Adam Terella

Viral RNA Mutations Are Region Specific and Increased by Ribavirin in a Full-Length Hepatitis C Virus Replication System

ABSTRACT High rates of genetic variation ensure the survival of RNA viruses. Although this variation is thought to result from error-prone replication, RNA viruses must also maintain highly conserved genomic segments. A balance between conserved and variable viral elements is especially important in order for viruses to avoid “error catastrophe.” Ribavirin has been shown to induce error catastrophe in other RNA viruses. We therefore used a novel hepatitis C virus (HCV) replication system to determine relative mutation frequencies in variable and conserved regions of the HCV genome, and we further evaluated these frequencies in response to ribavirin. We sequenced the 5′ untranslated region (5′ UTR) and the core, E2 HVR-1, NS5A, and NS5B regions of replicating HCV RNA isolated from cells transfected with a T7 polymerase-driven full-length HCV cDNA plasmid containing a cis-acting hepatitis delta virus ribozyme to control 3′ cleavage. We found quasispecies in the E2 HVR-1 and NS5B regions of untreated replicating viral RNAs but not in conserved 5′ UTR, core, or NS5A regions, demonstrating that important cis elements regulate mutation rates within specific viral segments. Neither T7-driven replication nor sequencing artifacts produced these nucleotide substitutions in control experiments. Ribavirin broadly increased error generation, especially in otherwise invariant regions, indicating that it acts as an HCV RNA mutagen in vivo. Similar results were obtained in hepatocyte-derived cell lines. These results demonstrate the potential utility of our system for the study of intrinsic factors regulating genetic variation in HCV. Our results further suggest that ribavirin acts clinically by promoting nonviable HCV RNA mutation rates. Finally, the latter result suggests that our replication model may be useful for identifying agents capable of driving replicating virus into error catastrophe.

Liver transplantation outcomes for early‐stage hepatocellular carcinoma: Results of a multicenter study

The incidence of hepatocellular carcinoma (HCC), a frequent and incurable complication of cirrhosis, continues to rise. Orthotopic liver transplantation (OLT) has been proposed as a treatment for unresectable, intrahepatic HCC limited in extent to the Milan criteria adopted by the United Network of Organ Sharing (UNOS) in 1998. More recently, somewhat less restrictive University of California, San Francisco (UCSF) 10 , criteria were proposed. To examine the long‐term outcomes of OLT for HCC patients and to assess the UNOS policy of assigning weighted allocation points to patients with HCC, we retrospectively analyzed 144 patients (113 after 1998) with HCC who underwent OLT over an 11‐year period at 3 institutions from UNOS Region 1. We compared their outcomes with 525 patients (272 after 1998) who underwent OLT for nonmalignant liver disease. The 1‐ and 5‐year survival rates were 80.3% and 46.7%, respectively, for patients with HCC and 81.5% and 70.6%, respectively, for patients without HCC (P = .020). However, there was no difference in survival between HCC and non‐HCC patients after implementation of disease‐specific allocation for HCC in 1998. A higher proportion of the HCC cohort was older and male and had chronic HCV infection and alcoholic liver disease. In univariate analysis, having alpha‐fetoprotein (AFP) levels of 10 ng/mL or less and meeting clinical and pathologic UCSF criteria were each significant predictors of improved survival (P = .005, P = .02, and P = .03, respectively). AFP greater than 10 ng/mL and exceeding pathologic UCSF criteria were also significant predictors of recurrence (P = .003 and P = .02, respectively). In conclusion, taken together, our data suggest that OLT is an acceptable option for patients with early HCC and that UCSF criteria predict outcome better than Milan or UNOS criteria. Regardless of which criteria are adopted to define eligibility, strict adherence to the criteria is important to achieve acceptable outcomes. (Liver Transpl 2004;10:1343–1354.)

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.

Multiple head injuries in rats: Effects on behavior

BACKGROUND Evidence suggests that mild head injuries in humans can result in cumulative damage. No investigation to date has considered the effects of multiple subacute mild head injuries in an animal model. METHODS Forty-one male Long-Evans hooded rats were trained in a Morris water maze. All animals were fitted with a hollow intracranial screw. Concussions were generated using a fluid percussion device. Animals were then evaluated in the water maze until performance returned to baseline. Control animals received no concussions. The remaining animals were randomized to receive one, two, or three concussions. Animals were allowed to return to baseline after each concussion and were then killed. Motor performance was evaluated on a balance beam both before and after concussions. RESULTS After one concussion, 85% of animals showed performance deviation from baseline as measured by time to reach the platform, returning to baseline within a mean of 14.0 trials. After two concussions, 48% of animals showed deviation, with a mean return to baseline of 6.8 trials. After three concussions, 25% of animals showed deviation, with a mean return to baseline of 2.3 trials. Of postconcussive animals, 42% developed new inconsistent baseline levels of performance. Balance beam performance was unaffected. CONCLUSION Multiple concussions cause immediate transient impairment in spatial recognition and have extended effects on baseline performance in rats. Motor performance is not affected.

