The lever arm ratio of the rotator cuff to deltoid muscle explains and predicts pseudoparalysis of the shoulder: the Shoulder Abduction Moment index.

AIMS: In patients with a rotator cuff tear, tear pattern and tendon involvement are known risk factors for the development of pseudoparalysis of the shoulder. It remains unclear, however, why similar tears often have very different functional consequences. The present study hypothesizes that individual shoulder anatomy, specifically the moment arms (MAs) of the rotator cuff (RC) and the deltoid muscle, as well as their relative recruitment during shoulder abduction, plays a central role in pseudoparalysis. MATERIALS AND METHODS: Biomechanical and clinical analyses of the pseudoparalytic shoulder were conducted based on the ratio of the RC/deltoid MAs, which were used to define a novel anatomical descriptor called the Shoulder Abduction Moment (SAM) index. The SAM index is the ratio of the radii of two concentric spheres based on the centre of rotation of the joint. One sphere captures the humeral head (numerator) and the other the deltoid origin of the acromion (denominator). A computational rigid body simulation was used to establish the functional link between the SAM index and a potential predisposition for pseudoparalysis. A retrospective radiological validation study based on these measures was also undertaken using two cohorts with and without pseudoparalysis and massive RC tears. RESULTS: Decreased RC activity and improved glenohumeral stability was predicted by simulations of SAM indices with larger diameters of the humeral head, being consequently beneficial for joint stability. Clinical investigation of the SAM index showed significant risk of pseudoparalysis in patients with massive tears and a SAM < 0.77 (odds ratio (OR) 11). CONCLUSION: The SAM index, which represents individual biomechanical characteristics of shoulder morphology, plays a determinant role in the presence or absence of pseudoparalysis in shoulders with massive RC tears.

Pull-out strength of patient-specific template-guided vs. free-hand fluoroscopically controlled thoracolumbar pedicle screws: a biomechanical analysis of a randomized cadaveric study.

PURPOSE: To assess the pull-out strength of thoracolumbar pedicle screws implanted via either a patient-specific template-guided or conventional free-hand fluoroscopically controlled technique in a randomized cadaveric study, and to evaluate the influence of local vertebral bone density, quantified by Hounsfield units (HU), on pedicle screw pull-out strength. METHODS: Thoracolumbar pedicles of three spine cadavers were instrumented using either a free-hand fluoroscopically controlled or a patient-specific template-guided technique. Preoperative bone density was quantified by HU measured on CT. Pedicle perforation was evaluated on postoperative CT scans by an independent and blinded radiologist. After dissected vertebrae were embedded in aluminum fixation devices, pull-out testing was initiated with a preload of 50 N and a constant displacement rate of 0.5 mm/s. Subgroup analyses were performed excluding pedicle screws with a pedicle breach (n = 47). RESULTS: Pull-out strength was significantly different with 549 ± 278 and 441 ± 289 N in the template-guided (n = 50) versus fluoroscopically controlled (n = 48) subgroups (p = 0.031), respectively. Subgroup analysis limited to screws with an intrapedicular trajectory revealed a tendency toward a higher pull-out strength in the template-guided (n = 30) versus fluoroscopically controlled screws (n = 21) with 587 ± 309 and 454 ± 269 N (p = 0.118), respectively. There was a trend toward a higher pull-out strength (709 ± 418 versus 420 ± 149 N) in vertebrae with a bone density of (>171 HU) versus (<133 HU), respectively (p = 0.061). CONCLUSIONS: There was a significantly higher pull-out strength of thoracolumbar pedicle screws when inserted via a patient-specific template-guided versus conventional free-hand fluoroscopically controlled technique, potentially associated with screw trajectory.

Activation of microglia cells is dispensable for the induction of rat retroviral spongiform encephalopathy.

