Genetic control of the CD4/CD8 T-cell ratio in humans.

We studied the genetic pattern of inheritance of the ratio between circulating CD4+ and CD8+ T lymphocytes in a population of healthy donors. The distribution of the CD4/CD8 ratio in males and females was significantly different and was significantly affected by age. In 46 randomly selected families, the parental CD4/CD8 ratio significantly influenced the ratio in offspring. Complex segregation analysis of the data rejected the non-genetic hypothesis; among the genetic models tested, a major recessive gene with a polygenic component and random environmental effects was the most parsimonious model. These findings indicate that the ratio of CD4+ and CD8+ T cells is genetically controlled in humans.

Tumor induction by murine sarcoma virus in AKR and C58 mice. Reduction of tumor regression associated with appearance of Gross leukemia virus pseudotypes.

Adult AKR and C58 mice injected intramuscularly with murine sarcoma virus, Moloney isolate (M-MSV), developed high incidence of nonregressing local tumors. Histologically, these tumors revealed the typical pleomorphism of M-MSV sarcomas; in some cases, however, neoplastic tissue showed a nodular or diffuse growth of monomorphic myoblastlike cells, reminiscent of clonal aggregates. No depression of immune reactivity was found in M-MSV-injected mice as evaluated by direct hemolytic plaque-forming cells against SRBC and by virus-neutralizing antibody production. The MSV recovered from the induced tumors proved to be, by neutralization assay, a Gross (G)-MSV pseudotype. Moreover, tumor cell suspensions absorbed out cytotoxic antibody directed against G-cell surface antigens. Therefore, the conclusion was drawn that MSV with envelope characteristics of endogenous G leukemia virus had formed in vivo through a phenotypic mixing phenomenon. The failure of tumors to regress has been interpreted as mainly due to the partial unresponsiveness of host immune reactivity towards G-MuLV specified antigens. Since MSV-tumors arose in AKR mice after a very long latent period, the possibility was considered that this relative resistance might depend on immunologic mechanisms. In fact, M-MSV-injected AKR mice immunodepressed by goat antimouse lymphocyte serum or rendered partially tolerant by neonatal M-MuLV inoculation developed sarcomas with higher incidence and with a shorter latency. Furthermore, the MSV recovered from these early tumors proved to be the original Moloney pseudotype.

Dynamics of viral replication in infants with vertically acquired human immunodeficiency virus type 1 infection.

About one-third of vertically HIV-1 infected infants develop AIDS within the first months of life; the remainder show slower disease progression. We investigated the relationship between the pattern of HIV-1 replication early in life and disease outcome in eleven infected infants sequentially studied from birth. Viral load in cells and plasma was measured by highly sensitive competitive PCR-based methods. Although all infants showed an increase in the indices of viral replication within their first weeks of life, three distinct patterns emerged: (a) a rapid increase in plasma viral RNA and cell-associated proviral DNA during the first 4-6 wk, reaching high steady state levels (> 1,000 HIV-1 copies/10(5) PBMC and > 1,000,000 RNA copies/ml plasma) within 2-3 mo of age; (b) a similar initial rapid increase in viral load, followed by a 2.5-50-fold decline in viral levels; (c) a significantly lower (> 10-fold) viral increase during the first 4-6 wk of age. All infants displaying the first pattern developed early AIDS, while infants with slower clinical progression exhibited the second or third pattern. These findings demonstrate that the pattern of viral replication and clearance in the first 2-3 mo of life is strictly correlated with, and predictive of disease evolution in vertically infected infants.

Large genomic deletions inactivate the BRCA2 gene in breast cancer families.

