Stability of 40 cytokines/chemokines in chronically ill patients under different storage conditions.

Numerous studies point to the utility of blood cytokine measurements in the diagnosis and treatment of human disease. Advances in detection allow robust multiplex analysis. However, cytokines are present at low levels and are produced and act in complex networks which can remain active in stored blood. A major barrier to the routine use of cytokines as clinical biomarkers is sample management prior to analysis. Studies on cytokine stability under storage frequently use 'spiked' normal control plasma or serum to generate detectable levels of the cytokines of interest. These conditions may oversimplify the reality of clinically complex samples and provide limited information regarding optimal management of whole blood samples prior to plasma separation. Cytokine stability has also been addressed previously using plasma from normal individuals under different conditions of anticoagulant use, storage time and temperature of storage. No studies have as yet been undertaken to address cytokine stability in critically ill patients which may differ from normal, healthy individuals due to underlying cofounders such as inflammation. To address these issues, we subjected samples from five patients exhibiting an inflammatory disease state to three storage extremes which might be encountered in a clinical setting, prior to analysis of 40 cytokines. Blood drawn into EDTA or ACD anticoagulant was immediately separated and plasma stored at -80 °C. Matched samples were stored as follows; whole blood overnight at room temperature, or whole blood or plasma stored 10 days at 4 °C. We used equivalence testing to determine the similarity of stored cytokine values to baseline values. In ACD plasma, Eotaxin, IL-6, IL-11, IL-15, IP10, MDC, MCP-1 met equivalence to baseline in all storage conditions while for EDTA plasma stored 10 days at 4 °C EGF, FGF2, Fractalkine, G-CSF, IL-1ß, IL-5, IL-6, IL-7, IL-11, IP-10, TGFα and TNFα showed equivalence to baseline measurements. Intraclass Correlation Coefficients (ICC) were calculated and the following cytokines showed good to excellent agreement across all 4 storage conditions: Eotaxin, IL-5, IL-6, IL-11, IL-13, IP-10, MCP-1 and TNFα when collected in EDTA, and Eotaxin, IL-5, IL-6, IL-11, IL12-p40, IL-15, IP-10 and MCP-1 when collected in ACD. Five plasma cytokines were identified as being the least stable in both ACD and EDTA: IL-7, IL-9, IL12p70, RANTES, sCD40L, while IL-1ß was identified as unstable stored in ACD plasma. This study identified several clinically important cytokines that are remarkably stable in blood and plasma, and some that stored poorly. To our knowledge, this is the first cytokine storage study to use medically unwell patient samples and equivalence testing to evaluate the stability of measured cytokine values after storage.

Differential red blood cell age fractionation and Band 3 phosphorylation distinguish two different subtypes of warm autoimmune hemolytic anemia.

Warm autoimmune hemolytic anemia (wAIHA) is a blood disorder characterized by the increased destruction of autologous red blood cells (RBCs) due to the presence of opsonizing pathogenic autoantibodies. Preliminary reports published more than three decades ago proposed the presence of two wAIHA subtypes: Type I, in which autoantibodies preferentially recognize the oldest, most dense RBCs; and Type II, characterized by autoantibodies that show no preference. STUDY DESIGN AND METHODS: We evaluated patients having wAIHA for Type I and II subtype using discontinuous Percoll gradient age fractionation and direct antiglobulin test (DAT). We performed Western immunoblotting and mass spectrometry to show autoantibody specificity for Band 3. We investigated Band 3 tyrosine phosphorylation in different Percoll fractions to determine aging associated with oxidative stress. RESULTS: We confirm the existence of two subtypes of wAIHA, Type I and Type II, and that autoantibodies recognize Band 3. Type I patients were characterized by five Percoll fractions, with a DAT showing IgG opsonization F1 < F5 and elevated Band 3 phosphorylation compared to healthy controls (HCs). In contrast, Type II wAIHA patients were characterized by three to four Percoll fractions, where the DAT IgG opsonization shows F1 ≥ F3/4 and Band 3 phosphorylation was absent or significantly decreased compared to HC. CONCLUSIONS: Type I patients have increased Band 3 tyrosine phosphorylation that may represent accelerated aging of their RBCs resulting in exacerbation of a pathologic form of RBC senescence. Type II patients show decreased Band 3 tyrosine phosphorylation and lack the oldest, most dense RBCs suggesting premature RBC clearance and a more severe wAIHA.

