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Biomedical subjects

Keith R Jerome

Publications and source records attributed to Keith R Jerome.

At least 19 recordsLinked to original sources
PubMed2006

Apoptosis and antigen receptor function in T and B cells following exposure to herpes simplex virus.

T cells are an essential component of the immune response against herpes simplex virus (HSV) infection. We previously reported that incubation of T cells with HSV-infected fibroblasts inhibits subsequent T cell antigen receptor signal transduction. In the current study, we found that incubation of T cells with HSV-infected fibroblasts also leads to apoptosis in exposed T cells. Apoptosis was observed in Jurkat cells, a T cell leukemia line, and also in CD4(+) cells isolated from human peripheral blood mononuclear cells. Direct infection of these cells with HSV also resulted in apoptosis. Clinical isolates of both HSV type 1 and 2 induced apoptosis in infected T cells at comparable levels to cells infected with laboratory strains of HSV, suggesting an immune evasion mechanism that may be clinically relevant. Further understanding of these viral immune evasion mechanisms could be exploited for better management of HSV infection.

Animals↗
PubMed2006

Multiplex real-time reverse transcription-PCR assay for determination of hepatitis C virus genotypes.

A variety of methods have been used to determine hepatitis C virus (HCV) genotypes. Because therapeutic decisions for chronic HCV-related hepatitis are made on the basis of genotype, it is important that genotype be accurately determined by clinical laboratories. Existing methods are often subjective, inaccurate, manual, time-consuming, and contamination prone. We therefore evaluated real-time reverse transcription-PCR (RT-PCR) reagents that have recently become commercially available (Abbott HCV Genotype ASR). The assay developed by our laboratory starts with purified RNA and can be performed in 4 to 5 h. An initial evaluation of 479 samples was done with a restriction fragment length polymorphism (RFLP) method and the RT-PCR assay, and discrepant samples were sequenced. An additional 1,200 samples were then tested, and data from all assays were used to evaluate the efficiency and specificity of each genotype-specific reaction. Good correlation between results by the two methods was seen. Discrepant samples included those indeterminate by the RT-PCR assay (n = 110) and a subset that were incorrectly called 2a by the RFLP method (n = 75). The real-time RT-PCR assay performed well with genotype 1, 2, and 3 samples. Inadequate numbers of samples were available to evaluate fully genotypes 4, 5, and 6. Analysis of each primer-probe set demonstrated that weak cross-reactive amplifications were common but usually did not interfere with the genotype determination. However, in about 1% of samples, two or more genotypes amplified at roughly equivalent amounts. Further studies are necessary to determine whether these mixed-genotype samples are true mixtures or a reflection of occasional cross-reactive amplifications.

Cross Reactions↗
PubMed2006

Inhibition of TCR signaling by herpes simplex virus.

T lymphocytes are an essential component of the immune response against HSV infection. We previously reported that T cells became functionally impaired or inactivated after contacting HSV-infected fibroblasts. In our current study, we investigate the mechanisms of inactivation. We report that HSV-infected fibroblasts or HSV alone can inactivate T cells by profoundly inhibiting TCR signal transduction. Inactivation requires HSV penetration into T cells but not de novo transcription or translation. In HSV-inactivated T cells stimulated through the TCR, phosphorylation of Zap70 occurs normally. However, TCR signaling is inhibited at linker for activation of T cells (LAT) and at steps distal to LAT in the TCR signal cascade including inhibition of calcium flux and inhibition of multiple MAPK. Inactivation of T cells by HSV leads to the reduced phosphorylation of LAT at tyrosine residues critical for TCR signal propagation. Treatment of T cells with tyrosine phosphatase inhibitors attenuates inactivation by HSV, and stimulus with a mitogen that bypasses LAT phosphorylation overcomes inactivation. Our findings elucidate a potentially novel method of viral immune evasion that could be exploited to better manage HSV infection, aid in vaccine design, or allow targeted manipulation of T cell function.

Calcium Signaling↗
PubMed2006

Altered CD4+/CD8+ T-cell ratios in cerebrospinal fluid of natalizumab-treated patients with multiple sclerosis.

