High prevalence of non-alcoholic fatty liver disease and metabolic risk factors in Guatemala: A population-based study.

BACKGROUND: There are no data on the prevalence of non-alcoholic fatty liver disease (NAFLD) in general population samples in Guatemala or in other Central American countries. The prevalence and distribution of NAFLD and its associated risk factors were evaluated in a population-based sample of adults in Guatemala. METHODS: Cross-sectional study of 411 men and women 40 years of age or older residing in urban and rural areas of Guatemala. Metabolic outcomes included obesity, central obesity, hypercholesterolemia, diabetes, and metabolic syndrome (MetS). Liver disease outcomes included elevated liver enzymes, elevated Fatty Liver Index (FLI), and elevated FIB-4 score. RESULTS: The overall prevalence of obesity, central obesity, diabetes, and MetS were 30.9, 74.3, 21.6, and 64.2%, respectively. The fully-adjusted prevalence ratios (95% CI) for obesity, central obesity, diabetes, and MetS comparing women to men were 2.83 (1.86-4.30), 1.72 (1.46-2.02), 1.18 (1.03-1.34), and 1.87 (1.53-2.29), respectively. The overall prevalence of elevated liver enzymes (ALT or AST), elevated FLI, and elevated FIB-4 scores were 38.4, 60.1, and 4.1%, respectively. The fully-adjusted prevalence ratios (95% CI) for elevated liver enzymes (either ALT or AST) and elevated FLI score comparing women to men were 2.99 (1.84-4.86) and 1.47 (1.18-1.84), respectively. CONCLUSIONS: The prevalence of metabolic abnormalities and liver outcomes in this general population study was very high. The prevalence of metabolic and liver abnormalities was particularly high among women, an observation that could explain the atypical 1:1 male to female ratio of liver cancer in Guatemala.

SOCS-1, a negative regulator of the JAK/STAT pathway, is silenced by methylation in human hepatocellular carcinoma and shows growth-suppression activity.

Hepatocellular carcinoma (HCC) is a major cause of cancer death, but the molecular mechanism for its development beyond its initiation has not been well characterized. Suppressor of cytokine signaling (SOCS-1; also known as JAB and SSI-1) switches cytokine signaling 'off' by means of its direct interaction with Janus kinase (JAK). We identified aberrant methylation in the CpG island of SOCS-1 that correlated with its transcription silencing in HCC cell lines. The incidence of aberrant methylation was 65% in the 26 human primary HCC tumor samples analyzed. Moreover, the restoration of SOCS-1 suppressed both growth rate and anchorage-independent growth of cells in which SOCS-1 was methylation-silenced and JAK2 was constitutively activated. This growth suppression was caused by apoptosis and was reproduced by AG490, a specific, chemical JAK2 inhibitor that reversed constitutive phosphorylation of STAT3 in SOCS-1 inactivated cells. The high prevalence of the aberrant SOCS-1 methylation and its growth suppression activity demonstrated the importance of the constitutive activation of the JAK/STAT pathway in the development of HCC. Our results also indicate therapeutic strategies for the treatment of HCC including use of SOCS-1 in gene therapy and inhibition of JAK2 by small molecules, such as AG490.

A human serum mannose-binding protein inhibits in vitro infection by the human immunodeficiency virus.

In vitro infection by the human immunodeficiency virus (HIV) of CD4+ H9 lymphoblasts is inhibited by a mannose-binding protein (MBP) purified from human serum. In addition, MBP is able to selectively bind to HIV-infected H9 cells and HIV-infected cells from the monocyte cell line U937. These results indicate MBP most likely recognizes high mannose glycans known to be present on gp120 in the domain that is recognized by CD4 and thereby inhibits viral entry to susceptible cells. In support of this contention, recombinant gp120 binds directly to MBP; the binding is saturable, mannan inhibitable, removed by N-glycanase treatment, and dependent on divalent cations.

RAFTK, a novel member of the focal adhesion kinase family, is phosphorylated and associates with signaling molecules upon activation of mature T lymphocytes.

