Antibodies against extractable nuclear antigen in non-Hodgkin lymphoma patients

Autoantibodies are found at higher frequency in malignant lymphoproliferative diseases and also the association of these diseases with autoimmunity is documented. However precise mechanisms are not yet understood beyond these findings. We measured anti-extractable nuclear antigen (ENA) antibodies in non-Hodgkin's lymphoma patients before, during and after chemotherapy and compared these values to healthy controls. Sixty six lymphoma patients' data were compared with 30 healthy patients' data. ENA levels were significantly elevated in untreated lymphoma patients compared with healthy controls (1.85 U/l versus 0.68 U/l, P < 0.05). This increase could be observed during and after treatment as well. Those patients who responded well to initial chemotherapy were demonstrated with gradually increasing ENA antibody titers compared with the rest of patients, where a gradual decrease in titer was found. These findings are not yet statistically significant, but may help us further understand immunological reactions beyond the treatment of malignant lymphomas.     

Time-dependent effect of cyclosporin-A on the TSH-receptor antibody synthesis in patients with Graves' disease

Two patients with hyperthyroidism and Graves' ophthalmopathy were treated with cyclosporin A (CyA), in addition to methimazole, after failure of steroid therapy. Eye disease showed favorable responses and TSH receptor antibody concentration showed precipitous decline in concentrations compared to a gradual linear decline in antibody concentrations observed in 10 patients not treated with CyA. These results prompted us to investigate the in vitro influence of CyA on the synthesis of TSH receptor antibody by a patient's lymphocytes (with highest antibody concentration) in response to thyroid membrane antigen. CyA caused a dose-dependent reduction of TSH receptor antibody synthesis compared to control cultures. The effect of CyA was more marked when added to lymphocyte culture at the same time rather than 24 h after addition of antigen, consistent with CyA's interference of early T cell triggering by antigen. This study emphasizes the importance of helper T cells in synthesis of TSH-receptor antibody by cells and suggests that the drug may be therapeutically beneficial in severe Graves' ophthalmopathy and/or Graves' hyperthyroidism resistant to conventional treatment.     

Improved detection of lymphoid cell surface antigens in tissues fixed in periodate-lysine-paraformaldehyde (PLP)

Stabilization of cell surface antigens and preservation of tissue morphologic characteristics are important for diagnostic immunologic studies. Current reports continue to regard unfixed frozen sections as the material of choice for immunoperoxidase studies of lymphoproliferative diseases. In this study, periodate-lysine-paraformaldehyde (PLP) is shown to be a valuable fixative for the improved detection of surface antigens in lymphoid tissue. In cases of non-Hodgkin's lymphoma and Hodgkin's disease, more frequent detection of diagnostic markers and ease of interpretation was demonstrated by use of PLP-fixed frozen tissue as compared with unfixed frozen tissue. Immunoglobulin staining was more easily interpreted in 30% of B-cell non-Hodgkin's lymphoma. In Hodgkin's disease, Ki-1 antigen, a diagnostic marker of Reed-Sternberg cells, was found in PLP-fixed tissue from two cases in which this antigen was not detected in corresponding unfixed frozen tissue. The authors have demonstrated that PLP-fixed tissue can be sent to a central reference laboratory at ambient or room temperature, avoiding the expense and inconvenience of transporting specimens on dry ice. The authors conclude that PLP fixation is the preferred method for immunopathologic study of human lymphomas.     

The effect of cyclosporin A on the interstitial mononuclear cell infiltration and the induction of Heymann''s nephritis.

Heymann's nephritis was induced with brush-border (BB) antigen. Interstitial mononuclear cell infiltration was studied with cytological examinations of fine-needle aspiration biopsies (FNAB), and with immunoperoxidase stains of frozen sections with monoclonal antisera. The effect of cyclosporin A (CyA), 20 mg/kg when administered intraperitoneally for 8 days in association with both initial immunization, and with the booster 4 weeks later, on the interstitial leukocyte infiltration and on the development of membranous glomerulonephritis (MGN) and proteinuria were investigated. Another group of rats was immunized, but not given CyA. Experimental animals were killed in groups 3, 6 and 20 weeks after initial immunization. CyA inhibited significantly the initial interstitial lymphocyte and blast cell response at 3 weeks (FNAB), but did not inhibit the secondary response after the booster. The anti-BB titre reacted in a similar fashion. Immunoperoxidase stains indicated a clearly suppressed T suppressor/cytolytic (T s/c) cell response. Glomerular basement membrane (GBM) deposits of IgG developed more slowly and were more scarce in the CyA-treated rats, when compared with the untreated group. Only one out of 15 CyA treated rats developed C3 deposits in the GBM during the course of the study, and none developed proteinuria, when most untreated rats (10/17) had C3 deposits and were nephrotic at 20 weeks. Thus, CyA depressed the initial interstitial cellular response after immunization with BB antigen, and also inhibited the development of antibody response, C3 deposits and proteinuria of Heymann nephritis. These effects of CyA may be contributed to an inhibited amplification of the autoimmune response associated with interstitial damage and continuous release of autoantigen.     

