Intracellular expression of antibody fragments directed against HIV reverse transcriptase prevents HIV infection in vitro.

We have tested a novel strategy of intracellular immunization to block human immunodeficiency virus (HIV) infection. The expression of a specific antibody within a cell was achieved by transduction of genes that encode for immunoglobulin chains with specificity to viral reverse transcriptase. We demonstrated that inhibition of this enzyme makes cells resistant to HIV infection by blocking an early stage of viral replication. If high efficiency transduction with a stable vector into lymphohaematopoietic stem cells or mature lymphocytes can be achieved, gene transfer-mediated intracellular immunization might be a feasible treatment strategy in AIDS.

Kaposi's sarcoma-associated human herpesvirus-8 encodes homologues of macrophage inflammatory protein-1 and interleukin-6.

Human herpesvirus-8 (HHV-8) has been detected in Kaposi's sarcoma (KS) lesions of all types (AIDS-related, classical and endemic), in body-cavity-based B-cell lymphomas (BCBLs) and in lesions of multicentric Castleman's disease (MCD). We have identified a major gamma-herpesvirus-divergent locus (DL-B) in HHV-8 DNA encoding several HHV-8 unique open reading frames (ORFs), including a homologue of interleukin-6 (IL-6) and two homologues of macrophage inflammatory protein MIP-1. We show that the HHV-8-encoded IL-6 homologue (vIL-6) shares functional properties with endogenous IL-6 proteins and that both vIL-6 and vMIP-1 transcripts are present at high levels following butyrate induction of an HHV-8' BCBL cell line. Low amounts of constitutive vIL-6, but not vMIP-1, mRNA were also detected. The presence of a functional IL-6 homologue encoded by HHV-8 may provide a mechanistic model for the hypothesized role of HHV-8 in KS, MCD and BCBL that involves the mitogenic effects of vIL-6 on surrounding cells. MIP-1 proteins may enhance these effects through the chemotactic recruitment of endogenous cytokine-producing cells into affected tissues and could potentially influence HIV disease progression in coinfected individuals through interactions with the HIV co-receptor CCR-5.

Long-term protection of chimpanzees against high-dose HIV-1 challenge induced by immunization.

A combination AIDS vaccine approach consisting of priming with adenovirus-HIV-1MN gp160 recombinants followed by boosting with HIV-1SF2 gp120 was evaluated in chimpanzees. Long-lasting protection, requiring only three immunizations, was achieved against a low-dose challenge with the SF2 strain of HIV-1 and a subsequent high-dose SF2 challenge administered 1 year later without an intervening boost. Notably, neutralizing antibody responses against both clinical and laboratory isolates developed in three chimpanzees and persisted until the time of high-dose challenge. The possibility that cytotoxic T-lymphocytes contribute to low-dose protection of a chimpanzee lacking neutralizing antibodies is suggested. Our results validate the live vector priming/subunit booster approach and should stimulate interest in assessing this combination vaccine approach in humans.

Configuration and expression of the T cell receptor beta chain gene in human T-lymphotrophic virus I-infected cells.

We studied the configuration and expression of the gene encoding the beta chain of the T cell receptor (TCR beta) in cell lines and primary tumor cells infected by the human T cell leukemia/lymphoma (lymphotrophic) virus type I (HTLV-I). Most of the cell lines and all the primary tumor cells showed rearrangement of the TCR beta gene, and in each case the rearrangement was distinct. The majority of cases examined were clonal with respect to a particular TCR beta gene rearrangement. Primary tumor cells from one case (SD) were found to have a tandem duplication of a portion of chromosome 7; this appears to have resulted in the presence of three alleles of the TCR beta gene, each of which is arranged differently. This suggests that the chromosomal abnormality, and possibly infection by HTLV-I, occurred before TCR beta gene rearrangement. Cell lines infected by HTLV-I express levels of TCR beta mRNA similar to PHA stimulated lymphocytes, suggesting that this gene is not transcriptionally activated as a result of infection by HTLV-I. Cloned T cells of known antigen specificity that are infected by HTLV-I in vitro show impairment of immune function, including loss of antigen-specific responsiveness and the acquisition of alloreactivity. Comparison of the configuration of the TCR beta gene before and after infection revealed no changes detectable by Southern blot analysis. Levels of expression of the TCR beta gene at the mRNA level and surface expression of the T3 complex were also not significantly altered, suggesting that changes in immune function cannot be attributed to quantitative changes in the TCR molecule. The configuration of the TCR beta gene in primary tumor cells infected by HTLV-I was compared with that in the derived cell lines. In all pairs examined, the configuration in the primary tumor cells was different from that in the cell lines, strongly suggesting that the cells that grow in culture are not the original neoplastic cells.

