Darinka Sakac

The human P-k histo-blood group antigen provides protection against HIV-1 infection

Several human histo-blood groups are glycosphingolipids, including P/P1/P(k). Glycosphingolipids are implicated in HIV-host-cell-fusion and some bind to HIV-gp120 in vitro. Based on our previous studies on Fabry disease, where P(k) accumulates and reduces infection, and a soluble P(k) analog that inhibits infection, we investigated cell surface-expressed P(k) in HIV infection. HIV-1 infection of peripheral blood-derived mononuclear cells (PBMCs) from otherwise healthy persons, with blood group P(1)(k), where P(k) is overexpressed, or blood group p, that completely lacks P(k), were compared with draw date-matched controls. Fluorescence-activated cell sorter analysis and/or thin layer chromatography were used to verify P(k) levels. P(1)(k) PBMCs were highly resistant to R5 and X4 HIV-1 infection. In contrast, p PBMCs showed 10- to 1000-fold increased susceptibility to HIV-1 infection. Surface and total cell expression of P(k), but not CD4 or chemokine coreceptor expression, correlated with infection. P(k) liposome-fused cells and CD4(+) HeLa cells manipulated to express high or low P(k) levels confirmed a protective effect of P(k). We conclude that P(k) expression strongly influences susceptibility to HIV-1 infection, which implicates P(k) as a new endogenous cell-surface factor that may provide protection against HIV-1 infection.

A novel soluble mimic of the glycolipid, globotriaosyl ceramide inhibits HIV infection

Objective:To determine the effect of a gp120 binding, non-cytotoxic soluble analogue of the glycosphingolipid (GSL), globotriaosyl ceramide (Gb3) on HIV infection in vitro. Design:HIV-1IIIB (X4 virus) infection in Jurkat and phytohaemagglutinin (PHA)/interleukin-2 (IL2) activated, peripheral blood mononuclear cells (PBMC), and HIV-1Ba-L (R5 virus) infection of PHA activated PBMC in vitro were assessed. We monitored cell surface markers, cell viability, and viral/host cell morphology to eliminate pleiotropic effects. Viral-host cell fusion was measured to further address any inhibitory mechanism. Methods:HIV infection was monitored by p24gag ELISA. CD4, CCR5, CXCR4 and apoptosis were determined by fluorescent antibody cell sorting. A model fusion system comprising a cell line transfected with either CD4 and CXCR4 or CCR5, cocultured with a cell line expressing gp120 from either X4-, R5-tropic HIV-1 or HIV-2 virions, was used. PHA/IL2 activated PBMC GSL synthesis was monitored by metabolic radiolabelling. Results:AdamantylGb3 blocked X4 and R5 virus infection with a 50% inhibitory concentration of approximately 150 μM. A reverse transcriptase and a protease-resistant X4 HIV-1 strain retained adamantylGb3 sensitivity. AdamantylGb3 had minimal effect on cell viability. Treated Jurkat cells showed a small increase in CCR5/CXCR4 expression and a slight, transient CD4 down-regulation, which was probably not related to the mechanism of inhibition. Electron microscopy showed normal viral and host cell morphology following adamantylGb3 treatment, and viral entry was blocked. AdamantylGb3 was able to prevent virus-host cell fusion irrespective of HIV strain or chemokine receptor preference. Conclusions:These results suggest that adamantylGb3 may provide a new basis for blocking HIV infections, irrespective of HIV envelope/chemokine co-receptor preference or resistance to other therapeutics.

Chemical compounds that target thiol-disulfide groups on mononuclear phagocytes inhibit immune mediated phagocytosis of red blood cells

BACKGROUND: Patients having immune cytopenias produce antibodies that target hematopoietic cells resulting in their phagocytosis and intracellular destruction. Early reports suggested that phagocytosis could be inhibited by interfering with membrane thiol (SH) groups on phagocytes. Thus, whether chemical compounds that interact with SH or disulfide (SS) groups on mononuclear phagocytes can inhibit phagocytosis of antibody‐coated cells was examined.

Lack of susceptibility of cells from patients with fabry disease to productive infection with R5 human immunodeficiency virus

A lack of viral replication after HIV-1Ba-L (R5) but not HIV-1IIIB (X4) infection was found using in-vitro activated peripheral blood-derived mononuclear cells from patients with Fabry disease, who have a defect in the catabolism of globotriaosylceramide. CCR5, but not CD4 or CXCR4 expression levels, were lower and the surface expression of globotriaosylceramide was negligible on activated patients’ cells. Our findings suggest a novel resistance mechanism to productive infection with R5 HIV-1 that potentially involves abnormal globotriaosylceramide catabolism.

