Summary:We investigated the use of 'prophylactic' donor lymphocyte infusions (DLI) containing 1 ؋ 10 7 CD3 ؉ cells, given at 30, 60 and 90 days post-allogeneic blood and marrow transplantation (BMT), following conditioning with fludarabine 30 mg/m 2 /4 days and melphalan 70 mg/m 2 /2 days. GVHD prophylaxis consisted of cyclosporin A (CsA) 2 mg/kg daily with early tapering by day 60. Our goals were the rapid achievement of chimerism and disease control, providing an immunological platform for DLIs to treat refractory patients with hematological malignancies. Twelve heavily pre-treated patients with life expectancy less than 6 months were studied; none were in remission. Diagnoses were AML (n ‫؍‬ 4), MDS (n ‫؍‬ 1), ALL (n ‫؍‬ 3), CML (n ‫؍‬ 3) and multiple myeloma (n ‫؍‬ 1). Response rate was 75%. Three patients are alive at a median of 450 days (range, 450-540). Two patients are in remission of CML in blast crisis and AML for more than 14 months. Median survival is 116 days (range, 25-648). Six patients received 12 DLIs; three patients developed acute GVHD after the first infusion and were excluded from further DLIs, but no GVHD occurred among patients receiving subsequent DLIs. One patient with CML in blast crisis went into CR after the first DLI. The overall incidence of acute GVHD was 70%. Primary causes of death were infections (n ‫؍‬ 3), acute GVHD (n ‫؍‬ 3), chronic GVHD (n ‫؍‬ 1) and disease relapse (n ‫؍‬ 2). We observed high response and chimerism rates at the expense of an excessive incidence of GVHD. DLI given at day ؉30 post BMT caused GVHD in 50% of the patients, and its role in this setting remains unclear. The therapeutic benefit of allogeneic BMT is in part related to an immunological graft-versus-leukemia (GVL) effect that frequently evolves in the context of graft-versus-host disease (GVHD). The ability of donor lymphocytes to induce remission in patients relapsing after allogeneic transplantation illustrates the potency of this effect.1 Establishing donor-recipient tolerance with less toxic regimens may provide the basis for further immunological manipulations in order to maximize the GVL effect. However, rapidly evolving diseases may not be amenable to this strategy, considering that the immune-mediated elimination of malignant cells may take weeks or months to occur. This fact suggests the need for strategies to reinforce the immune-mediated phenomena in the post-transplant period.Groups in Jerusalem and Houston pioneered the use of sub-lethal doses of fludarabine-based conditioning regimens. These regimens have been shown to be less toxic and to provide enough immunosuppression to prevent graft rejection and establish stable mixed or complete chimerism.2,3 The combination of melphalan and fludarabine has enabled allogeneic stem cell engraftment in the majority of patients treated, at least in the setting of HLA-identical transplantation. Patients with refractory relapses of advanced leukemias appear to benefit the least.

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.

Prostate cancer antigen 3 (PCA3) is a prostate-specific long noncoding RNA (lncRNA) involved in the control of prostate cancer (PCa) cell survival, through modulating androgen receptor (AR) signaling. To further comprehend the mechanisms by which PCA3 modulates LNCaP cell survival, we characterized the expression patterns of several cancer-related genes, including those involved in epithelial-mesenchymal transition (EMT) and AR cofactors in response to PCA3 silencing. We also aimed to develop a strategy to stably silence PCA3. Small interfering RNA (siRNA) or short hairpin RNA (shRNA) was used to knock down PCA3 in LNCaP cells. The expression of 84 cancer-related genes, as well as those coding for AR cofactors and EMT markers, was analyzed by quantitative real-time PCR (qRT-PCR). LNCaP-PCA3 silenced cells differentially expressed 16 of the 84 cancer genes tested, mainly those involved in gene expression control and cell signaling. PCA3 knockdown also induced the upregulation of several transcripts coding for AR cofactors and modulated the expression of EMT markers. LNCaP cells transduced with lentivirus vectors carrying an shRNA sequence targeting PCA3 stably downregulated PCA3 expression, causing a significant drop (60 %) in the proportion of LNCaP cells expressing the transgene. In conclusion, our data provide evidence that PCA3 silencing modulates the expression of key cancer-related genes, including those coding for AR cofactors and EMT markers. Transducing LNCaP cells with an shRNA sequence targeting PCA3 led to loss of viability of the cells, supporting the proposal of PCA3 knockdown as a putative therapeutic approach to inhibit PCa growth.

Cell therapy is a promising strategy to pursue the unmet need for treatment of spinal cord injury (SCI). Although several studies have shown that adult mesenchymal cells contribute to improve the outcomes of SCI, a descripton of the pro-regenerative events triggered by these cells is still lacking. Here we investigated the regenerative properties of human adipose tissue derived stromal cells (hADSCs) in a rat model of spinal cord compression. Cells were delivered directly into the spinal parenchyma immediately after injury. Human ADSCs promoted functional recovery, tissue preservation, and axonal regeneration. Analysis of the cord tissue showed an abundant deposition of laminin of human origin at the lesion site and spinal midline; the appearance of cell clusters composed of neural precursors in the areas of laminin deposition, and the appearance of blood vessels with separated basement membranes along the spinal axis. These effects were also observed after injection of hADSCs into non-injured spinal cord. Considering that laminin is a well-known inducer of axonal growth, as well a component of the extracellular matrix associated to neural progenitors, we propose that it can be the paracrine factor mediating the pro-regenerative effects of hADSCs in spinal cord injury.