搜索结果: 'cryostor'

PURPOSE We conducted an open-label,single-arm Phase I/II clinical trial in metastatic CRPC (mCRPC) patients eligible for docetaxel combined with treatment with autologous mature dendritic cells (DCs) pulsed with killed LNCaP prostate cancer cells (DCVAC/PCa). The primary and secondary endpoints were safety and immune responses,respectively. Overall survival (OS),followed as a part of the safety evaluation,was compared to the predicted OS according to the Halabi and MSKCC nomograms. EXPERIMENTAL DESIGN Twenty-five patients with progressive mCRPC were enrolled. Treatment comprised of initial 7 days administration of metronomic cyclophosphamide 50 mg p.o. DCVAC/PCa treatment consisted of a median twelve doses of 1 × 107 dendritic cells per dose injected s.c. (Aldara creme was applied at the site of injection) during a one-year period. The initial 2 doses of DCVAC/PCa were administered at a 2-week interval,followed by the administration of docetaxel (75 mg/m2) and prednisone (5 mg twice daily) given every 3 weeks until toxicity or intolerance was observed. The DCVAC/PCa was then injected every 6 weeks up to the maximum number of doses manufactured from one leukapheresis. RESULTS No serious DCVAC/PCa-related adverse events have been reported. The median OS was 19 months,whereas the predicted median OS was 11.8 months with the Halabi nomogram and 13 months with the MSKCC nomogram. Kaplan-Meier analyses showed that patients had a lower risk of death compared with both MSKCC (Hazard Ratio 0.26,95% CI: 0.13-0.51) and Halabi (Hazard Ratio 0.33,95% CI: 0.17-0.63) predictions. We observed a significant decrease in Tregs in the peripheral blood. The long-term administration of DCVAC/PCa led to the induction and maintenance of PSA specific T cells. We did not identify any immunological parameter that significantly correlated with better OS. CONCLUSIONS In patients with mCRPC,the combined chemoimmunotherapy with DCVAC/PCa and docetaxel was safe and resulted in longer than expected survival. Concomitant chemotherapy did not preclude the induction of specific anti-tumor cytotoxic T cells. View Publication

BACKGROUND Acute myeloid leukemia (AML) is initiated and maintained by a subset of self-renewing leukemia stem cells (LSCs),which contribute to the progression,recurrence and therapeutic resistance of leukemia. However,the mechanisms underlying the maintenance of LSCs drug resistance have not been fully defined. In this study,we attempted to elucidate the mechanisms of LSCs drug resistance. METHODS We performed reverse phase protein arrays to analyze the expression of anti-apoptotic proteins in the LSC-enriched leukemia cell line KG-1a. Immuno-blotting,cell viability and clinical AML samples were evaluated to verify the micro-assay results. The characteristics and transcriptional regulation of survivin were analyzed with the relative luciferase reporter assay,mutant constructs,chromatin immuno-precipitation (ChIP),quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR),and western blotting. The levels of Sp1,c-Myc,phospho-extracellular signal-regulated kinase (p-ERK),phospho-mitogen and stress-activated protein kinase (p-MSK) were investigated in paired CD34+ and CD34- AML patient samples. RESULTS Survivin was highly over-expressed in CD34 + CD38- KG-1a cells and paired CD34+ AML patients compared with their differentiated counterparts. Functionally,survivin contributes to the drug resistance of LSCs,and Sp1 and c-Myc concurrently regulate levels of survivin transcription. Clinically,Sp1 and c-Myc were significantly up-regulated and positively correlated with survivin in CD34+ AML patients. Moreover,Sp1 and c-Myc were further activated by the ERK/MSK mitogen-activated protein kinase (MAPK) signaling pathway,modulating survivin levels. CONCLUSION Our findings demonstrated that ERK/MSK/Sp1/c-Myc axis functioned as a critical regulator of survivin expression in LSCs,offering a potential new therapeutic strategy for LSCs therapy. View Publication

