Biomedical subjects

David F Stroncek

Publications and source records attributed to David F Stroncek.

27 records · Page 2Linked to original sources

PubMed2004

Improved gene transfer and normalized enzyme levels in primitive hematopoietic progenitors from patients with mucopolysaccharidosis type I using a bioreactor.

BACKGROUND: One of the major barriers to the clinical application of hematopoietic stem cell (HSC) gene therapy has been relatively low gene transfer efficiency. Other inadequacies of current transduction protocols are related to their multi-step procedures, e.g., using tissue-culture flasks, roller bottles or gas-permeable bags for clinical application. METHODS: In comparison with a conventional bag transduction protocol, a 'closed' hollow-fiber bioreactor system (HBS) was exploited to culture and transduce human peripheral blood CD34(+) progenitor cells (PBPC(MPS)) from patients with mucopolysaccharidosis type I (MPS I) using an amphotropic retroviral vector based on a murine Moloney leukemia virus LN prototype. Both short-term colony-forming cell (CFC) and long-term culture initiating cell (LTCIC) assays were employed to determine transduction frequency and transgene expression in committed progenitor cells and primitive progenitors with multi-lineage potentials. RESULTS: A novel ultrafiltration-transduction method was established to culture and transduce enzyme-deficient PBPC(MPS) over a 5-day period without loss in viability and CD34 identity (n = 5). Significantly higher transduction efficiencies were achieved in primary CFC that derived from the HBS (5.8-14.2%) in comparison with those from gas-permeable bags (undetectable to 1.7%; p < 0.01). Up to 15-fold higher-than-normal enzyme activity was found in selected PBPC(MPS)-LP1CD transductants. Moreover, higher gene transfer (4.4-fold) and expression in very primitive progenitors were observed in products from the HBS compared with bag experiments as indicated by CFC derived from primitive LTCIC. Remarkably, with relatively modest gene transfer levels in LTCIC from HBS experiments, the expression of the IDUA transgene corrected the enzyme-deficiency in 5-week long-term cultures (LTC). CONCLUSIONS: MPS I progenitor cells achieved normalized enzyme levels in LTC after transduction in a HBS system. These studies demonstrate the advantages of a bioreactor-transduction system for viral-mediated stem cell gene transfer.

Antigens, CD34↗

PubMed2004

Increasing hemoglobin oxygen saturation levels in sickle trait donor whole blood prevents hemoglobin S polymerization and allows effective white blood cell reduction by filtration.

UNLABELLED: BACKGROUND Red blood cell (RBC) components from donors with sickle cell trait (Hb AS) often occlude white blood cell (WBC) reduction filters. Techniques were investigated to successfully filter Hb AS donor blood by increasing the Hb oxygen saturation with storage bags and conditions suitable for transfusion products. STUDY DESIGN AND METHODS: Oxygenation kinetics were measured over 3 days in whole-blood units stored in standard-sized 600-mL polyvinylchloride (PVC) bags and whole-blood units divided into three equal parts and stored in standard-sized blood bags made from PVC, tri-2-(ethylhexyl)trimellitate (CLX) plastic, or Teflon. The filterability of Hb AS blood stored for 3 days was tested with whole-blood filters. RESULTS: Oxygen saturation levels did not increase in full whole-blood units from donors without sickle cell trait during 3 days of storage in 600-mL PVC bags. In divided Hb AS whole-blood units stored for 3 days, oxygen saturation levels increased from baseline levels of 45 to 56, 66, and 94 percent after storage in 600-mL PVC, CLX, and Teflon bags, respectively (n = 5, p < 0.02), and all components filtered completely. When full Hb AS whole-blood units from eight donors were stored for 3 days in 1.5-L CLX bags, all units filtered completely, but one had a high residual WBC count. CONCLUSION: Storage of Hb AS whole blood in large-capacity oxygen-permeable bags increases oxygen tension and allows more effective WBC reduction by filtration.

Benzoates↗

PubMed2003

Increasing oxygen tension improves filtration of sickle trait donor blood.

A major cause of filter failure of red cell (RBC) components from donors with sickle cell trait (HbAS) is the polymerization of haemoglobin. The oxygen saturation (sO2) of blood stored in various plastics and different volumes of air was assessed. Blood from 10 HbAS donors was collected and divided into two bags, one with air added, one without. Bags with added air had increased sO2 levels (from 49 +/- 10% to 76 +/- 6%). Filtration was successful for nine of 10 components with air, and one of 10 without air. Successful filtration of RBC components occurs when sO2 is increased.

