Volker Diehl

Establishment of a malignant, epstein-barr-virus (EBV)-negative cell-line from the pleura effusion of a patient with Hodgkin's disease

The pleura effusion was obtained from a 37-year-old woman with histologically proven Hodgkins disease, nodular sclerosing type, stage IVB, primarily diagnosed in 1972. In the 4 weeks before the last admission to our institution, the patient developed a severe eosinophilia (91 ~), the total WBC amounted to 26000/ram 3. The nodal and extranodal tumor progression did not respond to a polychemotherapy (MOPP) and the patient died due to a septical complication. The post mortem showed beside a disseminated lymphnode involvement extranodal invasion of the whole left-side chest-wall including the parietal pleura.

Long-term cultivation of plasma cell leukemia cells and autologous lymphoblasts (LCL) in vitro: a comparative study.

SummaryTwo long-term cell lines were established in vitro from the peripheral blood of a patient with plasma cell leukemia: one line with plasma cell proliferation, the other with lymphoblastoid cell proliferation (LCL).The 9-month-old plasma cell line showed the typical morphology of plasmoblasts. The cells neither had B-nor T-lymphocyte characteristics, were EBV negative, and showed aneuploidy with various marker chromosomes, including the 14 q+ marker. The cytogenetic findings indicate a monoclonal proliferation of the plasmacells. No tumor growth in thymusless nude mice could be induced upon intracranial inoculation with these cells.In contrast, the autologous LCL, cultured after addition of exogenous EBV, showed the characteristic markers of lymphoblastoid cells, with the typical morphology of pear and handmirror-shaped lymphoblasts, growing in clumps. They had C3- and Fc-receptors, surface-Ig, E-rosette-negativity, a diploid karyotype, and EBV dependent macromolecule synthesis. The lymphoblastoid cells produced intracranial tumors in nude mice in 8 out of 8 attempts.

Impact of risk factors on outcomes in early-stage Hodgkin's lymphoma: an analysis of international staging definitions

BACKGROUNDnIn early-stage Hodgkins lymphoma (HL), treatment according to the early favorable or unfavorable subgroup is guided by staging definitions, which differ between various study groups worldwide. We analyzed risk factors used in different international staging systems and their impact on the outcome of early-stage HL patients.nnnPATIENTS AND METHODSnIn 1173 early-stage HL patients treated homogenously within the German Hodgkin Study Group (GHSG) trials HD10 and HD11, the impact of three staging systems developed and used by the GHSG, the European Organization for Research and Treatment of Cancer (EORTC), and the National Comprehensive Cancer Network (NCCN) in discriminating risk groups for progression-free survival (PFS) and overall survival (OS) was assessed and the relevance of their single risk factors was investigated.nnnRESULTSnAll the three staging systems defined an unfavorable risk group out of early-stage patients of comparable size (56%, 55%, and 57%), having a significantly poorer PFS and OS as compared with the corresponding favorable group; 5-year differences between early favorable and early unfavorable in terms of PFS were 9.4% (HR 2.61, 95% CI 1.74-3.91), 6.7% (HR 2.10, 95% CI 1.41-3.13), and 8.6% (HR 2.14, 95% CI 1.45-3.16) with the GHSG, EORTC, and NCCN definition, respectively. Sensitivity was high for all systems (84%, 79%, and 83%); however, there was a low specificity with high rates of false-positive results (1-specificity 54%, 53%, and 55%, respectively). Models of high sensitivity included risk factors associated with large tumor burden and high tumor activity. Most risk factors for tumor-specific end points were also predictive of OS.nnnCONCLUSIONSnDifferentiating between a favorable and an unfavorable risk group has significant impact on PFS and OS in early-stage HL patients in the modern treatment era. Risk-adapted treatment strategies using new risk factors with higher specificity are needed.