The utility of a monocyte monolayer assay in the assessment of intravenous immunoglobulin-associated hemolysis.

BACKGROUND: Hemolysis following the administration of intravenous immunoglobulin (IVIG) is an important adverse event (AE). While the monocyte monolayer assay (MMA) has been used to predict in vivo hemolysis when serologically incompatible blood may be transfused, it has also been shown to correlate with IVIG-associated hemolysis. In this study, the MMA was examined for its utility in assessing the risk of hemolysis after IVIG. STUDY DESIGN AND METHODS: Forty-two non-blood group O patients receiving high-dose IVIG (≥2 g/kg) were examined using an autologous and allogeneic MMA. Hemolysis was defined by a drop in hemoglobin of ≥1 g/L, a positive direct antiglobulin test (DAT) and eluate, and a decrease in haptoglobin or increase in lactate dehydrogenase and/or reticulocytes. RESULTS: Forty-two patients provided 50 assessable postinfusion samples, with hemolysis observed in 20 (40%) of cases. Autologous MMA using post-IVIG red blood cells significantly correlated with clinical outcomes when compared to allogeneic MMA (P = .0320 vs .5806, t test). No significant difference in receiver operating characteristics was observed when comparing autologous MMA testing against DAT for the diagnosis of IVIG-associated hemolysis. However, when using samples collected 5 to 10 days after receipt of high-dose IVIG, the autologous MMA had higher sensitivity than the DAT. CONCLUSION: MMA testing with autologous monocytes collected 5 to 10 days after receipt of high-dose IVIG can be used for the diagnosis of IVIG-associated hemolysis and may be of particular value in cases in which the Day 5 to 10 DAT is negative.

Optimal conditions for the performance of a monocyte monolayer assay.

BACKGROUND: Various versions of the monocyte monolayer assay (MMA) have been used to assess clinical significance of red blood cell (RBC) alloantibodies in transfusion for more than 35 years. However, the optimal conditions, including anticoagulant used for whole blood samples, temperature and duration of storage, and optimal pH for assessing the response of monocytes to antibody-bound RBCs, have never been clearly delineated. STUDY DESIGN AND METHODS: Whole blood from healthy donors was collected in ACD, EDTA, or heparin and stored at room temperature (RT) versus 4°C for up to 2 days. pH was examined with and without buffers. Phagocytosis of anti-D-opsonized R2 R2 RBCs was used as the positive control for comparison studies. Whole blood was taken into ACD and kept at RT until testing, from patients with or without immune hemolytic anemia. RESULTS: No significant differences in the phagocytosis of the R2 R2 control RBCs were observed using ACD anticoagulant between freshly drawn or up to 36-hour-stored whole blood kept at RT, regardless of the donor. Physiologic pH during MMA was important for optimal monocyte interactions with antibody-opsonized RBCs. MMA results with patient samples, under optimal conditions, kept up to 30 hours in one instance of long-distance shipment, correlated with clinical hemolysis. CONCLUSION: MMA can be reliably performed on whole blood samples drawn into ACD and kept at RT for up to 36 hours and when physiologic pH is maintained during the assay. Future studies are required to confirm whether use of these conditions with patient monocytes can provide accurate determination of alloantibody significance in patients requiring blood transfusion.

Western immunoblotting as a new tool for investigating direct antiglobulin test-negative autoimmune hemolytic anemias.

BACKGROUND: Direct antiglobulin test-negative (DAT(-)) autoimmune hemolytic anemia, characterized by hemolysis without detectable immunoglobulin or complement on patient red blood cells (RBCs), poses a diagnostic challenge. To select therapy, classification of the hemolysis as immune- or non-immune-mediated is important. We developed a method using Western immunoblot (WB) to classify DAT(-) patients by measuring and comparing levels of RBC immunoglobulin (Ig)G to normal donors. STUDY DESIGN AND METHODS: RBC samples from 42 normal donors were made into ghosts and analyzed by WB and densitometry to establish a normal mean relative quantity of IgG (RQIgG) on the RBCs. RQIgG on eight DAT(-) and eluate-negative patients with hemolytic anemia was determined and compared to RQIgG on normal RBCs. RESULTS: RQIgG of 42 normal donors indicated a calculated mean ± SD of 0.0016 ± 0.0015 and we used a cutoff of 0.0047, the mean + 2SD. This was compared with a receiver operating curve cutoff of 0.0041 with 100% sensitivity and 93% specificity. Of the eight patients tested, three were classified as non-immune-mediated (one had pyruvate kinase deficiency) and five as immune-mediated. Two of the patients in the latter group underwent splenectomy, followed by remission. CONCLUSION: WB analysis is more sensitive than conventional test tube DAT or elution analysis. Our assay confirms: 1) previous studies showing normal RBCs are sensitized with IgG, perhaps due to natural autoantibody to senescence; 2) that some normal RBCs have increased levels of IgG without signs of disease; and 3) that WB distinguishes between non-immune- and immune-mediated hemolytic anemia in DAT(-) patients and may be useful for clinical diagnosis.

