An assessment of interactions between hepatitis C virus and herpesvirus reactivation in liver transplant recipients using molecular surveillance.

Hepatitis C virus (HCV) has been proposed to have immunomodulatory effects in transplant recipients and may promote herpesvirus reactivation. To assess this, we compared the incidence of herpesvirus reactivation in HCV-positive and HCV-negative liver transplant recipients. Quantitative viral load testing was performed at regular intervals posttransplantation for cytomegalovirus (CMV), Epstein-Barr virus (EBV), human herpesviruses (HHV) 6, 7, and 8, and varicella zoster virus (VZV) in 177 liver transplant patients who were HCV-positive (n=60) or HCV-negative (n=117). The incidence of CMV disease, CMV viremia, and the peak CMV viral load was not significantly different in HCV-positive vs. HCV-negative patients. Similarly, no differences in HHV-6 or EBV reactivation were observed. HHV-8 or VZV viremia was not detected in any patient in the study. A lower incidence of HHV-7 infection occurred in HCV-positive patients vs. HCV-negative patients (47.6% vs. 72.7%; P=0.006). In conclusion, these results suggest that HCV infection does not appear to promote herpesvirus reactivation after liver transplantation.

Association between toll-like receptor polymorphisms and the outcome of liver transplantation for chronic hepatitis C virus.

BACKGROUND: Experimental models suggest that immune cells recognize hepatitis C virus (HCV) through toll-like receptor (TLR)-2 and TLR4. We assessed the association between the single nucleotide polymorphism in genes that encode for these receptors and the outcome of liver transplantation for chronic HCV. METHODS: A historical cohort of 92 liver transplant patients with chronic HCV were screened for TLR2 Arg753Gln and TLR4 Asp299Gly and Thr399Ile polymorphisms. The results were correlated with the predefined composite primary outcome of cirrhosis, retransplantation, and death. Statistical analysis was performed using Kaplan-Meier estimation and Cox proportional hazard model. RESULTS: The mean patient age was 49+/-9 years. Sixty percent were male and 84% were white. Twelve (13%) patients had TLR2 Arg753Gln and 32 (35%) had TLR4 Asp299Gly and/or Thr399Ile polymorphism. During the mean follow-up period of 32 months after liver transplantation, the composite primary outcome occurred in 19 (24%) of 80 patients without TLR2 polymorphism, one (14%) of seven patients with heterozygous TLR2 polymorphism, and in all five (100%) patients with homozygous TLR2 polymorphism (P=0.0007). Time-to-event analysis showed a significant association between homozygous TLR2 polymorphism and the primary outcome (P<0.0001). After adjusting for donor age and azathioprine use, homozygous TLR2 mutation (RR 5.20 [1.65-13.9]; P=0.007) remained associated with the primary outcome. TLR4 polymorphisms were not associated with primary outcome. CONCLUSION: Homozygous TLR2 Arg753Gln polymorphism is associated with allograft failure and mortality after liver transplantation for chronic HCV. The potential clinical relevance of this observation should encourage studies to assess its biologic mechanism.

Relationship between Toll-like receptor 2 polymorphism and cytomegalovirus disease after liver transplantation.

