We tested the hypothesis that oral beclomethasone dipropionate (BDP) would control gastrointestinal graft-versus-host disease (GVHD) in patients with anorexia, vomiting, and diarrhea. Patients were ...randomized to prednisone for 10 days and either oral BDP 8 mg/d (n = 62) or placebo (n = 67) tablets for 50 days. At study day 10, prednisone was rapidly tapered while continuing study drug. On an intent-to-treat basis, the risk of GVHD-treatment failure was reduced for the BDP group at study day 50 (hazard ratio HR 0.63, 95% confidence interval CI 0.35-1.13) and at 30 days follow-up (HR 0.55, 95% CI 0.32-0.93). Among patients eligible for prednisone taper at study day 10, the risk of GVHD-treatment failure was significantly reduced at both study days 50 and 80 (HR 0.39 and 0.38, respectively). By day 200 after transplantation, 5 patients randomized to BDP had died compared with 16 deaths on placebo, a 67% reduction in the hazard of mortality (HR 0.33, P = .03). In 47 recipients of unrelated and HLA-mismatched stem cells, mortality at transplantation day 200 was reduced by 91% in the BDP group compared with placebo (HR 0.09, P = .02). The survival benefit was durable to 1 year after randomization. Oral BDP prevents relapses of gastrointestinal GVHD following tapering of prednisone; survival is statistically significantly better among patients receiving BDP.
B-cell activating factor (BAFF) single nucleotide polymorphisms (SNPs) are associated with autoimmune diseases. Because patients with classic and overlap chronic GVHD (cGVHD) have features of ...autoimmune diseases, we studied the association of recipient and/or donor BAFF SNPs with the phenotype of GVHD after allogeneic stem cell transplantation. Twenty tagSNPs of the BAFF gene were genotyped in 164 recipient/donor pairs. GVHD after day 100 occurred in 124 (76%) patients: acute GVHD (aGVHD) subtypes (n = 23), overlap GVHD (n = 29), and classic cGVHD (n = 72). In SNP analyses, 9 of the 20 tag SNPs were significant comparing classic/overlap cGVHD versus aGVHD subtypes/no GVHD. In multivariate analyses, 4 recipient BAFF SNPs (rs16972217 odds ratio = 2.72, P = .004, rs7993590 odds ratio = 2.35, P = .011, rs12428930 odds ratio2.53, P = .008, and rs2893321 odds ratio = 2.48, P = .009) were independent predictors of GVHD subtypes, adjusted for conventional predictors of cGVHD. This study shows that genetic variation of BAFF modulates GVHD phenotype after allogeneic stem cell transplantation.
Induced pluripotent stem cells (iPSCs) are becoming mainstream tools to study mechanisms of development and disease. They have a broad range of applications in understanding disease processes, in ...vitro testing of novel therapies, and potential utility in regenerative medicine. Although the techniques for generating iPSCs are becoming more straightforward, scientists can expend considerable resources and time to establish this technology. A major hurdle is the accurate determination of valid iPSC-like colonies that can be selected for further cloning and characterization. In this study, we describe the use of a gammaretroviral vector encoding a fluorescent marker, mRFP1, to not only monitor the efficiency of initial transduction but also to identify putative iPSC colonies through silencing of mRFP1 gene as a consequence of successful reprogramming.
Hematopoietic stem cells (HSCs) are the most well-characterized and studied stem cells. They have been used to treat various benign and malignant hematologic disorders. Most stem cell transplant ...recipients survive more than 5 years without any evidence of their original clinical disease. Early animal trials have demonstrated the ability to improve cardiac function by transfer of HSCs into the myocardium, and early human studies have demonstrated the feasibility and safety of this approach. Trials in patients after myocardial infarction and with chronic heart failure have seen limited and mixed success, probably because of the various cell types and methods used.
Mini-transplant has been used popularly in recent years. The intensity of conditioning regimens is less in mini-transplant, thus the anti-tumor effect is mainly carried by an immunological reaction, ...called graft-versus-leukemia (GVL)/graft-versus-tumor (GVT) effect. Thus, graft-versus-host disease (GVHD) in mini-transplant has drawn much attention as a surrogate marker for GVL/GVT effect. However, as the regimen-related toxicity is less in mini-transplant, it is also true that GVHD is the single most worrisome adverse event after mini-transplant. As opposed to early predictions, the incidence and severity of GVHD after mini-transplant does not seem to be much different from the one after conventional stem cell transplant (CST). In addition, researchers find that host antigen presenting cells (APC) may play an important role in the development of GVHD, and thus the existence of donor-host chimerism may be critical. As a result, the theory regarding GVHD after mini-transplant is getting more confusing. A comprehensive understanding of GVHD after mini-transplant is necessary.
We previously described the cloning and expression of canine methylguanine-DNA methyl transferase (MGMT) using monocistronic HIV-1 and gamma-retroviral vectors (Zaboikin M et. al. (2004) Hum Gene ...Ther 15:383-392). In that study, we showed that the canine MGMT was as efficient as the human MGMT for providing resistance to nitrosourea drugs such as BCNU. Here, we describe the creation and testing of dual-gene expression HIV-1 vectors encoding canine MGMT and EGFP under control of the EF1α promoter. The two genes were expressed either using encephalomyocarditis virus internal ribosome entry site (EMCV IRES) or the foot and mouth disease virus (FMDV) 2A cleavage factor. Vector stocks were prepared by transient transfection of 293T cells and used for infection of canine thyroid-adenocarcinoma cells. The cells were then subject to selection with O 6 -benzylguanine (BG) and BCNU. Flow cytometry of cells prior to and following drug selection indicated that both configurations of vector were able to provide drug resistance and allowed enrichment of transduced cells to greater than 84%. The expression of EGFP in the IRES containing vector (pN-EF1α-cMGMT-IRES-EGFP) was reduced compared to vector in which both transgenes were expressed using the FMDV 2A cleavage factor (pN-EF1α-cMGMT-2A-EGFP). These studies pave the way for utilizing canine MGMT encoding vectors for genetic correction in the dog model.