In the course of retroviral CNS infections, microglia activation has been observed frequently, and it has been hypothesized that activated microglia produce and secrete neurotoxic products like proinflammatory cytokines, by this promoting brain damage. We challenged this hypothesis in a rat model for neurodegeneration. In a kinetic study, we found that microglia cells of rats neonatally inoculated with neurovirulent murine leukemia virus (MuLV) NT40 became infected in vivo to maximal levels within 9-13 days postinoculation (d.p.i.). Beginning from 13 d.p.i., degenerative alterations, i.e., vacuolization of neurons and neuropil were found in cerebellar and other brain-stem nuclei. Elevated numbers of activated microglia cells--as revealed by immunohistochemical staining with monoclonal antibody ED1--were first detected at 19 d.p.i. and were always locally associated with degenerated areas but not with nonaltered, yet infected, brain regions. Both neuropathological changes and activated microglia cells increased in intensity and numbers, respectively, with ongoing infection but did not spread to other than initially affected brain regions. By ribonuclease protection assays, we were unable to detect differences in the expression levels of tumor-necrosis-factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and interleukin-6 (IL-6) in microglia cells nor in total brains from infected versus uninfected rats. Our results suggest that the activation of microglia in the course of MuLV neurodegeneration is rather a reaction to, and not the cause of, neuronal damage. Furthermore, overt expression of the proinflammatory cytokines TNF-alpha, IL-1beta, and IL-6 within the CNS is not required for the induction of retroviral associated neurodegeneration in rats.

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 Hodgkin's 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-kappa B mediated signaling at the step of I kappa B-alpha phosphorylation results in increased cell death with only a few typical RS cells resistant to overexpression of the dominant negative inhibitor I kappa B-alpha-N delta 54. However, dominant negative inhibition of NF-kappa B 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-kappa B mediated transcription, TRAF2 dependent signaling, and c-Jun activation act as a common pathogenetic denominator of both EBV negative and EBV positive HD.

Sequence polymorphisms between latent membrane proteins LMP1 and LMP2A do not correlate in EBV-associated reactive and malignant lympho-proliferations.

The latent membrane proteins LMP1 and LMP2A are co-expressed in most malignancies associated with Epstein-Barr virus (EBV). In contrast with the transforming LMP1 oncoprotein, LMP2A is expressed in lymphocytes of healthy EBV carriers and considered to maintain viral latency. Critical for these LMP2A functions are a transmembranous epitope recognized by specific cytotoxic T lymphocytes (CTLs) and the N-terminal immunoreceptor tyrosine-based activation motif (ITAM), blocking B-cell receptor signaling. To characterize ITAM and CTL motifs of LMP2A and to correlate them with C-terminal variants of LMP1 including the 30-bp deletion variant (LMP1delta), comparative sequence analysis was performed on 76 samples from patients with reactive and malignant lympho-proliferation (infectious mononucleosis, n=21; tonsillar hyperplasia, n=16, chronic lympho-proliferation, n = 9; Hodgkin's disease, n = 8; Non-Hodgkin's lymphoma, n = 5; AIDS-related large-cell lymphoma, n=17). The CTL motif was conserved in all but 2 cases (C426-->S). The ITAM motif was characterized by strictly conserved YXXL sequences in all cases, with a sequence polymorphism in between. The B95.8 prototype was found in 17% (13/76) of cases, while in 72% a variant with 3 point mutations (166796 C-->A, 166805 C-->A, 166810 C-->T) was detected; 11% had 1 or 2 of these mutations in addition to G-->A at 166793. In the C terminus of LMP1, a hypervariable region including LMP1delta was described in 61% of cases. There was no significant association of a particular LMP2A variant with either malignant phenotype or LMP1delta, demonstrating that the functional domains of LMP2A are conserved and that the sequence polymorphisms in LMP1 and LMP2A are independent.

Molecular and functional analysis of the Epstein-Barr virus LMP1 oncogene promoter in lymphoproliferative diseases.