BACKGROUND: BRCA1 and BRCA2 are the two major genes responsible for the breast and ovarian cancers that cluster in families with a genetically determined predisposition. However, regardless of the mutation detection method employed, the percentage of families without identifiable alterations of these genes exceeds 50%, even when applying stringent criteria for family selection. A small but significant increase in mutation detection rate has resulted from the discovery of large genomic alterations in BRCA1. A few studies have addressed the question of whether BRCA2 might be inactivated by the same kinds of alteration, but most were either done on a relatively small number of samples or employed cumbersome mutation detection methods of variable sensitivity. OBJECTIVE: To analyse 121 highly selected families using the recently available BRCA2 multiplex ligation dependent probe amplification (MLPA) technique. RESULTS: Three different large genomic deletions were identified and confirmed by analysis of the mutant transcript and genomic characterisation of the breakpoints. CONCLUSIONS: Contrary to initial suggestions, the presence of BRCA2 genomic rearrangements is worth investigating in high risk breast or ovarian cancer families.

Abelson murine leukemia virus-induced thymic lymphomas: transformation of a primitive lymphoid precursor.

For the investigation of whether Abelson murine leukemia virus (A-MuLV) is able to transform in vivo lymphocytes other than those of the B-cell lineage, newborn BALB/c and C57BL/6 mice were given an injection of A-MuLV directly into the thymus. Thymic lymphomas appeared with a short latent period of 4-5 weeks in BALB/c mice and 8 weeks in C57BL/6 mice. Cell lines derived from some thymic lymphomas presented a very immature phenotype and did not express cellular markers of either T-cells (Thy 1.2, Lyt 1.2, and Lyt 2.2) or B-cells (cytoplasmic IgM) even after treatment with several differentiation inducers. Molecular analysis showed that T-cell receptor (TCR) beta chain genes were never rearranged; in one case only, rearrangement of TCR gamma chain genes could be demonstrated, confirming the immaturity of the presumptive T-cell lines studied. Furthermore, the cell lines consistently carried diversity (D)-joining (J) but not variable (V)-D-J rearrangements of the immunoglobulin heavy chain genes. On the whole, these findings suggest that following intrathymic A-MuLV injection neoplastic transformation does involve lymphocytes possibly of T-cell lineage, at a very early stage of differentiation.

Immune reactivity in the Moloney strain of murine sarcoma virus oncogenesis: requirement of thymus-derived lymphocytes for in vivo protection.

To study the function of different lymphocyte populations in the Moloney strain of murine sarcoma virus (M-MuSV) tumorigenesis, we gave M-MuSV injections to CBA mice selectively deprived of thymus (T) lymphocytes by thymectomy, X-rradiation, and syngeneic bone marrow injection. Although no tumors appeared in the control group, 80% of the derived mice had tumors that grew progressively and ultimately killed them. In deprived mice, grafted with a syngeneic thymus (reconstituted mice) before or after an M-MuSV injection, tumors regressed or did not develop. Histologically, the lymph nodes and spleens of reconstituted mice, compared to those of deprived animals, showed repopulation of the thymus-dependent areas and prominent follicles in the cortex. Moreover, tumor tissue of reconstituted mice was extensively infiltrated by lymphocytes. To evaluate the number of lymphoid cells needed to prevent or regress M-MuSV tumors, we injected varying amounts of lymphoid cells into deprived mice. Even low lymphocyte numbers (10(6) cells) were sufficient to exert, in some cases, protection against M-MuSV tumorigenesis. This effect was not abolished by subsequent splenectomy or antilymphocyte serum treatment. Finally, deprived mice, given repeated injections of antiserum (hyperimmune) against M-MuSV, had tumors which appeared only after a prolonged latency. From these results, it is concluded that T-cell population integrity is important in affording total host protection against the M-MuSV tumors.

Secondary in vitro generation of cytolytic T-lymphocytes (CTL's) in the murine sarcoma virus system. Virus-specific CTL induction across the H-2 barrier.