Human T cell leukemia/lymphoma virus I infection and subsequent cloning of normal human B cells. Direct responsiveness of cloned cells to recombinant interleukin 2 by differentiation in the absence of enhanced proliferation.

A human T cell leukemia/lymphoma virus (HTLV)-I-infected B cell clone expressed Tac antigen on its cell surface and responded to recombinant interleukin 2 (IL-2) by increased production of IgM without any increase in proliferation. Anti-Tac antibody completely inhibited the IL-2-induced differentiation of this HTLV-I-infected B cell clone. This study demonstrates that HTLV-I can directly infect normal mature human B cells, and that the Tac antigen, which may be induced by infection with HTLV-I, is the functional receptor for IL-2-induced B cell differentiation. The availability of such cell lines and clones should provide useful tools to delineate precisely the differentiation step in the human B cell cycle.

Functional properties of antigen-specific T cells infected by human T-cell leukemia-lymphoma virus (HTLV-I).

Tetanus-toxoid specific helper-inducer T-cell clones, which had been infected and transformed by human T-cell leukemia-lymphoma virus (HTLV-I), were obtained from an antigen-specific human T cell line by using a limiting dilution technique in the presence of the virus. These HTLV-I-infected T-cell clones proliferated specifically in response to soluble tetanus toxoid but, unlike normal T cells, they could do so in the absence of accessory cells. The HTLV-I-infected T-cell clones did not present the antigen to autologous antigen-specific T cells that were not infected with HTLV-I. The capacity of helper-inducer T cells to retain antigen-specific reactivity after infection by HTLV-I, while losing the normal T-cell requirement for accessory cells, has clinical and theoretical implications.

Transformation and cytopathogenic effect in an immune human T-cell clone infected by HTLV-I.

Human T-cell leukemia-lymphoma virus (HTLV) is a human C-type retrovirus that can transform T lymphocytes in vitro and is associated with certain T-cell neoplasms. Recent data suggest that, in the United States, patients with acquired immunodeficiency syndrome (AIDS), homosexual men with lymphadenopathy, and hemophiliacs have had significant exposure rates to HTLV, whereas matched and unmatched control American subjects have rarely been exposed to this agent. In the present experiments, T cells specifically reactive against HTLV were propagated from a patient whose HTLV-bearing lymphoma was in remission. The T cells were cloned in the presence of the virus and an HTLV-specific cytotoxic T-cell clone was isolated. This clone was infected and transformed by the virus, with one copy of an HTLV-I provirus being integrated into the genome. This T-cell clone did not exhibit the normal dependence on T-cell growth factor (interleukin-2) and proliferated spontaneously in vitro. Exposure of the clone to HTLV-bearing, autologous tumor cells specifically inhibited its proliferation and resulted in its death. These results may have implications for HTLV-associated inhibition of T-cell responses.

Transforming potential of human c-sis nucleotide sequences encoding platelet-derived growth factor.

The nucleotide sequence of a transforming human c-sis complementary DNA shows an open reading frame 723 base pairs in length located downstream from an in-phase terminator thymine-guanine-adenine codon. Sequences within this region were identical to those previously determined for the exons of the normal human c-sis gene. Thus, the predicted transforming product, a protein of 27,281 daltons, may be the actual precursor for normal human platelet-derived growth factor chain A.

