Fourteen of these phage-peptide clones were identified

Fourteen of these phage-peptide clones were identified. using their known HIV-1 sequence and found to inhibit antiCGPIIIa49-66Cinduced platelet oxidation/fragmentation in vitro. Three rabbit antibodies raised against these peptides induced platelet oxidation/fragmentation in vitro and thrombocytopenia in vivo when passively transferred into mice. One of the peptides shared a known epitope region with HIV-1 protein nef and was derived from a variant region of the protein. These data provide strong support for molecular mimicry in HIV-1-immunologic thrombocytopenia within polymorphic regions of HIV-1 proteins. A known epitope of nef is particularly incriminated. Introduction Individuals with early HIV-1 illness generally develop an immunologic thrombocytopenia (HIV-1-ITP)1 with shortened platelet survival.2 Early onset HIV-1-ITP is clinically indistinguishable from vintage autoimmune idiopathic thrombocytopenia (AITP) and responsive to the same therapeutic agents used to treat AITP.1,3-6 However, HIV-1-ITP is different from AITP with respect to markedly elevated platelet-associated IgG, IgM, C3, and C4 and the presence of serum circulating immune complexes (CICs) composed of the same.3,4 These CICs also contain specific high-affinity IgG1 antibody (Ab) against the platelet glycoprotein IIIa (GPIIIa) integrin,7 its anti-idiotype blocking Ab,8 and platelet fragments.9 Unlike classic AITP in which multiple Abs have been explained with different specificities for platelet GPIIb, GPIIIa, and GPIb,6 patients with HIV-1-ITP have an IgG1 Ab against an immunodominant epitope, GPIIIa49-66 (CAPESIEFPVSEAREVLED). This Ab induces thrombocytopenia in mice (mouse GPIIIa offers 83% sequence similarity with human being GPIIIa and macrophages with Fc receptors capable of clearing human being IgG1) and correlates inversely with platelet count in HIV-1-ITP individuals (r =C0.71).7,10 Platelet affinity-purified antiplatelet Ab reacts predominantly with platelet GPIIIa following immunoprecipitation of 125I-labeled surface antigens from a platelet lysate, and more than 85% of platelet-reactive Ab is absorbed onto and eluted from a GPIIIa49-66 affinity column.10 This Ab is particularly unique in that it induces complement-independent platelet fragmentation in vitro and Rabbit polyclonal to HDAC5.HDAC9 a transcriptional regulator of the histone deacetylase family, subfamily 2.Deacetylates lysine residues on the N-terminal part of the core histones H2A, H2B, H3 AND H4. in vivo from the generation of peroxide through a newly explained platelet nicotinamide adenine dinucleotide phosphate (NADPH) oxidase,9 interacting with platelet 12-lipoxygenase to produce 12(S)-hydroxyeicosatetraenoic acid (HETE).11 It is therefore of particular interest to determine how this Abdominal is induced in HIV-1 infection. Molecular mimicry identifies antigen posting between sponsor and parasite. Because of the immunodominant specificity of this Ab we chose to examine whether molecular AZD1152 mimicry might be the mechanism for HIV-1-ITP. Using a filamentous phage surface display 7-mer peptide library we screened for peptides reactive having a rabbit as well as human being antiCGPIIIa49-66 Ab. The 10-mer HIV-1 peptides were synthesized from knowledge of the positive-reactive phage peptides. These peptides were then examined for their ability to inhibit human being antiCGPIIIa49-66Cinduced platelet oxidation/fragmentation and compared to Ab inhibition by its immunodominant epitope, GPIIIa49-66. They were also examined for their ability to raise rabbit Ab capable of inducing platelet fragmentation as well as passive Ab-induced transfer of thrombocytopenia in mice. Materials and methods Materials All reagents were from Sigma-Aldrich (St Louis, MO), unless otherwise indicated. 2,7-Dichlorodihydrofluorescein diacetate (DCFH-DA) was from Molecular Probes (Eugene, OR). The antiCHIV-1 sera immunoblot kit was from Immunetics (Boston, MA). Mice Female Balb/C mice were from Taconic Farms (Germantown, NY). Animal work was authorized by the New York University Medical Center Institutional Review Table. Antibodies Patient sera were from early onset HIV-1Cinfected thrombocytopenic individuals without AIDS and control subjects (healthy laboratory staff). These studies were authorized by the New York University or college Medical Center Institutional Review Table. Rabbit Ab against GPIIIa49-66 was produced as explained previously. Human being Ab was isolated from polyethylene glycol (PEG)Cprecipitable immune complexes,7 and affinity purified on a GPIIIa49-66 affinity column.9 Rabbit Ab was raised against mimicry HIV-1 peptides coupled AZD1152 to KLH or the phage itself. The sera were purified and affinity purified, as explained. Peptides Peptides were synthesized by Bio-synthesis (Lewisville, TX). Peptides not cited in Table 1 are a variant nef (60-71) peptide H19R3 (QEGEEVSFPVK) (accession no. “type”:”entrez-protein”,”attrs”:”text”:”CAA13487″,”term_id”:”3618046″,”term_text”:”CAA13487″CAA13487) and a variant gag (25-30) peptide PHC39 (GKNHYMLKHL) (GenBank accession no. AZD1152 “type”:”entrez-protein”,”attrs”:”text”:”AAL34661″,”term_id”:”17046632″,”term_text”:”AAL34661″AAL34661) as well as a relatively conserved irrelevant env peptide (517-526) Env (GIGALFLGFL) (GenBank accession no. “type”:”entrez-protein”,”attrs”:”text”:”AAQ98126″,”term_id”:”37682428″,”term_text”:”AAQ98126″AAQ98126). Table 1. Panning of PhD 7-phage peptide library PHB22 SIEFPTS None None PHC31 SKLFDEGLFN Envelope glycoprotein PHC32 YAPEFPL GEAPEFPSKQ pol PHC33 None None PHC34 VEFPVSD QEEEEVGFPVT nef protein PHC35 Same as PHC31 Same as PHC31 PHC36 TIIFPVE ELFNKTIIFP Envelope glycoprotein PHC37 Same as PHC31 Same as PHC31 PHC38 KWHWSDS None None PHC39 GKTHYMINPL gag protein HR1 VEFPVSD QEEEEVGFPVT nef protein H19R3 GIIFPVE EDEGIGFPVR nef protein H20R1 LVYPVSE FKLVPVSEAE nef protein H20R4 TPTTNLH SSNTPTTNAA nef protein Open in a separate windowpane GPIIIa49-66: CAPESIEFPVSEARVLED. Boldface type shows close sequence similarity with GPIIIa49-66; italics, related amino acid sequence among different clones; and underlining, homology with.

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