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Timothy L Pruett

Publications and source records attributed to Timothy L Pruett.

At least 19 recordsLinked to original sources

Comparison of clinical outcomes in chronic hepatitis B liver transplant candidates with and without hepatocellular carcinoma.

Patients with hepatocellular carcinoma (HCC) receive a higher MELD score and may undergo liver transplantation (OLT) earlier compared to patients with cirrhosis, potentially decreasing waiting list mortality. However, post-OLT survival may be reduced by recurrence of HCC. We compared clinical outcomes between patients with HBV-cirrhosis and no HCC and patients with HBV-HCC. A total of 279 patients (HBV-cirrhosis = 183; HBV-HCC = 96) in the US HBV-OLT study were followed for a median of 30.2 months from listing. Patients with HCC were older, more likely to be Asian, and had less severe liver impairment than patients with HBV-cirrhosis. Despite a higher rate of OLT in patients with HCC (78.1% vs. 51.4%; P < 0.001), intention-to-treat (ITT) survival (73% vs. 78%) and survival without OLT (82% vs. 79%) at 5 years were similar for patients with and without HCC. Cox regression analysis identified higher albumin, lower MELD, no HCC at listing, and being transplanted to be associated with better ITT survival. Ninety-four patients with HCC (including 19 new HCC) and 75 with HBV-cirrhosis underwent OLT. Post-OLT survival (83% vs. 90%) and HBV recurrence (11% vs. 10%) at 3 years were similar, while disease (HBV and/or HCC) recurrence (19% vs. 10%; P = 0.043) was higher in patients with HBV-HCC vs. HBV-cirrhosis. Disease recurrence was the only independent predictor of post-OLT survival. In conclusion, despite more advanced liver disease and a lower rate of transplantation, ITT survival of patients listed for HBV-cirrhosis was comparable to those with HBV-HCC, possibly related to beneficial effects of antiviral therapy.

Adult↗

Outcomes in hepatitis C virus-infected recipients of living donor vs. deceased donor liver transplantation.

In this retrospective study of hepatitis C virus (HCV)-infected transplant recipients in the 9-center Adult to Adult Living Donor Liver Transplantation Cohort Study, graft and patient survival and the development of advanced fibrosis were compared among 181 living donor liver transplant (LDLT) recipients and 94 deceased donor liver transplant (DDLT) recipients. Overall 3-year graft and patient survival were 68% and 74% in LDLT, and 80% and 82% in DDLT, respectively. Graft survival, but not patient survival, was significantly lower for LDLT compared to DDLT (P = 0.04 and P = 0.20, respectively). Further analyses demonstrated lower graft and patient survival among the first 20 LDLT cases at each center (LDLT 20; P = 0.002 and P = 0.002, respectively) and DDLT recipients (P < 0.001 and P = 0.008, respectively). Graft and patient survival in LDLT >20 and DDLT were not significantly different (P = 0.66 and P = 0.74, respectively). Overall, 3-year graft survival for DDLT, LDLT >20, and LDLT 20 were not significantly different. Important predictors of graft loss in HCV-infected patients were limited LDLT experience, pretransplant HCC, and higher MELD at transplantation.

Adult↗

Cost of Gram-negative resistance.

OBJECTIVE: It is unclear that infections with Gram-negative rods resistant to at least one major class of antibiotics (rGNR) have a greater effect on patient morbidity than infections caused by sensitive strains (sGNR). We wished to test the hypothesis that rGNR infections are associated with higher resource utilization. DESIGN: Retrospective observational cohort study of prospectively collected data. SETTING: University hospital surgical intensive care unit and ward. PATIENTS: Surgical patients with at least one GNR infection. MEASUREMENTS: We compared admissions treated for rGNR infection with those with sGNR infections. Primary outcomes were total hospital costs and hospital length of stay. Other outcomes included antibiotic treatment cost, in-hospital death, and intensive care unit length of stay. After univariate analysis comparing outcomes after rGNR infection with those after sGNR infection, multivariate linear regression models for hospital cost and length of stay were created to account for potential confounders. MAIN RESULTS: Cost data were available for 604 surgical admissions treated for at least one GNR infection (Centers for Disease Control and Prevention criteria), 137 (23%) of which were rGNR infections. Admissions with rGNR infections were associated with a higher severity of illness at the time of infection (Acute Physiology and Chronic Health Evaluation II score, 17.6 +/- 0.6 vs. 13.9 +/- 0.3), had higher median hospital costs ($80,500 vs. $29,604, p < .0001) and median antibiotic costs ($2,607 vs. $758, p < .0001), and had longer median hospital length of stay (29 vs. 13 days, p < .0001) and median intensive care unit length of stay (13 days vs. 1 day, p < .0001). Infection with rGNR within the first 7 days of admission was independently predictive of increased hospital cost (incremental increase in median hospital cost estimated at $11,075; 95% confidence interval, $3,282-$20,099). CONCLUSIONS: Early infection with rGNR is associated with a high economic burden, which is in part related to increased antibiotic utilization compared with infection with sensitive organisms. Efforts to control overuse of antibiotics should be pursued.