Following in vitro stimulation of murine sarcoma virus Moloney isolate (M-MuSV)-immune spleen cells with syngeneic antigenically related Moloney leukemia cells, highly efficient cytotoxic T-lymphocytes (CTL's) were generated. The cytotoxic effect was directed only against H-2-compatible target cells bearing M-MuSV tumor-associated antigens (TAA). However, in a cold target competition assay a weak but detectable capacity to block CTL activity was also obtained when allogeneic Moloney leukemia cells were added. Moreover, when M-MuSV-immune spleen cells from mice inoculated with virus 14 days previously were stimulated by allogeneic Moloney leukemia cells, a strong cytotoxic effect toward syngeneic and allogeneic tumor cells bearing M-MuSV TAA was elicited.

Spontaneous lymphomas in SJL/J-(v+) mice: ecotropic and dualtropic virus expression in normal and lymphomatous tissues.

Approximately 60% of inbred SJL/J-(v+) adult mice having high levels of ecotropic endogenous XC+ virus showed virus activation within the first month of life, while the others produced virus at comparable levels later on, in an attempt to correlate the time of virus activation with the incidence and latency of lymphomas, the tails of 57 1- and 2-month-old mice were tested for virus presence, and the mice were then observed for lymphoma appearance. While all 2-month-old mice expressed ecotropic virus, only 63% of the 1-month-old mice were virus-positive. However no relationship existed between early virus production (within 1 month) or late virus production and lymphoma latency, total lymphoma incidence, and histopathology. In contrast with high titers of XC+ virus in tail tissues of diseased mice, a markedly low virus content was found in lymphomatous organs. This difference was not due to selective growth of poor virus-producer cells or to inhibitory factors possibly released by the inflammatory cell component. Viral protein content and XC+ virus titer were not closely correlated in the neoplastic organs. Search for XC- viruses revealed that only 1 of 6 aged normal and 16 of 19 lymphomatous mice produced viruses that grew on mink lung cells. By use of a standard limiting dilution cloning procedure, four isolates were obtained that showed tropism for both murine and heterologous cells. Three of these isolates induced cytopathic changes similar to those induced by MCF viruses on mink lung cells but not on mouse cells. Interference and neutralization assays performed to better characterize the virus envelope properties further indicated that SJL/J isolates had features typical of MCF dualtropic viruses.

Human T-lymphotropic virus type I transcriptional regulation by methylation.

While human T-lymphotropic virus type I (HTLV-I) proviral genome is readily detected in leukemic lymphocytes from adult T-cell leukemia patients, viral antigens or viral RNA are not expressed unless these cells are cultured. To address the problem of possible restriction mechanism of HTLV-I replication, we studied the methylation state of provirus in four HTLV-I transformed cell lines. These cell lines are chronically infected with HTLV-I and carry several copies of proviruses but are characterized by different viral expression. Molecular analysis showed that in MT4 cell line, which expresses a low level of viral RNA and proteins, not only are the HTLV-I proviruses heavily methylated but methylation also involves large regions around and within the long terminal repeats. MT4 cells treatment with 5-azacytidine led to an increase in both total viral RNA and p24 gag protein expression. These findings indicate that proviral methylation is responsible for the low viral expression in MT4 cells and also suggest that this phenomenon may be relevant to HTLV-I in vivo latency.

Induction of Moloney murine sarcoma virus tolerance in adult mice by anti-CD4 monoclonal antibody treatment.

The role of CD4+ cells in the immune response to Moloney murine sarcoma/leukemia virus complex was studied in adult mice undergoing long-term CD4+ cell functional depletion by treatment with anti-CD4 H129.19 monoclonal antibody (immunoglobulin G2a, kappa). Adult mice given injections of Moloney murine sarcoma/leukemia virus complex 1 day or 2 wk after monoclonal antibody treatment died with progressing sarcomas at the inoculation site; mice challenged 4 wk after such treatment, when CD4+ cells recovered their functional activity, behaved like conventional mice; i.e., they spontaneously regressed the virus-induced sarcomas. Mice with progressing tumors did not generate virus-specific cytotoxic T-lymphocytes, despite a normal cytotoxic T-lymphocyte response to unrelated antigens, and they became virus carriers as demonstrated by Moloney murine leukemia virus antigen expression on their lymphoid cells a few days after virus injection. Moreover, the observed T-lymphocyte unresponsiveness was not due to the activity of specific suppressor T-cells. The findings indicate that the transient functional depletion of CD4+ cells at the time of virus administration provides appropriate environmental conditions for the spread of virus and facilitates tolerance induction.

