Claudia M. Gohr

Elimination of Intraluminal Colonization by Antibiotic Lock in Silicone Vascular Catheters

An in vitro model was designed to evaluate the efficacy of instilled antimicrobials to reduce or eliminate intraluminal microbial colonization. Minimal inhibitory concentration and minimal bactericidal concentration activity of appropriate test anti-infectives were determined using standard methodology against clinically derived and reference test strains commonly associated with catheter-related infection. Drug activity was validated by bioassay for the test anti-infectives. Reference and clinical test strains were inoculated to the intraluminal surface of silicone catheter segments and incubated for 30 min, after which the inoculum was replaced with total parenteral nutrition (TPN) solution and reincubated for 12 h. For 7 d, instillation of antibiotic and TPN solution was alternated every 12 h to simulate clinical conditions. On days 1, 4, and 7, catheter segments were rinsed, bisected, and sonicated for quantitative plate count to determine mean microbial counts per centimeter of catheter surface. Catheter segments were also prepared for scanning electron microscopy. A significant decrease in staphylococcal intraluminal colonization after instillation of nafcillin, ceftriaxone, gentamicin, and vancomycin was demonstrated (P < 0.001). Aztreonam, ceftriaxone, and gentamicin completely eliminated gram-negative catheter colonization (P < 0.001). Yeast was eradicated from the internal catheter surface after treatment with amphoteracin B, and fluconazole significantly decreased intraluminal colonization (P < 0.001). Results show a significant decrease in staphylococcal, gram-negative, and fungal intraluminal colonization after instillation of appropriate antimicrobial. In vitro results support early clinical success using this technique. Future studies are warranted to identify optimal drug concentrations and dosing intervals.

PROTEOMIC ANALYSIS OF ARTICULAR CARTILAGE VESICLES FROM NORMAL AND OSTEOARTHRITIC CARTILAGE

OBJECTIVE Articular cartilage vesicles (ACVs) are extracellular organelles found in normal articular cartilage. While they were initially defined by their ability to generate pathologic calcium crystals in cartilage of osteoarthritis (OA) patients, they can also alter the phenotype of normal chondrocytes through the transfer of RNA and protein. The purpose of this study was to analyze the proteome of ACVs from normal and OA human cartilage. METHODS ACVs were isolated from cartilage samples from 10 normal controls and 10 OA patients. We identified the ACV proteomes using in-gel trypsin digestion, nanospray liquid chromatography tandem mass spectrometry analysis of tryptic peptides, followed by searching an appropriate subset of the Uniprot database. We further differentiated between normal and OA ACVs by Holm-Sidak analysis for multiple comparison testing. RESULTS More than 1,700 proteins were identified in ACVs. Approximately 170 proteins satisfied our stringent criteria of having >1 representative peptide per protein present, and a false discovery rate of ≤5%. These proteins included extracellular matrix components, phospholipid binding proteins, enzymes, and cytoskeletal components, including actin. While few proteins were seen exclusively in normal or OA ACVs, immunoglobulins and complement components were present only in OA ACVs. Compared to normal ACVs, OA ACVs displayed decreases in matrix proteoglycans and increases in transforming growth factor β-induced protein βig-H3, DEL-1, vitronectin, and serine protease HtrA1 (P < 0.01). CONCLUSION These findings lend support to the concept of ACVs as physiologic structures in articular cartilage. Changes in OA ACVs are largely quantitative and reflect an altered matrix and the presence of inflammation, rather than revealing fundamental changes in composition.

Participation of transglutaminase in the activation of latent transforming growth factor β1 in aging articular cartilage

