Characteristics of antimicrobial stewardship programmes in rural East Texas hospitals.

INTRODUCTION: Antimicrobial stewardship programmes (ASPs) serve as the primary method to prevent and manage the development of antimicrobial resistance. Rural settings may lack the recommended personnel and resources needed to provide antimicrobial stewardship services. METHODS: An electronic survey was distributed via e-mail to pharmacy directors or antimicrobial stewardship programme directors of licensed hospitals within Public Health Region 4/5N of East Texas. RESULTS: Sixty percent of ASPs were established <12 months prior to the survey administration. All ASPs had pharmacist involvement, with only one (5%) having formal infectious diseases (ID) training through postgraduate education. Ninety percent of ASPs had a physician champion, with five (27.8%) physicians having formal ID training. Most institutions lacked one or more recommended antimicrobial stewardship practices. When compared with ASPs established for <12 months, ASPs existing for at least 12 months were more likely to have protocols to change antimicrobials from intravenous to enteral forms (100% vs 50%, P = 0.042), provide education to patients and families on appropriate antimicrobial use (87.5% vs 33.3%, P = 0.028), and track antimicrobial purchasing costs (87.5% vs 33.3%, P = 0.028). CONCLUSIONS: Institutions in rural settings require additional resources, personnel, and time to implement ASPs and perform various antimicrobial stewardship practices.

Recognition of the alternatively spliced segments of fibronectin by the RCJ 3.1C5.18 chondrocytic rat cell line.

OBJECTIVES: To define, for the C5.18 chondrocyte-restricted rat cell line, (1) the capacities for recognition of alternatively spliced segments of the adhesion protein fibronectin (FN), (2) the integrin subunits required for such recognition, and (3) differences in such FN recognition vs the multipotential chondroprogenitor line, RCJ 3.1. METHODS: C5.18 and RCJ 3.1 cells were tested for their capacities to adhere to recombinant alternatively spliced segments of rat FN, presented on plastic surfaces either in isolation or in partial FNs spanning the 7th through 15th type III repeats (III7-15 FNs). The effects on such adhesion of cations and integrin subunit-specific antibodies were tested. RESULTS: Despite significant augmentation in chondrocyte-specific gene expression in C5.18 relative to the RCJ 3.1 cells, the two lines exhibited similar recognition of FN spliced segments and partial isoforms. Specifically, both lines adhered to the extra type III repeat A (EIIIA) and V, but not extra type III repeat B (EIIIB), segments. There were different cation and integrin subunit requirements for adhesion to EIIIA vs V segments, and only the V segment was recognized in the context of a III7-15 FN. Such recognition was mediated via a "second" arginine-glycine-aspartic acid (RGD) sequence that is present in the V95 subsegment in rat, but not human, FN. CONCLUSION: The chondrocyte lineage-committed C5.18 cell line, similar to its multipotential chondroprogenitor, RCJ 3.1, recognizes the "cartilage-restricted" EIIIA and V segments of FN with cation, integrin, and molecular context requirements that are specific to each of these segments.

Cleavage of the MLL gene by activators of apoptosis is independent of topoisomerase II activity.

Exposure to topoisomerase II inhibitors is linked to the generation of leukemia involving translocations of the MLL gene, normally restricted to an 8.3 kbp tract, the breakpoint cluster region (BCR). Using an in vitro assay, apoptotic activators, including radiation and anti-CD95 antibody, trigger site-specific cleavage adjacent to exon 12 within the MLL BCR and promote translocation of the MLL gene in cells that can survive. To explore the mechanism of cleavage and rearrangement in more detail, the entire MLL BCR was placed into the pREP4 episomal vector and transfected into human lymphoblastoid TK6 cells. Episomes containing either the MLL BCR, or deletion constructs of 367 bp or larger, were cleaved at the same position as genomic MLL after exposure to apoptotic stimuli. Further analysis of sequence motifs surrounding the cleaved region of MLL showed the presence of both a predicted nuclear matrix attachment sequence and a potential strong binding site for topoisomerase II, flanking the site of cleavage. Inactivation of topoisomerase II by the catalytic inhibitor merbarone did not inhibit MLL cleavage, suggesting that the initial cleavage step for MLL rearrangement is not mediated by topoisomerase II.

BMP regulation of the mouse connexin43 promoter in osteoblastic cells and embryos.

