Julia C. Iezzoni

A pilot study of a thiazolidinedione, troglitazone, in nonalcoholic steatohepatitis

OBJECTIVES:Troglitazone is a thiazolidinedione and peroxisome proliferator–activated receptor gamma (PPARγ) ligand used to treat diabetes mellitus type II. Because hyperinsulinemia may be a factor in nonalcoholic steatohepatitis (NASH), we postulated that troglitazone could have beneficial effects in this disorder. Our study was initiated before reports of idiosyncratic hepatitis induced by this agent and was completed before its recent withdrawal from the market.METHODS:We studied 10 female patients (age 44 ± 16) with histological NASH. All but two were obese (mean body mass index, BMI = 38 ± 6). One had type 2 diabetes, and three had well-compensated cirrhosis with NASH. Troglitazone was given at a dose of 400 mg/day for ≤6 months. Responders (defined as normal ALT at the end of treatment) were rebiopsied. Paired specimens were compared in blinded fashion. Mitochondria were quantitated using ultrathin electron microscopy.RESULTS:Seven of ten patients responded with normal ALT at the end of treatment. One of three nonresponders initially normalized ALT but returned to pretreatment level at 3 months. In this patient, therapy was stopped, and the ALT has remained at the baseline level with no other clinical or laboratory findings. In the responders, ALT fell from 87 ± 38 before to 39 ± 9 at the end of treatment (p = 0.01), and AST decreased from 77 ± 23 to 30 ± 8 (p = 0.002). Biopsy comparisons before and after therapy showed persistent steatohepatitis in all cases, although four of seven showed a one-point improvement in the necroinflammatory grade. Electron microscopy revealed elongation of the mitochondria after therapy.CONCLUSIONS:Normal ALT was seen in 70% of NASH patients at the end of treatment, but this biochemical response was associated with only mild histological improvement, and all follow-up biopsies had evidence of NASH. Normalization of the liver enzymes in patients with NASH who are treated with thiazolidinediones should be viewed with reservation. Follow-up biopsy is essential to evaluate the efficacy of these agents, which, at the histological level, appears to be relatively modest.

Mitochondrial abnormalities in non-alcoholic steatohepatitis

BACKGROUND/AIMS We assessed mitochondrial morphology by electron microscopy and the prevalence of a mitochondrial gene deletion in patients with non-alcoholic steatohepatitis (NASH), alcohol-related liver disease and non-fatty liver diseases. Respiratory chain function using a cytoplasmic hybrid (cybrid) assay was further studied in NASH patients and healthy controls. METHODS Electron microscopy was performed in 26 specimens. Fifteen patients were studied by polymerase chain reaction to detect a 520-bp deletion product of the mitochondrial genome (dmtDNA). Cybrids were created by fusion of platelets with anaerobic neuroblastoma cells in six NASH patients and 12 controls. RESULTS Eight of ten NASH, one of seven alcoholics and two of nine other patients had linear crystalline inclusions in megamitochondria (p<0.05). Three of five patients with alcohol-related liver disease had dmtDNA compared to one of five NASH patients and one of five non-steatohepatitis controls. Cybrid respiratory chain function in platelets was not different from that of controls. CONCLUSIONS Respiratory chain dysfunction, if present in NASH, is not expressed in platelet-derived mitochondria. In contrast to alcohol-related liver disease with active drinking, NASH patients do not commonly express the 5-kb mitochondrial DNA gene deletion in liver tissue. As previously described in early alcohol-related liver disease, crystalline inclusions of unknown composition are seen in hepatic mitochondria in NASH. Their presence suggests either an adaptive process or mitochondrial injury.

The zonal distribution of megamitochondria with crystalline inclusions in nonalcoholic steatohepatitis

Megamitochondria with crystalline inclusions (MMC) have been previously described in nonalcoholic fatty liver; however, their distribution within hepatic zones is unknown. We sought to determine this distribution from the core liver biopsy specimens of 31 patients: 8 males and 23 females, age range 21 to 72 years. Twenty‐nine showed evidence of nonalcoholic steatohepatitis (NASH) on biopsy with steatosis, inflammation, varying degree of fibrosis, ballooned hepatocytes, and Mallory hyaline, and two patients had cryptogenic cirrhosis thought to represent “burned out” NASH. Identified by transmission electron microscopy, the abundance of MMC was compared between low‐stage (fibrosis stages 1 and 2) and high‐stage (fibrosis stages 3 and 4) groups and between zones with or without difference in fibrosis stage. Regardless of stage, the MMC were distributed equally in all zones and were abundant similarly in low‐ and high‐stage groups. This abundance did not correlate with the degree of oxidative stress (4‐hydroxynonenal staining) or with the abundance of ballooned hepatocytes. Consistent with age as a risk factor for more severe disease, the median age for the low‐stage group was significantly lower than that of the high‐stage group (P = .003). In conclusion, in NASH, the MMC seem to be distributed randomly among zones and without variation in abundance, regardless of the fibrosis stage. The exact function of these structures remains to be defined. In this study, their presence did not seem to correlate with the light microscopic injury pattern represented by ballooned hepatocytes or degree of oxidative stress defined by immunostaining for 4‐hydroxynonenal. (HEPATOLOGY 2004;39:1423–1429.)