B淋巴细胞增生性疑难病例中IgH基因克隆性重排的分析

目的 探讨IgH基因克隆性重排对B淋巴细胞增生性疑难病变的辅助诊断价值.方法 检测77例B淋巴细胞增生性疑难病例中IgH基因的克隆性重排情况,均采用BIOMED-2系统IgH克隆性试剂盒中FR1、FR2、FR3三组家族引物进行PCR及聚丙烯酰胺凝胶电泳,硝酸银染色后观察,并对照最终病理诊断进行分析.结果 77例病变的最终病理诊断:B淋巴细胞反应性增生12例,不能排除B淋巴细胞不典型增生或淋巴瘤20例,B细胞性淋巴瘤45例.三组中FR1、FR2和FR3至少有一个为阳性的比值分别为2/12、11/20(55%)和36/45(80%).B细胞性淋巴瘤中,FR1、FR2和FR3的阳性率分别为60%(27/45)、60%(27/45)、56%(25/45),其类型有边缘区B细胞性淋巴瘤20例(其中黏膜相关淋巴组织型结外边缘区淋巴瘤18例,结内边缘区淋巴瘤2例),弥漫性大B细胞淋巴瘤7例,滤泡性淋巴瘤7例,套细胞性淋巴瘤1例,Burkitt淋巴瘤1例,浆细胞瘤4例,不能分型5例.FR1、FR2和FR3三者检测均为阴性但仍诊断为淋巴瘤9例(20%),其中1例后来出现肝脏B细胞淋巴瘤.对IgH基因重排阳性的B淋巴细胞反应性增生和不典型增生14例的随访结果,4例重新取活检后诊断为B细胞性淋巴瘤,其中3例IgH基因重排检测为阳性.结论 联合检测IgH基因FR1、FR2和FR3克隆性重排对B淋巴细胞增生性疑难病变诊断有重要的辅助价值;对形态改变和免疫表型诊断淋巴瘤依据不足而基因重排阳性者,重取活检或随访有一定价值;对阴性病例有必要补充IgH基因重排及IgK和IgL基因重排的检测以提高检测敏感性.     

Expression of Epstein-Barr virus replicative proteins in AIDS-related non-Hodgkin's lymphoma cells.

Epstein-Barr virus (EBV) infection in lymphoproliferative lesions has been assumed to be strictly latent. In order to investigate the possible occurrence of EBV replication in AIDS-related lymphoma (ARL) cells, we studied 13 cases by immunohistology using monoclonal antibodies to the EBV-encoded switch-protein BZLF1, early antigens (EAs), late replicative proteins [virus capsid antigens (VCAs) and membrane antigens (MAs)], and to the latent proteins EB nuclear antigen 2 (EBNA 2) and latent membrane protein (LMP). EBV genomes were detected by in situ hybridization. EBV genomes and/or gene products were demonstrated in ten cases, including all immunoblast-rich lymphomas, two Burkitts lymphomas, and a T-cell anaplastic large-cell lymphoma. The BZLF1 protein, which disrupts latency in B cells, was identified in six (60 per cent), and EAs in four (40 per cent) of the EBV-positive ARL. Only one lymphoma (10 per cent) expressed VCAs and MAs. EBNA 2 and LMP were detected in three (30 per cent) and eight (80 per cent) of EBV-positive cases, respectively. EBV DNA was detected in lymphoma cells in 7 of 12 (58 per cent) cases. The most important finding of this study was frequent spontaneous activation of latent EBV in ARL. Production of complete virus, however, was either aborted, or tumour cells expressing late productive cycle proteins (VCA, MA) were rapidly cleared from tissues. It is suggested that host factors that normally inhibit replication of EBV are deficient in AIDS patients.     

