Jill Crouch

Defective Control of Latent Epstein-Barr Virus Infection in Systemic Lupus Erythematosus

EBV infection is more common in patients with systemic lupus erythematosus (SLE) than in control subjects, suggesting that this virus plays an etiologic role in disease and/or that patients with lupus have impaired EBV-specific immune responses. In the current report we assessed immune responsiveness to EBV in patients with SLE and healthy controls, determining virus-specific T cell responses and EBV viral loads using whole blood recall assays, HLA-A2 tetramers, and real-time quantitative PCR. Patients with SLE had an ∼40-fold increase in EBV viral loads compared with controls, a finding not explained by disease activity or immunosuppressive medications. The frequency of EBV-specific CD69+ CD4+ T cells producing IFN-γ was higher in patients with SLE than in controls. By contrast, the frequency of EBV-specific CD69+ CD8+ T cells producing IFN-γ in patients with SLE appeared lower than that in healthy controls, although this difference was not statistically significant. These findings suggest a role for CD4+ T cells in controlling, and a possible defect in CD8+ T cells in regulating, increased viral loads in lupus. These ideas were supported by correlations between viral loads and EBV-specific T cell responses in lupus patients. EBV viral loads were inversely correlated with the frequency of EBV-specific CD69+ CD4+ T cells producing IFN-γ and were positively correlated with the frequencies of CD69+ CD8+ T cells producing IFN-γ and with EBV-specific, HLA-A2 tetramer-positive CD8+ T cells. These results demonstrate that patients with SLE have defective control of latent EBV infection that probably stems from altered T cell responses against EBV.

Reduced incidence of Epstein-Barr virus-associated posttransplant lymphoproliferative disorder using preemptive antiviral therapy.

BACKGROUND Posttransplant lymphoproliferative disorder (PTLD) has been observed with increasing frequency consequent to the availability of more effective and potent immunosuppression. Prior work suggested that a peripheral blood monitoring strategy detecting peripheral B lymphoproliferation was effective in the early diagnosis of PTLD among 7 of 179 (3.9%) consecutive transplant recipients. Each of those seven patients received at least one course of antithymocyte globulin, Minnesota antilymphocyte globulin, or OKT3 before developing PTLD. METHODS To determine whether antiviral prophylaxis might reduce the incidence of PTLD, a subsequent group of 198 consecutive recipients received either ganciclovir or acyclovir during antilymphocyte antibody administration. When the donor or recipient were cytomegalovirus-seropositive, ganciclovir was given; acyclovir was used when both were cytomegalovirus-seronegative. Baseline and protocol posttransplant cell surface profiles were obtained using immunofluorescence and flow cytometry to detect T cells, lymphocyte activation markers, and the CD19 B cell antigen. RESULTS Demographic factors, including the incidence of recipients more than 50 years of age, non-Caucasians, previous transplantation, and diabetes mellitus, were similar in both groups. Additionally, the number of patients receiving antilymphocyte preparations was similar. However, only one patient (0.5%) from the latter group who received preemptive antiviral therapy developed PTLD. Although elevations in CD19+ B cells preceded clinical PTLD among each of the seven earlier patients, evidence of peripheral B cell proliferation was not demonstrated for the sole patient from the latter group, which suggests a possible effect of antiviral therapy. CONCLUSIONS Prophylactic antiviral therapy may reduce the sensitivity of peripheral monitoring for B lymphoproliferation, but the dramatic reduction in PTLD incidence strongly supports its use among transplant recipients at risk.

