Staphylococcus aureus is the principal causative agent of the inflammatory bone disease osteomyelitis. Unfortunately, the pathogenesis of this often chronic infection is poorly understood and is complicated by the recent observation that bone-forming osteoblasts can harbor S. aureus. Such an infection presents a significant challenge for the host immune response, because osteoblasts are not known to initiate protective cell-mediated immune responses. Cultured mouse and human osteoblasts infected with S. aureus were found to express high levels of interleukin (IL)-6 and IL-12p75, on the basis of complementary investigations demonstrating both S. aureus-induced up-regulation of expression of IL-6 and IL-12p40 mRNA and secretion of IL-6 and IL-12p75 by these cells. Additionally, a quantitative bioassay demonstrated that IL-12p75 secreted after infection was biologically active. These studies are the first to demonstrate induced IL-12p75 expression by osteoblasts and suggest a previously unrecognized role for osteoblasts in initiating immune responses after S. aureus infection.

.48), which negatively regulates insulin signaling by dephosphorylating the phosphotyrosine residues of the insulin receptor kinase activation segment. PTPN1 is located in 20q13, a genomic region linked to type 2 diabetes in multiple genetic studies. Surveys of the gene have previously identified only a few uncommon coding single nucleotide polymorphisms (SNPs). We have carried out a detailed association analysis of 23 noncoding SNPs spanning the 161-kb genomic region, which includes the PTPN1 gene. These SNPs have been assessed for association with type 2 diabetes in two independently ascertained collections of Caucasian subjects with type 2 diabetes and two control groups. Association is observed between multiple SNPs and type 2 diabetes. The most consistent evidence for association occurred with SNPs spanning the 3 end of intron 1 of PTPN1 through intron 8 (P values ranging from 0.043 to 0.004 in one case-control set and 0.038 -0.002 in a second case-control set). Analysis of the combined case-control data increased the evidence of SNP association with type 2 diabetes (P ‫؍‬ 0.005-0.0016). All of the associated SNPs lie in a single 100-kb haplotype block that encompasses the PTPN1 gene. Analysis of haplotypes indicates a significant difference between haplotype frequencies in type 2 diabetes case and control subjects (P ‫؍‬ 0.0035-0.0056), with one common haplotype (36%) contributing strongly to the evidence for association with type 2 diabetes. Odds ratios calculated from single SNP or haplotype data are in the proximity of 1.3. Haplotype-based calculation of population-attributable risk (PAR) results in an estimated PAR of 17-20% based on different models and assumptions. These results suggest that PTPN1 is a significant contributor to type 2 diabetes susceptibility in the Caucasian population. This risk is likely due to noncoding polymorphisms. Diabetes 53:3007-3012, 2004

P rotein phosphorylation at tyrosine is a key regulatory event that modulates intracellular signaling pathways involved in signal transduction. Protein tyrosine phosphatase (PTP)-1B is a ubiquitously expressed protein (1) that catalyzes the dephosphorylation of proteins at tyrosyl residues. PTP-1B has been implicated (2-4) in negatively regulating insulin signaling by dephosphorylating the phosphotyrosine residues of the insulin receptor kinase activation segment of the insulin receptor. In mouse models, disruption of the PTPN1 gene resulted in increased insulin sensitivity and resistance to diet-induced obesity (5,6). Further evidence for the role of PTP-1B in insulin sensitivity is seen in knockout mice, in which there was increased phosphorylation of the insulin receptor in liver and muscle tissue (5,6). These observations suggest that PTP-1B plays a role in modulating signal transduction, and defects in PTP-1B expression could lead to insulin resistance.The 10 exons of PTPN1 span Ͼ74 kb of chromosome 20q13.13, with the first intron containing Ͼ50 kb of the sequence. Several investigators (7-9) have searched PTPN1 for DNA sequence variants, e.g., single nucleotide polymorphisms (SNPs). Variation within the coding region of PTPN1 is relatively uncommon. Echwald et al. (7) identified a P387L variant that was found in 2.6% of type 2 diabetic individuals and 1% of healthy control subjects, which showed evidence of impaired in vitro serine phosphorylation of the PTP-1B peptide. Mok et al. (8) identified a 981C3 T polymorphism (5% minor allele frequency) that corresponded to a silent mutation in the PTP-1B protein