Pharmacological inhibition of in vitro infectivity of human T lymphotropic virus type I.

Human T lymphotropic virus type I (HTLV-I) is an exogenous RNA tumor virus etiologically linked to adult T cell leukemia and related diseases. In this paper, we describe that two 2',3'-dideoxynucleoside analogues, erythro 3'-azido-2',3'-dideoxythymidine (also called azidothymidine) and 2',3'-dideoxycytidine can inhibit the infectivity of HTLV-I against helper/inducer T cells in vitro. Both 2',3'-dideoxynucleoside analogues inhibited the overgrowth of target T cells, which was a consequence of virally mediated transformation, when they were exposed to the virus and cultured with the compounds. A profound decrease in the expression of HTLV-I gag-proteins was also observed. Moreover, we observed that the amount of proviral DNA detected in cellular DNA from the target T cells was substantially reduced when the cells were protected by the compounds against the virus and that at certain concentrations of the compounds the synthesis of viral DNA was completely suppressed. These results may be of value in developing a new pharmacological strategy for preventing the replication and possibly blocking the transmission of HTLV-I and related retroviruses in human beings.

Infection of human T lymphotropic virus-I-specific immune T cell clones by human T lymphotropic virus-I.

Human T lymphotropic virus-I (HTLV-I)-specific T cell lines were established and cloned. K5, an OKT8+ clone bearing multiple proviral integration sites, retained its HTLV-I-specific cytotoxicity and a normal dependence on interleukin 2 (IL-2), indicating that there is a finite number of transforming integration sites. R2, an OKT4+ HTLV-I-infected clone, initially mounted a proliferative response to HTLV-I; but then its IL-2-independent proliferation increased and the antigen specificity was lost. All HTLV-I-infected clones tested including K7, another OKT8+ transformed cytotoxic clone that had lost its reactivity, expressed comparable levels of T cell receptor beta-chain (TCR-beta) messenger (m)RNA. Although clones K5 and K7 had different functional properties, they had the same rearrangement of the TCR-beta gene, suggesting that they had the same clonal origin. These data indicate that HTLV-I-specific T cells retain their immune reactivity for variable periods of time following infection, but then usually lose it; in some cases, however, no alteration in function can be detected. The data also suggest that different consequences can take place in the same clone depending on the pattern of retroviral infection.

DNA methylation and expression of HLA-DR alpha.

B-cell lines established from two individuals with T-cell acute lymphocytic leukemia (T-ALL) express HLA-DR antigens, whereas the isogenic T-cells do not. The lack of expression correlates with a lack of detectable HLA-DR mRNA. All of the DR alpha DNA sequences detected by a cloned DR alpha cDNA probe are contained in a BglII fragment which varies slightly in size (4.0 to 4.8 kilobases) from one individual to another. In DNA from the T-cells not expressing DR alpha mRNA, all of the potential HpaII sites within the BglII fragment appeared to be methylated. In contrast, at least some of these sites were not methylated in DNA from the B-cells expressing high levels of DR alpha mRNA. Treatment of these T-cells with 5-azacytidine resulted in the induction of DR surface antigen expression, the appearance of DR alpha mRNA, and the partial demethylation of the DR alpha DNA sequences. T-cell lines established from human T-cell leukemia-lymphoma virus associated T-cell neoplasias, in contrast to the T-cell acute lymphocytic leukemia cell lines, expressed both DR antigens and DR alpha mRNA; the HpaII sites within the BglII fragment of DR alpha DNA of these human T-cell leukemia-lymphoma virus-positive T-cell lines were in all cases at least partially unmethylated. Uncultured peripheral blood T-cells from human T-cell leukemia-lymphoma virus-infected individuals expressed DR antigens at a low level, and the DR alpha locus was partially unmethylated. After 48 h in culture, DR antigen expression was substantially increased, but no significant changes were observed in methylation of the DR alpha locus or in the amount of DR mRNA which was present. This suggests that expression of DR antigens also can be modulated post-transcriptionally.

