Charles S., "Interleukin-1 polymorphisms associated with increased risk of gastric cancer" (2000). To evaluate dopaminergic cells of the dorsomedial cluster by tyrosine hydroxylase immunostaining, serial 4-mm sections were cut to include the entire brain. Immunopositive cells at the level of the giant interneuron commissure, posterior to the fan-shaped body, were counted in well oriented frontal sections at 1, 10, 30 and 60 days. At 1 day all control and experimental sections contained four or ®ve cells in the delineated region. At 30 and 60 days all controls showed four or ®ve cells. At 30 and 60 days all a-synucleinexpressing animals (a-synuclein, elav±GAL4 and a-synuclein, Ddc±GAL4 transheterozygotes) showed 0 or 1 tyrosine-hydroxylase-positive cell in the de®ned region. Tyrosinehydroxylase-positive cells outside the dorsomedial cluster were present, and served as internal controls for the immunostaining procedure. At least four, and usually between six and ten brains were examined for wild-type a-synuclein and each mutant a-synuclein. Controls included young and aged¯ies of the genotypes elav±GAL4/+ and Ddc±GAL4/+. We evaluated expression of a-synuclein and b-galactosidase on similar serial section preparations. Quanti®cation was simpli®ed in these experiments because no clear cellbody-associated a-synuclein or b-galactosidase immunoreactivity was observed in the aged a-synuclein transgenic¯ies at the times reported.For histological examination of retinas, heads were ®xed in glutaraldehyde and embedded in epon. Tangential retinal sections were prepared at a thickness of 1 mm and stained with toluidine blue (Fig 4).Standard electron microscopy was performed on brains from 25-day-old experimental (UAS±A30P a-synuclein/elav±GAL4) and control (elav±GAL4/+)¯ies. For immunoelectron microscopy, pre-embedding immunohistochemistry with an Hrp-congugated secondary antibody was performed on 60-day adult brains from experimental (UAS± A30P a-synuclein/elav±GAL4) and control (elav±GAL4/+)¯ies ®xed in 4% paraformaldehyde with 0.5% glutaraldehyde. Tissue was post-®xed in osmium and embedded in epon. Unstained ultrathin sections and ultrathin sections stained with uranyl acetate and lead citrate were examined. Climbing assayThe climbing assay was performed as described 19,20 . Forty¯ies were placed in a plastic vial, and gently tapped to the bottom of the vial. The number of¯ies at the top of the vial was counted after 18 s of climbing. Twenty trials were performed for each time point. The data shown represent results from a cohort of¯ies tested serially over 55 days. The experiment was repeated three times, with independently derived transgenic lines. Similar results were obtained from each experiment. The experiment was carried out under red light (Kodak Safelight Filter 1A). Control¯ies were of the genotype elav±GAL4/+. Experimental animals were of the following genotypes: (1) elav±GAL4/+; UAS±wild-type a-synuclein/+; (2) UAS±A30P a-synuclein/elav±GAL4; and (3) UAS±A53T a-synuclein/elav±GAL4.

Dendritic cells (DCs) play a key role in the activation and regulation of B and T lymphocytes. Production of indoleamine 2,3-dioxygenase (IDO) by macrophages has recently been described to result in inhibition of T cell proliferation through tryptophan degradation. Since DCs can be derived from monocytes, we sought to determine whether DCs could produce IDO which could potentially regulate T cell proliferation. Northern blot analysis of RNA from cultured monocyte-derived human DC revealed that IDO mRNA was induced upon activation with CD40 ligand and IFN-γ. IDO produced from activated DCs was functionally active and capable of metabolizing tryptophan to kynurenine. Activated T cells were also capable of inducing IDO production by DCs, which was inhibited by a neutralizing Ab against IFN-γ. DC production of IDO resulted in inhibition of T cell proliferation, which could be prevented using the IDO inhibitor 1-methyl-dl-tryptophan. These results suggest that activation of DCs induces the production of functional IDO, which causes depletion of tryptophan and subsequent inhibition of T cell proliferation. This may represent a potential mechanism for DCs to regulate the immune response.

SummaryWe previously reported that natural killer cell stimulatory factor (NKSF), a heterodimeric lymphokine purified from the conditioned medium of human B lymphoblastoid cell lines, induces interferon y (IFN-y) production from resting peripheral blood lymphocytes (PBL) and synergizes with interleukin 2 in this activity. In this study, we show that human NKSF induces IFN-y production from both resting and activated human PBL and from freshly isolated murine splenocytes . Human T and NK cells produce IFN-,y in response to NKSF, but resting PBL require the presence of nonadherent human histocompatibility leukocyte antigens DR' (HLA DR+) accessory cells to respond to NKSF. The mechanism(s) by which NKSF induces IFN-y production results in accumulation of IFN-y mRNA, is insensitive to cyclosporin A, and synergizes with those mediated by phytohemmagglutinin, phorbol diesters, anti-CD3 antibodies, and allogeneic antigens, but not by Ca 2+ ionophores. The ability of NKSF to directly induce IFN-,y production and to synergize with other physiological IFN-,y inducers, joined with the previously described ability to enhance lymphocyte cytotoxicity and proliferation, indicates that this lymphokine is a powerful immunopotentiating agent .

No abstract

The contribution of chemokines toward angiogenesis is currently a focus of intensive investigation. Certain members of the CXC chemokine family can induce bovine capillary endothelial cell migration in vitro and corneal angiogenesis in vivo, and apparently act via binding to their receptors CXCR1 and CXCR2. We used an RNAse protection assay that permitted the simultaneous detection of mRNA for various CXC chemokine receptors in resting human umbilical vein endothelial cells (HUVECs) and detected low levels of only CXCR4 mRNA. Stimulation of HUVECs with vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bFGF) up-regulated levels of only CXCR4 mRNA. CXCR4 specifically binds the chemokine stromal-derived factor-1alpha (SDF-1alpha). Competitive binding studies using 125I-labeled SDF-1alpha with Scatchard analysis indicated that VEGF or bFGF induced an average number of approximately 16,600 CXCR4 molecules per endothelial cell, with a Kd = 1.23 x 10(-9) mol/L. These receptors were functional as HUVECs and human aorta endothelial cells (HAECs) migrated toward SDF-1alpha. Although SDF-1alpha-induced chemotaxis was inhibited by the addition of a neutralizing monoclonal CXCR4 antibody, endothelial chemotaxis toward VEGF was not altered; therefore, the angiogenic effect of VEGF is independent of SDF-1alpha. Furthermore, subcutaneous SDF-1alpha injections into mice induced formation of local small blood vessels that was accompanied by leukocytic infiltrates. To test whether these effects were dependent on circulating leukocytes, we successfully obtained SDF-1alpha-induced neovascularization from cross sections of leukocyte-free rat aorta. Taken together, our data indicate that SDF-1alpha acts as a potent chemoattractant for endothelial cells of different origins bearing CXCR4 and is a participant in angiogenesis that is regulated at the receptor level by VEGF and bFGF.