BK virus (BKV) infection was studied prospectively in 50 unselected consecutive patients who had undergone kidney transplantation. Infection was monitored for one year after transplantation. Viral DNA in urine (viruria) and plasma (viremia) samples was detected by nested, qualitative polymerase chain reaction. BKV screening data was available for 92% (n = 46) of patients enrolled in the study. Four groups of patients were distinguished: uninfected patients (group 1, n = 30), patients with viruria (group 2, n = 3), patients with viremia (group 3, n = 6) and patients with developed BKV nephropathy (group 4, n = 7). Infection was observed starting form the first month, and the maximum number of patients with active BKV infection occurred at six months after transplantation. Five-year graft survival was 69% for patients with any evidence of BKV infection, compared with 80.0% (P = NS) for patients without BKV infection. The best graft function was observed in group one patient (mean serum creatinine 130 mkmol/l and glomerular filtration rate (GFR) 60.9 ml/min) and the worst in group 4 (mean serum creatinine 180 mkmol/l and GFR 52.31 ml/min) at five years after transplantation. Five-year patient survival was 82.6% and was not affected by presence of BKV infection.
Cellular Immunity in Human Herpes Viruses 6 and 7 Infected Gastrointestinal Cancer Patients
CD4+ T lymphocytes appear to be the preferential target for replication of HHV-6 (human herpes virus) as well as HHV-7 viruses in vivo. In addition, CD8+ T cells, monocytes/macrophages, natural killer cells, epithelial, endothelial, neural cells and fibroblasts may be infected. By definition, however, even a tumour designated by pathologists to be early stage may be late stage when considered by the immune system. Certainly, even early stage tumours have evaded immune control, suggesting that they have acquired many immunosuppressive characteristics. The aim of the study was to clarify the influence of beta-herpes viruses on cellular immune response. In 95 gastrointestinal cancer patients we determined the immunocompetent cell level CD3, CD4, CD8, CD19, CD38, CD95, CD25 using laser flow cytofluorimeter and B- herpes viruses HHV-6, HHV-7 presence using a nested polymerase chain reaction method. Our data showed no statistically significant difference in immunocompetent cell level between negative, latent and active HHV-6, HHV-7 infection. Patients with immunocompromised immune status (lymphopenia) had a tendency to decreased CD4+, CD19+ absolute count. It may be suggested that virus-mediated immune response inhibition seems to be similar to cancer mediated, but differences in immune response among the same group of individuals had no influence on the average number of the immunocompetent cells in the group. Therefore, to characterise host-virus-tumour interactions, individual interpretation of each case is needed.
Sensitivity and Reproducibility of Polymerase Chain Reaction Assays for Detection of Human Herpesviruses 6 and 7
Human herpesvirus 6 (HHV-6) and 7 (HHV-7) are ubiquitous viruses that undergo latency and may become reactivated leading to cytomegalovirus reactivation, bone marrow suppression, nervous system dysfunction, graft-versus-host disease and increased mortality. The aim of this study was to identify the most sensitive and reproducible nPCR for detection of HHV-6 and HHV-7 infection and to evaluate the reproducibility of these assays in different laboratories. The sensitivity of the six previously published HHV-6 (one targeting hypothetical protein Bgp009 gene, two — large tegument protein gene, one — major binding protein gene and two targeting hypothetical Bgp071 protein gene) and four HHV-7 (targeting nuclear phosphoprotein, tegument phosphoprotein, large tegument protein and immediately early A transactivator gene) nPCRs was determined. The most sensitive HHV-6 nPCR was targeted Bgp071 protein gene, which could detect 5 genomic copies of HHV-6. The most sensitive and reproducible HHV-7 nPCR assay, targeting nuclear phosphoprotein gene, could detect 1 genomic copy of HHV-7. The reproducibility of the selected HHV-6 and HHV-7 nPCRs was evaluated in five different laboratories. The results obtained in all laboratories were identical to our results, confirming that these nPCRs are useful as assays for molecular diagnostics of HHV-6 and HHV-7 infection.