血清TPS、NSE、CEA、β2MG水平与小细胞肺癌生物学关系的探讨

目的:探讨血清组织多肽特异性抗原(TPS)、神经元特异烯醇化酶(NSE)、癌胚抗原(CEA)和β2微球蛋白(β2-mG)水平与小细胞肺癌(SCLC)生物学行为的关系及其对肺癌的诊断价值。方法:采用ELISA和免疫放射分析法(IRMA)测定94例小细胞肺癌患者,86例肺良性疾病患者和89例正常对照组4项标志物的水平。结果:小细胞肺癌组血清TPS、NSE水平[(437.8±516.6)U/L、(76.8±91.4)μg/L]不但高于肺良性疾病组[(143.6±78.7)U/L、(13.3±10.8)μg/L]和正常对照组[(98.4±58.9)U/L、(10.1±5.7)μg/L)],P<0.01。CEA和β2-mG水平测定中,SCLC组亦高于良性疾病组和正常对照组(P<0.01),则以TPS、NSE水平为指标诊断SCLC的敏感性、特异性及准确性分别为84.4%、87.8%、83.6%和79.3%、93.7%、88.3%,高于CEA和β2-mG。此外,发生转移的SCLC血清中TPS、NSE水平高于未转移组,转移灶数目越多,则TPS、NSE水平越高。每天吸烟30～40支可达10～24倍,吸烟量与肺癌之间存在着量效关系。结论:血清TPS、NSE、CEA和β2-mG对于小细胞肺癌的早斯诊断有一定的价值,4项联合检测则有明显的互补性,其水平与肺癌转移密切相关。     

Analysis of T-cell subpopulations in T-cell non-Hodgkin's lymphoma of angioimmunoblastic lymphadenopathy with dysproteinemia type by single target gene amplification of T cell receptor- beta gene rearrangements

Angioimmunoblastic lymphadenopathy with dysproteinemia (AILD) is defined in the current lymphoma classifications as a T-cell non-Hodgkin's lymphoma. However, in approximately one third of the cases of this lymphoproliferative disease rearrangements of T-cell receptor (TCR) genes indicating clonal expansion of T cells are not detectable. It is currently believed that these cases may represent early stages of a lymphoma with a minor oligoclonal T-cell population. In the present study, 18 lymph nodes with the characteristic histology of AILD were investigated for clonal T-cell receptor gene rearrangements by analysis of DNA extracted from whole tissue sections. Dominant T-cell clones were detected in 12 of these cases. Single CD4(+) and CD8(+) T cells and proliferating Ki67(+) cells of seven cases were micromanipulated from frozen tissue sections. TCRbeta gene rearrangements were amplified from these cells by polymerase chain reaction and sequenced. In all informative cases, the clonal gene rearrangements were only detected among CD4(+), and not among CD8(+) T cells, indicating that the tumor clones in AILD usually derive from CD4(+) T cells. Minor clonal T-cell populations in those cases in which no clone was found by whole-tissue DNA analysis were not detectable even at single cell resolution. T-cell clones in 4 of 10 cases were found to express similar TCRbeta chains, indicating a potential role of (super) antigen triggering in at least some cases of AILD.     

血清HE4、TPS和CA125联检在卵巢癌诊断中的应用价值

目的：探讨血清人附睾分泌蛋白4（HE4）、组织特异性抗原（TPS）和CA125联检在卵巢癌诊断中的应用价值。方法：采用EIA、ELISA和ECLIA分别检测卵巢癌患者（n=30）、卵巢良性疾病患者（n=30）和健康对照者（n=31）的血清HE4、TPS和CA125血清水平。结果：卵巢癌组与卵巢良性疾病组、健康对照组、卵巢良性疾病组＋健康对照组比较,血清HE4、TPS和CA125的水平都有显著统计学差异（P〈0.01）。HE4在64.28pmol/L时约顿指数最大（0.667）,敏感性为70.0%,特异性为96.7%,ROC-AUC为0.878;TPS在63.20U/L时约顿指数最大（0.469）,敏感性为63.3%,特异性为83.6%,ROC-AUC为0.761;CA125在48.86U/ml时约顿指数最大（0.602）,敏感性为73.3%,特异性为86.9%,ROC-AUC为0.836;HE4＋CA125联检后敏感性为93.3%,特异性为82.0%,ROC-AUC为0.938;HE4＋CA125＋TPS联检后敏感性为93.3%,特异性为86.9%,ROC-AUC为0.941;HE4与CA125、HE4＋CA125、HE4＋CA125＋TPS比较ROC-AUC无显著差异,HE4和CA125联检后其ROC-AUC增大为0.938,与CA125、TPS二个单项指标相比,P值均〈0.05,与HE4、CA125、TPS三个指标联检后的ROC-AUC值0.941比较P〉0.05,不存在统计学差异。结论：血清HE4是卵巢癌诊断的良好指标,HE4和CA125联检是理想的组合方式,兼顾了敏感性和特异性,提高了对卵巢癌的诊断能力。     