Induction of HIV-1 resistance: cell susceptibility to infection is an inverse function of globotriaosyl ceramide levels

To examine the role of the glycosphingolipid (GSL), globotriaosylceramide (Gb(3), CD77, p(k) blood group antigen) in HIV-1 infection, we have pharmacologically modulated Gb(3) metabolism in an X4 HIV-1 infectable monocytic cell line (THP-1) that naturally expresses Gb(3) and in a Gb(3)-expressing glioblastoma cell line (U87) transfected to express both CD4 and CCR5 to permit R5 HIV-1 infection. THP-1 and U87 cells were treated with either a competitive inhibitor of alpha-galactosidase A, 1-deoxygalactonojirimycin (DGJ) to induce Gb(3) accumulation, or a glucosylceramide synthase inhibitor, phenyl-2-palmitylamino-3-pyrrolidino-1-propanol (P4) to deplete cells of Gb(3). HIV susceptibility was determined via measurement of p24(gag) antigen production by ELISA. In addition, total cellular Gb(3) content was determined using thin layer chromatography followed by Verotoxin1 overlay binding. The cell surface expression of Gb(3) was verified by FACS analysis. We found that DGJ significantly decreased THP-1 and U87 cell susceptibility to HIV-1(IIIB) and HIV-1(BaL) infection, respectively, at a concentration of approximately 100 microM. In contrast, P4 (2 microM) substantially increased cellular susceptibility to HIV-1 infection. Total cellular GSL analysis verified increased Gb(3) expression in cells treated with DGJ and considerable reduction of Gb(3) in P4-treated cells as compared to controls. These results show a reciprocal relationship between Gb(3) expression and infection with either X4 HIV-1(IIIB) or R5 HIV-1(Ba-L). These results support previous studies that Gb(3) provides resistance to HIV infection. Variable Gb(3) expression may provide a natural HIV resistance factor in the general population, and pharmacological manipulation of Gb(3) levels may provide an approach to induction of HIV resistance.

VPAC1 is a cellular neuroendocrine receptor expressed on T cells that actively facilitates productive HIV-1 infection.

Objective A lack of productive HIV-1 infection of Kit225 compared to Jurkat T cells, despite similar levels of CD4 and HIV-1 chemokine co-receptors, was found to correlate with the expression of vasoactive intestinal peptide/pituitary adenylate cyclase activating polypeptide receptor-1 (VPAC1). We therefore examined a role for this seven-transmembrane G protein-coupled neuroendocrine receptor in modulating HIV-1 infection. Methods Reverse transcription–PCR was used to show the level of VPAC1 expression in different T-cell lines. A signal-blocking antibody to VPAC1 was used to examine its inhibiting effect on HIV-1 infection. Transfection of VPAC1 cDNA in both sense and anti-sense orientation was used to assess the role of VPAC1 in HIV-1 infection. HIV-1 infection was monitored by gag p24 ELISA using HIV-1IIIB or by luciferase activity using pseudo envelope-typed HXB2-NL4-3-luciferase. Analysis of HIV-1 gag DNA and 2-LTR circles was utilized to examine a possible mechanism for the effect of VPAC1. Results Using VPAC1 signal blocking antibody, we showed that up to 80% of productive infection with HIV-1IIIB was inhibited. We also demonstrated that HIV-1 gp120 has sequence similarity to the natural ligand for VPAC1 and postulate that it can activate this receptor directly. Transfection of VPAC1 cDNA in the anti-sense orientation resulted in a significant loss, up to 50% of productive infection. In contrast, transfection of cells with VPAC1 in the sense orientation increased the productive infection by more than 15-fold and caused a profound increase in syncytium formation. Furthermore, stimulation of VPAC1 on primary cells facilitated in vitro infection with HIV-1 HXB2-NL4-3. Analysis of HIV-1 gag DNA indicated that VPAC1 does not affect viral entry; however, cells that show negligible expression of VPAC1 may not be productively infected as indicated by a lack of 2-LTR circle formation. Conclusion We have discovered a cellular receptor, VPAC1, that is a novel and potent facilitator of HIV-1 infection and thus, is a potentially important new target for therapeutic intervention.

Identification and characterization of five-transmembrane isoforms of human vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide receptors

The seven-transmembrane (7TM) G-protein-coupled neuroendocrine receptors VPAC1 (HGNC approved gene symbol VIPR1) and VPAC2 (HGNC approved gene symbol VIPR2) are expressed in different tissues and involved in the regulation of important biological functions. We now report the identification and characterization of novel five-transmembrane(5TM) forms of both human VPAC1 and human VPAC2. These alternatively spliced variant mRNAs result from the skipping of exons 10/11, spanning the third intracellular loop, the fourth extracellular loop, and the transmembrane regions 6 and 7, producing in-frame 5TM receptors predicted to lack a G-protein-binding motif. RT-PCR showed that these 5TM receptors are differentially expressed in transformed and normal cells. Translation of the 5TM protein was demonstrated by transfection and expression in CHO cells. Following agonist stimulation, differential signaling of the 7TM versus 5TM forms was shown both for the activation of adenylate cyclase and for tyrosine phosphorylation. The identification of these splice variants in various cells and their expression and differential signal transduction compared to the 7TM form suggest that these novel receptors have biological relevance.