The related adhesion focal tyrosine kinase (RAFTK), a recently discovered member of the focal adhesion kinase family, has previously been reported to participate in signal transduction in neuronal cells, megakaryocytes, and B lymphocytes. We have found that RAFTK is constitutively expressed in human T cells and is rapidly phosphorylated upon the activation of the T cell receptor (TCR). This activation also results in an increase in the autophosphorylation and kinase activity of RAFTK. After its stimulation, there was an increase in the association of the src cytoplasmic tyrosine kinase Fyn and the adapter protein Grb2. This association was mediated through the SH2 domains of Fyn and Grb2. RAFTK also co-immunoprecipitates with the SH2 domain of Lck and with the cytoskeletal protein paxillin through its COOH-terminal proline-rich domain. The tyrosine phosphorylation of RAFTK after T cell receptor-mediated stimulation was reduced by the pretreatment of cells with cytochalasin D, suggesting the role of the cytoskeleton in this process. These observations indicate that RAFTK participates in T cell receptor signaling and may act to link signals from the cell surface to the cytoskeleton and thereby affect the host immune response.

Proliferation of astroglia and oligodendroglia in response to human T cell-derived factors.

Human T lymphocytes transformed by human T cell leukemia-lymphoma viruses or activated by lectins were found to produce stimulating factors that promoted both proliferation and maturation of oligodendroglial and astroglial cells in vitro.

A new HTLV-III/LAV protein encoded by a gene found in cytopathic retroviruses.

The DNA of the HTLV-III/LAV group of retroviruses contains certain additional open reading frames that are not found in typical avian or mammalian retroviruses. The role of these sequences in encoding for gene products that may be related to pathogenesis remains to be resolved. An open reading frame whose 5' end overlaps with the pol gene, but is unrelated to the env gene, has been observed in HTLV-III/LAV and visna virus, both cytopathic mammalian retroviruses. Evidence presented here shows that this open reading frame is a bona fide coding sequence of HTLV-III/LAV and that its product, a protein with a molecular weight of 23,000, induces antibody production in the natural course of infection.

Blocking of HIV-1 infectivity by a soluble, secreted form of the CD4 antigen.

The initial event in the infection of human T lymphocytes, macrophages, and other cells by human immunodeficiency virus (HIV-1) is the attachment of the HIV-1 envelope glycoprotein gp120 to its cellular receptor, CD4. As a step toward designing antagonists of this binding event, soluble, secreted forms of CD4 were produced by transfection of mammalian cells with vectors encoding versions of CD4 lacking its transmembrane and cytoplasmic domains. The soluble CD4 so produced binds gp120 with an affinity and specificity comparable to intact CD4 and is capable of neutralizing the infectivity of HIV-1. These studies reveal that the high-affinity CD4-gp120 interaction does not require other cell or viral components and may establish a novel basis for therapeutic intervention in the acquired immune deficiency syndrome (AIDS).

Molecular characterization of human T-cell leukemia (lymphotropic) virus type III in the acquired immune deficiency syndrome.

The human T-cell leukemia (lymphotropic) virus type III (HTLV-III) appears to be central to the causation of the acquired immune deficiency syndrome (AIDS). Two full-length integrated proviral DNA forms of HTLV-III have now been cloned and analyzed, and DNA sequences of the virus in cell lines and fresh tissues from patients with AIDS or AIDS-related complex (ARC) have been characterized. The results revealed that (i) HTLV-III is an exogenous human retrovirus, approximately 10 kilobases in length, that lacks nucleic acid sequences derived from normal human DNA; (ii) HTLV-III, unlike HTLV types I and II, shows substantial diversity in its genomic restriction enzyme cleavage pattern; (iii) HTLV-III persists in substantial amounts in cells as unintegrated linear DNA, an uncommon property that has been linked to the cytopathic effects of certain animal retroviruses; and (iv) HTLV-III viral DNA can be detected in low levels in fresh (primary) lymphoid tissue of a minority of patients with AIDS or ARC but appears not to be present in Kaposi's sarcoma tissue. These findings have important implications concerning the biological properties of HTLV-III and the pathophysiology of AIDS and Kaposi's sarcoma.