Type 1 human T-cell leukemia virus small envelope protein expressed in mouse cells by using a bovine papilloma virus-derived shuttle vector.

In an attempt to express the small (transmembrane) envelope protein p21e of type 1 human T-cell leukemia (lymphotrophic) virus (HTLV-1) exclusive of other viral gene products, we have constructed a recombinant plasmid clone (pMBE-1) in a bovine papillomavirus-derived mammalian expression vector. Mouse C127 cells transfected with the pMBE-1 plasmid expressed the introduced HTLV-1 viral gene(s) as demonstrated by Northern blot and indirect immunofluorescence with natural human antisera. The transfected mouse cells were injected into BALB/c mice, and a monoclonal antibody was recovered which specifically recognizes a 21-kilodalton protein present in HTLV-1 virions, indicating that the pMBE-1 plasmid encodes the small envelope protein.

Isolation and characterization of an immortal neoplastic cell line (KS Y-1) from AIDS-associated Kaposi's sarcoma.

BACKGROUND: Acquired immunodeficiency syndrome (AIDS) is associated with the occurrence of tumors such as Kaposi's sarcoma (KS) and B-cell lymphoma. However, no evidence exists yet that human immunodeficiency virus type 1, the causative agent of AIDS, is directly responsible for cell transformation. It is also not clear whether KS lesions, which are of complex cellularity, contain tumor cells derived from a true monoclonal malignancy (originating from a single malignant cell) or whether the lesions are just polyclonally hyperplastic in nature (containing increased numbers of normal cells). In fact, the presence of malignant KS cells has never been unequivocally shown in AIDS-associated KS, and previously isolated KS cell cultures were not immortal or malignant. PURPOSE: Our purpose was to (a) utilize technology that could facilitate isolation and enrichment of tumor cells from AIDS-associated KS lesions, (b) establish and characterize an immortalized KS cell line, and (c) test the malignant potential of such a cell line in animal models. METHODS: Mononuclear cells were isolated from 2.5 L of pleural effusion from an AIDS-associated KS patient. T-lymphocytes, B-lymphocytes, monocytes/macrophages, and fibroblasts were removed by a cytotoxicity method, using monoclonal antibodies specific for cell surface markers and baby rabbit complement. KS cells were cultured in the absence of exogenous growth factors in an effort to select for transformed cells capable of self-sustained growth. The karyotype abnormalities were detected by G-banded marker studies, and phenotypic markers were determined by indirect immunofluorescence and immunocytochemical methods. Beige nude XID and severe combined immunodeficient mice were used to evaluate the tumorigenic, angiogenic, and metastatic potentials of cells. RESULTS: An immortalized cell line, named KS Y-1, was isolated. Its phenotype is similar to that of endothelial cells with positive CD34 and CD31 markers. Tetraploid chromosomal abnormalities were found in primary fresh KS tissue and in vitro passages of KS Y-1 cells. These cells promoted tumorigenesis, angiogenesis, and metastasis in immunodeficient mice. Tumors produced at the site of injection as well as metastases in the lung, spleen, pancreas, gastrointestinal tract, and skin showed a human tetraploid karyotype. KS Y-1 cells show high plating efficiency. CONCLUSION: The KS Y-1 cell line could be the first evidence of AIDS-associated KS cells that may develop clones with an indisputable malignant cell phenotype. IMPLICATIONS: KS Y-1 cells in the in vivo mouse model can be used to study the effects of therapeutic compounds in advanced KS.

Lack of expression of type C hamster virus after neoplastic transformation of hamster embryo fibroblasts by benzo(a)pyrene.