Abnormal splicing of SHP-1 protein tyrosine phosphatase in human T cells: Implications for lymphomagenesis

OBJECTIVE SHP-1 protein tyrosine phosphatase has been implicated in suppressing B-lymphocyte and myeloid cell malignancies; however, there are little data on this role of SHP-1 in T-lymphocyte malignancies. We examined malignant human T cells to identify any abnormalities of SHP-1 that would support a role for this molecule in suppressing T lymphomagenesis. MATERIALS AND METHODS Human T-lymphocyte cell lines and primary blood cells were used to examine the expression of SHP-1 mRNA and protein. Reverse transcriptase polymerase chain reaction was used to amplify particular portions of the SHP-1 mRNA for cloning and sequencing. Gene transfer was used to examine the effects of SHP-1 on cell growth and morphology. Glutathione S-transferase (GST) fusion proteins were generated and used to determine SHP-1-associated proteins. RESULTS Leukemia- and lymphoma-derived cell lines were identified that did not express SHP-1 protein. Examination of the mRNA from these and other T-cell lines, and from normal peripheral blood mononuclear cells (PBMCs), revealed three distinct transcripts by restriction enzymes, reverse transcriptase polymerase chain reaction, and Southern blot analysis. In addition to the expected wild-type transcript, two novel transcripts were identified. One was a deletion transcript found only in Jurkat leukemia-derived cells, predicted to encode for a 7-kDa protein containing most of the amino-terminal SH2 domain. The second contained an 88-nucleotide insert that is the unspliced second intron resulting in a frame shift and the formation of a noncoding transcript. This mRNA was found in all cells examined but was the only transcript detected in the cell lines lacking SHP-1 protein. Expressing wild-type SHP-1 in these cell lines resulted in a change in the morphology of the cells with a concomitant decrease in their growth. GST fusion constructs showed the 7-kDa variant able to associate with an identical array of proteins as wild-type SHP-1, suggesting that it could compete with the wild-type SHP-1 for substrates. This protein was detectable in the cell line expressing its corresponding mRNA and was able to induce significant changes in cell morphology when transfected into a cell line expressing wild-type SHP-1; however, it did not induce any changes in cell growth. CONCLUSIONS These data are the first to show the existence of multiple transcripts of SHP-1 in human transformed T lymphocytes and normal PBMCs and supports previous work showing that alternate forms of SHP-1 mRNA are a common finding in other cells. We also show the lack of splicing out of an intron as a novel mechanism of regulation of SHP-1 protein expression in both normal and transformed T cells. Moreover, we provide the first evidence showing a protein product detectable in cells that is translated from an alternatively spliced form of SHP-1 mRNA, a variant truncated SHP-1 protein having potential biologic relevance. This report provides evidence supporting the concept that SHP-1 can negatively regulate growth of malignant human T cells and that lack of SHP-1 protein or function may be associated with lymphomagenesis.

HIV‐1 infection is facilitated in T cells by decreasing p56lck protein tyrosine kinase activity

Several studies have suggested an important role for the protein tyrosine kinase p56lck (Lck) in HIV infection; however, the exact nature of this role remains unclear. Using a series of well characterized Jurkat‐derived cell lines having a wide range of Lck kinase activity, our results showed that, while the entry of HIV‐1 into these cell lines was similar, the kinetics of virus production by these cells were very different. Cells expressing a kinase‐inactive Lck showed accelerated viral replication, whereas, cells expressing Lck with normal or elevated enzymatic activity showed a delay in virus replication that was proportional to the initial level of endogenous Lck activity. The cell line having the highest initial Lck kinase activity showed the slowest rate of productive HIV‐1 infection. Analysis of 2‐LTR circles revealed that this inhibitory effect of Lck was not due to inhibition of reverse transcription of HIV‐1 genome or migration of the proviral DNA into the nuclei. This affect of Lck was confirmed in additional studies that used either the S1T cell line lacking completely Lck or where the Lck activity was altered in Jurkat cells prior to infection. S1T cells showed a 3‐ to 12‐fold increase in the level of infection compared to Jurkat cells despite similar CD4 and chemokine coreceptor expression and cell doubling times. Pretreatment of Jurkat with an antisense lck oligodeoxynucleotide inhibited the synthesis of functional Lck and facilitated the viral replication by the cells as did expressing a dominant‐negative mutant Lck which increased the productive infection>3‐fold. Conversely, whereas IL‐16 had no affect on productive infection in S1T cells that lack Lck, IL‐16 pretreatment of Jurkat cells resulted in an immediate (within 5 min) and sustained and gradual (over 5 h) increase in Lck activity that resulted in a reduction of HIV‐1 replication that paralleled the increasing Lck kinase activity. These results show that the enzymatic activity of Lck kinase can affect viral replication, that a lack of, or decreased Lck activity facilitates viral replication. Conversely, Lck can mediate a delay in HIV‐1 infection that is proportional to the initial endogenous Lck enzyme activity.

Desulfation of sulfoglycolipids by anchimeric assisted solvolysis

Abstract A mild method for acid-catalyzed desulfation of sulfoglycolipids in acetone is described. No ancillary degradation was observed. The method is suitable for glyceroglycolipids that might be deacylated under acid conditions. It is proposed that the formation of an acetone adduct during the reaction anchimerically assists the removal of the sulfate group.