Hanh Khuu

Granulocyte Colony-Stimulating Factor Mobilizes Functional Endothelial Progenitor Cells in Patients With Coronary Artery Disease

Objective— Endothelial progenitor cells (EPCs) that may repair vascular injury are reduced in patients with coronary artery disease (CAD). We reasoned that EPC number and function may be increased by granulocyte colony-stimulating factor (G-CSF) used to mobilize hematopoietic progenitor cells in healthy donors. Methods and Results— Sixteen CAD patients had reduced CD34+/CD133+ (0.0224±0.0063% versus 0.121±0.038% mononuclear cells [MNCs], P<0.01) and CD133+/VEGFR-2+ cells, consistent with EPC phenotype (0.00033±0.00015% versus 0.0017±0.0006% MNCs, P<0.01), compared with 7 healthy controls. Patients also had fewer clusters of cells in culture, with out-growth consistent with mature endothelial phenotype (2±1/well) compared with 16 healthy subjects at high risk (13±4/well, P<0.05) or 14 at low risk (22±3/well, P<0.001) for CAD. G-CSF 10 &mgr;g/kg per day for 5 days increased CD34+/CD133+ cells from 0.5±0.2/&mgr;L to 59.5±10.6/&mgr;L and CD133+/ VEGFR-2+ cells from 0.007±0.004/&mgr;L to 1.9±0.6/&mgr;L (both P<0.001). Also increased were CD133+ cells that coexpressed the homing receptor CXCR4 (30.4±8.3/&mgr;L, P<0.05). Endothelial cell-forming clusters in 10 patients increased to 27±9/well after treatment (P<0.05), with a decline to 9±4/well at 2 weeks (P=0.06). Conclusions— Despite reduced EPCs compared with healthy controls, patients with CAD respond to G-CSF with increases in EPC number and homing receptor expression in the circulation and endothelial out-growth in culture.

Magnetic Resonance Imaging and Confocal Microscopy Studies of Magnetically Labeled Endothelial Progenitor Cells Trafficking to Sites of Tumor Angiogenesis

AC133 cells, a subpopulation of CD34+ hematopoietic stem cells, can transform into endothelial cells that may integrate into the neovasculature of tumors or ischemic tissue. Most current imaging modalities do not allow monitoring of early migration and incorporation of endothelial progenitor cells (EPCs) into tumor neovasculature. The goals of this study were to use magnetic resonance imaging (MRI) to track the migration and incorporation of intravenously injected, magnetically labeled EPCs into the blood vessels in a rapidly growing flank tumor model and to determine whether the pattern of EPC incorporation is related to the time of injection or tumor size. Materials and Methods: EPCs labeled with ferumoxide–protamine sulfate (FePro) complexes were injected into mice bearing xenografted glioma, and MRI was obtained at different stages of tumor development and size. Results: Migration and incorporation of labeled EPCs into tumor neovasculature were detected as low signal intensity on MRI at the tumor periphery as early as 3 days after EPC administration in preformed tumors. However, low signal intensities were not observed in tumors implanted at the time of EPC administration until tumor size reached 1 cm at 12 to 14 days. Prussian blue staining showed iron‐positive cells at the sites corresponding to low signal intensity on MRI. Confocal microcopy showed incorporation into the neovasculature, and immunohistochemistry clearly demonstrated the transformation of the administered EPCs into endothelial cells. Conclusion: MRI demonstrated the incorporation of FePro‐labeled human CD34+/AC133+ EPCs into the neovasculature of implanted flank tumors.