Analysis of Variance↗

Systemic and cerebrospinal fluid T-helper cytokine responses in organ transplant recipients with Cryptococcus neoformans infection.

BACKGROUND: The role of Th1 and Th2 mediated cytokine responses in the pathogenesis of Cryptococcus neoformans infection in organ transplant recipients has not been defined. METHODS: We assessed cytokine levels in the sera and CSF collected prospectively at the time of diagnosis of infection in 25 transplant recipients with cryptococcosis. Serum levels were compared with those in healthy individuals and transplant recipients without cryptococcosis. IFN-gamma or IL-12 (Th1)/IL-10 (Th2) ratio < 1.0 was considered a dominant Th2 response. RESULTS: Cases had lower ratios of IFN-gamma/IL-10 (p = 0.03) and IL-12/IL-10 (p = 0.03) compared to healthy individuals. Cytokine responses, however, did not differ significantly for cases vs. transplant controls. Cases with fungemia compared to those without fungemia tended to have higher serum IL-10 levels (p = 0.07) and lower IL-12/IL-10 ratios (p = 0.06). CSF ratios of IFN-gamma/IL-10 (p = 0.04) and IL-12/IL-10 (p = 0.04) were lower in cases with cryptococcal meningitis compared to those without meningitis; 80% (8/10) of the cases with cryptococcal meningitis vs. 0% (4/4) of those without meningitis had CSF IFN-gamma/IL-10 ratio of < 1.0 (p = 0.015). The levels of IL-10 (p = 0.04) and IFN-gamma (p = 0.04) in the CSF in cases with cryptococcal meningitis were significantly higher than those in their serum, respectively. CONCLUSIONS: High expression of Th2 phenotype in cryptococcal meningitis and in fungemia suggests that Th dysregulation may contribute to the pathogenesis of cryptococcosis in organ transplant recipients.

Cryptococcus neoformans↗

Invasive aspergillosis in the recipients of liver retransplantation.

Retransplantation is a major risk factor for invasive aspergillosis in liver transplant recipients. However, the risk for invasive aspergillosis with time elapsed since retransplantation, clinical characteristics, and outcome of patients who develop this infection after retransplantation of the liver has not been defined. Patients comprised 17 liver retransplant recipients with invasive aspergillosis between 1990 and 2004. Retransplantation was considered early if it was performed within 30 days and late if performed after 30 days of the first or primary transplant. Retransplant recipients comprised 25% of all cases of invasive aspergillosis after liver transplantation. Fifty-three percent of the Aspergillus infections occurred within 30 days, and 76% within 90 days of retransplantation. In all, 53% (9/17) of the patients were late retransplant recipients. Late compared to early retransplant recipients with invasive aspergillosis were more likely to have central nervous system involvement with invasive aspergillosis (56% vs. 0%, P = 0.03). Mortality rate was 100% for late and 63% for early retransplant recipients with Aspergillus infections. In conclusion, time-varying risk for invasive aspergillosis after retransplantation has implications relevant for guiding antifungal prophylaxis. Given a greater risk for disseminated infection and poor outcome in late retransplant recipients with aspergillosis, potent and aggressive antifungal therapy should be considered upfront in these patients.

Adolescent↗

Can we define the ideal duration of antibiotic therapy?