Adolescent↗

PubMed2003

The matrix protein pp65(341-350): a peptide that induces ex vivo stimulation and in vitro expansion of CMV-specific CD8+ T cells in subjects bearing either HLA-A2402 or A0101 allele.

BACKGROUND: The stimulation of PBMNCs with HLA Class I restricted synthetic peptides derived from CMV phosphorylated matrix protein 65 (pp65) evokes CMV-specific cytotoxic T lymphocyte (CTL) activity, a necessary condition for initiating adoptive immunotherapy against CMV-related diseases in immune-compromised patients. It was previously demonstrated that the CMV decamer (10-mer) peptide pp65(341-350), QYDPVAALFF, was able to induce CMV-specific CTLs in HLA-A*2402 CMV-seropositive individuals. STUDY DESIGN AND METHOD: We investigated the ability of the peptide pp65(341-350) to reactivate memory CD8+ T cells in CMV-seropositive subjects bearing either the HLA-A24 or A1 allele. CTL responses were measured by IFN-gamma mRNA expression and IFN-gamma protein production as well as cytotoxic activity. RESULTS: In this study it was found that peptide pp65(341-350) induced a specific reactivation of memory CD8+ T cells from CMV-seropositive donors expressing either HLA-A*2402 and/or HLA-A*0101. Moreover, a pp65(341-350)-specific selection and expansion using PBMNCs of CMV-seropositive donors bearing both HLA-A*2402 and HLA-A*0101 alleles produced cytotoxic CTLs to both HLA-A24 and A1 peptide-pulsed and autologous CMV-infected target cells. CONCLUSION: The results demonstrate that pp65(341-350) induced a specific CTL activity at both molecular and protein levels and that the peptide is specifically processed, presented, and recognized by subjects bearing HLA-A*2402 and/or A*0101. These findings suggest that it may be possible to use this single immune dominant peptide to induce and expand CMV-reactive CTLs for the treatment of individuals with both HLA-A24 and A1 types.

Alleles↗

PubMed2002

Kinetics of G-CSF-induced granulocyte mobilization in healthy subjects: effects of route of administration and addition of dexamethasone.

BACKGROUND: Granulocyte donors are frequently given G-CSF with or without dexamethasone approximately 18 hours before apheresis to increase cell yields. The purpose of this study was to assess the kinetics of G-CSF plus dexamethasone neutrophil mobilization to determine whether the neutrophils can be mobilized and collected the same day. STUDY DESIGN AND METHODS: Sixteen subjects were given four separate mobilization regimens: IV G-CSF (5 microg/kg), subcutaneous G-CSF (5 microg/kg), IV G-CSF (5 microg/kg) plus oral dexamethasone (8 mg), and subcutaneous G-CSF (5 microg/kg) plus oral dexamethasone (8 mg). Blood cell counts were measured before and after G-CSF administration. RESULTS: Following all four mobilization regimens, neutrophil counts fell 0.5 hour after the mobilizing agents were given, rose above baseline levels at Hour 2, and increased further with each time interval to Hour 8. In the absence of dexamethasone at Hours 2 through 8, there was no difference in neutrophil counts by subcutaneous or IV G-CSF administration routes. The addition of dexamethasone enhanced mobilization of neutrophils from Hours 3 through 24. Through Hour 8, there was no difference in the degree of mobilization among the subcutaneous G-CSF plus dexamethasone and the IV G-CSF plus dexamethasone regimens. However, at Hour 24, neutrophil counts were sustained at higher levels with subcutaneous G-CSF plus dexamethasone than with IV G-CSF plus dexamethasone. CONCLUSIONS: Granulocyte mobilization response to subcutaneous G-CSF plus dexamethasone is sustained at peak levels for 8 to 24 hours after coadministration of the two drugs. There was no advantage to giving G-CSF intravenously.

Administration, Oral↗

PubMed2002

Detection of antibodies in acid eluates with the gel microcolumn assay.

