N-ras genes are not mutated in Hodgkin and Reed-Sternberg cells: results from single cell polymerase chain-reaction examinations.

Ras mutations play an important role in many human tumors. They usually occur at only three codons (12, 13 and 61) of the three ras gene family members and lead to altered proteins resulting in a constitutively activated downstream signal cascade. We have examined the N-ras gene status in Hodgkin's disease (HD). Little is known about the pathogenetic events leading to malignant phenotype in HD. Since Hodgkin and Reed-Sternberg (H and RD) cells comprise only a minority of the cellular infiltrate in HD-lymph nodes, molecular studies concerning the status of oncogenes have been difficult to perform and have yielded conflicting results. We have established a single cell PCR assay for N-ras analysis and have examined H and RS cells from 12 cases of HD by PCR amplification and direct sequencing. None of the single H and RS cells examined carried N-ras mutations at either codons 12/13 or 61. Therefore, N-ras mutations are not involved in the pathogenesis of HD.

Enrichment polymerase chain reaction for the detection of Ki-ras mutations: relevance of Taq polymerase error rate, initial DNA copy number, and reaction conditions on the emergence of false-positive mutant bands.

Screening for oncogene mutations as a marker for malignancy can be a powerful tool for the early diagnosis of cancer. The enrichment polymerase chain reaction (PCR) is a sensitive method for the detection of low-frequency mutations in small samples. However, false-positive results, caused by methodological errors, may have severe clinical implications. When applied to the detection of Ki-ras mutations in pancreatic secretions, the assay sensitivity is limited to approximately 1:1400. Our investigation of Ki-ras mutations in blood samples from patients with pancreatic carcinoma revealed PCR bands presumably derived from mutant Ki-ras in samples from healthy volunteers, while all blood samples of the patients with pancreatic carcinomas showed a wild-type band pattern. Mathematical modeling of the PCR reaction reveals that the rate of false positive PCR results depends on the initial amount of DNA, the Taq polymerase error rate, the number of PCR reaction cycles, reaction efficiency and the restriction endonuclease chosen. The overall error rate of false positive results of the enrichment PCR can be reduced to the square of the rate of a single-step analysis if repeated amplifications of the same DNA specimen show an identical result.

Detection of Epstein-Barr virus DNA in Hodgkin- and Reed-Sternberg-cells by single cell PCR.

The Epstein-Barr virus (EBV) can be detected in the majority of lymph nodes involved by Hodgkin's lymphoma using the highly sensitive polymerase chain reaction (PCR). However, the rate of EBV-DNA detection by in-situ hybridisation, which allows allocation of EBV to a defined cell population, i.e. the neoplastic H&RS-cells, is lower. In an attempt to combine the advantages of the high sensitivity of the PCR and the possibility of cellular allocation by in-situ hybridisation, we established a single-cell PCR of Hodgkin- and Reed-Sternberg (H&RS)-cells isolated by micromanipulation from biopsy tissues. We amplified EBV sequences from the BamW-region by single-cell PCR. Using this method we were able to detect EBV-DNA in the H&RS-cells from 4 of 6 patients. In EBV positive cases all H&RS-cells of a given patient were positive, proving the high sensitivity and reproducibility of the method. Other cells in the biopsy tissue involved by EBV-positive H&RS-cells were shown to be negative. This indicates that EBV may have a role in the pathogenesis of many but not all cases of Hodgkin's disease.

[Conventional x-ray study and computed tomography in the diagnosis of orbital fractures]. / Konventionelle Röntgenuntersuchung und Computertomographie bei der Diagnostik von Orbitafrakturen.