Two common pathogens of bone, Staphylococcus aureus and Salmonella, were investigated for their ability to induce chemokine expression in bone-forming osteoblasts. Cultured mouse or human osteoblasts could rapidly respond to bacterial infection by upregulating the mRNA encoding the chemokine, monocyte chemoattractant protein-1 (MCP-1). This rapid induction occurred on infection with either the gram-positive pathogen, S. aureus, or the gram-negative pathogen, Salmonella. Increased mRNA expression translated into MCP-1 secretion by cultured mouse or human osteoblasts in response to viable bacteria, whereas UV-killed bacteria were less effective in stimulating chemokine secretion. There was a dose-response relationship observed between the amount of input bacteria and increases in MCP-1 secretion. Immunohistochemical staining of infected osteoblasts indicated that the majority of cells could express MCP-1, with some osteoblasts having a higher intensity of staining than others. Organ cultures of mouse calvaria (skullcap) bone showed increases in MCP-1 immunostaining following bacterial infection. The immunoreactive MCP-1 in infected calvaria localized to areas containing active osteoblasts. Taken together, these studies demonstrate a conserved osteoblast-derived MCP-1 response to two very different pathogens of bone.

Staphylococcus aureus and Salmonella spp. are common causes of bone diseases; however, the immune response during such infections is not well understood. Colony-stimulating factors (CSF) have a profound influence on osteoclastogenesis, as well as the development of immune responses following infection. Therefore, we questioned whether interaction of osteoblasts with two very different bacterial pathogens could affect CSF expression by these cells. Cultured mouse and human osteoblasts were exposed to various numbers of S. aureus or Salmonella dublin bacteria, and a comprehensive analysis of granulocyte-macrophage (GM)-CSF, granulocyte (G)-CSF, macrophage (M)-CSF, and interleukin-3 (IL-3) mRNA expression and cytokine secretion was performed. Expression of M-CSF and IL-3 mRNAs by mouse osteoblasts was constitutive and did not increase significantly following bacterial exposure. In contrast, GM-CSF and G-CSF mRNA expression by mouse osteoblasts was dramatically upregulated following interaction with either viable S. aureus or Salmonella. This increased mRNA expression also translated into high levels of GM-CSF and G-CSF secretion by mouse and human osteoblasts following bacterial exposure. Viable S. aureus and Salmonella induced maximal levels of CSF mRNA expression and cytokine secretion compared to UV-killed bacteria. Furthermore, GM-CSF and G-CSF mRNA expression could be induced in unexposed osteoblasts separated by a permeable Transwell membrane from bacterially exposed osteoblasts. M-CSF secretion was increased in cultures of exposed human osteoblasts but not in exposed mouse osteoblast cultures. Together, these studies are the first to define CSF expression and suggest that, following bacterial exposure, osteoblasts may influence osteoclastogenesis, as well as the development of an immune response, via the production of these cytokines.

Staphylococcus aureus and Salmonella enterica serovar Dublin invade osteoblasts and are causative agents of human bone disease. In the present study, we examined the ability of S. aureus and Salmonella serovar Dublin to induce the production of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) by normal osteoblasts. Normal mouse and human osteoblasts were cocultured with S. aureus or Salmonella serovar Dublin at different multiplicities of infection. Following initial incubation and examination of TRAIL expression, extracellular bacteria were killed by the addition of media containing the antibiotic gentamicin. Lysates and conditioned media from osteoblast cultures were then collected at various times following invasion and analyzed. The results demonstrated that S. aureus and Salmonella serovar Dublin are potent inducers of TRAIL expression by osteoblasts. Mouse and human TRAIL mRNA expression was induced by bacterial infection and demonstrated a dose-dependent response. Analysis of kinetics suggested that TRAIL mRNA was induced within 30 min after exposure to bacteria and that its level of expression remained relatively constant over the time period examined. mRNA molecules encoding TRAIL receptors were constitutively expressed by osteoblasts. Furthermore, TRAIL protein was detected as early as 45 min and up to 24 h following infection. The quantity of TRAIL protein produced also increased in a dose-dependent manner. Collectively, these findings suggest a mechanism whereby bacterial pathogens mediate bone destruction via osteoblast apoptosis.

