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In JoVE (1)
Other Publications (79)
- AIDS (London, England)
- Experimental Hematology
- The Biochemical Journal
- The Journal of Biological Chemistry
- Journal of Clinical Virology : the Official Publication of the Pan American Society for Clinical Virology
- The Journal of Biological Chemistry
- AIDS (London, England)
- Transfusion Medicine Reviews
- Experimental Hematology
- International Immunology
- AIDS (London, England)
- Molecular and Cellular Endocrinology
- International Immunology
- Transfusion Medicine Reviews
- Transfusion and Apheresis Science : Official Journal of the World Apheresis Association : Official Journal of the European Society for Haemapheresis
- Current HIV Research
- Transfusion Medicine Reviews
- Experimental and Toxicologic Pathology : Official Journal of the Gesellschaft Fur Toxikologische Pathologie
- The Journal of Biological Chemistry
- Transfusion Medicine Reviews
- Cancer Biology & Therapy
- Glycoconjugate Journal
- Current Opinion in Hematology
- Transfusion Medicine Reviews
- Viral Immunology
- Discovery Medicine
- Medical Hypotheses
- Journal of Immunological Methods
- PloS One
- Transfusion Medicine Reviews
- Journal of Neuroinflammation
- Experimental Hematology
- PLoS Pathogens
- AIDS (London, England)
- Bioorganic & Medicinal Chemistry Letters
- Leukemia & Lymphoma
- Journal of Acquired Immune Deficiency Syndromes (1999)
- Bioorganic & Medicinal Chemistry
- Journal of Immunology (Baltimore, Md. : 1950)
- Nature Reviews. Immunology
- Endocrine-related Cancer
- PloS One
- Frontiers in Microbiology
- Current Pharmaceutical Design
- PLoS Neglected Tropical Diseases
- AIDS (London, England)
- Current Protocols in Immunology
- AIDS (London, England)
- Autoimmunity Reviews
Articles by Donald R. Branch in JoVE
Use of a Monocyte Monolayer Assay to Evaluate Fcγ Receptor-mediated Phagocytosis
Tik Nga Tong1, Donald R. Branch1,2
1Department of Laboratory Medicine and Pathobiology, University of Toronto, 2Centre for Innovation, Canadian Blood Services
Other articles by Donald R. Branch on PubMed
VPAC1 is a Cellular Neuroendocrine Receptor Expressed on T Cells That Actively Facilitates Productive HIV-1 Infection
AIDS (London, England). Feb, 2002 | Pubmed ID: 11834941
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.
Abnormal Splicing of SHP-1 Protein Tyrosine Phosphatase in Human T Cells. Implications for Lymphomagenesis
Experimental Hematology. Feb, 2003 | Pubmed ID: 12591278
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.
The POU Homeodomain Protein OCT3 As a Potential Transcriptional Activator for Fibroblast Growth Factor-4 (FGF-4) in Human Breast Cancer Cells
The Biochemical Journal. Oct, 2003 | Pubmed ID: 12841847
The POU (representing a homeodomain protein family of which the founder members are Pit-1, Oct-1/2 and Unc-86) homeodomain protein OCT3/Oct-3 (where OCT stands for octamer-binding protein) is an embryonic transcription factor expressed in oocytes, embryonic stem and embryonic carcinoma cells. We have demonstrated previously that human breast cancer cells regain the ability to express OCT3 mRNA [Jin, Branch, Zhang, Qi, Youngson and Goss (1999) Int. J. Cancer 81, 104-112]. Antibodies against human OCT3 were not available when this study was conducted. By using a human OCT3-glutathione S-transferase fusion protein to affinity purify a polyclonal antibody against the mouse Oct-3, we obtained an antibody that enabled us to detect OCT3 in human breast cancer cells by Western-blot analysis. Thus we have now confirmed that OCT3 is expressed in human breast cancer cells but not in normal human breasts and in three other organs. When breast cancer cell lines were treated with all- trans -retinoic acid, OCT3 expression was repressed, associated with decreased cell proliferation. Although another POU protein Brn-3 has been shown to be a repressor for BRCA1 (breast-cancer susceptibility gene 1), OCT3 does not repress human or mouse BRCA1/Brca-1 promoters. However, OCT3 is capable of activating a fusion promoter containing the fibroblast growth factor-4 (FGF-4) enhancer element. In addition, we documented for the first time that human breast cancer cells express FGF-4 protein, and its expression could be inhibited by all- trans -retinoic acid. Furthermore, overexpressing OCT3 stimulated endogenous FGF-4 expression in MCF7 breast cancer cell line. These observations indicate that OCT3 protein is selectively expressed in human breast cancer cells, and its expression may be implicated in mammary gland tumorigenesis via up-regulating FGF-4 expression.
The Journal of Biological Chemistry. Jun, 2004 | Pubmed ID: 15070900
Platelet activation triggers integrin alpha(IIb)beta(3)-dependent signals and the induction of tyrosine phosphorylation of the cytoskeletal protein alpha-actinin. We have previously reported that alpha-actinin is phosphorylated by the focal adhesion kinase (FAK). In this study, a phosphatase of 68 kDa that dephosphorylated alpha-actinin in vitro was isolated from platelet lysates by three sequential chromatography steps. The phosphatase was identified as SHP-1 by electrospray tandem mass spectrometry. alpha-Actinin was dephosphorylated in vitro by recombinant SHP-1 and by SHP-1 immunoprecipitated from unstimulated or thrombin-stimulated platelet lysates. SHP-1 immunoprecipitated from lysates of platelets adherent to fibrinogen, however, failed to dephosphorylate alpha-actinin. In contrast, the activity of SHP-1 against a synthetic substrate was not affected by the mode of platelet activation. The robust and sustained phosphorylation of alpha-actinin detected in platelets adherent to fibrinogen thus correlates with a decrease in the activity of SHP-1 toward it. Tyrosine phosphorylation of alpha-actinin is seen in vanadate-treated COS-7 cells that are co-transfected with alpha-actinin and wild type FAK. Triple transfection of the cells with cDNAs encoding for alpha-actinin, FAK, and wild type SHP-1 abolished the phosphorylation of alpha-actinin. The phosphorylation of FAK, however, was barely affected by the expression of wild type SHP-1. Both alpha-actinin and FAK were phosphorylated in cells co-expressing alpha-actinin, FAK, and a catalytic domain mutant (C453S) of SHP-1. These findings establish that SHP-1 can dephosphorylate alpha-actinin in vitro and in vivo and suggest that SHP-1 may regulate the tethering of receptors to the cytoskeleton and/or the extent of cross-linking of actin filaments in cells such as platelets.
Antibodies Reactive with C-terminus of the Second Conserved Region of HIV-1gp120 As Possible Prognostic Marker and Therapeutic Agent for HIV Disease
Journal of Clinical Virology : the Official Publication of the Pan American Society for Clinical Virology. Dec, 2004 | Pubmed ID: 15567092
It has been reported that antibodies reactive with peptide RSANFTDNAKTIIVQLNQSVEIN (peptide NTM) derived from the C-terminus of the second conserved domain of HIV-1 envelope glycoprotein gp120 could represent an important factor in control of the HIV disease. In order to check this notion we (i) tested reactivity with peptide NTM serum samples collected from 310 consecutive HIV-1 infected patients with a CD4(+) lymphocyte count ranging from 10 to 800/microL and (ii) performed the longitudinal study that included 107 sera samples collected from 29 HIV patients. Results of these studies demonstrated correlation between presence of anti-NTM antibodies in sera of HIV patients and disease progression measured by the CD4(+) cell count. Based on these findings we proposed the anti-NTM antibodies as useful prognostic marker for HIV disease.
