Biomedical subjects

E Daro

Publications and source records attributed to E Daro.

5 recordsLinked to original sources

PubMed2000

In vivo generation of human dendritic cell subsets by Flt3 ligand.

Dendritic cells (DCs) represent a family of ontogenically distinct leukocytes involved in immune response regulation. The ability of DCs to stimulate T-cell immunity has led to their use as vectors for immunotherapy vaccines. However, it is unclear whether and to what degree in vitro-generated DCs are representative of DCs that develop in vivo. Treatment of mice with human Flt3 ligand (FL) dramatically increases the number of DCs. We report here that administration of FL to healthy human volunteers increased the number of circulating CD11c(+ )IL-3Ralpha(low) DC (mean 44-fold) and CD11c(-) IL-3Ralpha(high) DC precursors (mean 12-fold). Moreover, the CD11c(+ )DCs were efficient stimulators of T cells in vitro. Thus, FL can expand the number of circulating, functionally competent human DCs in vivo.

Adjuvants, Immunologic↗

PubMed2000

Polyethylene glycol-modified GM-CSF expands CD11b(high)CD11c(high) but notCD11b(low)CD11c(high) murine dendritic cells in vivo: a comparative analysis with Flt3 ligand.

Dendritic cells (DC) are potent APCs that can be characterized in the murine spleen as CD11b(high)CD11c(high) or CD11b(low)CD11c(high). Daily injection of mice of Flt3 ligand (FL) into mice transiently expands both subsets of DC in vivo, but the effect of administration of GM-CSF on the expansion of DC in vivo is not well defined. To gain further insight into the role of GM-CSF in DC development and function in vivo, we treated mice with polyethylene glycol-modified GM-CSF (pGM-CSF) which has an increased half-life in vivo. Administration of pGM-CSF to mice for 5 days led to a 5- to 10-fold expansion of CD11b(high)CD11c(high) but not CD11b(low)CD11c(high) DC. DC from pGM-CSF-treated mice captured and processed Ag more efficiently than DC from FL-treated mice. Although both FL- and pGM-CSF-generated CD11b(high)CD11c(high) DC were CD8alpha-, a greater proportion of these DC from pGM-CSF-treated mice were 33D1+ than from FL-treated mice. CD11b(low)CD11c(high) DC from FL-treated mice expressed high levels of intracellular MHC class II. DC from both pGM-CSF- and FL-treated mice expressed high levels of surface class II, low levels of the costimulatory molecules CD40, CD80, and CD86 and were equally efficient at stimulating allogeneic and Ag-specific T cell proliferation in vitro. The data demonstrate that treatment with pGM-CSF in vivo preferentially expands CD11b(high)CD11c(high) DC that share phenotypic and functional characteristics with FL-generated CD11b(high)CD11c(high) DC but can be distinguished from FL-generated DC on the basis of Ag capture and surface expression of 33D1.

Animals↗

PubMed1999

RANK is essential for osteoclast and lymph node development.

The physiological role of the TNF receptor (TNFR) family member, RANK, was investigated by generating RANK-deficient mice. RANK(-/-) mice were characterized by profound osteopetrosis resulting from an apparent block in osteoclast differentiation. RANK expression was not required for the commitment, differentiation, and functional maturation of macrophages and dendritic cells from their myeloid precursors but provided a necessary and specific signal for the differentiation of myeloid-derived osteoclasts. RANK(-/-) mice also exhibited a marked deficiency of B cells in the spleen. RANK(-/-) mice retained mucosal-associated lymphoid tissues including Peyer's patches but completely lacked all other peripheral lymph nodes, highlighting an additional major role for RANK in lymph node formation. These experiments reveal that RANK provides critical signals necessary for lymph node organogenesis and osteoclast differentiation.

Animals↗

PubMed1997

Inhibition of endosome function in CHO cells bearing a temperature-sensitive defect in the coatomer (COPI) component epsilon-COP.

Recent evidence has suggested that subunits of the coatomer protein (COPI) complexes are functionally associated with endosomes in mammalian cells. We now provide genetic evidence that COPI plays a role in endocytosis in intact cells. The ldlF mutant CHO cell line bears a temperature-sensitive defect in the COPI subunit epsilon-COP. In addition to exhibiting conditional defects in the secretory pathway, we find that the cells are also defective at mediating endosome-associated functions. As found for cells microinjected with anti-COPI antibodies, ldlF cells at the restrictive temperature could not be infected by vesicular stomatitis (VSV) or Semliki Forest virus (SFV) that require delivery to acidic endosomes to penetrate into the cytosol. Although there was no temperature-sensitive defect in the internalization of receptor-bound transferrin (Tfn), Tfn recycling and accumulation of HRP were markedly inhibited at the restrictive temperature. Sorting of receptor-bound markers such as EGF to lysosomes was also reduced, although delivery of fluid-phase markers was only partially inhibited. In addition, lysosomes redistributed from their typical perinuclear location to the tips of the ldlF cells. Mutant phenotypes began to emerge within 2 h of temperature shift, the time required for the loss of detectable epsilon-COP, suggesting that the endocytic defects were not secondary to a block in the secretory pathway. Importantly, the mutant phenotypes were also corrected by transfection of wild-type epsilon-COP cDNA demonstrating that they directly or indirectly reflected the epsilon-COP defect. Taken together, the results suggest that epsilon-COP acts early in the endocytic pathway, most likely inhibiting the normal sorting and recycling functions of early endosomes.

Animals↗

PubMed1996

Rab4 and cellubrevin define different early endosome populations on the pathway of transferrin receptor recycling.

During receptor mediated endocytosis, at least a fraction of recycling cargo typically accumulates in a pericentriolar cluster of tubules and vesicles. However, it is not clear if these endosomal structures are biochemically distinct from the early endosomes from which they are derived. To better characterize this pericentriolar endosome population, we determined the distribution of two endogenous proteins known to be functionally involved in receptor recycling [Rab4, cellubrevin (Cbvn)] relative to the distribution of a recycling ligand [transferrin (Tfn)] as it traversed the endocytic pathway. Shortly after internalization, Tfn entered a population of early endosomes that contained both Rab4 and Cbvn, demonstrated by triple label immunofluorescence confocal microscopy. Tfn then accumulated in the pericentriolar cluster of recycling vesicles (RVs). However, although these pericentriolar endosomes contained Cbvn, they were strikingly depleted of Rab4. The ability of internalized Tfn to reach the Rab4-negative population was not blocked by nocodazole, although the characteristic pericentriolar location of the population was not maintained in the absence of microtubules. Similarly, Rab4-positive and -negative populations remained distinct in cells treated with brefeldin A, with only Rab4-positive elements exhibiting the extended tubular morphology induced by the drug. Thus, at least with respect to Rab4 distribution, the pathway of Tfn receptor recycling consists of at least two biochemically and functionally distinct populations of endosomes, a Rab4-positive population of early endosomes to which incoming Tfn is initially delivered and a Rab4-negative population of recycling vesicles that transiently accumulates Tfn on its route back to the plasma membrane.

Animals↗