Myocardial gene delivery using molecular cardiac surgery with recombinant adeno-associated virus vectors in vivo.

We use a novel technique that allows for closed recirculation of vector genomes in the cardiac circulation using cardiopulmonary bypass, referred to here as molecular cardiac surgery with recirculating delivery (MCARD). We demonstrate that this platform technology is highly efficient in isolating the heart from the systemic circulation in vivo. Using MCARD, we compare the relative efficacy of single-stranded (ss) adeno-associated virus (AAV)6, ssAAV9 and self-complimentary (sc)AAV6-encoding enhanced green fluorescent protein, driven by the constitutive cytomegalovirus promoter to transduce the ovine myocardium in situ. MCARD allows for the unprecedented delivery of up to 48 green fluorescent protein genome copies per cell globally in the sheep left ventricular (LV) myocardium. We demonstrate that scAAV6-mediated MCARD delivery results in global, cardiac-specific LV gene expression in the ovine heart and provides for considerably more robust and cardiac-specific gene delivery than other available delivery techniques such as intramuscular injection or intracoronary injection; thus, representing a potential, clinically translatable platform for heart failure gene therapy.

Biochemical characterization of glucosaminylmuramyldipeptide binding sites of murine macrophages.

By using radioligand analysis, murine peritoneal macrophages were shown to express several hundred high-affinity cell surface GMDP-binding sites (Ka 350 pM). Photoaffinity labeling followed by SDS-PAGE enabled us to identify 32-34 and 38 kDa proteins inside these cells that bound GMDP specifically.

Muramyl peptide-binding sites are located inside target cells.

Using flow cytometry and fluorescence polarization analysis, specific muramyl peptide-binding sites were shown to be located inside T-lymphocytes, macrophages and neuroblastoma cells, but not inside B-cells. No binding sites were found on the cell surface. The number of binding sites for each cell type was determined. Two types of binding sites were observed for myelomonocytic WEHI-3 cells with Kd values of 21 and 540 nM. Inhibition analysis demonstrated that for effective binding, an intact glycopeptide molecule and D-configuration of isoglutamine residue are important.

[Polypeptides from murine peritoneal macrophages recognizing glycosaminylmuramoyl dipeptide]. / Polipeptidy iz peritoneal'nykh makrofagov myshi, uznaiushchikh gliukozaminilmuramoildipeptidy.

By means of radioligand analysis, murine peritoneal macrophages were shown to express several hundreds cell surface high-affinity GMDP-binding sites with a binding constant 350 pM. Photoaffinity labeling followed by SDS-PAGE enabled us to identify inside these cells 32-34 and 38 kDa proteins, specifically binding GMDP. Proteins 32-34 kDa were also detected by Western blotting analysis using biotinylated conjugate of polyacrylamide with immobilized GMDP-Lys [(GMDP-Lys)-PAA-(Bi)] in cell lysate of murine peritoneal macrophages.

[Immunogenic properties of the colloidal gold]. / Immunogennye svoistva kolloidnogo zolota.

We studied the capacity of colloidal gold for enhancing specific and nonspecific immune response in laboratory animals (rabbits, rats, and mice) immunized with antigens of various nature. The antibody titers obtained with colloidal gold as a carrier were higher as compared to the standard immunization techniques (free antigen or Freund's adjuvant). Application of colloidal gold increased nonspecific immune responses as well: lysozyme concentration in the blood, activity of the complement system proteins, as well as phagocytic and bactericidal activities. The obtained antibodies were tested by immunodot assay using gold markers. Immunization of the animals with colloidal gold conjugates with haptens as well as complete antigens was shown to induce formation of highly active antibodies without using other antigens such as complete Freund's adjuvant. In addition, antigen quantities for animal immunization with colloidal gold was by one order of magnitude lower as compared to the complete Freund's adjuvant immunization. This fact can point to direct adjuvant activity of colloidal gold.

Use of dot-blot immunogold assay to identify a proliferative antigen in the initial cells of a wheat stem meristem.

We have devised a protocol for the isolation and identification of a proliferative antigen of the initial cells of wheat stem meristems (termed PAI). We have carried out a variety of immunochemical and immunocytochemical methods, using colloidal gold (CG) complexed with monospecific antibodies to PAI as the marker for the detection of PAI. We have been able to determine the effectiveness of immunoaffinity chromatography in isolating PAI from plant tissues and have shown the advantages of CG over enzyme labels for identification of the antigen. Finally, we have obtained a purified preparation of PAI and have determined its molecular mass (approximately 83 kDa).

Multiple effector functions mediated by human immunodeficiency virus-specific CD4(+) T-cell clones.

Mounting evidence suggests that human immunodeficiency virus type 1 (HIV-1) Gag-specific T helper cells contribute to effective antiviral control, but their functional characteristics and the precise epitopes targeted by this response remain to be defined. In this study, we generated CD4(+) T-cell clones specific for Gag from HIV-1-infected persons with vigorous Gag-specific responses detectable in peripheral blood mononuclear cells. Multiple peptides containing T helper epitopes were identified, including a minimal peptide, VHAGPIAG (amino acids 218 to 226), in the cyclophilin binding domain of Gag. Peptide recognition by all clones examined induced cell proliferation, gamma interferon (IFN-gamma) secretion, and cytolytic activity. Cytolysis was abrogated by concanamycin A and EGTA but not brefeldin A or anti-Fas antibody, implying a perforin-mediated mechanism of cell lysis. Additionally, serine esterase release into the extracellular medium, a marker for cytolytic granules, was demonstrated in an antigen-specific, dose-dependent fashion. These data indicate that T helper cells can target multiple regions of the p24 Gag protein and suggest that cytolytic activity may be a component of the antiviral effect of these cells.