The Sulfogalactose Moiety of Sulfoglycosphingolipids Serves As a Mimic of Tyrosine Phosphate in Many Recognition Processes. Prediction and Demonstration of Src Homology 2 Domain/sulfogalactose Binding
The Journal of Biological Chemistry. Apr, 2005 | Pubmed ID: 15634687
Multiple ligand co-recognition of 3'-sulfogalactosylceramide (SGC) and sulfotyrosine initiated the comparison of SGC and sulfotyrosine and, subsequently, phosphotyrosine (pY) binding. SGC is a receptor for ligands involved in cell adhesion/microbial pathology. pY forms a Src homology domain 2 recognition motif in intracellular signaling. Using hsp70, anti-SGC, and anti-pY antibodies, ligand binding is retained following phosphate/sulfate and tyrosine/galactose substitution in SGC and sulfate/phosphate exchange in pY. Remarkable lipid-dependent binding to phosphatidylethanolamine-conjugated sulfotyrosine suggests "microenvironmental" modulation of sulfotyrosine-containing receptors, similar to glycosphingolipids. Based on an aryl substrate-bound co-crystal of arylsulfatase A, a sulfogalactose and phosphotyrosine esterase, modeling provides a solvation basis for co-recognition. c-Src/Src homology domain 2:SGC/phosphogalactosylceramide binding confirms our hypothesis, heralding a carbohydrate-based approach to regulation of phosphotyrosine-mediated recognition.
Chemical Compounds That Target Thiol-disulfide Groups on Mononuclear Phagocytes Inhibit Immune Mediated Phagocytosis of Red Blood Cells
Transfusion. Mar, 2005 | Pubmed ID: 15752156
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
AIDS (London, England). Sep, 2005 | Pubmed ID: 16135910
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.
Transfusion Medicine Reviews. Oct, 2005 | Pubmed ID: 16214019
Experimental Hematology. Oct, 2005 | Pubmed ID: 16219538
To compare the cytotoxicity of KHYG-1 with other natural killer (NK)/NK T-cell lines and identify molecules that may be associated with enhanced cytotoxicity, thereby eventually leading to improved NK cell-mediated cancer immunotherapy.
B Cell Proliferation Following CD40 Stimulation Results in the Expression and Activation of Src Protein Tyrosine Kinase
International Immunology. Feb, 2006 | Pubmed ID: 16415104
Resting normal human B cells express negligible c-src mRNA or Src protein tyrosine kinase; however, upon induction of proliferation, these cells express high levels of both mRNA and protein and show a concomitant increase in tyrosine kinase activity of immunoprecipitated Src. Src expression was most pronounced upon stimulation with CD154, and to a lesser extent CD70, Staphylococcus aureus, Cowan strain I and phorbol ester, and correlated with the activation of the cells. Transfection of cDNA for human wild-type or kinase-dead Src into Raji B cells resulted in an increase and decrease, respectively, of the cell numbers in culture, showing a direct correlation of proliferation to the expression of Src that was corroborated using anti-sense oligodeoxynucleotides and chemical inhibitors. Furthermore, the human B cell lines, Namalwa, Daudi and Raji express low levels of Src but express very high levels of Src after stimulation with CD154 that showed a correlation with increased activation. This is the first report of Src detectable in normal B cells. The finding that Src expression is inducible and correlates with stimulation by CD154 and the proliferation of the B cells suggests that Src may play a specific role in normal and transformed B cell activation/proliferation pathways mediated primarily through CD40 stimulation.
AIDS (London, England). Feb, 2006 | Pubmed ID: 16439866
To determine the effect of a gp120 binding, non-cytotoxic soluble analogue of the glycosphingolipid (GSL), globotriaosyl ceramide (Gb3) on HIV infection in vitro.
Molecular and Cellular Endocrinology. Apr, 2006 | Pubmed ID: 16574312
A number of Hox and Hox-like homeodomain (HD) proteins have been previously shown to utilize members of the TALE HD protein family as co-factors in regulating gene expression. The caudal HD protein Cdx-2 is a transactivator for the proglucagon gene, expressed in pancreatic A cells and intestinal endocrine L cells. We demonstrate here that co-transfection of the TALE homeobox gene Pbx1 enhanced the activation of Cdx-2 on the proglucagon promoter in either the pancreatic A cell line InR1-G9 or BHK fibroblasts. The activation was observed for proglucagon promoter constructs with or without the binding motifs for Pbx1. Furthermore, mutating the penta-peptide motif (binding motif for TALE HD proteins) on Cdx-2 substantially attenuated its activation on proglucagon promoter, but not on the sucrase-isomaltase gene (SI) promoter, or its own (Cdx-2) promoter; suggesting that Cdx-2 utilizes Pbx1 as a co-factor for regulating the expression of selected target genes. Physical interaction between Cdx-2 and Pbx1 was demonstrated by co-immunoprecipitation as well as GST fusion protein pull-down. We suggest that this study reveals a novel function for Pbx1 in pancreatic islet physiology: regulating proglucagon expression by serving as a co-factor for Cdx-2.
International Immunology. Sep, 2006 | Pubmed ID: 16849396
The major mechanism for NK cell lysis of tumor cells is granule-mediated cytotoxicity. Polarization of granules is a prelude to the release of their cytotoxic contents in response to target-cell binding. We describe the novel observation of constitutive granule polarization in the cytotoxic NK cell line, KHYG-1. Continuous degranulation of KHYG-1 cells, however, does not occur and still requires target-cell contact. Disruption of microtubules with colcemid is sufficient to disperse the granules in KHYG-1 and significantly decreases cytotoxicity. A similar effect is not obtained by inhibiting extracellular signal-related kinase 2 (ERK2), the most distal kinase investigated in the cytolytic pathway. Disruption of microtubules significantly down-regulates activation receptors, NKp44 and NKG2D, implicating them as potential microtubule-trafficking receptors. Such changes in upstream receptor expression may have caused deactivation of ERK2, since NKG2D cross-linking also leads to receptor down-regulation and diminished ERK phosphorylation. Thus, a functional role for NKG2D in KHYG-1 cytotoxicity is demonstrated. Moreover, the novel primed state may contribute to the high cytotoxicity exhibited by KHYG-1.
Antenatal Administration of Rh-immune Globulin Causes Significant Increases in the Immunomodulatory Cytokines Transforming Growth Factor-beta and Prostaglandin E2
Transfusion. Aug, 2006 | Pubmed ID: 16934066
Production of specific cytokines in response to administration of Rh-immune globulin (RhIG) was examined to assess the mechanism of inhibition of the anti-D production and prevention of hemolytic disease of the newborn (HDN).
Identification and Characterization of Five-transmembrane Isoforms of Human Vasoactive Intestinal Peptide and Pituitary Adenylate Cyclase-activating Polypeptide Receptors
Genomics. Dec, 2006 | Pubmed ID: 16934434
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.
Transfusion Medicine Reviews. Oct, 2006 | Pubmed ID: 17008167
Detection of Antibodies Reacting with the Antithetical Duffy Blood Group Antigens Fy(a) and Fy(b) Using Recombinant Fusion Proteins Containing the Duffy Extracellular Domain
Transfusion and Apheresis Science : Official Journal of the World Apheresis Association : Official Journal of the European Society for Haemapheresis. Dec, 2006 | Pubmed ID: 17113828
Detecting blood group-specific antibodies in patient sera is essential to the management of blood transfusions or pregnancies. We produced the antithetical forms of the 65 amino acid extracellular domain (ECD) of the Duffy (Fy) blood group protein fused to glutathione sulfotransferase (GST): GST-Fy(a); and GST-Fy(b), differing only in Gly or Asp at position 44, respectively. The purified recombinant proteins were recognized more effectively by reference polyclonal or monoclonal antibodies than the antithetical Fy specificity by either ELISA or immunoblotting. Combined immunoblot and ELISA tests performed at 1:200 dilutions of sera using the recombinant proteins gave results in agreement with undiluted sera and agglutination for 17/19 alloimmunized patients. At 1:200, agglutination detected anti-Fy(a) or anti-Fy(b) in only three of 12 samples that were positive by ELISA. Recombinant ECD-Fy proteins are suitable and sensitive reagents for the detection of anti-Fy that use technology amenable to automation and/or miniaturization and avoid the need for intact red cells.