A Pilot Study of Consolidative Immunotherapy in Patients with High-Risk Pediatric Sarcomas

Purpose: Patients with metastatic or recurrent Ewing’s sarcoma family of tumors and alveolar rhabdomyosarcoma have <25% 5-year survival in most studies. This study administered a novel immunotherapy regimen aimed at consolidating remission in these patients. Experimental Design: Fifty-two patients with translocation positive, recurrent, or metastatic Ewing’s sarcoma family of tumors or alveolar rhabdomyosarcoma underwent prechemotherapy cell harvest via apheresis for potential receipt of immunotherapy. Following completion of standard multimodal therapy, 30 patients ultimately initiated immunotherapy and were sequentially assigned to three cohorts. All cohorts received autologous T cells, influenza vaccinations, and dendritic cells pulsed with peptides derived from tumor-specific translocation breakpoints and E7, a peptide known to bind HLA-A2. Cohort 1 received moderate-dose recombinant human interleukin-2 (rhIL-2), cohort 2 received low-dose rhIL-2, and cohort 3 did not receive rhIL-2. Results: All immunotherapy recipients generated influenza-specific immune responses, whereas immune responses to the translocation breakpoint peptides occurred in 39%, and only 25% of HLA-A2+ patients developed E7-specific responses. Toxicity was minimal. Intention-to-treat analysis revealed a 31% 5-year overall survival for all patients apheresed (median potential follow-up 7.3 years) with a 43% 5-year overall survival for patients initiating immunotherapy. Conclusions: Consolidative immunotherapy is a scientifically based and clinically practical approach for integrating immunotherapy into a multimodal regimen for chemoresponsive cancer. Patients receiving immunotherapy experienced minimal toxicity and favorable survival. The robust influenza immune responses observed suggest that postchemotherapy immune incompetence will not fundamentally limit this approach. Future studies will seek to increase efficacy by using more immunogenic antigens and more potent dendritic cells.

Donor demographic and laboratory predictors of allogeneic peripheral blood stem cell mobilization in an ethnically diverse population

A reliable estimate of peripheral blood stem cell (PBSC) mobilization response to granulocyte colony-stimulating factor (G-CSF) may identify donors at risk for poor mobilization and help optimize transplantation approaches. We studied 639 allogeneic PBSC collections performed in 412 white, 75 black, 116 Hispanic, and 36 Asian/Pacific adult donors who were prescribed G-CSF dosed at either 10 or 16 microg/kg per day for 5 days followed by large-volume leukapheresis (LVL). Additional LVL (mean, 11 L) to collect lymphocytes for donor lymphocyte infusion (DLI) and other therapies was performed before G-CSF administration in 299 of these donors. Day 5 preapheresis blood CD34(+) cell counts after mobilization were significantly lower in whites compared with blacks, Hispanics, and Asian/Pacific donors (79 vs 104, 94, and 101 cells/microL, P < .001). In addition, donors who underwent lymphapheresis before mobilization had higher CD34(+) cell counts than donors who did not (94 vs 79 cells/microL, P < .001). In multivariate analysis, higher post-G-CSF CD34(+) cell counts were most strongly associated with the total amount of G-CSF received, followed by the pre-G-CSF platelet count, pre-G-CSF mononuclear count, and performance of prior LVL for DLI collection. Age, white ethnicity, and female gender were associated with significantly lower post-G-CSF CD34(+) cell counts.

Differentiation of two types of mobilized peripheral blood stem cells by microRNA and cDNA expression analysis

BackgroundMobilized-peripheral blood hematopoietic stem cells (HSCs) have been used for transplantation, immunotherapy, and cardiovascular regenerative medicine. Agents used for HSC mobilization include G-CSF and the CXCR4 inhibitor AMD3100 (plerixafor). The HSCs cells mobilized by each agent may contain different subtypes and have different functions. To characterize mobilized HSCs used for clinical applications, microRNA (miRNA) profiling and gene expression profiling were used to compare AMD3100-mobilized CD133+ cells from 4 subjects, AMD3100 plus G-CSF-mobilized CD133+ cells from 4 subjects and G-CSF-mobilized CD34+ cells from 5 subjects. The HSCs were compared to peripheral blood leukocytes (PBLs) from 7 subjects.ResultsHierarchical clustering of miRNAs separated HSCs from PBLs. miRNAs up-regulated in all HSCs included hematopoiesis-associated miRNA; miR-126, miR-10a, miR-221 and miR-17-92 cluster. miRNAs up-regulated in PBLs included miR-142-3p, -218, -21, and -379. Hierarchical clustering analysis of miRNA expression separated the AMD3100-mobilized CD133+ cells from G-CSF-mobilized CD34+ cells. Gene expression analysis of the HSCs naturally segregated samples according to mobilization and isolation protocol and cell differentiation status.ConclusionHSCs and PBLs have unique miRNA and gene expression profiles. miRNA and gene expression microarrays maybe useful for assessing differences in HSCs.