BACKGROUND: Because of the increasing development of antimicrobial resistance, there is a greater responsibility within the medical community to limit the exposure of patients to antibiotics. We tested the hypothesis that shorter courses of antibiotics are associated with similar or better results than longer durations. We also sought to investigate the difference between a fixed duration of therapy and one based on physiologic measures such as fever and leukocytosis. METHODS: All infectious episodes on the general surgery units of the University of Virginia Health System from December 15, 1996, to July 31, 2003, were analyzed retrospectively for the relation between the duration of antibiotic therapy and infectious complications (recurrent infection with the same organism or at the same site). All infections associated with either fever or leukocytosis were categorized into quartiles on the basis of the absolute length of antibiotic administration or the duration of treatment following resolution of fever or leukocytosis. Multivariate logistic regression models were developed to estimate the independent risk of recurrence associated with a longer duration of antibiotic use. RESULTS: Of the 5,561 treated infections, 4,470 were associated with fever (temperature > or =38 degrees C) or leukocytosis (white blood cell count > or =11,000/mm(3)). For all infections, whether analyzed by absolute duration or time from resolution of leukocytosis or fever, the first or second quartiles (0-12 days, 0-9 days, 0-9 days, respectively) were associated with the lowest recurrence rates (14-18%, 17-23%, 18-19%, respectively). Individual analysis of intra-abdominal infections and pneumonia yielded similar results. The fixed-duration groups received fewer days of antibiotics on average, with outcomes similar to those in the physiologic parameters group. CONCLUSIONS: Shorter courses of antibiotics were associated with similar or fewer complications than prolonged therapy. In general, adopting a strategy of a fixed duration of therapy, rather than basing duration on resolution of fever or leukocytosis, appeared to yield similar outcomes with less antibiotic use.

Adult↗

Immunotherapy with tacrolimus (FK506) does not select for resistance to calcineurin inhibitors in Candida albicans isolates from liver transplant patients.

In Candida albicans, calcineurin mediates tolerance to azole antifungal drugs, survival in serum, and virulence. In this study, we examined 24 Candida isolates from liver transplant recipients receiving a calcineurin inhibitor as a component of their immunosuppressive therapy. We were unable to detect a difference in susceptibility to calcineurin inhibitors in combination with fluconazole, serum, or calcium in these isolates.

Calcineurin Inhibitors↗

Antifungal management practices and evolution of infection in organ transplant recipients with cryptococcus neoformans infection.

BACKGROUND: Therapeutic practices for Cryptococcus neoformans infection in transplant recipients vary, particularly with regards to antifungal agent employed, and duration of therapy. The risk of relapse and time to recurrence is not known. We assessed antifungal treatment practices for cryptococcosis in a cohort of prospectively followed organ transplant recipients. METHODS: The patients comprised 83 transplant recipients with cryptococcosis followed for a median of 2.1 and up to 5.2 years. RESULTS: Patients with central nervous system infection (69% vs. 16%, P = 0.00001), disseminated infection (82.7% vs. 20%, P = 0.00001), and fungemia (29% vs. 8%, P = 0.046) were more likely to receive regimens containing amphotericin B than fluconazole as primary therapy. The use of fluconazole, on the other hand, was more likely for infection limited to the lungs (64% vs. 14%, P = 0.00002). Survival at 6 months tended to be lower in patients whose CSF cultures at 2 weeks were positive compared to those whose CSF cultures were negative (50% vs. 91%, P = 0.06). Maintenance therapy was employed in 68% (54/79) of the patients who survived >3 weeks. The median duration of maintenance therapy was 183 days; 55% received maintenance for > or = 6 months and 25% for >1 year. Relapse was documented in 1.3% (1/79) of the patients. CONCLUSIONS: A majority of the organ transplant recipients with cryptococcosis receive maintenance antifungal therapy for 6 months with low risk of relapse. These data can assist in trials to assess the optimal therapeutic approach and duration of therapy for cryptococcosis in transplant recipients.

Adult↗

Allograft loss in renal transplant recipients with cryptococcus neoformans associated immune reconstitution syndrome.

This study describes the association of allograft loss and immune reconstitution syndrome (IRS) in the course of Cryptococcosis neoformans infection in renal transplant recipients. Patients comprised 54 renal allograft recipients with cryptococcosis in a prospective, multicenter study. IRS developed in 5.5% (3/54) of the renal transplant recipient with C. neoformans infection. The renal allograft was lost to chronic rejection in 66% (2/3) of the patients with cryptococcosis who developed IRS compared to 5.9% (3/51) of those who did not (P=0.012). Kaplan-Meier survival analysis showed that subsequent to cryptococcal infection the probability of allograft survival was significantly lower in patients who developed IRS compared to those who did not develop IRS (P=0.0004). Temporal association of graft loss with IRS suggests a common pathophysiologic basis for these entities with implications relevant for the optimal management of renal transplant recipients with cryptococcosis.