Cryopreservation is the use of low temperatures to preserve structurally intact living cells. The cells that survive the thermodynamic journey from the 37 °C incubator to the -196 °C liquid nitrogen storage tank are free from the influences of time. Thus,cryopreservation is a critical component of cell culture and cell manufacturing protocols. Successful cryopreservation of human cells requires that the cells be derived from patient samples that are collected in a standardized manner,and carefully handled from blood draw through cell isolation. Furthermore,proper equipment must be in place to ensure consistency,reproducibility,and sterility. In addition,the correct choice and amount of cryoprotectant agent must be added at the correct temperature,and a controlled rate of freezing (most commonly 1 °C/min) must be applied prior to a standardized method of cryogenic storage. This appendix describes how human primary cells can be frozen for long-term storage and thawed for growth in a tissue culture vessel. View Publication

Most patients with acute myelogenous leukemia (AML) relapse and die of their disease. Increasing evidence indicates that AML relapse is driven by the inability to eradicate leukemia stem cells (LSC). Thus,it is imperative to identify novel therapies that can ablate LSCs. Using an in silico gene expression-based screen for compounds evoking transcriptional effects similar to the previously described anti-LSC agent parthenolide,we identified AR-42 (OSU-HDAC42),a novel histone deacetylase inhibitor that is structurally similar to phenylbutyrate,but with improved activity at submicromolar concentrations. Here,we report that AR-42 induces NF-κB inhibition,disrupts the ability of Hsp90 to stabilize its oncogenic clients,and causes potent and specific cell death of LSCs but not normal hematopoietic stem and progenitor cells. Unlike parthenolide,the caspase-dependent apoptosis caused by AR-42 occurs without activation of Nrf-2-driven cytoprotective pathways. As AR-42 is already being tested in early clinical trials,we expect that our results can be extended to the clinic. View Publication

Renal cell therapy has shown clinical efficacy in the treatment of acute renal failure (ARF) and promise for treatment of end-stage renal disease (ESRD) by supplementing conventional small solute clearance (hemodialysis or hemofiltration) with endocrine and metabolic function provided by cells maintained in an extracorporeal circuit. A major obstacle in the widespread adoption of this therapeutic approach is the lack of a cryopreservable system to enable distribution,storage,and therapeutic use at point of care facilities. This report details the design,fabrication,and assessment of a Bioartificial Renal Epithelial Cell System (BRECS),the first all-in-one culture vessel,cryostorage device,and cell therapy delivery system. The BRECS was loaded with up to 20 cell-seeded porous disks,which were maintained by perfusion culture. Once cells reached over 5 A- 10(6) cells/disk for a total therapeutic dose of approximately 10(8) cells,the BRECS was cryopreserved for storage at -80°C or -140°C. The BRECS was rapidly thawed,and perfusion culture was resumed. Near precryopreservation values of cell viability,metabolic activity,and differentiated phenotype of functional renal cells were confirmed post-reconstitution. This technology could be extended to administer other cell-based therapies where metabolic,regulatory,or secretion functions can be leveraged in an immunoisolated extracorporeal circuit. View Publication

ONC201 is a first-in-class,orally active antitumor agent that upregulates cytotoxic TRAIL pathway signaling in cancer cells. ONC201 has demonstrated safety and preliminary efficacy in a first-in-human trial in which patients were dosed every 3 weeks. We hypothesized that dose intensification of ONC201 may impact antitumor efficacy. We discovered that ONC201 exerts dose- and schedule-dependent effects on tumor progression and cell death signaling in vivo. With dose intensification,we note a potent anti-metastasis effect and inhibition of cancer cell migration and invasion. Our preclinical results prompted a change in ONC201 dosing in all open clinical trials. We observed accumulation of activated NK+ and CD3+ cells within ONC201-treated tumors and that NK cell depletion inhibits ONC201 efficacy in vivo,including against TRAIL/ONC201-resistant Bax-/- tumors. Immunocompetent NCR1-GFP mice,in which NK cells express GFP,demonstrated GFP+ NK cell infiltration of syngeneic MC38 colorectal tumors. Activation of primary human NK cells and increased degranulation occurred in response to ONC201. Coculture experiments identified a role for TRAIL in human NK-mediated antitumor cytotoxicity. Preclinical results indicate the potential utility for ONC201 plus anti-PD-1 therapy. We observed an increase in activated TRAIL-secreting NK cells in the peripheral blood of patients after ONC201 treatment. The results offer what we believe to be a unique pathway of immune stimulation for cancer therapy. View Publication