BACKGROUND: Experimental data suggest that cytomegalovirus (CMV) initiates innate immunity through the activation of Toll-like receptor 2 (TLR2). To assess the clinical relevance of this experimental observation, we assessed the association between the specific single-nucleotide polymorphism that results in the substitution of arginine for glutamine in position 753 of TLR2 (the TLR2 Arg753Gln polymorphism) and CMV replication and disease after liver transplantation. METHODS: Ninety-two liver transplant recipients with chronic hepatitis C were screened for the presence of the TLR2 Arg753Gln polymorphism. CMV load was determined in serially collected blood samples using CMV DNA polymerase chain reaction. Kaplan-Meier estimation and univariable and multivariable stepwise Cox proportional hazard models were used to assess associations. RESULTS: The degree of CMV replication, as measured by CMV load, was significantly higher in patients who were homozygous (mean maximum viral load, 37,059 copies/mL) and heterozygous (mean maximum viral load, 29,718 copies/mL) for this polymorphism, compared with patients without the TLR2 Arg753Gln polymorphism (mean maximum viral load, 3252 copies/mL; P=.003). Kaplan-Meier survival analysis demonstrated an association between being homozygous for the TLR2 Arg753Gln polymorphism and CMV disease (P=.04). A multivariate Cox proportional hazard model demonstrated a trend towards a higher risk of CMV disease among patients who were homozygous for the TLR2 Arg753Gln polymorphism (hazard ratio, 1.91 [95% confidence interval, 0.91-3.40]; P=.08) after adjusting for patient age, CMV serostatus, and allograft rejection. CONCLUSIONS: TLR2 Arg753Gln polymorphism is possibly associated with CMV replication and disease after liver transplantation. This novel clinical observation supports the potential role of TLR2 in the immunologic control of CMV infection in humans.

Effacing of the T cell compartment by cardiac transplantation in infancy.

For cardiac transplantation in infants, T cells are depleted and the thymus is removed. These manipulations should cause profound defects in the T cell compartment. To test this concept, 20 subjects who underwent cardiac transplantation in infancy and healthy age-matched subjects were studied. The number of T cells in the blood was nearly normal in all subjects 1-10 years after surgery. However, newly generated T cells were undetectable in 10 recipients and 10-fold less than controls in 10, suggesting absence of thymic function. TCRbeta chain diversity, measured by a novel technique, was approximately 100-fold lower than controls. T cell function, deduced from levels of human herpesvirus 7 and response to hepatitis B immunization, were notably impaired. Yet cardiac transplant recipients were generally free of opportunistic infections. Our findings demonstrate a novel approach to measuring lymphocyte diversity and suggest that understanding how these subjects resist infection could yield important insights into immune fitness.

Stimulation of toll-like receptor 2 with bleomycin results in cellular activation and secretion of pro-inflammatory cytokines and chemokines.

The clinical use of bleomycin results in systemic and pulmonary inflammatory syndromes that are mediated by the production of cytokines and chemokines. In this study, we demonstrate that cell activation is initiated upon the recognition of bleomycin as a pathogen-associated molecular pattern by toll-like receptor (TLR) 2. The THP1 human monocytic cell line, which constitutively expresses high levels of TLR2, secretes interleukin (IL)-1beta, IL-8, and tumor necrosis factor (TNF)-alpha during bleomycin exposure. The TLR2-dependent nature of cell activation and cytokine secretion is supported by (1) the inability of TLR2-deficient human embryonic kidney (HEK) 293 cells to exhibit nuclear factor-kappa B (NF-kappaB) activation and secrete IL-8 in response to bleomycin; (2) the acquired ability of HEK293 to exhibit NF-kappaB activation and secrete IL-8 upon experimental expression of TLR2; and (3) the inhibition of cell activation in TLR2-expressing HEK293 and THP1 by anti-TLR2 monoclonal antibody. Collectively, these observations identify TLR2 activation as a critical event that triggers NF-kappaB activation and secretion of cytokines and chemokines during bleomycin exposure. Our in vitro findings could serve as a molecular mechanism underlying the pro-inflammatory toxicity associated with bleomycin. Whether bleomycin engages with other cellular receptors that results in activation of alternate signaling pathways and whether the TLR2-agonist activity of bleomycin contribute to its anti-neoplastic property deserve further study.

Herpesvirus infections in solid organ transplant patients at high risk of primary cytomegalovirus disease.

