Severe hepatocellular injury with apoptosis induced by a hepatitis C polymerase inhibitor - PubMed (original) (raw)

Case Reports

Severe hepatocellular injury with apoptosis induced by a hepatitis C polymerase inhibitor

Ariel Feldstein et al. J Clin Gastroenterol. 2009 Apr.

Abstract

Goals: To describe the mechanisms of severe hepatocellular injury with apoptosis in 2 patients receiving hepatitis C virus (HCV)-796.

Background: HCV-796 is a hepatitis C polymerase inhibitor approved by the US Food and Drug Administration for a phase 2 study of the treatment of hepatitis C in combination with PEG-Interferon and ribavirin.

Results: The injury occurred after more than 12 weeks of treatment, with a >20-fold increase in serum alanine aminotransferase and aspartate aminotransferase, and a marked increase in total (and direct) bilirubin in the absence of cholestasis. There was no evidence of autoimmune or viral hepatitis. Involvement of the mitochondrial apoptotic pathway was demonstrated by (1) release of cytochrome C into the cytosol; (2) association of cytochrome C with apoptotic protease activating factor-1 in the cytosol; (3) activation of initiator caspase 9; (4) activation of effector caspase 3; (5) increased serum caspase-3 cleaved cytokeratin-18 peptide; (6) nuclear fragmentation; (7) mitochondrial structural abnormalities; (8) expression of light chain 3 B, an indicator of autophagy; (9) probable autophagy of mitochondria by autophagosomes; and (10) probable phagocytosis of apoptotic hepatocytes by activated macrophages. Immunoglobulin G immune complexes were identified in the hepatocytes and localized to the endoplasmic reticulum and Golgi of these patients after the drug-induced liver disease, reflecting a primary or secondary target. Hepatitis C treatment was discontinued at weeks 15 and 19 in patients 1 and 2, respectively. After more than 6 months off the medication, both patients normalized the serum alanine aminotransferase, aspartate aminotransferase, and total bilirubin with undetectable HCV RNA.

Conclusions: HCV-796 may cause severe hepatocellular injury and apoptosis, with a marked immune reaction in susceptible patients.

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Conflict of interest statement

Potential conflict of interest: None

Figures

Figure 1.. Abnormalities in ALT, AST, Bilirubin and Albumin after the DILD.

Increase in serum ALT, AST, and total bilirubin (TB) and decrease in serum albumin (ALB) in patient 1 (A) and patient 2 (B). The horizontal red lines indicate the normal range for the laboratory values and the vertical blue lines indicate the start (day 1) and end of the HCV treatment (day 132 for patient 1, and day 105 for patient 2). The negative days reflect time before start of treatment, and the positive days reflect time after start of treatment. The data for patient 2 are interrupted before day −40 to allow inclusion of data before start of treatment.

Figure 2.. Severe Hepatocellular Injury and Phagocytosis of Apoptotic Hepatocytes after the DILD.

(A) Hematoxillin and eosin stain of liver biopsies in patient 1 and patient 2 (200x). In both biopsies there is panacinar hepatocellular injury with hepatocyte dropout and fibrosis in the perivenular zone. The hepatocytes are ballooned, with irregularly clumped cytoplasm. There is marked lobular inflammation and interface hepatitis. The infiltrate is mainly lymphohistiocytic with rare plasma cells. No evidence of canalicular or hepatocellular cholestasis was observed. There was no significant macro- or micro-vesicular steatosis. (B) Significant monocytes / macrophage infiltrate was identified by the expression of CD-68 within the fibrous septa and the lobule in patient 1 and patient 2 (upper panels; 200x**),** at times engulfing apoptotic hepatocytes (lower panels; 1000x**)**. (C) The monocytes / macrophage infiltrate, identified by confocal microscopy by the expression of CD-68 (in green), was increased in patient 1 and patient 2 in the liver biopsies Post-DILD compared to the liver biopsies Pre-DILD. Nuclei were identified with TO-PRO 3 (in blue).