HTLV-III in saliva of people with AIDS-related complex and healthy homosexual men at risk for AIDS.

Peripheral blood leukocytes and saliva from 20 individuals, including four with the acquired immune deficiency syndrome (AIDS), ten with AIDS-related complex (ARC), and six healthy homosexual males at risk for AIDS, were compared as sources of transmissible human T-cell leukemia (lymphotropic) virus type III (HTLV-III), the virus found to be the etiologic agent of AIDS. All of the AIDS and ARC patients and four of the six healthy homosexuals had evidence of prior exposure to HTLV-III as indicated by seropositivity for antibody to HTLV-III structural proteins. Infectious virus was isolated from the peripheral blood of one of the AIDS patients, four of the ARC patients, and two of the healthy homosexual males, consistent with previous reports. HTLV-III was also isolated from the saliva of four of the ARC patients and four of the healthy homosexuals. Virus was also observed by electron microscopy in material prepared by centrifugation of the saliva of one AIDS patient. Although AIDS does not appear to be transmitted by casual contact, the possibility that HTLV-III can be transmitted by saliva should be considered.

Virus envelope protein of HTLV-III represents major target antigen for antibodies in AIDS patients.

In this study, two glycoproteins (gp160 and gp120) that are encoded by human T-cell lymphoma virus type III (HTLV-III) were the antigens most consistently recognized by antibodies found in patients with the acquired immune deficiency syndrome (AIDS) and with the AIDS-related complex (ARC) and in healthy homosexual males. The techniques used to detect the glycoproteins were radioimmunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (RIP/SDS-PAGE). Although most antibody-positive samples from ARC patients and from healthy homosexual males also reacted with the virus core protein p24, less than half of the AIDS patients revealed a positive band with p24 under the same conditions. The ability to detect antibodies against a profile of both the major env gene encoded antigens and the gag gene encoded antigens suggests that the RIP/SDS-PAGE may be a valuable confirmatory assay for establishing the presence or absence of antibodies to HTLV-III in human serum samples.

HTLV-III in cells cultured from semen of two patients with AIDS.

Epidemiological results suggest that the etiological agent of the acquired immune deficiency syndrome (AIDS) is transmitted primarily through blood products, semen, and saliva. There is evidence that the human T-cell leukemia (lymphotropic) virus type III (HTLV-III) is this agent. HTLV-III has been isolated repeatedly from T cells obtained from peripheral blood or lymph node tissue of AIDS and pre-AIDS patients and of healthy people believed to have been exposed to the virus. In the present study, HTLV-III was detected in and isolated from T cells present in the seminal fluid of AIDS patients. Mononuclear cells from the semen of AIDS patients and normal individuals were cultured in the presence of T-cell growth factor (interleukin-2). After 6 to 8 days, HTLV-III antigens were transiently expressed by the cells from the AIDS patients but not by those from the normal individuals. When the mononuclear cells from the semen of AIDS patients were cocultured with a permissive human T-cell line, cell cultures were produced that expressed high levels of reverse transcriptase activity, showed retroviral particles by electron microscopy, and were positive for HTLV-III-specific antigens when tested by fixed-cell indirect immunofluorescence with the use of monoclonal antibodies to the p24 and p15 antigens of HTLV-III.

A new HTLV-III/LAV encoded antigen detected by antibodies from AIDS patients.

A newly identified protein from HTLV-III/LAV, the virus implicated as the etiologic agent of the acquired immune deficiency syndrome, was studied. This protein, which has a molecular weight of 27,000 (p27), was shown by amino acid sequencing to have a coding origin 3' to the env gene on the HTLV-III genome. The presence of antibodies to p27 in virus-exposed individuals indicated that this gene is functional in the natural host.