Thermodynamically-driven exfoliation and self-assembly of pristine graphene sheets is shown to provide thermally and electrically functional polymer composites. The spreading of graphene sheets at a high energy liquid/liquid interface is driven by lowering the overall energy of the system, and provides for the formation of water-in-oil emulsions stabilized by overlapping graphene sheets. Polymerization of the oil phase, followed by removal of the dispersed water phase, produces inexpensive and porous composite foams. Contact between the graphene-stabilized water droplets provides a pathway for electrical and thermal transport through the composite. Unlike other graphene foams, the graphite used to synthesize these composites is natural flake material, with no oxidation, reduction, sonication, high temperature thermal treatment, addition of surfactants, or high shear mixing required. The result is an inexpensive, low-density material that exhibits Joule heating and displays increasing electrical conductivity with decreasing thermal conductivity.

Hepatocyte nuclear factor 4␣ (HNF4A), the gene for the maturity-onset diabetes of the young type 1 monogenic form of type 2 diabetes, is within the type 2 diabeteslinked region on chromosome 20q12-q13.1 and, consequently, is a positional candidate gene for type 2 diabetes in the general population. Previous studies have identified only a few rare coding mutations. However, recent studies suggest that single nucleotide polymorphisms (SNPs) located near the P2 (␤-cell) promoter of HNF4A are associated with diabetes susceptibility. In this study, we evaluated 23 SNPs spanning 111 kb including the HNF4A gene for association with type 2 diabetes in a collection of Caucasian type 2 diabetic patients with end-stage renal disease (n ‫؍‬ 300) and control subjects (n ‫؍‬ 310). None of the individual SNPs were associated with type 2 diabetes in this collection of case subjects (P values ranging from 0.06 to 0.99). However, haplotype analysis identifies significant differences between haplotype frequencies in type 2 diabetic case and control subjects (P ‫؍‬ 0.013 to P < 0.001), with two uncommon "risk" haplotypes (2.4 and 2.2% of chromosomes) and two uncommon "protective" haplotypes (7.1 and 5.0% of chromosomes) accounting for the evidence of association. Our results suggest that type 2 diabetes linked to 20q12-13 is a heterogeneous disease in which different populations may have different type 2 diabetes susceptibility loci.Diabetes 54: 1185-1190, 2005 S everal studies have provided evidence for linkage of type 2 diabetes to the long arm of chromosome 20 in Caucasians (1-5) and Asians (6,7), suggesting that one or more type 2 diabetes susceptibility loci are located on chromosome 20q. Hepatocyte nuclear factor 4␣ (HNF4A) is a candidate gene within this linked region because mutations in HNF4A have been implicated in the maturity-onset diabetes of the young type 1 subtype of type 2 diabetes, a monogenic form of type 2 diabetes characterized by defective insulin secretion (8). HNF4A is a member of the steroid/thyroid hormone receptor superfamily of transcription factors (9), and it interacts with regulatory elements in promoters and enhancers of genes involved in cholesterol, fatty acid, and glucose metabolism (10). Thirteen exons have been identified in HNF4A, and alternative splicing of these exons results in at least nine isoforms of the gene. The transcription of three of these isoforms is driven by an alternate promoter known as P2, which is located ϳ45.5 kb upstream of the P1 promoter (11,12). Recent studies suggest that although both promoters function in pancreatic ␤-cells (13), it is the P2 promoter that primarily drives transcription in these cells (11,12). In previous studies (14,15), we surveyed the coding region of HNF4A and conserved enhancer-like elements in the distal HNF4A promoter, finding no evidence for significant association to type 2 diabetes in the general population. Recently, several groups have evaluated single nucleotide polymorphisms (SNPs) in the P2 region for association with type 2 diabetes. T...