The enhancer complex regulatory region at the 3' of the immunoglobulin heavy cluster (IgH3'EC) is duplicated in apes along with four constant genes and the region is highly conserved throughout humans. Both human IgH3'ECs consist of three loci high sensitive (HS) to DNAse I with enhancer activity. It is thus possible that the presence of structural divergences between the two IgH3'ECs and of relative polymorphisms correspond to functional regulatory changes. To analyse the polymorphisms of these almost identical regions, it resulted mandatory to identify the presence of divergent sequences, in order to select distinctive primers for specific PCR genomic amplifications. To this aim, we first compared the two entire IgH3'ECs in silicio, utilising the updated GenBank (GB) contigs, then we analysed the two IgH3'ECs by cloning and sequencing amplicons from independent genomes. In silicio analysis showed that several inversions, deletions and short insertions had occurred after the duplication. We analysed in detail, by sequencing specific regions, the polymorphisms occurring in enhancer HS1,2-A (which lies in IgH3'EC-1, 3' to the C alpha-1 gene) and in enhancer HS1,2-B (which lies in IgH3'EC-2, 3' to C alpha-2). Polymorphisms are due to the repetition (occurring one to four times) of a 38-bp sequence present at the 3' of the core of enhancers HS1,2. The structure of both human HS1,2 enhancers has revealed not yet described polymorphic features due to the presence of variable spacer elements separating the 38-bp repetitions and to variable external elements bordering the repetition cluster. We found that one of the external elements gave rise to a divergent allele 3 in the two clusters. The frequency of the different alleles of the two loci varies in the Italian population and allele 3 of both loci are very rare. The analysis of the Callicebus moloch, Gorilla gorilla and Pan troglodytes HS1,2 enhancers showed the transformation from the ancestral structure with the 31- to the 17-by external element in hominids.The relevance of the polymorphisms in the HS1,2 enhancers is due to the variable number of binding sites for the transcription factors: NF-kappa B, CMYB, BSAP1/2, AP1/4, E47, MyoD and mu E5 and thus to the possible influence of these variations on switch, production of Ig and on maturation of B cells. (c) 2004 Elsevier B.V. All rights reserved.

The present study describes the phenotypic and genotypic features of seven individual growth transformed, euploid-diploid EBV+ human B cell lines arisen spontaneously in vitro.The lines, obtained under general. and standard culture conditions (un-manipulated), from seven individual bone marrow samples of 18 healthy young adults, Caucasian, of both sexes, display many traits of normal B cells and represent a mixture of EBV infected latently (latency type 111) and producer cells (5-16% VCA(+) by immunofluorescence) releasing seven individual different viral strains [Fruscalzo et a]., 2001. DNA sequence heterogeneity within the Epstein-Barr virus family of repeats in the latent origin of replication. Gene 265, 165-173] similar to the B95-8 genotype as shown by results of Southern blot of BamHI-digested DNA fragment.These tests were planned to characterize more fully this panel of new bone marrow cell lines sharing normal B cell traits. (c) 2005 Elsevier B.V. All rights reserved.

BACKGROUND:Coeliac disease (CD) is characterized by increased immunological responsiveness to ingested gliadin in genetically predisposed individuals. This genetic predisposition is not completely defined. A dysregulation of immunoglobulins (Ig) is present in CD: since antiendomysium antibodies (anti-EMA) are of the IgA class. One polymorphic enhancer within the locus control region (LCR) of the immunoglobulin heavy chain cluster at the 3' of the C alpha-1 gene was investigated. The correlation of the penetrance of the four different alleles of the HS1,2-A enhancer of the LCR-1 3' to C alpha-1 in CD patients compared to a control population was analysed.METHODS:A total of 115 consecutive CD outpatients, on a gluten-free diet, and 248 healthy donors, age- and sex-matched, from the same geographical area were enrolled in the study. HS1,2-A allele frequencies were investigated by nested polymerase chain reaction (PCR).RESULTS:The frequency of allele 2 of the enhancer HS1,2-A gene was increased by 30.8% as compared to the control frequency. The frequency of homozygosity for allele 2 was significantly increased in CD patients. Crude odds ratio (OR) showed that those with 2/2 and 2/4 (OR 2.63, P < 0.001 and OR 2.01, P = 0.03) have a significantly higher risk of developing the disease. In contrast, allele 1/2 may represent a protective genetic factor against CD (OR 0.52, P = 0.01).CONCLUSIONS:These data provide further evidence of a genetic predisposition in CD. Because of the Ig dysregulation in CD, the enhancer HS1,2-A may be involved in the pathogenesis.

betaTrCP mediates the ubiquitination and subsequent degradation of several key molecules thereby playing a relevant role in different cellular processes during development and in the adult. In Xenopus embryo, betaTrCP acts as a negative regulator of Wnt signaling by interacting with beta-catenin. In this paper, we report results of the study on expression and regulation of the Xenopus betaTrCP gene. We found that xbetaTrCP is expressed in Xenopus oocytes as three transcripts, which very likely correspond to the previously identified localized mRNAs, and four isoforms. The xbetaTrCP promoter functional and structural analysis showed the presence of elements target of positive transcriptional control. Among them, we have identified a beta-catenin/Tcf signaling responsive region and a 45-bp element containing a sequence motif conforming to the SRF binding site, closer to the transcription initiation sites. There are also elements of transcriptional negative control.

