Molecular basis of platelet activation by an alphaIIbbeta3-CHAMPS peptide.

BACKGROUND: A novel method, known as computed helical anti-membrane protein (CHAMP), for the design of peptides that bind with high affinity and selectivity to transmembrane helices was recently described and illustrated using peptides that bind alphaIIb- and alphav-integrin subunits, which induce selective activation of integrins alphaIIbbeta3 and alphavbeta3, respectively. OBJECTIVES: In the present study, we have investigated the ability of an alphaIIb-CHAMPS peptide (termed integrin-activatory-peptide or IAP) to stimulate protein tyrosine phosphorylation and aggregation in human and mouse platelets. METHODS: The ability of IAP to stimulate platelet aggregation and dense granule secretion was measured in washed preparations of human and mouse platelets. Samples were taken for measurement of tyrosine phosphorylation. RESULTS: IAP stimulates robust tyrosine phosphorylation of the tyrosine kinase Syk and the FcR gamma-chain, but only weak phosphorylation of PLCgamma2. Aggregation to low but not high concentrations of IAP is reduced in the presence of the Src kinase inhibitor, PP1, or by inhibitors of the two feedback agonists, ADP and thromboxane A(2) (TxA(2)) suggesting that activation is reinforced by Src kinase-driven release of ADP and TxA(2). Unexpectedly, aggregation by IAP is only partially inhibited in human and mouse platelets deficient in integrin alphaIIbbeta3. Further, IAP induces partial aggregation of formaldehyde-fixed platelets. CONCLUSIONS: The present study demonstrates that the alphaIIb-CHAMPS peptide induces platelet activation through integrin alphaIIbbeta3-dependent and independent pathways with the former mediating tyrosine phosphorylation of FcR gamma-chain and Syk. The use of the alphaIIb-CHAMPS peptide to study integrin alphaIIbbeta3 function is compromised by non-integrin-mediated effects.

Bone marrow stem cell imaging after intracoronary administration.

Although feasibility and safety of autologous stem cells administration to the post-infarction heart has been proven it is not known what proportion of cells effectively do home at the damaged site. Therefore, we have labeled autologous bone marrow cells (ABMC's) by radioactive Indium and single photon emission computed tomography (SPECT) tissue distribution has been analyzed. It was detected that up to 10% of the cells were retained within the myocardium while their majority migrated or has been anchored at the spleen and liver. Comparing the number of homed cells to the total number of cells delivered one may postulate the indirect role for few hundred thousands ABMC's at heart regeneration.

Complex nature of the human antisperm antibody response in SCID mice.

Human peripheral blood mononuclear (PBMs) cells were introduced into the peritoneal cavity of severely-combined immunodeficient (SCID) mice in concentrations of 2.5-4.0 x 10(7) cells per mouse. Whole mononuclear cell suspensions were used either unstimulated or following primary in vitro culture with human spermatozoa. In some experiments, immunodepletion of CD8(+) cells was carried out prior to grafting. Lymphocytes were obtained from nonsensitized (to antigen) human subjects or from individuals who were primed in vivo (vasectomized individuals in case of sperm antigens). An enzyme-linked immunosorbent assay was employed to assess total human immunoglobulin (G or M) levels as well as the specificity of the antibodies generated. We have been successful by generating primary and secondary immune responses with 'naïve' human lymphocytes, challenged with chlamydia or ovalbumin but without adjuvant or CD8(+) immunodepletion; however, we were unable to induce specific antibodies to spermatozoa under this regime in SCID male mice. We then employed female SCID mice, treated with sperm antigen extracts (glycosylated or deglycosylated) encapsulated in liposomes and human lymphocytes obtained from 'naïve' or pre-sensitized in vivo subjects. It was found that the most pronounced humoral response to sperm antigens was obtained with deglycosylated antigens and PBMs from vasectomized (in vivo pre-primed to spermatozoa) individuals. A presented SCID mice model can be helpful at understanding of antisperm antibody development and the molecular nature of generated antibodies to modified sperm antigenic entities.

[Reconstruction of humoral response to sperm antigens in scid mice]. / Rekonstrukcja in situ ludzkiej odpowiedzi humoralnej w myszach z uposledzona odpornoscia wobec mieszaniny antygenów plemnikowych.

OBJECTIVE AND DESIGN: Production of specific human antisperm antibodies by using human-SCID mice model with deposited peripheral blood lymphocytes. MATERIALS AND METHODS: Human peripheral blood lymphocytes (PBL's; CD8(+)-negative cell fraction) were grafted to the peritoneal cavity of severely-combined immunodeficient (SCID) mice at concentration of 20-35 x 10(6) cells per mouse. Lymphocytes were obtained from non-sensitized individual (to sperm antigen) and from in vivo primed males (vasectomized). Two sets of experiments were carried out, with 'native' (glycosylated) and enzymatically deglycosylated sperm antigenic extracts. In all applied variants, sperm antigens were administered with Complete and then with Incomplete Freund adjuvant to improve an immune response. RESULTS AND CONCLUSION: This approach allowed us to obtain better pronounced humoral antisperm response, specific to sperm deglycosylated antigens when PBL's were obtained from individuals in vivo sensitized to sperm (after vasectomy).