Characterization of the Mycobacterium avium subsp. paratuberculosis laminin-binding/histone-like protein (Lbp/Hlp) which reacts with sera from patients with Crohn's disease

Mycobacterium avium subsp. paratuberculosis (Map) causes a chronic enteric disease in ruminants, called paratuberculosis or Johne's disease. The current model proposes that after ingestion by the host, Map crosses the intestinal barrier via internalization by the M cells. Experimental observations suggest, however, that Map may also transcytose the intestinal wall via the enterocytes, but the mechanisms involved in this process remain poorly understood. Cytoadherence assays performed on epithelial cells with Map revealed that the addition of laminin to the cell culture increases adhesion. A Map protein was isolated by heparin-Sepharose chromatography and identified as a laminin-binding protein like. The gene encoding this protein named Lbp/Hlp was identified in the Map genome sequence at locus MAP3024 (annotated Hup B). The deduced Map Lbp/Hlp amino acid sequence reveals 80% identity with that reported for other mycobacteria. The C-terminal domain involved in adhesion is mainly composed of arginine and lysine residues modified by methylation. In vitro tests demonstrated that recombinant Lbp/Hlp binds laminin, heparin, collagen and epithelial cells. Interestingly, we found that this adhesin corresponds to the antigen described as the target of pANCA and serum antibodies of patients with Crohn's disease.     

The glycan shield of HIV is predominantly oligomannose independently of production system or viral clade

The N-linked oligomannose glycans of HIV gp120 are a target for both microbicide and vaccine design. The extent of cross-clade conservation of HIV oligomannose glycans is therefore a critical consideration for the development of HIV prophylaxes. We measured the oligomannose content of virion-associated gp120 from primary virus from PBMCs for a range of viral isolates and showed cross-clade elevation (62-79%) of these glycans relative to recombinant, monomeric gp120 (～30%). We also confirmed that pseudoviral production systems can give rise to notably elevated gp120 oligomannose levels (～98%), compared to gp120 derived from a single-plasmid viral system using the HIV(LAI) backbone (56%). This study highlights differences in glycosylation between virion-associated and recombinant gp120.     

Ex vivo imaging of T cells in murine lymph node slices with widefield and confocal microscopes

Naïve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration of T cells into lymph nodes is a complex process which involves both cellular and chemical factors including chemokines. Recently, the use of two-photon microscopy has permitted to track T cells in intact lymph nodes and to derive some quantitative information on their behavior and their interactions with other cells. While there are obvious advantages to an in vivo system, this approach requires a complex and expensive instrumentation and provides limited access to the tissue. To analyze the behavior of T cells within murine lymph nodes, we have developed a slice assay ¹, originally set up by neurobiologists and transposed recently to murine thymus ². In this technique, fluorescently labeled T cells are plated on top of an acutely prepared lymph node slice. In this video-article, the localization and migration of T cells into the tissue are analyzed in real-time with a widefield and a confocal microscope. The technique which complements in vivo two-photon microscopy offers an effective approach to image T cells in their natural environment and to elucidate mechanisms underlying T cell migration.     

The relationship of the oral microbiotia to periodontal health and disease

The oral microbial community represents the best-characterized consortium associated with the human host. There are strong correlations between the qualitative composition of the oral microbiota and clinically healthy or diseased states. However, additional studies are needed to elucidate the mechanisms that define these microbial/host relationships.     

Independent roles of apical membrane antigen 1 and rhoptry neck proteins during host cell invasion by apicomplexa

During invasion, apicomplexan parasites form an intimate circumferential contact with the host cell, the tight junction (TJ), through which they actively glide. The TJ, which links the parasite motor to the host cell cytoskeleton, is thought to be composed of interacting apical membrane antigen 1 (AMA1) and rhoptry neck (RON) proteins. Here we find that, in Plasmodium berghei, while both AMA1 and RON4 are important for merozoite invasion of erythrocytes, only RON4 is required for sporozoite invasion of hepatocytes, indicating that RON4 acts independently of AMA1 in the sporozoite. Further, in the Toxoplasma gondii tachyzoite, AMA1 is dispensable for normal RON4 ring and functional TJ assembly but enhances tachyzoite apposition to the cell and internalization frequency. We propose that while the RON proteins act at the TJ, AMA1 mainly functions on the zoite surface to permit correct attachment to the cell, which may facilitate invasion depending on the zoite-cell combination.     

Structure of urban movements: polycentric activity and entangled hierarchical flows

The spatial arrangement of urban hubs and centers and how individuals interact with these centers is a crucial problem with many applications ranging from urban planning to epidemiology. We utilize here in an unprecedented manner the large scale, real-time 'Oyster' card database of individual person movements in the London subway to reveal the structure and organization of the city. We show that patterns of intraurban movement are strongly heterogeneous in terms of volume, but not in terms of distance travelled, and that there is a polycentric structure composed of large flows organized around a limited number of activity centers. For smaller flows, the pattern of connections becomes richer and more complex and is not strictly hierarchical since it mixes different levels consisting of different orders of magnitude. This new understanding can shed light on the impact of new urban projects on the evolution of the polycentric configuration of a city and the dense structure of its centers and it provides an initial approach to modeling flows in an urban system.     