Cyclosporin-induced endothelial cell injury

The administration of Cyclosporin-A (CyA) to animals and humans may induce an arteriolar damage. It has also been reported that CyA in some instances may cause an hemolytic uremic-like syndrome. This is a syndrome of vascular damage with thrombotic occlusions of the microcirculation. Endothelial injury is considered the first event in the pathogenetic cascade leading to hemolytic-uremic syndrome. We have used bovine aortic endothelial cells in culture to address the issue of CyA-induced arteriolar damage. Exposure of endothelial cells to different concentrations of CyA induced a time- and dose-dependent cell injury in vitro. The damage induced by CyA was characterized by an early cell detachment from culture substrate followed by cell lysis as documented by the increase in lactate dehydrogenase (LDH) and 51Cr release. Both detachment and lysis were negligible after short-term incubation of 1 microM CyA with endothelial cells. One micromolar CyA only induced lysis if incubations were prolonged above 6 hours. Ten and 50 microM CyA both induced marked endothelial cell detachment and lysis; lysis started 3 hours after incubation of endothelial cells with CyA and was maximal at the end of 24 hours incubation. CyA-induced injury was associated with dose- and time-dependent increase in prostacyclin and thromboxane A2 release by endothelial cells exposed to CyA independently from the concentrations of CyA used. CyA-induced generation of prostacyclin and thromboxane A2 was inhibited when the incubations were performed in the presence of aspirin (500 microM). These studies indicate that CyA exerts a direct cytotoxic effect on endothelial cells and might help in understanding the pathogenesis of CyA-induced vascular damage.     

Immunophenotypes in "well-differentiated" lymphoproliferative disorders, with emphasis on small lymphocytic lymphoma

The histologic, immunophenotypic, and clinical findings in 24 cases of "well-differentiated" lymphoproliferative disorders are presented, with an emphasis on small lymphocytic lymphoma (SLL; well-differentiated lymphocytic lymphoma, WDLL). SLL was diagnosed in 18 patients, Waldenstr?m's macroglobulinemia in one, mantle zone lymphoma (MZL) in four, and intermediate differentiated lymphocytic lymphoma (IDL) in one. Immunophenotypic analysis revealed one SLL to be of T-cell derivation, while a monoclonal B-cell phenotype was found in the remaining 23 patients; 20 of these neoplasms co-expressed the pan-T-cell antigen Leu-1. This included three of the four patients with MZL and the patient with IDL. All SLLs with pseudofollicular growth centers stained for transferrin receptor. Additionally, a survey of 248 non-Hodgkin's lymphomas of all types revealed four of 70 patients with B-cell large cell lymphoma (LCL; diffuse histiocytic lymphoma, DHL) that also co-expressed the same Leu-1 antigen. Immunoperoxidase study of both frozen tissue sections and cytocentrifuge preparations eliminated the limitations of faint antigen expression and/or interstitial immunoglobulin staining. In addition to detailing the immunophenotype of SLL, the immunologic kinship of MZL/IDL to SLL and the uncommon co-expression of Leu-1 in B-cell LCLs are demonstrated.     

Performance evaluation of three automated human immunodeficiency virus antigen-antibody combination immunoassays

Three fourth-generation antigen/antibody combination assays (Elecsys, AxSYM, Architect HIV) and two third-generation (AxSYM, Centaur) HIV antibody immunoassays were evaluated. The evaluation panel of 479 samples included: nine tissue culture derived HIV-1 strains at four different p24 antigen concentrations (n=36), a p24 antigen sensitivity panel (n=10), 149 HIV-1 or HIV-2 confirmed antibody positive samples, ten anti-HIV-1 positive low titer samples, three seroconversion panels (n=21), and 253 HIV negative samples. The Architect had the best sensitivity for detection of HIV-1 antigen across eight HIV-1 subtypes, followed by the AxSYM while the Elecsys could not detect the highest antigen concentration evaluated (25 pg/mL) for eight of nine virus isolates. All assays showed 100% sensitivity for detection of HIV-1, group M, group O, and HIV-2 antibody positive samples. The Architect Ag/Ab Combo assay detected the first positive bleed of the three seroconversion panels and detected infection 4-26 days earlier than the third generation assays. Based on evaluation of 253 negative samples, assay specificity varied from 98.0% to 99.6%. The Architect HIV Ag/Ab Combo exhibited the best performance for specificity and detection of p24 antigen leading to closure of seroconversion window and demonstrating its utility for early diagnosis of HIV infection.     