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.

Repair of a calvarial defect with biofactor and stem cell-embedded polyethylene glycol scaffold.

OBJECTIVE Segmental bony defects resulting from congenital facial anomalies, facial trauma, infection, or oncologic surgical resection represent a common and significant clinical problem. Currently, these defects are reconstructed with autologous or allogeneic bone grafts or prosthetic devices. These options are limited by bone supply for grafting, donor site morbidity, risk of infection, and extrusion. This study investigated the in vivo osteogenic capability of polyethylene glycol-diacrylate (PEG-DA) and a protease-sensitive PEG matrix metalloproteinases (PEG-MMP), photoencapsulated with mesenchymal stem cells (MSCs) and bone morphogenetic protein (BMP)-2, in healing a critical-size rat calvarial defect. METHODS Both PEG-DA and PEG-MMP scaffolds photoencapsulated with rat MSCs (rMSCs) and/or BMP-2 were implanted into a critical-size defect. Microcomputed-tomographic (micro-CT) analysis was completed 1, 4, and 8 weeks after implantation. Bone growth was histologically evaluated. The micro-CT data were analyzed using ASPIProVM software to calculate the percentage of closure of cranial defects. RESULTS Both PEG-MMP and PEG-MMP + BMP2 showed significantly enhanced bone compared with controls. Polyethylene glycol-diacrylate seemed to inhibit bone growth regardless of biofactor and rMSCs. The addition of rMSCs did not enhance bone regeneration. CONCLUSION Polyethylene glycol sensitive to proteolysis significantly improved bone repair in a critical-size calvarial defect.

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.

Complications in Periorbital Surgery

Comprehensive rejuvenation of the periorbital region commonly involves management of the brow, as well as the upper and lower eyelids. Browlifting, upper and lower blepharoplasty, fat transfer, and neuromodulators are frequently utilized with excellent results. However, surgery in this region can be fraught with potential complications ranging from a poor cosmetic outcome to orbital hematoma and vision loss. Although avoidance of complications is preferred, it is incumbent on the surgeon to have a detailed understanding of the pathophysiology, prevention, and management of these complications. The authors examine the more common complications of periorbital surgery.

Plexiform fibrohistiocytic tumor: ultrastructural studies may aid in discrimination from cellular neurothekeoma.

ABSTRACT Plexiform fibrohistiocytic tumor is a low-grade soft tissue malignancy that can at times be difficult to differentiate from the less biologically aggressive cellular neurothekeoma. The two entities, which may display identical clinical and histological features, cannot be distinguished by immunohistochemical or molecular diagnostic means. Electron microscopy may enable the accurate identification of problematic examples and thus aid in resolving these occasionally occurring diagnostic dilemmas. To illustrate typical variations in the ultrastructural appearance of plexiform fibrohistiocytic tumor, the authors present two diagnostically noncontroversial examples, and to demonstrate the potential diagnostic utility of electron microscopy in this setting, they present an example of plexiform fibrohistiocytic tumor that could not otherwise have been distinguished from cellular neurothekeoma.

Recurrent allograft HCV presenting as acute cellular rejection: successful management with interferon and ribavirin alone

Abstract:  Hepatitis C virus (HCV) is currently the leading indication worldwide for orthotopic liver transplantation. However, the majority of patients receiving transplant for HCV eventually develop histopathologic evidence of recurrent allograft HCV and approximately 10% die or require retransplantation within the first 5 post‐operative years because of accelerated graft injury and cirrhosis. Traditional induction immunosuppressive regimens and intensive immunosuppression used to treat episodes of acute cellular rejection (ACR) are associated with enhanced viral replication and higher likelihood and severity of recurrent HCV. At our institution, therefore, we have used low‐dose steroid therapy in an effort to limit HCV replication. However, this practice has been associated with frequent early presentations consistent with ACR. Here, we present three cases consistent with histologic ACR treated with conventional antirejection therapy that improved transiently, but evolved rapidly to progressive HCV. A fourth patient with a similar presentation experienced dramatic improvement in aminotransferases when treated solely with interferon and ribavirin. We propose that histologic characteristics traditionally associated with ACR may, in fact, represent early recurrent HCV as both processes share common immunopathogenetic mechanisms, or alternatively, that both ACR and recurrent HCV may be present simultaneously. We conclude that in cases suggestive of ACR, careful consideration should be given to treatment for recurrent HCV in lieu of or in concert with intensive immunosuppression.