Binding of bovine parathyroid hormone to surface receptors of cultured B-lymphocytes

Binding of parathyroid hormone onto B-lymphocytes is detected by the utilization of the labelled antibody membrane assay. The amount of parathyroid hormone bound to the receptor sites was depending on the quantity of cells in the incubation milieu. Each cell line showed typical characteristics in time course of parathyroid hormone binding and maximal receptor capacity. Fragmentation of intact parathyroid hormone, also varying with the cell line tested, was very rapid, even at 24 degrees C. Within 20 min most of the cell lines destroyed 20% of the native hormone in the incubation mixture, indicating a fragmentation rate of up to 2.25 ng/min at 37 degrees C. Bmax and KD for the different lymphocytes was 5.3--19 . 10(11) M and 1.8--18,5 . 10(11) M, respectively. These values are in the range of reported plasma concentrations and may therefore represent more physiological values for the capacity and affinity of membrane receptors.

Sister chromatid exchange in cell lines from malignant lymphomas (lymphoma lines)

SummaryThe frequency of sister chromatid exchanges (SCEs) was studied in cells from three freshly established lymphoma lines, derived from two patients with Hodgkins disease and one patient with non-Hodgkin lymphoma. These values were compared to SCE rates found in cells from two long-established lymphoma lines (Raji and BJAB) and to those recorded in control cell lines of normal human donors. The highest SCE levels were demonstrated in the freshly established lymphoma lines, the lowest SCE values separated the lymphoblastoid cell lines from healthy controls, and the older lymphoma lines Raji and BJAB presented rates in between. The influences of BUDR concentration and of the duration of BUDR treatment on the frequency of SCEs were tested. Furthermore, the dependence of the SCE rate on the time interval between establishment of the cell line and its SCE investigation was considered. The connection between elevated SCE rates and the neoplastic nature of lymphoma lines is discussed.

Correlation of chromosomal aberrations in a myeloma cell line with tumorigenicity in nude mice

SummaryA human myeloma cell line (L 363) exhibiting several markers of malignancy including a 14q+ chromosome marker was not tumorigenic in nude mice. Long-term cultivation in vitro resulted in new subclones with slight, well-defined chromosomal differences to the original line. Certain subclones turned out to have a growth advantage in vitro and became tumorigenic after transplantation in nude mice. A strong correlation between changes in the chromosome 1 constellation resulting in the threefold dose of long arm-material and the increased proliferative potential in both experimental systems was found.

Loss of heterozygosity in the Hodgkin-Reed Sternberg cell line L1236

Hodgkin-Reed Sternberg cells are derived from germinal centre B-cells in most cases. Somatic mutations affecting their rearranged immunoglobulin genes were detected, rendering potential functional rearrangements non-functional. Under physiological conditions such cells would be designated to undergo apoptosis within the germinal centre. In search for the specific transforming event that prevents Hodgkin-Reed Sternberg cells from programmed cell death, cytogenetic analyses were broadly performed but did not reveal specific chromosomal aberrations. Analysis of these cells on the molecular level is difficult to perform due to the scarcity of the cells in the lymphoma tissue and the given limitations of in situ studies. To overcome these limitations, the cell line L1236, known to be derived from Hodgkin-Reed Sternberg cells in situ, was chosen for allelotype analysis. Using a panel of microsatellite loci assigned to nearly all chromosomal arms, regions of loss of heterozygosity were detected on chromosomal arms 6p, 9q and 17p. The size of lost segments was estimated by amplification of additional microsatellite loci mapped to the respective regions. Further analyses of single Hodgkin-Reed Sternberg cells will reveal whether LOH affecting these regions is a recurrent event in HD and to which extent the smallest commonly affected region can be estimated.

Establishment of Peripheral Lymphoid Cell Cultures from Patients with Hodgkin's Disease Depending on Epstein-Barr-Virus-Reactivity and Cellular Immunity

