Synthesis and evaluation of the cardiovascular effects of two, membrane impermeant, macromolecular complexes of dextran-testosterone

The incidence of cardiovascular disease is greater in men than in premenopausal women. Testosterone has been considered a significant risk factor for cardiovascular disease, but testosterone's mechanism of action and its cellular site of action are still not clear. However, it is likely that non-genomic extracellular effects of the hormone are involved. With the aim of providing further information about this phenomenon, two membrane impermeant, macromolecular complexes of testosterone were synthesized and their cardiovascular effects were evaluated. We covalently bound testosterone (through carbon 3 or C-17 functional groups) to dextran (2 MDa) and evaluated its effects on isolated and perfused rat hearts (Langerdorff model). Our results showed that the macromolecular complexes increased vascular resistance similarly to free testosterone and blocked adenosine-induced vasodilatation. These effects were exerted rapidly and possibly through a non-genomic mechanism. Blockade of C-3 or C-17 functional groups by binding to macromolecular dextran induced no qualitative and/or quantitative changes in testosterone-induced effects.     

Monocyte-derived dendritic cells induce a house dust mite-specific Th2 allergic inflammation in the lung of humanized SCID mice: involvement of CCR7

In rodents, airway dendritic cells (DCs) capture inhaled Ag, undergo maturation, and migrate to the draining mediastinal lymph nodes (MLN) to initiate the Ag-specific T cell response. However, the role of human DCs in the pathogenesis of the Th2 cell-mediated disease asthma remains to be clarified. Here, by using SCID mice engrafted with T cells from either house dust mite (HDM)-allergic patients or healthy donors, we show that DCs pulsed with Der p 1, one of the major allergens of HDM, and injected intratracheally into naive animals migrated into the MLN. In the MLN, Der p 1-pulsed DCs from allergic patients induced the proliferation of IL-4-producing CD4(+) T cells, whereas those from healthy donors induced IFN-gamma-secreting cells. In reconstituted human PBMC-reconstituted SCID mice primed with pulsed DCs from allergic patients, repeated exposure to aerosols of HDM induced 1) a strong pulmonary inflammatory reaction rich in T cells and eosinophils, 2) an increase in IL-4 and IL-5 production in the lung lavage fluid, and 3) increased IgE production compared with that in mice primed with unpulsed DCs. All these effects were reduced following in vivo neutralization of the CCR7 ligand secondary lymphoid tissue chemokine. These data in human PBMC-reconstituted SCID mice show that monocyte-derived DCs might play a key role in the pathogenesis of the pulmonary allergic response by inducing Th2 effector function following migration to the MLN.     

Coelomocyte locomotion in the sipunculan Themiste petricola induced by exogenous and endogenous chemoattractants: role of a CD44-like antigen--HA interaction

Cell migration is a key event in the invertebrate immuno-defense system. Microbial products like lipopolysacharide (LPS) and formyl-methyl-leucyl-phenylalanine (fMLP) promote cell recruitment to sites of infection. In mammals, complement activation by factors such as zymosan induces C5a production, which influences leukocyte migration. The endogenous factor hyaluronic acid (HA), an extracellular matrix component, also promotes cell migration through its receptor CD44. We evaluated whether coelomocytes from the sipunculan worm T. petricola migrated towards LPS, fMLP, or zymosan treated plasma (ZTP) and if HA was involved in coelomocyte migration and adhesion. We also evaluated if antibodies specific for mouse HA receptor CD44 inhibited any of the effects induced by HA. Using microchemotaxis chambers we found that coelomocytes migrated towards exogenously and endogenously derived chemoattractants. We also observed that HA was a potent chemotactic signal and that coelomocytes adhered strongly to plates coated with LMW-HA but not with HMW-HA. In addition we found that these HA mediated effects were blocked by the monoclonal antibody IM7 directed to mouse CD44, suggesting that a CD44-like cross-reactive antigen might play a role in HA mediated coelomocyte locomotion.     

An essential role for Prox1 in the induction of the lymphatic endothelial cell phenotype

The process of angiogenesis has been well documented, but little is known about the biology of lymphatic endothelial cells and the molecular mechanisms controlling lymphangiogenesis. The homeobox gene Prox1 is expressed in a subpopulation of endothelial cells that, after budding from veins, gives rise to the mammalian lymphatic system. In Prox1(-)(/-) embryos, this budding becomes arrested at around embryonic day (E)11.5, resulting in embryos without lymphatic vasculature. Unlike the endothelial cells that bud off in E11.5 wild-type embryos, those of Prox1-null embryos did not co-express any lymphatic markers such as VEGFR-3, LYVE-1 or SLC. Instead, the mutant cells appeared to have a blood vascular phenotype, as determined by their expression of laminin and CD34. These results suggest that Prox1 activity is required for both maintenance of the budding of the venous endothelial cells and differentiation toward the lymphatic phenotype. On the basis of our findings, we propose that a blood vascular phenotype is the default fate of budding embryonic venous endothelial cells; upon expression of Prox1, these budding cells adopt a lymphatic vasculature phenotype.     