37 cases of skull fractures with involvement of the orbit were reviewed retrospectively. The value of plain films, tomography and computed tomography was analysed. Combined use of 28 degrees Caldwell and water views revealed 96% of all orbital floor fractures. Orbital emphysema in facial bone fractures nearly almost (in 14 of 15 cases) indicated involvement of the medial orbital wall. CT--performed on a biplane basis--showed best diagnostic accuracy in evaluating orbital fractures. Axial CT scans revealed only 70% of all orbital floor fractures. Because of that coronal scans are especially necessary for evaluating the orbital floor and the orbital roof. CT is necessary for evaluating the medial orbital wall since conventional radiology only shows 15% of all medial orbital wall fractures.

Improved method for the cyanogen bromide activation of agarose beads.

The significant new feature of the procedure is the reaction control of the BrCN activation merely by the slow transit of BrCN from a dispersed organic phase to the aqueous phase containing agarose beads in concentrated buffer. The product thus obtained was applied in a model immunoaffinity chromatographic separation. Experimental conditions are given for the control of the degree of activation and of the multiplicity of attachment of the protein ligand, for optimizing the immunological reactivity of the immunosorbent and for minimizing leakage of covalently bound protein from the resin.

Influence of subclass-specific anti-idiotypic antibodies on the kinetics of the immune response to BCG.

The role of anti-idiotypic subclass-specific antibodies was analysed in the regulation of the immune response to BCG in the guinea-pig. The idiotypes to BCG were separated into subclasses and anti-idiotypes were carried out by immunizing with the purified IgG1 and IgG2 anti-idiotypes. The in vivo T cell response was recorded by tuberculin skin testing, and the in vitro response by lymphocyte stimulation testing with tuberculin. A suppressive effect was detected in cases where the animals were preimmunized with anti-idiotypic IgG2 against anti-BCG IgG1. In the B cell response, the anti-BCG IgG1 and IgG2 subclasses were also quantified by a solid-phase radioimmunoassay. There were 10 times more IgG2 antibodies than IgG1 against BCG in the guinea-pig, and this major idiotypic subclass was suppressed by the IgG2 anti-idiotype raised against anti-BCG IgG2. The minor component anti-BCG IgG1 was slightly stimulated by both IgG2 anti-idiotypes.

A fatal case of severe SLE complicated by invasive aspergillosis.

We report on the case of a 25-year-old female with severe systemic lupus erythematosus (SLE) who presented with pancytopenia, fever, arthralgia and abdominal pain. After antibiotic treatment, the patient was afebrile for 3 days before her temperature rose again. Dyspnoea and cough pointed towards pneumonia which was confirmed by X-ray. Different antibiotics and the antimycotic agent fluconazol were given. The lupus flare was treated with high-dose prednisolone. After a couple of days, the dyspnoea increased and mechanical ventilation became necessary. Bronchoscopy and transbronchial biopsy revealed the diagnosis of invasive aspergilloses. Despite of an immediate treatment with amphotericin B, the patient died because of respiratory insufficiency. The literature on aspergillosis in SLE is reviewed and prophylactic, diagnostic and therapeutic options are discussed for this infectious complication which has an 80% mortality in patients with SLE.

Detection of the t(2;5)-associated NPM/ALK fusion cDNA in peripheral blood cells of healthy individuals.

The translocation t(2;5), which leads to the fusion of the nucleophosmin gene (NPM) on chromosome 5q35 to the receptor kinase ALK on chromosome 2p23, is found in CD30+ anaplastic large cell lymphomas and some cases of B-cell lymphoma. Hodgkin's disease (HD) is a malignant lymphoma characterized by large multinucleated tumour cells, Hodgkin and Reed-Sternberg (H&RS) cells, surrounded by a dense lymphohistiocytic infiltrate. Our group recently demonstrated NPM/ALK fusion cDNAs by single-cell RT-PCR in < 3% of CD30+ tumour cells in 2/9 cases of HD. To further delineate the relevance of this finding for HD, we studied the occurrence of NPM/ALK fusion genes in peripheral blood cells of healthy donors by RT-PCR. NPM/ALK fusion cDNAs were found by RT-PCR in 14/29 healthy individuals and confirmed by hybridization with a breakpoint-specific oligonucleotide. Due to the low rate of NPM/ALK-positive cells in the peripheral blood of positive individuals, an assignment to a defined cellular subpopulation was not possible. We conclude that NPM/ALK fusion genes are present in peripheral blood cells of healthy donors. After t(14;18) and t(9;22), t(2;5) represents the third example of tumour-associated translocation products in blood cells of apparently healthy donors. The implications of this finding are discussed.