The epidemiology of infections with 5 human herpesviruses (HHVs) (HHV-6, HHV-7, HHV-8, varicella zoster virus [VZV], and Epstein-Barr virus [EBV]) was investigated during the first year after solid organ transplantation in 263 patients who received oral ganciclovir or valganciclovir prophylaxis. HHV-6B DNAemia was uncommon, HHV-6A DNAemia was not observed, and HHV-7 DNAemia was prevalent. HHV-6 and HHV-7 DNAemia were not significantly associated with cytomegalovirus (CMV) disease, although a trend toward higher incidence of CMV disease was observed in HHV-6 DNAemic patients. VZV and HHV-8 DNAemia were not detected. EBV infection was common, although incidence of high-level EBV DNAemia was low, especially in patients who received valganciclovir prophylaxis. EBV-related posttransplant lymphoproliferative disease was not observed up to 12 months after transplantation. Compared with historic data, data from the present study suggest that antiviral prophylaxis may lower the incidence, prevalence, or level of DNAemia for infection with HHV-6, HHV-8, VZV, and EBV but not for infection with HHV-7.

A longitudinal molecular surveillance study of human polyomavirus viremia in heart, kidney, liver, and pancreas transplant patients.

In this study of 263 heart, kidney, liver, and pancreas transplant patients, BK virus (BKV) and JC virus (JCV) DNAemia were observed most commonly in kidney and/or pancreas transplant patients (26%), although they were also observed, to a lesser extent, in heart (7%) and liver (4%) transplant patients. The majority of episodes of polyomavirus DNAemia were subclinical, although, in some cases, BKV DNAemia was associated with kidney rejection, and JCV DNAemia was accompanied by nonspecific symptoms. Hence, BKV and JCV DNAemia are not uncommon during the first year after kidney, heart, liver, and pancreas transplantation, and they could be associated with certain clinical syndromes in transplant patients.

A surveillance study of adenovirus infection in adult solid organ transplant recipients.

Little is known about adenovirus infections in adult organ transplant recipients. We prospectively assessed adenovirus infection in 263 transplant recipients using polymerase chain reaction (PCR) on plasma samples at regular intervals post-transplant. Adenovirus DNA was detected in 19 of 263 patients (7.2%). Viremia by transplant type was: liver (n = 10 of 121 [8.3%]), kidney (n = 6 of 92 [6.5%]) and heart (n = 3 of 45 [6.7%]). Time to viremia onset was within 10 days post-transplant (n = 4), on day 28 (n = 1), on day 100 (n = 7) and between months 6 and 12 (n = 7). At the time of viremia, 11 of 19 (58%) patients had no symptoms, 2 of 19 (10.5%) had gastrointestinal (GI) symptoms, 2 of 19 (10.5%) had respiratory symptoms and 4 patients (21%) had vague/non-specific symptoms. All patients recovered spontaneously. Only 1 of 19 (5%) patients had subsequent acute rejection. Adenovirus viremia is relatively common in adult liver, kidney and heart transplant recipients and most infections are asymptomatic, transient and self-limited. No serious clinical sequelae or effects on subsequent acute rejection were observed.

Nystatin induces secretion of interleukin (IL)-1beta, IL-8, and tumor necrosis factor alpha by a toll-like receptor-dependent mechanism.

Nystatin is an antifungal compound with potent proinflammatory properties. Herein, we demonstrate that nystatin induces interleukin (IL)-1beta, IL-8, and tumor necrosis factor alpha secretion through its activation of toll-like receptor 1 (TLR1) and TLR2. Hence, a TLR-dependent mechanism could serve as the molecular basis for the proinflammatory properties of nystatin.

Human immunodeficiency virus reactivation by phorbol esters or T-cell receptor ligation requires both PKCalpha and PKCtheta.

