Characterization of CD20-transduced T lymphocytes as an alternative suicide gene therapy approach for the treatment of graft-versus-host disease.

We have previously proposed the CD20 molecule as a novel suicide gene for T lymphocytes in the context of allogeneic bone marrow transplantation, because CD20 can be used both as a selection marker and as a killer gene after exposure to the anti-CD20 therapeutic antibody rituximab. We now report on preclinical studies using this novel system, in which the best transduction protocol, reproducibility, yield, feasibility, and functionality of the transduced T lymphocytes have been investigated with a large donor series. Wild-type human CD20 cDNA was transduced into human T lymphocytes, using a Moloney-derived retroviral vector. Alternative protocols were tested by employing either one or four spinoculations (in which cells are centrifuged in the presence of retroviral vector supernatant) and stimulating T cells with phytohemagglutinin (PHA) or anti-CD3/CD28. One spinoculation alone was sufficient to obtain approximately 30% CD20-positive cells within four experimental days. Four spinoculations significantly increased transduction to 60%. A small difference in transduction efficiency was observed between the two stimulation methods, with PHA being superior to anti-CD3/CD28. Transduced cells could be purified on immunoaffinity columns, with purity reaching 98% and yield being on average 50%. Finally, 86-97% of immunoselected T lymphocytes could be killed in vitro with rituximab and complement. More importantly, the CD20 transgene did not alter the functionality of T lymphocytes with respect to allogeneic recognition and cytotoxic response, anti-Epstein-Barr virus cytotoxic response, antigenic response to tetanus toxoid antigen, interleukin 2 (IL-2), IL-4, and interferon gamma production; chemotaxis in the presence of stromal cell-derived factor 1, phenotype for several activation markers including HLA-DR, CD25, CD69, and CD95, and T cell repertoire.

Transforming growth factor beta2 inhibition of hepatocyte growth factor-induced endothelial proliferation and migration.

Angiogenesis is a highly controlled event which depends on the proper equilibrium of activators and inhibitors present within the microenvironment. Hepatocyte Growth Factor (HGF) activates migration and proliferation of endothelial cells and is angiogenic, acting through the tyrosine kinase receptor encoded by the Met protooncogene. To get insights into the molecular mechanisms involved in HGF-induced angiogenesis, we searched for cDNAs differentially expressed in human endothelial cells exposed to HGF, a potent angiogenic factor. We found that HGF-treated endothelial cells upregulated the expression of Transforming Growth Factor (TGF) beta2. To understand the significance of this finding, we cultured endothelial cells with HGF and TGF beta2 simultaneously. We found that TGF beta2 impairs HGF-dependent proliferative and migratory responses. TGF beta2 did not prevent the tyrosine phosphorylation of Met, but it inhibited some signalling pathways activated by HGF. We show that endothelial proliferation induced by HGF required the activation of the MAPK cascade, while HGF-induced endothelial migration was dependent on the tyrosine phosphorylation of Src. Indeed, TGF beta2 inhibited HGF effects because it prevented HGF-induced MAP kinase activation and tyrosine phosphorylation of Src. We suggest that the induction of TGF beta2 by HGF in endothelial cells may represent a physiologic mechanism to counterbalance HGF angiogenic activity.

Modulation of WHSC2 expression in human endothelial cells.

WHSC2, a novel gene recently isolated within the critical region of Wolf-Hirschhorn syndrome, is expressed in endothelial cells. WHSC2 is downregulated by HIV-1 Tat, whereas it is not modulated by angiogenic and pro-differentiative molecules. WHSC2 encodes a basic polypeptide of 528 amino acids. The in vitro translated protein shows a molecular weight of 57 kDa. WHSC2 has two nuclear translocation sequences which actively mediate its transport to the nucleus, as shown in whsc2-GFP-transfected NIH-3T3. We also found a helix-loop-helix (HLH) motif in region 130-185. Since members of the HLH family control differentiation and cell cycle progression, we hypothesize that WHSC2 may function as a transcriptional repressor.

[A rare congenital malformation: genu recurvatum. Presentation of a case]. / Una rara malformazione congenita: il genu recurvatum. Presentazione di un caso.

The authors report a case of congenital genu recurvatum: a rare malformation characterized for abnormal hyperextension of knee and wide limitation of flexion. Pathogenesis, clinical pictures and therapy are described.