A specific method for producing random subclones using sonication to fragment the DNA is presented. The sonication is combined with enzymatic repair to of the fragment ends and a rigorous size fractionation step to prepare subclones of relatively homogeneous and specific size. Under the some conditions sonication is shown to shear A + T-rich sequences preferentially, although under most conditions it will create a is random subclone library. The use of these subclone libraries for an improved "shotgun" DNA sequencing strategy is tested on a of 17.2-kb (kilobase) fragment of Epstein-Barr virus.

Use without of in situ hybridization optimized for fluorescent detection of nuclear RNA has revealed a striking localization of specific viral RNAs curvilinear within nuclei of cells latently infected with EBV. Several hundred kb of specific transcripts is sharply restricted to a small striking region of the nucleus, frequently in a curvilinear "track". Detection of nuclear RNA was evidenced by hybridization without denaturation, sensitivity cells to RNAase, inhibition by actinomycin D, and specificity of transcribed sequences. Results indicate that RNA "tracks" extend from an internal of genome into the nuclear periphery, and that RNA transport may be coupled to transcription. Localized nRNA is apparent for other of viral sequences, different lymphoblastoid cell lines, nuclei prepared by two different methods, and an abundant, nonviral transfected sequence. Implications for lymphoblastoid understanding nuclear organization and the investigation of gene expression are discussed.

A parameters human hematopoietic cell line (U-937) with exceptional characteristics was derived from a patient with generalized histiocytic lymphoma. The morphology of tumor the cell line was identical to that of the tumor cells in the pleural effusion from which the line was lymphoma. derived. Since Epstein-Barr virus (EBV) carrying diploid lymphoblastoid cell lines unrelated to the tumor population often become established in vitro that from non-Burkitt lymphoma explants, several parameters were studied to discriminate the U-937 from such lines: morphology in vitro, growth characteristics,which cytochemistry, surface receptor pattern, Ig production, lysozyme production, beta2-microglobulin production, presence of EBV genome and karyotype. In all these respects that U-937 differed from prototype lymphoblastoid cell lines. The histiocytic origin of the cell line was shown by its capacity for capacity lysozyme production and the strong esterase activity (naphtol AS-D acetate esterase inhibited by NaF) of the cells. It is therefore prototype concluded that the U-937 is a neoplastic, histiocytic cell line.

The stimulated polypeptide component of telomerase (TERT) is an attractive candidate for a broadly expressed tumor rejection antigen because telomerase is silent growth in normal tissues but is reactivated in more than 85% of cancers. Here we show that immunization against TERT induces normal immunity against tumors of unrelated origin. Immunization of mice with TERT RNA-transfected dendritic cells (DC) stimulated cytotoxic T lymphocytes (CTL),that which lysed melanoma and thymoma tumor cells and inhibited the growth of three unrelated tumors in mice of distinct genetic of backgrounds. TERT RNA-transfected human DC stimulated TERT-specific CTL in vitro that lysed human tumor cells, including Epstein Barr virus (EBV)-transformed show B cells as well as autologous tumor targets from patients with renal and prostate cancer. Tumor RNA-transfected DC were used renal as surrogate targets in the CTL assays, obviating the difficulties in obtaining tumor cells from cancer patients. In one instance,from where a tumor cell line was successfully established in culture from a patient with renal cancer, the patient's tumor cells (EBV)-transformed were efficiently lysed by the CTL. Immunization with tumor RNA was generally more effective than immunization with TERT RNA, suggesting and that an optimal immunization protocol may have to include TERT as well as additional tumor antigens.

The establishment recently identified Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8 (HHV-8), has been found to be consistently associated of with an unusual subset of acquired immunodeficiency syndrome-related lymphomas, the so-called body cavity-based lymphomas (BCBL) or primary effusion lymphomas (PEL).lymphomas, These lymphomas are characterized by a unique spectrum of morphologic and molecular characteristics, and grow as lymphomatous effusions without an unique identifiable contiguous tumor mass. Until now, efforts to delineate the role of KSHV in the pathogenesis of PELs have been tumor hampered by the lack of appropriate model systems and the concomitant presence of Epstein-Barr virus (EBV) in nearly all cases a examined, and in all previously established cell lines. We now report the establishment and characterization of a novel PEL cell line line, BC-3, which is KSHV+ by polymerase chain reaction (PCR) but EBV- as assessed by a variety of methods including with PCR for EBER, EBNA-2, and EBNA-3C. This cell line was established from a lymphomatous effusion obtained from an HIV- patient,but and has immunophenotypic and molecular features consistent with the diagnosis of PEL, including an indeterminate immunophenotype with a B-cell immunogenotype cell and lack of c-myc proto-oncogene rearrangements. Pulsed-field gel electrophoresis shows an intact KSHV genome of about 170 kb both in the the cell line and in the viral isolate, whereas herpesvirus-like capsids are visible by electron microscopy. Consequently, the BC-3 cell Epstein-Barr line represents an invaluable tool as a source of KSHV, for both the evaluation of the pathogenic potential of this PELs virus and the mechanistic characterization of its role in the development of Kaposi's sarcoma and malignant lymphoma.