It has been suggested that an atypical course of primary infection by EBV and the reactivation of EBV infection in transplanted patients may induce hepatitis. We explored the possibility to dissect the infectious activity from the ability to promote B lymphocyte proliferation in vivo by injecting in nu/nu mice a low number (2 × 106–0.05 × 106) of cells from CE a normal human bone marrow–derived B cell line. This line carries an endogenous EBV in episomal and linear forms. Twenty nu/nu mice were inoculated subcutaneously with the B cell line CE and a matched group with the cell line RAG obtained by EBV in vitro infection of normal human peripheral blood. The mice injected with the CE line did not develop a lymphoproliferative disease, but 5 of them displayed typical histopathological lesions of chronic hepatitis without involvement of other organs. Similar results were obtained in 2 out of 20 animals in the RAG group. A close association between liver lesions and a previous EBV infection, by putative circulating B lymphoblastoid cells releasing their EBV, was established by PCR and by in situ hybridization with BamHI “W” DNA probe. This latter probe detected the presence of about 15% of positive cells only in affected livers. In addition, the rare detection in some hepatocytes of “A” type Cowdry bodies would suggest the occurrence of continuous EBV replication although at a very low level. These data show that we succeeded in dissecting the infectious from the proliferative activity of the endogenous EBV carrier CE cell line. This provides in addition a promising model for chronic EBV‐associated hepatitis. J. Med. Virol. 66:70–81, 2002. © 2002 Wiley‐Liss, Inc.

To detect the presence of variability in the tandemly repeated sequences of the Epstein–Barr virus latent origin of replication, we analyzed the length of the family of repeats in 14 lymphoblastoid and Burkitt's lymphoma cell lines by PCR amplification. The gel electrophoresis analysis of the PCR products revealed a broad banding pattern, characteristic of each line, consisting of several fragments, sometimes smeared, of variable length. This finding was interpreted as a result of the hairpin-like structures generated by the palindrome within the family of repeats, able to originate artefacts. Since the banding pattern was different only in strictly non-correlated cell lines, we supposed that the sequence of the repeat units was polymorphic. We therefore sequenced the family of repeats in three healthy bone marrow derived lymphoblastoid cell lines carrying an endogenous EBV as well as in a B95-8 infected cell line as control. The sequence analysis revealed that each line is different both in the number and in the sequence of repeats. At the 3′ end of the family of repeats the B95-8 virus was found to have a 252 bp region missing in the GenBank standard sequence. This one is probably a partial sequence since it was shorter than the control specimens obtained from different sources of B95-8 DNA analyzed by Southern blot hybridization. The length analysis of the family of repeats can be used to characterize EBV strains by PCR.

A highly spread polymorphism flanking the 3· Cα1 human IG heavy chain gene was identified. This polymorphism allowed the detection of an internal duplication within the 3′ flanking region of both Cα1 and Cα2. This region has a regulatory function with four enhancer structures also present at the 3′ end of the human Cα2 as well as in that of mouse and rat single Cα genes. The 5682-bp sequence of clone λpl8 described here starts 3′ of Cα1 and presents three open reading frames; one of them contains part of the tandem repeats with the 20-bp consensus described previously that is expressed in a poly(A)+ RNA and found in three dbEST clones of the human tonsillar cDNA library. Here, we demonstrate that in the CLF1 B lymphoblastoid cell line, this transcript is associated with polysomes. We also discuss the possibility of the presence of a new regulatory gene that does not encode an immunoglobulin and maps in the human IG heavy chain gene cluster.

BACKGROUND AND OBJECTIVE:Primary effusion lymphomas (PELs) containing Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8/HHV-8) DNA sequences represent a distinct but heterogeneous group of rare non-Hodgkin's lymphomas of null-cell phenotype/B-cell origin. We aimed to describe the clinicopathologic features of two human immunodeficiency virus (HIV)-related PELs occurring in homosexual men with Kaposi's sarcoma (KS). DESIGN AND METHODS:Thoracentesis was followed by morphologic plus immunophenotypic studies and molecular analysis of tumor cell DNA by means of combination of polymerase chain reaction and Southern blot analysis. RESULTS:Patients developed recurrent lymphomatous effusions lacking tissue involvement, in the context of severe immunodepression (CD4 count < 60/microL) and anti-retroviral therapy. The effusions disclosed an immunoblast-like population CD45/CD30+, but B-cell- and T-cell-associated antigen negative, showing clonal immunoglobulin heavy chain gene rearrangements and harbouring HHV-8 DNA sequences. One case contained Epstein-Barr virus genome with no evidence of c-myc, bcl-2 and bcl-6 gene alterations. Both patients had aggressive disease. INTERPRETATIONS AND CONCLUSIONS:These cases represent additional examples of PEL associated with HHV-8 and confirm that the group of HIV-positive homosexual men may be at highest risk for PEL.