Neutralization of the AIDS retrovirus by antibodies to a recombinant envelope glycoprotein.

Mammalian cell lines have been engineered to produce a secreted form of the AIDS retrovirus envelope glycoprotein. The recombinant protein has been isolated from growth-conditioned culture media and used to immunize animals. Antibodies directed against the recombinant molecule were found to react with the envelope glycoprotein produced in virus-infected cells. Furthermore, these antibodies were able to directly inactivate the AIDS retrovirus in a neutralization assay in vitro. The expression system reported here should provide sufficient quantities of the AIDS retrovirus envelope protein for biological and vaccination studies.

HTLV-III infection in brains of children and adults with AIDS encephalopathy.

Unexplained debilitating dementia or encephalopathy occurs frequently in adults and children with the acquired immune deficiency syndrome (AIDS). Brains from 15 individuals with AIDS and encephalopathy were examined by Southern analysis and in situ hybridization for the presence of human T-cell leukemia (lymphotropic) virus type III (HTLV-III), the virus believed to be the causative agent of AIDS. HTLV-III DNA was detected in the brains of five patients, and viral-specific RNA was detected in four of these. In view of these findings and the recent demonstration of morphologic and genetic relatedness between HTLV-III and visna virus, a lentivirus that causes a chronic degenerative neurologic disease in sheep, HTLV-III should be evaluated further as a possible cause of AIDS encephalopathy.

Evaluation of novel cell cycle inhibitors in mantle cell lymphoma.

Signature abnormalities in the cell cycle and apoptotic pathway have been identified in mantle cell lymphoma (MCL), affording the opportunity to develop targeted therapies. In this study, we tested a novel class of kinase inhibitors, styryl sulfones, which differ from prior cell cycle inhibitors in that they are not related to purines or pyrimidines. We observed that two closely related compounds, ON013100 and ON01370, altered the growth and cell cycle status of MCL lines and potently inhibited the expression of several important molecules, including cyclin-dependent kinase 4, p53, mouse double minute 2 (MDM2), and cyclin D as well as increased cyclin B expression. Using both terminal deoxy transferase uridine triphosphate nick end-labelling and poly ADP-ribose polymerase assays, we found that these compounds caused apoptosis in MCL cells. In addition, using molecular analyses, we observed the modulation of caspase-3 activity but not the expression of B-cell lymphoma family molecules. Next, we investigated the cytotoxicity of the MCL lines upon treatment with styryl sulfone compounds in combination with other currently used chemotherapeutic agents, such as doxorubicin (DOX) or vincristine (VCR). We found that the combination of DOX plus styryl sulfone or VCR plus styryl sulfone increased cytotoxicity by one log scale, compared with the single styryl sulfone compound. Thus, styryl sulfones alone, or in combination with chemotherapeutic agents, present attractive opportunities for new drug development in MCL.

Thrombopoiesis. Capturing the unicorn.

The elusive factor that stimulates megakaryocytes to produce platelets has at last been found; as well as its physiological interest, this factor--thrombopoietin--may be of considerable therapeutic importance.

Characterization of adhesive interactions between human endothelial cells and megakaryocytes.

Cell-cell adhesion is essential for many immunological functions and is believed to be important in the regulation of hematopoiesis. Adhesive interactions between human endothelial cells and megakaryocytes were characterized in vitro using the CMK megakaryocytic cell line as well as marrow megakaryocytes. Although there was no adhesion between unactivated human umbilical vein endothelial cells (HUVEC) and megakaryocytes, treatment of HUVEC with inflammatory cytokines such as IL-1 beta, tumor necrosis factor alpha, INF-gamma, or the phorbol ester phorbol myristate acetate (PMA) resulted in a time- and dose-dependent increase in adhesion. Stimulation of marrow megakaryocytes or CMK cells with the cytokines IL-1 beta, GM-CSF, IL-6, IL-3, or PMA augmented their adhesion to endothelium. Monoclonal antibodies against the LFA-1 subunit of the leukocyte adherence complex CD18 inhibited the binding of marrow megakaryocytes or CMK cells to HUVEC. Adhesion blocking experiments also demonstrated that the VLA-4/VCAM-1 pathway was important for megakaryocyte attachment to HUVEC. Adhesion promoted maturation of megakaryocytic cells as measured by increased expression of glycoproteins GpIb and GpIIb/IIIa and by increased DNA content. These observations suggest that alterations in megakaryocyte adhesion may occur during inflammatory conditions, mediated by certain cytokines, resulting in augmented megakaryocyte maturation.