OBJECTIVE Transglutaminase (TGase) catalyzes the calcium-dependent crosslinking of polypeptide chains, resulting in posttranslational protein modifications that affect both intracellular and extracellular processes. We previously demonstrated a dramatic elevation of TGase activity levels in aging articular chondrocytes and postulated a role for TGase in the pathologic processes common in aging joints. In several cell systems, TGase participates in the activation of latent transforming growth factor beta (LTGFbeta). Since TGFbeta is a key factor in age-related cartilage diseases, the purpose of the present study was to determine whether TGase from aging articular chondrocytes participates in LTGFbeta activation. METHODS We measured the ability of old and young porcine articular chondrocytes to activate 10 ng/ml of LTGFbeta1 in the presence and absence of TGase inhibitors. The activity of plasmin, another key participant in LTGFbeta activation, was also measured. RESULTS Old chondrocytes activated 11+/-1.8% (mean +/- SD) of exogenous LTGFbeta1 at 6 hours, while young chondrocytes activated 4.2+/-0.5% of exogenous LTGFbeta1. The addition of 3 different TGase inhibitors suppressed active TGFbeta1 in the cell layer to levels that were 35-69% of control values in old chondrocytes and had no effect on young chondrocytes. The ability to suppress TGFbeta activation correlated with the ability of each of the TGase inhibitors to inhibit TGase activity. The activity of plasmin, which enzymatically activates LTGFbeta1, did not differ between young and old chondrocytes and was unaffected by TGase inhibition. CONCLUSION We report here a novel pathologic function for TGase in aging articular cartilage. This work supports a role for elevated TGase activity in age-related arthritis based in part on its participation in the activation of the critical growth factor TGFbeta in articular cartilage.

Promotion of articular cartilage matrix vesicle mineralization by type I collagen

OBJECTIVE Calcium pyrophosphate dihydrate (CPPD) and basic calcium phosphate (BCP) crystals occur in up to 60% of osteoarthritic joints and predict an increased severity of arthritis. Articular cartilage vesicles (ACVs) generate CPPD crystals in the presence of ATP and BCP crystals with added beta-glycerophosphate. While ACVs are present in normal articular cartilage, they mineralize primarily in cartilage from osteoarthritic joints. The aim of this study was to explore the hypothesis that ACV mineralization is regulated by components of the surrounding extracellular matrix. METHODS Porcine ACVs were embedded in agarose gels containing type II and/or type I collagen and/or proteoglycans. Mineralization was measured as (45)Ca accumulation stimulated by ATP or beta-glycerophosphate and reflects both nucleation and growth. Synthetic CPPD and BCP crystals were embedded in similar gels to isolate the effect of matrix components on crystal growth. RESULTS After establishing baseline responsiveness of ACVs to ATP and beta-glycerophosphate in agarose gels, we examined the ability of ATP and beta-glycerophosphate to stimulate mineral formation in gels containing various matrix components. Type II collagen suppressed the ability of ATP to stimulate mineralization, while a combination of type II plus type I collagen increased the effect of ATP and beta-glycerophosphate on mineralization. Type I collagen affected ACV mineralization in a dose-responsive manner. Neither type of collagen significantly affected crystal growth or levels of mineralization-regulating enzymes. Proteoglycans suppressed mineral formation by ACVs in gels containing both type I and type II collagen. CONCLUSION Cartilage matrix changes that occur with osteoarthritis, such as increased quantities of type I collagen and reduced proteoglycan levels, may promote ACV mineralization.

Characterization of articular calcium-containing crystals by synchrotron FTIR

OBJECTIVE Sixty percent of synovial fluids from patients with severe osteoarthritis (OA) contain calcium pyrophosphate dihydrate (CPPD) or basic calcium phosphate (BCP) crystals. These bioactive crystals can be particularly difficult to accurately identify in complex biologic systems, such as in vitro models of crystal formation. We sought to determine if synchrotron Fourier Transform Infrared spectroscopy (sFTIR) could be used to identify and characterize calcium-containing crystals in mineralization models. METHODS CPPD and BCP crystals from porcine models of crystal formation were examined with an FTIR Microscope attached to a synchrotron light source. As a comparison, crystals from human synovial fluids were also examined. The sFTIR spectra generated were compared with known spectra of multiple forms of BCP and CPPD crystals, as well as spectra generated by synthetic CPPD and BCP crystals and cartilage proteoglycans, alone and in mixtures. RESULTS sFTIR readily identified CPPD and BCP crystals in porcine models as well as in fresh synovial fluids. Brushite was also present in human and porcine samples, and whitlockite was seen in some porcine samples. Mixtures of minerals were commonly found in a single crystal aggregate in both human and porcine samples. In spectra from many CPPD crystals, the peak at the 1134 cm(-1) found on the standard spectrum for CPPD was diminished. Addition of spectra from cartilage proteoglycans to those of synthetic CPPD crystals dampened the peak at this frequency region, much as this peak was diminished in biologically derived CPPD crystals. CONCLUSION sFTIR analysis allows for accurate identification of CPPD and BCP crystals generated in vitro and will be a useful research tool to study articular crystals.