Characterization of SHP-1 protein tyrosine phosphatase transcripts, protein isoforms and phosphatase activity in epithelial cancer cells.

We identified 7 SHP-1 (PTPN6) transcripts using epithelial cancer-derived cell lines. Four were shown to utilize the epithelial promoter 1 to transcribe a full-length, a partial (exon 3) or complete (exons 3 & 4) deletion of the N-SH2 domain, and also a non-coding transcript having a stop codon caused by a frame shift due to intron 2 retention. Three additional transcripts were shown to utilize the hematopoietic promoter 2 to transcribe a full-length, a partial (exon 3) deletion of the N-SH2 domain and a non-coding transcript with intron 2 retention. We show that endogenous proteins corresponding to the open-reading-frame (ORF) transcripts are produced. Using GST-fusion proteins we show that each product of the ORF SHP-1 transcripts has phosphatase activity and isoforms with an N-SH2 deletion have increased phosphatase activity and novel protein-protein interactions. This study is the first to document utilization of promoter 2 by SHP-1 transcripts and a noncoding transcript in human epithelial cells.

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 synthetic globotriaosylceramide analogue inhibits HIV-1 infection in vitro by two mechanisms.

Previously, it was shown that the cell-membrane-expressed glycosphingolipid, globotriaosylceramide (Gb(3)/P(k)/CD77), protects against HIV-1 infection and may be a newly described natural resistance factor against HIV infection. We have now investigated the potential of a novel, water soluble, non-toxic and completely synthetic analogue of Gb(3)/P(k) (FSL-Gb(3)) to inhibit HIV-1 infection in vitro. A uniquely designed analogue, FSL-Gb(3), of the natural Gb(3)/P(k) molecule was synthesized. HIV-1(IIIB) (X4 virus) and HIV-1(Ba-L) (R5 virus) infection of PHA/interleukin-2-activated, peripheral blood mononuclear cells (PBMCs) and Jurkat T cells in vitro was assessed, as well as infection of U87.CD4.CCR5 by various clinical R5 tropic viruses after treatment with FSL-Gb(3). We monitored Gb(3), CD4 and CXCR4 expression by fluorescent antibody cell sorting and viral replication by p24(gag) ELISA. Total cellular Gb(3) was examined by glycosphingolipid extraction and thin layer chromatography. In vivo toxicity was monitored in mice by histological assessment of vital organs and lymphoid tissue. FSL-Gb(3) blocked X4 and R5 of both lab and clinical viral strains in activated PBMCs or the U87.CD4.CCR5 cell line with a 50% inhibitory concentration (IC(50)) of approximately 200-250 microM. FACS and TLC overlay showed that FSL-Gb(3) can insert itself into cellular plasma membranes and that cellular membrane-absorbed FSL-Gb(3) is able to inhibit subsequent HIV-1 infection. There was no effect of FSL-Gb(3) on cell surface levels of CD4 or CXCR4. Thus, FSL-Gb(3) can inhibit HIV-1 by two mechanisms: direct inhibition of virus and inhibition of viral entry. Infusion of FSL-Gb(3) into laboratory mice at doses well in excess of theoretical therapeutic doses was tolerated with no untoward reactions. Our results demonstrate the potential utility of using a completely synthetic, water soluble globotriaosylceramide analogue, FSL-Gb(3), having low toxicity, for possible future use as a novel therapeutic approach for the systemic treatment of HIV/AIDS.

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.

Reduction of disulfide bonds within anti-D results in enhanced Fcgamma receptor blockade.