Latently human immunodeficiency virus (HIV)-infected memory CD4(+) T cells represent the major obstacle to eradicating HIV from infected patients. Antigens, T-cell receptor (TCR) ligation, and phorbol esters can reactivate HIV from latency in a protein kinase C (PKC)-dependent manner; however, it is unknown which specific PKC isoforms are required for this effect. We demonstrate that constitutively active (CA) forms of both PKCtheta, PKCthetaA148E, and PKCalpha, PKCalphaA25E, induce HIV long terminal repeat (LTR)-dependent transcription in Jurkat and primary human CD4(+) T cells and that both PKCthetaA148E and PKCalphaA25E cause HIV reactivation in J1.1 T cells. Suppression of both PKCalpha and PKCtheta with short hairpinned (sh) RNA inhibited CD3/CD28-induced HIV LTR-dependent transcription and HIV reactivation in J1.1 T cells. Both prostratin and phorbol myristate 13-acetate induced HIV LTR-dependent transcription and HIV reactivation in J1.1 T cells that was blocked by shRNA against either PKCalpha or PKCtheta. Since suppression of PKCalpha and PKCtheta together has no greater inhibitory effect on HIV reactivation than inhibition of PKCalpha alone, our data confirm that PKCalpha and PKCtheta act in sequence. The requirement for PKCalpha and PKCtheta for prostratin-induced HIV reactivation and the ability of selective PKCalpha or PKCtheta agonists to induce HIV transcription indicate that these PKC isoforms are important targets for therapeutic drug design.

Pharmacodynamics of oral ganciclovir and valganciclovir in solid organ transplant recipients.

BACKGROUND: A randomized, double-blind study was conducted to evaluate the pharmacokinetics of ganciclovir following oral administration of ganciclovir or valganciclovir for prophylaxis of cytomegalovirus (CMV) disease in solid organ transplant recipients (n = 240/372). METHODS: The correlations between individual exposure to ganciclovir during prophylaxis, with CMV viremia incidence during and after treatment, CMV disease up to 12 months posttransplant, and hematological toxicity were assessed. RESULTS: Mean daily areas under the curve (AUCs) of ganciclovir from valganciclovir and oral ganciclovir were 46.3 +/- 15.2 and 28.0 +/- 10.9 microg.h/ml (mean +/- SD), respectively. Viremia was suppressed during prophylaxis when exposure to ganciclovir was 40-50 microg.h/ml, AUCs typical of those achieved in valganciclovir-treated patients. The development of viremia 1 month after ending prophylaxis was also reduced with higher ganciclovir AUC (median predicted incidence, 20% and 10% at AUCs of 33 and 50 microg h/ml, respectively). The development of CMV disease within 1 year of transplant was 17.6% and independent of prophylactic exposure to ganciclovir. There was only a weak tendency to increased neutropenia and leukopenia with higher ganciclovir exposure. CONCLUSIONS: The greater systemic exposure to ganciclovir delivered by valganciclovir was associated with delayed development of viremia. There was only a weak association between AUC and hematological toxicity.

Clinical utility of cytomegalovirus (CMV) serology testing in high-risk CMV D+/R- transplant recipients.

Late-onset cytomegalovirus (CMV) disease is a significant problem in D+/R- solid organ transplant (SOT) patients who receive antiviral prophylaxis. We assessed the clinical utility of CMV IgG and IgM serology testing for predicting late-onset CMV disease. We evaluated 352 D+/R- transplant recipients who participated in a trial comparing 100 days of ganciclovir versus valganciclovir prophylaxis. CMV serology was assessed on day 28, 56, 100, and 6 and 12 months post-transplant. IgG seroconversion occurred in 26.9% of patients by day 100, and in 63.4% and 75.3% by 6 and 12 months, respectively. IgM seroconversion occurred in 8.3%, 41.8% and 54.9% by day 100, month 6 and month 12, respectively. Seroconversion by day 100 (end of prophylaxis) was not predictive of subsequent CMV disease (CMV disease 13.3% if seropositive vs. 17.8% if seronegative; p = NS). However, at 6 months post-transplant, IgG serostatus was predictive of subsequent CMV disease between month 6 and 12 (CMV disease 1.3% if seropositive vs. 10.0% if seronegative; p = 0.002). In D+/R- patients, CMV serology testing is for the most part not clinically useful for predicting subsequent disease. However, seroconversion by 6 months may be useful for identifying patients at risk of late-onset CMV disease.

Secretion of proinflammatory cytokines and chemokines during amphotericin B exposure is mediated by coactivation of toll-like receptors 1 and 2.

