EBV+ B Lymphoma Cell Lines from Patients with Post-Transplant Lymphoproliferative Disease Are Resistant to TRAIL-Induced Apoptosis

Lymphomas associated with post-transplant lymphoproliferative disease (PTLD) represent a significant complication of immunosuppression in transplant recipients. In immunocompetent individuals, EBV-specific cytotoxic T lymphocytes (CTL) prevent the outgrowth of activated B lymphoblasts through apoptosis induction. Soluble versions of TNF-related apoptosis-inducing ligand/Apo2 ligand (TRAIL) can induce apoptosis in numerous tumor cell types. Given the therapeutic potential of TRAIL, we examined the sensitivity of EBV⁺ spontaneous lymphoblastoid cell lines (SLCL) derived from patients with PTLD to treatment with soluble TRAIL. Despite abundant expression of TRAIL receptors (TRAIL-R), resistance to TRAIL-induced apoptosis was observed in all SLCL examined. This resistance could not be overcome by concomitant treatment with several pharmacological agents. Unlike BJAB positive control cells, for each SLCL tested, cleavage and activation of caspase 8 was inhibited due to failed recruitment of FADD and caspase 8 to TRAIL receptors upon stimulation. Further indicative of a proximal defect, TRAIL receptor aggregation could not be detected on the cell surface of SLCL following ligand engagement. These results suggest that the use of TRAIL for eliminating PTLD-associated tumors may be of limited clinical utility, and illustrate another mechanism by which EBV⁺ B lymphoma cells can evade tumor surveillance at the level of death receptor signaling.     

Establishment and characterization of a chronic infectious mononucleosislike syndrome in common marmosets

Epstein-Barr virus (EBV) was inoculated into two species of marmosets. Successful infection was established in the majority of the animals of one species, Callithrix jacchus, as evidenced by the development of high, persistent levels of antibody against virus-specific capsid and early nonstructural proteins. Antibodies also were produced against the major membrane antigen and, in some animals, against EBV nuclear antigen (EBNA) 2 but not against EBNA 1. This is the antibody profile normally noted in individuals with chronic infectious mononucleosis (IM). EBV-induced lymphoproliferation was not seen, and EBV-specific proteins were not detected in the peripheral blood lymphocytes of infected animals. Hence, EBV infection in C. jacchus apparently does not generally include extensive B-cell involvement. However, the marmosets clearly are useful as a model for EBV primary infection and also possibly for chronic IM.     

Post-Transplant Lymphoproliferative Disorders Occurring After Renal Transplantation in Adults: Report of 230 Cases From the French Registry

Post-transplant lymphoproliferative disorders (PTLD) are a rare but serious complication after organ transplantation. A French Registry of PTLD was set up in a nationwide population of kidney transplant recipients. We prospectively enrolled all adult kidney recipients developing PTLD between January 1, 1998, and December 31, 2003. We analyzed the incidence, risk and prognostic factors of PTLD by Kaplan-Meier and Cox analyses. Totally 230 cases of PTLD were referred to the French Registry. Cumulative incidence was 1.18% after 5 years. Older age (per year, AHR = 2.19, CI = 1.22-3.94) and recipient Epstein-Barr virus seronegativity (AHR = 3.01, CI = 1.57-5.08) were associated with an increased risk of PTLD. Patients with PTLD had a reduced survival rate (61% at 5 years). Graft PTLD had the best prognosis with an 81% survival rate after 5 years. Infection with hepatitis C or B virus (HCV or HBV), late-onset PTLD, multiple sites involvement and high Ann Arbor staging were risk factors for patient death. Use of azathioprine was associated with a poorer survival rate. PTLD incidence and risk factors in French recipients are in line with the international or American PTLD series. We highlighted the role of HBV or HCV in patient mortality and described the relevant prognosis factors for patients with post-transplant lymphoproliferations.     

Bilateral facial palsy: Epstein–Barr virus, not Lyme disease

Bilateral facial palsy is frequently linked with lyme disease. We report a patient with bilateral facial palsy due to Epstein-Barr virus infection but with Borrelia burgdorferi IgM in serum caused by polyclonal B-lymphocyte stimulation.     