BACKGROUND: We have investigated whether chemicals known to disrupt disulfide bonds are capable of altering immunoglobulin anti-D structure resulting in an increased efficacy of the chemically modified anti-D to inhibit Fcgamma receptor (FcgammaR)-mediated phagocytosis. If successful, this would provide a rationale to explore this mechanism of enhancing FcgammaR blockade for future use in immunoglobulin therapies for immune cytopenias. STUDY DESIGN AND METHODS: Anti-D that was shown to block 50 percent of the FcgammaR-mediated phagocytosis of opsonized red blood cells (RBCs) using a monocyte monolayer assay (MMA) was combined with two different thiol-containing compounds, dithiothreitol (DTT) or p-toluenesulfonylmethyl mercaptan, with or without treatment with iodoacetamide, and allowed to react. Excess chemical was removed by extensive dialysis. FcgammaR blockade was assessed by MMA with dialyzed, untreated, or chemically treated anti-D using both D+ and D- opsonized target RBCs. Toxicity was determined by fluorescence-activated cell sorting. Aggregates and oligomerization of chemically treated anti-D were examined using gel filtration-high pressure liquid chromatography. RESULTS: Using disulfide-reducing compounds to chemically modify anti-D significantly increases the efficacy of the anti-D to induce an FcgammaR blockade and decrease phagocytosis in vitro of opsonized D+ or D- RBCs. This effect was shown not due to unbound residual chemical, toxicity, or formation of immunoglobulin G oligomers. S-alkylation was required when using low concentrations of reducing compound. CONCLUSION: Our results demonstrate that irreversible reduction of interchain disulfide bonds within the immunoglobulin anti-D results in a significantly increased efficacy to inhibit FcgammaR-mediated phagocytosis regardless of opsonized target cell. With the use of this strategy, more effective and less expensive immunoglobulin treatment for immune cytopenias such as immune thrombocytopenic purpura or autoimmune hemolytic anemia may be developed.

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-1(IIIB) (X4 virus) infection in Jurkat and phytohaemagglutinin (PHA)/interleukin-2 (IL2) activated, peripheral blood mononuclear cells (PBMC), and HIV-1(Ba-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 p24(gag) 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 microM. 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.

c-SRC protein tyrosine kinase regulates early HIV-1 infection post-entry.

OBJECTIVE: We investigated whether HIV-1 inhibition by SRC-family kinase inhibitors is through the non-receptor tyrosine kinase pp60 (c-SRC) and its binding partner, protein tyrosine kinase 2 beta (PTK2B). DESIGN: CD4 T-lymphocytes were infected with R5 (JR-FL) or X4 (HXB2) HIV-1. We used SRC-family kinase inhibitors or targeted siRNA knockdown of c-SRC and PTK2B, then monitored effects on the early HIV-1 lifecycle. METHODS: Four SRC-family kinase inhibitors or targeted siRNA knockdown were used to reduce c-SRC or PTK2B protein expression. Activated CD4 T-lymphocytes were infected with recombinant, nef-deficient, or replication-competent infectious viruses. Knockdown experiments examined early infection by monitoring: luciferase activity, expression of host surface receptors, reverse transcriptase activity, p24 levels and qPCR of reverse transcripts, integrated HIV-1, and two-long terminal repeat (2-LTR) circles. RESULTS: All SRC-family kinase inhibitors inhibited R5 and X4 HIV-1 infection. Neither c-SRC nor PTK2B siRNA knockdown had an effect on cell surface receptors (CD4, CXCR4, and CCR5) nor on reverse transcriptase activity. However, using JR-FL both decreased luciferase activity while increasing late reverse transcripts (16-fold) and 2-LTR circles (eight-fold) while also decreasing viral integration (four-fold). With HXB2, c-SRC but not PTK2B siRNA knockdown produced similar results. CONCLUSIONS: Our results suggest c-SRC tyrosine kinase is a major regulator of HIV-1 infection, participating in multiple stages of infection post-entry: Reduced proviral integration with increased 2-LTR circles is reminiscent of integrase inhibitors used in combination antiretroviral therapy. Decreasing c-SRC expression and/or activity provides a new target for antiviral intervention and the potential for repurposing existing FDA-approved kinase inhibitors.

Extracellular histones identified in crocodile blood inhibit in-vitro HIV-1 infection.

