Biomedical subjects

P A Bulur

Publications and source records attributed to P A Bulur.

5 recordsLinked to original sources

PubMed2006

Testing the safety of clinical-grade mature autologous myeloid DC in a phase I clinical immunotherapy trial of CML.

BACKGROUND: We conducted a phase I clinical immunotherapy trial of CML to evaluate the safety of a clinical-grade leukemic DC product standardized for purity and mature phenotype. METHODS: We injected autologous DC into patients in late chronic or accelerated phases of CML. The patients received mature CD83+ and bcr-abl+ DC prepared from CD14+ cells. Two cohorts of three patients received four injections each of 3 x 10(6) DC and 15 x 10(6) DC/injection, respectively. The first patient was studied before imatinib mesylate (IM) was available, four patients were treated concurrently with IM therapy and one did not tolerate the IM and was off the drug at the time of DC therapy. IM effects on WBC counts precluded DC preparation in numbers sufficient for further dose escalation. The first patient received DC s.c. and all subsequent patients received DC into a cervical lymph node under ultrasound guidance. RESULTS: DC injections were well tolerated. We observed no clinical responses. T cells drawn later in the course of therapy were more sensitive to stimulation by CML DC in vitro. DISCUSSION: The increase in T-cell sensitivity to CML-specific stimulation that accompanied active immunization by CML DC justifies further clinical studies, possibly with modifications such as an increased frequency and number of DC injections.

Aged↗

PubMed2001

Maturation of dendritic cells infected by recombinant adenovirus can be delayed without impact on transgene expression.

Adenovirus-mediated gene transfer to dendritic cells is highly efficient and often used, but the relationship among cell maturation, viral infection and expression of a transferred gene remains unclear. To study this relationship, we introduced a recombinant replication-defective adenovirus encoding the gene for green fluorescent protein to normal human immature myeloid dendritic cells. We induced maturation by the addition of TNF-alpha, IL-1beta, IL-6 and prostaglandin E2 to the medium and assessed cell maturity by the levels of the secreted p40 subunit of IL-12 and of membrane-bound CD83. We quantified the efficiency of gene expression by GFP fluorescence and analyzed the data by a mixed-model analysis of variance; the model explained more than 97% of the effects. CD83 expression and p40 secretion depended solely on incubation time and maturation medium. The cells cultured in the absence of maturation medium remained immature and maintained the ability to respond to the later addition of the maturation irrespective of adenovirus infection and transferred gene expression. This expression was independent of cell maturation. In comparison with mature cells, the transferred gene was expressed in immature dendritic cells with a lag compatible with the less effective initial step (infection and/or gene transfer) in the absence of the maturation medium rather than less effective later GFP synthesis. Expression of CD83 and p40 were unaffected by adenovirus infection and transferred gene expression. Thus, immature dendritic cells infected with recombinant adenoviruses can be matured when desired after transferred gene expression.

Adenoviridae↗

PubMed2001

Transgenic interleukin 2 secreted by CML dendritic cells stimulates autologous T(H)1 T cells.

BACKGROUND: We investigated if dendritic cells (DC), derived from patients suffering from chronic myeloid leukemia (CML) could be modified by recombinant replication-defective adenoviruses to express functional interleukin 2 (IL-2). Such modification might confer onto antigen-presenting cells the ability to stimulate expansion of effector cells. METHODS: To quantify the infection efficiency of CML dendritic cells (CML-DC) by recombinant adenovirus, we measured the expression of green fluorescent protein (GFP) gene contained in the virus. In CML-DC infected with an adenovirus containing the IL-2 gene, we evaluated their ability to secrete IL-2 and stimulate proliferation of autologous T cells. RESULTS: Uninfected CML-DC and normal DC secreted similar amounts of IL-12 and stimulated similarly efficient autologous mixed leukocyte reaction. Immature CML-DC infected by an adenovirus containing the gene for IL-2 secreted large amounts of IL-2 and stimulated proliferation of autologous T cells more efficiently than the corresponding CML-DC alone. High levels of interferon eta, but not of IL-4, in cell culture supernates indicated that the proliferating cells were T(H)1. Infected mature CML-DC were more effective than infected immature CML-DC, showing that T cell stimulation by mature DC and by IL-2 was additive. DISCUSSION: CML-DC can be modified genetically and functionally by recombinant replication-defective adenoviruses, providing new possibilities for clinical trials in dendritic cell-based immunotherapy.

Adenoviridae↗

PubMed2000

Maturation of human monocyte-derived dendritic cells studied by microarray hybridization.

We compared the transcript profiles of human myeloid immature dendritic (IDC) cells and mature dendritic cells (MDC) by hybridization of cell-derived cDNA to DNA probes immobilized on microarrays. The microarrays contained probes for 4110 known genes. We report maturation-dependent changes in transcription of clusters of differentiation, cytokines, cytokine receptors, chemokines, chemokine receptors, neuropeptides, adhesion molecules, and other genes. We identified 1124 transcripts expressed in IDC and 1556 transcripts expressed in MDC. Maturation increased the levels of 291 transcripts twofold or more and reduced the levels of 78 transcripts to one-half or less than in IDC. We identified a concerted maturation-stage-dependent transcription of the variable chains of the members of the gamma-chain-cytokine receptor family IL-4R, IL-7R, and IL-15R. Also, we found the reversal of the ratio of transcripts for galectin-3 and galectin-9 upon maturation. We identified maturation-dependent changes in the levels of transcripts for numerous genes encoding proteins previously undetected in dendritic cells such as indoleamine 2,3-deoxygenase, Epstein-Barr virus induced protein 3 and kinesin-2. Moreover, MDC transcribed and translated insulin like growth factor-1 receptor, transforming growth factor alpha, and neuropeptide Y.

Antigens, CD↗

PubMed2000

Optimizing preparation of normal dendritic cells and bcr-abl+ mature dendritic cells derived from immunomagnetically purified CD14+ cells.

The goal of this work was to optimize dendritic cell (DC) preparations obtained from patients suffering from chronic myeloid leukemia (CML) and compare them with DC prepared from normal CD14+ mononuclear cells (MNC). We studied normal DC and bcr-abl+ leukemic DC (CML-DC) yields, expression of membrane molecules, differentiation status, and ability to stimulate T cells. We isolated DC precursors from PBMC by CD14-specific immunoadsorption and cultured them for 7 days in GM-CSF and IL-4, followed by a 3-day incubation to fully differentiate the cells. We evaluated cultures of CML-DC using RPMI 1640 medium supplemented with FBS and X-VIVO 15 medium containing human AB serum. In contrast to cells matured in RPMI 1640, virtually all cells incubated in X-VIVO 15 expressed CD83, a marker of mature DC. CML-DC and normal DC were indistinguishable in expression of CD83, resulting in the highest percentage reported so far. The yields of normal DC and CML-DC from CD14+ cells were indistinguishable. The percentage of bcr-abl+ cells in PBMC varied among patients between 65% and 97% and the final CML-DC preparations were >98% bcr-abl+ the highest purity of bcr-abl+ cells to date. Normal DC and CML-DC were equally effective in stimulating proliferation of allogeneic and autologous T cells. These techniques provide highly enriched, mature, functional CML-DC.

Cell Division↗