Drug resistance is often a limiting factor in successful chemotherapy. Our laboratory has been interested in studying mechanisms of resistance to drugs that are targeted to the thymidylate biosynthesis pathway especially those that target thymidylate synthase (TS) and dihydrofolate reductase (DHFR). We have used leukemia as a model system to study resistance to methotrexate (MTX) and colorectal cancer as the model system to study 5-fluorouracil (5-FU) resistance. In leukemias, we and others have shown that transport, efflux, polyglutamylation and hydrolase activities are major determinants of MTX resistance. We have further reported that some leukemic cells have an increase in DHFR gene copy number possibly contributing to the resistant phenotype. Recently, we have begun to study in detail the molecular mechanisms that govern translational regulation of DHFR in response to MTX as an additional resistance mechanism. Studies thus far involving colorectal tumors obtained from patients have focused predominantly on the predictive value of levels of TS expression and p53 mutations in determining response to 5-FU. Although the predictive value of these two measures appears to be significant, given the variety of resistance to 5-FU observed in cell lines, it is not likely that these are the only measures predictive of response or responsible for acquired resistance to this drug. The enzyme uridine-cytidine monophosphate kinase (UMPK) is an essential and rate-limiting enzyme in 5-FU activation while dihydropyrimidine dehydrogenase (DPD) is a catabolic enzyme that inactivates 5-FU. Alterations in UMPK and DPD may therefore explain failure of 5-FU response in the absence of alterations in TS or p53. Transcription factors that regulate TS may also influence drug sensitivity. We have found that mRNA levels of the E2F family of transcription factors correlates with TS message levels and are higher in lung metastases than in liver metastases of colorectal cancers. Moreover, gene copy number of the E2F-1 gene appears to be increased in a significant number of samples obtained from metastases of colorectal cancer. We have also generated mutants of both DHFR and TS that confer resistance to MTX as well as 5-FU by random as well as site-directed mutagenesis. These mutants used alone or as fusion cDNAs of the mutants have proven to be useful in transplant studies where transfer of these mutant cDNAs to bone marrow cells have been shown to confer drug resistance to recipients. The fusion cDNAs of DHFR such as the DHFR-herpes simplex virus type 1 thymidine kinase (HSVTK) are also useful for regulation of gene expression in vivo using MTX as the small molecule regulator that can be monitored by positron emission tomography (PET) scanning or by optical imaging using a fusion construct such as DHFR-EGFP.

Graft-versus-host disease (GVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (HSCT).Migration of donor-derived T cells into GVHD target organs plays a critical role in the development of GVHD and chemokines and their receptors are important molecules involved in this process. Here, we demonstrate in murine bone marrow transplantation models that the expression of the inflammatory CC chemokine receptor 2 (CCR2) on donor-derived CD8 ؉ T cells is relevant for the control of CD8 ؉ T-cell migration and development of GVHD. Recipients of CCR2-deficient (CCR2 ؊/؊ ) CD8 ؉ T cells developed less damage of gut and liver than recipients of wild-type CD8 ؉ T cells, which correlated with a reduction in overall GVHD morbidity and mortality. Assessment of donor CD8 ؉ T-cell target organ infiltration revealed that CCR2 ؊/؊ CD8 ؉ T cells have an intrinsic migratory defect to the gut and liver. Other causes for the reduction in GVHD could be excluded, as alloreactive proliferation, activation, IFN-␥ production and cytotoxicity of CCR2 ؊/؊ CD8 ؉ T cells were intact. Interestingly, the graft-versustumor effect mediated by CCR2 ؊/؊ CD8 ؉ T cells was preserved, which suggests that interference with T-cell migration by blockade of CCR2 signaling can separate GVHD from GVT activity. ( IntroductionAllogeneic hematopoietic stem cell transplantation (HSCT) is a well-established therapy for a variety of malignant and nonmalignant disorders of the hematopoietic system and for certain solid tumors. 1 A major complication limiting the success and wider application of allogeneic HSCT is the occurrence of acute graft-versus-host disease (GVHD), which is a rapidly progressive illness with immunosuppression, cachexia and specific target organ damage of liver, intestines, skin, and lung. 2 Donor-derived alloreactive T cells play a major role in the pathogenesis of GVHD and depletion of T cells from the donor cell inoculum remains the most effective approach to prevent the development of disease. 3 However, alloreactive donor T cells also display graft-versus-tumor (GVT) activity, which is increasingly being recognized as an important component of the overall antitumor effect of an allogeneic HSCT. 4 Recent murine bone marrow transplantation studies suggest that specifically interfering with T-cell migration represents an attractive therapeutic approach toward the goal of amelioration of GVHD without reducing GVT activity. 5,6 It is known that 3 families of migration molecules (selectins, chemokines, integrins, and their respective ligands and receptors) control T-cell migration in homeostasis and inflammation 7 and members of all 3 families have been identified as important players during GVHD. 5 CC chemokine receptor 2 (CCR2) and its main ligand chemokine ligand 2 (CCL2) are among the chemokine receptor-ligand pairs that control leukocyte migration during inflammatory processes. 8,9 CCL2 (originally termed monocyte chemoattractant protein-1 [MCP-1]) belongs to the family of inflammatory CC chemokines and was one...