Virology. May, 2007 | Pubmed ID: 17257640
Successful HIV-1 infection requires a number of specific stages leading to integration of the provirus. We previously suggested that members of the VPAC neuroendocrine receptor family may play a role in HIV-1 infection. We now show that stimulation of the VPAC2 receptor with specific agonists provides strong resistance to HIV-1 infection. Daily stimulation of VPAC2, but not VPAC1 or PAC1, resulted in up to 90% inhibition of X4 or R5 productive infections in either cell lines or PBMCs. VPAC2 agonist stimulation had no effect on cell surface co-receptors, the rate of apoptotic cells, or HIV-1 entry or reverse transcription of viral RNA. However, we provide evidence that VPAC2-specific agonists inhibit HIV-1 infection through an inhibitory effect on the ability of the HIV-1 cDNA to integrate into the host DNA. These data reveal that VPAC2 agonists are appropriate candidates for further study as possible treatments aimed at the amelioration of HIV/AIDS.
Structure-function Studies for in Vitro Chemical Inhibition of Fc Gamma Receptor-mediated Phagocytosis
Transfusion. Feb, 2007 | Pubmed ID: 17302776
Previous studies [Transfusion 2005;45:384] showed that certain chemical compounds containing sulfur-reactive groups can inhibit Fcgamma receptor (FcgammaR)-mediated phagocytosis in vitro. These studies, however, did not prove that only sulfur functionality-induced reactivity was efficacious. In an effort to develop a drug-based approach for the future treatment of immune-mediated cytopenias, these earlier findings have now been extended and this chemically induced interference with FcgammaR-mediated phagocytosis of anti-D-coated red cells (RBCs) was examined to assess the optimal structural requirements for the inhibitory effect.
Chemical Treatment of Anti-D Results in Improved Efficacy for the Inhibition of Fcgamma Receptor-mediated Phagocytosis
Transfusion. Dec, 2007 | Pubmed ID: 17714414
This study investigated whether treatment of immunoglobulins anti-D or intravenous immune globulin (IVIG) with chemicals previously shown to inhibit phagocytosis could result in an enhancement of Fcgamma receptor (FcgammaR) blockade in vitro. If successful, this approach may provide the possibility of targeting these chemicals to monocyte-macrophages for increased efficacy of immunoglobulin-based therapies in vivo.
The Presence of Antibodies Recognizing a Peptide Derived from the Second Conserved Region of HIV-1 Gp120 Correlates with Non-progressive HIV Infection
Current HIV Research. Sep, 2007 | Pubmed ID: 17896963
The C-terminus of the second conserved region of HIV-1 gp120 represents a functionally important domain, as it encompasses amino acids directly involved in the binding to the CD4 receptor and in post-receptor binding events. Previous studies have suggested that antibodies with specific affinity to a 23 amino acids-long NTM polypeptide, derived from this HIV-1 gp120 domain, may be involved in the control of HIV disease progression. In the current work, we searched for NTM-recognizing antibodies in specific cohorts of HIV-1 infected individuals, including long-term nonprogressors (LTNP) and progressors. For this purpose, we employed a previously defined bioinformatics criterion for design of an NTM peptide mimetic to select an octapeptide, NTMs (FTDNAKTI), which is more suitable for use in a solid-state enzyme-linked immunosorbent assay (ELISA). Our results show that NTMs-reactive antibodies are significantly more prevalent (p < 0.01) in LTNP as compared to progressors and healthy control subjects, indicating their association with non-progressive infection. The presence of antibodies recognizing the second conserved region of the HIV-1 gp120 derived peptide, NTMs, in LTNP sera suggest that these antibodies could be of considerable interest for development of anti-HIV immune-based therapies and vaccines.
Transfusion Medicine Reviews. Oct, 2007 | Pubmed ID: 17900493
Experimental and Toxicologic Pathology : Official Journal of the Gesellschaft Fur Toxikologische Pathologie. Mar, 2009 | Pubmed ID: 18771903
A recent report shows a correlation of the historical use of thimerosal in therapeutic immunizations with the subsequent development of autism; however, this association remains controversial. Autism occurs approximately four times more frequently in males compared to females; thus, studies of thimerosal toxicity should take into consideration gender-selective effects. The present study was originally undertaken to determine the maximum tolerated dose (MTD) of thimersosal in male and female CD1 mice. However, during the limited MTD studies, it became apparent that thimerosal has a differential MTD that depends on whether the mouse is male or female. At doses of 38.4-76.8mg/kg using 10% DMSO as diluent, seven of seven male mice compared to zero of seven female mice tested succumbed to thimerosal. Although the thimerosal levels used were very high, as we were originally only trying to determine MTD, it was completely unexpected to observe a difference of the MTD between male and female mice. Thus, our studies, although not directly addressing the controversy surrounding thimerosal and autism, and still preliminary due to small numbers of mice examined, provide, nevertheless, the first report of gender-selective toxicity of thimerosal and indicate that any future studies of thimerosal toxicity should take into consideration gender-specific differences.
Induction of HIV-1 Resistance: Cell Susceptibility to Infection is an Inverse Function of Globotriaosyl Ceramide Levels
Glycobiology. Jan, 2009 | Pubmed ID: 18842961
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.
Blood. May, 2009 | Pubmed ID: 19139081
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.
Transfusion. May, 2009 | Pubmed ID: 19170994
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.
Protein-tyrosine Phosphatase-alpha and Src Functionally Link Focal Adhesions to the Endoplasmic Reticulum to Mediate Interleukin-1-induced Ca2+ Signaling
The Journal of Biological Chemistry. Jul, 2009 | Pubmed ID: 19497848
Calcium (Ca2+) signaling by the pro-inflammatory cytokine interleukin-1 (IL-1) is dependent on focal adhesions, which contain diverse structural and signaling proteins including protein phosphatases. We examined here the role of protein-tyrosine phosphatase (PTP) alpha in regulating IL-1-induced Ca2+ signaling in fibroblasts. IL-1 promoted recruitment of PTPalpha to focal adhesions and endoplasmic reticulum (ER) fractions, as well as tyrosine phosphorylation of the ER Ca2+ release channel IP3R. In response to IL-1, catalytically active PTPalpha was required for Ca2+ release from the ER, Src-dependent phosphorylation of IP3R1 and accumulation of IP3R1 in focal adhesions. In pulldown assays and immunoprecipitations PTPalpha was required for the association of PTPalpha with IP3R1 and c-Src, and this association was increased by IL-1. Collectively, these data indicate that PTPalpha acts as an adaptor to mediate functional links between focal adhesions and the ER that enable IL-1-induced Ca2+ signaling.
Blood Group Antigens and Normal Red Blood Cell Physiology: a Canadian Blood Services Research and Development Symposium
Transfusion Medicine Reviews. Oct, 2009 | Pubmed ID: 19765518
Improved Mouse Models for the Study of Treatment Modalities for Immune-mediated Platelet Destruction
Transfusion. Jun, 2010 | Pubmed ID: 20088841
We found when using a mouse model of immune thrombocytopenia (ITP) that platelet (PLT) nadir could not be maintained in the face of daily PLT antibody, making interpretation of treatment modalities difficult. This finding was documented to be at least in part due to increased thrombopoiesis as a result of a compensated thrombocytolytic state. Thus, it was important to develop an improved mouse model of human ITP so as to maintain PLT nadir over time.
MDM2 Antagonist Nutlin Plus Proteasome Inhibitor Velcade Combination Displays a Synergistic Anti-myeloma Activity
Cancer Biology & Therapy. Jun, 2010 | Pubmed ID: 20418664
Mutliple myeloma (MM) has one of the poorest prognosis of the hematological malignancies and novel therapeutic approaches are needed. Therapeutic induction of p53 might be important to evaluate the drugs either individually or in combination. Direct inhibition of MDM2 function by an MDM2 antagonist nutlin or blocking proteasomal degradation of p53 by a selective proteasome inhibitor velcade can stabilize p53 and activate the p53 apoptotic signaling pathway. We examined if inhibition of p53-MDM2 interaction by nutlin might potentiate the cytotoxic effects of velcade in MM cell lines and primary MM samples. Nutlin or velcade resulted in a reduction in cell proliferation or viability in MM cells harboring wild type p53. Nutlin plus velcade showed a synergistic anti-myeloma activity as evidenced by a significant increase of cytotoxicity with respect to each agonist alone. These effects were accompanied by accumulation of p53 and its two immediate downstream targets, p21 and MDM2, as well as caspase activation and induction of proapoptotic targets, PUMA, BAX and BAK. The induction of p53 target genes induced by nutlin and/or velcade was further validated by gene expression profiling and expression of some selective targets was quantified by qRT-PCR. These preclinical studies provide the framework for clinical trial of nutlin, alone and in combination with conventional and novel therapies such as velcade to increase efficacy and improve patient outcome in MM.