Hepatitis C virus (HCV) infection may be complicated by non-Hodgkin's lymphoma through yet unknown pathogenetic mechanisms. We describe the case of a patient with HCV-related cirrhosis who developed a primary effusion lymphoma (PEL) of Burkitt's type confined to the peritoneal cavity, in the absence of immunodeficiency or autoimmunity. Paracentesis followed by immunophenotyping, karyotyping, and molecular studies allowed us to diagnose a small non-cleaved B-cell lymphoma (CD20+, CD24+, CD10+, CD5−, CD23−, λ+) with the t(8;22) (q24; q11) translocation and clonal rearrangement of the immunoglobulin heavy chain gene. HCV-RNA, Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus were not identified within lymphoma cells. The finding of HCV-RNA in the ascitic fluid suggests a link between HCV and development of lymphoma with HCV playing the role of persistent antigenic stimulation to intraperitoneal B-cell clonal expansion(s).

On the assumption that EBV integration into the genome of human B lymphocyte might lead to the inactivation of a hypothetical gene determining the limited duplicative capacity and consequently participate to the cell immortalization, a search for the human-virus junction was done. This led to the identification of a site of integration in the central part of the heavy chain of the immunoglobulin region. The coincidence of the involvement of the site in lymphomatogenesis with the first complete characterization of an integration site led to the speculation that the heavy chain gene itself might be an important controller of cell duplication in the B lymphocyte.

Epstein-Barr virus (EBV) DNA sequences were detected in four established monoblast or early monocytic cell lines (CM-S, ROV-S, CV-S and AD-S) obtained from bone marrow of children suffering from maturation defects of haematopoiesis. EBV is present in these cells in a latent state. The viral DNA in these cell lines was analysed by Southern blot hybridization, using a set of cloned EBV DNA fragments from the EBV strain B95-8 as probes. A common spectrum of highly related but distinguishable EBV DNA restriction enzyme sequences was found, suggesting some genomic diversity. Propagation of the cells in long-term culture revealed a gradual decrease of EBV copies per cell in all lines with some minor changes in the restriction pattern of the EBV DNA. These findings demonstrate that human precursor monocyte cells may be susceptible to infection by EBV.

Some hematological disorders of either proliferative or aplastic type are at present treated with bone marrow transplantation. In the absence of adverse reactions, such a s BVHD and infections, successful graft implantation and remission from these diseases are obtained in a large fraction of cases. Herpesviruses reactivation from the latent chronic infection can actually be a major complication as a consequence of immunodeficiency status of the host (1). In addition, EBV has been postulated to cause secondary immunoproliferative disorders in bone marrow transplant patients (2). It can be hypothesized that the onset of clinical findings of the EBV infection in the transplanted hosts could also be caused by an engraft of EBV infected cells, exogenously added with the donor marrow (3, 4). Whether in EBV seropositive healthy individuals bone marrow is a reservoir of infected cells is on the other hand, not clearly established.

Five human lymphoblastoid cell lines immortalized in vitro with the B95-8 EBV strain, chosen to have a low number of copies of EBV genome, were examined to detect variations in electrophoretic mobility of viral restriction fragments and in the karyotype. Patterns of mobility detected with different viral probes are always the same as those obtained with fragments from purified virus-plasmidic DNA, with one exception. This “non-plasmidic” pattern occurs with a probe containing the termini of the linear virion DNA and consists in an increase of the molecular weight and in the appearance of more than one band. Cytogenetic studies carried on the same cell populations used as source of DNA, early after immortalization, showed a diploid modal chromosome number and no G banding rearrangements.

A characteristic DNA rearrangement, the loss of an EcoRI cleavage site next to the 3'-end of the human c-mos gene, has been found to be frequently present in DNA from transformed hematopoietic cells of the myeloid lineage but not in DNA from either normal or transformed cells of different tissue types. Three established cell lines, respectively a pro-monocytic line (CM-S) and two precursor granulocytic lines (My/K1 and My/K5), carry the same genome rearrangement, but not fibroblasts obtained from the marrow of the same patients. This DNA rearrangement is maintained in three different hybridomas derived by fusion of CM-S cells with normal human embryo hepatocytes.

Our aim was to verify whether differentiated murine cells grafted into mouse blastocysts would follow the same fate as teratocarcinoma cells which lose their malignant character and participate in the development of the host embryo yielding chimaeric animals. Two combinations of host embryo-cancer cells were used. Animals were examined after birth or by pregnancy interruption at various stages of development. More than 350 animals obtained from cancer-cells-injected embryos failed to show direct evidence of loss of malignancy.

To test the ability of cloned committed erythroleukemic cells to participate in development we have injected. Friend leukemia cells (FLC) into C57Bl/6 mouse blastocysts together with Friend leukemia virus (FLV) and we have examined the newborn individuals derived from them. Five animals out of 32 born have FLC-derived neoplasia. The incidence of neoplasia is increased as compared with other similar experiments without the virus. In two of the animals with the FLC neoplasia the disease manifestation is an erythroid leukemia similar to the one obtained directly with the virus in normal DBA/2 mice.