Amphotericin B (AmB) is a ligand of toll-like receptor 2 (TLR2). Here, we demonstrate the participation of TLR1 in AmB-induced cell activation that led to the secretion of tumor necrosis factor alpha, interleukin 6 (IL-6), and IL-8. Hence, TLR2-TLR1 coactivation serves as the underlying mechanism for the proinflammatory toxicities associated with AmB.

Valganciclovir for the prevention and treatment of cytomegalovirus disease in immunocompromised hosts.

Valganciclovir (Valcyte, Roche), a prodrug of the deoxyguanosine analog ganciclovir (Cytovene, Roche), is indicated for induction and maintenance treatment of cytomegalovirus retinitis in patients with AIDS and for prevention of cytomegalovirus disease in selected high-risk solid organ transplant recipients. After oral administration, valganciclovir is rapidly absorbed and converted to ganciclovir by intestinal and hepatic esterases. Valganciclovir is a highly recognized substrate of the intestinal peptide transporter PEPT1, which underlies the tenfold higher bioavailability of ganciclovir after valganciclovir compared to oral ganciclovir administration. At oral dose of 900 mg, valganciclovir provides a systemic ganciclovir exposure that is comparable to intravenous ganciclovir, at the standard dose of 5 mg/kg of body weight. Subsequent phosphorylation of ganciclovir, which occurs preferentially within cytomegalovirus-infected cells, results in the active metabolite, ganciclovir triphosphate, which is responsible for suppressing viral DNA synthesis by competitively inhibiting the incorporation of the natural substrate deoxyguanosine into viral DNA and thereby, terminating cytomegalovirus replication.

Human immunodeficiency virus-induced apoptosis of human hepatocytes via CXCR4.

Human immunodeficiency virus (HIV) infection is commonly associated with liver dysfunction. The X4 HIV glycoprotein 120 envelope (env) induces apoptosis in T cells and neurons via the HIV coreceptor CXCR4. Therefore, we investigated whether hepatocyte death could result from the HIV env signaling through CXCR4 on the hepatocyte. We demonstrated that hepatocytes in humans express CXCR4 on the cell surface. Furthermore, we established that the X4 HIV env and the entire HIV virion signal hepatocyte apoptosis through CXCR4. The apoptotic process is dependent on G(ialpha) protein signaling, yet it is independent of caspase cascade activation. Thus, HIV can directly cause hepatocyte death in humans by signaling through CXCR4, without infecting the cell.

Protein kinase Calpha (PKCalpha) acts upstream of PKCtheta to activate IkappaB kinase and NF-kappaB in T lymphocytes.

NF-kappaB is an ubiquitous transcription factor that is a key in the regulation of the immune response and inflammation. T-cell receptor (TCR) cross-linking leads to NF-kappaB activation, an IkappaB kinase (IKK)-dependent process. However, the upstream kinases that regulate IKK activity following TCR activation remain to be fully characterized. Herein, we demonstrate using genetic analysis, pharmacological inhibition, and RNA interference (RNAi) that the conventional protein kinase C (PKC) isoform PKCalpha, but not PKCbeta1, is required for the activation of the IKK complex following T-cell activation triggered by CD3/CD28 cross-linking. We find that in the presence of Ca(2+) influx, the catalytically active PKCalphaA25E induces IKK activity and NF-kappaB-dependent transcription; which is abrogated following the mutations of two aspartates at positions 246 and 248, which are required for Ca(2+) binding to PKCalpha and cell membrane recruitment. Kinetic studies reveal that an early phase (1 to 5 min) of IKK activation following TCR/CD28 cross-linking is PKCalpha dependent and that a later phase (5 to 25 min) of IKK activation is PKCtheta dependent. Activation of IKK- and NF-kappaB-dependent transcription by PKCalphaA25E is abrogated by the PKCtheta inhibitor rottlerin or the expression of the kinase-inactive form of PKCtheta. Taken together, our results suggest that PKCalpha acts upstream of PKCtheta to activate the IKK complex and NF-kappaB in T lymphocytes following TCR activation.

