Progression of Fibrosis during Chronic Hepatitis C Is Associated with Rapid Virus Evolution

ABSTRACT Hepatic fibrosis is the primary mediator of disease due to chronic infection with hepatitis C virus (HCV). HCV exists as a quasispecies in each infected individual, and longitudinal viral sequence changes may reveal viral dynamics and the selection pressures applied by the host immune system. Thus, we hypothesized that patterns of sequence change might reveal the immunopathogenesis of fibrosis progression. We tested this hypothesis by studying individuals enrolled in a prospective study of chronic HCV-related hepatic fibrosis with little or no fibrosis at first biopsy (stage 0 or 1) and a second planned liver biopsy sample obtained 4 years later. Serum was obtained from five individuals with fast progression (FP; defined as a >2-stage change between visits) and 10 carefully matched individuals with slow progression (SP; defined as a <2-stage change between visits). We sequenced multiple cloned hemigenomic cDNAs from each person spanning six genes (core through NS3). Phylogenetic analysis revealed temporal shifts in phylogenetic clustering over time, suggesting frequent quasispecies replacement rather than simple diversification. In addition, mixed infections were detected in three subjects, with coexistence in two subjects (one FP, one SP) of subtypes 1a and 1b throughout the 4-year biopsy interval. Subjects with FP had a higher rate of evolution than subjects with SP, with a preponderance of synonymous changes, suggesting purifying selection, except in hypervariable region 1, where positive selection pressure is frequently detected. Thus, in a small but carefully matched cohort we found evidence for rapid neutral evolution of HCV in persons with rapid progression of hepatic fibrosis, suggesting higher turnover of infected cells.

Autoantibodies against subunits of pyruvate dehydrogenase and citrate synthase in a case of paediatric biliary cirrhosis

In a newborn girl with a history of connatal liver damage, histological examination of a liver biopsy sample taken during the seventh week of life revealed incipient destruction of bile ducts. Very high titres of antimitochondrial antibodies were later detected in the plasma. As the hepatic injury tended towards fibrosis, the histological diagnosis became primary biliary cirrhosis. Autoantibodies against E1α, E2, and E3 subunits and protein X component of pyruvate dehydrogenase complex, and against citrate synthase were detected on western immunoblotting in a 1 in 1000 dilution of the patient’s serum. The patient died of her illness at 11 years of age. In liver specimens obtained at autopsy human immunoglobulin deposition was detected on the surface of almost all hepatic cells by immunohistology. As there is a physical and functional interaction between pyruvate dehydrogenase and citrate synthase within the mitochondria, the presence of autoantibodies against certain proteins in the patient suggests that in this form of the disease the molecular recognition and then the autoimmunisation process could be directed against a mitochondrial enzyme cluster containing both pyruvate dehydrogenase and citrate synthase.

Biopsy Mineral Analysis by Inductively Coupled Plasma—Atomic Emission Spectroscopy with Ultrasonic Nebulization

Liver copper concentration is generally considered the best measure of copper nutritional status in cattle. Ultrasonic nebulization in conjunction with inductively coupled plasma-atomic emission spectroscopy (ICP—AES) was investigated as a method to provide adequate sensitivity to allow accurate simultaneous measurement of copper and 14 additional elements from needle biopsy samples. The element concentration frequency distribution profile of 12 elements routinely present in liver was compared to profiles of the elements in fat, muscle, vena cava, kidney, and clotted blood. The profiles could be used to confirm the authenticity of the liver biopsy sample. Element concentrations in biopsy samples taken in triplicate from the five lobes of a bovine liver were compared to those from triplicate wedge sections taken adjacent to the biopsies and analyzed by conventional ICP—AES. Precision between biopsies was equal to or better than precision between wedge samples. Some element concentrations determined by the biopsy procedure differed statistically from those determined by the wedge procedure, but differences were not sufficient to influence clinical interpretation of data.