BACKGROUND: Treatment with natalizumab, a monoclonal antibody against the adhesion molecule very late activation antigen 4, an alpha4beta(1) integrin, was recently associated with the development of progressive multifocal leukoencephalopathy, a demyelinating disorder of the central nervous system caused by JC virus infection. OBJECTIVE: To test the effect of natalizumab treatment on the CD4(+)/CD8(+) T-cell ratios in cerebrospinal fluid (CSF) and peripheral blood. DESIGN: Prospective longitudinal study. SETTING: Academic and private multiple sclerosis centers. PATIENTS: Patients with multiple sclerosis (MS) treated with natalizumab, untreated patients with MS, patients with other neurologic diseases, and human immunodeficiency virus-infected patients. MAIN OUTCOME MEASURES: CD4(+) and CD8(+) T cells were enumerated in CSF and peripheral blood. The mean fluorescence intensity of unbound alpha4 integrin on peripheral blood CD4(+) and CD8(+) T cells was analyzed before and after natalizumab therapy. RESULTS: Natalizumab therapy decreased the CSF CD4(+)/CD8(+) ratio of patients with MS to levels similar to those of human immunodeficiency virus-infected patients. CD4(+)/CD8(+) ratios in peripheral blood in patients with MS progressively decreased with the number of natalizumab doses, but they remained within normal limits. Six months after the cessation of natalizumab therapy, CSF CD4(+)/CD8(+) ratios normalized. The expression of unbound alpha4 integrin on peripheral blood T cells decreases with natalizumab therapy and was significantly lower on CD4(+) vs CD8(+) T cells. CONCLUSIONS: Natalizumab treatment alters the CSF CD4(+)/CD8(+) ratio. Lower expression of unbound alpha4 integrin on CD4(+) T cells is one possible mechanism. These results may have implications for the observation that some natalizumab-treated patients with MS developed progressive multifocal leukoencephalopathy.

Adolescent↗
PubMed2006

Immune surveillance in multiple sclerosis patients treated with natalizumab.

OBJECTIVE: Our objective was to test whether natalizumab, an antibody against very late activating antigen (VLA)-4, interferes with central nervous system immune surveillance as assessed by leukocyte cell numbers and cellular phenotypes in cerebrospinal fluid (CSF) and peripheral blood. METHODS: Cell numbers and cellular phenotypes in CSF and peripheral blood were analyzed in multiple sclerosis (MS) patients treated with natalizumab, untreated MS patients, and patients with other neurological disease (OND). JC virus DNA in the CSF and peripheral blood was quantified by kinetic polymerase chain reaction. RESULTS: CSF leukocyte counts, CD4(+) and CD8(+) T cells, CD19(+) B cells, and CD138(+) plasma cells were significantly lower in natalizumab-treated MS patients compared with OND patients and untreated MS patients. JC virus DNA was not detected in CSF or peripheral blood from natalizumab-treated patients. Six months after cessation of natalizumab therapy, low lymphocyte counts in the CSF persisted. The patient with the highest total leukocyte and CD4(+) and CD8(+)T-cell counts in the CSF experienced a clinical relapse. INTERPRETATION: These data suggest that natalizumab treatment results in a prolonged decrease of lymphocytes in the CSF and are consistent with the hypothesis that natalizumab impairs immune surveillance of the central nervous system.

Adult↗
PubMed2006

Herpes simplex virus genes Us3, Us5, and Us12 differentially regulate cytotoxic T lymphocyte-induced cytotoxicity.

Many viruses, including Herpes Simplex Virus (HSV), have developed strategies to avoid detection by cytotoxic T lymphocytes (CTLs). In this article, we evaluated the role of individual HSV-1 genes in preventing cytolysis and apoptosis, and in decreasing viral yield after CTL exposure of HSV-infected fibroblasts, using viruses deleted for the immune evasion gene Us12 or one of the two antiapoptotic genes Us3 and Us5. To evaluate CTL-mediated apoptosis, we used a flow cytometry assay measuring active caspase-3 in target cells. This assay was more sensitive than the chromium release assay used to evaluate cytolysis, and measured a different aspect of CTL cytotoxicity. Although virus with deletion of Us12 was markedly defective in the ability to prevent lysis of target fibroblasts, it retained most of its ability to protect target fibroblasts from CTL-induced apoptosis. Virus with deletion of Us3 was also defective in the ability to prevent lysis of target fibroblasts, yet such virus protected target fibroblasts from CTL-induced apoptosis as well as wild-type viruses. In contrast, Us5-deleted virus showed defects in the ability to protect target fibroblasts from both cytolysis and apoptosis after CTL attack. In addition, the replication of Us12-deleted virus was reduced compared with wild-type virus in fibroblasts subjected to CTL attack 6 h after infection, but showed equivalent replication when CTL attack occurred later. In contrast, Us3- or Us5-deleted virus showed no measurable defect in their ability to replicate in fibroblasts under CTL attack. Our data suggest that cytolysis, apoptosis, and viral yield do not necessarily correlate in infected cells under CTL attack. Furthermore, the Us3, Us5, and Us12 viral genes each have unique inhibitory effects on the different T lymphocyte cytotoxic effects. Taken together, these results suggest that HSV evasion of cellular immunity is multifacterial and complex, and relies on the partially redundant activities of various individual HSV proteins.