The expression of the Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) oncogene is tightly regulated by viral and cellular factors. LMP1 is present in the majority of nasopharyngeal carcinoma tumor cells and in Reed-Sternberg cells from Hodgkin's lymphoma, in which the only EBV nuclear antigen detected is EBNA1. The aim of this study was to test whether mutations affecting LMP1 gene expression were present in lymphoproliferative disorders, and, if so, whether their presence correlated with the clinical course of the disease. For this purpose we characterized the LMP1 promoter region from seven cases including two patients with aggressive Hodgkin's disease and two with atypical lymphoproliferative syndromes. Our results show that the sequences -298 to +29 relative to the transcription start site diverged up to 9.3% when compared with the prototype EBV strain B95-8. The cAMP responsive-like element (CRE), located at positions -37 to -44, was found to be mutated in 3 of the 7 cases. Functional analysis of transfected human embryonic kidney 293 cells using the firefly luciferase reporter gene revealed that mutations within the CRE site led to a 70% mean decrease in reporter activity. Our analysis indicates that in lymphoproliferative disorders, naturally occurring LMP1 variants that exhibit weak promoter activity are still associated with clinically progressive disease.

Natural 30 base pair and 69 base pair deletion variants of the LMP1 oncogene do stimulate NF-kappaB-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 Burkitt's 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-kappaB. 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-kappaB responsive elements. We tested the NF-kappaB inducing activity of four naturally occurring LMP1 deletion variants. Our results show that these deletion variants activate NF-kappaB to the same level as the wild-type form, indicating that the crucial residues for NF-kappaB activation are conserved among the variants isolated and lie within the last 32 amino acids of the C-terminal domain of the LMP1 oncogene.

Glycosylation analysis and protein structure determination of murine fetal antigen 1 (mFA1)--the circulating gene product of the delta-like protein (dlk), preadipocyte factor 1 (Pref-1) and stromal-cell-derived protein 1 (SCP-1) cDNAs.

By means of sequence analysis, murine fetal antigen 1 (mFA1) isolated from Mus musculus amniotic fluid was shown to be the circulating protein of the delta-like protein, stromal-cell-derived protein 1 (SCP-1) and preadipocyte factor 1 (Pref-1) gene products. The protein contains 36 cysteine residues arranged in six epidermal-growth-factor-like domains. The purification of several C-terminal peptides of varying lengths showed mFA1 to be C-terminal heterogeneous. O-linked glycosylations of the NeuNAc alpha2-3Gal beta1-3(NeuNAc alpha2-6)GalNAc type were present on all C-terminal peptides at residues Thr235, Thr244 and Thr248, although glycosylation on Thr244 was only partial. Three N-linked glycosylations were localized in mFA1 (Asn77, Asn142 and Asn151), two of which (Asn142 and Asn151) were in the unusual Asn-Xaa-Cys motif. Fucosylated biantennary complex-type and small amounts (less than 5%) of triantennary complex-type structures were identified on the glycosylated asparagine residues using sequential exoglycosidase and endoglycosidase digestions combined with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The presence of O-linked monosaccharides (glucose attached to Ser71, Ser193 and fucose at Thr201) was tentatively ascertained by combining Edman degradation and MALDI-MS. The results presented shows mFA1 to be the circulating heterogeneous cleavage products of the membrane-bound protein encoded by the murine cDNAs dlk, pref-1 and SCP-1.

Expression of the LMP1 oncoprotein in the EBV negative Hodgkin's disease cell line L-428 is associated with Reed-Sternberg cell morphology.

The latent membrane protein 1 (LMP1) oncogene is one of the major proteins synthesized by the Epstein-Barr virus (EBV) and is expressed in Reed-Sternberg (RS) cells of EBV-associated Hodgkin's disease (HD). We have studied the effect of this oncoprotein on the formation of RS cells in the EBV-negative HD cell lines L-428 and KM-H2 as well as on the formation of multinucleated cells in the mononuclear human embryonic kidney cell line 293. LMP1 prototype (B95-8) and its naturally occurring carboxy terminal 30 base pair deletion variant LMP1-del were transfected into the cell lines and cytocentrifuge preparations were analysed after 24, 48, 72, 144, 216, and 240 h. While no oncoprotein expression was seen in the KM-H2 cell lines, expression of LMP1 and LMP1-del was observed in the L-428 and 293 cell lines. In the HD cell line L-428 oncoprotein expression was infrequent but when observed was very strong and preferentially associated with multinucleated RS cell morphology (71% of LMP1 positive cells). This is in contrast with the untransfected or transfected but not expressing cells where intermediate mononuclear elements predominated over multinucleated RS cells (< 3%). Frequent oncoprotein expression was observed in the 293 cells and again was associated with multinuclearity. These LMP1 expressing 293 giant cells showed strong expression of ICAM-1(CD54), not detectable in the untransfected cells. In the LMP1-del transfectants giant cells with more than four nuclei were frequently observed. However, giant cells were much less frequent in 293 cells transfected with the amino terminal deletion variant of LMP1 or the lytic form of LMP1, known to induce low NF-kappa B activation compared to the LMP1 prototype. Therefore, LMP1 mediated NF-kappa B activation appears to be involved in polycaria formation. The strong association of LMP1 expression with multinuclearity in a genetically unstable condition -the L-428 and 293 cells show multiple chromosomal abnormalities-suggests that this oncoprotein including its naturally occurring carboxy terminal deletion variant promote the formation of multinuclear cells, in particular of RS cells in EBV-associated HD.