BACKGROUND: Gel microcolumns can be used to detect unexpected serum antibodies and to determine ABO blood group and Rh phenotype. DATs can also be performed with this system. The purpose of this study was to compare the gel microcolumn to the tube IAT using anti-IgG for the detection of antibodies eluted from RBCs. STUDY DESIGN AND METHODS: Acid eluates were prepared from 30 peripheral blood and 41 umbilical cord blood samples. Twelve of the 71 eluates made were from control samples (known DAT negative). Specificities of eluted antibodies were determined by both tube and gel assays with a three-cell screen plus A1 and B cells, as determined by blood type. RESULTS: Ten of 30 peripheral blood eluates were reactive in both assays. Eighteen were nonreactive in both assays, and two from patients with autoimmune hemolytic anemia were reactive by gel assays and nonreactive by tube assays. Thirty three of the 41 cord blood eluates were reactive in both assays. Eluates from 2 of the 35 DAT-positive samples reacted with A1 and B cells by the tube method but were nonreactive by the gel method. Of the 33 cord blood eluates that were reactive by both assays, antibody specificity differed for two samples. When tested by tube assay, these eluates reacted with both A1 and B cells, whereas the same eluates tested by gel assay showed one reacting with only A1 cells and the other with only B cells. CONCLUSIONS: Results of testing eluates in gel assays were similar to those obtained in tube assays. The gel assays may be better at detecting antibodies eluted from RBCs from patients with autoimmune hemolytic anemia, and tube assays may be better at detecting isohemagglutinins eluted from umbilical cord blood.

Adult↗

PubMed2002

Sickle Hb polymerization in RBC components from donors with sickle cell trait prevents effective WBC reduction by filtration.

BACKGROUND: RBC components collected from donors with sickle cell trait frequently occlude WBC-reduction filters. In vitro, sickle trait RBCs have the potential for sickle Hb (Hb S) polymerization at low oxygen saturations and high Hb concentrations. STUDY DESIGN AND METHOD: To determine if the low pH and high osmolarity of the CP2D used in the collection contributed to filter failures, the filterability of sickle trait donor RBCs collected in CP2D was compared with RBCs from the same donors collected in heparin. RESULTS: Five of six sickle trait components collected in CP2D did not complete filtration, but all six RBC components collected in heparin filtered completely. RBC components collected in CP2D from four other sickle trait donors were divided in two, and one-half was treated with carbon monoxide to convert Hb S to its liganded form to prevent Hb S polymerization. All four carbon monoxide-treated components filtered within 9 minutes, but only one untreated component filtered completely. RBC components collected by apheresis contained less CP2D, and five of seven sickle trait apheresis components filtered completely; four of the five filtered rapidly (<15 min) and one filtered in 100 minutes. Hb oxygen saturation was greater in the four rapidly filtering apheresis RBC components (68 +/- 9%) than in the three that filtered slowly or incompletely (37 +/- 5%, p = 0.03). CONCLUSIONS: Hb S polymerization appears responsible for RBC WBC-reduction filter failures. Citrate anticoagulant and low oxygen saturation are responsible in part for Hb S polymerization in this setting.

Anticoagulants↗

PubMed2002

Anemia in parasite- and recombinant protein-immunized aotus monkeys infected with Plasmodium falciparum.

Plasmodium falciparum-induced anemia was characterized in Aotus monkeys repeatedly immunized by infection with P. falciparum (FVO strain) parasites, then cross-challenged with CAMP strain, or in monkeys receiving blood stage challenges as part of malaria vaccine trials. In 4 studies, 25 (30.5%) of 82 monkeys had at least a 50% reduction in hematocrit; mean day of maximum parasitemia was 12.5, whereas the mean day of minimum hematocrit was 18.8 (P < 0.0009). Decreased hematocrit levels were not associated with reticulocytosis until parasite densities decreased significantly from peak levels. Direct antibody tests to detect IgG and C3d on the surface of erythrocytes were negative. Nonantibody/noncomplement-mediated lysis of uninfected erythrocytes seems to be the principal cause of the anemia, and it also seems that bone marrow suppression and lysis of infected erythrocytes contributed to the anemia. Partial immunity-whether induced by repeated immunization with whole parasites or with vaccine-seems important to the development of anemia.

Anemia↗

PubMedDate not supplied

Advancing cancer biotherapy with proteomics.

Proteomics is becoming increasingly important for cancer biotherapy. The development of high-throughput platforms now allows the analysis of multiple proteins from small quantities of material. While these techniques are being used to discover new biomarkers, they are particularly important for assessing complex biologic processes such as immunotherapy for cancer. Recent advances in this field are reviewed, as well as the use of proteomics to assess the effectiveness and toxicities of high-dose IL-2 cancer therapy. Proteomics is becoming useful in assessing cancer biotherapies and in unraveling their mechanisms of action. High-throughput proteomic technologies have now advanced to a stage where they have the potential to become effective discovery tools for biomarkers/predictors of disease, disease recurrence, and response to therapy.

Humans↗