Glycoconjugate Journal. Jul, 2010 | Pubmed ID: 20582467
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.
Current Opinion in Hematology. Nov, 2010 | Pubmed ID: 20739878
Histo-blood group antigens belonging to the P1PK and GLOB blood group systems are involved in bacterial infections, but a substantial body of evidence is emerging that some of these glycosphingolipids play a role in HIV infection. These recent findings have raised additional questions regarding the possible role of the P/Gb3 histo-blood group antigen in HIV-1 infection.
Transfusion-related Acute Lung Injury (TRALI): A Canadian Blood Services Research and Development Symposium
Transfusion Medicine Reviews. Oct, 2010 | Pubmed ID: 20851333
Since the first description of transfusion-related acute lung injury (TRALI) more than 2 decades ago, we have only recently begun to learn how this disorder may occur and how to prevent it. Scientists from around the world have made great strides in identifying the possible causes of this condition. Blood banks and transfusion services have risen to the challenges of prevention. Recent introduction of restricting most plasma products to those obtained from male donors only has greatly reduced the incidence of TRALI worldwide. Scientists have recently identified the gene and protein for the human neutrophil antigen-3a associated with most mortality due to TRALI, and this presents an opportunity for a screening assay to prevent future TRALI-associated deaths. Finally, animal models of TRALI have provided insight into the possible mechanisms of this disorder and can be used to explore potential treatment modalities.
Unexpected Suppression of Anti-Fya and Prevention of Hemolytic Disease of the Fetus and Newborn After Administration of Rh Immune Globulin
Transfusion. Apr, 2011 | Pubmed ID: 20946183
Rh immune globulin (RhIG) has been used successfully for many years for the antenatal suppression of anti-D in D- mothers carrying D+ babies to prevent hemolytic disease of the fetus and newborn. Although the mechanism of RhIG-induced immunosuppression remains unknown, a recent report (TRANSFUSION 2006;46:1316-22) has shown that women receiving RhIG produce elevated levels of transforming growth factor (TGF)β-1, a powerful immunosuppressant cytokine. It was suggested that induction of TGFβ-1 and immunosuppression may be independent of cognate antigen recognition by RhIG. Herein, we present a description of a mother and baby that supports this hypothesis.
A Bioinformatics Approach to Identify Natural Autoantibodies from Healthy Blood Donors' Sera Reactive with the HCV NS5A-derived Peptide by Immunoassay
Viral Immunology. Apr, 2011 | Pubmed ID: 21449717
Natural autoantibodies (NAbs) are continually produced throughout life and have an ability to recognize self and altered self, as well as foreign antigens, by recognizing cellular pattern recognition receptors. Sometimes NAb specificity demonstrates overlap between human and pathologic proteomes. This information can be useful in selecting target sequences for screening purposes. In this study we undertook a multi-step bioinformatics search to predict a virus-derived peptide that can be recognized by NAbs in sera of uninfected individuals. We selected protein hepatitis C virus (HCV) NS5A as a target sequence, motivated by the fact that the HCV proteome is characterized by extensive sequence similarities to the human proteome, and because screening for anti-HCV antibodies, including anti-NS5A, is important clinically, particularly in screening of potential blood donors. The virus-specific peptide P1, and the homologous human peptide derived from enzyme-inducible nitric oxide synthase (iNOS), P2, exhibiting not only simple homology, but also complementarities of physicochemical patterns, were synthesized and 80 HCV-negative and 50 HCV-positive blood donor sera were tested by ELISA. These peptides reacted similarly (p<0.001) with HCV-negative sera, and in several cases the measured reactivity was significantly above the cut-off value of commercial anti-HCV screening assays. In HCV-positive sera, the titers of antibodies reactive with analyzed HCV NS5A peptide were not significantly increased (p<0.001) compared to host peptide, the implications of which are unclear, but may be consistent with these antibodies being "naturally produced." Finally, we extended our bioinformatics analyses to the dataset of human self-binding sequences, and propose a general approach for the selection of specific diagnostic and screening antigens for use in immunoassays.
Discovery Medicine. Apr, 2011 | Pubmed ID: 21524384
Much remains unknown about basic aspects of HIV-1 infection and cell susceptibility. Glycosphingolipid (GSL) binding by the HIV-1 adhesin gp120 has long been implicated in the infection of non-lymphoid cells, as well as CD4(+) T cells and monocytes, the primary targets of HIV-1 infection. We have identified the P(k) blood group antigen (a GSL) globotriaosylceramide (Gb(3)) as a new resistance effector against HIV-1 infection. Significantly, the α-galactosyltransferase (A4GALT, Gb(3) synthase) responsible for the synthesis of Gb(3) is included among markers genetically linked to HIV-1 resistance. Other GSLs, including GalCer and GM3, have been implicated as facilitators of HIV infection. This review will address the role of GSLs in HIV/AIDS but focus on the role of Gb(3) as a newly described natural resistance factor for the prevention of HIV infection and examine potential therapies that would utilize soluble analogues of this unique GSL.
Can Natural Antibodies to VIP or VIP-like HIV-1 Glycoprotein Facilitate Prevention and Supportive Treatment of Breast Cancer?
Medical Hypotheses. Sep, 2011 | Pubmed ID: 21684085
The incidence of non-AIDS-defining cancer is remarkably higher in HIV-infected than in the general population. In contrast, breast cancer risk is significantly reduced in the HIV-infected population. The molecular mechanisms underlying the phenomenon of suppression of breast cancer in the HIV-infected population may serve as a basis for development of a new platform for prevention and treatment of breast cancer.
Journal of Immunological Methods. Aug, 2011 | Pubmed ID: 21726561
The cell surface-expressed glycosphingolipid (GSL), globotriaosylceramide (Gb(3)), is becoming increasingly important and is widely studied in the areas of verotoxin (VT)-mediated cytotoxicity, human immunodeficiency virus (HIV) infection, immunology and cancer. However, despite its diverse roles and implications, an optimized detection method for cell surface Gb(3) has not been determined. GSLs are differentially organized in the plasma membrane which can affect their availability for protein binding. To examine various detection methods for cell surface Gb(3), we compared four reagents for use in flow cytometry analysis. A natural ligand (VT1B) and three different monoclonal antibodies (mAbs) were optimized and tested on various human cell lines for Gb(3) detection. A differential detection pattern of cell surface Gb(3) expression, which was influenced by the choice of reagent, was observed. Two mAb were found to be suboptimal. However, two other methods were found to be useful as defined by their high percentage of positivity and mean fluorescence intensity (MFI) values. Rat IgM anti-Gb(3) mAb (clone 38-13) using phycoerythrin-conjugated secondary antibody was found to be the most specific detection method while the use of VT1B conjugated to Alexa488 fluorochrome was found to be the most sensitive; showing a rare crossreactivity only when Gb(4) expression was highly elevated. The findings of this study demonstrate the variability in detection of Gb(3) depending on the reagent and cell target used and emphasize the importance of selecting an optimal methodology in studies for the detection of cell surface expression of Gb(3).
Physical Activity and Natural Anti-VIP Antibodies: Potential Role in Breast and Prostate Cancer Therapy
PloS One. 2011 | Pubmed ID: 22140573
There is convincing evidence from numerous clinical and epidemiological studies that physical activity can reduce the risk for breast and prostate cancer. The biological mechanisms underlying this phenomenon remain elusive. Herein we suggest a role for naturally produced antibodies reactive with the vasoactive intestinal peptide (VIP) in the suppression of breast and prostate cancer, which we believe could offer a possible molecular mechanism underlying control of these cancers by physical exercise.