Identification of seven new BRCA1 germline mutations in Italian breast and breast/ovarian cancer families.

We analyzed 16 Italian breast and breast/ovarian cancer families for BRCA1 germline mutations using a combination of the protein truncation test (PTT) and the single-strand conformation polymorphism techniques. Genomic DNA from the affected proband of each family was analyzed by applying the PTT to exon 11 of the BRCA1 gene. This initial screening led to the identification of truncated protein products in three families that were shown to carry three different frameshift mutations. In the families that scored negative in the PTT, single-strand conformation polymorphism analysis of the entire coding sequence of the gene revealed four additional mutations consisting of one nonsense, one in-frame deletion, one frameshift, and one missense mutation (in a family with a case of male breast cancer). The four frameshift mutations resulted in a decreased expression of the mutant allele, whereas no loss of transcript was associated with the other three mutations. All mutant alleles were shown to cosegregate with the cancer phenotype within the families, and none have previously been reported.

Effects of angiostatin gene transfer on functional properties and in vivo growth of Kaposi's sarcoma cells.

Gene transfer delivery of endogenous angiogenesis inhibitors such as angiostatin would circumvent problems associated with long-term administration of proteins. Kaposi's sarcoma (KS), a highly vascular neoplasm, is an excellent model for studying tumor angiogenesis and antiangiogenic agent efficacy. We investigated the effects of angiostatin gene transfer in in vitro and in vivo models of KS-induced neovascularization and tumor growth. A eukaryotic expression plasmid and a Moloney leukemia virus-based retroviral vector for expression of murine angiostatin were generated harboring the angiostatin cDNA with cleavable leader signals under the control of either the strong cytomegalovirus promoter/enhancer or the Moloney leukemia virus long terminal repeat. Angiostatin secretion was confirmed by radioimmunoprecipitation and Western blot analysis. Supernatants of angiostatin-transfected cells inhibited endothelial cell migration in vitro. Stable gene transfer of the angiostatin cDNA by retroviral vectors in KS-IMM cells resulted in sustained angiostatin expression and delayed tumor growth in nude mice, which was associated with reduced vascularization. These findings suggest that gene therapy with angiostatin might be useful for treatment of KS and possibly other highly angiogenic tumors.

Identification of a 3 kb Alu-mediated BRCA1 gene rearrangement in two breast/ovarian cancer families.

Most of the hereditary breast cancers are attributed to constitutive alterations of either BRCA1 or BRCA2 genes; nonetheless, germline mutations of these genes in 'high risk' families are found less frequently than expected from linkage data. Recent findings suggest that major genomic rearrangements of the BRCA1 gene might account for at least some of the apparently mutation negative cases. We studied 60 affected probands belonging to families with a strong history of breast and/or ovarian cancer who scored negative for BRCA1 gene mutations by PTT and SSCP analysis. DNA was analysed by the Southern blotting procedure using three different restriction enzymes, and two probes obtained by RT-PCR of the 5' and 3' BRCA1 coding sequence. A 3 kb deletion encompassing exon 17 and causing a frameshift mutation was identified in two independently ascertained families. RT-PCR and long-range DNA PCR were employed to characterize the rearrangement that was finally shown to be the result of a recombination between two very similar Alu repeats. This type of mutation is not identified by the conventional methods of mutation detection which are based on PCR amplification of single exons. Therefore, further search for gene rearrangements is needed to better define the proportion of 'high risk' families that might be explained by gross genomic alterations of the BRCA1 gene.

Mitochondrial targeting of the p13II protein coded by the x-II ORF of human T-cell leukemia/lymphotropic virus type I (HTLV-I).

