Bernhard F. Odermatt

Detection of Epstein-Barr virus DNA by polymerase chain reaction in lymph node biopsies from patients with angioimmunoblastic lymphadenopathy.

Summary. Epstein‐Barr virus DNA was detected by the polymerase chain reaction (PCR) in five lymph node biopsies from eight patients with diagnosis of angioimmunoblastic lymphadenopathy (AILD). Three pairs of specific primers detected EBV DNA sequences near the 5’end (Bam W region), the middle (BMRF 1 region) and the 3’end (Eco RI D region) of the viral genome with equal accuracy when 1 μg of DNA and 30 amplification cycles were used. When only 100 ng of DNA were screened with the BMRF 1 set of primers, a specific amplification product was still identifiable. The 593 base pair amplification product obtained using the Eco RI D set of primers was shown to contain an expected Sma I site at position 215, confirming the viral origin of sequence. Our findings indicate that lymph nodes of AILD patients frequently harbour the entire EBV genome at a high percentage of at least 1 viral copy per 15 000 human cells.

The Role of Epstein-Barr Virus in Neoplastic Transformation

In this review, we focus on new data from basic, translational and clinical research relating to the Epstein-Barr virus (EBV). Beside its well-known tropism for B lymphocytes and epithelial cells, EBV also infects T lymphocytes, monocytes and granulocytes. After primary infection, EBV persists throughout the life span in resting memory B cells, from where it is reactivated upon breakdown of cellular immunity. In the process of neoplastic transformation, the EBV-encoded latent membrane protein 1 (LMP1) oncogene represents the major driving force. LMP1 acts like a constitutively activated receptor of the tumor necrosis factor receptor family and allows the amplification or bypassing of physiological regulatory signals through direct and indirect interactions with proteins of the tumor necrosis factor receptor-associated factor (TRAF) family. TRAF2-mediated NF-ĸB activation, AP-1 induction and JAK3/STAT activation may result in sustained proliferation leading to lymphoma. The ability of LMP1 to suppress germinal center formation and its capacity to mediate its own transcriptional activation shed new light on the pathogenesis of EBV-associated latency type II lymphoproliferations like Hodgkin’s disease and angioimmunoblastic lymphadenopathy. The carboxy terminus of LMP1 is also a reliable marker for individual EBV strain identification and thus offers new possibilities in tracing the molecular events leading to posttransplant lymphoproliferative disorders (PTLDs). Cytotoxic T lymphocytes directed against well-characterized epitopes of EBV latency genes represent an already successful and promising therapeutic approach to EBV-associated lymphomas, in particular PTLDs.

Differentiation of mononuclear blood cells into macrophages, fibroblasts and endothelial cells in thrombus organization

The organization of thrombi and emboli may be performed exclusively by mononuclear blood cells which represent precursors of various mesenchymal cell types. Between the 10th and 20th day after the onset of blood clotting, mononuclear cells within the fibrinous matrix differentiate into (1) macrophages responsible for hematoclasia and hematophagocytosis, (2) endothelial cells lining autolytic slits in the fibrinous matrix and forming new capillaries, and (3) fibroblasts and even smooth muscle cells building up a young mesenchymal connective tissue.

Natural 30 base pair and 69 base pair deletion variants of the LMP1 oncogene do stimulate NF-κB-mediated transcription

An increasing number of reports shows a link between the Epstein-Barr virus (EBV) and lymphoid neoplasia. The latent membrane protein 1 (LMP1) is likely to play a determinant role in this process since this EBV encoded protein has oncogenic properties and is usually expressed in EBV-associated lymphoproliferative diseases (LPD), except Burkitts lymphoma. We previously identified in LPD patients mutational hot spots and a 30 bp or 69 bp deletion in the LMP1 gene region coding for the C-terminal domain. These deletions are located in an area shown to be important for the activation of the transcription factor NF-κB. These findings lead us to test whether these natural deletion variants may have a functional effect. We measured the stimulation of their activity using a luciferase reporter plasmid containing NF-κB responsive elements. We tested the NF-κB inducing activity of four naturally occurring LMP1 deletion variants. Our results show that these deletion variants activate NF-κB to the same level as the wild-type form, indicating that the crucial residues for NF-κB activation are conserved among the variants isolated and lie within the last 32 amino acids of the C-terminal domain of the LMP1 oncogene.