Advancing chimeric antigen receptor (CAR)-engineered adoptive T cells for the treatment of solid cancers is a major focus in the field of immunotherapy,given impressive recent clinical responses in hematological malignancies. Prostate cancer may be amenable to T cell-based immunotherapy since several tumor antigens,including prostate stem-cell antigen (PSCA),are widely over-expressed in metastatic disease. While antigen selectivity of CARs for solid cancers is crucial,it is problematic due to the absence of truly restricted tumor antigen expression and potential safety concerns with on-target off-tumor" activity. Here View Publication

Nonhuman primate (NHP) models are more predictive than rodent models for developing induced pluripotent stem cell (iPSC)-based cell therapy,but robust and reproducible NHP iPSC-cardiomyocyte differentiation protocols are lacking for cardiomyopathies research. We developed a method to differentiate integration-free rhesus macaque iPSCs (RhiPSCs) into cardiomyocytes with {\textgreater}85{\%} purity in 10 days,using fully chemically defined conditions. To enable visualization of intracellular calcium flux in beating cardiomyocytes,we used CRISPR/Cas9 to stably knock-in genetically encoded calcium indicators at the rhesus AAVS1 safe harbor locus. Rhesus cardiomyocytes derived by our stepwise differentiation method express signature cardiac markers and show normal electrochemical coupling. They are responsive to cardiorelevant drugs and can be successfully engrafted in a mouse myocardial infarction model. Our approach provides a powerful tool for generation of NHP iPSC-derived cardiomyocytes amenable to utilization in basic research and preclinical studies,including in vivo tissue regeneration models and drug screening. View Publication

CD34 is a transmembrane phosphoglycoprotein used to selectively enrich bone marrow in hematopoietic stem cells for transplantation. Treating rats with CD34+ cells derived from human umbilical cord blood before or after heat stroke has been shown to promote survival. We investigated whether CD34- human placenta-derived stem cells (PDMSCs) could improve survival following heat stroke in rats. Rats were subjected to heat stress (42°C for 98 min) to induce heat stroke. Intravenous administration of PDMSCs 1 day before or immediately after the onset of heat stroke improved survival by 60{\%} and 20{\%},respectively. Pre-treatment with CD34- PDMSCs protected against heat stroke injury more effectively than that treatment after injury. PDMSCs treatment attenuated cerebrovascular dysfunction,the inflammatory response,and lipid peroxidation. These data suggest human PDMSCs protect against heat stroke injury in rats. Moreover,these effects do not require the presence of CD34+ cells. View Publication

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) grown in engineered heart tissue (EHT) can be used for drug screening,disease modeling,and heart repair. However,the immaturity of hiPSC-CMs currently limits their use. Because mechanical loading increases during development and facilitates cardiac maturation,we hypothesized that afterload would promote maturation of EHTs. To test this we developed a system in which EHTs are suspended between a rigid post and a flexible one,whose resistance to contraction can be modulated by applying braces of varying length. These braces allow us to adjust afterload conditions over two orders of magnitude by increasing the flexible post resistance from 0.09 up to 9.2 mu$N/mu$m. After three weeks in culture,optical tracking of post deflections revealed that auxotonic twitch forces increased in correlation with the degree of afterload,whereas twitch velocities decreased with afterload. Consequently,the power and work of the EHTs were maximal under intermediate afterloads. When studied isometrically,the inotropy of EHTs increased with afterload up to an intermediate resistance (0.45 mu$N/mu$m) and then plateaued. Applied afterload increased sarcomere length,cardiomyocyte area and elongation,which are hallmarks of maturation. Furthermore,progressively increasing the level of afterload led to improved calcium handling,increased expression of several key markers of cardiac maturation,including a shift from fetal to adult ventricular myosin heavy chain isoforms. However,at the highest afterload condition,markers of pathological hypertrophy and fibrosis were also upregulated,although the bulk tissue stiffness remained the same for all levels of applied afterload tested. Together,our results indicate that application of moderate afterloads can substantially improve the maturation of hiPSC-CMs in EHTs,while high afterload conditions may mimic certain aspects of human cardiac pathology resulting from elevated mechanical overload. View Publication