Cryptococcosis↗

Primary human hepatocytes in spheroid formation to study hepatitis C infection.

BACKGROUND: Hepatitis C (HCV) is a worldwide health problem, affecting nearly 170 million people. Current models for studying Hepatitis C have focused primarily on the use of poorly permissive cell lines and viral constructs, because of the lack of a suitable animal model or an in vitro system for studying functional infection. As hepatocytes are the primary reservoir for the virus in vivo, we report on a model using primary human hepatocytes cultured in spheroid formation. MATERIALS AND METHODS: The hepatocytes were harvested from uninfected liver resections and cultured as spheroids (that promotes a differentiated phenotype) or monolayers. Spheroids expressed the putative receptors CD81 and human scavenger receptor B1 in a variable pattern throughout the culture period. Samples were inoculated with infectious HCV serum, and HCV RNA was detected using RT-PCR. RNA was detected in the cells and culture medium by 3 days and 5 days after inoculation, respectively. Selection of HVR1 variants occurred in a differential pattern based on culture technique, suggesting that viral selection was dependent on host phenotype. Detection of NS5A by Western blot analysis of infected samples and immunofluorescence for HCV core protein was seen only in infected spheroids. CONCLUSION: The use of spheroid formation to study Hepatitis C is associated with the establishment of HVR1 selection and functional infection. This represents a promising alternative model to study Hepatitis C.

Antigens, CD↗

Magnetic resonance urography for the assessment of potential renal donors: comparison of the RARE technique with a low-dose gadolinium-enhanced magnetic resonance urography technique in the absence of pharmacological and mechanical intervention.

The aim of this study was to determine whether magnetic resonance urography without pharmacological (diuretic) stimulation and mechanical compression allows conclusive evaluation of the urinary system in potential renal donors. In 28 consecutive patients magnetic resonance urography (MRU) was performed on a 1.5-T system. Two techniques, rapid acquisition with relaxation enhancement (RARE) and a gadolinium (Gd)-enhanced 3D fast low angle shot (FLASH) sequence were compared in the absence of adjunctive measures. Two reviewers assessed image quality, presence of artifacts and completeness of visualization of the collecting systems and ureters. Among the 53 MR urograms, there was no difference in image quality and presence of artifacts between RARE and Gd-MRU. Despite high image quality, visualization of the urinary collecting system was insufficient. Continuous visualization from the collecting system to the distal ureter was demonstrated bilaterally in only 14% of the RARE and 26% of Gd-enhanced MR urograms, respectively. Overall, Gd-enhanced MRU was superior to the RARE technique in displaying the segments of the urinary collecting system, but this difference was not found to be statistically significant. Neither the RARE technique nor the gadolinium-enhanced MRU technique is accurate enough to allow the evaluation of the collecting system and ureters in potential renal donors in the absence of pharmacological intervention and compression.

Adult↗

An immune reconstitution syndrome-like illness associated with Cryptococcus neoformans infection in organ transplant recipients.

BACKGROUND: We describe an immune reconstitution syndrome (IRS)-like entity in the course of evolution of Cryptococcus neoformans infection in organ transplant recipients. METHODS: The study population comprised a cohort of 83 consecutive organ transplant recipients with cryptococcosis who were observed for a median of 2 years in an international, multicenter study. RESULTS: In 4 (4.8%) of the 83 patients, an IRS-like entity was observed a median of 5.5 weeks after the initiation of appropriate antifungal therapy. Worsening of clinical manifestations was documented, despite cultures being negative for C. neoformans. These patients were significantly more likely to have received tacrolimus, mycophenolate mofetil, and prednisone as the regimen of immunosuppressive therapy than were all other patients (P = .007). The proposed basis of this phenomenon is reversal of a predominantly Th2 response at the onset of infection to a Th1 proinflammatory response as a result of receipt of effective antifungal therapy and a reduction in or cessation of immunosuppressive therapy. CONCLUSIONS: This study demonstrated that an IRS-like entity occurs in organ transplant recipients with C. neoformans infection. Furthermore, this entity may be misconstrued as a failure of therapy. Immunomodulatory agents may have a role as adjunctive therapy in such cases.