We examined BMP regulation of the gap junction gene Gjal (Cx43alpha1) using a series of lacZ reporter constructs containing up to 6.7 kbs of mouse Cx43alpha1 promoter sequence. Using transient transfection assays, we showed that BMP2, BMP4, and GDF5, but not BMP6 or BMP7, can modulate Cx43alpha1 promoter activity in the osteosarcoma cell line ROS17/2.8. Positive regulatory elements were found at the proximal and distal ends of the 6.7 kb promoter fragment, while negative regulatory elements were found in the intervening region. Comparison of Cx43alpha1 promoter sequences from the human vs. mouse showed five regions with significant sequence conservation, two of which contained Smad binding elements in conjunction with a BMP response element. Analysis of a transgenic mouse line containing a Cx43alpha1 promoter driven lacZ reporter construct revealed lacZ expression in the developing joints, an expression pattern similar to that previously reported for Gdf5. LacZ expression was also observed in axial regions of the skeletal anlage, which in situ hybridization analysis confirmed as sites of Gdf5 transcript expression. When the Cx43alpha1 promoter driven lacZ transgene was bred into the brachypodism mouse Gdf5(bpJ)(bp) harboring a Gdf5 loss of function mutation, lacZ expression was extinguished. This was observed in homozygous and heterozygous bp animals, suggesting that Cx43alpha1 promoter regulation by GDF5 is subject to haploinsufficiency. Overall, these observations are consistent with recent studies by others indicating a role for Cx43alpha1 in osteogenesis and osteoblastic function during mouse development.

Effect of cartilage oligomeric matrix protein on mesenchymal chondrogenesis in vitro.

OBJECTIVE: Cartilage oligomeric matrix protein (COMP) mutations have been identified as responsible for two arthritic disorders, multiple epiphyseal dysplasia (MED) and pseudoachondroplasia (PSACH). However, the function of COMP in chondrogenic differentiation is largely unknown. Our investigation focuses on analyzing the function of normal COMP protein in cartilage biology. METHODS AND RESULTS: To explore the function of COMP we make use of an in vitro model system for chondrogenesis, consisting of murine C3H10T1/2 mesenchymal cells maintained as a high-density micromass culture and stimulated with bone morphogenetic protein 2 (BMP-2). Under these culture conditions, C3H10T1/2 cells undergo active chondrogenesis in a manner analogous to that of embryonic limb mesenchymal cells, and have been shown to serve as a valid model system to investigate the mechanisms regulating mesenchymal chondrogenesis. Our results indicate that ectopic COMP expression enhances several early aspects of chondrogenesis induced by BMP-2 in this system, indicating that COMP functions in part to positively regulate chondrogenesis. Additionally, COMP has inhibitory effects on proliferation of cells in monolayer. However, at later times in micromass culture, ectopic COMP expression in the presence of BMP-2 causes an increase in apoptosis, with an accompanying reduction in cell numbers in the micromass culture. However, the remaining cells retain their chondrogenic phenotype. CONCLUSIONS: These data suggest that COMP and BMP-2 signaling converge to regulate the fate of these cells in vitro by affecting both early and late stages of chondrogenesis.

Development of an evolutionarily novel structure: fibroblast growth factor expression in the carapacial ridge of turtle embryos.

The turtle shell, an evolutionarily novel structure, contains a bony exoskeleton that includes a dorsal carapace and a ventral plastron. The development of the carapace is dependent on the carapacial ridge (CR), a bulge in the dorsal flank that contains an ectodermal structure analogous to the apical ectodermal ridge (AER) of the developing limb (Burke. 1989a. J Morphol 199:363-378; Burke. 1989b. Fortschr Zool 35:206-209). Although the CR is thought to mediate the initiation and outgrowth of the carapace, the mechanisms of shell development have not been studied on the molecular level. Here, we present data suggesting that carapace formation is initiated by co-opting genes that had other functions in the ancestral embryo, specifically those of limb outgrowth. However, there is divergence in the signaling repertoire from that involved in limb initiation and outgrowth. In situ hybridizations with antisense riboprobes derived from Trionyx spiniferous fibroblast growth factor-10 (tfgf10) and Trachemys scripta (T. scripta) fibroblast-growth factor 8 (tfgf8) cDNAs were performed on sections of early T. scripta embryos (< 30 days). Expression of tfgf10 was localized to the mesenchyme subjacent to the ectoderm of the CR. In the chick limb bud, FGF10 is known to be expressed in the early limb-forming mesenchyme and is capable of inducing FGF8 in the AER to initiate the outgrowth of the limb bud. Although the expression of tfgf8 was found in the AER of the developing turtle limb, it was not seen in the CR. Thus, the initiation of the carapace is in agreement with FGF10 expression in the CR, but FGF8 does not appear to have a role in mediating early carapace outgrowth.

Morphogenesis of the turtle shell: the development of a novel structure in tetrapod evolution.

The turtle shell is an evolutionary novelty that is synapomorphic for chelonians. The carapace is initiated by the entrapment of the ribs by the carapacial ridge (CR), a lateral bulge of the dorsal ectoderm and dermal mesoderm. The mechanisms by which the CR is initiated, the ribs entrapped and the dorsal dermis ossified, remains unknown. Similarly, the formation of the plastron remains unexplained. Here, we present a series of anatomical investigations into plastron and carapace formation in the red-eared slider, Trachemys scripta, and the snapping turtle, Chelydra serpentina. We document the entrapment of the ribs by the CR and the formation of the plastron and carapacial bones by intramembranous ossification. We note the formation of the ossification centers around each rib, which suggest that the rib is organizing dermal ossification by secreting paracrine factors. The nuchal ossification center is complex and appears to involve multiple bone-forming regions. Individual ossification centers at the periphery of the carapace form the peripheral and pygial bones. The intramembranous ossification of the plastron proceeds from nine distinct ossification centers, and there appear to be interactions between the spicules of apposing centers as they draw near each other.

