Sequential deglycosylation and utilization of the N-linked, complex-type glycans of human alpha1-acid glycoprotein mediates growth of Streptococcus oralis

Streptococcus oralis is the agent of a large number of infections in immunocompromised patients, but little is known regarding the mechanisms by which this fermentative organism proliferates in vivo. Glycoproteins are widespread within the circulation and host tissues, and could provide a source of fermentable carbohydrate for the growth of those pathogenic organisms with the capacity to release monosaccharides from glycans via the production of specific glycosidases. The ability of acute phase serum alpha1-acid glycoprotein to support growth of S.oralis in vitro has been examined as a model for growth of this organism on N-linked glycoproteins. Growth was accompanied by the production of a range of glycosidases (sialidase, N-acetyl-beta-D-glucosaminidase, and beta-D-galactosidase) as measured using the 4-methylumbelliferone-linked substrates. The residual glycoprotein glycans remaining during growth of this organism were released by treatment with hydrazine and their analysis by HPAEC-PAD and MALDI demonstrated extensive degradation of all glycan chains with only terminal N-acetylglucosamine residues attached to asparagines of the protein backbone remaining when growth was complete. Monosaccharides were released sequentially from the glycans by S.oralis glycosidases in the order sialic acid, galactose, fucose, nonterminal N-acetylglucosamine, and mannose due to the actions of exo-glycosidic activities, including mannosidases which have not previously been reported for S.oralis. All released monosaccharides were metabolized during growth with the exception of fucose which remained free in culture supernatants. Direct release of oligosaccharides was not observed, indicating the absence of endo-glycosidases in S.oralis. We propose that this mechanism of deglycosylation of host glycoproteins and the subsequent utilization of released monosaccharides is important in the survival and persistence of this and other pathogenic bacteria in vivo.     

Th2-induced airway mucus production is dependent on IL-4Ralpha, but not on eosinophils

Mucus hyperproduction in asthma results from airway inflammation and contributes to clinical symptoms, airway obstruction, and mortality. In human asthmatics and in animal models, excess mucus production correlates with airway eosinophilia. We previously described a system in which TCR transgenic CD4 Th2 cells generated in vitro were transferred into recipient mice and activated in the respiratory tract with inhaled Ag. Th2 cells stimulated airway eosinophilia and a marked increase in mucus production, while mice that received Th1 cells exhibited airway inflammation without eosinophilia or mucus. Mucus could be induced by IL-4-/- Th2 cells at comparable levels to mucus induced by IL-4+/+ Th2 cells. In the current studies we dissect further the mechanisms of Th2-induced mucus production. When IL-4-/- Th2 cells are transferred into IL-4Ralpha-/- mice, mucus is not induced, and BAL eosinophilia is absent. These data suggest that in the absence of IL-4, IL-13 may be critical for Th2-induced mucus production and eosinophilia. To determine whether eosinophils are important in mucus production, IL-5-/- Th2 cells were transferred into IL-5-/- recipients. Eosinophilia was abolished, yet mucus staining in the epithelium persisted. These studies show definitively that IL-5, eosinophils, or mast cells are not essential, but signaling through IL-4Ralpha is critically important in Th2 cell stimulation of mucus production.     

Vascular endothelial growth factor (VEGF) induces remodeling and enhances TH2-mediated sensitization and inflammation in the lung

Exaggerated levels of VEGF (vascular endothelial growth factor) are present in persons with asthma, but the role(s) of VEGF in normal and asthmatic lungs has not been defined. We generated lung-targeted VEGF(165) transgenic mice and evaluated the role of VEGF in T-helper type 2 cell (T(H)2)-mediated inflammation. In these mice, VEGF induced, through IL-13-dependent and -independent pathways, an asthma-like phenotype with inflammation, parenchymal and vascular remodeling, edema, mucus metaplasia, myocyte hyperplasia and airway hyper-responsiveness. VEGF also enhanced respiratory antigen sensitization and T(H)2 inflammation and increased the number of activated DC2 dendritic cells. In antigen-induced inflammation, VEGF was produced by epithelial cells and preferentially by T(H)2 versus T(H)1 cells. In this setting, it had a critical role in T(H)2 inflammation, cytokine production and physiologic dysregulation. Thus, VEGF is a mediator of vascular and extravascular remodeling and inflammation that enhances antigen sensitization and is crucial in adaptive T(H)2 inflammation. VEGF regulation may be therapeutic in asthma and other T(H)2 disorders.     