SummaryPeripheral blood lymphocytes from 43 patients with Hodgkins disease were studied for spontaneous growth in longterm cultures in vitro. The rate of culture establishment in Hodgkins patients was dependant on a positive Epstein-Barr-Virus (EBV)-seroreactivity and intact delayed hypersensitivity reaction to tuberculin.Localized and inactive disease, as well as the absence of atypical mononuclear cells in the peripheral blood had a favourable influence on the longterm in vitro growth.The overall establishment rate in Hodgkin patients was 18 out of 60 attempts (30%), 16 out of 34 (47%) in patients without treatment, only 2 out of 26 (7.7%) attempts during treatment. These results were compared with culture attempts of peripheral blood cells from healthy individuals and umbilical cord blood lymphocytes. Only 12 out of 60 attempts in healthy donors (18.2%) and 0 out of 49 attempts with umbilical cord blood lymphocytes were successful.ZusammenfassungIn vitro-Langzeitwachstum peripherer Blutlymphozyten von 43 Hodgkin-Patienten war abhängig von einer positiven Seroreaktivität gegenüber Epstein-Barr-Virus (EBV) und einer intakten Immunreaktion vom verspäteten Typ.Lymphozyten von Patienten in lokalisiertem Stadium ohne Krankheitsaktivität, und Fehlen von atypischen Lymphozyten im peripheren Blut dieser Patienten zeigten einen positiven Einfluß auf das Langzeitwachstum in vitro. Das Resultat der Kulturversuche war wie folgt:18 Kulturansätze von insgesamt 60 (30%) zeigten ein positives Ergebnis, während 16 von 34 (47%) Kulturversuche von unbehandelten Patienten, und nur 2 von 26 (7,7%) unter Therapie erfolgreich waren.Die Kulturergebnisse mit Lymphozyten von Hodgkin-Patienten wurden verglichen mit denen von gesunden Erwachsenen und Nabelschnurblutlymphozyten:12 von 60 Versuchen varen positiv (18,2%) mit Blutzellen von Erwachsenen, und O von 49 Versuchen mit Nabelschnurblutlymphozyten.

Monoclonal glucose-oxidase-anti-glucose-oxidase (GAG) immunosandwich assay for the detection of monoclonal antibodies on routine hematological smears

SummaryA murine monoclonal antibody specific for aspergillus niger glucose oxidase has been prepared and used in an unlabeled antibody bridge technique for the detection of monoclonal antibodies. This procedure — the monoclonal glucose oxidase anti-glucose oxidase (GAG) immunosandwich assay — provides excellent immunocytochemical labeling of routine hematological films in combination with optimal preservation of cellular details. In contrast to conventional immunofluorescence procedures, routine hematological films can be used, and these can be stored before and after the immunolabeling. Compared with other immunoenzyme techniques such as those using alkaline phosphatase or peroxidase, the GAG assay is as sensitive and has the advantage that no problems with endogenous enzyme activity are encountered. The availability of alcohol-resistant disclosing reagents allows for routine hematological counterstaining which provides a very clear visualization of both the immunoreaction and the individual morphology of the blood cells.

Immunologischer Status bei Hodgkin-Patienten: Korrelation zu Epstein-Barr-Virus(EBV)-Titern

Zusammenfassung1.Immunologische in vitro- und in vivo-Parameter haben prognostische Aussage-Wertigkeit bei der Hodgkinschen Erkrankung.2.Der Ausfall der Hauttestreaktionen korreliert am besten zur Prozentzahl an peripheren T-Lymphozyten und zu der Lymphozytenstimulierbarkeit durch das Lektin Concanavalin A.3.Concanavalin A scheint von den bisher angewandten in vitro-Mitogenen am empfindlichsten einen latenten Immundefekt aufzuzeigen.4.Patienten mit Hodgkinscher Erkrankung in Langzeitremission nach intensiver Tumorreduktionstherapie haben eine qualitativ und quantitativ geschwÄchte Lymphozytenreaktion.5.Antikörpertiter gegen Herpesviren sind nur für das EBV signifikant bei Hodgkin-Patienten erhöht. Die Titererhöhung geht einher mit einer verminderten T-Zellzahl und -funktion.Summary1.In Hodgkins disease patients immunological in vitro and in vivo parameters are of prognostic importance.2.Skin test reactivity correlates to peripheral T-lymphocyte counts and Con A induced lymphoblastogenesis.3.Con A is the most sensitive in vitro indicator for detecting latent immunodeficiency.4.Hodgkin patients in long term remission after tumor reductive therapy exhibit a qualitative and quantitative lymphocyte defect.5.In Hodgkin patients Herpes virus related antibody titers are elevated against Epstein Barr virus (EBV). The elevation coincides with a decreased T-cell number and function. Antibodies against other Herpes viruses (HSV, CMV, VZV) are in the normal range, when tested by the complement fixation method.