Direct transcriptional regulation of RelB by 1alpha,25-dihydroxyvitamin D3 and its analogs: physiologic and therapeutic implications for dendritic cell function.

The nuclear factor-kappaB (NF-kappaB) protein RelB plays a unique role in dendritic cell (DC) function and, as such, is an important regulator of antigen presentation and immune regulation. In this study, inhibition of RelB expression in DCs exposed to an analog of the active form of vitamin D3 (1alpha,25-dihydroxyvitamin D3 (1alpha,25-(OH)2D3)) was observed and shown to be mediated by the vitamin D receptor (VDR). Potential vitamin D response elements were identified within promoter regions of human and mouse relB genes. In gel shift experiments, these motifs specifically bound VDR.retinoid X receptor-alpha complexes. Reporter assays confirmed that transcriptional activity of human and mouse relB promoters was inhibited by 1alpha,25-(OH)2D3 agonists in a DC-derived cell line. The inhibition was abolished by mutagenesis of the putative vitamin D response elements and was enhanced by overexpression of VDR. Mutagenesis of NF-kappaB response elements within the relB promoter did not affect the magnitude of 1alpha,25-(OH)2D3 analog-mediated inhibition, ruling out an indirect effect on NF-kappaB signaling. Glucocorticoid caused additional inhibition of relB promoter activity when combined with the 1alpha,25-(OH)2D3 analog. This effect was dependent on the integrity of the NF-kappaB response elements, suggesting separate regulatory mechanisms for the two steroid pathways on this promoter. We conclude that relB is a direct target for 1alpha,25-(OH)2D3-mediated negative transcriptional regulation via binding of VDR.retinoid X receptor-alpha to discrete DNA motifs. This mechanism has important implications for the inhibitory effect of 1alpha,25-(OH)2D3 on DC maturation and for the potential immunotherapeutic use of 1alpha,25-(OH)2D3 analogs alone or combined with other agents.

Dynamics of cytomegalovirus replication during preemptive therapy with oral ganciclovir.

The degree and dynamics of cytomegalovirus (CMV) replication were investigated in blood samples that were prospectively collected in the context of a placebo-controlled study evaluating the efficacy of preemptive oral ganciclovir for the prevention of CMV disease after liver transplantation. The degree of viral replication was strongly associated with progression to CMV disease or viremia (risk ratio, 8.8 and 51.5 among patients with virus loads < or =2860 and >2860 copies/10(6) peripheral blood leukocytes, respectively). Preemptive oral ganciclovir therapy diminished the incidence of CMV disease or viremia but did not completely suppress higher levels of CMV replication. Six (21%) of 29 patients had persistent CMV replication during preemptive oral ganciclovir therapy; 2 patients subsequently developed "breakthrough" CMV syndrome. This study identifies a relative cutoff virus load that predicts subsequent development of CMV disease and highlights the inability of oral ganciclovir to suppress CMV replication in a subset of patients.

Detection of JNK and p38 activation by flow cytometry analysis.

JNK and p38 protein kinases are involved in the signal transduction of apoptotic stimulus. JNK and p38 are activated by dual phosphorylation on threonine and tyrosine residues. Different techniques such as Western blotting (WB) and confocal microscopy analysis have been developed to detect the activation by using antibodies that recognize the phosphorylated forms of both enzymes. However, these techniques are time consuming, not quantitative, and dependent on subjective interpretation. Herein, we describe a flow cytometry-based analysis to detect JNK and p38 activation. Using human primary lymphocytes and Jurkat CD4(+) T cells stimulated with PMA/ionomycin, we demonstrate activation (phosphorylation) of JNK and p38, which is further confirmed by two additional established techniques (WB and confocal microscopy). Flow cytometry analysis is shown to be more sensitive than WB to detect JNK and p38 activation, which can be quantitated and enables us to study their activation within cell populations.