Red Blood Cell Storage Lesions and Related Transfusion Issues: a Canadian Blood Services Research and Development Symposium
Transfusion Medicine Reviews. Jan, 2012 | Pubmed ID: 21871777
For centuries, man has been trying to figure out how to revive sick and traumatized individuals using fluids of various types, even from animals. In the 17th century, it was determined that blood was the best fluid to use and, in the early 1900s, after the discovery of the ABO blood groups, human blood was found to provide significant benefit for patients with shock and/or anemia. In the 1950s and 1960s, various ways to obtain, process, and store human blood were developed. It soon became apparent that storage of human blood for transfusion was problematic because red cells, as they aged in vitro, underwent a multitude of physicochemical changes that greatly affected their shelf life, the so-called storage lesion. More recently, the question has arisen as to the potential detrimental effects of the storage lesion and suggestions that older blood may induce increased morbidity and even mortality despite its acceptable in vivo survival. To address this issue of the efficacy and safety of transfusion of aged stored blood, a number of controlled clinical trials have been instituted to determine if older blood is significantly detrimental compared with fresher blood in transfusion recipients.
Sialylation-independent Mechanism Involved in the Amelioration of Murine Immune Thrombocytopenia Using Intravenous Gammaglobulin
Transfusion. Aug, 2012 | Pubmed ID: 22257295
Sialylation of the N-linked glycan on asparagine-297 within the Fc region of intravenous gammaglobulins (IVIgs) was shown to be necessary for efficacy of IVIg in the amelioration of experimental inflammatory arthritis. To test the role for Fc sialylation of IVIg in immune modulating therapies beyond the K/BxN arthritis model, we examined the efficacy of sialylated compared to nonsialylated IVIg for the ability to attenuate immune thrombocytopenia (ITP) in a mouse model that approximates the clinical setting of human ITP.
Mouse Background and IVIG Dosage Are Critical in Establishing the Role of Inhibitory Fcγ Receptor for the Amelioration of Experimental ITP
Blood. May, 2012 | Pubmed ID: 22508937
A recognized paradigm for the therapeutic action of intravenous immunoglobulin (IVIG) in immune thrombocytopenia (ITP) involves up-regulation of the inhibitory Fcγ receptor (FcγRIIB) in splenic macrophages. However, published data have indicated that opposing results are obtained when using FcγRIIB-deficient mice on different strain backgrounds. Herein we show BALB/c FcγRIIB(-/-) and wild-type, with or without spleens, all recover ITP with similar dynamics after IVIG (1 g/kg) treatment; however, this was not the case for C57BL/6 (B6) FcγRIIB(-/-). In investigating this conundrum, we found that wild-type B6 mice are much less sensitive than BALB/c to IVIG-mediated amelioration of ITP, requiring approximately 2- to 2.5-fold more IVIG than BALB/c. When using 2.5 g/kg IVIG in FcγRIIB(-/-) B6 mice, amelioration of ITP was as in wild-type in all animals. Our findings led us to the conclusion that different strains of mice respond differently to IVIG and that FcγRIIB plays no role in the mechanism of effect of IVIG in experimental ITP.
Evaluating the Role of IL-11, a Novel Cytokine in the IL-6 Family, in a Mouse Model of Spinal Cord Injury
Journal of Neuroinflammation. Jun, 2012 | Pubmed ID: 22715999
Spinal cord injury (SCI) is a devastating condition with substantial functional and social morbidity. Previous research has established that the neuroinflammatory response plays a significant role in cord damage post-SCI. However, global immunosuppressive therapies have demonstrated mixed results. As a result, more specific therapies modulating inflammation after injury are needed. In this regard, research into cytokine signaling has demonstrated that cytokines of the gp130 family including IL-6 and leukemia inhibitory factor (LIF) play key roles in mediating damage to the spinal cord. Since members of the gp130 family all share a common signal transduction pathway via the JAK/STAT system, we performed the first study of a relatively new member of the gp130 family, IL-11, in SCI.
Experimental Hematology. Dec, 2012 | Pubmed ID: 22960265
SHP-1, encoded by the PTPN6 gene, is a protein tyrosine phosphatase with two src-homology-2 (SH2) domains that is implicated as providing suppression of hematopoietic malignancies. A number of reports have shown protein-protein interactions between SHP-1 SH2 domains and tyrosine-phosphorylated proteins. However, despite its having three proline-rich, potential SH3-binding motifs, no reports of protein-protein interactions through src-homology-3 (SH3)-binding domains with SHP-1 have been described. Herein we show that the SH3 domain-containing CT10 regulator of kinase-like (CrkL) adaptor protein associates with SHP-1. We also provide results that suggest this association is due to CrkL binding to PxxP domains located at amino acid residues 158-161 within the SHP-1 C-terminal SH2 domain, and amino acid residues 363-366 within its phosphatase domain. This study is the first to identify and define an interaction between SHP-1 and an SH3 domain-containing protein. Our findings provide an alternative way that SHP-1 can be linked to potential substrates.
Immunotherapy. Sep, 2012 | Pubmed ID: 23046234
Evaluation of: Stephen J, Cairns LS, Pickford WJ, Vickers MA, Urbaniak SJ, Barker RN. Identification, immunomodulatory activity and immunogenicity of the major helper T cell epitope on the K blood group antigen. Blood 119(23), 5563-5574 (2012). Alloimmunization to blood group antigens is a major concern in transfusion medicine. This occurs when antigen-mismatched blood is transfused into a recipient lacking a red blood cell antigen that is expressed on the donor red blood cells. Alloimmunization in this case can result in future problems in finding compatible blood for transfusion and can cause hemolytic transfusion reactions. Alloimmunization can also occur in instances where a mother lacks a red blood cell antigen that is carried by the fetus. In these cases, alloimmunization can result in an antibody that can cross the placenta and cause moderate-to-severe problems in the fetus or newborn due to hemolytic anemia and/or inhibition of hematopoiesis. This is called hemolytic disease of the fetus and newborn. Stephen et al. describe a unique approach to producing a peptide tolerogen to prevent alloimmunization to a specific blood group antigen, K, in the Kell blood group system. They identify an immunodominant K peptide and use this peptide to show that it strongly stimulates human T helper cells from K-immunized people in vitro and that it shows efficacy when used as a nasal tolerogen to suppress immunization with K protein in a mouse model. These results open the door for therapies aimed at the prevention and/or treatment of alloimmunization in both a transfusion setting and, importantly, in hemolytic disease of the fetus and newborn.
ABO Blood Groups Influence Macrophage-mediated Phagocytosis of Plasmodium Falciparum-infected Erythrocytes
PLoS Pathogens. 2012 | Pubmed ID: 23071435
Erythrocyte polymorphisms associated with a survival advantage to Plasmodium falciparum infection have undergone positive selection. There is a predominance of blood group O in malaria-endemic regions, and several lines of evidence suggest that ABO blood groups may influence the outcome of P. falciparum infection. Based on the hypothesis that enhanced innate clearance of infected polymorphic erythrocytes is associated with protection from severe malaria, we investigated whether P. falciparum-infected O erythrocytes are more efficiently cleared by macrophages than infected A and B erythrocytes. We show that human macrophages in vitro and mouse monocytes in vivo phagocytose P. falciparum-infected O erythrocytes more avidly than infected A and B erythrocytes and that uptake is associated with increased hemichrome deposition and high molecular weight band 3 aggregates in infected O erythrocytes. Using infected A(1), A(2), and O erythrocytes, we demonstrate an inverse association of phagocytic capacity with the amount of A antigen on the surface of infected erythrocytes. Finally, we report that enzymatic conversion of B erythrocytes to type as O before infection significantly enhances their uptake by macrophages to observed level comparable to that with infected O wild-type erythrocytes. These data provide the first evidence that ABO blood group antigens influence macrophage clearance of P. falciparum-infected erythrocytes and suggest an additional mechanism by which blood group O may confer resistance to severe malaria.