The aim of this study was to investigate the possibility of using monocytes/macrophages as mediators in human herpesvirus-6 (HHV-6) infection of thyroid gland tissues in autoimmune thyroiditis (AIT). Seventy-three AIT patients were enrolled in this study. The control group consisted of 80 blood donors. Monocyte/macrophage isolation for AIT patient samples was performed by adherence. HHV-6 was detected in peripheral blood mononuclear cell (PBMC) DNA samples using nested polymerase chain reaction (nPCR). Gene expression of HHV-6 active infection marker (U79/80) and chemokine receptors (U12, U51) in patient monocyte/macrophage samples and blood donor PBMC samples was detected using reverse-transcription PCR. HHV-6 viral load was detected by using quantitative-PCR technique. The HHV-6 genomic sequence was found significantly more frequently among AIT patient than control group samples. Markers of active infection were found in 8 AIT patient monocyte/macrophage samples (11%) and in none of control group PBMC samples. HHV-6 U51 mRNA expression was detected only in AIT patient samples (2/24 previously positive for HHV-6). Since HHV-6 genomic sequences were found significantly more frequently in AIT patient samples and active infection markers were found in patient monocytes/macrophages, our results suggest that monocytes/macrophages may be used by HHV-6 as mediators for thyroid gland infection.
Viral infections have been frequently cited as important environmental factors implicated in autoimmune thyroiditis (AIT) development, although no specific virus has yet been conclusively associated with the disease. Some evidence implicates human herpesvirus-6 (HHV-6) in this disease. The aim of this study was to investigate the role of the HHV-6 U83 gene expression in autoimmune thyroiditis development. Fifty-one patients with AIT following thyroidectomy and a control group of 30 autopsied subjects without thyroid pathologies for comparing virology results and 30 healthy blood donors for comparing serology results were enrolled in this study. HHV-6 U83 gene expression was determined using nested PCR with complementary DNA as the template acquired from thyroid gland extracted RNA. Plasma samples of AIT patients and blood donors were tested for IL-2, IL-4, IL-10, sTNF-RII and IL-1beta levels by ELISA. Virology results were compared with pro- and anti-inflammatory cytokine levels to determine possible interaction of HHV-6 with host immune response. HHV-6 U83 gene expression was found only in 24% (12/49) of AIT patient thyroid gland tissue samples and in none of the control group individuals, showing possible involvement of this gene in AIT development. However, no interaction between HHV-6 and changes in cytokine levels was found.
Human herpesviruses HHV-6 and HHV-7 reactivation in transplantation is associated with indirect immunomodulatory effects, such as cytomegalovirus (CMV) disease, increased opportunistic infections, graft dysfunction and acute rejection (AR). In this study, we analysed the clinical and immunological outcomes in renal transplant recipients (RTR) with active HHV-6 and HHV-7 infection. Between January 2007 and December 2007, clinical, virological and immunological tests were carried out in 46 RTR. The patients were divided into three groups: with active HHV-6 infection; with active HHV-7 infection; and without infection (control). The mean follow-up was 14 ± 2.5 months. At three months after renal transplantation (RT), active CMV infection was present in 12 (26%); HHV-6 in four (8.6%); and HHV-7 in nine (19.5%) of RTR. Active ß-herpesviruses infection was not associated with more frequent AR and worsening of graft function in recipients at different times after RT. The lymphocyte subsets (CD3+; CD4+ and CD8+ cell count) were considerably lower in RTR before RT. At 3 months after RT CD19+ and CD25+ cell counts were significantly increased in the HHV-7 group compared with the control group (P < 0.05). Significant differences were not found in clinical and immunological outcomes between patients with active ß-herpesviruses infection and those without active ß-herpesviruses infection.
The Förster Resonance Energy Transfer (FRET) method has wide application in modern science for studying protein–protein interactions and conformational changes. FRET allows to assess molecular interactions by measuring energy transfer between acceptor and donor fluorophores coupled to the molecule(s) of interest. The method demands high precision in experimental design, experimental settings and correct data interpretation. Therefore, we tested several parameters to estimate FRET measurement accuracy in our Nikon wide-field fluorescence FRET system. The experiments were performed in a HEK-293 cell line transfected with DNA constructs expressing Calcium Release-Activated Channel (CRAC) subunits STIM1 and ORAI1 coupled to donor fluorophore Cyan Fluorescent Protein (CFP) and acceptor fluorophore Yellow Fluorescent Protein (YFP), respectively. Exposure time and approach of data analysis varied throughout experiments in order to optimise FRET data quality. Dependence of FRETeff values on measurement quality and donor/acceptor fluorophore ratio in the cells was estimated. We demonstrated that, using the wide-field fluorescence FRET system, minimising the exposure of fluorophores before measurement using neutral density (ND) filters considerably minimises undesirable photo-bleaching of the fluorophores. There was a strong correlation between the CFP/YFP ratio in the cells and the observed FRET level, suggesting that only cells with certain donor/acceptor ratio might be comparable. We also showed impact of FRET measurement quality, defined as accordance of FRET pixels to Gaussian distribution, on FRET artefacts. Knowledge obtained during our experiments may be important for approbating similar wide-field fluorescence FRET systems to study two separate molecule interactions and for understanding the correct setup of the experiments and data interpretation.