OBJECTIVE: It has been reported that crocodile blood contains potent antibacterial and antiviral properties. However, its effects on HIV-1 infection remain unknown. DESIGN: We obtained blood from saltwater crocodiles to examine whether serum or plasma could inhibit HIV-1 infection. We purified plasma fractions then used liquid chromatography-mass spectrometry to identify the inhibitory protein factor(s). We then analyzed the ability of recombinant proteins to recapitulate HIV-1 inhibition and determine their mechanism of action. METHODS: Crocodylus porosus plasma was tested for inhibition of Jurkat T-cell HIV-1 infection. Inhibitor(s) were purified by reverse-phase chromatography then identified by protein liquid chromatography-mass spectrometry. Anti-HIV-1 activity of purified plasma or recombinant proteins were measured by p24 enzyme-linked immunosorbent assay and luciferase readouts, and mechanism of action was determined by measuring HIV-1 RNA, cDNA and transcription (using 1G5 cells). RESULTS: Crocodile plasma contains potent inhibitors of HIV-1IIIB infection, which were identified as histones. Recombinant human histones H1 and H2A significantly reduced HIV-1JR-FL infection (IC50 of 0.79 and 0.45 µmol/l, respectively), whereas H4 enhanced JR-FL luciferase activity. The inhibitory effects of crocodile plasma, recombinant H1 or recombinant H2A on HIV-1 infection were during or post-viral transcription. CONCLUSION: Circulating histones in crocodile blood, possibly released by neutrophil extracellular traps, are significant inhibitors of HIV-1 infection in-vitro. Extracellular recombinant histones have different effects on HIV-1 transcription and protein expression and are downregulated in HIV-1 patients. Circulating histones may be a novel resistance factor during HIV-1 infection, and peptide versions should be explored as future HIV-1 therapeutics that modulate viral transcription.

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.

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.

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.

c-Src and Pyk2 protein tyrosine kinases play protective roles in early HIV-1 infection of CD4+ T-cell lines.

BACKGROUND: During early HIV-1 infection of CD4 T-lymphocytes, many host protein tyrosine kinases become activated within minutes, including phosphoprotein pp60 (c-Src) and the focal adhesion kinase family member, proline-rich tyrosine kinase 2 (Pyk2). Whether their activation facilitates or impedes infection remains to be determined. METHODS: c-Src kinase inhibitors (SU6656, PP1, and PP2), adenovectors [wild-type and dominant-negative (DN) c-src] or siRNA (targeting c-src or pyk2) were used to inhibit, compete with or knockdown c-Src in Jurkat C, Jurkat E6-1, Hut 78 or Kit225 T-cell lines. Cells were then infected with HIV-1 luciferase reporter virus expressing VSV-G or HXB2(X4) envelope, and luciferase activity was measured after 2 days. Reverse transcriptase activity and viral cDNA were measured 1 hour after infection, whereas integrated virus was measured 12 hours after infection. RESULTS: Pretreating Jurkat T-cells with SU6656 led to increased VSV-G luciferase activity. In the adenovector experiments, T-cells overexpressing dominant-negative c-Src, but not wild-type c-Src, showed increased luciferase activity after VSV-G infection. siRNA knockdown of c-Src or Pyk2, followed by HXB2 infection in Jurkat T-cells, lead to increased reverse transcriptase activity, viral cDNA, integrated virus, and increased luciferase activity. CONCLUSIONS: Pyk2 is known to interact with c-Src. Thus, Pyk2 activation that coincides with increased c-Src activity during HIV-1 infection could be responsible for c-Src activation. Reduced c-Src activation increases HIV-1 reverse transcription, integration, and/or transcription, suggesting the high c-Src activity seen early in HIV-1 infection may be a cellular response to slow or prevent early infection in CD4 T-cells.

CD4(+) T-cells are unable to express the HIV natural resistance factor globotriosylceramide.

Globotriaosylceramide (Gb(3)) is a cell surface-expressed natural resistance factor for HIV infection, but, its expression in human T-cells remains unknown. Therefore, Gb(3) in resting or activated CD4(+) T-cells was assessed by flow cytometry and thin layer chromatography of cell extracts. We found the majority of CD4(+) T-cells, whether resting or activated, do not express Gb(3) at significant levels (<2% positive cells). Thus, HIV treatment or prevention strategies must focus on development of soluble Gb(3) analogues for inhibition of HIV infection.