Toxins. Dec, 2012 | Pubmed ID: 23242319
Our previous genetic, pharmacological and analogue protection studies identified the glycosphingolipid, Gb(3) (globotriaosylceramide, Pk blood group antigen) as a natural resistance factor for HIV infection. Gb(3) is a B cell marker (CD77), but a fraction of activated peripheral blood mononuclear cells (PBMCs) can also express Gb(3). Activated PBMCs predominantly comprise CD4+ T-cells, the primary HIV infection target. Gb(3) is the sole receptor for Escherichia coli verotoxins (VTs, Shiga toxins). VT1 contains a ribosome inactivating A subunit (VT1A) non-covalently associated with five smaller receptor-binding B subunits. The effect of VT on PHA/IL2-activated PBMC HIV susceptibility was determined. Following VT1 (or VT2) PBMC treatment during IL2/PHA activation, the small Gb(3)+/CD4+ T-cell subset was eliminated but, surprisingly, remaining CD4+ T-cell HIV-1(IIIB) (and HIV-1(Ba-L)) susceptibility was significantly reduced. The Gb(3)-Jurkat T-cell line was similarly protected by brief VT exposure prior to HIV-1(IIIB) infection. The efficacy of the VT1A subunit alone confirmed receptor independent protection. VT1 showed no binding or obvious Jurkat cell/PBMC effect. Protective VT1 concentrations reduced PBMC (but not Jurkat cell) proliferation by 50%. This may relate to the mechanism of action since HIV replication requires primary T-cell proliferation. Microarray analysis of VT1A-treated PBMCs indicated up regulation of 30 genes. Three of the top four were histone genes, suggesting HIV protection via reduced gene activation. VT blocked HDAC inhibitor enhancement of HIV infection, consistent with a histone-mediated mechanism. We speculate that VT1A may provide a benign approach to reduction of (X4 or R5) HIV cell susceptibility.
AIDS (London, England). Mar, 2013 | Pubmed ID: 23380967
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.
Bioorganic & Medicinal Chemistry Letters. Apr, 2013 | Pubmed ID: 23489619
Immune thrombocytopenia (ITP) is caused by production of an autoantibody to autologous platelets. ITP can be treated either by reducing platelet destruction or by increasing platelet production. Fcγ receptor mediated phagocytosis of the opsonized blood cells is a well-accepted mechanism for the underlying pathogenesis of ITP and inhibition of this phagocytosis process with small molecules is a potential strategy for the development of drugs against ITP. A broad screen indicated that 4-methyl-1-phenyl-pyrazole derivative (1) could inhibit the phagocytosis of opsonized blood cells with weak potency. We reveal here the discovery of the polysulfide products, synthesis of various 1-phenyl-pyrazole derivatives, and the biological evaluation of pyrazole derivatives as inhibitors of phagocytosis for potential use as therapeutics for ITP. Substitution at C4 of the pyrazole moiety in the disulfide-bridged dimers influenced the potency in the increasing order of 10 ~/= 11~/= 16 < 19 < 20. A novel scaffold, 20 with an IC(50) of 100 nM inhibiting opsonized blood cell phagocytosis was identified as a potential candidate for further studies. Confirmation of the disulfide bridge additionally provides clues for the non-thiol or non-disulfide bridge carrying ligands targeting ITP and other similar disorders.
Characterization of SHP-1 Protein Tyrosine Phosphatase Transcripts, Protein Isoforms and Phosphatase Activity in Epithelial Cancer Cells
Genomics. Nov-Dec, 2013 | Pubmed ID: 24100145
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.
Acute Hemolysis After Intravenous Immunoglobulin Amid Host Factors of ABO-mismatched Bone Marrow Transplantation, Inflammation, and Activated Mononuclear Phagocytes
Transfusion. Mar, 2014 | Pubmed ID: 23829192
Hemolysis may follow intravenous immunoglobulin (IVIG), with product, dosing, and host factors contributing. The importance of recipient features remains unclear.
Isolated Bone Marrow Hodgkin Lymphoma in a Human Immunodeficiency Virus-negative Patient: a Second Case
Leukemia & Lymphoma. Jul, 2014 | Pubmed ID: 24070425
C-Src and Pyk2 Protein Tyrosine Kinases Play Protective Roles in Early HIV-1 Infection of CD4+ T-cell Lines
Journal of Acquired Immune Deficiency Syndromes (1999). Jun, 2014 | Pubmed ID: 24413044
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.
Structure-activity Relationships of Pyrazole Derivatives As Potential Therapeutics for Immune Thrombocytopenias
Bioorganic & Medicinal Chemistry. May, 2014 | Pubmed ID: 24685704
Idiopathic or immune thrombocytopenia (ITP) is a serious clinical disorder involving the destruction of platelets by macrophages. Small molecule therapeutics are highly sought after to ease the burden on current therapies derived from human sources. Earlier, we discovered that dimers of five-membered heterocycles exhibited potential to inhibit phagocytosis of human RBCs by macrophages. Here, we reveal a structure-activity relationship of the bis-pyrazole class of molecules with -C-C-, -C-N- and -C-O- linkers, and their evaluation as inhibitors of phagocytosis of antibody-opsonized human RBCs as potential therapeutics for ITP. We have uncovered three potential candidates, 37, 47 and 50, all carrying a different linker connecting the two pyrazole moieties. Among these compounds, hydroxypyrazole derivative 50 is the most potent compound with an IC50 of 14 ± 9 μM for inhibiting the phagocytosis of antibody-opsonized human RBCs by macrophages. None of the compounds exhibited significant potential to induce apoptosis in peripheral blood mononuclear cells (PBMCs). Current study has revealed specific functional features, such as up to 2-atom spacer arm and alkyl substitution at one of the N(1) positions of the bivalent pyrazole core to be important for the inhibitory activity.
Therapeutic Effect of IVIG on Inflammatory Arthritis in Mice is Dependent on the Fc Portion and Independent of Sialylation or Basophils
Journal of Immunology (Baltimore, Md. : 1950). Jun, 2014 | Pubmed ID: 24760152
High-dose i.v. Ig (IVIG) is used to treat various autoimmune and inflammatory diseases; however, the mechanism of action remains unclear. Based on the K/BxN serum transfer arthritis model in mice, IVIG suppression of inflammation has been attributed to a mechanism involving basophils and the binding of highly sialylated IgG Fc to DC-SIGN-expressing myeloid cells. The requirement for sialylation was examined in the collagen Ab-induced arthritis (CAbIA) and K/BxN serum transfer arthritis models in mice. High-dose IVIG (1-2 g/kg body weight) suppressed inflammatory arthritis when given prophylactically. The same doses were also effective in the CAbIA model when given subsequent to disease induction. In this therapeutic CAbIA model, the anti-inflammatory effect of IVIG was dependent on IgG Fc but not F(ab')2 fragments. Removal of sialic acid residues by neuraminidase had no impact on the anti-inflammatory activity of IVIG or Fc fragments. Treatment of mice with basophil-depleting mAbs did not abrogate the suppression of either CAbIA or K/BxN arthritis by IVIG. Our data confirm the therapeutic benefit of IVIG and IgG Fc in Ab-induced arthritis but fail to support the significance of sialylation and basophil involvement in the mechanism of action of IVIG therapy.
Nature Reviews. Immunology. May, 2014 | Pubmed ID: 24762829
Relaxin Receptor Antagonist AT-001 Synergizes with Docetaxel in Androgen-independent Prostate Xenografts
Endocrine-related Cancer. Jun, 2014 | Pubmed ID: 24812057
Androgen hormones and the androgen receptor (AR) pathway are the main targets of anti-hormonal therapies for prostate cancer. However, resistance inevitably develops to treatments aimed at the AR pathway resulting in androgen-independent or hormone-refractory prostate cancer (HRPC). Therefore, there is a significant unmet need for new, non-androgen anti-hormonal strategies for the management of prostate cancer. We demonstrate that a relaxin hormone receptor antagonist, AT-001, an analog of human H2 relaxin, represents a first-in-class anti-hormonal candidate treatment designed to significantly curtail the growth of androgen-independent human prostate tumor xenografts. Chemically synthesized AT-001, administered subcutaneously, suppressed PC3 xenograft growth by up to 60%. AT-001 also synergized with docetaxel, standard first-line chemotherapy for HRPC, to suppress tumor growth by more than 98% in PC3 xenografts via a mechanism involving the downregulation of hypoxia-inducible factor 1 alpha and the hypoxia-induced response. Our data support developing AT-001 for clinical use as an anti-relaxin hormonal therapy for advanced prostate cancer.
