progressively enlarged and irregularly distributed. L. Ag expression was parallel to the morphological changes. Portal distribution was significant at the 4th day and on later stages centrilobular localization became predominant. Spiral forms suggestive of intact leptospires were initially found but, chiefly at the 6th day, L. Ag was seen in granules, probably resulting from phagocytosis. GLP staining was similar to granular L. Ag in morphology, and distribution. Cytokeratin condensation was seen in liver cells with acidophilic necrosis and was marked in areas of disorganization of cell plates. Our findings lead us to hypothesize a direct leptospiral cytotoxic effect on endothelial and on liver-cell membranes. At first, leptospires themselves would induce subcellular changes acting mainly on membrane permeability. Afterwards, their granular forms, including GLP, would act as adjuvant factors. These findings demonstrate that the disarray of liver cell plates at the late phase of the disease is genuine.

Leptospiral antigens (L. interrogans serogroup ictero-haemorrhagiae) in the kidney of experimentally infected guinea pigs and their relation to the pathogenesis of the renal injury.

The search for leptospiral antigens (L. interrogans serogroup icterohaemorrhagiae) was carried out in 24 guinea pigs experimentally inoculated with 1 ml of culture containing 10(7)-10(8) leprospires and sequentially sacrificed from the first until the 6th day of infection. Semiquantitative analysis of histopathological variables comprising kidney interstitium, tubules and glomeruli was done in 1 micron sections of tissue embedded in glycolmetacrylate. Leptospiral antigen (LAg) and its glycolipoprotein (GLP) expression were detected through PAP in paraffin embedded tissue. The mild interstitial involvement of the kidney, manifested chiefly by oedema and focal interstitial nephritis seen at the 4th day, progressed to tubular damage at the 6th day, characterized by either swelling or cytoplasmic acidophilia of epithelial cells with loss of cell cohesion and sloughing of cells into the tubular lumina. Brush border alterations and mitochondrial changes were observed. Endothelial cell injury was noted in the interstitial vessels. LAg expression was parallel to the kidney changes: small deposits of elongated forms of LAg were detected at the 4th day either within the vascular lumen or free in the interstitium. A rise in the antigen expression was observed at the 5th day when it was seen either around tubules or in their walls. LAg was detected inside the tubular lumina at the 6th day of infection when granular LAg and GLP were abundant. This sequence reproduces the pathway of leptospires in the kidney and the crescent amounts of antigens detected toward the end of the experiment, with antigen concentration in cases of major tissue damage suggesting a direct action of the microorganisms and/or their products in the pathogesis of the lesions.

Anti-liver-kidney microsome antibody-positive autoimmune hepatitis presenting as fulminant liver failure.

A 17-month-old girl presented with acute hepatitis, which took a fulminant course leading to death 2 months after onset. No known cause of fulminant liver failure could be identified. Postmortem examination of the liver showed massive multilobular necrosis and areas of severe piecemeal necrosis. A high level of total serum gamma-globulins raised the possibility of autoimmune hepatitis. Search for anti-liver-kidney microsome antibody in the patient's serum was positive by immunofluorescence and enzyme-linked immunosorbent assay. Western blot analysis showed reactivity of the antibody with a 50-kDa protein identical to that observed in children with autoimmune hepatitis. This patient's history strongly suggests that autoimmune hepatitis can present as fulminant liver failure in children. Early diagnosis in such a patient could lead to early immunosuppressive therapy.

Cryptogenic cirrhosis: clinico-biochemical comparison with alcoholic and viral etiologies.

Cryptogenic cirrhosis defines a group of undetermined etiology that would either be caused by factors as alcohol, virus and others or be due to still unknown etiological factors. To test these hypotheses we have looked for similarities or differences in clinico-biochemical presentation of 196 cases of alcoholic, viral and cryptogenic cirrhosis. Age, jaundice, spiders, palmar erythema and factor V showed a statistically significant difference of the cryptogenic cirrhosis when compared with both alcoholic and viral etiologies. On clinico-biochemical grounds it could be suggested that cryptogenic cirrhosis would constitute a discrete group, based on the following parameters: predominance of females, a more advanced age, less marked peripheral signs of chronic hepatic failure (jaundice, spiders and palmar erythema) besides milder alterations of laboratory liver function tests.

[High resolution light microscopy: adaptation of the method and its use in the study of experimental leptospirosis in guinea pigs]. / Microscopia óptica de alta resolução: adaptação do método e sua aplicação ao estudo da leptospirose experimental em cobaias.

Morphological lesions in parenchimal and mesenchimal structures of liver and kidney were studied in guinea-pigs experimentally infected with Leptospira interrogans serogroup icterohaemorrhagiae in comparison with a group of non-infected guinea-pigs. All specimens were submitted to conventional light microscopy as well as to high resolution light microscopy, in one micrometer sections of tissue embedded in glycolmethacrylate. High resolution light microscopy, applied for the first time in leptospirosis, was proved very useful, since it enabled us to visualize cellular structures in the same slide used for panoramic view. Cell cohesion, brush borders, pynocytotic vesicles and organellae distributions were parameters especially suitable for analysis at this low-cost, highly precise procedure in microscopy.

Hepatobiliary alterations in massive biliary ascariasis. Histopathological aspects of an autopsy case.

Hepatobiliary alterations found in an autopsy case of massive Biliary Ascariasis, are reported on histological grounds. Severe cholangitis was the main finding, but other changes were also detected, such as pyloric and intestinal metaplasia, hyperplasia of the epithelial lining, with intraductal papillomas and adenomatous proliferation. Remnants of the worm were observed tightly adhered to the epithelium, forming microscopic intrahepatic calculi. Mucopolysaccharides, especially acid, showed to be strongly positive on the luminal border, and in proliferated glands around the ducts. The authors discuss the similarity between such findings and Oriental Cholangio-hepatitis, and suggest that inflammation and the presence of the parasitic remnants are responsible for the hyperplastic and metaplastic changes, similarly with what occurs in chlonorchiasis, fascioliasis and schistosomiasis.

[Recombinant alfa interferon in the treatment of chronic B and non-A, non-B hepatitis: preliminary results]. / Alfa-interferon recombinante no tratamento das hepatities crônicas B e não-A, não-B: resultados preliminares.

Recombinant alpha-interferon (IFN-R) was given to 17 patients with non-A, non-B chronic hepatitis (NANB-CH) and to 11 patients with B chronic hepatitis (B-CH). Fever (100.4 to 102.2 Fahrenheit) was observed in every patient during the early phase of treatment. Other side-effects included rigors, myalgia, headache and laboratory changes such as leucopenia, neutropenia and, in some cases, thrombocytopenia. However, the tolerance was considered acceptable and treatment had to be interrupted in only one patient presenting generalized mucosal lesions attributed to a hypersensitivity reaction. The response to IFN-R in NANB-CH was considered positive when serum aminotransferase levels became normal or below two times the upper normal limit. Out of eight patients who completed the treatment, four were considered as responders but one of them, treated during five months, showed a relapse after three months. On the other hand, in one patient treated for twelve months, a persistent normalization of serum amino-transferases was observed: a liver biopsy showed a striking decrease of the inflammatory changes. As to the B-CH. 3 out of 8 patients who completed the treatment showed a disappearance of HBeAg and DNA-polymerase and were considered as responders. These preliminary results show that IFN-R is a promising drug but only multicenter controlled trials will establish its value in the treatment of viral chronic hepatitis.