Deborah Casson

Ex vivo analysis of human memory CD4 T cells specific for hepatitis C virus using MHC class II tetramers

Containment of hepatitis C virus (HCV) and other chronic human viral infections is associated with persistence of virus-specific CD4 T cells, but ex vivo characterization of circulating CD4 T cells has not been achieved. To further define the phenotype and function of these cells, we developed a novel approach for the generation of tetrameric forms of MHC class II/peptide complexes that is based on the cellular peptide-exchange mechanism. HLA-DR molecules were expressed as precursors with a covalently linked CLIP peptide, which could be efficiently exchanged with viral peptides following linker cleavage. In subjects who spontaneously resolved HCV viremia, but not in those with chronic progressive infection, HCV tetramer-labeled cells could be isolated by magnetic bead capture despite very low frequencies (1:1,200 to 1:111,000) among circulating CD4 T cells. These T cells expressed a set of surface receptors (CCR7+CD45RA-CD27+) indicative of a surveillance function for secondary lymphoid structures and had undergone significant in vivo selection since they utilized a restricted Vbeta repertoire. These studies demonstrate a relationship between clinical outcome and the presence of circulating CD4 T cells directed against this virus. Moreover, they show that rare populations of memory CD4 T cells can be studied ex vivo in human diseases.

Screening for Wilson Disease in Acute Liver Failure: A Comparison of Currently Available Diagnostic Tests

Acute liver failure (ALF) due to Wilson disease (WD) is invariably fatal without emergency liver transplantation. Therefore, rapid diagnosis of WD should aid prompt transplant listing. To identify the best method for diagnosis of ALF due to WD (ALF‐WD), data and serum were collected from 140 ALF patients (16 with WD), 29 with other chronic liver diseases and 17 with treated chronic WD. Ceruloplasmin (Cp) was measured by both oxidase activity and nephelometry and serum copper levels by atomic absorption spectroscopy. In patients with ALF, a serum Cp <20 mg/dL by the oxidase method provided a diagnostic sensitivity of 21% and specificity of 84% while, by nephelometry, a sensitivity of 56% and specificity of 63%. Serum copper levels exceeded 200 μg/dL in all ALF‐WD patients measured (13/16), but were also elevated in non‐WD ALF. An alkaline phosphatase (AP) to total bilirubin (TB) ratio <4 yielded a sensitivity of 94%, specificity of 96%, and a likelihood ratio of 23 for diagnosing fulminant WD. In addition, an AST:ALT ratio >2.2 yielded a sensitivity of 94%, a specificity of 86%, and a likelihood ratio of 7 for diagnosing fulminant WD. Combining the tests provided a diagnostic sensitivity and specificity of 100%. Conclusion: Conventional WD testing utilizing serum ceruloplasmin and/or serum copper levels are less sensitive and specific in identifying patients with ALF‐WD than other available tests. More readily available laboratory tests including alkaline phosphatase, bilirubin and serum aminotransferases by contrast provides the most rapid and accurate method for diagnosis of ALF due to WD. (HEPATOLOGY 2008.)

Hepatitis C virus is independently associated with increased insulin resistance after liver transplantation.

Background and Aims. There is a strong epidemiologic association between diabetes mellitus (DM) and hepatitis C virus (HCV) infection. However, the pathogenetic basis for this association has not been established. We sought to evaluate the association between insulin resistance (IR), &bgr;-cell dysfunction, and HCV among orthotopic liver transplant (OLT) recipients. Methods. We performed a cross sectional analysis comparing 39 HCV(+) with 60 HCV(−) OLT recipients. IR and &bgr;-cell function were calculated using validated measures and were correlated with clinical variables. Results. By multivariate analysis of the entire cohort, HCV infection and body mass index (BMI) were independent predictors of IR (P =0.04 and 0.0006, respectively). HCV infection was associated with 35% increase in IR. Because the model used to calculate IR was derived from nondiabetic subjects, we performed additional analysis of patients who did not meet criteria for diabetes at the time of their study evaluation. In this analysis, HCV(+) subjects had greater fasting insulin and homeostasis model assessment (HOMA) IR (15.3 &mgr; U/mL and 3.8) compared with HCV(−) patients (10.7 &mgr; U/mL and 2.5) (P =0.03, 0.03). There was no difference in &bgr;-cell function or hepatic insulin extraction between the HCV (+) and (−) groups. HCV (P =0.0005), BMI (P <0.0001), and high high-density lipoprotein (P =0.039) were the only independent predictors of IR. The presence of HCV infection and a 10-fold increase in HCV RNA were associated with a 62% and 8% increase in IR, respectively. Conclusions. HCV is independently associated with increased IR after OLT. These findings provide a possible pathogenetic basis for the association of DM with HCV.

