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Background: Cryopreserved donor lymphocyte infusion (DLI) products are manufactured and administered to treat relapse after allogeneic hematopoietic stem cell transplantation. Reported clinical responses to DLIs vary broadly, even within the same group of patients. While there is an implicit recognition of the fact that different manufacturing protocols may have specific effects on different cell types, cryopreservation protocols are frequently derived from our experience in the cryopreservation of stem cell products and do not account for the heterogeneous functional nature of DLI T-cell populations. Here, we report the results of a prospective, multicenter trial on the effect of four different cryopreservation solutions that were used to freeze DLIs compared to control DLIs that were refrigerated overnight.Study Design and Methods: Cryopreserved postthawed and refrigerated specimens were analyzed side by side for their T-cell subpopulation content and viability, as well as T-cell proliferation, cytokine secretion, and cytotoxic activities.Results: This study indicates that "homemade" 10% dimethyl sulfoxide (DMSO) results in reduced viability of different CD4+ T-cell populations, including T-helper, T-cytotoxic, and T-regulatory populations, and a decrease in their proliferative and cytotoxic response to immunologically relevant stimuli, while the use of solutions containing 5% DMSO with intracellular-like cryoprotectant stabilizers maintains T-cell function at levels similar to refrigerated control samples.Conclusion: This study has important implications in determining the best cryoprotectant solution for specific clinical applications in allogeneic immunotherapy. [ABSTRACT FROM AUTHOR]

Background: Granulocyte transfusion from healthy donors is used in the treatment of patients with granulocyte function defects, or transient neutropenia and severe bacterial or fungal infections resistant to maximal antimicrobial treatment.Study Design and Methods: This study evaluated the performance and safety of the newly developed granulocyte collection protocol of the Spectra Optia in a prospective, multicenter, open-label, randomized, paired crossover trial compared with the COBE Spectra apheresis system in a population of 32 evaluable healthy subjects. All subjects received granulocyte-colony-stimulating factor and dexamethasone before collection.Results: Granulocyte procedures from Spectra Optia apheresis procedures had an approximately 23% higher polymorphonuclear (PMN) collection efficiency (CE) than the COBE Spectra collections (mean, 53.7% vs. 43.2%; p < 0.01), while the platelet CE (10.9% vs. 10.8%, respectively) and hematocrit (7.4% vs. 7.4%) were comparable between collections on both devices. Spectra Optia collections generated a higher total PMN yield per liter of blood processed than those produced by the COBE Spectra (with means of 8.6 × 10(10) vs. 6.8 × 10(10) , respectively). Granulocyte viability was more than 99% with both devices, and chemotaxic and bacterial killing activities of circulating versus collected granulocytes were similarly preserved. Fewer operator adjustments were required with Spectra Optia and there was no significant difference in the number or intensity of adverse events between instruments.Conclusion: CE of the granulocyte collection procedure with the Spectra Optia was approximately 10 percentage points higher than with the COBE Spectra, required less operator involvement, and is safe for clinical implementation. [ABSTRACT FROM AUTHOR]

BACKGROUND: Availability of platelets (PLTs) is severely limited by shelf life in some settings. Our objective was to determine and compare to Food and Drug Administration (FDA) criteria the PLT recovery and survival of autologous PLTs cryopreserved at −65°C or less in 6% dimethyl sulfoxide (DMSO) reconstituted with a no-wash method (cryopreserved PLTs [CPPs]) compared to autologous fresh PLTs. STUDY DESIGN AND METHODS: This was a randomized, Phase I study analyzing PLT viability and in vitro function in consenting healthy subjects. Apheresis PLTs (APs) were collected in plasma. APs were suspended in 6% DMSO, concentrated, and placed at not more than −65°C for 7 to 13 days. Frozen CPPs were thawed at 37°C and resuspended into 25 mL of 0.9% NaCl. Control PLTs (fresh autologous) and CPPs were labeled with 111In or 51Cr, and recovery and survival after reinfusion were determined using standard methods. A panel of in vitro assays was completed on APs and CPPs. RESULTS: After frozen storage, CPPs retained 82% of AP yield and showed increased PLT associated P-selectin and reduced responses to agonists. CPP 24-hour recovery (41.6 ± 9.7%) was lower than for fresh PLTs (68.4 ± 8.2%; p < 0.0001) and did not meet the current FDA criterion. CPPs had diminished survival compared to fresh PLTs (7.0 ± 2.1 days vs. 8.4 ± 1.2 days, respectively; p = 0.018), but did meet and exceed the FDA criterion for survival. CONCLUSION: While 24-hour recovery does not meet FDA criteria for liquid-stored PLTs, the CPP survival of circulating PLTs was surprisingly high and exceeded the FDA criteria. These data support proceeding with additional studies to evaluate the clinical effectiveness of CPPs. [ABSTRACT FROM AUTHOR]