The X region of the HTLV-I genome contains four major open reading frames (ORFs), two of which, termed x-I and x-II, are of still undefined biological significance. By indirect immunofluorescence and dual labeling with marker proteins, we demonstrate that p13II, an 87-amino acid protein coded by the x-II ORF, is selectively targeted to mitochondria. Mutational analysis revealed that mitochondrial targeting of p13II is directed by an atypical 10-amino acid signal sequence that is not cleaved upon import and is able to target the Green Fluorescent Protein to mitochondria. Expression of p13II results in specific alterations of mitochondrial morphology and distribution from a typical string-like, dispersed network to round-shaped clusters, suggesting that p13II might interfere with processes relying on an intact mitochondrial architecture. Functional studies of mitochondria with the cationic fluorochrome tetramethylrhodamine revealed that a subpopulation of the cells with p13II-positive mitochondria show a disruption in the mitochondrial inner membrane potential (Apsi), an early event observed in cells committed to apoptosis. Taken together, these results suggest novel virus-cell interactions that might be important in HTLV-I replication and/or pathogenicity.

Studies on the mechanisms of HTLV-I leukemogenesis.

The factors that regulate low viral expression and long latency after HTLV-I infection are poorly understood. To study the possible mechanisms involved in the regulation of gene expression and cell transformation, we studied whether (1) methylation could play a role in viral transcription, and (2) tax product could favor chromosomal instability. The results indicate that methylation of HTLV-I LTRs blocks their transcriptional activity and that tax protein triggers DNA damage.

Chromosome damage induced in cord blood T-lymphocytes infected in vitro by HTLV-I.

Experiments were carried out to investigate whether the human T-lymphotropic virus type I (HTLV-I), alone or in combination with a chemical mutagen such as mitomycin C (MMC), has the capacity to damage host chromosomes. Cord-blood T lymphocytes (CBL) were infected by co-cultivation with lethally irradiated HTLV-I-producing cells. Infected and immortalized CBL were then studied for frequencies of sister chromatid exchanges (SCE), chromosome breaks and micronuclei. HTLV-I-infected cells had statistically higher baseline SCE, chromosome aberrations and micronucleus values than the uninfected control CBL. While MMC treatment further augmented these values both in control and in infected lymphocytes, the latter did not show dose-related increases, most likely because of the more pronounced MMC-induced delaying effect on cell progression to mitosis. In view of similar previous observations in mouse lymphocytes carrying the Moloney murine leukemia virus, it is suggested that expression of a common retrovirus gene product, such as the pol endonuclease, might be responsible for the cytogenetic abnormalities observed. In addition to the IL-2 autocrine loop, the direct induction of chromosome damage by HTLV-I in target lymphocytes may be related to the pathogenesis of malignancies associated with HTLV-I infection.

Genetic instability of a dinucleotide repeat-rich region in three hematologic malignancies.

Microsatellite instability is a newly identified mechanism of mutation that occurs in some heritable neurological and muscular disorders, as well as in an increasing number of human cancers. To extend previous data, we examined the genetic instability of a human genomic region, termed S3/1, which we isolated from a human DNA library. The S3/1 sequence contains a stretch with exceptionally high numbers of (GA)n and (CA)n dinucleotide repeats. An interesting rearranged pattern emerged from Southern blot analysis of genomic DNA from three patients with different hematopoietic proliferative diseases out of 69 analyzed (one case of essential thrombocytosis (ET), one of chronic myelogenous leukemia (CML) and one of acute myelogenous leukemia (AML)). The CML and ET patients showed a deletion of 300 to 400 base pairs (bp), and the AML an insertion of about 600 bp, involving the S3/1 locus. Amplification of the rearranged fragments confirmed these observations, and enabled a precise analysis of the region involved. In normal individuals, no gross rearrangements involving this region could be detected. Analysis of DNA from three consecutive bone marrow biopsies of the CML patient disclosed that the genetic alteration affecting S3/1 was no longer detectable following alpha 2-interferon therapy, neither by Southern blot nor by polymerase chain reaction (PCR), thus confirming the tumor-specificity of the alteration; in the same patient, moreover, two out of five other analyzed microsatellites showed tumor-specific alleles, suggesting a more generalized genetic instability in the leukemic cells. These results demonstrate genetic instability of a region containing high numbers of short dinucleotide repeats in a small percentage (4%) of human hematopoietic proliferative disorders.