[Sequential high-dose cytarabine therapy in combination with asparaginase in acute myeloid leukemia]. / Sequentielle hochdosierte Cytarabin-Therapie in Kombination mit Asparaginase bei der akuten myeloischen Leukämie.

Between 1984 and 1987 14 patients with acute non-lymphocytic leukemia were treated with sequential high-dose cytosine arabinoside in combination with asparaginase. Twelve patients were suffering from refractory leukemia; in these patients complete remissions were achieved in 58%. The efficacy of this schedule was much better in patients with substantial leukemia cell reduction due to antecedent conventional therapy and no more than 25% blast cells in the bone marrow. In this subgroup complete remissions were achieved in 75% and 86% respectively, taking into account only the completed treatment courses. Beside the well-known side-effects such as alopecia, nausea, vomiting and hepatotoxicity, we observed an increase in severe infections. Three patients died of pulmonary mycosis.

Assessment of clonality of rosetting T lymphocytes in Hodgkin's disease by single-cell polymerase chain reaction: detection of clonality in a polyclonal background in a case of lymphocyte predominance Hodgkin's disease.

Rosetting of CD4+ T cells around the neoplastic Hodgkin and Reed-Sternberg (H&RS) cells is a characteristic feature of Hodgkin's disease (HD). To answer the question whether this phenomenon is solely due to chemokine-mediated attraction of T cells or whether the rosetting T cells in addition recognize antigens presented by the H&RS cells, we examined the T cells adherent to H&RS cells. Cells from five cases of HD [four classic HD and one lymphocyte-predominant (LP) HD] were examined by single-cell analysis for the T-cell receptor (TCR) gamma gene. Between 5 and 17 rosettes containing one to ten rosetting lymphocytes and the corresponding H&RS cells were amplified in separate plastic tubes. Of the resulting 119 TCRgamma polymerase chain reaction (PCR) products, 87 were sequenced. While no evidence of a clonal expansion was obtained in the lymph nodes from four of five patients with classic HD, clonal TCRgamma sequences were found in the lymph node from the patient within LPHD in two independent experiments analyzing seven and ten different rosetting complexes, respectively. Of 13 products, 11 showed identical Vgamma9 sequences. Unrelated products were found in all other TCRgamma family subgroups in this case. Single H&RS cells picked as controls were negative for TCRgamma rearrangements. Our results demonstrate that clonal proliferations on a polyclonal background can occur among the T cells forming rosettes with Hodgkin cells and lend support to the view that Hodgkin cells may also function as cells presenting antigens to the adhering T cells.

The value of magnetic resonance imaging at 1.5 T in the evaluation of the scrotal content.

50 patients with a broad variety of scrotal disorders were evaluated in form of a prospective study by high-resolution scrotal sonography and magnetic resonance imaging at 1.5 T with high-resolution surface coils. Basic anatomic correlations to normal structures of the scrotum were studied in 4 fresh cadaver testicles without pathology. Magnetic resonance imaging is sensitive and specific, but time-consuming and expensive.

Lymphocytic plasmocytoid lymphoma with a three-banded gammopathy: reactivity of one of these paraproteins with cytomegalovirus.