Chronically inflamed livers up-regulate expression of inhibitory B7 family members.

Hepatitis B virus, hepatitis C virus, autoimmune hepatitis, and nonalcoholic fatty liver disease can induce chronic liver disease. The Programmed Death‐1 (PD‐1) inhibitory pathway assists in T cell response regulation during acute and chronic inflammation and participates in the progression of inflammatory liver disease. To examine whether PD‐1 and its ligands, B7‐H1 and B7‐DC, are modulated during chronic necroinflammatory liver disease, we investigated expression profiles in normal patients and patients with the aforementioned conditions. Relative to liver biopsies from normal individuals, those from patients with chronic necroinflammatory liver diseases (hepatitis B virus, hepatitis C virus, and autoimmune hepatitis) contain increased numbers of PD‐1–expressing lymphocytes. Kupffer cells, liver sinusoidal endothelial cells, and leukocytes express PD‐1 ligands. We also detect PD‐1 ligands on hepatocytes within biopsies and on isolated cells. All forms of chronic necroinflammatory liver disease examined correlate with increased B7‐H1 and B7‐DC expression on Kupffer cells, liver sinusoidal epithelial cells, and leukocytes. The degree of necroinflammation correlates with expression levels of PD‐1 family members. Conclusion: These results demonstrate that expression of PD‐1/PD‐1 ligands links more directly with the degree of inflammation than with the underlying etiology of liver damage. The PD‐1 pathway may assist the liver in protecting itself from immune‐mediated destruction. (HEPATOLOGY 2009.)

Has natural selection in human populations produced two types of metabolic syndrome (with and without fatty liver)

Fatty liver is closely related to the development of the insulin resistance syndrome that largely results from abnormal insulin signaling in three major organs: (i) skeletal muscle in which insulin sensitivity depends on fat content and metabolic activity (exercise); (ii) adipose tissue, which serves as a reservoir of energy in the form of triglycerides; and (iii) the liver, which variably serves as a source or storage site of carbohydrates and lipids. In many respects, the fatty liver resembles a mixture of brown adipose tissue (microvesicular steatosis) and white adipose tissue (macrovesicular steatosis) including the stages of fatty droplet accumulation, and the expression of uncoupling proteins and perilipin‐like substances. Furthermore, the development of an inflammatory infiltrate and the increased production of cytokines as occurs in adipose tissue, suggest that the liver in some individuals serves as an extension of adipose tissue. Moreover, current evidence indicates that these morphological changes represent altered gene expression similar to that of adipocytes. However, fatty liver does not appear to be a uniform feature of the metabolic syndrome and there is substantial variation in humans in the development of fatty liver independent of insulin resistance. In this regard, the variable development of fatty liver in Palmipedes (migratory fowl) and its close relationship to skeletal muscle utilization of fatty acids, lipoprotein metabolism and thermoregulation are instructive. The predilection to non‐alcoholic fatty liver disease among some varieties of Palmipedes suggests that the development of fatty liver represents an adaptive process, closely integrated with skeletal muscle fat utilization and adipose tissue distribution, and facilitates survival in a very cold, resource‐scarce environment. Variation in human populations with metabolic syndrome likewise suggests that the trait evolved in populations exposed in ancient times to different environmental challenges and, because the liver plays a central role in lipid metabolism, the presence or absence of fatty liver is likely to be integrated with insulin sensitivity in other target organs and with lipoprotein metabolism.

E2 Quasispecies Specificity of Hepatitis C Virus Association With Allografts Immediately After Liver Transplantation

It is unknown whether all hepatitis C virus (HCV) quasispecies variants found within patient serum have equal capacity to associate with the liver after transplantation; however, in vitro models of HCV infection suggest that variations in the hypervariable region 1 (HVR1) of the second envelope protein (E2) may be important in infectivity. The hypothesis of the current study is that the two hypervariable regions (HVR1 and HVR2) within E2 are important in the initial virus–liver interaction, and, therefore, certain HCV quasispecies variants will be isolated from the liver after reperfusion. In 8 patients with end‐stage liver disease secondary to HCV infection, HCV envelope quasispecies were determined from intraoperative serum samples obtained before the anhepatic phase of transplantation and from liver biopsies 1.5 to 2.5 hours after the transplanted liver was perfused. Explanted (native) liver biopsies were taken as a control. Sequence analysis was performed on clones of specific HCV reverse transcriptase‐polymerase chain reaction products spanning HVR1 and HVR2 of the E2 protein. HVR1 was more variable than HVR2 for all samples. Quasispecies isolated from postperfusion liver differed more from serum than did explanted liver quasispecies at HVR1 (P = 0.03) but not at HVR2 (P = 0.2). Comparison of HVR1 sequences from postperfusion liver versus serum revealed significantly less HVR1 genetic complexity and diversity (P = 0.02 and P = 0.04, respectively). Immediately after transplantation but before actual infection, liver allografts select out from the infecting serum inoculum a less heterogeneous, more closely related population of quasispecies variants. (Liver Transpl 2004;10:208–216.)

