Edith Bachmann

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.

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.

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.

EBV‐associated lymphoproliferative syndrome with a distinct 69 base‐pair deletion in the LMP‐1 oncogene

Summary. We describe an immunocompetent 12‐year‐old boy with chronic EBV infection and lymphoid interstitial pneumonitis. Lymph node biopsies showed effacement of the architecture with polymorphic cellular infiltrates, consisting predominantly of T cells and natural killer cells. No clonal rearrangement of TCR or immunoglobulin genes was seen. DNA was extracted from hilar lymph nodes; sequencing of the carboxy terminal region of the latent membrane protein 1 (LMP‐1) oncogene revealed a 69 base‐pair deletion and four point mutations. Immunosuppressive treatment with prednisone and cyclosporine reversed the lymphadenopathy.

Expression of human recombination activating genes (RAG-1 and RAG-2) in lymphoma.

Two recently discovered genes, the recombination activating genes 1 and 2 (RAG-1 and RAG-2), are necessary to perform variable (V), diversity (D), and joining (J) recombination. They synergistically activate VDJ recombination to generate immunocompetent lymphocytes. Disruption of either gene results in a maturation arrest at a very early B and T cell progenitor stage. Expression and downregulation of RAGs are closely associated with interleukin 7, sIgM and TCR-CD3 complex, respectively. Assessment of RAG mRNA expression is a valuable marker in identifying the genotypic maturation status of leukemias and lymphomas. Persistent RAG expression in otherwise mature lymphoid proliferations may explain puzzling biological and clinical observations such as multiple rearrangements in lymphomas with a mature phenotype. Lack of RAG expression in Hodgkins disease with abundant Reed-Sternberg cells is consistent with a mature phenotype of the latter. Availability of a anti-RAG-1 monoclonal antibody in the near future will facilitate RAG analysis of lymphomas.

Expression of human recombination activating genes (RAG‐1 and RAG‐2) in angioimmunoblastic lymphadenopathy and anaplastic large cell lymphoma of T‐type

Summary. In order to determine the genotypic maturation status of the proliferating lymphoid cells in angioimmunoblastic lymphadenopathy (AILD) and in anaplastic large cell lymphoma of T‐type (T‐ALC), recombinase activating gene (RAG‐1 and RAG‐2) expression was assessed in six AILD and five T‐ALC cases using a sensitive reverse transcriptase (RT) and competitive (C) polymerase chain reaction (PCR). RAG transcripts were not detectable in nine cases with high proliferating activity, suggesting that in most cases the proliferating cells are derived from mature (rearranged) lymphocytes. However, low levels of RAG transcripts were detected in one AILD and one T‐ALC case and are consistent with either an involvement of immature lymphoid precursors in the proliferating pool or a deregulated T‐cell maturation pathway with persistence of RAG expression. An association between RAG gene expression and poor response to therapy is possible but has to be tested in larger prospective series.

Semiquantitative Analysis of Epstein-Barr Virus DNA by Polymerase Chain Reaction in Clinical Samples of Lymphoproliferative Disorders

Epstein-Barr virus (EBV) DNA was detected and semiquantitatively analyzed in clinical samples from patients with Hodgkin’s disease (HD), angioim-munoblastic lymphadenopathy (AILD), hairy cell leukemia (HCL), T-lymphoblastic lymphoma (TLL), familial gastric lymphoma, lymphocytic/ mixed thymoma, and reactive lymph node hyperplasia (HR). High numbers of viral DNA copies were detected in HD and AILD, whereas no or very few viral DNA copies were found in HCL and thymoma. One case of TLL and two cases of HR contained relatively high numbers of EBV DNA copies. In the case of TLL, the viral genomes probably originated from accessory B cells. The high amount of EBV DNA identified in the HD and AILD cases is not associated with the Bam W/Bam Z rearrangement known to disrupt viral latency. Technical details of amplification, oligonucleotide hybridization, and semiquantitative EBV DNA analysis are described. A sensitive detection system of the Bam W/Bam Z rearrangement is also presented.

Significance of the Detection of Epstein—Barr Virus DNA In Lymph Nodes in Patients with Hodgkin's Disease

Epstein-Barr virus (EBV) DNA is frequently identified in benign and malignant lymphoproliferative conditions. As shown by in situ hybridization studies viral DNA is localized within malignant cells as well as benign lymphocytes. Clonal and nonclonal EBV genomes are present in Hodgkins disease (HD), lymphomas of the immunocompromised host and reactive lymph node hyperplasia. Lytic infection with formation of linear genomes is observed in the same conditions but appears to be infrequent in HD as shown by quantitation of mRNA coding for viral capsid antigen. Expression of the oncogene LMP (latent membrane protein) is seen in Sternberg-Reed (SR) cells and immunoblasts of AIDS-related lymphoma and infectious mononucleosis (IM). In HD, the region of the BNLF1 oncogene coding for the amino terminal and transmembrane domains (associated with oncogenic function) of LMP appears to be homogeneous whereas the region coding for the intracytoplasmic (carboxy terminal) domain of LMP is heterogeneous. Cytological similarities between SR cells and immunoblasts of IM and AIDS-related lymphomas are consistent with the hypothesis that the BNLF1 oncogene is one possible inducer of morphological features of SR cells. Whether chromosomal integration of EBV DNA is an important factor in activation of such a transforming activity remains to be elucidated. EBV DNA positive and negative HD cases with numerous SR cells lack significant mRNA expression of the two recombinase activating genes (RAG-1 and RAG-2). Therefore the SR cells appear to be derived from lymphocytes beyond the pre-B-cell or common thymocyte stage which may or may not subsequently become infected by EBV.