Mouse fetal antigen 1 (mFA1), the circulating gene product of mdlk, pref-1 and SCP-1: isolation, characterization and biology.

The mouse homologue to human fetal antigen 1 (hFA1) was purified from mouse amniotic fluid by cation exchange chromatography and immunospecific affinity chromatography. Mouse FA1 (mFA1) is a single chain glycoprotein with an M(r) of 42-50 kDa (SDS-PAGE). The N-terminal amino acid sequence (39 residues) revealed 74% identity to hFA1 and 100% identity to the translated cDNAs referred to as mouse dlk, pref-1 and SCP-1. mFA1 is the secreted processed molecule encoded by the mRNA defined by these identical mouse cDNAs. Monospecific rabbit anti-mFA1 antibodies, purified by ammonium sulfate precipitation and immunospecific affinity chromatography, were used for immunohistochemical and quantitative ELISA techniques. The indirect immunoperoxidase technique demonstrated mFA1 within the endocrine structures of adult mouse pancreas, whereas the exocrine tissue remained unstained. FA1-positive staining was also seen in the pituitary gland and the mouse adrenal gland. The concentration of mFA1 in a pool of amniotic fluid was assessed at 25 micrograms ml-1 and the serum concentration in normal nonpregnant adult mice (n = 28) was 11.3 +/- 5.0 ng ml-1 (2 SD). During pregnancy the concentration of mFA1 in maternal serum increased above the nonpregnant reference value at midpregnancy, reaching a maximum concentration (> 0.35 microgram ml-1) 2 days prepartum. The maternal serum concentration was positively correlated with the number of fetuses. After delivery the rate of disappearance of mFA1 in maternal serum was very fast with a t1/2 < 1 h. The concentration of mFA1 in newborn mice was about 15 micrograms ml-1 and did not reach normal adult values until the age of > 50 days.

Deletion variants within the NF-kappa B activation domain of the LMP1 oncogene prevail in acquired immunodeficiency syndrome-related large cell lymphomas and human immunodeficiency virus-negative atypical lymphoproliferations.

This sequencing study of 17 acquired immunodeficiency syndrome-related lymphomas (9 primary brain, 8 systemic) and 8 human immunodeficiency virus-negative atypical lymphoproliferations expressing large amounts of the latent membrane protein 1 (LMP1) of Epstein-Barr virus was performed to characterize the carboxy terminal NF-kappa B activation domain of LMP1 at the molecular level in an immunocompromised host. In-frame deletions within the NF-kappa B activation domain were identified in all but 2 primary brain lymphomas, 4 systemic lymphomas, and 4 atypical lymphoproliferations, ie, in 60% of cases. In addition, non silent point mutations (range 1 to 5, mean 3.3) were detected in all cases. Although all changes occurred within the first 100 nucleotides of the carboxy terminal NF-kappa B activation domain--a critical sequence for the protein half-life--not a single point mutation was found in the remaining 62 nucleotides, necessary for malignant transformation. Such a clustering of nonrandom sequence variations, associated with a high oncoprotein expression in immunocompromised hosts, suggests that this part of the LMP1 oncogene behaves as a hypervariable region with natural selection of growth-promoting variants through prolongation of the protein half-life.

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

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.