The application of RNA interference (RNAi) to stem cell-based therapies will require highly specific and lineage-restricted gene silencing. Here we show the feasibility and therapeutic potential of coregulating transgene expression and RNAi in hematopoietic stem cells. We encoded promoterless small-hairpin RNA (shRNA) within the intron of a recombinant gamma-globin gene. Expression of both gamma-globin and the lariat-embedded small interfering RNA (siRNA) was induced upon erythroid differentiation, specifically downregulating the targeted gene in tissue- and differentiation stage-specific fashion. The position of the shRNA within the intron was critical to concurrently achieve high-level transgene expression, effective siRNA generation and minimal interferon induction. Lentiviral transduction of CD34(+) cells from patients with sickle cell anemia led to erythroid-specific expression of the gamma-globin transgene and concomitant reduction of endogenous beta(S) transcripts, thus providing proof of principle for therapeutic strategies that require synergistic gene addition and gene silencing in stem cell progeny.

In general, tuberculosis (Tb) is rarely seen in allogeneic stem cell transplant (alloSCT) recipients, but this observation has been challenged in developing countries such as Turkey, where Tb infection is more prevalent than in Europe and the US. In this retrospective study, we report on the incidence of Tb infections in 351 alloSCT recipients at 4 bone marrow transplantation units in Turkey over the last 10 years. The frequency of Tb in alloSCT recipients after allografting (5 of 351) was far greater than that in the general population (35.4 per 100,000). Of the 351 patients who underwent alloSCT, 77 who received isoniazid (INH) chemoprophylaxis for 6 months did not develop posttransplantation Tb. However, 5 of the remaining 274 patients who received no chemoprophylaxis developed Tb a median of 12 months (range, 10-47 months) after allografting. Antituberculosis therapy resulted in complete recovery in all cases. In 2 additional patients who were found to have active pulmonary Tb at the time of transplantation, alloSCT was delayed until the infections were treated. Infections of mycobacteria other than Mycobacterium tuberculosis were not observed. The number of patients who received and tolerated INH may not be sufficient for firm conclusions, but the data suggest that, in countries where Tb is prevalent, pre- and posttransplantation follow-up for Tb and the use of INH prophylaxis should be considered.

