Surface immunoglobulin-deficient Epstein-Barr virus-infected B cells in the peripheral blood of pediatric solid-organ transplant recipients

Epstein-Barr virus (EBV), a ubiquitous human herpesvirus, normally causes an asymptomatic latent infection with very low levels of circulating virus in the peripheral blood of infected individuals. However, EBV does have pathogenic potential and has been linked to several diseases, including posttransplant lymphoproliferative disease (PTLD), which involves very high circulating viral loads. As a consequence of immunosuppression associated with transplantation, children in particular are at risk for PTLD. Even in the absence of symptoms of PTLD, very high viral loads are often observed in these patients. EBV-infected B cells in the circulations of 16 asymptomatic pediatric solid-organ transplant recipients from Children's Hospital of Pittsburgh were simultaneously characterized for their surface immunoglobulin (sIg) isotypes and EBV genome copy numbers. Patients were characterized as having high and low viral loads on the basis of their stable levels of circulating virus. Patients with high viral loads had both high- and low-copy-number cells. Cells with a high numbers of viral episomes (>20/cell) were predominantly Ig null, and cells with low numbers of episomes were predominantly sIgM positive. Patients with low viral loads carried the vast majority of their viral load in low-copy-number cells, which were predominantly IgM positive. The very rare high-copy-number cells detected in carriers with low viral loads were also predominantly Ig-null cells. This suggests that two distinct types of B-lineage cells contribute to the viral load in transplant recipients, with cells bearing high genome copy numbers having an aberrant Ig-null cellular phenotype.     

B-cell lymphoproliferative disorders in solid-organ transplant patients: detection of Epstein-Barr virus by in situ hybridization.

B-cell lymphoproliferative disorders (BLPDs) occur in approximately 2% of transplant recipients and are frequently fatal. Indirect serologic evidence has implicated Epstein-Barr virus (EBV) as an etiologic factor in these lesions. Direct evidence of the presence of EBV in these lesions has been obtained in relatively few cases. We used in situ hybridization (ISH) with a probe for the BamHI-W region of the EBV genome to study 52 tissue specimens from 28 solid-organ transplant patients who had BLPD. Epstein-Barr virus-infected lymphoid cells were identified in 26 of these 28 patients. The two patients without ISH evidence of EBV infection showed no distinctive clinical, morphologic, or serologic features. Previous filter-hybridization studies of these two patients had demonstrated evidence of EBV infection. Seven additional transplant patients without evidence of BLPD were studied as controls and showed no evidence of EBV in their lymphoid cells by ISH. These data provide further support for the etiologic role of EBV in the pathogenesis of posttransplantation lymphoproliferative disorders.     

用荧光原位杂交从母血中检测胎儿细胞

目的 从母血中分离胎儿细胞并确定其来自胎儿。方法 从孕早、中期各２０名、分娩后１５名母血中富集并分离有核细胞。用Ｙ特异性探针（ＰＹ３．４）行荧光原位杂交,从中识别胎儿细胞。结果 孕早、中期孕妇各怀１５名男胎。阳性细胞比例是１：６５２８．０及１：２７３．８。与同期１０名女胎阳性细胞相比,差别有高度显著性。分娩１周内的３名,阳必民孕中期的差别没有显著性。分娩３个月后的阳性率与比,差别有高度显著性。分娩     

肺癌组织中EB病毒感染的检测

目的探讨原发性肺癌中EB病毒（Epstein—Barr virus，EBV）的存在情况及EBV与原发性肺癌的关系。方法唐山市人民医院和开滦医院病理科储存的2001--2006年肺癌手术切除石蜡包埋肺癌组织108份，癌旁组织22份，以EBV阳性鼻咽癌组织为阳性对照，用原位杂交法（ISH）检测肺癌患者石蜡包埋组织标本中EBV编码的小RNA（EBERl），并采用图像分析法进行形态学定量。结果癌组织及癌旁组织EBERl的阳性率分别为33．3％（36／108）和4．5％（1／22），二者间差异有统计学意义（P〈0．01）。鳞癌、腺癌、小细胞癌及大细胞癌中EBV感染率分别是35．9％（14／39）、31．6％（12／38）、31．0％（9／29）和1／2。EBV感染与患者年龄、性别和组织学类型无关，但与肺癌的部位、癌组织分化程度有关，右肺明显高于左肺，中低分化癌明显高于高中分化癌。结论唐山地区原发性肺癌组织中EBV感染率为33．3％，EBV感染可能是肺癌的潜在病因之一，在癌组织分化的不同阶段有不同的作用。     

High levels of Epstein-Barr virus DNA in blood of solid-organ transplant recipients and their value in predicting posttransplant lymphoproliferative disorders