Cytokine Profiles in Mouse Models of Experimental Immune Thrombocytopenia Reveal a Lack of Inflammation and Differences in Response to Intravenous Immunoglobulin Depending on the Mouse Strain
Transfusion. Nov, 2014 | Pubmed ID: 24826802
Mouse models of human immune thrombocytopenia (ITP) have been used for years to investigate the mechanism of intravenous immunoglobulin (IVIG) to ameliorate ITP; however, how closely these experimental mouse models mirror the human autoimmune inflammatory disease is unclear. The aim of this study was to assess the cytokine profiles in experimental ITP with and without IVIG treatment.
Optimal Attenuation of Experimental Autoimmune Encephalomyelitis by Intravenous Immunoglobulin Requires an Intact Interleukin-11 Receptor
PloS One. 2014 | Pubmed ID: 25078447
Intravenous immunoglobulin (IVIg) has been used to treat a variety of autoimmune disorders including multiple sclerosis (MS); however its mechanism of action remains elusive. Recent work has shown that interleukin-11 (IL-11) mRNAs are upregulated by IVIg in MS patient T cells. Both IVIg and IL-11 have been shown to ameliorate experimental autoimmune encephalomyelitis (EAE), an animal model of MS. The objective of this study was to determine whether the protective effects of IVIg in EAE occur through an IL-11 and IL-11 receptor (IL-11R)-dependent mechanism.
Virtual Screen for Repurposing Approved and Experimental Drugs for Candidate Inhibitors of EBOLA Virus Infection
F1000Research. 2015 | Pubmed ID: 25717373
The ongoing Ebola virus epidemic has presented numerous challenges with respect to control and treatment because there are no approved drugs or vaccines for the Ebola virus disease (EVD). Herein is proposed simple theoretical criterion for fast virtual screening of molecular libraries for candidate inhibitors of Ebola virus infection. We performed a repurposing screen of 6438 drugs from DrugBank using this criterion and selected 267 approved and 382 experimental drugs as candidates for treatment of EVD including 15 anti-malarial drugs and 32 antibiotics. An open source Web server allowing screening of molecular libraries for candidate drugs for treatment of EVD was also established.
Frontiers in Microbiology. 2015 | Pubmed ID: 25745423
The worst Ebola virus (EV) outbreak in history has hit Liberia, Sierra Leone and Guinea hardest and the trend lines in this crisis are grave, and now represents a global public health threat concern. Limited therapeutic and/or prophylactic options are available for people suffering from Ebola virus disease (EVD) and further complicate the situation. Previous studies suggested that the EV glycoprotein (GP) is the main determinant causing structural damage of endothelial cells that triggers the hemorrhagic diathesis, but molecular mechanisms underlying this phenomenon remains elusive. Using the informational spectrum method (ISM), a virtual spectroscopy method for analysis of the protein-protein interactions, the interaction of GP with endothelial extracellular matrix (ECM) was investigated. Presented results of this in silico study suggest that Elastin Microfibril Interface Located Proteins (EMILINs) are involved in interaction between GP and ECM. This finding could contribute to a better understanding of EV/endothelium interaction and its role in pathogenesis, prevention and therapy of EVD.
Current Pharmaceutical Design. 2015 | Pubmed ID: 25777762
Immune thrombocytopenia or ITP is a debilitating and life-threatening disorder affecting more than 4 in every 10, 000 adults annually. Following a basic understanding of the immunopathology underlying ITP, namely that production of anti-platelet antibodies results in accelerated platelet clearance and thrombocytopenia, animal models of ITP were quickly developed. Rodent models that develop ITP spontaneously or by passive transfer of anti-platelet sera or antibodies have become instrumental in investigating the mechanisms responsible for the breakdown of tolerance in human ITP, understanding the immunopathology that underlies the progression of platelet destruction, elucidating the mechanism(s) of therapeutic amelioration of the ITP, and driving the development of new therapeutic modalities. This review aims to capture the development history and methodology of currently available ITP disease models, and review their advantages and limitations in the study of various aspects of ITP. We also review how closely the various ITP models reflect the pathobiology of human ITP and their usefulness in advancing the development of new therapeutics, which are particularly needed to address the unmet need of patients who are refractory to the currently available repertoire of interventions.
Western Immunoblotting As a New Tool for Investigating Direct Antiglobulin Test-negative Autoimmune Hemolytic Anemias
Transfusion. Jun, 2015 | Pubmed ID: 25808506
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.
In Silico Analysis Suggests Repurposing of Ibuprofen for Prevention and Treatment of EBOLA Virus Disease
F1000Research. 2015 | Pubmed ID: 26167272
The large 2014/2015 Ebola virus outbreak in West Africa points out the urgent need to develop new preventive and therapeutic approaches that are effective against Ebola viruses and can be rapidly utilized. Recently, a simple theoretical criterion for the virtual screening of molecular libraries for candidate inhibitors of Ebola virus infection was proposed. Using this method the 'drug space' was screened and 267 approved and 382 experimental drugs as candidates for treatment of the Ebola virus disease (EVD) have been selected. Detailed analysis of these drugs revealed the non-steroidal anti-inflammatory drug ibuprofen as an inexpensive, widely accessible and minimally toxic candidate for prevention and treatment of EVD. Furthermore, the molecular mechanism underlying this possible protective effect of ibuprofen against EVD is suggested in this article.
Transfusion. Jul, 2015 | Pubmed ID: 26174900
Intravenous immune globulin (IVIG) therapy has shown great success in a number of autoimmune and inflammatory conditions and its use continues to increase worldwide. There is growing awareness of significant side effects of high-dose IVIG: however, particularly severe hemolysis in patients that are non-group O. It has been proposed that IVIG-associated hemolysis may be heralded by an existing inflammatory condition. In the work presented herein, we have provided a review of the pathophysiology of inflammation, particularly as it applies in immune-mediated red blood cell hemolysis, and a summary of previous publications that suggest an association between IVIG-mediated hemolysis and a state of existing inflammation. In addition, preliminary results from a prospective study to address the mechanism of IVIG-associated hemolysis are provided. These preliminary data support the idea of an existing inflammatory condition preceding overt hemolysis after high-dose IVIG therapy that: 1) is restricted to non-group O patients, 2) is seen when using IVIG doses of more than 2 g/kg, 3) involves an activated mononuclear phagocyte system, 4) may be presaged by a significant increase in the anti-inflammatory cytokine interleukin-1 receptor agonist, and 5) is independent of secretor status.
Transfusion. Jul, 2015 | Pubmed ID: 26174901
Intravenous immunoglobulin (IVIG) is made from thousands of donors having a variety of blood groups. All of the donors being used for IVIG production, with the exception of group AB donors, have in their plasma antibodies of variable titer commonly known as isohemagglutinins or ABO antibodies. As blood groups O and A are the most commonly found in the world population, most of the plasma used in IVIG production is from donors having these blood groups, with group B and group AB donors being fewer in number. Consequently, all batches of IVIG contain antibodies that are reactive with individuals of group A, group B, and group AB. These antibodies were originally discovered by Dr Karl Landsteiner in the early 1900s and are now known to consist of immunoglobulin (Ig)M, IgG, and IgA classes. As the process for producing IVIG results in almost exclusively IgG, isohemagglutinins contained in IVIG are of this immunoglobulin class. ABO antibodies are highly clinically significant and, because of this, blood bank cross-matching is done to ensure that blood of the correct type is transfused into recipients to avoid a so-called major mismatch or major incompatibility that can cause significant morbidity and often death. Administration of IVIG, which contains ABO antibodies, is often infused into individuals who have the corresponding ABO antigens, commonly called a minor mismatch, and although not as significant as a major mismatch, the isohemagglutinins contained in the IVIG have some risk for a significant transfusion reaction due to the ABO incompatibility. Indeed, currently there is no way to match IVIG to recipients according to blood type, so when IVIG is administered to group A, B, or AB recipients, there is potential for transfusion reactions analogous to a blood transfusion mismatch. For this reason, strict guidelines have been put into place to restrict the titers of the ABO antibodies contained in IVIG. This review will provide background information about the discovery and biochemistry of the ABO antigens and discuss the various isohemagglutinins that are found in plasma of the different ABO blood types and their potential clinical significance. In addition, a brief discussion of the controversial topic of the origins of these antibodies will conclude this review.