BACKGROUND Prostate tumor-initiating cells (TICs) have intrinsic resistance to current therapies. TICs are commonly isolated by cell sorting or dye exclusion, however, isolating TICs from limited primary prostate cancer (PCa) tissues is inherently inefficient. We adapted the collagen adherence feature to develop a combined immunophenotypic and time-of-adherence assay to identify human prostate TICs. METHODS PCa cells from multiple cell lines and primary tissues were allowed to adhere to several matrix molecules, and fractions of adherent cells were examined for their TIC properties. RESULTS Collagen-I rapidly-adherent PCa cells have significantly higher clonogenic, migration, and invasion abilities, and initiated more tumor xenografts in mice when compared to slowly-adherent and no-adherent cells. To determine the relative frequency of TICs among PCa cell lines and primary PCa cells, we utilized zebrafish xenografts to define the tumor initiation potential of serial dilutions of rapidly-adherent α2β1hi/CD44hi cells compared to non-adherent cells with α2β1low/CD44low phenotype. Tumor initiation from rapidly-adherent α2β1hi/CD44hi TICs harboring the TMPRSS2:ERG fusion generated xenografts comprising of PCa cells expressing Erg, AMACR, and PSA. Moreover, PCa-cell dissemination was consistently observed in the immune-permissive zebrafish microenvironment from as-few-as 3 rapidly-adherent α2β1hi/CD44hi cells. In zebrafish xenografts, self-renewing prostate TICs comprise 0.02–0.9% of PC3 cells, 0.3–1.3% of DU145 cells, and 0.22–14.3% of primary prostate adenocarcinomas. CONCLUSION Zebrafish PCa xenografts were used to determine that the frequency of prostate TICs varies among PCa cell lines and primary PCa tissues. These data support a paradigm of utilizing zebrafish xenografts to evaluate novel therapies targeting tumor initiating cells in prostate cancer.

Summary. Fifty-seven adult patients with idiopathic thrombocytopenic purpura (ITP) were treated with either conventional-dose prednisolone (CDP) (1 mg/kg/d, 36 patients) or high-dose methylprednisolone (HDP) (30 mg/ kg/d, 21 patients), as first-line treatment. Patients in the HDP arm responded more rapidly (4·7 v 8·4 d), with a higher response rate (80% v 52·7%), and without severe sideeffects. One quarter of the patients (3/12) who were nonresponsive to CDP achieved complete remission when they were treated with HDP. The findings suggest that HDP may be a more effective first-line treatment than CDP for adult ITP, and it may also be preferred for life-threatening cases of ITP. However, these results must be confirmed by a randomized study prior to any change in the current practice of employing CDP as first-line treatment for adult ITP.

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mRNA transfection is a useful approach for temporal cell reprogramming with minimal risk of transgenemediated mutagenesis. We applied this to redirect lymphocyte cytotoxicity toward malignant cells. Using the chimeric immune receptor (CIR) constructs anti-CD19 CIR and 8H9 CIR, we achieved uniform expression of CIRs on virtually the entire population of lymphocytes. We reprogrammed CD3 + CD8 + , CD3 + CD4 + , and natural killer (NK ) cells toward autologous and allogeneic targets such as B cells, Daudi lymphoma, primary melanoma, breast ductal carcinoma, breast adenocarcinoma, and rhabdomyosarcoma. The reprogramming procedure is fast. Although most of the experiments were performed on lymphocytes obtained after 7-day activation, only 1-day activation of T cells with anti-CD3, anti-CD28 antibodies, and interleukin-2 is sufficient to develop both lymphocyte cytotoxicity and competence for mRNA transfer. The entire procedure, which includes lymphocyte activation and reprogramming, can be completed in 2 days. The efficiency of mRNA-modified human T cells was tested in a murine xenograft model. Human CD3 + CD8 + lymphocytes expressing anti-CD19 CIR mRNA inhibited Daudi lymphoma growth in NOD=SCID mice. These results demonstrate that a mixed population of cytotoxic lymphocytes, including T cells together with NK cells, can be quickly and simultaneously reprogrammed by mRNA against autologous malignancies. With relatively minor modifications the described method of lymphocyte reprogramming can be scaled up for cancer therapy.