Analysis of Killer Cell Activities in Epstein-Barr Virus Infections

Using spontaneously established autologous lymphoblastoid B cell lines (LCL), killer cell activities were studied in children with severe infectious mononucleosis (IM), chronic IM, and acute IM, and compared with those in EBV-seropositive normal controls. Natural killer (NK) cell activity of fresh peripheral blood mononuclear cells (PBMC) was normal in acute IM patients, but it was low in four of six patients with severe or chronic IM. Recombinant inter-leukin 2 (rIL-2)-activated PBMC from normal controls showed lymphokine-activated killer (LAK) cell activity against the respective autologous LCL. The levels of LCL lysis by LAK cells were significantly higher in acute IM patients, lower in chronic IM patients, and much lower in severe IM patients. In contrast to the fact that PBMC stimulated in vitro with autologous LCL (IVS cells) from normal controls and acute IM patients showed potent killing of autologous LCL, IVS cells from severe or chronic IM patients showed lower levels of LCL lysis, which were markedly augmented in three patients by rIL-2 addition to the cultures. These killer cell dysfunctions appear to be responsible for the severe or chronic course of EBV infection.     

Early Impairment of CD8+ T Cells Immune Response Against Epstein–Barr Virus (EBV) Antigens Associated with High Level of Circulating Mononuclear EBV DNA Load in HIV Infection

Immunodeficiency related to HIV may increase the incidence of EBV-associated lymphomas, by altering EBV-specific immune control and consequently favoring EBV reactivation. The aim of the present study was to assess the relationship between the decrease of EBV-specific cellular immunity and the increase of EBV reactivation in a prospective cohort of 72 unselected HIV-infected individuals. EBV-specific immunity was evaluated by a highly sensitive IFN-gamma ELISPOT assay using 22 peptides mimicking latent and lytic antigens, and circulating mononuclear (PBMC) EBV DNA load was quantified by real-time quantitative PCR. The mean circulating cell-associated EBV DNA load was higher in HIV-infected patients (639 copies/10(6) PBMC) than in healthy controls (21, n = 14) ( P = 0.005) and was higher in patients with CD4(+) T-cell count below 350/microL than that in patients harboring higher CD4(+) T-cell count (1112 vs. 389, P = 0.003). The mean intensity of EBV-specific cellular responses was lower in HIV-infected patients than in controls ( P = 0.001), even in patients with CD4(+) T-cell count above 350/-microL ( P = 0.007). The number of EBV peptides recognized was lower in HIV-infected patients than in controls (frequency: 0.44 vs. 0.67; P = 0.02), indicating reduced polyclonality in HIV-infected patients. The polyclonality was 1.5-fold lower in HIV-infected patients with CD4(+) T-cell count below 350/-microL ( P =0.007). For EBV load >1000 copies/10(6) PBMC, the levels of cell-associated EBV DNA and those of EBV-specific cellular immunity, either in intensity or in polyclonality, or both, were inversely correlated. These findings demonstrate early impairment of the EBV-specific cellular immune control with progressive increase of EBV reactivation in the course of HIV infection. These observations likely provide a basis for appreciating the risk to develop non-Hodgkin's lymphomas in HIV-infected individuals.     

The effect of phenytoin in vitro on normal human mononuclear cells and on human lymphoblastoid B cell lines of different Ig isotype specificities

The anticonvulsant drug phenytoin, in less than cytotoxic concentrations, caused significant reductions in Ig secretion by unstimulated or EBV-stimulated normal MNC, as measured by PFC or secretion of Ig into the culture medium. Isotype-specific LBL varied in their sensitivity, the secretion of IgA (1 line) and IgG (3 lines) being reduced by phenytoin near therapeutic concentrations, whereas that of IgM (1 line) was resistant. Six-day exposure of MNC to phenytoin caused no selective depletion of or enrichment for B cells, monocytes or T cell subsets. The results suggest that the reduction in serum Ig levels reported in phenytoin-treated epileptic patients is, at least in part, due to a direct effect of the drug on the B lymphocyte. However, among EBV-activated normal MNC, those secreting IgA were no more sensitive to the drug than those secreting IgG or IgM, and other factors may, therefore, operate to cause the preferential reduction in serum IgA in phenytoin-treated patients.     