Epstein-Barr virus (EBV) DNA was quantitated in peripheral blood mononuclear cells (PBMC) from 25 healthy subjects, 105 asymptomatic solid-organ transplant (SOT) recipients, and 15 SOT recipients with symptomatic EBV infections by using a newly developed quantitative-PCR technique. Patients with symptomatic EBV infections had significantly higher (P < 0.001) median EBV DNA levels than asymptomatic SOT recipients and immunocompetent individuals. In SOT recipients, the positive predictive value of EBV DNA levels of >1, 000 genome equivalents (GE)/0.5 microg of total PBMC DNA was 64.7% for symptomatic EBV infection, while the negative predictive value was 96.1%. In 19 of 32 (59.3%) asymptomatic SOT recipients, EBV DNA levels were consistently below 1,000 GE for as long as 18 months, while 10 of 32 (31.2%) patients had 1,000 to 5,000 EBV GE at least once during follow-up. In a minority of patients (3 of 32; 9.3%), >/=5,000 GE could be detected at least once during follow-up. Reduction of immunosuppressive treatment decreased EBV DNA levels by >/=1 log(10) unit in patients with symptomatic EBV infections. Quantification of EBV DNA is valuable for the diagnosis and monitoring of symptomatic EBV infections in SOT recipients.     

Detection of Epstein-Barr virus DNA in sera from transplant recipients with lymphoproliferative disorders

Early diagnosis of Epstein-Barr Virus (EBV)-associated posttransplant lymphoproliferative disease (PTLD) is important because many patients respond to reduction in immunosuppression, especially if PTLD is detected at an early stage. Previous studies have found elevated EBV DNA levels in blood from patients with PTLD, but these assays required isolation of cellular blood fractions and quantitation. We evaluated the presence of cell-free EBV DNA in serum from solid-organ transplant recipients as a marker for PTLD. Five of 6 transplant recipients with histopathologically documented PTLD had EBV DNA detected in serum at the time of diagnosis (sensitivity = 83%), compared with 0 of 16 matched transplant recipients without PTLD (specificity = 100%) (P < 0.001 [Fisher's exact test]). Furthermore, EBV DNA was detected in serum 8 and 52 months prior to the diagnosis of PTLD in two of three patients for whom stored sera were analyzed. Detection of EBV DNA in serum appears to be a useful marker for the early detection of PTLD in solid-organ transplant recipients. Further studies to define the role of such assays in evaluating solid-organ transplant patients at risk for PTLD are warranted.     

Detection of the Epstein-Barr virus in primary gastric lymphoma by in situ hybridization

The Epstein-Barr virus (EBV) has been shown to be associated with numerous human malignancies including Burktt's lymphoma and nasopharyngeal lymphoepithelioma. In addition, some typical gastric adenocarcinomas were also recently reported to demonstrate EBV relevance. The present study was designed to detect EBV in primary gastric lymphoma, using the in situ hybridization (ISH) method, in which oligo-nucleotide probes for the EBERl RNA and the EBV DNA W region have been used. Of the 49 cases of primary gastric lymphoma studied, which all showed B cell immunopheno-type, EBER1 sequences could only be found in four cases, including two low-grade cases and two high-grade cases of histological subtypes while the number of positive cells was less than 50% of the tumor cells. In one case of low-grade mucosa associated lymphoid tissue (MALT) lymphoma, the EBER1 -positive neoplastic cells were found in the regional lymph node, but the primary site of the stomach showed no positive signals. The EBV presence was further confirmed by the EBV DNA ISH. Using the ISH method, rare or occasional positive lymphoid cells (probably non-tumorous bystander cells) could be detected in 10 other cases including all histological subtypes. The present study shows that only a small proportion of primary gastric lymphoma is associated with EBV, and such positive cases could be found in both high- and low-grade histological subtypes. It is also suggested that the EBV presence in the neoplastic cells of some cases of primary gastric lymphoma is most likely a secondary phenomenon.     

Pediatric solid-organ transplant recipients carry chronic loads of Epstein-Barr virus exclusively in the immunoglobulin D-negative B-cell compartment

Solid-organ transplant recipients are at risk for development of lymphoproliferative diseases. The purpose of this study was to examine the distribution of Epstein-Barr virus (EBV) load in the peripheral blood of pediatric transplant recipients who had become chronic viral load carriers (>8 copies/10(5) lymphocytes for >2 months). A total of 19 patients with viral loads ranging from 20 to 5,000 viral genome copies/10(5) lymphocytes were studied. Ten patients had no previous diagnosis of posttransplant lymphoproliferative disease (PT-LPD), while nine had recovered from a diagnosed case of PT-LPD. No portion of the peripheral blood viral load was detected in the cell-free plasma fraction. Viral DNA was found in a population of cells characterized as CD19(hi) and immunoglobulin D negative, a phenotype that is consistent with the virus being carried exclusively in the memory B-cell compartment of the peripheral blood. There was no difference in the compartmentalization based upon either the level of the viral load or the past diagnosis of an episode of PT-LPD. These results have implications for the design of tests to detect EBV infection and for the interpretation and use of positive EBV PCR assays in the management of transplant recipients.     

