SS-56, a novel cellular target of autoantibody responses in Sjögren syndrome and systemic lupus erythematosus.

Certain autoimmune disorders, including Sjögren syndrome (SS) and systemic lupus erythematosus (SLE), are characterized by autoantibodies against the Ro/SSA and La/SSB cellular antigens. Although the implication of these autoantibodies in disease pathogenesis is still unclear, it is believed that the aberrant responses against autoantigens may extend to other proteins that are not yet well defined. In an attempt to analyze the regulated gene expression in lymphocytes by an HIV-suppressive immunomodulator, we have identified and cloned a novel gene encoding a 56-kDa protein, named SS-56, which is structurally related to the 52-kDa Ro/SSA antigen. The new protein showed primarily perinuclear cytoplasmic localization, and recombinant SS-56 was found to react in ELISA with sera from most patients with SS or SLE. Western blot analysis confirmed the autoantigenic nature of native SS-56 in extracts from HeLa cells. Interestingly, the incidence of antibodies to SS-56 was associated with visceral complications in SLE, and roughly half of the 17 SS or SLE patients with no detectable antibodies to SSA and SSB antigens presented measurable antibodies against recombinant SS-56. Thus, SS-56 represents a new member of the SS family of autoantigens and could become an additional and important diagnostic marker for SS and SLE.

A major human immunodeficiency virus type 1-initiated killing pathway distinct from apoptosis.

We have investigated the relative contribution of apoptosis or programmed cell death (PCD) to cell killing during acute infection with T-cell-tropic, cytopathic human immunodeficiency virus type 1 (HIV-1), by employing diverse strategies to inhibit PCD or to detect its common end-stage sequelae. When Bcl-2-transfected cell lines were infected with HIV-1, their viability was only slightly higher than that of control infections. Although the adenovirus E1B 19-kDa protein has been reported to be a stronger competitor of apoptosis than Bcl-2, it did not inhibit HIV-mediated cell death better than Bcl-2 protein. Competition for Fas ligand or inactivation of the Fas pathway secondary to intracellular mutation (MOLT-4 T cells) also had modest effects on overall cell death during acute HIV infection. In contrast to these observations with HIV infection or with HIV envelope-initiated cell death, Tat-expressing cell lines were much more susceptible (200% enhancement) to Fas-induced apoptosis than controls and Bcl-2 overexpression strongly (75%) inhibited this apoptotic T-cell death. PCD associated with FasR ligation resulted in the cleavage of common interleukin-1beta-converting enzyme (ICE)-protease targets, poly(ADP-ribose) polymerase (PARP) and pro-ICE, whereas cleaved products were not readily detected during HIV infection of peripheral blood mononuclear cells or T-cell lines even during periods of extensive cell death. These results indicate that one important form of HIV-mediated cell killing proceeds by a pathway that lacks the characteristics of T-cell apoptosis. Our observations support the conclusion that at least two HIV genes (env and tat) can kill T cells by distinct pathways and that an envelope-initiated process of T-cell death can be discriminated from apoptosis by many of the properties most closely associated with apoptotic cell death.

Human immunodeficiency virus 1 envelope-initiated G2-phase programmed cell death.

Despite intensive investigation, no clearly defined mechanism explaining human immunodeficiency virus (HIV)-induced cell killing has emerged. HIV-1 infection is initiated through a high-affinity interaction between the HIV-1 external envelope glycoprotein (gp120) and the CD4 receptor on T cells. Cell killing is a later event intimately linked by in vitro genetic analyses with the fusogenic properties of the HIV envelope glycoprotein gp120 and transmembrane glycoprotein gp41. In this report, we describe aberrancies in cell cycle regulatory proteins initiated by cell-cell contact between T cells expressing HIV-1 envelope glycoproteins and other T cells expressing CD4 receptors. Cells rapidly accumulate cyclin B protein and tyrosine-hyperphosphorylated p34cdc2 (cdk1) kinase, indicative of cell cycle arrest at G2 phase. Moreover, these cells continue to synthesize cyclin B protein, enlarge and display an abnormal ballooned morphology, and disappear from the cultures in a pattern previously described for cytotoxicity induced by DNA synthesis (S phase) inhibitors. Similar changes are observed in peripheral blood mononuclear cells infected in vitro with pathogenic primary isolates of HIV-1.