巢式PCR检测龈下菌斑中HCMV、EBV、HSV-1与慢性牙周炎活动性的关系

目的　建立临床检测龈下菌斑标本中人巨细胞病毒 (HCMV)、Epstein Barr病毒 (EBV)、单纯疱疹病毒 1型 (HSV 1 )巢式PCR方法 ,研究这 3种病毒与慢性牙周炎活动性的关系。方法　收集6 2例慢性牙周炎患者 (男性 2 7例、女性 35例 ,平均年龄 5 3岁 )的牙周炎活动部位、牙周炎静止部位的龈下菌斑 ,提取DNA后使用巢式PCR检测HCMV、EBV、HSV 1 ,比较分析其在同一病人不同部位的检出率。结果　牙周炎活动部位的HCMV检出率为 38.7%,EBV的检出率为 5 8%,HSV 1的检出率为30 .6 %,两种以上病毒合并感染的检出率为 4 0 .3%;牙周炎静止部位的HCMV检出率为 1 4 .5 %,EBV为 2 2 .6 %,HSV 1为 1 1 .3%,两种以上病毒合并感染的检出率为 1 1 .3%。这 3种病毒及其合并感染在牙周炎活动部位的检出率均高于牙周炎静止部位 ,差异有统计学意义 (P <0 .0 5 )。结论　提示HC MV、EBV、HSV 1与慢性牙周炎的活动性相关。     

Characterization of a monoclonal anti-Bw4 antibody (Tü109): Evidence for similar epitopes on the Bw4 and Bw6 antigens

The production and serologic, as well as immunochemical properties of a cytotoxic murine IgG monoclonal antibody (Tü109) that precipitates HLA-class I molecules, are described. In the microcytotoxicity assay Tü109 supernatant was demonstrated on a panel of 424 HLA-ABC, -DR, -DQ, -MT typed normal Caucasian blood donors to define an epitope on HLA-B locus molecules in great association with the supertypic specificity Bw4. Reactivity of supernatant showed MHC linked inheritance of the Tü109 determinant and discriminated the HLA-Bw4/Bw6 associated HLA-B locus split antigens. Weak or lack of binding on lymphocytes from some HLA-Bw4 heterozygous individuals, particularly typing for HLA-Bw44, appeared to be due to qualitative and/or quantitative variations of HLA-B locus molecules on the cell surface. With Tü109 ascites fluid, however, extra-reactivity on all HLA-Bw6⁺ cells was demonstrated. Preferential binding of supernatant to HLA-Bw4, but reactivity of ascites fluid with HLA-Bw6⁺ molecules in addition, was furthermore confirmed by IEF analysis of antigens immunoprecipitated with Tü109 from cell lysates. Thus the antibody may help to analyze the evolutionary relationship of the diallelic specificities Bw4 and Bw6.     

EB病毒LMP2A特异性CTL的体外诱导及分析

用EBV LMP2A重组痘苗病毒 (rVV LMP2A )转染人树突状细胞 (DC ) ,转染后的DC分别在第 1、 7、 14天刺激相同MHC背景的T细胞 ,在IL 2作用下诱导LMP2A特异性CTL。用LDH释放法检测CTL杀伤活性 ;流式细胞术 (FACS )检测CTL诱导分化过程中CD3+ 、CD4 + 、CD8+ 、CD5 6 + 等细胞的分群变化 ;RT PCR检测细胞分化过程中FasLmRNA表达 ;生物活性法检测功能性细胞因子IFN γ的分泌。结果显示本法诱导的CTL对靶细胞有特异性杀伤活性 ,第 2次和第 3次DC刺激后杀伤活性有所上升 ;在CTL诱导分化的第 7、 14、 2 1天细胞分群以CD4 + 、CD8+ 细胞为主 ;RT PCR证实所诱导的细胞内有FasLmRNA的表达 ;随细胞培养天数的增加IFN γ分泌增加 ,在第 14天达到较高水平。研究表明重组痘苗病毒载体rVV LMP2A转染的DC刺激T细胞可诱导出EBV LMP2A特异性CTL。