Adult↗

A randomized, open-label study to evaluate the safety and pharmacokinetics of human hepatitis C immune globulin (Civacir) in liver transplant recipients.

Chronic hepatitis C is the most common indication for liver transplantation, but viral recurrence is universal and progressive graft injury occurs in most recipients. Our aim was to assess the safety, pharmacokinetics (PK), and antiviral effects of high doses of a human hepatitis C antibody enriched immune globulin product (HCIG) in patients undergoing liver transplantation for chronic hepatitis C. This was a multicenter, randomized, open-label, controlled trial conducted at 4 transplant centers in the United States. A total of 18 patients with chronic hepatitis C, who underwent liver transplantation, were randomized to receive low-dose HCIG (75 mg/kg) or high-dose HCIG (200 mg/kg), or no treatment. A total of 17 infusions of HCIG were administered in each treated patient over 14 weeks using a time-dependent dosing strategy based on the PK of anti-hepatitis B immune globulin in liver transplant recipients. Hepatitis C virus levels, liver enzymes, and liver biopsies were obtained serially throughout the study period. PK profiles of HCV antibodies were determined on days 4, 10, and 98. HCIG infusions were safe and tolerated. The infusion rate could not be maximized because of symptoms for 18% to 30% of the doses. The half-life of HCIG was extremely short immediately after transplantation but was gradually prolonged. In the high-dose group, serum alanine aminotransferase (ALT) levels normalized in most subjects and no patient developed hepatic fibrosis. However, serum HCV RNA levels were not suppressed at either dose. In conclusion, HCIG, an anti-HCV enriched immune globulin product, appears to be safe in patients with chronic hepatitis C undergoing liver transplantation. Further studies are required to determine whether the drug has beneficial effects in this group of patients.

Adult↗

Nature of gram-negative rod antibiotic resistance during antibiotic rotation.

PURPOSE: The aim of this study was to characterize the evolution of gram-negative antibiotic resistance during a study of empiric antibiotic rotation. METHODS: We showed previously that quarterly rotation of a single antibiotic class is inferior to cycling two antibiotics per quarter for empiric treatment of gram-negative rod (GNR) infections, as evidenced by increased incidence of antibiotic-resistant GNR (rGNR) infections. Resistance patterns were examined by quantifying GNRs resistant to one or more of the following drug classes: Aminoglycosides, cephalosporins, carbapenems, fluoroquinolones, or piperacillin-tazobactam. For all rGNR isolates, the mean number of antibiotic classes to which an organism was resistant was calculated per quarter, as was the number of rGNR species. RESULTS: Single-antibiotic rotation (SAR) was associated with significant increases in the incidence of piperacillin-tazobactam (p < 0.0005) and cephalosporin (p = 0.003) resistance, reaching nearly 25% and 30% of rGNR isolates respectively, most notably during the quarter of designated cephalosporin use (VI). Multi-drug resistance emerged over time; resistant classes/resistant GNR isolates ranged from 1.2 in the dual-antibiotic rotation (DAR) to 1.9 in the SAR period (p = 0.02). Resistance was evident in an increasing number of unique GNR species. On average, 1.3 species were isolated per month in the DAR period and 3.0/month in the SAR period (p = 0.004), but proportionally, no single GNR species became significantly more resistant across time. Compared to only 5.8% in the DAR period, 29% noncompliance was observed in the SAR, with a six-fold increase in the use of nonscheduled empiric antibiotics due to the presence of an organism resistant to the scheduled rotation drug. CONCLUSIONS: A single-antibiotic rotation is associated with increased incidence and heterogeneity of resistant GNR isolates, as well as increased multiple-drug-class resistance. The attenuation of resistance observed in the single-antibiotic rotation may reflect the effect of unintended antibiotic heterogeneity driven by increasing resistance to the antibiotic class recommended for use each quarter. This suggests that reliance on a single antibiotic class for empiric treatment of GNR infection exerts sufficient pressure within the environment to encourage the development of diversified resistance, as well as cross-resistance over antibiotic classes, thus narrowing the availability of effective antibiotic treatment.

