Systematic evasion of the restriction-modification barrier in bacteria.

Bacteria that are recalcitrant to genetic manipulation using modern in vitro techniques are termed genetically intractable. Genetic intractability is a fundamental barrier to progress that hinders basic, synthetic, and translational microbiology research and development beyond a few model organisms. The most common underlying causes of genetic intractability are restriction-modification (RM) systems, ubiquitous defense mechanisms against xenogeneic DNA that hinder the use of genetic approaches in the vast majority of bacteria and exhibit strain-level variation.

Restriction-modification mediated barriers to exogenous DNA uptake and incorporation employed by Prevotella intermedia.

Prevotella intermedia, a major periodontal pathogen, is increasingly implicated in human respiratory tract and cystic fibrosis lung infections. Nevertheless, the specific mechanisms employed by this pathogen remain only partially characterized and poorly understood, largely due to its total lack of genetic accessibility. Here, using Single Molecule, Real-Time (SMRT) genome and methylome sequencing, bisulfite sequencing, in addition to cloning and restriction analysis, we define the specific genetic barriers to exogenous DNA present in two of the most widespread laboratory strains, P.

DC-STAMP Is an Osteoclast Fusogen Engaged in Periodontal Bone Resorption.

Dendritic cell-specific transmembrane protein (DC-STAMP) plays a key role in the induction of osteoclast (OC) cell fusion, as well as DC-mediated immune regulation. While DC-STAMP gene expression is upregulated in the gingival tissue with periodontitis, its pathophysiological roles in periodontitis remain unclear. To evaluate the effects of DC-STAMP in periodontitis, anti-DC-STAMP-monoclonal antibody (mAb) was tested in a mouse model of ligature-induced periodontitis ( n = 6-7/group) where Pasteurella pneumotropica ( Pp)-reactive immune response activated T cells to produce receptor activator of nuclear factor kappa-B ligand (RANKL), which, in turn, promotes the periodontal bone loss via upregulation of osteoclastogenesis.

Modulation of infection-mediated migration of neutrophils and CXCR2 trafficking by osteopontin.

Osteopontin (OPN) is a pro-inflammatory protein that paradoxically protects against inflammation and bone destruction in a mouse model of endodontic infection. Here we have tested the hypothesis that this effect of OPN is mediated by effects on migration of innate immune cells to the site of infection. Using the air pouch as a model of endodontic infection in mice, we showed that neutrophil accumulation at the site of infection with a mixture of endodontic pathogens is significantly reduced in OPN-deficient mice.

Osteopontin in Immune-mediated Diseases.

Since its initial identification as one of the genes most highly upregulated upon T-cell activation, osteopontin (or Eta-1, as it was designated then) has been demonstrated to have many roles in the regulation of the immune response on multiple levels. It contributes to the development of immune-mediated and inflammatory diseases, and it regulates the host response to infection. In some cases, the mechanisms of these effects have been elucidated, while other mechanistic functions of the protein remain obscure.

Early Cytokine Response to Infection with Pathogenic vs Non-Pathogenic Organisms in a Mouse Model of Endodontic Infection.

Using the subcutaneous chamber model of infection, we showed previously that a mixture of four endodontic pathogens (EP: P. intermedia, F. nucleatum, S.

Osteopontin binding to the alpha 4 integrin requires highest affinity integrin conformation, but is independent of post-translational modifications of osteopontin.

Osteopontin (OPN) is a ligand for the α4ß1 integrin, but the physiological importance of this binding is not well understood. Here, we have assessed the effect of post-translational modifications on OPN binding to the α4 integrin on cultured human leukocyte cell lines and compared OPN interaction with α4 integrin to that of VCAM and fibronectin. Jurkat cells, whose α4 integrins are inherently activated, adhered to different preparations of OPN in the presence of Mn(2+): the EC50 of adhesion was not affected by phosphorylation or glycosylation status.

Hindlimb-unloading suppresses B cell population in the bone marrow and peripheral circulation associated with OPN expression in circulating blood cells.

Rodent hindlimb unloading (HU) by tail-suspension is a model to investigate disuse-induced bone loss in vivo. Previously, we have shown that osteopontin (OPN, also known as Spp1) is required for unloading-induced bone loss. However, how unloading affects OPN expression in the body is not fully understood.

Suppression of tumour growth by orally administered osteopontin is accompanied by alterations in tumour blood vessels.

Background: The integrin-binding protein osteopontin is strongly associated with tumour development, yet is an abundant dietary component as a constituent of human and bovine milk. Therefore, we tested the effect of orally administered osteopontin (o-OPN) on the development of subcutaneous tumours in mice.

Methods: Bovine milk osteopontin was administered in drinking water to tumour-bearing immune-competent mice.

Osteopontin regulates interleukin-17 production in hepatitis.

The overexpression of osteopontin is associated with various inflammatory liver diseases. Interestingly, each of these diseases is also associated with IL-17 expression. Therefore, we sought to determine whether there is any mechanistic link between osteopontin and IL-17.

Osteopontin Deficiency Suppresses Tumor Necrosis Factor-α-Induced Apoptosis in Chondrocytes.

Objective: Apoptosis of chondrocytes in articular cartilage has been observed in rheumatoid arthritis patients. However, molecules involved in such chondrocyte apoptosis in arthritic joints have not been fully understood. We previously observed that apoptosis of chondrocytes is enhanced in a murine arthritis model induced by injection with anti-type II collagen antibodies and lipopolysaccharide (mAbs/LPS), and osteopontin (OPN) deficiency suppresses chondrocyte apoptosis in this arthritis model in vivo.

Osteopontin in macrophage function.