IL-13-induced chemokine responses in the lung: role of CCR2 in the pathogenesis of IL-13-induced inflammation and remodeling

IL-13 stimulates inflammatory and remodeling responses and contributes to the pathogenesis of human airways disorders. To further understand the cellular and molecular events that mediate these responses, we characterized the effects of IL-13 on monocyte chemotactic proteins (MCPs) and compared the tissue effects of transgenic IL-13 in mice with wild-type (+/+) and null (-/-) CCR2 loci. Transgenic IL-13 was a potent stimulator of MCP-1, -2, -3, and -5. This stimulation was not specific for MCPs because macrophage-inflammatory protein (MIP)-1alpha, MIP-1beta, MIP-2, MIP-3alpha, thymus- and activation-regulated chemokine, thymus-expressed chemokine, eotaxin, eotaxin 2, macrophage-derived chemokines, and C10 were also induced. The ability of IL-13 to increase lung size, alveolar size, and lung compliance, to stimulate pulmonary inflammation, hyaluronic acid accumulation, and tissue fibrosis, and to cause respiratory failure and death were markedly decreased, whereas mucus metaplasia was not altered in CCR2(-/-) mice. CCR2 deficiency did not decrease the basal or IL-13-stimulated expression of target matrix metalloproteinases or cathepsins but did increase the levels of mRNA encoding alpha1-antitrypsin, tissue inhibitor of metalloproteinase-1, -2, and -4, and secretory leukocyte proteinase inhibitor. In addition, the levels of bioactive and total TGF-beta(1) were decreased in lavage fluids from IL-13 transgenic mice with -/- CCR2 loci. These studies demonstrate that IL-13 is a potent stimulator of MCPs and other CC chemokines and document the importance of MCP-CCR2 signaling in the pathogenesis of the IL-13-induced pulmonary phenotype.     

Pulmonary type II cell hypertrophy and pulmonary lipoproteinosis are features of chronic IL-13 exposure

Interleukin (IL)-13, a key mediator of Th2-mediated immunity, contributes to the pathogenesis of asthma and other pulmonary diseases via its ability to generate fibrosis, mucus metaplasia, eosinophilic inflammation, and airway hyperresponsiveness. In these studies, we compared surfactant accumulation in wild-type mice and mice in which IL-13 was overexpressed in the lung. When compared with littermate controls, transgenic animals showed alveolar type II cell hypertrophy under light and electron microscopy. Over time, their alveoli also filled with surfactant in a pulmonary alveolar proteinosis pattern. At the same time, prominent interstitial fibrosis occurs. Bronchoalveolar lavage fluid from these mice had a three- to sixfold increase in surfactant phospholipids. Surfactant proteins (SP)-A, -B, and -C showed two- to threefold increases, whereas SP-D increased 70-fold. These results indicate that IL-13 is a potent stimulator of surfactant phospholipid and surfactant accumulation in the lung. IL-13 may therefore play a central role in the broad range of chronic pulmonary conditions in which fibrosis, type II cell hypertrophy, and surfactant accumulation occur.     

Pathogenicity of Haemoproteus danilewskyi,Kruse, 1890, in blue jays (Cyanocitta cristata)

Although the impact of blood parasite infections on passerine birds is potentially great, little is known of their pathologic effects. We studied Haemoproteus danilewskyi in experimentally infected captive and naturally infected free-ranging blue jays (Cyanocitta cristata) to determine patterns of infection and examine the pathologic effects of the parasite on the host. Physiologic changes, such as elevated numbers of lymphocytes, heterophils, basophils, eosinophils, and monocytes and decreased packed cell volume in the peripheral blood were associated with the erythrocytic phase of experimental infections of captive juvenile jays. Sublethal pathologic changes associated with the pre-erythrocytic phase of infections were observed in the liver, lung, and spleen. Schizonts were observed in the pulmonary capillaries of a 1 yr old jay necropsied 31 days post-inoculation, but not in 20 juvenile jays necropsied 57 days post-inoculation. In free-ranging naturally infected jays plasma protein concentration increased with density of natural infections.     

