In vivo Downregulation of MHC Class I Molecules by HCMV Occurs During All Phases of Viral Replication but Is Not Always Complete.

Based on cell culture data, MHC class I downregulation by HCMV on infected cells has been suggested as a means of immune evasion by this virus. In order to address this issue in vivo, an immunohistochemical analysis of tissue sections from biopsy and autopsy materials of HCMV infected organs was performed. HCMV antigens from the immediate early, early, and late phase of viral replication, and cellular MHC class I molecules were detected simultaneously or in serial sections by immuno-peroxidase and immuno-alkaline phosphatase techniques. Investigated organs included lung, gastrointestinal tract, and placenta. Colocalization of MHC molecules with sites of viral replication as well as MHC expression in individual infected cells were analyzed. To detect immune effector cells at sites of viral replication, leukocytes, CD8+ lymphocytes, and HCMV antigens were stained in serial sections. While strong MHC class I expression was detected in the cells surrounding infected cells, it appeared downregulated in the majority of infected cells themselves, particularly in the late replication phase. Despite significantly reduced MHC class I signals on infected cells, sites of infection were infiltrated by inflammatory cells that consisted predominantly of CD8+ lymphocytes. The extent of inflammatory infiltrates was negatively correlated with the extent of HCMV infected cells. Taken together, our findings indicate that HCMV can downmodulate MHC class I expression in vivo, whereas cytokines originating from infiltrating immune effector cells probably up regulates MHC class I expression in noninfected bystander cells. The presence of cytotoxic lymphocytes in close contact to infected cells may reflect control of viral spread by these cells despite MHC class I downmodulation.

Personalized healthcare in clotting disorders.

In terms of managing thrombotic disorders, genotype-based individualized patient care emerged as early as 1994 when the association of factor V Leiden (G1691A), and later, prothrombin (G20210A), with thrombotic phenotypes were discovered. Since then, genetic tests for specific thrombophilic SNPs have been routinely incorporated into daily practices in both thrombotic risk assessment and clinical decision-making with respect to prophylactic anti-thrombotic therapy. Recently, the area of pharmacogenomics in major anti-thrombotic drugs, such as warfarin and clopidogrel, has been the principal driver for personalized therapy based on one's own individual characteristics.

Peritubular capillary C4d staining in late acute renal allograft rejection--is it relevant?

BACKGROUND: In the early post-transplant period, renal allograft rejection with diffuse peritubular capillary (PTC) C4d deposition predicts poor graft survival. In the late post-transplant setting, that is, one or more yr after transplantation, the implication of diffuse PTC C4d deposition is still a topic of debate. The purpose of our study was to see if diffuse PTC C4d deposition, in late acute rejection (LAR), occurring more than one yr post-transplant, has any impact on graft survival and function. METHODS: We selected cases, both cadaveric as well as living donor renal transplant recipients, in whom acute rejection with PTC C4d deposition was first detected after the first year post-transplant. Recipients with multiple acute rejection episodes during the first year post-transplant were excluded from the study. The first biopsy diagnosed with LAR was considered the index biopsy (n = 40). We formed two groups: group 1, C4d-positive LAR (n = 20), and group 2, C4d-negative LAR (n = 20). Groups were matched for maintenance and post-rejection immunosuppressive therapy, baseline serum creatinine levels before the time of the index biopsy, time from transplant to index biopsy, as well as chronic allograft damage index (CADI) score in the index biopsies. We compared the rate of graft loss, and the graft function of the surviving grafts at the end of the study period, as well as histologic parameters in the index biopsy specimens between the two groups. The mean follow-up period was 20 months. RESULTS: No significant differences in the rate of graft loss or graft function were found between groups 1 and 2 at the end of the follow-up period. Histologically, PTC margination and transplant glomerulopathy were more common in the C4d-positive group, and this difference was statistically significant. There was no statistically significant difference in the degree of plasma cell infiltrates. CONCLUSIONS: Unlike in the acute setting, the presence or absence of PTC C4d staining in renal allografts with LAR may not have a predictive value regarding graft outcome.

The re-emerging concept of personalized healthcare.

Personalized healthcare has regained momentum through the unprecedented surge of research in the genomics field and related areas of biology. These new insights compel the optimization of healthcare and therapy for individual subjects. While the potential benefits are large, substantial obstacles need to be overcome for attaining clinical utility in general medial practice. These range from scientific hurdles emerging from biological and genetic complexity, to cultural, economic, legal, regulatory and ethical issues. This article addresses, in broad strokes, these intercalated issues, discussing recent advances, remaining questions and how to move forward. It also highlights recent developments at academic centers devoted to promoting personalized healthcare as an avenue with great potential for improving our healthcare system.

Lipopolysaccharide, tumor necrosis factor alpha, or interleukin-1beta triggers reactivation of latent cytomegalovirus in immunocompetent mice.