Syrian hamster embryo fibroblasts transformed in vitro with benzo(a)pyrene were analyzed for the presence of type C viral components, including extra- and intracellular reverse transcriptase activity, intracellular type C hamster virus-related RNA, and cellular hamster virus group-specific antigen. No evidence could be obtained for the presence of any of these components, although they were easily detectable in hamster fibroblasts producing either B-34 virus (a hamster virus pseudotype of Harvey murine sarcoma virus which contains an excess of helper type C hamster virus) or Harvey virus itself. In addition, intracellular viral RNA could not be detected in normal hamster embryo fibroblasts, in hamster fibroblasts transformed with simian virus 40, or in newborn hamster kidney and liver. Thus the detectable expression of the indigenous hamster type C virus is not required to maintain the transformed phenotype of these cells.

Restricted expression of retrovirus nucleic acids and proteins in primate type C virus (gibbon ape leukemia virus-simian sarcoma virus)-initiated human B-lymphoblast cultures.

Fresh human B-lymphoblasts established in culture following exposure of adult peripheral blood leukocytes to type C retroviruses of the simian sarcoma virus/simian sarcoma-associated virus-gibbon ape leukemia virus group were analyzed in detail for the presence of the infecting virus. Viral expression ranged from production of low levels of intact virus in a few cultures to the presence of viral RNA and protein in the absence of detectable of levels of complete virus in the majority of the cultures. In situ molecular hybridization assays using 3H-labeled complementary DNA and indirect immunofluorescence assays using antibody to purified viral protein indicated that the expression of viral RNA and proteins are preferentially expressed in only a fraction of the cells in some cultures. If expression of the infecting viral sequences is necessary for the sustained growth of these cells, then those cells detectably synthesizing viral RNA and proteins may be influencing the growth of the remaining virus-negative cells. The lack of virus production in cultures synthesizing viral RNA and protein indicate that these human B-lymphocytes restrict the life cycle of these viruses at some step(s) after transcription of viral RNA or translation of viral protein.

New insight on the role of extrachromosomal retroviral DNA.

During infection with different retroviruses, high levels of unintegrated extrachromosomal DNA accumulate in infected cells. While extrachromosomal linear DNA is the immediate precursor of the integrated provirus, the function, if any, of extrachromosomal circular DNA has been unclear. Several groups have attempted to address the possible function, activity, and importance of this unintegrated DNA during the life cycle of retroviruses and the course of retroviral-associated diseases. This review summarizes recent work in this field and tries to analyze some aspects of extrachromosomal forms of retroviral DNA and their possible application as a molecular biological tool.

cDNA sequencing confirms HTLV-I expression in adult T-cell leukemia/lymphoma and different sequence variations in vivo and in vitro.

Human T-lymphotropic virus type I (HTLV-I) is the etiological agent of adult T-cell leukemia/lymphoma (ATL) and of tropical spastic paraparesis/HTLV-I-associated myelopathy (TSP/HAM). In both diseases, expression of viral message can generally only be demonstrated by the reverse transcriptase-polymerase chain reaction (RT-PCR) technique. We have previously reported on the the expression of at least four types of alternatively spliced pX mRNAs in vitro and in vivo (1). The sequence variation between HTLV-I pX cDNAs cloned from two different HTLV-I-infected cell lines and from uncultured primary peripheral blood mononuclear cells (PBMC) from two ATL patients was examined. None of the cDNA clones from one of the ATL samples was completely identical to any of the previously cloned cell line messages, establishing that the demonstration of HTLV-I mRNA in ATL is not the result of PCR contamination. Sequence analysis showed that differences between samples can be clustered according to their geographic origin. Cell line cDNAs showed a more marked sequence drift than ATL cDNAs, especially in the long terminal repeat (LTR), demonstrating association of intrastrain variability with culture in vitro. Intrastrain cDNA variability in vivo also suggests ongoing viral replication in infected individuals. A premature stop codon in the pX-II open reading frame (orf) was a common finding, suggesting that the complete putative pX-II protein is not essential for T-cell immortalization or HTLV-I replication.

Suppression of T-cell responsiveness by inducible cAMP early repressor (ICER).