The establishment of a bank of stored clinical bone marrow stromal cell products

BackgroundBone marrow stromal cells (BMSCs) are being used to treat a variety of conditions. For many applications a supply of cryopreserved products that can be used for acute therapy is needed. The establishment of a bank of BMSC products from healthy third party donors is described.MethodsThe recruitment of healthy subjects willing to donate marrow for BMSC production and the Good Manufacturing Practices (GMP) used for assessing potential donors, collecting marrow, culturing BMSCs and BMSC cryopreservation are described.ResultsSeventeen subjects were enrolled in our marrow collection protocol for BMSC production. Six of the 17 subjects were found to be ineligible during the donor screening process and one became ill and their donation was cancelled. Approximately 12 ml of marrow was aspirated from one posterior iliac crest of 10 donors; one donor donated twice. The BMSCs were initially cultured in T-75 flasks and then expanded for three passages in multilayer cell factories. The final BMSC product was packaged into units of 100 × 106 viable cells, cryopreserved and stored in a vapor phase liquid nitrogen tank under continuous monitoring. BMSC products meeting all lot release criteria were obtained from 8 of the 11 marrow collections. The rate of growth of the primary cultures was similar for all products except those generated from the two oldest donors. One lot did not meet the criteria for final release; its CD34 antigen expression was greater than the cut off set at 5%. The mean number of BMSC units obtained from each donor was 17 and ranged from 3 to 40.ConclusionsThe production of large numbers of BMSCs from bone marrow aspirates of healthy donors is feasible, but is limited by the high number of donors that did not meet eligibility criteria and products that did not meet lot release criteria.

Sterility testing of cell therapy products: parallel comparison of automated methods with a CFR-compliant method

BACKGROUND: Automated blood culture systems are not FDA‐approved for sterility testing of human cells, tissues, or cellular‐ or tissue‐based products. It was previously demonstrated that BacT/ALERT (bioMérieux) and Bactec (Becton Dickinson) were superior to the manual CFR method described in the general biologics regulations, in rates of detection and time to detection of organisms seeded into mock mononuclear cell products with a variety of background media and antibiotics. In this study, the two automated systems were compared to the CFR method for sterility testing of actual cell therapy products manufactured in our facility.

Catastrophic failures of freezing bags for cellular therapy products: description, cause, and consequences

BACKGROUND Container integrity is critical for maintaining sterility of cryopreserved cellular therapy products. We investigated a series of catastrophic bag failures, first noticed in early 2001. METHODS Process records were reviewed for all PBPC and lymphocyte products cryopreserved in bags from January 2000 through April 2002. Patient charts were also reviewed. RESULTS One thousand two hundred and four bags were removed from storage for infusion to 261 patients. All products had been cryopreserved in Cryocyte poly(ethylene co-vinyl acetate) (EVA) bags in either 10% DMSO or 5% DMSO and 6% pentastarch. Product volumes were 25-75 mL, and bags were stored with overwrap bags in a liquid nitrogen tank. From January 2000 to April 2001, failure occurred in 10 of 599 (1.7%) bags. From May 2001 to April 2002, 58 of 605 (9.6%) bags failed, typically with extensive fractures that were visible before thaw. Of the 58 that failed, 24 were salvaged by aseptic methods and infused to patients under antibiotic coverage; 10 of those 24 (42%) had positive bacterial cultures. Bag failures were not related to product type, cryoprotectant solution, liquid versus vapor storage, or freezer location. Failures were linked to use of four Cryocyte bag lots manufactured in 2000 and 2001. After replacing these lots with a 1999 Cryocyte lot and with KryoSafe polyfluoroethylene polyfluoropropylene (FEP) bags, no more failures occurred in 75 and 102 bags, respectively, thawed through April 2002. DISCUSSION High rates of bag failure were associated with four Cryocyte bag lots. No serious adverse patient effects occurred, but bag failures led to microbial contamination, increased product preparation time, increased antibiotic use, and increased resource expenditure to replace products.

