Biomedical subjects

M A Velleca

Publications and source records attributed to M A Velleca.

4 recordsLinked to original sources

PubMed2001

Ex vivo evaluation of PBMNCs collected with a new cell separator.

BACKGROUND: This study reports on an evaluation of the ability of a cell separator (Amicus, Baxter Healthcare) and the integral MNC computer software program to collect a variety of MNC subsets. The collection efficiency (CE) of the Amicus for these MNC subsets was compared to that of another cell separator (CS-3000 Plus, Baxter). The collected MNCs were also assayed ex vivo to determine if these cells remained functional. STUDY DESIGN AND METHODS: Healthy volunteer blood donors were recruited to provide PBMNCs for the isolation of CD3+, CD4+, CD8+, CD19+, NK, and gammadelta+ cells and monocytes. Cells were collected with an Amicus (test arm; n = 16) or a CS-3000 Plus (control arm; n = 11) cell separator. Cells were counted on a flow cytometer and CEs were calculated. For functional studies, the Amicus-collected MNC data were compared to CS-3000 Plus historical data. Functional studies performed included surface antigen expression assays (CD8+), proliferation assays (CD4+ and CD8+ cells), NK cytotoxicity assays for K562 and HUVE cells, and E-selectin induction on endothelial cells through NK+ contact dependency. Dendritic cells (DCs) were generated from CD34+ cells collected on the Amicus, positively selected by the use of antibody-bound, magnetic bead technology, and then cultured ex vivo with a combination of growth factors to generate the DCs. RESULTS: CEs were higher on the Amicus than on the CS-3000 Plus for CD3+ (68 vs. 54%), CD4+ (70 vs. 56%), CD8+ (68 vs. 52%), and CD19+ (60 vs. 48%) cells (p<0.05). For the two separators, CEs were equivalent for monocytes, NK+, and gammadelta+ cells. The Amicus separator collected significantly fewer platelets than did the CS-3000 Plus (p<0.00001). CD4+, CD8+, and NK cells proliferated normally. NK cells appropriately stimulated E-selectin expression on endothelial cells. Culture-generated DCs obtained by using Amicus-collected CD34+ cells expressed appropriate cell surface markers. CONCLUSION: The Amicus separator is acceptable for the collection of PBMNC subsets. The device collects CD3+, CD4+, CD8+, and CD19+ T- and B-cell subsets with greater efficiency and collects MNCs with significantly fewer contaminating platelets than does the CS-3000 Plus. Cells collected on the Amicus are suitable for use in a variety of research and clinical immunobiologic studies.

Blood Donors↗

PubMed2000

Large-scale culture and selective maturation of human Langerhans cells from granulocyte colony-stimulating factor-mobilized CD34+ progenitors.

Dendritic cells (DCs) play a critical role as APCs in the induction of the primary immune response. Their capacity for Ag processing and presentation is tightly regulated, controlled by a terminal developmental sequence accompanied by striking changes in morphology, organization, and function. The maturation process, which converts DCs from cells adapted for Ag accumulation to cells adapted for T cell stimulation, remains poorly understood due in part to difficulties in the culture and manipulation of DCs of defined lineages. To address these issues, we have devised conditions for the culture of a single DC type, Langerhans cells (LCs), using CD34+ cells from G-CSF-mobilized patients. Homogenous populations of LCs, replete with abundant immunocytochemically demonstrable Birbeck granules, could be stably maintained as immature DCs for long periods in culture. Unlike other human DC preparations, the LCs remained fully differentiated after cytokine removal. Following exposure to TNF-alpha, LPS, or CD40 ligand, the LCs could be synchronously induced to mature. Depending on the agent used, distinct types of LCs emerged differing in their capacity for T cell stimulation, IL-12 production, intracellular localization of MHC products, and overall morphology. Most interestingly, the expression of different sets of Toll family receptors is induced or down-regulated according to the maturation stimulus provided. These results strongly suggest that different proinflammatory stimuli might drive distinct developmental events.

Adult↗

PubMed1994

A novel synapse-associated noncoding RNA.

Synaptic nuclei of innervated muscle transcribe acetylcholine receptor (AChR) genes at a much higher level than extrasynaptic nuclei. To isolate candidate synaptic regulatory molecules responsible for the unique transcriptional potential of synaptic nuclei, we have taken a subtractive hybridization approach. Here, we report the cloning and characterization of a novel synapse-associated RNA, 7H4. 7H4 is expressed selectively in the endplate zone of skeletal muscle and is upregulated during early postnatal development and after denervation. Interestingly, the 7H4 gene has no introns, and yet two different-size RNAs with identical polyadenylated 3' ends are generated. Most intriguingly, the nucleotide sequence does not contain any significant open reading frames, suggesting that 7H4 may function as a noncoding RNA.

Animals↗

PubMed1989

Molecular cloning and sequence analysis of the Chlamydomonas gene coding for radial spoke protein 3: flagellar mutation pf-14 is an ochre allele.

Chlamydomonas reinhardtii flagellar motility mutant pf-14 fails to assemble radial spokes because of a deficiency for assembly-competent radial spoke protein 3 (Huang, B., G. Piperno, Z. Ramanis, and D. J. L. Luck. 1981. J. Cell Biol. 88:80-88). Here, we raise an antiserum to protein 3 and use it to isolate the corresponding structural gene from an expression library. Southern blot analysis indicates that the gene is single copy and has not undergone major rearrangement in mutant pf-14 cells. Northern blot analysis suggests that wild-type amounts of an apparently normal 2.3-kb transcript accumulate in mutant cells during flagellar regeneration. When this mutant RNA is hybrid selected and translated in vitro, however, it produces a slightly truncated polypeptide 3 with an altered charge. The mutant protein 3 fails to assemble into pf-14 flagella and is maintained within a cytoplasmic pool of unassembled radial spoke polypeptides, as indicated by immunoblot analysis of proteins from whole cells and isolated axonemes using antisera to several radial spoke polypeptides. Interestingly, amounts of the mutant protein are greatly diminished relative to other spoke components. Complete genomic and cDNA nucleotide sequences were determined, and the pf-14 mutation was identified. It is a C-to-T transition near the 5' end of the protein coding region, which changes codon 21 to the ochre termination signal UAA. The size and charge of the mutant protein, and its reduced levels in cells, suggest that it is produced by relatively inefficient translational initiation as codon 42. The unphosphorylated isoform of radial spoke protein 3 is identified, and the sequence similarities between intervening sequences of the radial spoke protein 3 gene and a conserved intervening sequence of the two Chlamydomonas beta-tubulin genes (Youngblom, J., J. A. Schloss, and C. D. Silflow. 1984. Mol. Cell. Biol. 4:2686-2696) are reported.

Alleles↗