Distribution pattern of tenascin‐C in normal and neoplastic mesenchymal tissues

Descriptions for tenascin‐C distribution are largely restricted to epithelial tumours. The present study utilized newly developed and characterized monoclonal (hT191) and polyclonal antibodies to investigate the distribution pattern of tenascin‐C in a panel of mesenchymal tumours, which was contrasted with normal tissue. The specific antibodies recognized the distinctive star‐like hexabrachion protein isolated from transformed cell‐culture medium and serum from normal individuals. In normal tissues, a strong tenascin‐C expression in the extracellular matrix was largely restricted to basement‐membrane regions of epithelium and tonsilar sinusoids, pericellularly within smooth‐muscle bundles, associated with perimysial, ‐chondrial, ‐neurial and ‐tendon surfaces, and diffusely within vascular adventitia. It was found in the corresponding tumours of the neural sheath (schwannoma) and smooth muscle (leiomyosarcoma), and was abundantly present around certain blood vessels of mesenchymal tumours. Although not detected in normal muscle, or in adipose or fibrous connective tissue, neo‐expression of tenascin‐C was shown in more than half of the rhabdomyosarcomas, fibromas and liposarcomas, with an increased positive percentage in variably malignant myxoid liposarcomas compared with lipoma‐like sarcomas. Tenascin‐C was typically found in the extracellular matrix of soft‐tissue tumours, but was notably absent from the epithelial‐cell components of mixed epithelial/mesenchymal tumours. Its apparently enhanced expression in soft‐tissue tumours differs from that of most other large extracellular‐matrix proteins, suggesting possible functional involvement of the cell‐adhesion molecule, tenascin‐C, in the neoplastic phenotype. Int. J. Cancer 72:217–224, 1997.

Latent membrane protein 1 associated signaling pathways are important in tumor cells of Epstein-Barr virus negative Hodgkin's disease.

The latent membrane protein 1 (LMP1) oncogene of Epstein-Barr virus (EBV) is selectively expressed in the Reed-Sternberg (RS) cells of EBV-associated Hodgkins disease (HD). However, no differences in clinical presentation and course are found between EBV positive and EBV negative forms of HD suggesting a common pathogenetic mechanism. We have studied the LMP1 associated signaling pathways and their dominant negative inhibition in the myelomonocytic HD-MyZ and the B-lymphoid L-428 HD cell lines. In both EBV negative cell lines expression of LMP1 is associated with the formation of multinuclear RS cells. Dominant negative inhibition of NF-κB mediated signaling at the step of IκB-α phosphorylation results in increased cell death with only a few typical RS cells resistant to overexpression of the dominant negative inhibitor IκB-α-NΔ54. However, dominant negative inhibition of NF-κB mediated signaling at the early step of TRAF2 interaction results in the formation of multinuclear cells in both cell lines and, in addition, in clusters of small mononuclear cells in the HD-MyZ cell line. In HD-MyZ cells overexpression of the powerful JBD-inhibitor of the JNK signal transduction pathway is restricted to small cells and never observed in RS cells. These small cells undergo apoptosis as shown by the TUNEL technique. Apoptosis of small cells is still observed after co-transfection of JBD and LMP1 but in addition a few apoptotic HD-MyZ cells with large fused nuclear masses are identified suggesting that specific inhibition of JNK leads also to apoptosis of LMP1 induced RS cells. Thus, activation of the JNK signaling pathway is also important in the formation of Reed-Sternberg cells. Our findings are consistent with a model where all three LMP1 associated functions, i.e. NF-κB mediated transcription, TRAF2 dependent signaling, and c-Jun activation act as a common pathogenetic denominator of both EBV negative and EBV positive HD.

