MAB1951F Sigma-AldrichAnti-Integrin β1 Antibody, clone P4G11, FITC conjugated

Rickettsia conorii, a member of the spotted fever group (SFG) of the genus Rickettsia and causative agent of Mediterranean spotted fever, is an obligate intracellular pathogen capable of infecting various mammalian cell types. SFG rickettsiae express two major immunodominant surface cell antigen (Sca) proteins, OmpB (Sca5) and OmpA (Sca0). While OmpB-mediated entry has been characterized, the contribution of OmpA has not been well defined. Here we show OmpA expression in Escherichia coli is sufficient to mediate adherence to and invasion of non-phagocytic human endothelial cells. A recombinant soluble C-terminal OmpA protein domain (954-1735) with predicted structural homology to the Bordetella pertussis pertactin protein binds mammalian cells and perturbs R. conorii invasion by interacting with several mammalian proteins including β1 integrin. Using functional blocking antibodies, small interfering RNA transfection, and mouse embryonic fibroblast cell lines, we illustrate the contribution of α2β1 integrin as a mammalian ligand involved in R. conorii invasion of primary endothelial cells. We further demonstrate that OmpA-mediated attachment to mammalian cells is in part dependent on a conserved non-continuous RGD motif present in a predicted C-terminal 'pertactin' domain in OmpA.Our results demonstrate that multiple adhesin-receptor pairs are sufficient in mediating efficient bacterial invasion of R. conorii.

Previous studies had shown important functions of galectin 3 (Gal-3) in endometrium during embryo implantation, in regulation of endometrial cell proliferation and adhesion by interacting with integrin ?3. In this study, we investigated hormonal regulation of Gal-3 in trophoblasts and its extracellular effects on endometrium. We used BeWo and RL95-2 cells as a model of trophoblastic and endometrial epithelial cells, respectively, to create an in vivo model of embryo implantation. Our results indicated that 17?-estradiol (E2), progesterone (P4), and human chorionic gonadotropin (hCG) induced the expression of Gal-3 and promoted its secretion from BeWo cells. The exogenous Gal-3 inhibited cell proliferation and induced apoptosis of endometrial cells (RL95-2 cells) through activation of integrin ?1. We further validated the proapoptotic effect of Gal-3 secreted by trophoblastic cell on endometrial cells by culturing RL95-2 cells with Bewo cells and measuring the apoptotic rate. Our analysis provides new insight into the critical roles of Gal-3 in embryo implantation.

To explore the role of integrin alpha5beta1 in proliferative vitreoretinopathy (PVR) pathogenesis by evaluating the expression alpha5beta1 on ARPE-19 cells and patient proliferative membranes, quantifying the inhibitory effects of JSM6427 (a small molecule alpha5beta1 inhibitor) on ARPE-19 cell adhesion and migration, and assessing the therapeutic potential of JSM6427 in a rabbit retinal detachment model.Expression of alpha5beta1 was evaluated on activated ARPE-19 cells by flow cytometry and on PVR membranes by immunohistochemistry. ARPE-19 cells were used in fibronectin-dependent adhesion and migration assays with various concentrations of JSM6427; IC(50) was calculated. In the rabbit model, eyes were intravitreally injected with vehicle or JSM6427 on day 0 or 1 after retinal detachment; BrdU was administered intravitreally on day 3, and retinal tissues were harvested on day 3 (4 hours later) or 7. Retinal scarring, cellular proliferation, and inflammatory responses were quantified, and retinal morphology was analyzed in retinal sections.Activated ARPE-19 cells and PVR membranes expressed high levels of alpha5beta1; expression was low in control eyes. JSM6427 provided a dose-dependent blockade of ARPE-19 cell adhesion to fibronectin (IC(50), 7.1 +/- 2.5 microM) and inhibition of migration (IC(50), 6.0 +/- 4.5 microM). In the rabbit model, intravitreal injection of JSM6427 provided significant inhibition of proliferation of retinal cells (Müller cells, microglia, and macrophages) on days 3 and 7 after detachment and inhibition of inflammatory response and retinal scarring on day 7 after detachment.JSM6427 is a promising treatment for PVR, with data suggesting that inhibition of alpha5beta1-fibronectin interactions addresses multiple pathways involving retinal pigment epithelial, glial, and inflammatory cells.