We report the case of a 70-year-old woman suffering from small lymphocytic, plasmocytoid lymphoma with abdominal lymphomas and infiltration of the lung and the bone marrow. A three-banded, IgG lambda, IgM lambda and IgA lambda-paraproteinemia was determined using immunofixation. Because of the patient's high antibody titre against cytomegalovirus (CMV), the possible reactivity of these paraproteins with CMV was studied. The immunoglobulins were transferred to nitrocellulose sheets by a contact diffusion blotting system. CMV was applied to these sheets and the IgG lambda-paraprotein was shown to bind CMV. The reactivity of only one of the paraproteins with CMV suggests an oligoclonal origin of this gammopathy. In addition to the malignant disease an abnormal immune response to a CMV infection could be the cause of this three-banded gammopathy.

Detection of immunoglobulin heavy-chain gene rearrangement at the single-cell level in malignant lymphomas: no rearrangement is found in Hodgkin and Reed-Sternberg cells.

Hodgkin and Reed-Sternberg (HRS) cells, the neoplastic cells of Hodgkin's disease (HD), represent only a minority of the cellular infiltrate in affected tissue. Therefore, rearrangements of the immunoglobulin heavy-chain (IgH) gene detected in DNA extracted from an entire Hodgkin's lymph node cannot be attributed to the HRS cells and cannot be used as an argument for the B-cell origin of HRS cells. We developed a new method for the amplification of rearranged DNA of the IgH gene from single cells. Using 6 "forward primers" which were constructed corresponding to consensus sequences of the 6 known families of the IgH variable (V) region (framework region I) and a mix of 2 "reverse primers" corresponding to consensus sequences of the different joining (J) segments, rearrangements of all 6 V-families were detected in human peripheral blood lymphocytes. Rearranged IgH DNA could be amplified from single cells of B-cell lymphoma-cell lines and from 13 patients with B-cell non-Hodgkin's lymphomas. However, analysis of HRS cells isolated from lymph nodes of 13 patients with Hodgkin's disease did not show any rearrangement of the IgH gene locus. These findings, obtained by polymerase chain reaction (PCR) on isolated single HRS cells, contrast with previous studies that used Southern-blot analysis of entire tissues affected by Hodgkin's disease. We conclude that the neoplastic HRS cells in Hodgkin's disease--with the possible exception of the nodular paragranuloma subtype--are probably not derived from B cells.

[Transitory oligoclonal paraproteinemia with virus infection and malignant lymphatic disease]. / Transitorische oligoklonale Paraproteinämie bei Virusinfektion und maligner lymphatischer Erkrankung.

Oligoclonal paraproteinaemia occurred in two patients with malignant lymphocytic disease (highly malignant non-Hodgkin lymphoma, B-cell type of acute lymphocytic leukaemia), in one case during a cytomegalovirus infection and in the other during an infectious mononucleosis. At that time both patients were in complete remission. Paraproteinaemia in the first case disappeared within a year where the transformation from an initially four-banded paraproteinaemia (2 IgM-lambda and 2 IgG-lambda) into a three-banded paraproteinaemia (IgG-lambda) could be observed. In the second patient the concentration of the paraprotein decreased considerably. Because both patients were no longer under cytostatic treatment after manifestation of the paraproteinaemia, and were in complete remission during the whole of the observation period (4 years and 1 year), a direct relationship with the primary disease is unlikely. The transitory paraproteinaemia appears to be rather the result of an acquired defect of the immune system.

Regional rates of myocardial fatty acid metabolism: comparison with coronary angiography and ventriculography.

In 50 patients, 1 mCi 123I phenylpentadecanoic acid (IPPA) was injected at peak ergometric stress and 1500 frames were acquired (1 frame/s) with a high count rate gamma camera. Parametric images of rates of decrease and increase for different time intervals after stress were compared with coronary angiography and LV ventriculography, separately evaluating the 3 main coronary territories: 18/150 territories supplied by normal coronaries presented rather homogeneous regional clearing rates, whereas a gradual decrease in clearing rates towards the end of the territory (frequently with peripheral defects) was seen in all 87/150 territories with significant coronary narrowing. In local correspondence to clearing defects, initial IPPA accumulations could be observed with later onset of clearing between 10 and 25 min. 44/150 territories presented abnormal clearing rates, mostly with a patchy pattern, with normal coronary anatomy, but all except one had LV dysfunction and a clinical diagnosis of cardiomyopathy, diabetes mellitus or hypertensive disease. Twenty four of the 41 patients with CAD had, in correspondence to a prior myocardial infarction, minimum or missing metabolic activity frequently in circumscribed zones, partly separated by bridges of still viable tissue with preserved but reduced clearing rates.

