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Liver histology and hepatic mitochondrial function in HIV-1-infected patients on antiretroviral therapy with chronic transaminase elevation
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Reported by Jules Levin
15th CROI, Feb 200, Boston
Valantin M.-A.1, Ingiliz P.2, Charlotte F.3, Medja F.4, Dominguez S.1, Tubiana R.1, Duvivier C.2, Lombes A4, Benhamou Y.2, Katlama C.1
1Infectious diseases department, 2Hepato-gastroenterology department, 3Anatomopathology department, 4Inserm, UMR582, Pitie-Salpetriere hospital, Paris, F-75013 France
OBJECTIVE
The objectives of this study were to describe liver lesions of HIV-infected patients with unexplained chronic ALT/AST elevation and to identify factors potentially linked to these abnormalities. In addition, the role of a possible mitochondrial dysfunction was evaluated.
AUTHOR CONCLUSIONS
HIV-positive patients with chronic ALT elevation have liver lesions at a high rate.
The most common histological finding in this study was steatohepatitis consistent with the diagnosis of NASH.
NASH lesions were significantly correlated with hyperglycemia and hyperinsulinemia.
In our study, the evaluation of the mitochondrial respiratory chain differed from the pattern observed in typical mitochondrial toxicity. The activity of the respiratory complex IV and amount of mtDNA as observed by quantitative PCR was significantly increased in HIV-infected patients compared to the control group whereas the activity of complex I was decrease.
This finding seems to remove the hypothesis of the mitochondrial DNA toxicity in the genesis of the observed histologic liver abnormalities.
ABSTRACT
Background: Few data exist on liver histology and mitochondrial function in HIV-infected patients with chronic ALT/AST elevation of unknown origin.
Methods: We investigated prospectively mitochondrial function and mitochondrial DNA (mtDNA) content in liver biopsies from HIV-infected pts with ALT elevation>ULN for more than 6 months and treated with cART. Pts with chronic alcohol abuse, HCV or HBV-coinfection, autoimmune or genetic liver diseases were excluded. In a subgroup of sixteen pts with available liver biopsies, mitochondrial respiratory chain (complexes I, II, and IV) and citrate synthase activities were evaluated in liver homogenates using spectrophotometric assays while the mtDNA was quantified by real-time PCR.
Results: Thirty pts were included with a median age of 46 yrs [31-67], a median BMI of 23 kg/m2 [22-24], a viral load<200 copies/mL [200-94600] in 19 (63%) pts and a median CD4 of 365/mm3 [204-921] at time of biopsy. The median duration of HIV infection was 13 yrs [9-15].
The median exposure time to cART was: 118 months [83-133] for NRTIs, 41 months [22-46] for NNRTIs and 53 months [36-90] for PIs.
The median ALT level was 80 U/l [68-135]. Histologic abnormalities were found in 20/30 (67%) pts. Steatosis was present in 18/30(60%) pts (mild (6%-30%) in 9 pts and severe (>30%) in 9 patients) and was associated with concomitant lobular inflammation and/or hepatocellular ballooning in 16/18 pts consistent with the diagnosis of NASH according to the NAS-score.
Liver fibrosis was found in 18/30 pts: 13 pts as F1 and 5 pts as ≥F2, including 3 pts with cirrhosis (Metavir-score). Compared to pts without NASH, pts with NASH had significantly higher fasting glucose levels (median 5.2 [5-5.5]mmol/l NASH vs 4.9 [4.4-5.3]mmol/l, p=0.01) and fasting insulin levels (median 19.4 [14.7 32.8]mU/l vs 10.9 [8.7-14.4]mU/l, p=0.007).
In the subgroup of sixteen patients, 9 were classified as NASH. Between patients with and without NASH respectively, no significant difference in mitochondrial function (complex IV activity: 94[43-136] vs 76[26-92]nanomoles/min.mg of protein) and liver mtDNA amount (median 6920[3684-19893] vs 5584[4113 27472]copies/cells) was observed.
