Heat shock proteins purified from autologous tumors using antibody-based affinity chromatography.

Heat shock proteins (HSP) isolated from autologous tumors have become a promising tool for active-specific anticancer immunotherapy due to their properties as carriers of antigenic peptides on one hand and as immunostimulatory adjuvants on the other. Rapid and efficient isolation of HSP-peptide complexes from a patient's tumor is fundamental for their clinical application. Herein, we describe the purification of the HSP Gp96 and Hsc70/Hsp70 from human autologous tumor sources by one-step antibody-based affinity chromatography. Recombinant anti-Gp96 and anti-Hsp70 single-chain Fv antibodies are covalently coupled to a chromatographic bead resin to obtain highly specific affinity matrices. Chromatographic columns are assembled and then used to simultaneously isolate various HSP from the supernatant of lysates of human tumor samples of different origin in a single chromatographic step.

Induction of therapeutic neoangiogenesis using in vitro-generated endothelial colony-forming cells: an autologous transplantation model in rat.

BACKGROUND: Accumulating evidence shows the potential of bone marrow-derived endothelial colony-forming cells (bmECFCs) as promising tools for vascular repair. However, knowledge about their in vitro expansion, characterization, and functional behavior is still controversial. We demonstrate the in vitro generation of rat bmECFCs and analyze their ability to promote tissue reperfusion in a chronic hind-limb ischemia model. METHODS: Either in vitro-generated and characterized autologous bmECFCs or placebo was injected into ischemic hind limbs of Sprague-Dawley rats. Tissue perfusion was quantified by laser Doppler, in perfusion units (PU), at days 0, 15, and 30. RESULTS: Rat bmECFCs acquire a typical phenotype (CD34(+)VEGFR2(+)CD133(+)CXCR4(+)CD45(-)), culture, and functional behavior (Dil-ac-LDL+) in vitro. Injection of autologous bmECFCs improves tissue perfusion in ischemic hind limbs (183.5 ± 3.29 PU(bmECFCs/day 30)versus 131 ± 3.9 PU(controls/day 30), P < 0.001). CONCLUSIONS: We conclude that rat bmECFCs promote ischemic tissue reperfusion and their proangiogenic properties are a potential mechanism for this effect.

[Experimental model of in vitro generation of human dendritic cells]. / Obtinerea si cultivarea celulelor dendritice umane in vitro. Model experimental.

INTRODUCTION: Dendritic cells (DCs) are considered the most powerful antigen-presenting cells (APC) playing a strategic role in triggering the immune response. Monocytes are the physiological precursors of myeloid DCs. AIMS: This paper aims to develop an in vitro experimental model of human dendritic cells generation and phenotypic and functional analysis starting from CD14+ monocyte precursors isolated from peripheral blood. MATERIALS AND METHODS: CD14+ monocytes were isolated from venous peripheral blood which was harvested from consenting healthy donors. By the means of GM-CSF and IL-4 immature DCs were generated from monocytes. In the sixth day we added TNF-alpha and PGE-2 maturation factors and, after 48 hours we harvested mature DCs. Their morphology was monitored by optical microscopy, while the phenotypes were determined by flow cytometry. RESULTS: After three days of culture, the monocytes developed a specific morphology, gathering in clusters; cells presenting specific features start to detach in the sixth day. Treatment with TNF-alpha and PGE-2 induces the maturation of DCs and up-regulation of CD80, CD83 and CD86 expressions. Phenotypic analysis of mature DCs was HLA-DR+++/CD86- CD80+++/CD14(-)/CD11a+/CD83+++. DCs obtained are able to stimulate in vivo allolymphocyte capacity. Functional analysis test showed 87% CD4+ CFSE+(DCs+PBMC) cells vs CD4+ CFSE+(PBMC) 15% cells. CONCLUSIONS: This experiment developed an effective and viable way to generate DCs from PBMCs exposed to GM-CSF and IL-4. The cells obtained presented morphological, phenotypic and functional features as DCs described in the literature.