Kinetics of cytomegalovirus load decrease in solid-organ transplant recipients after preemptive therapy with valganciclovir.

The availability of valganciclovir (VGCV) has significantly simplified the treatment of human cytomegalovirus (HCMV) infection after solid-organ transplantation. We show that there was no difference in the kinetics of the decrease in HCMV load after preemptive therapy with VGCV in 22 solid-organ transplant recipients (T1/2=2.16 days), compared with that in 23 patients treated with intravenous ganciclovir (GCV) (T(1/2) = 1.73 days; P=.63). Preemptive therapy with VGCV provides control of HCMV replication that is comparable to that achieved with preemptive intravenous therapy with GCV.

Cytomegalovirus-specific cellular immune responses and viremia in recipients of allogeneic stem cell transplants.

The immune suppression inherent in allogeneic stem cell transplantation (SCT) offers a favorable environment for infection by opportunistic agents, such as human cytomegalovirus (CMV). Despite the application of potent antiviral prophylaxis, patients remain at risk for CMV infection until adequate immunity is restored. CMV-specific CD8(+) T cell counts were monitored, using HLA-A2 tetrameric complexes, to establish the level of immune response to the viral phosphoprotein UL83 in patients after allogeneic SCT. Correlating this with viral replication and clinical status shows that the level of tetramer-positive T cells provides an assessment of CMV immune reconstitution after stem cell transplantation. Most patients with seropositive donors did reconstitute long-term CMV immunity, unless prolonged immunosuppression to control graft-versus-host disease was induced. Together with polymerase chain reaction testing, this technique provides measurable parameters that can be a guide to therapeutic decision making and can form the basis of CMV immunotherapy.

Quantity of cytomegalovirus DNA in different leukocyte populations during active infection in vivo and the presence of gB and UL18 transcripts.

The quantity of human cytomegalovirus (HCMV) DNA in the blood of immunocompromised individuals correlates with the development of HCMV disease. We wished to determine which leukocytes harboured DNA and whether this represented active viral replication. Magnetic bead separation techniques were used to obtain pure polymorphonuclear leukocyte (PMNL), monocyte, B and T cell fractions, and RT-PCR and quantitative-competitive PCR (QC-PCR) to detect HCMV glycoprotein B (gB; UL55) transcripts and quantify HCMV DNA levels, respectively, in each cell fraction. QC-PCR revealed that PMNLs contribute the greatest to the overall viral load in blood (median viral load: PMNLs, 10(5.37) genomes/ml of blood; monocytes, 10(4.40) genomes/ml; B cells, 10(3.70) genomes/ml; and T cells, 10(4.08) genomes/ml). However, monocytes have a viral burden of 0.65 genomes/monocyte which is greater than that within the other leukocyte populations (0.11 genomes/PMNL, 0.23 genomes/B cell, and 0.20 genomes/T cell). Glycoprotein B transcripts were detected in all four cell populations: 3/10 PMNL fractions, 6/13 monocyte fractions, 5/13 B cell fractions, and 4/13 T cell fractions. The data show that productive infection of these leukocyte subpopulations, including PMNLs, can occur in vivo. Furthermore, transcripts of gpUL18, the putative natural killer (NK) cell decoy, were detected in 2/6 monocyte fractions with active replication, and 1/4 T cell fractions but not in the other leukocyte fractions. The transient nature of UL18 gene expression, and the low abundance of the transcript relative to gB were confirmed.

The minisatellite of the GPI/AMF/NLK/MF gene: interspecies conservation and transcriptional activity.

Minisatellites are tandemly repeated DNA sequences found throughout the genomes of all eukaryotes. They are regions often prone to instability and hence hypervariability; thus repeat unit sequence is generally not conserved beyond closely related species. We have studied the minisatellite located in intron 9 of the human glucose phosphate isomerase (GPI) gene (also known as neuroleukin, autocrine motility factor, maturation and differentiation factor) and have found, by Zoo blotting coupled with PCR amplification and DNA sequencing, that similar repeat units are present in seven other species of mammal. There is also evidence for the presence of the minisatellite in chicken. The repeat unit does not appear to be present at any other locus in these genomes. Minisatellite DNA has been reported to be involved in recombination activity, control of gene expression of nearby gene(s) (both transcriptional and translational), whilst others form protein coding regions. The high level of conservation exhibited by the GPI minisatellite, coupled with the unique location, strongly suggests a functional role. Our results from transient and stable transfections using luciferase reporter constructs have shown that the GPI minisatellite region can act to increase transcription from the SV40 promoter, CMV promoter and the human GPI promoter.