The secreted phosphorylated protein osteopontin (OPN) is expressed in a variety of tissues and bodily fluids, and is associated with pathologies including tissue injury, infection, autoimmune disease and cancer. Macrophages are ubiquitous, heterogeneous cells that mediate aspects of cell and tissue damage in all these pathologies. Here, the role of OPN in macrophage function is reviewed.

Osteopontin deficiency enhances parathyroid hormone/ parathyroid hormone related peptide receptor (PPR) signaling-induced alteration in tooth formation and odontoblastic morphology.

Parathyroid hormone/parathyroid hormone-related protein receptor (PPR) signaling is known to be involved in tooth development. In bone, extracellular matrix protein osteopontin (OPN) is a negative regulator of PPR signaling in bone formation. However, the role of OPN in modulation of PPR action in tooth development is not understood.

Osteopontin expressed in tubular epithelial cells regulates NK cell-mediated kidney ischemia reperfusion injury.

Renal ischemia reperfusion injury (IRI) occurs after reduced renal blood flow and is a major cause of acute injury in both native and transplanted kidneys. Studies have shown diverse cell types in both the innate and the adaptive immune systems participate in kidney IRI as dendritic cells, macrophages, neutrophils, B cells, CD4(+) NK(+) cells, and CD4(+) T cells all contribute to this form of injury. Recently, we have found that NK cells induce apoptosis in tubular epithelial cells (TECs) and also contribute to renal IRI.

Osteopontin deficiency impairs wear debris-induced osteolysis via regulation of cytokine secretion from murine macrophages.

Objective: To investigate the molecular mechanisms underlying particle-induced osteolysis, we focused on osteopontin (OPN), a cytokine and cell-attachment protein that is associated with macrophage chemoattractant and osteoclast activation.

Methods: We compared OPN protein levels in human periprosthetic osteolysis tissues with those in osteoarthritis (OA) synovial tissues. To investigate the functions of OPN during particle-induced osteolysis in vivo, titanium particles were implanted onto the calvaria of OPN-deficient mice and their wild-type (WT) littermates.

Regulatory role of DC-derived osteopontin in systemic allergen sensitization.

Osteopontin (OPN) is a secreted phosphoglycoprotein with a wide range of functions, and is involved in various pathophysiological conditions. However, the role of OPN in IgE and Th2-associated allergic responses remains incompletely defined. The aim of this study was to elucidate the role of OPN in systemic allergen sensitization in mice.

Protective role of osteopontin in endodontic infection.

Endodontic infections are polymicrobial infections resulting in bone destruction and tooth loss. The host response to these infections is complex, including both innate and adaptive mechanisms. Osteopontin (OPN), a secreted, integrin-binding protein, functions in the regulation of immune responses and enhancement of leucocyte migration.

Bone sialoprotein, but not osteopontin, deficiency impairs the mineralization of regenerating bone during cortical defect healing.

Bone healing is a complex multi-step process, which depends on the position and size of the lesion, and on the mechanical stability of the wounded area. To address more specifically the mechanisms involved in cortical bone healing, we created drill-hole defects in the cortex of mouse femur, a lesion that triggers intramembranous repair, and compared the roles of bone sialoprotein (BSP) and osteopontin (OPN), two proteins of the extracellular matrix, in the repair process. Bone regeneration was analyzed by ex vivo microcomputerized X-ray tomography and histomorphometry of bones of BSP-deficient, OPN-deficient and wild-type mice.

Impaired angiogenic response in the corneas of mice lacking osteopontin.

Purpose: To investigate the effects of loss of osteopontin (OPN) in the development of neovascularization in corneal stroma in mice. Cell culture study was also conducted to clarify the effects of OPN in transforming growth factor (TGF) beta1-driven cell signaling and expression of vascular endothelial growth factor (VEGF).

Methods: Ocular fibroblasts from wild-type and OPN-null mice were used to study the role of OPN in TGFbeta1 signal and VEGF expression.

Accelerated development of aging-associated and instability-induced osteoarthritis in osteopontin-deficient mice.

Objective: To investigate the role of osteopontin (OPN) in the development of osteoarthritis (OA) under in vivo and in vitro conditions.

Methods: Both instability-induced and aging-associated OA models were generated using OPN-deficient (OPN-/-) and control wild-type (WT) mice. An in vitro cartilage degradation model was also used, to evaluate the effect of OPN on proteoglycan loss from joint cartilage.

Transforming growth factor-beta1 regulation of ATF-3 and identification of ATF-3 target genes in breast cancer cells.

Transforming growth factor-beta1 (TGF-beta1) is a crucial molecule for stimulation of breast cancer invasion and formation of bone metastases. The molecular mechanisms of how TGF-beta1 mediates these effects have yet to be completely determined. We have found that activating transcription factor-3 (ATF-3) is strongly stimulated and its level is sustained by TGF-beta1 in highly invasive and metastatic human breast cancer (MDA-MB231) and in mouse mammary pad tumor cells (r3T).

Both cell-surface and secreted CSF-1 expressed by tumor cells metastatic to bone can contribute to osteoclast activation.

Tumors metastatic to the bone produce factors that cause massive bone resorption mediated by osteoclasts in the bone microenvironment. Colony stimulating factor (CSF-1) is strictly required for the formation and survival of active osteoclasts, and is frequently produced by tumor cells. Here we hypothesize that the CSF-1 made by tumor cells contributes to bone destruction in osteolytic bone metastases.

Effect of osteopontin on diarrhea duration and innate immunity in suckling mice infected with a murine rotavirus.

We studied the role of osteopontin (OPN) in host responses against rotavirus (RV) infection. OPN knockout (OPN-KO) suckling mice were more susceptible to RV (strain EW) infection and showed prolonged diarrhea duration compared to wild-type (WT) suckling mice. OPN in the small intestine of WT mice was expressed after 48 h post-infection.