Detection of fetal erythroid cells from maternal blood using fluorescence in situ hybridization and liquid culture

Fetal nucleated erythrocytes circulating in maternal blood are a potential source of fetal DNA for noninvasive prenatal genetic diagnosis. However, the estimated ratio of fetal to maternal cells is extremely small. In order to enrich these cells, we performed direct culture using a two-phase liquid system. Mononuclear cells were obtained from maternal blood samples at 8-10(+3) weeks of gestation and cultured in the first phase. After 4-5 days, the nonadherent cells were harvested and recultured with erythropoietin in the second phase for another 3-5 days. We examined cellular morphology, and counted the number of benzidine- positive cells and the percentage of glycophorin A/CD71 positive erythroid cells. We also did Kleihauer-Betke stain for Hb F, polymerase chain reaction (PCR) for SRY/DYZ1, chromosome analysis, and fluorescence in situ hybridization (FISH). The number of total erythroid cells reached about 0.1 x 10(6)-1.0 x 10(6)/mL with a purity of 84.0-97.3%. Hb F stain showed total erythroid cells of approximately 0.4 x 10(4)-9.8 x 10(4)/mL. Male DNA was detected in one case by PCR. In this case, the XY karyotype was confirmed by FISH and amniocentesis. This approach provides enriched source of fetal cells for further prenatal genetic analysis without complicated separation or sorting procedures.     

Detection of Epstein-Barr virus infection in the epithelial cells and lymphocytes of non-neoplastic tonsils by in situ hybridization and in situ PCR

Summary.  Non-neoplastic tonsils were analyzed for detection of Epstein-Barr virus (EBV)-positive cells by in situ hybridization and in situ PCR. EBV-encoded small nuclear RNA 1(EBER1)-positive cells were found in 28.2% of the tonsils and were evenly localized in the extrafollicular area and within germinal centers. Latent membrane protein 1 (LMP1)-positive cells were also dispersed in the extrafollicular and germinal center. Using in situ DNA-DNA hybridization, the EBV-positive signals were observed in the upper epithelial cell layers of the tonsils. In addition, in situ PCR detected EBV DNA-positive cells in the lower epithelial cell layers and lymphoid cells. Accepted December 1, 1997 Received September 16, 1997     

The detection of Epstein-Barr virus in hairy cell leukemia cells by in situ hybridization.

Epstein-Barr virus (EBV) has been implicated in the pathogenesis of several B-cell lymphoid proliferations. Because patients with hairy cell leukemia (HCL) have a high incidence of seropositivity for EBV antigens, we studied the cells of HCL for evidence of EBV infection using in situ hybridization techniques. EBV mRNA was detected in the tumor cells in four of six cases using a radiolabeled RNA probe. Confirmatory serologic data were available in three cases in which the viral DNA was detected and in one negative case. Our results suggest that EBV infection may have a pathogenetic role in this disorder.     

套式PCR和原位杂交技术检测肝病患者单个核细胞内TTV DNA

目的　了解慢性乙型肝炎(中度)和原发性肝癌(HBsAg阳性)患者PBMC内TTVDNA存在情况。方法　采用套式PCR以及原位杂交技术检测外周血单个核细胞(PBMC)内TTVDNA。结果　套式PCR检测26例慢性乙型肝炎(中度)患者PBMC内TTVDNA阳性7例,阳性率269%,非常显著高于健康对照(χ2=143,P<0.001);21例原发性肝癌(HBsAg阳性)患者PBMC内TTVDNA阳性4例,阳性率190%,显著高于健康对照(χ2=486,P<0.05)。7例PBMC内TTVDNA阳性的慢性乙型肝炎(中度)患者PBMC原位杂交,其中4例细胞内阳性。结论　慢性乙型肝炎(中度)和原发性肝癌(HBsAg阳性)患者PBMC细胞浆内存在TTVDNA,且阳性率相当高。     

Detection of Epstein-Barr virus DNA in mouthwashes by hybridization.

An assay for the presence of Epstein-Barr virus (EBV) DNA was developed by using a cloned EBV DNA probe. After preliminary testing showed the assay to be sensitive and specific, it was applied to 135 mouthwashes from bone marrow transplant recipients, and 21 of these tests were positive. The concentration of EBV DNA in mouthwashes in some cases was as high as 10(8) genome equivalents per ml. When compared with the lymphocyte transformation assay on the same samples, the sensitivity was 75% and the specificity was 97%. In contrast to the lymphocyte transformation assay, the hybridization was semiquantitative and yielded results in 72 h. Potential applications include monitoring the effects of various interventions, such as immunosuppressive and antiviral chemotherapy, on EBV shedding.     

