聚合酶链反应产物特异性的阳性证实

以聚合酶链反应(PCR)法在mRNA水平检测T淋巴细胞受体α链可变区基因表达为例,介绍用~(32)P标记的人工合成寡核苷酸探针对PCR产物特异性作阳性证实的方法。该法以干琼脂糖凝胶作为支持物、相对较为简便和省财。用Ca探针以干凝胶作支持物的杂交结果,证实29个Vα基因的PCR扩增中物均为特异性的,放射自显影的带型与位置和溴乙锭染色所示完全吻合。     

Surface immunoglobulin density in the differential diagnosis of B-cell chronic lymphocytic leukemia and leukemic immunocytoma.

Using flow cytometry peripheral blood samples of 37 consecutive patients with B-cell chronic lymphocytic leukemia (B-CLL) and 17 consecutive patients with leukemic immunocytoma (IC) were studied in order to determine quantitative differences in the surface immunoglobulin (slg) density. In 8/37 (21.6%) cases of B-CLL and 1/17 (5.9%) cases of IC slg staining remained in the control level. Analysis of slg-positive cases demonstrated a close association between the amount of slg and diagnosis: per case the mean calculated fluorescence intensity for IC lymphocytes was 209.7 arbitrary linear intensity units (IU) (median: 156.4, standard error of the mean (SEM): 53.7) and for B-CLL lymphocytes 10.8 IU (median: 7.3, SEM: 1.1; p less than 0.0001). Altogether, 94.6% of all B-CLL patients and 76.5% of all IC patients were correctly classified when a cut-off point was fixed at a mean fluorescence intensity value of 20.0 IU. The percentage of leukemic cells as characterized by CD19 and HLA-DR reactivity was significantly lower in cases of IC (p less than 0.03 and p less than 0.01, respectively). In both entities disease progression occurred more frequently in advanced stages (II-IV) according to the Rai classification (p less than 0.01). In progressive disease rather than in stable disease circulating T lymphocytes were shown to express decreased amounts of surface CD3 antigen (p less than 0.02). We conclude that the quantitative assessment of surface antigens in addition to their qualitative characterization provides accurate information. In particular, the diagnostic discrimination between B-CLL and IC may be improved by determining the lymphocytes' slg amount.     

Methodological aspects of DNA image cytometry in formalin-fixed paraffin-embedded material from pancreatic adenocarcinoma

Deparaffinized and disintegrated material from conventionally formalin-fixed and paraffin-embedded surgical specimens of 100 cases of ductal adenocarcinoma of the pancreas was Feulgen-stained, and the cytochemical DNA distribution patterns of at least 100 single tumour cells and 50 "control" cells (fibrocytes) were assessed by means of image cytometry (ICM). In 77 cases a sufficient number of neoplastic cells could be obtained for these DNA assessments. The fairly high number (23) of cases that had to be excluded due to too small amounts of disintegrated cells or cell nuclei may be explained by the high content of connective tissue stroma in these pancreatic adenocarcinomas. The tumour cell nuclei in 76 of these 77 cases showed cytochemically a clear-cut "non-diploid" DNA distribution pattern. This observation reflects the well-known highly malignant growth potential of this carcinoma. Despite the fact that about 1/4 of the tumours had to be excluded, the main result of our methodological study is, after all that conventionally formalin-fixed paraffin-embedded specimens of most pancreatic adenocarcinomas can be successfully used for the deparaffinization-disintegration procedure preceding the nuclear DNA assessments by means of ICM. Additional studies are, however, required to obtain the diagnostic and prognostic impact of the results of such cytochemical analyses of the DNA distribution pattern in adenocarcinomas of the pancreas.     

Investigation of nuclear c-MYC oncoprotein expression in human hematopoiesis: suitability of a rapid and reliable semiquantitative evaluation system.