CD8+ cytotoxic lymphocyte responses against cytomegalovirus after liver transplantation: correlation with time from transplant to receipt of tacrolimus.

The effects of the immunocompromised state after liver transplantation on the frequency of cytomegalovirus-specific cytotoxic T lymphocytes (CTL) were investigated in 93 patients by using HLA class I tetrameric complexes corresponding to HLA-A*0201, HLA-B*0702, HLA-B*0801, and HLA-B*3501 refolded with peptides from the ppUL83 matrix protein. ppUL83 CTL frequencies were suppressed during the first 6 months after transplantation. Patients with >1 HLA-restricted response detected had high correlation among ppUL83 CD8(+) CTL frequencies restricted by different HLA haplotypes (Spearman's rho=.67; P<.0001). There was an inverse correlation among levels of the calcineurin inhibitor, tacrolimus, and ppUL83 CD8(+) CTL frequencies (r=-.31; P=.005), which is consistent with the presence of a large proportion (70%) of activated (CD38(+)) ppUL83 CD8(+) CTL within the population of HLA class I tetramer-positive cells.

Application of viral-load kinetics to identify patients who develop cytomegalovirus disease after transplantation.

BACKGROUND: Cytomegalovirus (CMV) continues to be a major problem post-transplantation; early markers for predicting patients at risk of CMV disease are needed. Peak CMV load in the blood correlates with CMV disease but frequently occurs too late to provide prognostic information. METHODS: 359 transplant recipients (162 liver, 87 renal, and 110 bone marrow) were prospectively monitored for CMV DNA in the blood with qualitative and quantitative PCR. 3873 samples were analysed. The CMV load in the first PCR-positive sample and the rate of increase in CMV load in blood during the initial phase of replication were assessed as risk factors for CMV disease using logistic regression. FINDINGS: 127 of the 359 patients had CMV DNA in the blood and 49 developed CMV disease. Initial viral load correlated significantly with peak CMV load (R2=0.47, p=<0.001) and with CMV disease (odds ratio 1.82 [95% CI 1.11-2.98; p=0.02; 1.34 [1.07-1.68], p=0.01, and 1.52 [1.13-2.05], p=0.006, per 0.25 log10 increase in viral load for liver, renal, and bone-marrow patients, respectively). The rate of increase in CMV load between the last PCR-negative and first PCR-positive sample was significantly faster in patients with CMV disease (0.33 log10 versus 0.19 log10 genomes/mL daily, p<0.001). In multivariate-regression analyses, both initial CMV load and rate of viral load increase were independent risk factors for CMV disease (1.28 [1.06-1.52], p=0.01, per 0.25 log10 increase in CMV load and 1.52 [1.06-2.17], p=0.02, per 0.1 log10 increase in CMV load/mL daily, respectively). INTERPRETATION: CMV load in the initial phase of active infection and the rate of increase in viral load both correlate with CMV disease in transplant recipients; in combination, they have the potential to identify patients at imminent risk of CMV disease.

Prospective study of human betaherpesviruses after renal transplantation: association of human herpesvirus 7 and cytomegalovirus co-infection with cytomegalovirus disease and increased rejection.