Platelet PlA2 polymorphism enhances risk of neurocognitive decline after cardiopulmonary bypass

BACKGROUND Neurocognitive decline, often produced by atherosclerotic plaque embolization, remains a frequent complication of cardiopulmonary bypass. Plaque fragments may initiate local thrombosis, which, in turn, aggravates the embolic insult. Prothrombotic genetic factors may exacerbate this process. We investigated whether the PlA2 polymorphism of platelet GPIIIa, a prothrombotic risk factor in other cardiovascular settings, is associated with early neurocognitive decline after cardiopulmonary bypass. METHODS Neurocognitive changes were evaluated by the Mini-Mental State Examination administered preoperatively and on postoperative day 4 and the PlA genotype determined in 70 patients undergoing cardiopulmonary bypass. RESULTS Forty-nine patients were PlA1/A1, and 21 were PlA1/A2 or PlA2/A2. Fifty-two patients (74%) demonstrated post-cardiopulmonary bypass neurocognitive decline, of which 34 were PlA1/A1 and 18 were PlA1/A2 or PlA2/A2 Multivariate analysis revealed that the PlA2 genotype and baseline Mini-Mental State Examination were significantly associated with greater neurocognitive decline (decreased Mini-Mental State Examination scores, p = 0.036 and 0.024, respectively). CONCLUSIONS This study demonstrates a link between the PlA2 allele of platelet GPIIIa and more severe neurocognitive decline after cardiopulmonary bypass. Although the mechanism is unknown, it could represent exacerbation of platelet-dependent thrombotic processes associated with plaque embolism.

Endocarditis Caused by Culture-Negative Organisms Visible by Brown and Brenn Staining: Utility of PCR and DNA Sequencing for Diagnosis

ABSTRACT Two cases of culture-negative endocarditis with cocci seen in valve vegetations are presented. The organisms were identified by molecular analysis using broad-range PCR primers complementary to the 16S rRNA gene, sequencing, and database search using BLAST software. The results and utility of this method are discussed.

Platelet PlA2 polymorphism and platelet activation are associated with increased troponin I release after cardiopulmonary bypass.

Background The PlA2 polymorphism of platelet glycoprotein IIIa has been identified as a prothrombotic risk factor in a number of cardiovascular settings. The aim of this study was to determine whether the PlA2 polymorphism of platelet glycoprotein IIIa and degree of platelet activation were associated with more severe myocardial injury as indicated by troponin I release following cardiopulmonary bypass. Methods The PlA genotype was determined in 66 patients undergoing elective coronary artery bypass grafting requiring cardiopulmonary bypass. Troponin I concentrations and the percentage of circulating, activated (CD62P+) platelets were measured at predetermined intervals perioperatively. Results Forty-six patients were PlA1,A1, and 20 were PlA1,A2 or PlA2,A2. Patients with at least one PlA2 allele had significantly greater postoperative troponin I concentrations than PlA1 homozygotes (P = 0.006, analysis of variance). Peak troponin I concentrations also correlated significantly with the increase in circulating, activated platelets (P = 0.02, Spearman rank correlation). Conclusions The PlA2 allele of platelet glycoprotein IIIa is associated with higher troponin I concentrations following cardiopulmonary bypass surgery, suggesting that this platelet polymorphism contributes to perioperative myocardial injury.

Epstein-Barr virus suspension cell assay using in situ hybridization and flow cytometry

We have developed a procedure for quantitative assay of Epstein-Barr virus (EBV)-infected cells in suspension in either latent or replicative phase using in situ hybridization and flow cytometry. The cells were hybridized with EBV-specific digoxigenin or biotin-labeled oligonucleotide probes, followed by binding to fluorescein-conjugated anti-digoxigenin or phycoerythrin-conjugated streptavidin, respectively. The cells hybridizing to the specific probes were quantitated by flow cytometry. A strong shift in fluorescence intensity (20-fold) was observed when the EBV-positive culture cells were hybridized with a specific EBER1 antisense probe. The sensitivity of the assay was at least one positive cell out of 9,000 beyond the normal control mean +/- 2 S.D. We performed two-color in situ hybridization/flow cytometry using probes to an EBV replication phase-specific mRNA and EBER1 on B95-8 cells in which a small portion (2-4%) of cells induce spontaneously into the replicative phase. In addition, we have developed a method for simultaneous analysis of the cell surface phenotype and EBV nucleic acid content in individual cells.