Targeted cancer immunotherapy with irradiated,granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting,allogeneic cancer cell lines has been an effective approach to reduce tumor burden in several patients. It is generally assumed that to be effective,these cell lines need to express immunogenic antigens coexpressed in patient tumor cells,and antigen-presenting cells need to take up such antigens then present them to patient T cells. We have previously reported that,in a phase I pilot study (ClinicalTrials.gov NCT00095862),a subject with stage IV breast cancer experienced substantial regression of breast,lung,and brain lesions following inoculation with clinical formulations of SV-BR-1-GM,a GM-CSF-secreting breast tumor cell line. To identify diagnostic features permitting the prospective identification of patients likely to benefit from SV-BR-1-GM,we conducted a molecular analysis of the SV-BR-1-GM cell line and of patient-derived blood,as well as a tumor specimen. Compared to normal human breast cells,SV-BR-1-GM cells overexpress genes encoding tumor-associated antigens (TAAs) such as PRAME,a cancer/testis antigen. Curiously,despite its presumptive breast epithelial origin,the cell line expresses major histocompatibility complex (MHC) class II genes (HLA-DRA,HLA-DRB3,HLA-DMA,HLA-DMB),in addition to several other factors known to play immunostimulatory roles. These factors include MHC class I components (B2M,HLA-A,HLA-B),ADA (encoding adenosine deaminase),ADGRE5 (CD97),CD58 (LFA3),CD74 (encoding invariant chain and CLIP),CD83,CXCL8 (IL8),CXCL16,HLA-F,IL6,IL18,and KITLG. Moreover,both SV-BR-1-GM cells and the responding study subject carried an HLA-DRB3*02:02 allele,raising the question of whether SV-BR-1-GM cells can directly present endogenous antigens to T cells,thereby inducing a tumor-directed immune response. In support of this,SV-BR-1-GM cells (which also carry the HLA-DRB3*01:01 allele) treated with yellow fever virus (YFV) envelope (Env) 43-59 peptides reactivated YFV-DRB3*01:01-specific CD4+ T cells. Thus,the partial HLA allele match between SV-BR-1-GM and the clinical responder might have enabled patient T lymphocytes to directly recognize SV-BR-1-GM TAAs as presented on SV-BR-1-GM MHCs. Taken together,our findings are consistent with a potentially unique mechanism of action by which SV-BR-1-GM cells can act as APCs for previously primed CD4+ T cells. View Publication

Traumatic brain injury (TBI) is soon predicted to become the third leading cause of death and disability worldwide. After the primary injury,a complex set of secondary injuries develops hours and days later with prolonged neuroinflammation playing a key role. TBI and other inflammatory conditions are currently being treated in preclinical and clinical trials by a number of cellular therapies. Mesenchymal stem cells (MSC) are of great interest due to their widespread usage,safety,and relative ease to isolate and culture. However,there has been a wide range in efficacy reported using MSC clinically and in preclinical models,likely due to differences in cell preparations and a significant amount of donor variability. In this study,we seek to find a correlation between in vitro activity and in vivo efficacy. We designed assays to explore the responsiveness of MSC to immunological cues to address the immunomodulatory properties of MSC,one of their primary modes of therapeutic activity in TBI. Our results showed intrinsic differences in the immunomodulatory capacity of MSC preparations from different bone marrow and amniotic fluid donors. This difference mirrored the therapeutic capacity of the MSC in an experimental model of TBI,an effect confirmed using siRNA knockdown of COX2 followed by overexpressing COX2. Among the immunomodulatory factors assessed,the therapeutic benefit correlated with the secretion of prostaglandin E2 (PGE2 ) by MSC prior to treatment,suggesting that measurement of PGE2 could be a very useful potency marker to create an index of predicted efficacy for preparations of MSC to treat TBI. Stem Cells 2017;35:1416-1430. View Publication