Susceptibility to T cell-mediated injury in immune complex disease is linked to local activation of renin-angiotensin system: the role of NF-AT pathway

FcR provides a critical link between ligands and effector cells in immune complex diseases. Emerging evidence reveals that angiotensin (Ang)II exerts a wide variety of cellular effects and contributes to the pathogenesis of inflammatory diseases. In anti-glomerular basement membrane Ab-induced glomerulonephritis (GN), we have previously noted that FcR-deficient mice (gamma(-/-)) surviving from lethal initial damage still developed mesangial proliferative GN, which was drastically prevented by an AngII type 1 receptor (AT1) blocker. We further examined the mechanisms by which renin-Ang system (RAS) participates in this immune disease. Using bone marrow chimeras between gamma(-/-) and AT1(-/-) mice, we found that glomerular injury in gamma(-/-) mice was associated with CD4(+) T cell infiltration depending on renal AT1-stimulation. Based on findings in cutaneous delayed-type hypersensitivity, we showed that AngII-activated renal resident cells are responsible for the recruitment of effector T cells. We next examined the chemotactic activity of AngII-stimulated mesangial cells, as potential mechanisms coupling RAS and cellular immunity. Chemotactic activity for T cells and Th1-associated chemokine (IFN-gamma-inducible protein-10 and macrophage-inflammatory protein 1alpha) expression was markedly reduced in mesangial cells from AT1(-/-) mice. Moreover, this activity was mainly through calcineurin-dependent NF-AT. Although IFN-gamma-inducible protein-10 was NF-kappaB-dependent, macrophage-inflammatory protein 1alpha was dominantly regulated by NF-AT. Furthermore, AT1-dependent NF-AT activation was observed in injured glomeruli by Southwestern histochemistry. In conclusion, our data indicate that local RAS activation, partly via the local NF-AT pathway, enhances the susceptibility to T cell-mediated injury in anti-glomerular basement membrane Ab-induced GN. This novel mechanism affords a rationale for the use of drugs interfering with RAS in immune renal diseases.     

3'-azido-3'-deoxy-5'-O-isonicotinoylthymidine, a new prodrug of zidovudine. Synthesis, solid state characterization, and anti HIV-1 activity

Synthesis, solid state characterization and anti HIV-1 activity of 3'-azido-3'-deoxy-5'-O-isonicotinoylthymidine (2), a new prodrug of zidovudine (AZT, 1), are described. Two solid forms of 2 prepared by crystallization from ethyl acetate-petroleum ether (form alpha) and from a melt sample of form alpha (amorphous form) were characterized by X-ray diffractometry, infrared spectroscopy, differential scanning calorimetry (DSC) and thermogravimetry (TGA) techniques. The novel nucleoside exhibited antiviral activity against standard and resistant strain panels of HIV-1 as well as cytotoxicity similar to that of AZT.     

Ontogeny of semaphorins 3A and 3F and their receptors neuropilins 1 and 2 in the kidney

Semaphorins 3A and 3F are axon guidance proteins during nervous system development. Their expression pattern and function outside the nervous system are unknown. Neuropilin 1 and 2 (NP-1, NP-2) are natural ligands for semaphorins 3A and 3F, respectively. NP-1 is also a co-receptor for vascular endothelial growth factor (VEGF) required for normal vascular development. We showed that VEGF is a direct chemoattractant for glomerular endothelial cells towards developing nephrons. To examine whether semaphorins could modulate VEGF endothelial cell guidance cues in the developing kidney, we studied the expression of semaphorin 3A and semaphorin 3F and their receptors NP-1 and NP-2 in the kidney during ontogeny using Northern blot analysis, in situ hybridization, Western blot analysis and immunohistochemistry. All four genes are developmentally regulated, with abundant expression during organogenesis and downregulation in the adult kidney. Semaphorin 3A and 3F are expressed by podocytes and tubules whereas their receptors NP-1 and NP-2 are localized to endothelial cells. In vitro, renal tubular epithelial cell lines (tsMPT, IRPT and MDCK) and glomerular endothelial cells express both semaphorins and their receptors, suggesting the presence of an autocrine system. The distribution of the receptors NP-1 and NP-2 in endothelial cells and developing vessels is complementary to that of the ligands in adjacent epithelial cells during kidney development. The sum of the guidance cues provided by VEGF and semaphorins 3A and 3F may be important determinants of the pattern of endothelial cell migration during kidney morphogenesis.     

Chronic bronchial allergic inflammation increases alveolar liquid clearance by TNF-alpha -dependent mechanism

Bronchial inflammation in allergic asthma is associated with active exudation from the bronchial tree into the interstitial space of both mucosa and submucosa. The aim of this study was to evaluate epithelial and endothelial permeability as well as alveolar fluid movement in a model of chronic allergic inflammation in Brown-Norway rats sensitized and challenged with ovalbumin (OA). Control groups were challenged with saline solution (C), and rats were immunized by OA but not challenged (Se). Lung sections showed a marked inflammatory infiltrate associated with perivascular and peribronchiolar edema in OA. To measure alveolar liquid clearance, a 5% bovine albumin solution with 1 microCi of (125)I-labeled human albumin was instilled into the air spaces. Alveolar-capillary barrier permeability was evaluated by intravascular injection of 1 microCi of (131)I-labeled albumin. Endothelial permeability was significantly increased in OA, from 0.08 +/- 0.01 in the C group to 0.19 +/- 0.03 in OA group (P < 0.05). Final-to-initial protein ratio was also statistically higher in OA (1.6 +/- 0.05) compared with C (1.38 +/- 0.03, P = 0.01) and Se groups (1.42 +/- 0.03, P = 0.04). Administration of anti-tumor necrosis factor-alpha antibodies within the instillate significantly decreased this ratio (1.32 +/- 0.08, P = 0.003 vs. OA). To conclude, we demonstrated a tumor necrosis factor-alpha-dependent increase in alveolar fluid movement in a model of severe bronchial allergic inflammation associated with endothelial and epithelial leakage.