Surgical sepsis: Constancy of antibiotic susceptibility of causative organisms

BACKGROUND It is well documented that antibiotic therapy exerts selective pressure on bacteria. Conversion of bacteria from susceptible to resistant to antibiotics has been observed often during antimicrobial therapy. It has been postulated that human intestinal reservoirs facilitate communication of transposons that can transfer resistance determinants among various bacterial species. METHODS This study examined the susceptibilities of organisms isolated from infected abdomens to a number of antibiotic agents during a 12-year time interval. Analysis included 1102 isolates recovered from 255 specimens, representing the following genera: Bacteroides, Clostridium, Gemella, Fusobacterium, Peptostreptococcus, Porphyromonas, Prevotella, Enterococcus, Staphylococcus, Streptococcus, Pseudomonas, and Enterobacteriaceae. Strains were tested against beta-lactam agents, beta-lactams in combination with beta-lactamase inhibitors, first, second, and third generation cephalosporins, aminoglycosides, clindamycin, metronidazole, chloramphenicol, and imipenem. RESULTS The results indicated that during a time period of more than a decade essentially no change occurred in the antibiotic susceptible fraction of all species tested. CONCLUSIONS Abdominal sepsis is caused by leakage of endogenous intestinal flora. This study suggests that the intestinal flora is not permanently affected by short-term antibiotic therapy and that older antibiotics are appropriate first-line therapeutic agents for community-acquired infections caused by normal intestinal flora.

The progressive ankylosis gene product ANK regulates extracellular ATP levels in primary articular chondrocytes

IntroductionExtracellular ATP (eATP) is released by articular chondrocytes under physiological and pathological conditions. High eATP levels cause pathologic calcification, damage cartilage, and mediate pain. We recently showed that stable over-expression of the progressive ankylosis gene product, ANK, increased chondrocyte eATP levels, but the mechanisms of this effect remained unexplored. The purpose of this work was to further investigate mechanisms of eATP efflux in primary articular chondrocytes and to better define the role of ANK in this process.MethodsWe measured eATP levels using a bioluminescence-based assay in adult porcine articular chondrocyte media with or without a 10 minute exposure to hypotonic stress. siRNAs for known ATP membrane transporters and pharmacologic inhibitors of ATP egress pathways were used to identify participants involved in chondrocyte eATP release.ResultseATP levels increased after exposure to hypotonic media in a calcium-dependent manner in monolayer and 3-dimensional agarose gel cultures (p < 0.001). A potent transient receptor potential vanilloid 4 (TRPV4) agonist mimicked the effects of hypotonic media. ANK siRNA suppressed basal (p < 0.01) and hypotonically-stressed (p < 0.001) ATP levels. This effect was not mediated by altered extracellular pyrophosphate (ePPi) levels, and was mimicked by the ANK inhibitor, probenecid (p < 0.001). The P2X7/4 receptor inhibitor Brilliant Blue G also suppressed eATP efflux induced by hypotonic media (p < 0.001), while ivermectin, a P2X4 receptor stimulant, increased eATP levels (p < 0.001). Pharmacologic inhibitors of hemichannels, maxianion channels and other volume-sensitive eATP efflux pathways did not suppress eATP levels.ConclusionsThese findings implicate ANK and P2X7/4 receptors in chondrocyte eATP efflux. Understanding the mechanisms of eATP efflux may result in novel therapies for calcium crystal arthritis and osteoarthritis.