BACKGROUND: Galectin-3 (gal-3) is a beta-galactoside-binding protein which can be detected in endometrium. The study was designed to investigate synergism of gal-3 and integrinbeta3 in endometrial cell proliferation and adhesion in an in vitro model of endometrial receptivity. METHODS: The RL95-2 cell line was employed as an in vitro model for receptive endometrium. Cells transfected with gal-3 siRNA or treated with exogenous gal-3 were incubated with or without function-blocking integrinbeta1/3 antibody for evaluating synergism of gal-3 and integrins on cell proliferation and adhesion. Proliferation was measured by BrdU incorporation, and adhesion to fibronectin (FN) was determined by an adhesion assay. Integrin expression was analyzed by Flow Cytometry and western blots. Bewo spheroids were co-cultured with the RL95-2 monolayer to mimic the blastocyst-endometrial interaction, and colocalization of gal-3, integrinbeta3 and FN at the interface was observed by confocal microscopy. RESULTS: The knock-down of gal-3 inhibited RL95-2 cell proliferation and adhesion. However, a reduction of proliferation and adhesion was also observed in presence of exogenous gal-3, and this was further reduced by a functional block to integrinbeta3. Moreover, gal-3 knock-down significantly increased integrinbeta3 expression, however, the colocalization of integrinbeta3 and FN was not increased. As expected, the colocalization of integrinbeta3 was decreased with the knock-down of gal-3. CONCLUSIONS: This study has provided an in vitro model for the complex interactions between gal-3 and integrinbeta3 in the regulation of endometrial cell proliferation and adhesion.

To study a step of the very complex processes of the formation of the neuromuscular junction (NMJ), we have analysed the clustering of acetylcholine receptors (AChR) and acetylcholinesterase (AChE) in myotubes cultured in various conditions. On the surface of rat myotubes cultured in the presence of spinal cord cells from embryonic rat, numerous AChE clusters appeared. Such clusters are always co-localized with AChR clusters, but the reverse is not true: the number of AChR clusters largely exceeds that of AChE clusters. Very few AChE clusters formed when such co-cultures were treated with monoclonal antibodies (mAbs) against the main immunogenic region (MIR) of the AChR, which provoke internalization and degradation of the AChRs of the muscular membrane. The total levels of AChE and proportions of molecular forms were unaffected. We also used non-innervated myotubes in which addition of agrin, a protein normally synthesized by motoneurons, transported to nerve terminals and inserted into the synaptic basal lamina, induces the formation of small clusters of AChE. When added to rat myotubes devoid of membrane AChR, agrin-induced AChE clusters did not form. Finally, we analysed the capacity of the variant of the C2 mouse muscle cell line deficient in AChR (1R-) to form clusters of AChE in co-cultures with spinal cord cells from rat: no formation of AChE clusters could be observed. In all these different systems of cultures, the conditions which prevented clustering of AChR (anti-AChR antibodies, deficiency of the variant C2 cell line) also suppressed AChE clustering. We concluded that clustering of AChR is a prerequisite for clustering of AChE, so that NMJ formation implies the sequential accumulation of these two components.

The cutaneous T cell lymphomas (CTCL), typified by mycosis fungoides, and several chronic T cell mediated dermatoses are characterized by the migration of T lymphocytes into the epidermis (epidermotropism). Alternatively, other types of cutaneous inflammation (malignant cutaneous B cell lymphoma, CBCL, or lymphocytoma cutis, non-malignant T or B cell type) do not show evidence of epidermotropism. This suggests that certain T lymphocyte subpopulations are able to interact with and penetrate the epidermal basement membrane. We show here that T lymphocytes derived from patients with CTCL (HUT 78 or HUT 102 cells), adhere to the detergent-insoluble extracellular matrix prepared from cultured basal keratinocytes (HFK ECM). HUT cell adhesion to HFK ECM was inhibitable with monoclonal antibodies (mAbs) directed to the alpha 3 (P1B5) or beta 1 (P4C10) integrin receptors, and could be up-regulated by an activating anti-beta 1 mAb (P4G11). An inhibitory mAb, P3H9-2, raised against keratinocytes identified epiligrin as the ligand for alpha 3 beta 1 positive T cells in HFK ECM. Interestingly, two lymphocyte populations could be clearly distinguished relative to expression of alpha 3 beta 1 by flow cytometry analysis. Lymphokine activated killer cells, alloreactive cytotoxic T cells and T cells derived from patients with CTCL expressed high levels of alpha 3 beta 1 (alpha 3 beta 1high). Non-adherent peripheral blood mononuclear cells, acute T or B lymphocytic leukemias, or non-cutaneous T or B lymphocyte cell lines expressed low levels of alpha 3 beta 1 (alpha 3 beta 1low). Resting PBL or alpha 3 beta 1low T or B cell lines did not adhere to HFK ECM or purified epiligrin. However, adhesion to epiligrin could be up-regulated by mAbs which activate the beta 1 subunit indicating that alpha 3 beta 1 activity is a function of expression and affinity. In skin derived from patients with graft-vs.-host (GVH) disease, experimentally induced delayed hypersensitivity reactions, and CTCL, the infiltrating T cells could be stained with mAbs to alpha 3 or beta 1 and were localized in close proximity to the epiligrin-containing basement membrane. Infiltrating lymphocytes in malignant cutaneous B disease (CBCL) did not express alpha 3 beta 1 by immunohistochemical techniques and did not associate with the epidermal basement membrane. The present findings clearly define a function for alpha 3 beta 1 in T cells and strongly suggest that alpha 3 beta 1 interaction with epiligrin may be involved in the pathogenesis of cutaneous inflammation.