A Rapid Screening Assay Identifies Monotherapy with Interferon-ß and Combination Therapies with Nucleoside Analogs As Effective Inhibitors of Ebola Virus
PLoS Neglected Tropical Diseases. Jan, 2016 | Pubmed ID: 26752302
To date there are no approved antiviral drugs for the treatment of Ebola virus disease (EVD). While a number of candidate drugs have shown limited efficacy in vitro and/or in non-human primate studies, differences in experimental methodologies make it difficult to compare their therapeutic effectiveness. Using an in vitro model of Ebola Zaire replication with transcription-competent virus like particles (trVLPs), requiring only level 2 biosafety containment, we compared the activities of the type I interferons (IFNs) IFN-α and IFN-ß, a panel of viral polymerase inhibitors (lamivudine (3TC), zidovudine (AZT) tenofovir (TFV), favipiravir (FPV), the active metabolite of brincidofovir, cidofovir (CDF)), and the estrogen receptor modulator, toremifene (TOR), in inhibiting viral replication in dose-response and time course studies. We also tested 28 two- and 56 three-drug combinations against Ebola replication. IFN-α and IFN-ß inhibited viral replication 24 hours post-infection (IC50 0.038μM and 0.016μM, respectively). 3TC, AZT and TFV inhibited Ebola replication when used alone (50-62%) or in combination (87%). They exhibited lower IC50 (0.98-6.2μM) compared with FPV (36.8μM), when administered 24 hours post-infection. Unexpectedly, CDF had a narrow therapeutic window (6.25-25μM). When dosed >50μM, CDF treatment enhanced viral infection. IFN-ß exhibited strong synergy with 3TC (97.3% inhibition) or in triple combination with 3TC and AZT (95.8% inhibition). This study demonstrates that IFNs and viral polymerase inhibitors may have utility in EVD. We identified several 2 and 3 drug combinations with strong anti-Ebola activity, confirmed in studies using fully infectious ZEBOV, providing a rationale for testing combination therapies in animal models of lethal Ebola challenge. These studies open up new possibilities for novel therapeutic options, in particular combination therapies, which could prevent and treat Ebola infection and potentially reduce drug resistance.
AIDS (London, England). Mar, 2016 | Pubmed ID: 26807966
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).
Current Protocols in Immunology. Apr, 2016 | Pubmed ID: 27038460
Immune thrombocytopenia (ITP) is a debilitating, life-threatening autoimmune disorder affecting more than 4 in every 100,000 adults annually, stemming from the production of antiplatelet antibody resulting in accelerated platelet destruction and thrombocytopenia. Numerous animal models of ITP have been developed that contributed to the basic understanding of the underlying mechanisms of ITP onset, progression, and maintenance. Rodent models that develop ITP spontaneously, or by passive transfer of an antiplatelet sera or antibody, play an instrumental role in the investigation of ITP mechanisms responsible for the breakdown of tolerance in human ITP, in studies of the immunopathology underlying the progression of platelet destruction, and in elucidation of the mechanisms of therapeutic amelioration of ITP by existing and new therapeutic modalities. This unit captures the protocols for the implementation and readout of passive antibody transfer mouse models of ITP, established by the infusion of a commercially-available monoclonal rat anti-mouse CD41 platelet antibody.
AIDS (London, England). Aug, 2016 | Pubmed ID: 27191975
It has been reported that crocodile blood contains potent antibacterial and antiviral properties. However, its effects on HIV-1 infection remain unknown.
Autoimmunity Reviews. Aug, 2016 | Pubmed ID: 27296447
Immune cytopenias are conditions characterized by low blood cell counts, such as platelets in immune thrombocytopenia (ITP) and red blood cells in autoimmune hemolytic anemia (AIHA). Chronic ITP affects approximately 4 in 100,000 adults annually while AIHA is much less common. Extravascular phagocytosis and massive destruction of autoantibody-opsonized blood cells by macrophages in the spleen and liver are the hallmark of these conditions. Current treatment modalities for ITP and AIHA include the first-line use of corticosteroids; whereas, IVIg shows efficacy in ITP but not AIHA. One main mechanism of action by which IVIg treatment leads to the reduction in platelet destruction rates in ITP is thought to involve Fcγ receptor (FcγR) blockade, ultimately leading to the inhibition of extravascular platelet phagocytosis. IVIg, which is manufactured from the human plasma of thousands of donors, is a limited resource, and alternative treatments, particularly those based on bioavailable small molecules, are needed. In this review, we overview the pathophysiology of ITP, the role of Fcγ receptors, and the mechanisms of action of IVIg in treating ITP, and outline the efforts and progress towards developing novel, first-in-class inhibitors of phagocytosis as synthetic, small molecule substitutes for IVIg in ITP and other conditions where the pathobiology of the disease involves phagocytosis.
Transfusion. Aug, 2016 | Pubmed ID: 27546234
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.
Blood. Sep, 2016 | Pubmed ID: 27688803
RBC alloimmunization is a serious complication of transfusion or pregnancy. Despite the widespread use of RhIg to prevent pregnancy associated anti-D alloimmunization, its mechanism of action remains elusive. We have previously described a murine model in which immunoprophylaxis with polyclonal anti-KEL sera prevents alloimmunization in wild type recipients transfused with transgenic murine RBCs expressing the human KEL glycoprotein. To investigate the mechanism of action, we have now evaluated the outcome of immunoprophylaxis treatment in mice lacking Fcγ receptors (FcγR), complement (C3), both, or none. Whereas polyclonal anti-KEL sera completely prevented alloimmunization in wild type and single knock-out (KO) mice lacking FcγR or C3, double KO mice lacking both FcγR and C3 became alloimmunized despite immunoprophylaxis. Rapid clearance of essentially all transfused RBCs with detectable KEL glycoprotein antigen occurred within 24 hours in wild type and single KO recipients treated with immunoprophylaxis, with the transfused RBCs remaining in circulation having minimal KEL glycoprotein antigen detectable by flow cytometry or western blot. In contrast, transfused RBCs with the KEL glycoprotein antigen fully intact continued to circulate for days in double KO mice despite treatment with immunoprophylaxis. Further, in-vitro phagocytosis assays showed no consumption of opsonized murine RBCs by double KO splenocytes. Taken in combination, our data suggest that modulation of the KEL antigen (and potentially RBC clearance) by redundant recipient pathways involving both FcγR and C3 may be critical to the mechanism of action of polyclonal anti-KEL immunoprophylaxis. These findings could have implications for the development of immunoprophylaxis programs in humans.
Virology. Dec, 2016 | Pubmed ID: 28013103
Stress granules (SGs) are dynamic cytoplasmic aggregates of translationally silenced mRNAs that assemble in response to environmental stress. SGs appear to play an important role in antiviral innate immunity and many viruses have evolved to block or subvert SGs components for their own benefit. Here, we demonstrate that intracellular Ebola virus (EBOV) replication and transcription-competent virus like particles (trVLP) infection does not lead to SG assembly but leads to a blockade to Arsenite-induced SG assembly. Moreover we show that EBOV VP35 represses the assembly of canonical and non-canonical SGs induced by a variety of pharmacological stresses. This SG blockade requires, at least in part, the C-terminal domain of VP35. Furthermore, results from our co-immunoprecipitation studies indicate that VP35 interacts with multiple SG components, including G3BP1, eIF3 and eEF2 through a stress- and RNA-independent mechanism. These data suggest a novel function for EBOV VP35 in the repression of SG assembly.