This study aimed to determine peripheral blood mononuclear cells’ (PBMC) proliferative response to parvovirus B19 (B19) antigens in rheumatoid arthritis (RA) patients and possible changes in proliferative response due to chemotherapy. Serum and blood samples of 52 RA patients and 25 sex and age matched healthy individuals were examined for the presence of anti-B19 IgG and IgM class antibodies and virus specific DNA sequence by the recomLine B19 test and nested polymerase chain reaction, respectively. The PBMC proliferative activity was estimated on the 3rd and 6th day of PBMC cultivation in the presence of virus and B19 VP1/VP2 peptide, using thymidine incorporation assay. On the 3rd day, PBMC response to B19 antigens was detected in 74.1% RA patients with active, in 44.8% - with remote and in 40% - with latent stage of persistent B19 infection, while in the control group the response was observed only in two individuals with active viral infection. On the 6th day, the response was found in 50% RA patients with active, 68.9% - with remote and in 80% - with latent stage of latent persistent infection as well as in 41.1% remotely infected control individuals. The highest PBMC mean stimulation indices were detected in the RA patients and control persons with active infection as well as in RA patients with latent stage of persistent viral infection. On the 3rd and 6th day, strong proliferative response was significantly more frequently observed in RA patients not receiving methotrexate treatment, compared to the patients receiving methotrexate treatment in different combinations with other drugs. RA patients had more frequent and faster response to B19 antigens than apparently healthy persons.
The relationship between HHV-6 and HHV-7 reactivation and development of post-autologous peripheral stem cell transplantation complications was examined. The presence of viral genomic sequences in whole peripheral blood and cell free plasma was determined by nested PCR, HHV-6 and HHV-7 load by real-time PCR, virus specific antibodies and cytokines in serum by ELISA, and HHV-6 variants by restriction endonuclease analysis. Clinical features, reactivation of viruses and serum TNF- α, and IL-6 concentrations were determined in seventy-six patients with Roseolovirus infection before and after transplantation. Anti-HHV-6 antibodies were found in 62 of 76 (81.6%) patients before transplantation. A significantly higher rate of single HHV-7 infection was found in patients with viral infection in comparison with single HHV-6 infection (p = 0.0003) and concurrent (HHV-6 and HHV-7) infection (p = 0.0017). Complications after transplantation developed in 30.3% of patients and reactivation of viruses was detected in all of these patients. Significant increase of HHV-6 and HHV-7 reactivation with simultaneous increase of pro-inflammatory cytokines serum levels suggests that both viruses may be involved in the development of complications after autologous peripheral blood stem cell transplantation via their immunomodulatory ability. The kinetics of the Roseolovirus reactivation may reflect the potential role of HHV-7 as a co-factor for HHV-6 activation.
Microvascular free flap surgery is a complex method of wound closure for large wounds. Tissue trauma, surgical stress and general anaesthesia are known immunosuppressors that may exacerbate postoperative infections. Beta-herpesviruses HHV-6 and HHV-7 are immunomodulating viruses highly prevalent in the population of healthy individuals, which can interfere with the function of the host immune system. These viruses can be reactivated in immunosuppressed conditions. The aim of this study was to monitor the potential effects of two different anaesthesia techniques - general anaesthesia (GA) and regional anaesthesia (RA) - on the activation of HHV-6 and HHV-7 infection in relation to changes in the total lymphocyte count and peripheral immune cell distribution after microvascular free flap surgery. We found significant increase in the frequency of active HHV-7 infection after surgery (p < 0.05) in the GA group. In the RA group changes were not significant. The activation of HHV-7 infection was associated with decrease in the total lymphocyte count post-operatively in patients from the GA group. The data of our study show that reconstructive flap surgery under GA is linked with more frequent postoperative lymphopenia, which is a potential post-operative immunosuppressor that probably triggers the activation of HHV-6 and HHV-7 infection