We have previously shown that cytomegalovirus (CMV) can reactivate in lungs of nonimmunosuppressed patients during critical illness. Our recent work has shown that polymicrobial bacterial sepsis can trigger reactivation of latent murine CMV (MCMV). We hypothesize that MCMV reactivation following bacterial sepsis may be caused by inflammatory mediators. To test this hypothesis, BALB/c mice latently infected with Smith strain MCMV received sublethal intraperitoneal doses of lipopolysaccharide (LPS), tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), or saline. Lung tissue homogenates were evaluated for viral reactivation 3 weeks after mediator injection. Because LPS is known to signal via Toll-like receptor 4 (TLR-4) in mice, further studies blocking this signaling mechanism were performed using monoclonal MTS510. Finally, mice were tested with intravenous TNF-alpha to determine whether this would cause reactivation. All mice receiving sublethal intraperitoneal doses of LPS, TNF-alpha, or IL-1beta had pulmonary reactivation of latent MCMV 3 weeks following injection, and LPS caused MCMV reactivation with kinetics similar to those for sepsis. When TLR-4 signaling was blocked, exogenous LPS did not reactivate latent MCMV. Intravenous TNF-alpha administration at near-lethal doses did not reactivate MCMV. Exogenous intraperitoneal LPS, TNF-alpha, and IL-1beta are all capable of reactivating CMV from latency in lungs of previously healthy mice. LPS reactivation of MCMV appears dependent on TLR-4 signaling. Interestingly, intravenous TNF-alpha did not trigger reactivation, suggesting possible mechanistic differences that are discussed. We conclude that inflammatory disease states besides sepsis may be capable of reactivating CMV from latency.

Pulmonary cytomegalovirus reactivation causes pathology in immunocompetent mice.

OBJECTIVE: Cytomegalovirus (CMV) is a ubiquitous herpes virus that persists in the host in a latent state following primary infection. We have recently observed that CMV reactivates in lungs of critically ill surgical patients and that this reactivation can be triggered by bacterial sepsis. Although CMV is a known pathogen in immunosuppressed transplant patients, it is unknown whether reactivated CMV is a pathogen in immunocompetent hosts. Using an animal model of latency/reactivation, we studied the pathobiology of CMV reactivation in the immunocompetent host. DESIGN: Laboratory study. SETTING: University laboratory. SUBJECTS: Cohorts of immunocompetent BALB/c mice with or without latent murine CMV (MCMV+/MCMV-). INTERVENTIONS: Mice underwent cecal ligation and puncture. Lung tissue homogenates were evaluated after cecal ligation and puncture for tumor necrosis factor-alpha, interleukin-1beta, neutrophil chemokine KC, and macrophage inflammatory protein-2 messenger RNA by polymerase chain reaction and real-time quantitative reverse transcription-polymerase chain reaction. Because pulmonary tumor necrosis factor-alpha expression is known to cause pulmonary fibrosis, trichrome-stained sections of lung tissues were analyzed using image analysis to quantitate pulmonary fibrosis. In a second experiment, a cohort of MCMV+ mice received ganciclovir (10 mg/kg/day subcutaneously) following cecal ligation and puncture. Tumor necrosis factor-alpha messenger RNA and pulmonary fibrosis were evaluated as described previously. MEASUREMENTS AND MAIN RESULTS: All MCMV+ mice had CMV reactivation beginning 2 wks after cecal ligation and puncture. Following reactivation, these mice had abnormal tumor necrosis factor-alpha, interleukin-1beta, neutrophil chemokine KC, and macrophage inflammatory protein-2 messenger RNA expression compared with controls. Image analysis showed that MCMV+ mice had significantly increased pulmonary fibrosis compared with MCMV- mice 3 wks after cecal ligation and puncture. Ganciclovir treatment following cecal ligation and puncture prevented MCMV reactivation. Furthermore, ganciclovir-treated mice did not demonstrate abnormal pulmonary expression of tumor necrosis factor-alpha messenger RNA. Finally, ganciclovir treatment prevented pulmonary fibrosis following MCMV reactivation. CONCLUSIONS: This study shows that CMV reactivation causes abnormal tumor necrosis factor-alpha expression, and that following CMV reactivation, immunocompetent mice have abnormal pulmonary fibrosis. Ganciclovir blocks MCMV reactivation, thus preventing abnormal tumor necrosis factor-alpha expression and pulmonary fibrosis. These data may explain a mechanism by which critically ill surgical patients develop fibroproliferative acute respiratory distress syndrome. These data suggest that human studies using antiviral agents during critical illness are warranted.

Human cytomegalovirus protein pp71 disrupts major histocompatibility complex class I cell surface expression.