Fatty acid oxidation and its impact on response of spontaneously hypertensive rat hearts to an adrenergic stress: benefits of a medium-chain fatty acid

The spontaneously hypertensive rat (SHR) is a model of cardiomyopathy characterized by a restricted use of exogenous long-chain fatty acid (LCFA) for energy production. The aims of the present study were to document the functional and metabolic response of the SHR heart under conditions of increased energy demand and the effects of a medium-chain fatty acid (MCFA; octanoate) supplementation in this situation. Hearts were perfused ex vivo in a working mode with physiological concentrations of substrates and hormones and subjected to an adrenergic stimulation (epinephrine, 10 microM). (13)C-labeled substrates were used to assess substrate selection for energy production. Compared with control Wistar rat hearts, SHR hearts showed an impaired response to the adrenergic stimulation as reflected by 1) a smaller increase in contractility and developed pressure, 2) a faster decline in the aortic flow, and 3) greater cardiac tissue damage (lactate dehydrogenase release: 1,577 +/- 118 vs. 825 +/- 44 mU/min, P < 0.01). At the metabolic level, SHR hearts presented 1) a reduced exogenous LCFA contribution to the citric acid cycle flux (16 +/- 1 vs. 44 +/- 4%, P < 0.001) and an enhanced contribution of endogenous substrates (20 +/- 4 vs. 1 +/- 4%, P < 0.01); and 2) an increased lactate production from glycolysis, with a greater lactate-to-pyruvate production ratio. Addition of 0.2 mM octanoate reduced lactate dehydrogenase release (1,145 +/- 155 vs. 1,890 +/- 89 mU/min, P < 0.001) and increased exogenous fatty acid contribution to energy metabolism (23.7 +/- 1.3 vs. 15.8 +/- 0.8%, P < 0.01), which was accompanied by an equivalent decrease in unlabeled endogenous substrate contribution, possibly triglycerides (11.6 +/- 1.5 vs. 19.0 +/- 1.2%, P < 0.01). Taken altogether, these results demonstrate that the SHR heart shows an impaired capacity to withstand an acute adrenergic stress, which can be improved by increasing the contribution of exogenous fatty acid oxidation to energy production by MCFA supplementation.     

Collagen and elastin cross-linking: a mechanism of constrictive remodeling after arterial injury

Constrictive remodeling after arterial injury is related to collagen accumulation. Cross-linking has been shown to induce a scar process in cutaneous wound healing and is increased after arterial injury. We therefore evaluated the effect of cross-linking inhibition on qualitative and quantitative changes in collagen, elastin, and arterial remodeling after balloon injury in the atherosclerotic rabbit model. Atherosclerotic-like lesions were induced in femoral arteries of 28 New Zealand White rabbits by a combination of air desiccation and a high-cholesterol diet. After 1 mo, balloon angioplasty was performed in both femoral arteries. Fourteen rabbits were fed beta-aminopropionitrile (beta-APN, 100 mg/kg) and compared with 14 untreated animals. The remodeling index, i.e., the ratio of external elastic lamina at the lesion site to external elastic lamina at the reference site, was determined 4 wk after angioplasty for both groups. Pyridinoline was significantly decreased in arteries from beta-APN-treated animals compared with controls, confirming inhibition of collagen cross-linking: 0.30 (SD 0.03) and 0.52 (SD 0.02) mmol/mol hydroxyproline, respectively (P = 0.002). Scanning and transmission electron microscopy showed a profound disorganization of collagen fibers in arteries from beta-APN-treated animals. The remodeling index was significantly higher in beta-APN-treated than in control animals [1.1 (SD 0.3) vs. 0.8 (SD 0.3), P = 0.03], indicating favorable remodeling. Restenosis decreased by 33% in beta-APN-treated animals: 32% (SD 16) vs. 48% (SD 24) (P = 0.02). Neointimal collagen density was significantly lower in beta-APN-treated animals than in controls: 23.0% (SD 3.8) vs. 29.4% (SD 4.0) (P = 0.004). These findings suggest that collagen and elastin cross-linking plays a role in the healing process via constrictive remodeling and restenosis after balloon injury in the atherosclerotic rabbit model.     

Mitochondrial respiratory chain and NAD(P)H oxidase are targets for the antiproliferative effect of carbon monoxide in human airway smooth muscle

Carbon monoxide (CO), one of the end products of heme oxygenase activity, inhibits smooth muscle proliferation by decreasing ERK1/2 phosphorylation and cyclin D1 expression, a signaling pathway that is known to be modulated by reactive oxygen species (ROS) in airway smooth muscle cells (ASMCs). Two important sources of ROS involved in cell signaling are the membrane NAD(P)H oxidase and the mitochondrial respiratory chain. Thus, that CO could modulate redox signaling in ASMCs by interacting with the heme moiety of NAD(P)H oxidase and/or the respiratory chain is a plausible hypothesis. Here we show that a recently identified carbon monoxide-releasing molecule, [Ru(CO)3Cl2]2 (or CORM-2) 1) inhibits NAD(P)H oxidase cytochrome b558 activity, 2) increases oxidant production by the mitochondria, and 3) inhibits ASMC proliferation and phosphorylation of the ERK1/2 mitogen-activated protein kinase and expression of cyclin D1, two critical pathways involved in muscle proliferation. No such effects were observed with the negative control (Ru(Me2SO)4Cl2), which does not contain CO groups. Because both diphenylene iodinium or apocynin (inhibitors of NAD(P)H oxidase) and rotenone (a molecule that increases mitochondrial ROS production by blocking the respiratory chain) mimicked the effect of CORM-2 on cyclin D1 expression and ASMC proliferation, the antiproliferative effect of CORM-2 is probably related to inhibition of cytochromes on both NAD(P)H oxidase and the respiratory chain. The involvement of increased mitochondria-derived oxidants is substantiated by the findings showing that the antioxidant N-acetylcysteine partially inhibited the effects of CORM-2. This study provides a new mechanism to explain redox signaling by CO.