Depending on the nature of the costimulation of T lymphocytes, expression of regulatory cytokines and chemokines is either susceptible or resistant to cyclic AMP (cAMP)-mediated inhibition. Our data show that cAMP-mediated inhibition of endogenously expressed cytokines, which is characteristic for T helper (Th) 1- and Th 2-like phenotypes, correlates with the induction of a potent transcriptional repressor, inducible cAMP early repressor (ICER), in both subsets of T cells activated under conditions of suboptimal interleukin-2 (IL-2) expression. Importantly, Th-specific expression of certain chemokines is also susceptible to cAMP-mediated transcriptional attenuation. To determine whether ICER per se, rather than forskolin-mediated elevation of intracellular cAMP, is responsible for the observed inhibitory effect, we generated transgenic mice expressing ICER under the control of a lymphocyte-specific lck promoter. On stimulation, transgenic thymocytes overexpressing ICER exhibited reduced levels of IL-2 and interferon (IFN)-gamma and failed to express the macrophage inflammatory protein (MIP)-1alpha and MIP-1beta genes. Splenic T cells from ICER-transgenic mice showed a defect in proliferation and lacked a mixed lymphocyte reaction response, implying that ICER-mediated inhibition of cytokine and chemokine expression might play an important role in T-cell inactivation.

No detection of HTLV-I DNA in punch skin biopsies from patients with cutaneous T-cell lymphoma by the polymerase chain reaction.

In this study we have tried to take advantage of the high genetic homology between conserved regions of human T-cell lymphotropic virus (HTLV) types I and II, hoping that a possible retrovirus in patients with cutaneous T-cell lymphoma (CTCL) would have regions with homology to types I/II. DNA was extracted from punch skin biopsies from 21 patients and subjected to the polymerase chain reaction (PCR), using primer sets designed to match conserved regions in the HTLV-I/II genome. The PCR products were subjected to agarose gel electrophoresis with subsequent Southern blotting and hybridization to an HTLV-I probe. No bands of exogenous origin were seen on the agarose gel or by hybridization. If a retrovirus is present in the skin in CTCL patients, it is either not related to HTLV-I/II, present at a copy number below the PCR detection limit, or has been cleared from the skin before the clinical symptoms appear.

Chronic dietary L-arginine prevents endothelial dysfunction secondary to environmental tobacco smoke in normocholesterolemic rabbits.

Our goal was to determine whether environmental tobacco smoke causes endothelial dysfunction in the absence of hypercholesterolemia and whether such an effect can be prevented by supplementation with L-arginine. Environmental tobacco smoke exposure is associated with an increase in coronary artery disease events and mortality. We have previously demonstrated that environmental tobacco smoke causes endothelial dysfunction and atherosclerosis in rabbits with diet-induced hypercholesterolemia and atherosclerosis and that chronic dietary L-arginine supplementation prevents this. The effects of L-arginine supplementation (2.25% solution ad libitum) and environmental tobacco smoke (smoking chambers for 10 weeks) were examined with a 2 x 2 design in 32 rabbits fed a normal diet. Acetylcholine, calcium ionophore A23187, and nitroglycerin-induced vasorelaxation were assessed in aortic rings precontracted with phenylephrine. Endothelial L-arginine levels were measured by chromatography. Chronic L-arginine supplementation increased serum (P < .001) and endothelial (P = .003) L-arginine levels. Environmental tobacco smoke reduced endothelium-dependent acetylcholine-induced relaxation, and L-arginine blocked this adverse effect (P = .04). Environmental tobacco smoke tended to increase phenylephrine-induced contraction (P = .06). Neither environmental tobacco smoke nor L-arginine influenced A23187-induced relaxation nor endothelium-independent nitroglycerin-induced relaxation. Endothelial dysfunction secondary to environmental tobacco smoke may occur in the absence of diet-induced hypercholesterolemia and atherosclerosis. Chronic dietary supplementation with a nitric oxide donor such as L-arginine offsets the endothelial dysfunction associated with environmental tobacco smoke in normocholesterolemic rabbits, possibly through substrate loading of the nitric oxide pathway.