A CD30-positive T cell line established from an aggressive anaplastic large cell lymphoma, originally diagnosed as Hodgkin's disease.

Ten months following the diagnosis of Hodgkin's disease (HD), a 46-year-old woman presented cutaneous and leukemic involvement by CD30+ anaplastic large cells, from which a continuously growing, exogenous growth factor-independent T cell line was established. The cultured cells are phenotypically and genotypically T cell in type, negative for EBV, HTLV-I and HTLV-II viral sequences, and release soluble CD30 into the supernatant. Karyotype analysis disclosed several chromosomal abnormalities, but none on chromosome 5q. The involvement of the short arm of chromosome 17 prompted us to investigate the TP53 gene by means of the polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) analysis, but no alterations were found in exons 5-8.

Morphological and phenotypical changes in EBV positive lymphoblastoid cells infected by HIV-1.

Human Immunodeficiency Virus type 1 (HIV-1) infects CD4+ T lymphocytes and various other cell types, including B cells. Since HIV-1 seropositive individuals have high numbers of B cells carrying Epstein-Barr Virus (EBV), and are at high risk for development of EBV-associated lymphoproliferative diseases, we studied the mode of HIV-1 infection in four EBV-positive lymphoblastoid B-cell lines (LCLs) as well as some molecular and biological features of the B cells infected by both viruses. We found that LCL cells were successfully infected in vitro by HIV-1, despite the lack of CD4 antigen expression on the cell membrane. LCL cells displayed a persistent, productive, and non-cytopathic infection. Moreover, HIV-1 infection induced reactivation of EBV latent genomes in one cell line. Following HIV-1 infection, LCL cells showed a decrease in B-cell activation markers CD23 and CD39, and an increase in CD10 immature B-cell antigen. Not all cells in each LCL expressed HIV-1 antigens, but all CD10+ cells also co-expressed the HIV-1 envelope protein gp 120. Furthermore, HIV-1 infected LCL cells grew as disperse suspensions, and formed more agar colonies than control, non-HIV-1-infected LCLs. These findings raise the possibility that HIV-1 might play a role in EBV reactivation, and in B-cell lymphoma pathogenesis in AIDS patients.

Tumor outgrowth in peripheral blood mononuclear cell-injected SCID mice is not associated with early Epstein-Barr virus reactivation.

Epstein-Barr virus (EBV)-positive B-cell lymphoproliferative disease develops in severe combined immunodeficient (SCID) mice inoculated with peripheral blood mononuclear cells (PBMC) from EBV(+) individuals (SCID/hu mice). In this study, we investigated the contribution of EBV reactivation and de novo infection of B lymphocytes to tumor outgrowth in SCID/hu mice. Evaluation of BZLF-1, an early EBV activation transcript, in cells recovered from the mouse peritoneal cavity within 16 days following PBMC transfer did not reveal EBV reactivation, while BZLF-1 expression was only detected in tumor masses or in vitro established lymphoblastoid cell lines. To confirm these data by a different strategy, we coinjected PBMC from seropositive donors with purified B cells from seronegative donors of different sex. Fluorescence in situ hydridization analysis of the resulting tumor masses disclosed that the overwhelming majority of lymphoma cells originated from the seropositive donor, implying that no substantial in vivo production and transmission of virus had occurred. Further, treatment of SCID/hu mice with ganciclovir did not prevent lymphoma development. Our results suggest that in the SCID/hu mouse, early EBV replication and secondary infection of bystander B cells does not occur, and that the direct outgrowth of the transformed B lymphocytes present within the PBMC inoculum is the predominant mechanism, which leads to lymphoma generation in this experimental model.