Polymerase chain reaction amplified HTLV-I, HIV-1 and HIV-2 DNA fragments in subjects with mixed retroviral infections.

Serum and peripheral blood mononuclear cells from eight patients from the Ivory Coast with positive screening test results for retroviral infections were studied by serology (ELISA, Western blot (WB), synthetic peptide test), cell co-culture, and polymerase chain reaction (PCR). Two HIV-2 infections with indeterminate interpretation on HIV-1 WB were detected, two were clear dual HIV-1/HIV-2 infections, three were ambiguous mixed HIV-1/HIV-2 infections, and one was a triple retroviral infection by HTLV-I, HIV-1 and HIV-2. Four slow/low HIV-1 strains were isolated at the expense of HTLV-I and HIV-2 strains. The ELISA tests were found to be very sensitive. Indeterminate WB interpretations were frequent (HTLV-I, four; HIV-1, three; HIV-2, two). PCR provided clear evidence of multiple retroviral infections in three cases and enabled interpretation of indeterminate WB samples in three cases. One sample presented a puzzling pattern with positive PCR results for HIV-1 and HIV-2 associated with negative or indeterminate serological results. Thus, our data emphasise the need to analyse serological as well as virological markers to gain better insight on mixed retroviral infections, especially in endemic areas such as West Africa.

Regulatory elements in the human immunodeficiency virus type 1 long terminal repeat LTR (HIV-1) responsive to steroid hormone stimulation.

Within the long terminal repeat (LTR) of the human immunodeficiency virus type 1 (HIV-1) provirus there exists a steroid hormone-responsive element corresponding to the TGTTCT sequence identified as the glucocorticoid receptor binding element within the LTR of mouse mammary tumor virus. We have used an LTR(HIV-1)-chloramphenicol acetyl transferase (CAT) plasmid construct to transfect infected H9V3 and noninfected H9 cells. Four hours before harvest the cells were divided into two parts and half was treated with hydrocortisone (10(-7) M). The cells were harvested and washed, and the CAT activity was measured. In eight repeat experiments an increased expression of the CAT gene has consistently been observed in H9V3 cells in response to the glucocorticoid but no significant effect of the steroid was observed in noninfected cells. Double transfection of LTR(HIV-1)-TAT and LTR(HIV-1)-CAT into noninfected H9 cells results in a cell population in which the CAT gene was responsive to glucocorticoid stimulation. A time course and dose response for the steroid effect have been determined and the binding of steroid receptor fo the LTR-DNA characterized by gel retardation experiments.

Variable stringency hybridization of polymerase chain reaction amplified HIV-1 DNA fragments.

DNA isolated from the peripheral blood mononuclear cells of HIV-1 seropositive individuals was used for polymerase chain reaction (PCR) amplification of gag and envelope regions. Eight aliquots of the amplified DNA fragments have been subjected to Southern/dot blot analysis, hybridizing with 32P-labelled-BH10 (HIV-1 strain IIIB) at low stringency. After the filters had been autoradiographed, they were cut so that each hybridized band/dot could be subject to variable stringency washing using various ionic concentrations at a fixed temperature. The filter was reconstructed so that the effect of the variable stringency wash might be visualized following a second exposure to Kodak film. The level of activity for each band/dot was measured by counting the 32P or by densitometry analysis of the photographic record. The results allow a plot to be made of the decrease in bound radioactivity against ionic strength. By comparison with a standard curve obtained for HIV-1 strain IIIB amplified fragments subject to similar hybridization and analysis, an estimation of the degree of nucleotide mismatch relative to the BH10 DNA probe can be obtained. The technique provides a rapid means of characterizing PCR amplified fragments.