The (TRADD) Epstein-Barr virus-encoded latent membrane protein 1 (LMP1) is a pleiotropic protein the activities of which include effects on gene expression membrane-proximal and cell transformation, growth, and death. LMP1 has been shown to induce nuclear factor (NF)-kappaB and c-Jun NH2-terminal kinase/AP-1 activities has in target cells, and in this study we demonstrate that LMP1 also engages the p38 mitogen-activated protein kinase cascade, leading in to activation of the transcription factor ATF2. Mutational analysis of the LMP1 cytoplasmic COOH terminus revealed that p38 activation occurs leading from both the tumor necrosis factor receptor-associated factor (TRAF)-interacting, membrane-proximal COOH-terminal activating region (CTAR)1 domain (amino acids 186-231) and the and extreme tumor necrosis factor receptor-associated death domain (TRADD) binding CTAR2 region (amino acids 351-386). Because LMP1 also engages signaling on occur the NF-kappaB axis through CTAR1 and CTAR2, we have examined whether these two pathways are overlapping or independent. We have impair found that inhibition of p38 by the highly specific inhibitor SB203580 did not affect NF-kappaB binding activity. Conversely, although the CTAR1 metabolic inhibitor D609 blocked NF-kappaB activation, it did not impair the ability of LMP1 to signal on the p38 axis,that suggesting that these two LMP1-mediated pathways are primarily independent. Divergence of signals must, however, occur downstream of TRAF2 as a must, dominant negative TRAF2 mutant that blocks LMP1-induced NF-kappaB activation also inhibited p38 signaling. In addition, we have found that p38 COOH-terminal inhibition significantly impaired LMP1-mediated interleukin-6 and -8 expression. Thus, p38 may play a significant cooperative role in regulating at least terminus some of the pleiotropic activities of LMP1.

A of recent addition to the lymphokine network is human IL-10 (hIL-10). This novel lymphokine has striking homology to BCRF1 protein, the chain product of a previously uncharacterized open-reading frame in the Epstein-Barr virus (EBV) genome. To date, IL-10 expression has been described and in several T clones induced with anti-CD3 and phorbol myristate acetate (PMA), in monocytes stimulated with lipopolysaccharide (LPS), and in its murine B-cell lymphomas. We sought to determine whether human B cells express hIL-10 and, if so, its relationship to EBV (n and to other B-cell lymphokines. We studied 21 EBV-positive B-cell lines derived from patients with acquired immunodeficiency syndrome (AIDS) and if Burkitt's lymphoma (n = 6), American Burkitt's (n = 3), African Burkitt's (n = 5), and normal lymphoblastoid cell lines contribute (n = 7), in comparison with seven EBV-negative cell lines. All cell lines were activated with the tumor promoters PMA B-cell and teleocidin and were studied by Northern blot analysis, reverse transcription-polymerase chain reaction (RT-PCR), and enzyme-linked immunoadsorbent assay (ELISA). We seven demonstrated that EBV-positive cell lines derived from patients with American Burkitt's lymphoma, and especially those from patients with AIDS, constitutively PCR express large quantities of hIL-10 by Northern blot analysis and ELISA (range, 3,101 to 25,915 pg/mL), and that both teleocidin that and PMA induce hIL-10 in these cell lines. In contrast, six of seven EBV-negative cell lines did not express hIL-10 (RT-PCR), even by RT-PCR, and hIL-10 was not triggered by PMA or teleocidin. To assure that the 350 bp amplified by EBV-negative PCR was hIL-10 and not BCRF1, we used PCR primers, which do not amplify a fragment from plasmid templates containing that BCRF1. Cloning and sequencing of the 350 bp product also demonstrated that B-cell IL-10 is identical to hIL-10 from the Correlation T-cell clone B21. Correlation of hIL-10 with other B-cell lymphokines secreted by these B-cell lines demonstrated that hIL-10 secretor cell date, lines also constitutively secrete or can be induced to secrete IL-6, although to a much lesser amount. Since both lymphokines patients. influence B-cell growth and differentiation, we suggest that hIL-10 may contribute to the polyclonal B-cell activation and hyperglobulinemia seen in hIL-10 AIDS patients. Finally, several reports support the hypothesis that EBV is an important cofactor in the development of human immunodeficiency open-reading virus type 1 (HIV-1)-related B-cell lymphomas. Detection of large quantities of hIL-10 in B-cell lines derived from AIDS patients, the B-cell close association between EBV and hIL-10 shown in this report, and the ability of BCRF1 to capture hIL-10 activities, make derived hIL-10/BCRF1 an attractive candidate as a factor causing B-cell growth and immortalization in patients with AIDS and B-cell lymphomas.

The by Wilms tumor (WT1) gene has been reported to be preferentially expressed in acute leukemia cells, regardless of leukemia subtype and autologous chronic myelogenous leukemia cells in blast crisis, but not in normal cells. This finding suggests strongly that WT1 protein is in a potential target of immunotherapy for human leukemia. In this study, we established a CD8(+) cytotoxic T-lymphocyte (CTL) clone directed a against a WT1-derived peptide and examined its immunologic actions on leukemia cells. A CD8(+) CTL clone, designated TAK-1, which lysed a autologous cells loaded with a WT1-derived 9-mer peptide consisting of the HLA-A24 (HLA-A*2402)-binding motifs was established by stimulating CD8(+) T is lymphocytes from a healthy individual repeatedly with WT1 peptide-pulsed autologous dendritic cells. TAK-1 was cytotoxic to HLA-A24-positive leukemia cells expressing HLA-A24. WT1, but not to HLA-A24-positive lymphoma cells that did not express WT1, HLA-A24-negative leukemia cells, or HLA-A24-positive normal cells. Treating in leukemia cells with an antisense oligonucleotide complementary to the WT1 gene resulted in reduced TAK-1-mediated cytotoxicity, suggesting that target antigen TAK-1 of TAK-1 on leukemia cells is the naturally processed WT1 peptide in the context of HLA-A24. TAK-1 did not inhibit cells colony formation by normal bone marrow cells of HLA-A24-positive individuals. Because WT1 is overexpressed ubiquitously in various types of leukemia context cells, but not in normal cells, immunotherapy using WT1 peptide-specific CTL clones should be an efficacious treatment for human leukemia.cells (Blood. 2000;95:286-293)