The human cytomegalovirus tegument protein pp71 is the product of the UL82 gene. Roles for pp71 in stimulating gene transcription, increasing infectivity of viral DNA, and the degradation of retinoblastoma family proteins have been described. Here we report a novel function for pp71 in limiting accumulation of cell surface major histocompatibility complex (MHC) class I complexes. MHC molecules were analyzed in glioblastoma cells exposed to a replication-defective adenovirus expressing UL82 (Adpp71) or after transient transfection of the UL82 gene. Accumulation of cell surface MHC class I levels diminished in a specific and dose-dependent manner after exposure to Adpp71 but not after exposure to an adenovirus expressing beta-galactosidase (Adbeta gal). UL82 expression did not interfere with accumulation of either MHC class I heavy-chain transcript or protein, nor did UL82 expression correlate with markers of apoptosis. Rather, UL82 expression correlated with an increased proportion of MHC class I molecules exhibiting sensitivity to endoglycosidase H treatment. Finally, we show that, in cells infected with recombinant virus strain missing all of the unique short region MHC class I evasion genes, disruption of UL82 expression by short, interfering RNAs led to increased accumulation of cell surface MHC class I complexes. These findings support a novel role for HCMV pp71 in disruption of the MHC class I antigen presentation pathway.

The association of prior cytomegalovirus infection with neovascular age-related macular degeneration.

PURPOSE: To determine if prior exposure to pathogens associated with vascular disease, cytomegalovirus, Chlamydia pneumoniae, and Helicobacter pylori correlates with neovascular age-related macular degeneration (AMD). DESIGN: An experimental study. SETTING: Institutional. Bascom Palmer Eye Institute, October 2001 to December 2002. PATIENT POPULATION: 150 patients (47 neovascular amd, 36 dry amd, and 67 non-amd controls) were included in the study. exclusion criteria included hiv infection, malignancy, recent acute illness requiring hospitalization within 6 months, or immunosuppressive illness. PROCEDURE: Serum samples were obtained for analysis of cytomegalovirus, chlamydia pneumoniae, and helicobacter pylori igg antibody titers by elisa. MAIN OUTCOME MEASURE: Comparison of the distribution of igg titers between patients with wet amd, dry amd, and controls. RESULTS: The average cytomegalovirus IgG titer was higher in patients with wet AMD versus controls (p = 0.02, Student t-test, two-tailed) and patients with dry AMD (p = 0.06). Twenty-six (55%) of 47 subjects with wet AMD had high cytomegalovirus IgG titers compared with 14 (39%) of 36 patients with dry AMD (odds ratio [OR] = 2.23, 95% confidence interval [CI] = 0.77 to 6.44) and 23 (34%) of 67 control patients (OR = 2.49, 95% CI = 0.98 to 6.33). There was no major difference in the distribution of titers for Chlamydia pneumoniae IgG and Helicobacter pylori IgG in wet and dry AMD patients. Five of 47 patients with wet AMD (11%) had high antibody titers to all three pathogens, compared with only 1 of 36 patients with dry AMD (3%) (OR = 4.17, 95% CI = 0.46 to 37.36). CONCLUSIONS: There was a significant association of high cytomegalovirus IgG titer with neovascular AMD compared with dry AMD and control patients. Chronic infection with cytomegalovirus may be a novel risk factor for the progression from dry to neovascular AMD.

Analysis of the molecular quality of human tissues: an experience from the Cooperative Human Tissue Network.

The scientific usefulness of the data obtained from tissue analysis is related to specimen quality, which may be affected by conditions that may contribute to the degradation of the specimen before processing and analysis. We determined the usability of nucleic acids extracted from banked human tissues for further molecular analyses. We assayed 151 tissue specimens, storedfor various times at 4 divisions of the Cooperative Human Tissue Network, National Cancer Institute, Bethesda, MD, for DNA and RNA degradation. Simple electrophoresis, polymerase chain reaction (PCR), reverse-transcriptase (RT)-PCR, and Northern blot analysis were compared to determine the optimal quality control procedure. In addition, a time course degradation procedure was performed on human lung tissue. Gel electrophoresis was as informative as PCR, RT-PCR, and Northern blot analysis in determining the molecular usefulness of the human tissues. Overall, 80% of the stored human tissues had good-quality DNA, and 60% had good-quality RNA. Electrophoresis procedures for DNA and RNA offer a quick and valuable measure of the molecular quality of stored human tissues. The DNA and RNA degradation of one tissue type (lung) was stable for both nucleic acids for up to 5 hours after excision.

Parvovirus B19 associated adult Henoch Schönlein purpura.