Animals↗
PubMed2005

A phase II multicenter study of visilizumab, humanized anti-CD3 antibody, to treat steroid-refractory acute graft-versus-host disease.

Results of a previous phase I study suggested that a single 3 mg/m2 dose of the humanized non-FcR-binding anti-CD3 monoclonal antibody visilizumab (Nuvion) was well tolerated and had efficacy for the treatment of steroid-refractory acute graft-versus-host disease (GVHD). We now report results of a multicenter phase II study in which visilizumab was given to 44 participants with steroid-refractory acute GVHD. Eighty-two percent of the participants had visceral involvement, and 86% had overall grade III or IV acute GVHD at study entry. The respective complete and overall response rates were 14% and 32% at 42 days. Plasma Epstein-Barr virus DNA increased to more than 1000 copies per milliliter in 19 subjects. Seventeen received rituximab, and no fatal lymphoproliferative disorders were observed. Survival at 180 days was 32% (95% confidence interval, 18%-46%). The administration of visilizumab as used in this study seems to be sufficiently safe and effective to warrant further assessment for treatment or prevention of GVHD.

Acute Disease↗
PubMed2005

Polyomavirus nephropathy in native kidneys of non-renal transplant recipients.

Chronic renal dysfunction is common in non-renal solid organ (SOT) and hematopoietic stem cell transplant (HSCT) recipients and is commonly attributed to calcineurin inhibitor toxicity, often without renal histopathologic evaluation. Polyomavirus nephropathy (PVN) is an important cause of allograft dysfunction in kidney transplant recipients but has rarely been reported in native kidneys of non-renal transplant recipients. We report the clinical, pathologic and virologic features of PVN in native kidneys of two allograft recipients. In both, severe renal dysfunction was accompanied by histopathologic evidence of PVN, including characteristic viral inclusions by routine stains, immunohistochemistry and electron microscopy. High levels of BK virus (BKV) DNA were detected in kidney tissue of patients using BKV-specific polymerase chain reaction (PCR). In 1 patient, high levels of BKV DNA were detected in plasma and urine, and administration of low-dose cidofovir was associated with clearance of BK viremia. These results extend the populations in which PVN has been documented in native kidneys to include heart and stem cell transplant recipients, and suggest that cidofovir has activity against BKV in vivo. Studies to define the incidence and potential contribution of PVN to chronic renal dysfunction commonly attributed to calcineurin inhibitor toxicity in non-renal transplant recipients are warranted.

Adult↗
PubMed2005

Calibration curves for real-time PCR.

BACKGROUND: Despite the increasing use of real-time PCR in the diagnosis and management of viral infections, there are no published studies adequately addressing the optimum number of calibrators, the number of replicates of each calibrator, and the frequency with which calibration needs to be repeated. This study was designed to address these issues. METHODS: Cycle threshold data (ABI 7700) was collected from >50 consecutive real-time PCR runs for hepatitis B and Epstein-Barr viruses. Our routine calibration curve made from serial 10-fold dilutions run in duplicate was compared with alternative options, including duplicate 100-fold dilutions, inclusion of a low-copy calibrator, and omission of the duplicate determination. Control data were used to examine the use of an average calibration curve made from multiple runs. RESULTS: Use of duplicate serial 10-fold dilutions led to the least imprecision, duplicate 100-fold dilutions had slightly higher imprecision, and calibration curves obtained with singlet measurements showed the greatest imprecision. For patient data, the duplicate 100-fold dilution calibration curve produced results that best matched those from the routine calibration curve. Use of singlet dilutions or inclusion of a low-copy calibrator produced poorer agreement. Variability in controls was lower with a daily calibration curve than with an average calibration curve. CONCLUSIONS: Duplicate 100-fold dilution calibration curves produced equivalent results and the same imprecision as curves with more calibrators, and thus are a valid alternative. Laboratories should carefully evaluate the variability resulting from the use of average calibration curves before adopting this approach.

Calibration↗
PubMed2004

The danger within.

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Adjuvants, Immunologic↗
PubMed2004

Cytomegalovirus quantitation by real-time PCR is unaffected by delayed separation of plasma from whole blood.

A previous study suggested that delayed sample preparation can cause a false-positive plasma cytomegalovirus (CMV) PCR. We evaluated 78 samples submitted for CMV testing, and we report that a 24-h delay in plasma separation did not affect viral quantitation by real-time PCR. Laboratories should evaluate specimen handling requirements for their own PCR technology.

Blood Specimen Collection↗
PubMed2004

Use of the MagNA pure LC automated nucleic acid extraction system followed by real-time reverse transcription-PCR for ultrasensitive quantitation of hepatitis C virus RNA.