ATP and UTP activate calcium-mobilizing P2U-like receptors and act synergistically with interleukin-1 to stimulate prostaglandin E2 release from human rheumatoid synovial cells.

OBJECTIVE: To pharmacologically and functionally characterize calcium-mobilizing purine receptors on adherent human rheumatoid synovial cells. METHODS: Fura-2-loaded synovial cells were screened for changes in cytosolic calcium concentration after the addition of purine receptor agonists. Release of interleukin-1 (IL-1) and prostaglandin E2 (PGE2) was assessed by enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay, respectively. The effect of IL-1 prestimulation on purine-mediated PGE2 release was determined. RESULTS: ATP (1-100 microM) and UTP (1-100 microM), but not 2-methylthio-ATP or adenosine, stimulated mobilization of calcium from intracellular stores in synovial cells. ATP and UTP stimulated a small, but significant, increase in PG release from resting synoviocytes and a dramatic increase in PG release from synoviocytes prestimulated with recombinant human IL-1alpha. Neither ATP nor UTP stimulated synoviocyte release of IL-1 as measured by specific ELISA. The effects of ATP and UTP on PG secretion were mimicked by phorbol 12-myristate 13-acetate and thapsigargin, and blocked by BAPTA buffering of cytosolic calcium. CONCLUSION: Adherent human rheumatoid synovial cells mobilize intracellular calcium via a P2U-like purine receptor. P2U receptor agonists stimulate PGE2 release from synoviocytes, an effect that is greatly enhanced by IL-1alpha prestimulation and blocked by intracellular calcium buffering.

Regulation of glycosaminoglycan metabolism by bone morphogenetic protein-2 in equine cartilage explant cultures.

OBJECTIVE: To investigate whether recombinant human bone morphogenetic protein-2 (rhBMP-2) regulates glycosaminoglycan (GAG) synthesis and release from equine articular cartilage explant cultures. DESIGN: Equine articular cartilage explants were maintained in vitro for 7 days in the presence of 0 (control), 1, 10, or 100 ng of rhBMP-2/ml. Synthesis and release of GAG were assessed as measures of production and degradation of the extracellular matrix, respectively. ANIMALS: 6 horses (age range, 2 to 25 years old) without clinically detectable musculoskeletal abnormalities. PROCEDURE: Rate of synthesis of GAG was assessed by incorporation of [36S]sulfate during the final 24 hours of the 7-day incubation period. Release of GAG was assessed on days 3, 6, and 7, using 1,9-dimethylmethylene blue. RESULTS: Explants from all 6 horses had a significant (P = 0.05) increase in release of GAG in response to incubation with 100 ng of rhBMP-2/ml. There was a significant (P = 0.05) decrease in GAG synthesis in explants from only 2 of the 6 horses at the same concentration of rhBMP-2. There was no significant age correlation between responsive and nonresponsive horses. CONCLUSIONS: A concentration of 100 ng of rhBMP-2/ml stimulates GAG release from explant cultures of equine articular cartilage. The data suggest that bone morphogenetic proteins may be potential regulators of equine cartilage degradation and repair. CLINICAL RELEVANCE: Surgical procedures that damage subchondral bone may stimulate generation of improved cartilage-like tissue. It is, therefore, crucial to understand how bone-derived factors may influence cartilage metabolism in horses.

Influence of alginate polysaccharide composition and culture conditions on chondrocytes in three-dimensional culture.

Alginate-embedded chondrocytes have been used for experimental analysis of cartilage matrix metabolism and as a potential bioartificial system for repairing cartilage defects. Alginates are linear copolymers composed of 1,4-linked beta-n-mannuronic acid and 1,4 linked alpha-L-guluronic acid, which occur as regions made up exclusively of one unit or the other, or as regions in which the monomers approximate an alternating sequence. Data presented here demonstrate that the mannuronic to guluronic acid (M/G) ratio and molecular weight of the alginate utilized effects the tissue-culture handling properties of the resultant gel and the activity of embedded chondrocytes. In experiments comparing chondrocyte survival and matrix synthesis, optimal conditions were obtained with an alginate of both high mannuronic acid content and high molecular weight. Chondrocytes survived and proliferated when maintained in unsupplemented media, in media supplemented with fetal calf serum, and in media supplemented with the defined serum replacement ITS+ (6.25 microg/ml insulin, 6.25 microg/ml transferrin, 6.25 ng/ml selenous acid, 1.25 mg/ml bovine serum albumin, 5.35 microg/ml linoleic acid). High cell survival rate and increase in cell number was obtained in the absence of serum. In contrast, long-term matrix synthesis and deposition required media supplementation as indicated by uptake of [(35)S]sulfate into glycosaminoglycans and by immunofluorescence using antibodies specific for cartilage matrix molecules.