The mechanism of the antiherpetic activity of zinc sulphate.

The molecular mechanism of the effects of zinc ions against herpes simplex virus (HSV) infection was investigated. Zinc sulphate (100 microM) in the culture medium of an HSV-infected African green monkey kidney cell line did not block viral DNA synthesis and, at this concentration, only moderate cytotoxic effects were observed in uninfected cells. Nevertheless, virus yields were reduced to less than 1% of the control. Thus the long standing hypothesis that zinc might block multiplication of HSV by selective intranuclear inhibition of the viral DNA polymerase apparently has lost its validity. Inhibition of virus growth in the absence of severe cytotoxicity must therefore result from other effects of ZnSO4. Free virus is inactivated by 15 mM-ZnSO4 within a few hours of its addition. The inactivated virus is defective in the glycoprotein-dependent functions of penetration and, to some extent, adsorption. Electron micrographs show massive deposition of zinc onto virion components. In a virion, transmembrane transport of zinc ions is not expected and the established antiviral effect is therefore explained by an inhibition of virion glycoprotein function after non-specific accumulation of zinc into many virion membrane components.

Immunoglobulin and T cell receptor gene rearrangements in lymphoproliferative disorders.

Thirty-one samples representing Hodgkin's and non-Hodgkin's lymphomas, angioimmunoblastic lymphadenopathy (AILD), and benign follicular hyperplasia in HIV infections were examined for rearrangements of the immunoglobulin (Ig) and T cell receptor (TcR) beta-chain gene loci. In 11 of 12 non-Hodgkin's lymphomas (classified as Burkitt lymphoma (2), centrocytic lymphoma (1), centrocytic-centroblastic lymphoma (5), centroblastic lymphoma (3], only rearranged Ig genes could be detected. The exceptional case was an unclassified high-grade lymphoma, which represented a rearrangement of the TcR beta-chain. We also examined DNA from lymphoid neoplasms in which the lineage of the malignant cell was still controversial. Rearrangement of the TcR could exclusively be demonstrated in all 3 cases of AILD. One Ig gene rearrangement and 4 TcR beta-chain rearrangements were found in 13 samples of Hodgkin's lymphomas (11 lymph nodes, 1 pleura effusion and 1 bone biopsy with proven infiltration). Examination of 3 cases of benign follicular hyperplasia in HIV infection represented one Ig rearrangement.

Analysis of the soluble cytoplasmic components of Mycobacteria and Nocardia by crossed immunoelectrofocusing.

Soluble cytoplasmic components of Mycobacterium tuberculosis, strain H 37 Ra, and Washington II, Mycobacterium bovis, strain BCG and Nocardia asteroides were analysed by isoelectric focusing, crossed immunoelectrophoresis, and crossed immunoelectrofocusing. Using con A-affinity chromatography con A-reactive polysaccharides could be separated from the cytoplasmic fraction which improved the focusing effect. The isoelectric focusing patterns of the various mycobacterial strains were similar, however, concentration differences of the respective components might occur. N. asteroides had a different pattern. By crossed immunoelectrofocusing it was shown that most of the bands of the focusing pattern had antigenic character. Con A-reactive polysaccharides were located near to the cathode, and the con A-nonbound antigens occured close to the anode. Cross-reaction between mycobacteria and N. asteroides was remarkable. In crossed-line immunoelectrophoresis four line in N. asteroides antigens were identical with the highest peaks of M. bovis, strain BCG antigens.