Detection of subtle reciprocal translocations by fluorescence in situ hybridization

Three different subtle reciprocal translocations were detected on long, well-banded chromosomes. The same translocations were examined using fluorescence in situ hybridization (FISH) with chromosome-specific libraries and unique DNA sequences. Our findings show that FISH allows rapid and unequivocal detection and characterization of this type of chromosome rearrangement. This approach is especially useful for prenatal diagnosis when one of the parents is a balanced carrier of such small fragment translocations.     

Detection of Epstein-Barr virus in lymphoid tissues of patients with infectious mononucleosis by in situ hybridization.

Although the immunological response during infectious mononucleosis (IMN) has been studied in detail, little is known about the spread of Epstein-Barr virus (EBV) in lymphoid organs or the topographical distribution of the infected cells. In this study, EBV was detected in 11 lymph nodes, 4 tonsils, and 1 spleen of 16 patients with IMN. The predominant cell type positive for the EBV genome was identified as small lymphocytes localized chiefly within typical T areas, preferentially in perifollicular and interfollicular regions of the lymph node. A few endothelia of epithelioid venules were also found to be positive. Furthermore, a small number of sinus lining cells of lymph nodes exhibited labelling. Altogether, only a small number of cells, not exceeding 1 per cent of all cells, were infected with EBV. Our results show that only a small number of lymphocytes carry the EBV and that besides B lymphocytes, other cell constituents of lymphatic tissues are infected by EBV during IMN.     

套式PCR和原位杂交技术检测肝病患者单个核细胞？…

目的 了解慢性乙型肝炎（中度）和原发性肝癌（ＨＢｓＡｇ阳性）患者ＰＢＭＣ内ＴＴＶ ＤＮＡ存在情况。方法 采用套式ＰＣＲ以及原位杂交技术检测外周血单个核细胞（ＰＢＭＣ）内ＴＴＶ ＤＮＡ。结果 套式ＰＣＲＳＷＩＭ２６例慢性乙型肝炎（中度）患者ＰＢＭＣ内ＴＴＶ ＤＮＡ阳性７例。阳性率２６．９％,非常显著高于健康对照（Ｘ＾１２＝１４．３,Ｐ〈０．００１）;２１例原发性肝癌（ＨＢｓＡｇ阳性）虱ＰＢＭＣ内ＴＴ     

Detection of Epstein-Barr virus genomes by in situ DNA hybridization with a terminally biotin-labeled synthetic oligonucleotide probe from the EBV Not I and Pst I tandem repeat regions

We describe a case of acute, disseminated Epstein-Barr virus (EBV) infection which was analyzed for the cellular distribution of viral replication by automated, colorimetric in situ DNA hybridization using a single, synthetic, terminally biotin-labeled oligonucleotide probe composed of 23 consecutive nucleotides selected from the EBV Not I region. The GC-rich, Not I region is a 125-base pair sequence that is repeated in tandem an average of 12.6 times in the EBV genome. The synthetic sequence had 91% base homology with another EBV genomic tandem repeat, the 102-base pair Pst I region, which is also GC-rich, has an overall 70% homology with the Not I region, and is reiterated about 25 times in the viral DNA. Disseminated EBV infection was detected in nuclei of atypical lymphocytes in several organs, including lung, bronchus, trachea, spleen, liver, and stomach, with the probes. In addition, the synthetic oligomer compared favorably with a significantly more expensive, nick translated, biotinylated probe cloned from the BAM HI-V (W), large internal repeat region. This 3.0-kilobase pair (kbp) sequence is repeated an average of 11 times in EBV. Although both probes identified regions repeated multiple times in the virus, and each confirmed an identical tissue distribution for the infection, the signal obtained with the Not I/Pst I probe was more intense and confined to the nuclei of fewer lymphocytes than the general, more weakly distributed signal obtained with the probe from the large internal repeat region. Consistent positive cellular staining was obtained with the Not I/Pst I probe in both EBV-infected control tissue culture cells and in formalin-fixed, paraffin-embedded tissue sections.(ABSTRACT TRUNCATED AT 250 WORDS)     

Partial elimination of latent Epstein-Barr virus genomes from virus-producing cells by cyclohexamide.

EBV-producing P3 HRI cells were converted to a nonproductive state by treatment with cyclohexamide, and the number of EBV genomes decreased from 350 to 7 genomes per cell. Clones isolated from soft agar showed variations in the number of viral genomes per cell, ranging from 1 to 20. IUDR induced viral antigens and produced infectious viruses in all the clones except one, clone #9 carrying a single copy of EBV genome per cell. Growth characteristics of the clones were the same regardless of the number of EBV genomes present. The role of cyclohexamide on elimination of viral genomes and the significance of isolation of a clone with only a single copy of EBV genome per cell are discussed.