The time course of development and impact from viral resistance against ganciclovir in cytomegalovirus infection.

(Val)ganciclovir is used to treat cytomegalovirus (CMV) infection following solid organ (SOT) or hematopoietic stem cell (HSCT) transplantation. Treatment failures occur, but the contribution from 39 known ganciclovir-related mutations (GRMs) in the CMV-UL97 gene remains controversial. We propose a categorization of these GRMs potentially useful when interpreting sequence analyses in clinical settings. The UL97 gene was sequenced from first/recurrent CMV infections among consecutive SOT or HSCT recipients during 2004-2009. GRMs were categorized as: Signature GRM (sGRM) if in vitro ganciclovir IC(50) ratio for mutated versus wild-type virus >2 (n = 24); polymorphic GRM (pGRM) if ratio <2 (n = 15). (Val)ganciclovir treatment failure was defined as persistent viremia for 30 days or switch to foscarnet within this period. Of 99 (49 HSCT and 50 SOT) recipients with one CMV infection episode, 15 (13 HSCT and 2 SOT) experienced a total of 19 recurrent infection episodes. The prevalence of sGRM was 0% at start of first episode, whereas at start of recurrent episodes, prevalence was 37%. Only one sGRM was present at a time in individual patients. Patients with CMV containing an sGRM (vs. wild type)-but not with a pGRM-were at excess risk of treatment failure (odds ratio = 70.6 [95% CI:8.2-609.6]; p < 0.001). sGRMs emerged only following longer termed use of antiherpetic drugs and usually in recurrent CMV infection episodes. Risk of ganciclovir treatment failure was raised if an sGRM was detected.

Identification of gene expression patterns in superficial and invasive human bladder cancer.

Multiple transcriptional events take place when normal urothelium is transformed into tumor tissue. These can now be monitored simultaneously by the use of oligonucleotide arrays, and expression patterns of superficial and invasive tumors can be established. Single-cell suspensions were prepared from bladder biopsies (36 normal, 29 tumor). Pools of cells were made from normal urothelium and from pTa grade I and II and pT2 grade III and IV bladder tumors. From these suspensions, and from 10 single-tumor biopsies, labeled cRNA was hybridized to oligonucleotide arrays carrying probes for 6500 genes. The obtained expression data were sorted according to a weighting scheme and were subjected to hierarchical cluster analysis of tissues and genes. Northern blotting was used to verify the array data, and immunohistology was used to correlate between RNA and protein levels. Hierarchical clustering of samples correctly identified the stage using both 4076 genes and a subset of 400 genes covarying with the stages and grades of tumors. Hierarchical clustering of gene expression levels identified several stage-characteristic, functionally related clusters, encoding proteins that were related to cell proliferation, oncogenes and growth factors, cell adhesion, immunology, transcription, proteinases, and ribosomes. Northern blotting correlated well with array data. Immunohistology showed a good concordance between transcript level and protein staining. The study indicates that gene expression patterns may be identified in bladder cancer by combining oligonucleotide arrays and cluster analysis. These patterns give new biological insight and may form a basis for the construction of molecular classifiers and for developing new therapy for bladder cancer.

Clinical Application of Variation in Replication Kinetics During Episodes of Post-transplant Cytomegalovirus Infections.

BACKGROUND: Cytomegalovirus (CMV) infection in transplant recipients is reported to replicate with a doubling time of 1.2-2 days, and weekly screening is recommended for early diagnosis. We re-evaluated these features in our cohort of transplant recipients. METHODS: The CMV doubling time of the first CMV infection in the first year post-transplant could be calculated for 193 recipients of haematopoietic stem cell or solid organ transplantation. Factors determining the proportion of recipients with a high diagnostic CMV viral load (≥ 18,200 IU/mL) were explored using mathematical simulation. FINDINGS: The overall median doubling time was 4.3 days (IQR 2.5-7.8) and was not influenced by prior CMV immunity, or type of transplantation (p > 0.4). Assuming a fixed doubling time of 1.3 days and screening intervals of 7 or 10 days, 11.1% and 33.3% were projected to have a high CMV viral load at diagnosis, compared to 1.4% and 4.3% if the doubling time varies as observed in our cohort. Consistently, 1.9% of recipients screened weekly had a high diagnostic virus load. INTERPRETATION: Screening intervals can be extended to 10 days in cohorts with comparable CMV doubling time, whereas shorter than 7 days is required in cohorts with shorter doubling times to maintain pre-emptive screening quality.

The effects of oxidative stress in in vitro cultured astroglial cells.

It has been suggested that glial cells in the central nervous system might function as a buffer and protect neurons and synapses. Associated with such a function, glial cells might be affected in degenerative diseases, for example, Alzheimer's disease and Parkinson's disease, due to generation of free-radicals. Free-radicals might be generated during the metabolic transformation of xenobiotics. The purpose of the present study was to determine whether a xenobiotic (in this case, paraquat), is metabolised in glial cells during the generation of free-radicals. Furthermore, this study determined whether free-radicals can induce DNA fragmentation and whether this fragmentation can be repaired. The data produced indicated that astroglial cells contain P450-reductase which transforms paraquat into a pyridium free-radical. In turn, this causes a dose-dependent DNA fragmentation, as determined by using single-cell gel electrophoresis. The dose-dependent effect was valid up to 80µM paraquat. The oxidative stress induced in the astroglial cells was also associated with a maximum 15% increase in the anti-oxidative enzyme, glutathione peroxidase. After exposure to 40µM paraquat, followed by growth of the cells in a paraquat-free medium, DNA repair was shown to be rather slow, and was only obvious two hours after exposure to paraquat. This might be related the shuttle in which paraquat/P450-reductase is implicated, which causes a protracted generation of free-radicals. The data are discussed in relation to the available literature.

Transcriptional profiling of myotubes from patients with type 2 diabetes: no evidence for a primary defect in oxidative phosphorylation genes.

AIMS/HYPOTHESIS: Microarray-based studies of skeletal muscle from patients with type 2 diabetes and high-risk individuals have demonstrated that insulin resistance and reduced mitochondrial biogenesis co-exist early in the pathogenesis of type 2 diabetes independently of hyperglycaemia and obesity. It is unknown whether reduced mitochondrial biogenesis or other transcriptional alterations co-exist with impaired insulin responsiveness in primary human muscle cells from patients with type 2 diabetes. METHODS: Using cDNA microarray technology and global pathway analysis with the Gene Map Annotator and Pathway Profiler (GenMapp 2.1) and Gene Set Enrichment Analysis (GSEA 2.0.1), we examined transcript levels in myotubes established from obese patients with type 2 diabetes and matched obese healthy participants, who had been extensively metabolically characterised both in vivo and in vitro. We have previously reported reduced basal lipid oxidation and impaired insulin-stimulated glycogen synthesis and glucose oxidation in these diabetic myotubes. RESULTS: No single gene was differently expressed after correction for multiple testing, and no biological pathway was differently expressed using either method of global pathway analysis. In particular, we found no evidence for differential expression of genes involved in mitochondrial oxidative metabolism. Consistently, there was no difference in mRNA levels of genes known to mediate the transcriptional control of mitochondrial biogenesis (PPARGC1A and NRF1) or in mitochondrial mass between diabetic and control myotubes. CONCLUSIONS/INTERPRETATION: These results support the hypothesis that impaired mitochondrial biogenesis is not a primary defect in the sequence of events leading to insulin resistance and type 2 diabetes.