Feasibility of a Tetracycline-Binding Method for Detecting Synovial Fluid Basic Calcium Phosphate Crystals

OBJECTIVE Basic calcium phosphate (BCP) crystals are common components of osteoarthritis (OA) synovial fluid. Progress in understanding the role of these bioactive particles in clinical OA has been hampered by difficulties in their identification. Tetracyclines stain calcium phosphate mineral in bone. The aim of this study was to investigate whether tetracycline staining might be an additional or alternative method for identifying BCP crystals in synovial fluid. METHODS A drop of oxytetracycline was mixed with a drop of fluid containing synthetic or native BCP, calcium pyrophosphate dihydrate (CPPD), or monosodium urate (MSU) crystals and placed on a microscope slide. Stained and unstained crystals were examined by light microscopy, with and without a portable broad-spectrum ultraviolet (UV) pen light. A small set of characterized synovial fluid samples were compared by staining with alizarin red S and oxytetracycline. Synthetic BCP crystals in synovial fluid were quantified fluorimetrically using oxytetracycline. RESULTS After oxytetracycline staining, synthetic and native BCP crystals appeared as fluorescent amorphous aggregates under UV light. Oxytetracycline did not stain CPPD or MSU crystals or other particulates. Oxytetracycline staining had fewer false-positive test results than did alizarin red S staining and could provide estimates of the quantities of synthetic BCP crystals in synovial fluid. CONCLUSION With further validation, oxytetracycline staining may prove to be a useful adjunct or alternative to currently available methods for identifying BCP crystals in synovial fluid.

TRANSGLUTAMINASE CONTRIBUTES TO CPPD CRYSTAL FORMATION IN OSTEOARTHRITIS

Calcium pyrophosphate dihydrate (CPPD) crystals are common components of osteoarthritic joints and correlate with a poor prognosis. Transglutaminase (Tgase) enzymes have been implicated in pathologic mineralization in cartilage; yet, definitive studies linking Tgase activity to CPPD crystal formation in osteoarthritic articular cartilage are lacking. We measured in-vivo Tgase activity in osteoarthritic and normal human cartilage, and explored the effect of Tgase inhibitors on CPPD crystal formation by normal chondrocytes. Osteoarthritic articular cartilage from was obtained from specimens discarded at the time of knee replacement surgery. Normal adult cartilage samples from a tissue bank were used as controls. Tgase-specific isopeptide (epsilon-(gamma-glutamyl) lysine) bonds were measured in cartilage extracts by HPLC. Tgase-specific crosslinks were localized in osteoarthritic cartilage by immunohistochemistry. The effect of Tgase inhibition was determined in an in-vitro model of CPPD crystal formation. Tgase-specific crosslink levels were 1.55 +/- 0.3 picomoles/ng protein in normal human adult articular cartilage and 4.74 +/- 0.7 picomoles/ng protein in osteoarthritic human cartilage (p less than 0.001). Immunostaining confirmed the presence of Tgase crosslinks in the pericellular matrix of chondrocytes at potential sites of CPPD crystal formation. Tgase inhibitors suppressed CPPD crystal formation by porcine chondrocytes. These findings support a role for Tgase in CPPD crystal formation in aging or degenerated cartilage.

Hyperglycemia Reduces Proteoglycan Levels in Tendons

Rationale: Diabetic tendinopathy is characterized by increased stiffness, thickness, and excess calcification of affected tendons. We investigated the hypothesis that proteoglycans (PGs), as key regulators of tendon structure and calcification, are altered in diabetic tendons. Methods: Adult porcine patellar tendons were incubated in iso-osmolar media with high or normal glucose levels for 2 weeks. The PG fraction was isolated and analyzed. Protein and mRNA levels of five PGs were measured. PG production was assessed in primary tenocyte monolayers by 35S-sulfate labeling in high and normal glucose conditions with and without exposure to advanced glycation end-products (AGEs). Levels of transforming growth factor β, which commonly mediates some effects of hyperglycemia, were also measured and the effects of free radical scavengers on 35S-sulfate incorporation were determined. Results: PG levels were significantly decreased in tendons exposed to high glucose media compared with tendons in iso-osmolar control media. Relative quantities of individual PGs were unchanged by exposure to hyperglycemia and mRNAs for PGs were variably affected. High glucose media decreased PG production by tenocytes as measured by 35S-sulfate labeling, whereas AGE-modified type I collagen and free radical scavengers had no effects. Hyperglycemic conditions increased levels of transforming growth factor β1 in an AGE-independent manner. Conclusions: Hyperglycemia produces a reduction in PG levels related to decreased synthesis or sulfation of glycosaminoglycans, which may contribute to the tendon pathology observed clinically in diabetes.