Anti-Bacterial Agents↗

Comparison of fungal and nonfungal infections in a broad-based surgical patient population.

BACKGROUND: Our aim was to compare fungal and nonfungal infections among a diverse surgical patient population. METHODS: Data on all hospital-acquired infectious episodes among surgical intensive care unit and ward patients were collected prospectively over six years at a single university hospital. The relationships between fungal and nonfungal infection and over 100 variables were examined using univariate and multiple logistic regression analysis. RESULTS: During the study period, 3,980 infectious episodes were identified; 554 were associated with fungal infection. Multiple logistic regression analysis demonstrated that markers of severity of acute illness (higher APACHE II scores and white blood cell counts, greater transfusion of cellular blood products, mechanical ventilator dependency, and prior infection) predicted fungal infection, whereas markers of chronic illness (comorbidities) did not independently predict either fungal or nonfungal infection. Patients with fungal infection were treated with more antibiotics for longer periods of time, had prolonged lengths of stay, and more often died compared with nonfungal infection patients. A separate multiple logistic regression analysis demonstrated that both fungal infection and the number of fungal sites of infection independently predicted mortality. Of all fungal isolates, only Candida albicans and Aspergillus spp. independently predicted mortality. CONCLUSIONS: Fungal infections differ significantly in character and outcomes from nonfungal infections among surgical patients.

APACHE↗

Outcomes of 385 adult-to-adult living donor liver transplant recipients: a report from the A2ALL Consortium.

OBJECTIVE: The objective of this study was to characterize the patient population with respect to patient selection, assess surgical morbidity and graft failures, and analyze the contribution of perioperative clinical factors to recipient outcome in adult living donor liver transplantation (ALDLT). SUMMARY BACKGROUND DATA: Previous reports have been center-specific or from large databases lacking detailed variables. The Adult-to-Adult Living Donor Liver Transplantation Cohort Study (A2ALL) represents the first detailed North American multicenter report of recipient risk and outcome aiming to characterize variables predictive of graft failure. METHODS: Three hundred eighty-five ALDLT recipients transplanted at 9 centers were studied with analysis of over 35 donor, recipient, intraoperative, and postoperative variables. Cox regression models were used to examine the relationship of variables to the risk of graft failure. RESULTS: Ninety-day and 1-year graft survival were 87% and 81%, respectively. Fifty-one (13.2%) grafts failed in the first 90 days. The most common causes of graft failure were vascular thrombosis, primary nonfunction, and sepsis. Biliary complications were common (30% early, 11% late). Older recipient age and length of cold ischemia were significant predictors of graft failure. Center experience greater than 20 ALDLT was associated with a significantly lower risk of graft failure. Recipient Model for End-stage Liver Disease score and graft size were not significant predictors. CONCLUSIONS: This multicenter A2ALL experience provides evidence that ALDLT is a viable option for liver replacement. Older recipient age and prolonged cold ischemia time increase the risk of graft failure. Outcomes improve with increasing center experience.

Adult↗

HCV infection of the transplanted liver: changing CD81 and HVR1 variants immediately after liver transplantation.

The second envelope protein at hypervariable region 1 (HVR1) has been implicated in contributing to hepatitis C virus (HCV)-host cell interactions and CD81 (a multifunctional protein) has been demonstrated to act as a cell surface receptor for HCV and may interact directly with HVR1. The purpose of the current study was to determine if certain HVR1 quasispecies variants more effectively associate with and infect allografts after liver transplantation than other HVR1 variants and whether CD81 receptor expression changes after transplantation. Blood and allograft samples were obtained from the peritransplant period in seven patients. Clones of RT-PCR product were directly sequenced to identify HVR1 quasispecies variants. Explanted liver and serial allograft biopsies in recipients with HCV were examined by immunohistochemistry (IHC) for CD81 expression. Examination of HVR1 sequences demonstrated that only a fraction of the quasispecies variants recovered from each patient's blood sampled immediately prior to transplantation associated with and infected the allografts. Genetic diversity at HVR1 decreased with reperfusion but did not significantly decrease with infection. Expression of CD81 varied during the immediate post-transplant period. In conclusion, HVR1 quasispecies variants differentially associate with, and infect allografts, after liver transplantation. Additionally, allografts express variable amounts of CD81 after transplantation.

Amino Acid Sequence↗