Phospholipids and lung surfactant proteins are known to be recycled within the lung alveolus mainly by uptake into type II epithelial cells that secrete lipid-enriched lung surfactant. Dipalmitoyl phosphatidylcholine (DPPC) is the major component of lung surfactant lipids and cholesterol is the second most abundant. However, cholesterol turnover in vivo has not been measured and it is not known how long steroidal compounds persist in the lung in intact animals. Here we report on experiments in which radiolabeled cholesterol was instilled into the lungs of rats, then at various postinstillation periods, radioactive sterols in lavage fluid, and in postlavage whole lungs were measured in individual animals. Radioactive sterols in the lungs remained high for a week and were still detectable 46 days later. The clearance rate during the initial postinstillation week was approximately 10% per day. Both radioactive free and esterified sterols were recovered from bronchoalveolar lavage fluid and postlavage lungs. [ABSTRACT FROM AUTHOR]

Background Systemic in vivo gene therapy has resulted in widespread correction in animal models when treated at birth. However, limited improvement was observed in postnatally treated animals with mainly targeting to the liver and bone marrow. It has been shown that an O6-methylguanine-DNA-methyltransferase variant (MGMTP140K) mediated in vivo selection of transduced hematopoietic stem cells (HSC) in animals. Methods We investigated the feasibility of MGMTP140K-mediated selection in primary hepatocytes from a mouse model of mucopolysaccharidosis type I (MPS I) in vitro using lentiviral vectors. Results We found that multiple cycles of O6-benzylguanine (BG)/1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) treatment at a dosage effective for ex vivo HSC selection led to a two-fold increase of MGMT-expressing primary hepatocytes under culture conditions with minimum cell expansion. This enrichment level was comparable to that obtained after selection at a hepatic maximal tolerated dose of BCNU. Similar levels of increase were observed regardless of initial transduction frequency, or the position of MGMT (upstream or downstream of internal ribosome entry site) in the vector constructs. In addition, we found that elongation factor 1α promoter was superior to the long-terminal repeat promoter from spleen focus-forming virus with regard to transgene expression in primary hepatocytes. Moreover, the levels of therapeutic transgene expression in transduced, enzyme-deficient hepatocytes directly correlated with the doses of BCNU, leading to metabolic correction in transduced hepatocytes and metabolic cross-correction in neighbouring non-transduced MPS I cells. Conclusions These results demonstrate that MGMTP140K expression confers successful protection/selection in primary hepatocytes, and provide 'proof of concept' to the prospect of MGMTP140K-mediated co-selection for hepatocytes and HSC using BG/BCNU treatment. Copyright © 2007 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

An abstract of the article "In Vivo Adult Stem Cell Gene Transfer in Mice by In Situ Delivery of a Self-Inactivating Lentiviral Vector Using Intrafemoral Injection," by D. Nicole Worsham, Kimberly Bohn, David Kuhel, David A. Williams, Christof von Kalle and Dao Pan is presented.

Background: Systemic in vivo gene therapy has resulted in widespread correction in animal models when treated at birth. However, limited improvement was observed in postnatally treated animals with mainly targeting to the liver and bone marrow. It has been shown that an O(6)-methylguanine-DNA-methyltransferase variant (MGMT(P140K)) mediated in vivo selection of transduced hematopoietic stem cells (HSC) in animals., Methods: We investigated the feasibility of MGMT(P140K)-mediated selection in primary hepatocytes from a mouse model of mucopolysaccharidosis type I (MPS I) in vitro using lentiviral vectors., Results: We found that multiple cycles of O(6)-benzylguanine (BG)/1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) treatment at a dosage effective for ex vivo HSC selection led to a two-fold increase of MGMT-expressing primary hepatocytes under culture conditions with minimum cell expansion. This enrichment level was comparable to that obtained after selection at a hepatic maximal tolerated dose of BCNU. Similar levels of increase were observed regardless of initial transduction frequency, or the position of MGMT (upstream or downstream of internal ribosome entry site) in the vector constructs. In addition, we found that elongation factor 1alpha promoter was superior to the long-terminal repeat promoter from spleen focus-forming virus with regard to transgene expression in primary hepatocytes. Moreover, the levels of therapeutic transgene expression in transduced, enzyme-deficient hepatocytes directly correlated with the doses of BCNU, leading to metabolic correction in transduced hepatocytes and metabolic cross-correction in neighbouring non-transduced MPS I cells., Conclusions: These results demonstrate that MGMT(P140K) expression confers successful protection/selection in primary hepatocytes, and provide 'proof of concept' to the prospect of MGMT(P140K)-mediated co-selection for hepatocytes and HSC using BG/BCNU treatment., (Copyright (c) 2007 John Wiley & Sons, Ltd.)