Full-Breadth Analysis of CD8+ T-Cell Responses in Acute Hepatitis C Virus Infection and Early Therapy

ABSTRACT Multispecific CD8+ T-cell responses are thought to be important for the control of acute hepatitis C virus (HCV) infection, but to date little information is actually available on the breadth of responses at early time points. Additionally, the influence of early therapy on these responses and their relationships to outcome are controversial. To investigate this issue, we performed comprehensive analysis of the breadth and frequencies of virus-specific CD8+ T-cell responses on the single epitope level in eight acutely infected individuals who were all started on early therapy. During the acute phase, responses against up to five peptides were identified. During therapy, CD8+ T-cell responses decreased rather than increased as virus was controlled, and no new specificities emerged. A sustained virological response following completion of treatment was independent of CD8+ T-cell responses, as well as CD4+ T-cell responses. Rapid recrudescence also occurred despite broad CD8+ T-cell responses. Importantly, in vivo suppression of CD3+ T cells using OKT3 in one subject did not result in recurrence of viremia. These data suggest that broad CD8+ T-cell responses alone may be insufficient to contain HCV replication, and also that early therapy is effective independent of such responses.

Prospective study of liver transplant recipients with HCV infection: evidence for a causal relationship between HCV and insulin resistance.

An association between hepatitis C virus (HCV) infection and insulin resistance (IR) has been recently reported. However, causality has not been established. The cross‐sectional nature of most reported studies and varying degrees of fibrosis have limited definitive conclusions about the independent role of HCV in development of IR. We sought to evaluate whether HCV induces IR by prospectively analyzing a cohort of adult liver transplant (LT) recipients. A total of 34 adults (14 HCV(+) and 20 HCV(−)) who underwent consecutive LT were followed during the first year posttransplantation. IR was estimated using the homeostasis model assessment (HOMA). Univariate and multivariate repeated measures analyses and Cox regression models were used. There were no significant differences between the groups with respect to age, body mass index (BMI), family history of diabetes, alcohol consumption, or laboratory indices. The cohort had no or minimal fibrosis. There was lower prednisone use in the HCV(+) group, and no difference in the use of tacrolimus between the two groups was found. IR was 77% higher in HCV(+) subjects during the first year post‐LT when controlling for BMI (P = 0.035). Subjects with high HCV ribonucleic acid (RNA) levels reached high HOMA‐IR significantly earlier than those with lower HCV RNA (P = 0.03). Following the first month post‐LT, HCV(+) subjects were 4 times more likely to become diabetic than HCV(−) controls (P < 0.01). In conclusion, there is significantly higher IR in the HCV(+) group during the first year post‐LT. This cannot be explained by differences in BMI, medications used, alcohol consumption, or degree of fibrosis. Higher HCV RNA levels were associated with earlier elevations in HOMA‐IR. Collectively, these results provide strong evidence that HCV induces the development of IR. Liver Transpl, 2007.

Elevated troponin I levels in acute liver failure: Is myocardial injury an integral part of acute liver failure?

Although rare instances of cardiac injury or arrhythmias have been reported in acute liver failure (ALF), overall, the heart is considered to be spared in this condition. Troponin I, a sensitive and specific marker of myocardial injury, may be elevated in patients with sepsis and acute stroke without underlying acute coronary syndrome, indicating unrecognized cardiac injury in these settings. We sought to determine whether subclinical cardiac injury might also occur in acute liver failure. Serum troponin I levels were measured in 187 patients enrolled in the US Acute Liver Failure Study Group registry, and correlated with clinical variables and outcomes. Diagnoses were representative of the larger group of >1000 patients thus far enrolled and included 80 with acetaminophen‐related injury, 26 with viral hepatitis, 19 with ischemic injury, and 62 others. Overall, 74% of patients had elevated troponin I levels (>0.1 ng/ml). Patients with elevated troponin I levels were more likely to have advanced hepatic coma (grades III or IV) or to die (for troponin I levels >0.1 ng/ml, odds ratio 3.88 and 4.69 for advanced coma or death, respectively). Conclusion: In acute liver failure, subclinical myocardial injury appears to occur more commonly than has been recognized, and its pathogenesis in the context of acute liver failure is unclear. Elevated troponin levels are associated with a significant increase in morbidity and mortality. Measurement of troponin I levels may be helpful in patients with acute liver failure, to detect unrecognized myocardial damage and as a marker of unfavorable outcome. (HEPATOLOGY 2007;45:1489–1495.)

Spontaneous resolution of chronic hepatitis C virus disease after withdrawal of immunosuppression

Approximately 85% of acute cases of hepatitis C infection result in chronic hepatitis. Spontaneous clearance of hepatitis C virus has been thought to occur exclusively after acute infection and is associated with a robust cellular immune response. We describe here a case of a renal transplant recipient who acquired posttransplant hepatitis C virus infection with rapid histological progression but who subsequently experienced spontaneous viral clearance along with histological remission after removal of immunosuppression. Immunologic studies showed persistently strong cellular immune responses. This case underscores the importance of restoration of the immune system in the control of hepatitis C virus viremia and disease progression and the need to minimize or obviate immunosuppression in organ transplant recipients.