Comparison of automated culture systems with a CFR/USP-compliant method for sterility testing of cell-therapy products

BACKGROUND Although widely used, commercially available automated culture methods are not US Food and Drug Administration-approved for sterility testing of cell-therapy products. For cell-therapy products regulated under Section 351 of the Public Health Service Act, sterility testing must be performed by the methods described in 21 CFR 610.12 and USP <71> (CFR/USP method), or by methods demonstrated to be equivalent. METHODS Two automated methods, BacT/Alert (BTA; bioMerieux) and Bactec (Becton Dickinson), were compared with the CFR/USP method. Representative mononuclear cell (MNC) products were formulated using six different product media. MNC product aliquots containing 10-50 x 10(6) cells in a 0.5 mL volume were seeded with organisms, and cultured for 14 days in aerobic and anaerobic bottles of each system. Ten different organisms at target concentrations of 10 and 50 colony-forming units (CFU) per bottle were tested. RESULTS Positives were detected in a mean (range) of 72% (7-100%) of cultures for CFR/USP, 82% (0-100%) for BTA, and 93% (57-100%) for Bactec. For nine of the 10 organisms tested, overall detection rates for BTA and Bactec were equivalent to or higher than CFR/USP. Of the six product media tested, detection of organisms was impaired only by the medium containing multiple antibiotics: this occurred in all three systems. Both BTA and Bactec had shorter times to detection than the CFR/USP method, with overall means (ranges) of 87 (24-264) h for CFR/USP, 24 (12-54) h for BTA, and 33 (12-80) h for Bactec. Detection occurred consistently within 7 days for both BTA and Bactec, but not for CFR/USP. DISCUSSION Both BTA and Bactec are superior to the CFR/USP method for overall detection and time to detection of organisms in MNC products suspended in commonly used media. These data support general use of either BTA or Bactec for sterility testing of a variety of cell-therapy products, and suggest that a 7-day culture period is sufficient to detect clinically relevant organisms. These results confirm the need for bacteriostasis and fungistasis testing of antibiotic-containing products, even when antibiotic-binding substances are used.

Differences in the Phenotype, Cytokine Gene Expression Profiles, and In Vivo Alloreactivity of T Cells Mobilized with Plerixafor Compared with G-CSF

Plerixafor (Mozobil) is a CXCR4 antagonist that rapidly mobilizes CD34+ cells into circulation. Recently, plerixafor has been used as a single agent to mobilize peripheral blood stem cells for allogeneic hematopoietic cell transplantation. Although G-CSF mobilization is known to alter the phenotype and cytokine polarization of transplanted T cells, the effects of plerixafor mobilization on T cells have not been well characterized. In this study, we show that alterations in the T cell phenotype and cytokine gene expression profiles characteristic of G-CSF mobilization do not occur after mobilization with plerixafor. Compared with nonmobilized T cells, plerixafor-mobilized T cells had similar phenotype, mixed lymphocyte reactivity, and Foxp3 gene expression levels in CD4+ T cells, and did not undergo a change in expression levels of 84 genes associated with Th1/Th2/Th3 pathways. In contrast with plerixafor, G-CSF mobilization decreased CD62L expression on both CD4 and CD8+ T cells and altered expression levels of 16 cytokine-associated genes in CD3+ T cells. To assess the clinical relevance of these findings, we explored a murine model of graft-versus-host disease in which transplant recipients received plerixafor or G-CSF mobilized allograft from MHC-matched, minor histocompatibility–mismatched donors; recipients of plerixafor mobilized peripheral blood stem cells had a significantly higher incidence of skin graft-versus-host disease compared with mice receiving G-CSF mobilized transplants (100 versus 50%, respectively, p = 0.02). These preclinical data show plerixafor, in contrast with G-CSF, does not alter the phenotype and cytokine polarization of T cells, which raises the possibility that T cell–mediated immune sequelae of allogeneic transplantation in humans may differ when donor allografts are mobilized with plerixafor compared with G-CSF.