We have tested primary canine marrow mononuclear cells (MNC) as a potential target for gene therapy of hemophilia A by investigating their ability to support the expression of functional transgenic ...factor VIII (FVIII). . To this end, B-domain deleted human FVIII (BDFVIII) transgene was expressed in HIV-1 vectors from the viral LTR promoter or constitutively active internal cellular promoters: the mouse phosphoglycerate kinase (PGK) or elongation factor 1α (EF1α) promoters. The vector that expressed BDFVIII from the LTR also encoded a functional single-exon Tat protein (Tat72) Srinivasakumar and Schuening (2000) J. Virol. 74:6659-6668. The promoters expressed transgenes at high levels in hematopoietic cells, as has been previously shown Srinivasakumar et. al., (2002). J. Virol. 76:7334-7342. Vector stocks for each of the above vectors were prepared by transient transfection of 293T cells Srinivasakumar (2002) Methods Mol. Med. 69:275-302 and their titers were determined by real-time quantitative PCR of genomic DNA isolated from vector infected indicator cells. Each vector stock was then used for infection of primary canine marrow MNC by three spin-infections over 2 days on CH296-coated 6-well plates. The medium was replaced at the end of the transduction procedure. Conditioned medium was harvested 3-days after transduction and assayed for functional FVIII activity using the Coamatic Factor VIII kit (Chromogenix, Monza, Italy). Measured factor VIII activity in conditioned medium was normalized to input vector titer. The results, shown in the figure below, indicate that vector-derived FVIII could be detected in the supernatants of canine marrow MNCs transduced with any of the three HIV-1 vectors. Highest levels of functional FVIII were obtained with the Tat-encoding bicistronic HIV-1 vector. These results demonstrate that canine marrow MNC support expression and secretion of functional human FVIII. Further experiments are warranted to determine if the levels of FVIII observed in vitro will translate into therapeutic benefit in vivo in canine models of hemophilia A. (See Figure)
Since their discovery in 2006, induced pluripotent stem cells (iPSCs) with their ES cell-like self-renewal and differentiation capability, are set to revolutionize the field of regenerative medicine. ...There is tremendous interest in the field of hematology for derivation of hematopoietic stem cells (HSCs) and hematopoietic progenitors (HPCs) by in vitro differentiation of IPSCs. IPSCs can be differentiated into HSC/HPCs by coculture on feeder cells, such as OP9, or by using stepwise differentiation protocols on defined media. Neither approach produces high yields of HSCs or HPCs. With an intention to improve this, we systematically investigated various parameters for in vitrodifferentiation of iPSCs into HPCs.
iPSCs were derived from human adult dermal fibroblasts by transduction with the Yamanaka retroviral vectors (encoding human Klf4, Oct3/4, Sox2 and cMyc) or by electroporation with the Yamanaka Epstein–Barr virus-based episomal plasmid vectors (encoding Klf4, Oct3/4, Sox2, L-Myc and p53 targeting shRNA). One iPSC clone of each variety was then subjected to a stepwise differentiation protocol described by Niwa and coworkers PLoSOne. (2011); 6(7):e22261 followed by hematopoietic colony forming (CFU) assays in MethoCult (STEMCELL Technologies, Vancouver, Canada). The original protocol calls for the use of Stemline II serum-free medium (Sigma, St. Louis, MO) supplemented with various growth factors/cytokines. We investigated the use of APEL medium described by Ng and coworkers Nature Protocols. (2008); 3(5): 768 as a possible substitute for Stemline II. We also tested the effect of varying the number of colonies seeded in 6-well plates and the efficiency of hematopoietic differentiation after seeding iPSCs as single cells. The results, based on the number of hematopoietic colonies obtained in MethoCult following differentiation, showed that the APEL medium (>100 CFU/100,000 cells) was a superior substitute to the Stemline II medium (<10 CFU/100,000). When IPSCs were seeded as single cells, at initial densities of 10, 100 or 1,000 cells/cm2 in the presence of Y-27632 Rock inhibitor, only the cells at starting density higher than 1,000 per cm2survived but did not yield hematopoietic CFUs in MethoCult. When seeded as colony fragments, lower density of seeding in 6-well plates (< 20 colonies/well) was superior to higher density (>50 colonies/well) for obtaining HPCs. Other parameters that can affect differentiation, such as bone-morphopoietic protein (BMP) and O2 concentration, are being investigated.
Figure A. Cellular markers detected at different time points of stepwise hematopoietic differentiation. B. CFUs in MethoCult. Display omitted
No relevant conflicts of interest to declare.
Between January 1990 and April 2001, 115 patients received high-dose chemotherapy (HDT) followed by autologous stem cell transplantation (ASCT) for relapsed or refractory Hodgkin lymphoma (HL). With ...a median follow-up of 58 months (range, 1 - 175 months), 5-year progression-free survival (PFS) and overall survival (OS) were 46% and 58%, respectively. Twelve patients with primary refractory disease had a 5-year PFS of 41% and OS of 58%, not significantly different from those of the remaining cohort. Early and overall regimen related mortality were 7% and 16%, respectively. Male gender (P = 0.04) and a time to relapse (TTR) < 12 months (P = 0.03) were associated with decreased OS by univariate analysis. In multivariate analysis, TTR < 12 months remained statistically significant (P = 0.04). We have confirmed that HDT and ASCT result in long-term survival for a proportion of patients with relapsed or refractory HL. All patients, including those with primary refractory disease, benefited from HDT and ASCT.
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