1-Thio-beta-D-galactofuranosides: synthesis and evaluation as beta-D- galactofuranosidase inhibitors

Beta-D-galactofuranosidase is a good chemotherapeutic target for the design of inhibitors, since beta-D-galactofuranose is a constituent of important parasite glycoconjugates but is not present in the host mammals. With this aim, we have synthesized for the first time alkyl, benzyl and aryl 1-thio-beta-D-galactofuranosides by condensation of penta-O-benzoyl-alpha,beta-D-galactofuranose with the corresponding thiols, in the presence of SnCl4as catalyst. The complete chemical and spectroscopical characterization of these compounds showed that the reaction was stereoselective. Debenzoylation with sodium methoxide afforded the beta-S-galactofuranosides in high yield. The thioglycosides were tested as inhibitors of the beta-D- galactofuranosidase of Penicillium fellutanum, using for the first time 4-nitrophenyl-beta-D-galactofuranoside as chromogenic substrate. The 4- aminophenyl-1-thio-beta-D-galactofuranoside, obtained by catalytic hydrogenation of the nitrophenyl derivative, was the best inhibitor being then an adequate ligand for the preparation of an affinity phase aimed at the isolation of beta-d-galactofuranosidases from different sources. Also the inhibitory activity of d-galactono-1, 4-lactone was shown.      

Characterization of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> Beijing isolates from the Mediterranean area

Background  

The Beijing lineage of Mycobacterium tuberculosis is causing concern due to its global distribution and its involvement in severe outbreaks. Studies focused on this lineage are mainly restricted to geographical settings where its prevalence is high, whereas those in other areas are scarce. In this study, we analyze Beijing isolates in the Mediterranean area, where this lineage is not prevalent and is mainly associated with immigrant cases.     

Increased hyperoxia-induced mortality and acute lung injury in IL-13 null mice

IL-13 is a critical effector at sites of Th2 inflammation and remodeling. As a result, anti-IL-13-based therapies are being actively developed to treat a variety of diseases and disorders. However, the beneficial effects of endogenous IL-13 in the normal and diseased lung have not been adequately defined. We hypothesized that endogenous IL-13 is an important regulator of oxidant-induced lung injury and inflammation. To test this hypothesis, we compared the effects of 100% O(2) in mice with wild-type and null IL-13 loci. In this study, we demonstrate that hyperoxia significantly augments the expression of the components of the IL-13R, IL-13Ralpha1, and IL-4Ralpha. We also demonstrate that, in the absence of IL-13, hyperoxia-induced tissue inflammation is decreased. In contrast, in the IL-13 null mice, DNA injury, cell death, caspase expression, and activation and mortality are augmented. Interestingly, the levels of the cytoprotective cytokines vascular endothelial cell growth factor, IL-6, and IL-11 were decreased in the bronchoalveolar lavage fluid. These studies demonstrate that the expression of the IL-13R is augmented and that the endogenous IL-13-IL-13R pathway contributes to the induction of inflammation and the inhibition of injury in hyperoxic acute lung injury.     

Inhibition of NF-kappaB activation reduces the tissue effects of transgenic IL-13

IL-13 is a major Th2 cytokine that is capable of inducing inflammation, excessive mucus production, airway hyperresponsiveness, alveolar remodeling, and fibrosis in the murine lung. Although IL-13 through its binding to IL-4Ralpha/IL-13Ralpha1 uses the canonical STAT6-signaling pathway to mediate these tissue responses, recent studies have demonstrated that other signaling pathways may also be involved. Previous studies from our laboratory demonstrated that IL-13 mediates its tissue effects by inducing a wide variety of downstream genes many of which are known to be regulated by NF-kappaB. As a result, we hypothesized that NF-kappaB activation plays a critical role in the pathogenesis of IL-13-induced tissue alterations. To test this hypothesis, we compared the effects of transgenic IL-13 in mice with normal and diminished levels of NF-kappaB activity. Three pharmacologic approaches were used to inhibit NF-kappaB including 1) PS1145, a small molecule inhibitor of IkappaBalpha kinase (IKK2), 2) antennapedia-linked NF-kappaB essential modulator-binding domain (NBD) peptide (wild-type NBD), and 3) an adenoviral construct expressing a dominant-negative version of IKK2. We also crossed IL-13-transgenic mice with mice with null mutations of p50 to generate mice that overproduced IL-13 in the presence and absence of this NF-kappaB component. These studies demonstrate that all these interventions reduced IL-13-induced tissue inflammation, fibrosis and alveolar remodeling. In addition, we show that both PS1145 and wild-type NBD inhibit lung inflammatory and structural cell apoptosis. PS1145 inhibits caspase activation and up-regulates inhibitor of apoptosis protein cellular-inhibitor of apoptosis protein 1 (c-IAP-1). Therefore, NF-kappaB is an attractive target for immunotherapy of IL-13-mediated diseases.