BACKGROUND: Human herpesvirus 6 (HHV-6) and HHV-7 are two lymphotropic herpesviruses, which, like cytomegalovirus (CMV), have the potential to be pathogenic in immunocompromised individuals. We have conducted a prospective investigation to compare the natural history of HHV-6 and HHV-7 infection with that of CMV after renal transplantation. METHODS: Polymerase chain reaction was used to identify infections and quantify the viral load of CMV, HHV-6, and HHV-7 in peripheral blood samples from 52 renal transplant recipients. Betaherpesvirus infections were related to defined clinical criteria obtained by detailed examination of the clinical records of each patient for the immediate 120-day posttransplant period. RESULTS: CMV was the most commonly detected virus after transplant (58% of patients), followed by HHV-7 (46%) and HHV-6 (23%). Examining the time to first polymerase chain reaction positivity, HHV-7 infection was detected earlier than CMV (P=0.05). The median maximum CMV viral load was significantly higher than those for HHV-6 (P=0.01) and HHV-7 (P<0.0001) and a trend for HHV-7 viral load to be greater than HHV-6 (P=0.08). Clinicopathological analyses revealed that, in those patients with rejection, HHV-7 was associated with more episodes of rejection (P=0.02). In addition, there was a significant increase in CMV disease occurring in patients with CMV and HHV-7 co-infection compared to those with CMV infection only (P=0.04). CONCLUSIONS: HHV-7 should be further investigated as a possible co-factor in the development of CMV disease in renal transplant patients and may potentially exacerbate graft rejection. No clear pathological role was observed for HHV-6.

Quantity of human cytomegalovirus (CMV) DNAemia as a risk factor for CMV disease in renal allograft recipients: relationship with donor/recipient CMV serostatus, receipt of augmented methylprednisolone and antithymocyte globulin (ATG).

A prospective longitudinal study of 87 renal allograft recipients identified 31 patients with cytomegalovirus (CMV) viraemia. Previous studies have identified CMV viraemia, donor positivity, and CMV load in urine as independent risk factors for disease following renal transpl antation. We used quantitative-competitive polymerase chain reaction (QC-PCR) to quantify the CMV DNA load in blood from these patients, and report that it is a significant and independent risk factor for CMV disease. Patients with symptomatic CMV infection had significantly higher maximum CMV loads than those with no disease (P = .0003). We also found that peak loads were significantly higher in individuals experiencing primary CMV infection (P < .01), and CMV re-infection (P < .05) compared with recipients reactivating endogenous CMV. Univariate analysis revealed that CMV DNA load in blood, donor seropositivity, and receipt of antithymocyte globulin (ATG) were all significantly associated with disease (P = .005, .04, and .05, respectively). However, the association of donor/recipient serostatus, and receipt of ATG became nonsignificant in multivariate analyses whereas the significance of the quantity of CMV DNAemia was maintained, illustrating that CMV load plays a central role in the pathogenesis of CMV disease.

Diagnostic approaches to cytomegalovirus infection in bone marrow and organ transplantation.

Cytomegalovirus (CMV) continues to be a clinical problem, impairing the overall success rate of transplantation, either through direct involvement of a variety of end-organs or by inducing indirect effects such as graft rejection. We review here how the virus manages to evade host immune responses and replicate extensively in allograft recipients. Recent studies show that the quantity of CMV (viral load) is related directly to the development of CMV disease. We review how clinically significant levels of CMV viral load can be defined and summarize the results of studies showing that a high CMV viral load is the major determinant of CMV disease, explaining the previously reported risk factors of pre-transplant serostatus and the post-transplant detection of CMV viremia.Note

Transcription of the human cytomegalovirus natural killer decoy gene, UL18, in vitro and in vivo.

Multiplex RT-PCR analysis of human cytomegalovirus (HCMV) replication in human fibroblasts showed transcription of the natural killer (NK) cell decoy gene, UL18, from 72 h onwards. Transcription of glycoprotein B (gpUL55; a late gene) occurred from early time-points and peaked at 24 h post-infection. UL18 mRNA was also detected in the peripheral blood mononuclear cells of organ transplant recipients with HCMV viraemia, especially those with HCMV DNA virus loads greater than 10(5) genomes/ml whole blood. Thus, UL18 is produced via a low abundance transcript late during the infectious cycle at a time coincidental with the increased risk of NK cell lysis as a consequence of class I HLA down-regulation.

Characterization of cDNAs coding for glucose phosphate isomerase and phosphoglycerate kinase in Chinese hamster ovary cell line CHO-K1 and identification of defects in R1.1.7, a glycolysis-deficient variant of CHO-K1.

Full-length cDNAs for glucose phosphate isomerase (GPI) and phosphoglycerate kinase (PGK) of the Chinese hamster ovary cell line CHO-K1 have been characterized using RT-PCR and cycle sequencing of the PCR-amplified templates. Mutations in both genes have been identified in a glycolysis-deficient Chinese hamster ovary cell line, R1.1.7, derived from CHO-K1 cells.