Figure 3.. Severe Liver Fibrosis and Activation of Hepatic Stellate Cells.

(A) Mallory’s trichrome and (200x) (B) Gomori’s reticulin stain (200x) of liver biopsies in patient 1 (left panels) and patient 2 (right panels). The Metavir stage for fibrosis was 3 (severe bridging fibrosis and portal-portal septae) in patient 1, and 4 (cirrhosis) in patient 2. Some sinusoidal fibrosis with minor collapse of the architecture is seen in (B). (C) There was a moderate degree of stellate cell activation, identified by the expression of α-SMA (in red; 100x) in both patients.

Figure 4.. Phagocytosis of Apoptotic Hepatocytes with Mitochondrial Swelling and Autophagy.

Transmission electron microscopy (TEM) was performed as described in Methods. Photographs are representative of findings in both patients. (A). Activated macrophages (M) were often observed engulfing hepatocytes (H), and in close proximity to activated stellate cells (HSC) (bar= 5μm). (B) In addition, TEM showed mitochondria changes from normal (left panel; bar= 1μm**)** to vesicular and swelling (right panel; (bar= 0.5μm**)** morphology. C). Occasional mitochondria autophagy by autophagosomes (bar= 1μm) was observed.

Figure 5.. Activation of Hepatocyte Mitochondrial Apoptotic Pathway after the DILD.

We assessed by confocal scanning microscopy the mitochondrial apoptotic pathway as described in Methods. (A) The cytochrome C was detected in the cytosol (in green) and associated with APAF-1 (in red), as indicated by the co-localization (yellow) in patient 1 and patient 2 but not in a control sample (250x). (B) Activation of the initiator caspase 9 (in green) was identified in patient 1 and patient 2 in the Post-DILD biopsies but not in the Pre-DILD biopsies. Nuclei were identified with TO-PRO 3 (in blue). Only background staining was observed when omitting the first antibody. Photographs are representative of findings in both patients (250x).

Figure 6.. Activation of Caspase 3 and Autophagy after the DILD

We assessed by confocal scanning microscopy the mitochondrial apoptotic pathway as described in Methods. (A) Active caspase 3 (in green) was detected in patient 1 and patient 2 but not in a control sample. Nuclei were identified with TO-PRO 3 (in blue) (250x). (B) Expression of the modulator of autophagy LC3B (in green) was detected Post-DILD but not Pre-DILD, in the liver of patient 1 and patient 2. Nuclei were identified with TO-PRO 3 (in blue). Only background staining was observed when omitting the first antibody. Photographs are representative of findings in both patients (250x).

Figure 7.. Immune Reaction in Hepatocytes after the DILD.

We assessed by confocal scanning microscopy the immune reaction as described in Methods. (A) IgG complexes (in green) were detected in the liver Post-DILD but not Pre-DILD in patient 1 and patient 2 (250x). (B) IgM complexes (in green) were detected in the liver after the DILD in patient 1 and patient 2, but not in a control liver. Nuclei were identified with TO-PRO 3 (in blue). Only background staining was observed when omitting the first antibody. Photographs are representative of findings in both patients (250x).

Figure 8.. The Immune Reaction was Targeted to the ER and Golgi in Hepatocytes after the DILD.

We assessed by confocal scanning microscopy the immune reaction as described in Methods. (A) The IgG complexes (in green) were detected in the hepatocytes and associated with an ER marker (in red), as indicated by the co-localization (yellow) Post-DILD but not Pre-DILD in patient 1 and patient 2 (250x). (B) The IgG complexes (in green) were detected in the hepatocytes after the DILD and associated with a Golgi marker (in red), as indicated by the co-localization (yellow) in patient 1 and patient 2, but not in a control liver. Nuclei are identified with TO-PRO-3 (blue). Only background staining was observed when omitting the first antibody. Photographs are representative of findings in both patients (250x).

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Severe hepatocellular injury with apoptosis induced by a hepatitis C polymerase inhibitor - PubMed (original) (raw)