Polyclonal rearrangements of the T-cell receptor beta-chain in fatal angioimmunoblastic lymphadenopathy

Genomic rearrangement of germline T‐cell antigen receptor (TcR) and immunoglobulin (Ig) genes was studied by Southern blot analysis in seven patients with angioimmunoblastic lymphadenopathy (AILD). In three cases clinically suspected of transformation into malignant lymphoma, hybridization with the TcRβ probe showed markedly dimished intensity in the 11.5 kb germline band after Eco RI digestion and normal germline configuration after Hind III and Bam HI digestion, indicating polyclonal T cell rearrangements. A clonal rearrangement of the TcRβ gene was detected in only one case at initial biopsy. No monoclonal rearrangement of Ig genes was observed. These data show that in some cases of AILD disease progression is indicated by polyclonal TcR rearrangements and not by outgrowth of a malignant clone, supporting the concept of AILD as an immunoregulatory disorder.

Diagnostic and prognostic value of monoclonal antibodies in immunophenotyping of angioimmunoblastic lymphadenopathy/lymphogranulomatosis X

Summary. Immunophenotyping of frozen lymph node sections from seven patients with morphologically defined angioimmunoblastic lymphadenopathy (AILD) was performed with a panel of 20 monoclonal antibodies (MoAb). MoAb for identification of dendritic reticulum cells showed remnants of follicular structures in all cases. In four cases these ‘burnt out’ follicular structures were associated with clusters of polyclonal B‐cells. Vascular proliferation and small amounts of intercellular collagenous fibrils were reliably revealed by MoAb CIV22 against basement membrane collagen. Cellular infiltrates of T4 + cells prevailed in four cases, of T8+ cells in one case. In one case conventionally identified blasts were of T‐and B‐cell origin. Expression of cell proliferation associated nuclear antigen identified by MoAb Ki‐67 was present in more than 25% of lymph node cells in three patients with fatal outcome, but in less than 10% of cells in two patients with a better clinical course. Therefore MoAb Ki‐67 may represent a valuable prognostic marker in AILD.

Monoclonal Antibodies to Human Cytokeratins: Application to Various Epithelial and Mesothelial Cells

Immunohistological analysis of human tissue using monoclonal antibodies against cytokeratins, which are confined to cells of epithelial origin, is a valuable technique. Using human epidermal keratins as antigen, we prepared monoclonal antibodies against cytokeratins (ZK1, ZK7, ZK61 and ZK99) and against a desmosomal protein (ZK31). Immunohistochemical staining of human skin sections using these antibodies showed a specific reaction with the epidermis: ZK1 stained the entire epidermis, ZK7 only the basal layer, ZK61 and ZK99 the suprabasal layers, and ZK31 the cellular interfaces. In order to test for antibody specificity, immunoblots with human epidermal and amnion epithelial cytokeratin polypeptides, as well as immunofluorescence microscopy of simple epithelia (glandular and simple columnar epithelia) were performed. ZK1, ZK61 and ZK99 reacted preferentially with cytokeratin polypeptides of stratified squamous epithelia and ZK7 recognized cytokeratins of stratified and simple epithelia. When the ZK antibodies were tested on mesothelial cells in pleural effusions, only ZK7 reacted with these cells. Biochemical analysis of cytokeratin accumulation in cells of primary and long-term cultures indicated that the cytokeratin pattern of mesothelial cells was quite unstable, while that of amnion epithelial cells showed only minor quantitative changes. The use of these antibodies to determine the epithelial origin of cells present in pleural effusions is proposed.

Loss of type VI collagen in experimental and most spontaneous human fibrosarcomas

Expression of type VI collagen, an adhesive protein of mesenchymal tissues, is significantly down‐regulated upon viral transformation of fibroblasts. Likewise, most cell lines derived from spontaneous mesenchymal tumors, including fibrosarcomas, rhabdomyosarcomas, leiomyosarcomas, chondrosarcomas and liposarcomas, do not synthesize type VI collagen because they are not capable of expressing all 3 of the polypeptide chains required for the assembly of a functional heterotrimeric molecule. When injected into nude mice, neither fibrosarcoma cells (HT1080) nor rhabdomyosarcoma cells (A204) initiate the synthesis of type VI collagen, suggesting that the inhibition is not caused by deficiency of a paracrine factor. Immuno‐histochemical studies further illustrate that 15 of 17 spontaneous adult fibrosarcomas lack type VI collagen in the tumor stroma. The absence of this important adhesion protein may contribute to tumorigenicity, invasiveness and/or metastasis of mesenchymal tumor cells. Int. J. Cancer 86:331–336, 2000.