Conclusions: HIV infected pts on cART with chronic ALT elevation have a high rate of liver lesions with predominant histologic patterns of NASH related to insuline resistance. Despite long exposure to cART, in our population, the liver abnormalities may not account to mitochondrial dysfunction.
PATIENTS AND METHODS
Patients with ALT/AST elevation of more than the upper limit of normal (ULN) at least twice in the past 6 months were selected on the basis of the following criteria:
documented HIV infection, no alcohol abuse (≦ 20g alcohol daily, no history of chronic alcohol consumption), no history of hereditary and autoimmune liver disease, no evidence of hemochromatosis (normal ferritinemia and saturation coefficient), negative HCV RNA, and negative serum HBV DNA. Patients with any contra-indication for liver biopsy were excluded. All selected patients provided an informed consent form.
Data on demographics, drug or alcohol use and history of liver diseases were obtained at first visit. History of HIV infection was obtained by chart review information. A month-by-month documentation of the prescribed drug combination allowed a calculation of each patientLs individual cumulative drug exposure. Blood samples were taken from all selected patients for laboratory parameters before liver biopsy to exclude viral, hereditary or autoimmune liver disease.
LIVER BIOPSY
· Slides were evaluated by a single pathologist (F. Charlotte), deemed adequate based on specimen size and number of portal tracts and scored according to the Metavir scoring system: no fibrosis, F0; fibrosis without septae, F1; fibrosis with some septae, F2; fibrosis with numerous septae, F3; cirrhosis, F4.
· Hepatic steatosis was classified on a 4-point scale: 0, none; 1, steatosis involving ≦ 5% of the hepatocytes; 2, 6%-30%; 3, >30%.
· Intralobular necrosis was graded 0 (absent) to 3 (1, less than one; 2, one or two; 3, more than two hepatocyte necrosis injuries per lobule). Hepatocyte ballooning was graded 0 (absent) to 2 (1, ballooned hepatocytes in less than 50% of lobules; 2, ballooned hepatocytes in more than 50% of lobules).
NASH was defined by the presence of steatosis > 10% associated with lobular necrosis (grade 1 or more) and/or hepatocyte ballooning (grade 1 or more), with or without fibrosis (grade 1 or more).
STUDY POPULATION
Between July 2003 and march 2007, 33 HIV-positive patients were eligible to undergo liver biopsy.
Three patients were excluded from further observation:
· In one case, liver biopsy showed zones of centrolobular hepatocytic atrophy with regenerative hepatocytic activity consistent with regenerative nodular hyperplasia.
· A second patient retrospectively admitted chronic alcohol consumption.
· The third patient had a history of several cholestatic infections and was found by chart review to have HIV-associated cholangiopathy.
MITOCHONDRIAL ANALYSIS
19 HIV-infected patients were compared to a control group for the mitochondrial analyses.
Controls consisted in a group of patients with no liver disease who underwent hepatic resection for liver metastasis. Normal liver sample was taken during surgery for the mitochondrial analyses and were assessed using the same procedure as in HIV infected patients. All the controls had normal liver histological examination.
A fragment of the liver biopsy was snap frozen in liquid nitrogen and kept at 80C before use. A part of the fragment was used for spectrophometric assays of the mitochondrial activity on liver homogenates. They included the activities of respiratory chain complex II (CII) and citrate synthase (CS) and the activities of respiratory chain complexes I and IV (CI and CIV)
Another part of the fragment was used for an extraction of total liver DNA using a routine procedure based on SDS-proteinase K digestion, followed by isopropanol precipitation. The amounts of nuclear and mitochondrial DNA were quantified by real time PCR using LightCycler FastStart Reaction SYBR Green I kit (Roche). The amount of nuclear DNA was quantified by amplification of a fragment of the 28S ribosomal gene using as standard serial dilutions of total DNA from control fibroblasts while the amount of mitochondrial DNA was quantified by amplification of a fragment of the 12S ribosomal gene using as standard serial dilutions of a linearized plasmid containing the entire 12S gene as an insert.
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