A deletion mutant of the LMP1 oncogene of Epstein-Barr virus is associated with evolution of angioimmunoblastic lymphadenopathy into B immunoblastic lymphoma.

The latent membrane protein 1 (LMP1) oncogene is one of the major proteins synthesized by the Epstein-Barr virus (EBV). It is expressed in Reed-Sternberg cells of Hodgkin's disease (HD), tumor cells of nasopharyngeal carcinoma (NPC), and immunoblasts of angioimmunoblastic lymphadenopathy (AILD). A particular LMP1 deletion mutant was recently identified in NPC and clinically and histologically aggressive HD. We studied two patients with AILD that subsequently progressed into immunoblastic lymphoma (IBL) in order to investigate whether the LMP1 deletion mutant was implicated in progression of AILD into IBL. Immunohistology and in situ hybridization were performed on diagnostic biopsies. DNA extracted from fresh frozen material was used for rearrangement studies and polymerase chain reaction (PCR) based amplification and sequencing of portions of the LMP1 gene. Immunohistochemistry revealed B cell origin of both cases of IBL. In the first patient clonal rearrangement of the immunoglobulin heavy-chain gene was present in IBL but not in AILD. In this patient, scattered immunoblasts of AILD and numerous tumor cells of B-IBL were shown to contain EBV transcripts (EBER1) and to express LMP1. Sequence analysis of the LMP1 gene from AILD and IBL in the first, and from IBL in the second patient, revealed identical deletions and point mutations. This LMP1 deletion mutant is identical to those which have been reported in HD and NPC. Its association with evolution of AILD into B-IBL, aggressive HD and NPC, suggests that this particular mutant is more widespread than originally thought and is clinically relevant.

Mutational hot spots within the carboxy terminal region of the LMP1 oncogene of Epstein-Barr virus are frequent in lymphoproliferative disorders.

We have recently identified in Epstein-Barr virus (EBV) positive Hodgkin's disease (HD) a variant of the latent membrane protein 1 (LMP1) oncogene characterized by four point mutations and a 30 base pair deletion. These findings led us to test whether such mutants were also present in other lymphoproliferative disorders (LPD). We analysed 98 EBV DNA positive cases (67 LPD, 15 benign conditions, 16 lymphoblastoid cell lines) by PCR for deletions within the LMP1 gene. DNA sequencing of the region coding for the carboxy terminal protein domain was performed on 24 cases. In 13 cases the same combination of 4 point mutations at positions 168,320, 168,308, 168,295 and 168,225 was identified. Of these cases, 12 had an additional point mutation at position 168,357 and eight at position 168,355, and nine had a 30 base pair deletion including nucleotides 168,285 to 168,256. These deletion mutants were identified in HD, angioimmunoblastic lymphadenopathy, B-immunoblastic lymphoma, peripheral T-cell lymphoma, and two lymphoblastoid cell lines. Our findings reveal a high frequency of non-random point mutations at preferential sites within the 3' (carboxy terminal) region of the LMP1 oncogene. The association of these mutational hot spots with LPD suggests that they are involved in EBV related lymphomagenesis and that they define a clinically relevant EBV strain.

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 RAG's 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 Hodgkin's 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.

Persistence of the same viral strain in early and late relapses of Epstein-Barr virus-associated Hodgkin's disease.

Twelve cases of relapsing Hodgkin's disease were investigated for the presence of Epstein-Barr virus (EBV). Of these, 7 cases contained EBV gene products (LMP1, EBER RNA) in the diagnostic Reed-Sternberg cells and variants at first presentation and at relapse(s), whereas 5 cases were negative at both first diagnosis and relapse. Among the 7 EBV-positive cases, material for DNA extraction was available in 2 cases at both diagnosis and relapse(s). Ig and T-cell receptor gene rearrangements displayed a germline configuration in the 2 cases. However, Southern blot analysis of the terminal repeats (TR) of EBV genome showed that, in 1 of the 2 cases, the fragment was of the same size at diagnosis and in the subsequent two relapses (1 early and 1 late). The second case contained monoclonal EBV genome at diagnosis, but the Southern analysis of the TR was negative at relapse. The latent membrane protein (LMP1) sequence analysis confirmed the persistence of a distinctive viral strain in each of the 2 cases with individual abnormalities within the carboxy terminal region (5 point mutations and a 30-bp deletion for the first case and 6 point mutations for the second case). The persistence of a given strain in early and late relapses is evidence towards the view that in Hodgkin's disease such relapses are related to a single residual tumor cell clone.