Bacterial Etiology for Chronic Villitis Is Not Supported by Polymerase Chain Reaction for 16S rRNA DNA

Chronic villitis is characterized by chorionic villi infiltrated by lymphocytes, histiocytes, and sometimes plasma cells. In a small percentage of cases, an infectious agent can be demonstrated within areas of chronic villitis. However, the pathogenesis of most lesions is idiopathic. Chronic villitis may represent the direct spread of chronic endometrial infection by bacterial organisms that are particularly problematic for culture. To test this hypothesis, polymerase chain reaction (PCR) using primers for the universal bacterial 16S rRNA DNA was performed on DNA extracted from areas of chronic villitis selected from placentas in the Yale Pathology database. Specific areas of chronic villitis were first confirmed by examination of sections stained with hematoxylin and eosin and then removed from archived paraffin blocks. Control tissue spiked with known bacterial counts was also prepared to test the sensitivity of the experiment. All tissue was deparaffinized, dehydrated, and digested with proteinase K. DNA extraction was performed with the Gentra Puregene kit. PCR was done using primers p11 and p13 for the 16S rRNA DNA. The 233-bp amplified target product was identified by agarose gel electrophoresis. Nineteen specimens with multifocal chronic villitis without confinement to anchoring villi were studied. None of the chronic villitis specimens had a demonstrable product using the PCR primers for 16S rRNA DNA, despite adequate DNA in the samples and controls. The assay was sensitive down to approximately 1500 bacteria per specimen. In conclusion, these data do not support a bacterial etiology for chronic villitis.

Systemic Tropheryma whippleii Infection Associated With Monoclonal B-Cell Proliferation: A Helicobacter pylori–Type Pathogenesis?

We report a case of Whipple disease in a 55-year-old woman who presented with arthralgia, weight loss, and lymphadenopathy. Tropheryma whippleii bacilli were identified in the mesenteric lymph nodes by diastase-resistant periodic acid-Schiff stain and confirmed by electron microscopy. Retrospectively, previous biopsy specimens from the duodenum and right axillary lymph node of this patient, which were initially considered to demonstrate reactive changes, also showed features consistent with involvement by Whipple disease. At the time of presentation, a large kappa-restricted monoclonal B-cell population with the phenotype CD20+CD19+CD5-CD10- was identified in the patients peripheral blood, lymph nodes, and bone marrow by flow cytometry study. The monoclonality of the mesenteric lymph node B cells was confirmed by immunohistochemical stain for kappa chain after antigen retrieval and also by polymerase chain reaction with the primer set targeting FR2-V(H). Routine cytogenetic study failed to reveal any chromosomal abnormalities, and polymerase chain reaction for Bcl-2 major and minor breakpoint cluster of t(14:18) was not detected. The monoclonal B cells have persisted in blood for the entire follow-up period (10 months). The possibility of reactive monoclonal B-cell proliferation versus Whipple disease-related B-cell lymphoma is discussed.

Epstein-Barr virus RNA detection and glandular differentiation in nasopharyngeal carcinoma: report of 2 cases.

Most tumors arising in the nasopharynx are either squamous cell carcinoma or so-called undifferentiated carcinoma of the nasopharyngeal type. Primary adenocarcinomas of the nasopharynx are rare, and glandular differentiation in undifferentiated carcinoma of the nasopharyngeal type has not been reported to date. We report 2 cases of undifferentiated carcinoma of the nasopharyngeal type that show distinct glandular differentiation by light microscopy, histochemistry, immunohistochemistry, and ultrastructure. Both tumors showed equal positivity for Epstein-Barr virus latent membrane protein and in situ hybridization for Epstein-Barr virus genome in the undifferentiated areas of the tumor and those featuring glandular differentiation.