Hepatitis C virus (HCV) infection is an increasing health problem worldwide. Quantitative assays for HCV viral load are valuable in predicting response to therapy and for following treatment efficacy. Unfortunately, most quantitative tests for HCV RNA are limited by poor sensitivity. We have developed a convenient, highly sensitive real-time reverse transcription-PCR assay for HCV RNA. The assay amplifies a portion of the 5' untranslated region of HCV, which is then quantitated using the TaqMan 7700 detection system. Extraction of viral RNA for our assay is fully automated with the MagNA Pure LC extraction system (Roche). Our assay has a 100% detection rate for samples containing 50 IU of HCV RNA/ml and is linear up to viral loads of at least 10(9) IU/ml. The assay detects genotypes 1a, 2a, and 3a with equal efficiency. Quantitative results by our assay correlate well with HCV viral load as determined by the Bayer VERSANT HCV RNA 3.0 bDNA assay. In clinical use, our assay is highly reproducible, with high and low control specimens showing a coefficient of variation for the logarithmic result of 2.8 and 7.0%, respectively. The combination of reproducibility, extreme sensitivity, and ease of performance makes this assay an attractive option for routine HCV viral load testing.

Automation↗
PubMed2003

CTL are inactivated by herpes simplex virus-infected cells expressing a viral protein kinase.

Numerous cell-to-cell signals tightly regulate CTL function. Human fibroblasts infected with HSV type 1 or 2 can generate such a signal and inactivate human CTL. Inactivated CTL lose their ability to release cytotoxic granules and synthesize cytokines when triggered through the TCR. Inactivation requires cell-to-cell contact between CTL and HSV-infected cells. However, inactivated CTL are not infected with HSV. The inactivation of CTL is sustainable, as CTL function remains impaired when the CTL are removed from the HSV-infected cells. IL-2 treatment does not alter inactivation, and the inactivated phenotype is not transferable between CTL, distinguishing this phenotype from traditional anergy and T regulatory cell models. CTL inactivated by HSV-infected cells are not apoptotic, and the inactivated state can be overcome by phorbol ester stimulation, suggesting that inactivated CTL are viable and that the signaling block is specific to the TCR. HSV-infected cells require the expression of U(S)3, a viral protein kinase, to transmit the inactivating signal. Elucidation of the molecular nature of this signaling pathway may allow targeted manipulation of CTL function.

Apoptosis↗
PubMed2003

Treatment of refractory BK virus-associated nephropathy with cidofovir.

BK virus-associated nephropathy (BKVN) has become recognized as an important cause of allograft dysfunction in renal transplant recipients and despite reduction in immunosuppression, 30-40% of recipients ultimately progress to allograft loss. Cidofovir is an antiviral agent that demonstrates in vitro activity against murine polyomavirus and has been proposed for treatment of BKVN in renal allograft recipients. We describe the clinical course, renal function, serial renal histology and urine and blood viral load measurements in two consecutive patients with refractory BKVN who were treated with low-dose cidofovir (0.25 mg/kg IV). In each case, renal dysfunction and BK viral load progressed despite reduced immunosuppression, and persistent BK virus infection was documented in serial renal allograft biopsy specimens. Administration of low-dose cidofovir was associated with clearance of BK virus DNA from blood and allograft, and stabilization of renal function in both patients, without significant toxicity. These preliminary data suggest that low-dose cidofovir may be tolerated, even among renal transplant recipients with significant renal dysfunction due to BKVN. Prospective, controlled trials are warranted to further define the optimal dose, toxicity and potential role of cidofovir in renal transplant recipients with BK virus nephropathy.

Adult↗
PubMed2003

Evaluation of real-time PCR versus PCR with liquid-phase hybridization for detection of enterovirus RNA in cerebrospinal fluid.

A LightCycler and two TaqMan real-time PCR assays were evaluated against an older PCR with liquid-phase hybridization method for the detection of enterovirus RNA in 74 patient samples. The two-step LightCycler and the two-step TaqMan formats correlated well with each other (r(2) = 0.90) and were equally sensitive compared to the liquid-phase hybridization method, whereas the one-step recombinant Tth DNA polymerase format was rather insensitive, detecting enterovirus RNA in only about one-half of those patient samples previously positive by liquid-phase hybridization. The two-step TaqMan method was optimized utilizing 10 micro l of cDNA and demonstrated the highest degree of analytical sensitivity among the methods evaluated in our study, being able to reproducibly quantify down to 510 copies of enteroviral RNA/ml of cerebrospinal fluid. This new assay can be performed in 4 h, is much less labor intensive, and showed less cross-reactivity with rhinovirus than the liquid-phase hybridization assay. Thus, the two-step TaqMan assay should prove useful in the diagnosis of enteroviral meningitis versus bacterial meningitis, thereby resulting in timely and appropriate clinical management that can amount to significant cost savings to the patient and health care system.

Computational Biology↗