[Internal deletions of the latent membrane protein oncogenes of Epstein-Barr virus in Hodgkin's disease are almost identical with those of Asiatic nasopharyngeal carcinoma]. / Deletionen innerhalb des latenten Membran-Protein-Onkogens des Epstein-Barr Virus bei Morbus Hodgkin sind weitgehend identisch mit denjenigen beim asiatischen nasopharyngealen Karzinom.

The latent membrane protein (LMP) oncogene of the Epstein-Barr virus (EBV) is 1300 base pairs (bp) long and expressed in Hodgkin and Reed-Sternberg (HRS) cells of about 50% of Hodgkin's lymphomas and in tumor cells of about 60% of nasopharyngeal carcinomas (NPC). The LMP sequences of EBV variants isolated from two NPC (NPC 1510 and NPC CAO) have recently been published. Compared to the standard EBV sequence (EBV B95-8) they both show deletions of 30 bp near the 3' end. These mutations render the LMP oncogene more aggressive in NPC. In 52 Hodgkin's lymphomas expressing the LMP oncogene was amplified the coding sequence by the polymerase chain reaction. In five tumors deletions within the coding region for the intracytoplasmic LMP domain have been found. DNA sequencing revealed three 30 bp deletions almost identical to those observed in NPC 1510 and NPC CAO. In a forth case, a 70 bp deletion encompassing the regions of the 30 bp deletions was found. Histologically, all five tumors with such deletions showed abundant HRS cells. It is likely that these mutations of the LMP oncogene in a similar way as in NPC also favour the proliferation of HRS cells in Hodgkin's disease.

Deletions within the LMP1 oncogene of Epstein-Barr virus are clustered in Hodgkin's disease and identical to those observed in nasopharyngeal carcinoma.

This study of 52 European patients with Hodgkin's disease (HD) expressing the latent membrane protein 1 (LMP1) oncogene within diagnostic Hodgkin and Reed-Sternberg (HRS) cells was performed to detect LMP1 isolates carrying deletions and to characterize them at a molecular and histologic level. Deletions were identified in 5 cases, clustered near the 3' end of the LMP1 gene, and histologically associated with numerous HRS cells. DNA sequencing showed homology with the deletions seen in the Asian nasopharyngeal carcinoma (NPC) isolates CAO and 1510. Our findings suggest that partial deletions of the LMP1 oncogene, associated with aggressive behavior in NPC CAO and NPC 1510, occur at a particular localization and confer a proliferative phenotype to lymphoid cells in HD.

Demonstration of Epstein-Barr virus replication in Reed-Sternberg cells of Hodgkin's disease.

Epstein-Barr virus (EBV) is detectable in approximately 40% of cases of Hodgkin's disease (HD). The viral genomes remain latent but positive staining with anti-ZEBRA antibody in a small fraction of Reed-Sternberg (RS) cells of some cases of HD would suggest possible activation of EBV replication within these cells. We report the investigation of 40 cases of EBV-associated HD (including 5 human immunodeficiency virus [HIV]-positive cases) using anti-ZEBRA antibodies. Positive staining was found in only three (HIV-negative) cases. One of these three cases showed approximately 1% of ZEBRA-positive tumor cells, whereas the other two cases showed rare positive cells. In the case with 1% ZEBRA-positive cells, a strong signal was obtained with anti-EA-R antibody and BHLF1 oligoprobes, which indicated early gene expression. EBV replication could be shown in this case by nonisotopic in situ DNA-DNA hybridization, which showed markedly increased numbers of EBV genomes in a few RS cells. Viral replication was confirmed using reverse transcriptase and polymerase chain reaction that detected transcripts from the BLLF1 gene encoding for the membrane antigen gp350/220. EBV replication in RS cells seems to be an exceptional event but may provide clues to mechanisms of control of viral latency and assume clinical implications in the future.

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

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.