BACKGROUND: Parvovirus B19 has recently been implicated in various vasculitic syndromes including Henoch Schönlein purpura (HSP), Wegener's granulomatosis and microscopic polyarteritis. The association was established through serology, the identification of DNA in the peripheral blood and affected tissues and more recently by RNA localization to cutaneous capillary endothelium. However, direct localization of the viral DNA to the glomerular and cutaneous endothelium in HSP in correlation with the histopathologic findings has not been demonstrated. METHODS: Skin and kidney biopsy tissues were processed for hematoxylin and eosin, immunofluorescent, polymerase chain reaction (PCR) and reverse transcriptase in situ PCR studies. CASE PRESENTATION: A 64-year-old-female presented with palpable purpura and nephrotic range proteinuria. Kidney and skin biopsies showed IgA-associated mesangioproliferative glomerulonephritis and IgA-associated leukocytoclastic vasculitis, respectively. A diagnosis of HSP was rendered. Her clinical course was refractory to prednisone. Parvovirus B19 DNA and tumor necrosis factor alpha DNA were identified in the dermal and glomerular capillary endothelial cells and surrounding dermal inflammatory cells. CONCLUSION: This is the first documentation of B19 localization to dermal and glomerular capillary endothelium in HSP. It is important to recognize parvovirus B19-associated adult HSP cases, as the treatment of choice is intravenous gamma globulin in concert with anti-TNFalpha therapy. In contrast immunosuppressive therapy may lead to a persistent and/or worsening disease course.

The expression of the cytomegalovirus chemokine receptor homolog US28 sequesters biologically active CC chemokines and alters IL-8 production.

We hypothesized that US28, a cytomegalovirus (CMV) CC chemokine receptor homolog, plays a role in modulating the host antiviral defense. Monocyte chemotaxis was induced by supernatants from fibroblasts infected with a US28 deletion mutant of CMV (CMV Delta US28) due to endogenously produced CC chemokines MCP-1 and RANTES. However, these chemokines were sequestered from the supernatants of CMV-infected cells that did express US28. US28 was also capable of sequestering exogenously added RANTES. Surprisingly, cells infected with CMV Delta US28 transcribed and secreted increased levels IL-8, a CXC chemokine, when compared to CMV-infected cells. Finally, because chemokines are potent mediators of immune cell migration through the endothelium, we characterized the CC chemokine binding potential of CMV-infected endothelial cells. We propose that US28 functions as a 'chemokine sink' by sequestering endogenously and exogenously produced chemokines and alters the production of the CXC chemokine IL-8, suggesting that CMV could significantly alter the inflammatory milieu surrounding infected cells.

Human cytomegalovirus disrupts constitutive MHC class II expression.

CD8(+) and CD4(+) T lymphocytes are important in controlling human CMV (HCMV) infection, but the virus has evolved protean mechanisms to inhibit MHC-based Ag presentation and escape T lymphocyte immunosurveillance. Herein, the interaction of HCMV with the MHC class II Ag presentation pathway was investigated in cells stably transfected with class II transactivator. Flow cytometry experiments demonstrate that HCMV infection decreases cell-surface MHC class II expression. HCMV down-regulates MHC class II surface expression without a significant effect on class II RNA or steady-state protein levels. SDS-stability and confocal microscopy experiments demonstrate normal levels of steady-state peptide-loaded class II molecules in infected cells and that class II molecules reach late endosomal and HLA-DM positive peptide-loading compartments. However, MHC class II positive vesicles are retained in an abnormal perinuclear distribution. Finally, experiments with a mutant HCMV strain demonstrate that this novel mechanism of decreased MHC class II expression is not mediated by one of the known HCMV immunomodulatory genes. These defects in MHC class II expression combined with previously identified CMV strategies for decreasing MHC class I expression enables infected cells to evade T lymphocyte immunosurveillance.

Intra-abdominal bacterial infection reactivates latent pulmonary cytomegalovirus in immunocompetent mice.

Critically ill surgery patients are susceptible to pulmonary reactivation of latent cytomegalovirus (CMV), but what triggers this reactivation is unknown. Immunosuppression and bacterial sepsis are thought to stimulate reactivation of CMV, and in this study it was hypothesized that immunosuppressive effects of surgery with or without concomitant bacterial infection may reactivate latent CMV. Mice infected with CMV were allowed to develop latent infections. Latently infected mice underwent a laparotomy with cecal ligation and puncture (CLP; n=30), a laparotomy alone (sham; n=10), or no surgery (control; n=5). Lung tissue homogenates were evaluated for viral activity, and, 2 and 3 weeks after CLP, lungs of 7 of 7 and 5 of 5 mice, respectively, showed reactivation of latent CMV. In contrast, lungs from all sham-operated animals and controls showed no viral reactivation. These findings demonstrate that surgery with subsequent intra-abdominal bacterial infection reactivated CMV in lungs of latently infected mice. The mechanism of this reactivation is unknown but likely involves cytokines induced by sepsis.