Predictive value of actin‐free Gc‐globulin in acute liver failure

Serum concentrations of the actin scavenger Gc‐globulin may provide prognostic information in acute liver failure (ALF). The fraction of Gc‐globulin not bound to actin is postulated to represent a better marker than total Gc‐globulin but has been difficult to measure. We tested a new rapid assay for actin‐free Gc‐globulin to determine its prognostic value when compared with the Kings College Hospital (KCH) criteria in a large number of patients with ALF. A total of 252 patients with varying etiologies from the U.S. ALF Study Group registry were included; the first 178 patients constituted the learning set, and the last 74 patients served as the validation set. Actin‐free Gc‐globulin was determined with a commercial enzyme‐linked immunosorbent assay kit. The median (range) actin‐free Gc‐globulin level at admission for the learning set was significantly reduced compared with controls (47 [0‐183] mg/L vs. 204 [101‐365] mg/L, respectively, P < 0.001). Gc‐globulin levels were significantly higher in spontaneous survivors than in patients who died or were transplanted (53 [0‐129] mg/L vs. 37 [0‐183] mg/L, P = 0.002). A receiver operating characteristic curve analysis showed that a 40 mg/L cutoff level carried the best prognostic information, yielding positive and negative predictive values of 68% and 67%, respectively, in the validation set. The corresponding figures for the KCH criteria were 72% and 64%. A new enzyme‐linked immunosorbent assay for actin‐free Gc‐globulin provides the same (but not optimal) prognostic information as KCH criteria in a single measurement at admission. Liver Transpl 13:1324–1329, 2007.

Pilot study of postexposure prophylaxis for hepatitis C virus in healthcare workers.

BACKGROUND AND OBJECTIVE Hepatitis C virus (HCV) transmission occurs in 0.2%-10% of people after accidental needlestick exposures. However, postexposure prophylaxis is not currently recommended. We sought to determine the safety, tolerability, and acceptance of postexposure prophylaxis with peginterferon alfa-2b in healthcare workers (HCWs) exposed to blood from HCV-infected patients. DESIGN Open-label pilot trial of peginterferon alfa-2b for HCV postexposure prophylaxis. SETTING Two academic tertiary-referral centers. METHODS HCWs exposed to blood from HCV-infected patients were informed of the availability of postexposure prophylaxis. Persons who elected postexposure prophylaxis were given weekly doses of peginterferon alfa-2b for 4 weeks. RESULTS Among 2,702 HCWs identified with potential exposures to bloodborne pathogens, 213 (7.9%) were exposed to an HCV antibody-positive source. Of 51 HCWs who enrolled in the study, 44 (86%) elected to undergo postexposure prophylaxis (treated group). Seven subjects elected not to undergo postexposure prophylaxis (untreated group). No cases of HCV transmission were observed in either the treated or untreated group, and no cases occurred in the remaining 162 HCWs who did not enroll in this study. No serious adverse events related to a peginterferon alfa-2b regimen were recorded, but minor adverse events were frequent. CONCLUSION In this pilot study, there was a lower than expected frequency of HCV transmission after accidental occupational exposure. Although peginterferon alfa-2b was safe, because of the lack of HCV transmission in either the treated or untreated groups there is little evidence to support routine postexposure prophylaxis against HCV in HCWs.

The role of etiology in the hyperamylasemia of acute liver failure.

OBJECTIVES:Hyperamylasemia (HA) is often reported in patients with acute liver failure (ALF). Direct toxic effects of acetaminophen on the pancreas have been postulated, but the occurrence of HA in other etiologies raises the question of whether multiorgan failure is part of the pathogenesis of HA in this setting. Our main aim was to describe and analyze the incidence, clinical characteristics, and outcomes of HA in ALF of different etiologies.METHODS:Patients enrolled in the Acute Liver Failure Study Group registry with an admission amylase value available were included. For the purpose of this analysis, HA was defined as ≥3× upper limits of normal. Patients were classified as having acetaminophen (APAP)- or non-APAP-induced ALF, and by amylase group: normal (<115), mildly elevated (115–345), or HA (>345). Significant variables identified by univariate analysis were added to a multiple linear regression model. The primary outcome was overall survival.RESULTS:In total, 622 eligible patients were identified in the database, including 287 (46%) with APAP-induced ALF; 76 (12%) patients met the criteria for HA. Among patients with HA, 7 (9%) had documented clinical pancreatitis. The incidence of HA was similar among APAP (13%) and non-APAP (12%) patients. Although HA was associated with renal failure and greater Model for End-stage Liver Disease scores for both groups, HA was not an independent predictor of mortality in multivariate analysis.CONCLUSIONS:Although not an independent predictor of mortality, HA in ALF was present in all etiologies and was associated with diminished overall survival. HA appeared to be related to renal dysfunction in both groups and multiorgan failure in non-APAP ALF.