The biologic functions mediated by the nuclear protooncogene, c-MYC are correlated to gene dosage. Since automated quantification programs are expensive, time-consuming and not easily available, and since analysis by flow cytometry is difficult in the case of nuclear antigens, we examined the suitability and reproducibility of a semiquantitative in situ evaluation system. This system was based on the percentage of nuclear area staining positively, and comprised the following categories: 0: negative, 1+: single scattered grains of the immunocytochemical staining product, 2+: confluence of grains to patches but less than 50% nuclear area positive, and 3+: greater than 50% positive nuclear area. In addition, sensitivity and specificity of two anti-c-MYC antibodies were investigated. Although both antibodies differed slightly in staining pattern and sensitivity, the four quantification categories were applicable for immunostainings of both antisera and highly reproducible when re-evaluated by the same observer (r = 0.98; p = 0.0001) or a second investigator (rAb155 = 0.98, rAb DCPm = 0.96; p = 0.0001), both reading blindly and independently. Comparing our semiquantitative evaluation categories and results of computer-assisted image analysis, the percentage of positive nuclear area (p less than 0.0001), the median staining intensity (p less than 0.0001), and the product of both (p less than 0.0001) differed significantly in the four evaluation categories. This result still held true after correction for nuclear size, which differed appreciably in various cell types (p less than 0.0001). The product of positive nuclear area, staining intensity and nuclear size (microns 2), which best approximates the absolute amount of c-MYC within a certain cell, was clearly different within the four staining categories (p less than 0.0001) and did not depend on cellular morphology within the staining categories 0 to 2. Also, the immunocytochemical technique proved highly reproducible (median day/day variance 0.65% (0-13); r = 0.995). The practicability of this system for semiquantification was demonstrated by (a) correlation of H score values of immunocytochemical stainings with densitometric scans of Western blots and (b) by the fact that peripheral blood lymphocytes, Phytohemagglutinin stimulated blasts, 13 cases of multiple myeloma and HL-60 cells differed concerning their estimated c-MYC amounts (p = 0.0125). This confirms on the effector molecule level results previously reported from mRNA in situ and Northern blotting analyses. We conclude that a simple and highly reproducible evaluation system can be used for in situ comparison of nuclear oncogene dosage.     

Sequential activity of gamma-delta T-cell receptor bearing lymphocytes, specific T-cell factors, and alpha-beta T-cell receptor bearing T-cells: a hypothesis

The cell-mediated immune response against cell-bound antigens are biphasic responses. The 'classical' components of cell-mediated immunity such as delayed type hypersensitivity (DTH) and cytotoxic T-cells, are preceded by antigen-specific T-cell factor production. These antigen-specific T-cell factors can be detected in the serum 1-2 days after immunization, which suggests that these antigen-specific T-cell factors play an important role in the initiation of cell-mediated immune responses. Factor-producing lymphocytes can be detected already 1 day after immunization at the site of the antigen challenge, and at first 4-5 days after immunization in the peripheral lymphoid organs. The phenotype of these factor-producing lymphocytes is Thy-1+, CD3+, L3T4 (CD4)-, Lyt2 (CD8)-, whereas the T-lymphocytes inducing the DTH response are Thy 1+, CD3+, L3T4 (CD4)+, Lyt2 (CD8)-. This suggests that these factor-producing lymphocytes are gamma-delta bearing T-lymphocytes. gamma-delta TCR bearing T-cells can develop independently of the thymus, and are present in athymic nude mice. Immunization of athymic (nude) mice induces antigen-specific T-cell factor production as well. This suggests that the gamma-delta bearing T lymphocytes present in nude mice can react to cell-bound antigens and produce these factors.     

Encoded latent membrane protein 1 of Epstein-Barr virus on follicular dendritic cells in residual germinal centres in Hodgkin's disease.

AIMS--To determine if there is an association between Epstein-Barr virus (EBV) infection and Hodgkin''s disease. METHODS--Fifty cases of Hodgkin''s disease and 25 reactive lymph nodes were screened for the presence of EBV-RNA (EBER) using in situ hybridisation, and for the expression of EBV encoded latent membrane protein 1 (LMP-1) by immunohistochemistry. RESULTS--In 42% of the cases of Hodgkin''s disease, EBER was detected in the nuclei of the malignant cells, and in LMP-1 expression was found 36%. Both EBER and LMP-1 positivity were seen in 34% of the cases. An additional finding was the presence of LMP-1 on follicular dendritic cells in residual germinal centres in two cases of Hodgkin''s disease. EBER was not detected in these germinal centres. In reactive lymph nodes only occasional EBER positive, small, lymphoid cells were found, without LMP-1 expression. CONCLUSIONS--These results show a strong correlation between the presence of EBER and the LMP-1 expression in the Reed-Sternberg cells. They corroborate a role for EBV in at least some cases of Hodgkin''s disease. LMP-1 is probably presented as an immune complex in the germinal centres, as part of an immune response against EBV.