Troglitazone-associated hepatic failure.

though no signs of breast cancer recurrence were observed during the follow-up period, a HCC recurred in S6 and the patient died of deteriorated liver function. Hormonal imbalances, including increased estrogens and decreased testosterone, are postulated to play an important etiological role in the development of breast cancer in men (1–3). Although a hormonal evaluation was not performed in our patient with liver cirrhosis, the existence of gynecomastia strongly suggested an underlying hormonal imbalance. Liver cirrhosis is considered to be a risk factor for male breast cancer (1, 2). However, very few cases of male breast cancer have been documented in cirrhotics. Specifically, to our knowledge, no case of breast cancer in a male cirrhotic patient with HCC has been reported. We believe this to be the first reported case of male breast cancer developed in a patient with liver cirrhosis, complicated with HCC.

Nonneoplastic Endometrial Signet-Ring Cells Vacuolated Decidual Cells and Stromal Histiocytes Mimicking Adenocarcinoma

We describe 5 patients (mean age, 50 years; all had uterine bleeding) whose routine endometrial biopsy and curettage specimens contained prominent signet-ring cells. Each specimen contained loose aggregates of signet-ring cells scattered within the endometrial stroma that were characterized by peripherally displaced, small, uniform nuclei with indistinct nucleoli and showed no mitotic activity. The central portion of the cytoplasm was occupied by single or multiple cytoplasmic vacuoles. In all cases, the signet-ring cells were reactive for vimentin and negative for epithelial membrane antigen and cytokeratin. Four cases were focally positive for muscle-specific actin or smooth muscle actin and negative for CD68, Mac387, periodic acid-Schiff, mucicarmine, and alcian blue. In these 4 cases, the surrounding endometrial stroma showed decidual changes, and the signet-ring cells demonstrated a morphologic continuum with more typical decidualized stroma. As such, the signet-ring cells in these cases were vacuolated, decidualized endometrial stromal cells. In the remaining case, the vacuolar contents of the signet-ring cells were periodic acid-Schiff-positive and resistant to diastase predigestion, and the cells reacted with Mac387 and CD68. The surrounding stroma showed no decidual reaction. Thus, the signet-ring cells in this case were of histiocytic differentiation. Endometrial stroma occasionally may contain nonneoplastic signet-ring cells that closely mimic adenocarcinoma. At least 2 directions of differentiation, decidual and histiocytic, are possible.

Thiazolidinediones for the Treatment in Nash: Sustained Benefit After Drug Discontinuation?

Background Although of unproven benefit for nonalcoholic steatohepatitis (NASH), thiazolidinediones (TZDs) have emerged as a promising therapy. Little evidence exists, however, regarding sustained effects of TZDs in NASH after drug discontinuation. A recent clinical study suggests that relapse of NASH pathophysiology is inevitable and that lifelong therapy may be needed to maintain histologic response. Goal We reviewed extended follow-up data of NASH patients previously treated with troglitazone to evaluate the influence of weight and physical activity on clinical and histologic parameters related to NASH recurrence. Study After medical record review, 9 of 10 patients had complete data for follow-up and 5 had an extended follow-up biopsy 3 years or more after discontinuing troglitazone therapy. Results In contrast to recent work showing relapse of NASH to be nearly universal after discontinuation of TZD therapy, 2 of our patients had 1-stage improvement in fibrosis, normal aminotransferases, and absence of diabetes after median follow-up of 37 months after discontinuation of troglitazone. Both patients had sustained exercise programs and interval body mass index reduction. In contrast, active steatohepatitis, progression of fibrosis, and requirement of antidiabetic medications were seen in patients unable to achieve lifestyle modifications. Conclusions We conclude that sustained histologic response after short-term TZD therapy for NASH is not invariably lost after medication discontinuation but rather is intimately related to sustained changes in lifestyle, particularly physical activity. Activity and diet in the setting of thiazolidinedione or other drug therapy of NASH is an essential consideration that warrants careful incorporation into future drug trials.

CASE REPORT: Marked Transaminase Elevation in Anorexia Nervosa

Anorexia nervosa, a syndrome most commonly affecting young women, is characterized by voluntary weight loss of at least 25% of body weight, distorted body image, and fear of becoming obese (1). Its mortality of up to 9% is one of the highest among the psychiatric disorders (2). Many organ systems are involved, resulting in amenorrhea, bradycardia, hypotension, hirsutism, skin dryness, impaired renal concentration, and decreased immunoglobulins, complement and endocrine hormones (3, 4). Constipation is a common gastrointestinal manifestation (5), but a long list of uncommon complications have been reported (6–17). Liver involvement, although common in other forms of protein-energy malnutrition such as marasmus and kwashiorkor (18– 24), has been infrequently reported in anorexia nervosa (3–5, 26–34) in the Americas and Europe. We report the case of a young woman with anorexia nervosa who presented with marked and persistent transaminase elevation in the absence of other identifiable factors, which normalized solely with improved nutrition and weight gain.