Production of tumor necrosis factor alpha and interleukin 1 beta by monocytic cells infected with human immunodeficiency virus.

The production of tumor necrosis factor alpha (TNF alpha) and IL-1 beta by the monocytic cell line THP-1, productively infected with HIV-1, was investigated using specific RIA and Northern blot analysis. HIV-infected cells, like uninfected cells, did not constitutively produce any detectable amounts of protein or mRNA for TNF alpha or IL-1 beta. After stimulation with LPS or a combination of LPS plus IFN-gamma, TNF alpha and IL-1 beta were detected in tissue culture supernatants and cell lysates and transcripts for both cytokines were seen on Northern blots. No significant difference in production of these two cytokines was observed between uninfected and chronically infected cells. Acutely HIV-infected cells, however, showed phenotypic changes compatible with maturation and an increase in TNF alpha and IL-1 beta mRNA production, and released significantly higher levels of TNF alpha and IL-1 beta compared with chronically infected or uninfected cells. Furthermore, LPS stimulation of HIV-infected cells increased virus production. These results suggest that HIV-infected monocytic cells may produce increased amounts of TNF alpha and IL-1 beta in response to stimuli that could be present in vivo.

Cytokine signaling through the novel tyrosine kinase RAFTK in Kaposi's sarcoma cells.

A number of cytokines, including basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), oncostatin M (OSM), IL-6, and tumor necrosis factor alpha (TNF-alpha), have been postulated to have a role in the pathogenesis of Kaposi's sarcoma (KS). The proliferative effects of bFGF and OSM may be via their reported activation of the c-Jun NH2-terminal kinase (JNK) signaling pathway in KS cells. We now report that KS cells express a recently identified focal adhesion kinase termed RAFTK which appears in other cell systems to coordinate surface signals between cytokine and integrin receptors and the cytoskeleton as well as act downstream to modulate JNK activation. We also report that the tyrosine kinase receptor FLT-4, present on normal lymphatic endothelium, is robustly expressed in KS cells. Treatment of KS cells with VEGF-related protein (VRP), the ligand for the FLT-4 receptor, as well as with the cytokines bFGF, OSM, IL-6, VEGF, or TNF-alpha resulted in phosphorylation and activation of RAFTK. Following its activation, there was an enhanced association of RAFTK with the cytoskeletal protein paxillin. This association was mediated by the hydrophobic COOH-terminal domain of the kinase. Furthermore, JNK activity was increased in KS cells after VEGF or VRP stimulation. We postulate that in these tumor cells RAFTK may be activated by a diverse group of stimulatory cytokines and facilitate signal transduction to the cytoskeleton and downstream to the growth promoting JNK pathway.

Genotyping by mass spectrometric analysis of short DNA fragments.

A method has been developed to produce small DNA fragments from PCR products for analysis of defined DNA variations by mass spectrometry. The genomic region to be analyzed is PCR-amplified with primers containing a sequence for the type IIS restriction endonuclease Bpml. Bpml digestion of the resultant PCR products yields fragments as small as seven bases, which are then analyzed by electrospray ionization mass spectrometry. The approach was validated using seven different variants within the APC tumor suppressor gene, in which a perfect correlation was obtained with DNA sequencing. Both the sense and antisense strands were analyzed independently, and several variants can be analyzed simultaneously. These results provide the basis for a generally applicable and highly accurate method that directly queries the mass of variant DNA sequences.