Matrix metalloproteinase-2, squamous cell carcinoma antigen, and tissue polypeptide-specific antigen expression in Egyptian patients with cervical carcinoma: Relationship with prognosis

Matrix metalloproteinases (MMPs), a family of proteolytic enzymes produced by both stromal and tumor cells, appear to have a key role in the events leading to local invasion and metastasis by malignant neoplasms. In the present study, we evaluated the role of MMP-2, squamous cell carcinoma antigen (SCCA), and tissue polypeptide - specific antigen (TPS) in cervical neoplasia. Using Western blotting and enzyme immunoassay (EIA), we analyzed 50 patients with cervical carcinoma (CC) and 25 normal controls for expression of MMP-2 in tissue cell lysates. We also quantified SCCA and TPS with microparticle immunoassay and EIA, respectively. The results were correlated with human papilloma virus (HPV) infection, clinicopathological findings, and disease outcome. The cutoff point for each marker was estimated from receiver operating characteristic curves. Logistic regression analysis was performed to estimate the odds ratio (OR) and 95% confidence interval (CI) for each marker. MMP-2, SCCA, and TPS protein expression were significantly higher in patients with CC than in normal controls. While TPS was the best marker for discriminating between patients and controls, MMP-2 was associated with an advanced tumor stage (OR, 13.9 [95% CI, 1.4-133.9]) and poor histological grade (OR, 10.2 [95% CI, 1.7-60.5]). Moreover, independent of the effect of an advanced CC stage and grade, the patients' age, and the presence of HPV infection, MMP-2 was considered a strong predictor for CC recurrence (OR, 8.1 [95% CI, 1.3- 49.1]). Tissue markers may be used to select high-risk patients for early detection of and adjuvant therapy for recurrence. Our MMP-2 findings are particularly relevant to the development of protease inhibitors as a new cancer therapy approach.     

Cyclosporine A and prednisolone inhibit lectin- and alloantigen-induced release of sCD8: correlation with proliferative responses

It has been shown previously that there is a strong correlation between the in vitro release of soluble CD8 glycoprotein (sCD8) and CD8+ T lymphocyte activation. In the present study, the lectin stimulation of peripheral blood mononuclear cells (PBMC) induced a dose-dependent release of sCD8 which correlated with the magnitude of CD8 lymphocyte activation as measured by the expression of the interleukin 2 (IL-2) receptor and HLA-DR antigen and of the T cell proliferative responses. Both the proliferative responses and the release of sCD8 were inhibited by cyclosporine A (CyA) and prednisolone (PRED) in a concentration-dependent manner. When the immunosuppressants were present for only 60 min before the initiation of the cultures, an inhibitory effect was also seen, but this was maximal only when the agents were added at the initiation of the culture period; when the addition of CyA or PRED was delayed for either 24 or 48 hr after the initiation of the culture, the degree of inhibition of the proliferative response was greatly reduced. However, there was a significant inhibition of sCD8 release by CyA even when it was added 48 hr after the culture initiation. The addition of recombinant IL-2 did not affect the lectin-induced sCD8 release. The inhibition of the lectin-induced proliferative response and sCD8 release by PRED, but not that by CyA, was reversed by the recombinant IL-2. Alloantigen stimulation also induced sCD8 release and this release was inhibited both by CyA and by PRED. These data, together with the known effects of CyA on differentiation, clonal amplification, and activation of CD8 T lymphocytes, suggest that in vitro sCD8 release occurs during the early stages of activation of CD8+ cytotoxic T cells.     

Rapid immunodiagnosis of active cytomegalovirus infection by monoclonal antibody staining of blood leucocytes

The appearance of cytomegalovirus (CMV) antigen positive blood leucocytes (CMV antigenaemia) was investigated in 52 renal transplant recipients during the first three months after transplantation. Using a mixture of three monoclonal antibodies, CMV (immediate early) antigens were detected in cytocentrifuged blood leucocytes within 3-5 h after sampling. The results were related to virus isolation from buffy coats (CMV viraemia), serology with a sensitive enzyme-linked immunosorbent assay (ELISA), and clinical symptoms of CMV disease. The antigen test was positive in all 14 patients with CMV viraemia, in 25 out of 27 of patients with serological evidence of primary or secondary CMV infection, and in 2 out of 25 patients without active infection. In patients with a clinical CMV syndrome the presence of CMV antigen (CMV-Ag) positive blood cells correspond with the period of signs and symptoms. CMV antigens were not detected in 23 out of 25 patients without active infection, nor in healthy controls and patients with other herpesvirus infections. CMV-Ag positive blood cells appeared, on average, nine days before serological signs of active infection. This method provides a rapid and sensitive approach to CMV detection, enabling early clinical diagnosis and subsequent tapering of immunosuppression or commencement of antiviral therapy.