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.

Thyroxine stimulates transglutaminase activity in articular chondrocytes.

OBJECTIVES: Thyroid hormones induce features of the hypertrophic phenotype in mature articular chondrocytes as well as in growth plate chondrocytes. Hypertrophic chondrocytes are responsible for extracellular matrix mineralization, with formation of bone mineral in growth plate cartilage and pathologic calcium crystals in aging articular cartilage. Elevated activity levels of the two transglutaminase (Tgase) enzymes (type II Tgase and Factor XIIIA (FXIIIA)) have recently been described as additional features of hypertrophic growth plate chondrocytes. Because Tgases may participate in pathologic mineralization in aging cartilage, we explored the effects of thyroid hormones on Tgase activity in articular chondrocytes. METHODS: Adult porcine articular chondrocytes were incubated with or without 250-750nM L-thyroxine (T4) or 10-100 nM 3,3',5-tri-iodothyronine (T3). Tgase activity was measured with a standard radiometric assay. The effects of thyroid hormones on protein and mRNA levels of type II Tgase and FXIIIA were determined. As Tgase activity can be stimulated by proteases, endoproteinase levels were also measured. The mechanisms of these effects were explored. RESULTS: T4 (750 nM) or T3 (100 nM) stimulated Tgase activity by twofold in articular chondrocytes at 4h and increased the percentage of Tgase activity in the extracellular matrix. Chondrocytes rapidly converted T4 to T3, but the time course suggests similar mechanisms for T4 and T3. T4-induced Tgase activity was suppressed with cycloheximide and protein kinase C inhibitors. The effects of T4 on type II Tgase and FXIIIA levels were modest, but T4 strongly induced endoproteinase activity in chondrocytes. CONCLUSIONS: We report in this study that thyroid hormones increase Tgase activity in articular chondrocytes via a non-genomic mechanism, which may involve increased endoproteinase secretion.

The transglutaminase, Factor XIIIA, is present in articular chondrocytes.

OBJECTIVES: The transglutaminase (TGase) family includes seven different enzymes that catalyse a protein cross-linking reaction resulting in structural and functional alterations in substrate proteins. TGase activity is easily measureable in mature articular cartilage where it may contribute to CPPD deposition disease through its actions on growth factors, crystal components or extracellular matrix proteins. In contrast, low levels of TGase activity are found in chondrocytes from young animals. We previously demonstrated type II TGase protein in articular chondrocytes. Earlier work also suggested the presence of another form of TGase in chondrocytes. We sought to determine if articular chondrocytes contain the TGase, Factor XIIIA (FXIIIA). METHODS: Western blots with FXIIIA antibody were used to detect FXIIIA in young and old porcine articular chondrocytes and articular cartilage vesicles (ACVs). The presence of FXIIIA mRNA was confirmed by RT-PCR. RESULTS: Old chondrocyte conditioned medium, cytosol, and membrane fractions contained FXIIIA protein on Western blots, while less FXIIIA was detectable in cell fractions or media from young chondrocytes. ACVs also contained FXIIIA. FXIIIA mRNA was demonstrated by PCR in old and young chondrocytes. CONCLUSIONS: FXIIIA is present in articular chondrocytes. FXIIIA levels correlate with TGase activity in chondrocytes. The presence of two forms of TGase in articular chondrocytes suggest an important function for this enzyme family in articular cartilage.

Transglutaminase participates in the incorporation of latent TGFbeta into the extracellular matrix of aging articular chondrocytes.

TGFbeta1 is a multifunctional peptide growth factor that promotes processes associated with age-related degenerative diseases in articular cartilage. Large quantities of TGFbeta1 are stored in cartilage extracellular matrix (ECM) in a latent form (LTGFbeta1), and yet little is known about the factors that participate in the incorporation of LTGFbeta1 into the highly specialized cartilage ECM. We previously demonstrated high levels of the protein cross-linking enzyme transglutaminase (TGase) in aging articular chondrocytes and showed that this enzyme participated in LTGFbeta1 activation. This work explores the hypothesis that extracellular TGase participates in LTGFbeta1 incorporation into ECM in aging chondrocytes. We studied the effects of TGase inhibitors on TGFbeta1 levels in ECM of old and young porcine articular chondrocytes. TGase inhibitors decreased the quantity of LTGFbeta1 in the ECM in old but not in young chondrocytes to 60-70% of control values (p<.05). Fibronectin, an extracellular TGase competitive substrate, also decreased LTGFbeta1 levels in ECM (p<.01). Levels of activated TGFbeta1 also decreased in the presence of TGase inhibitors, as did levels of latent TGFbeta binding protein 1 in the cell layer. Extracellular TGase activity was present in old but not young chondrocyte cultures. These findings support a role for extracellular TGase in the incorporation of LTGFbeta1 in the ECM of aging chondrocytes.

Participation of transglutaminase in the activation of latent transforming growth factor beta1 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.

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.

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.

Microflora of abdominal sepsis by locus of infection.

Specimens from 152 abdominal infections were examined to determine which groups of endogenous bacteria participate in infection emanating from different sites in the gastrointestinal tract. A notable finding was the predominance of anaerobic microflora from infections of ischemic versus perforated small bowel. Empiric antibiotic treatment for ischemic bowel should include focused coverage for anaerobes.

Microbroth versus agar dilution for susceptibility testing of fluoroquinolones and other agents against anaerobic bacteria.

Agar dilution and microbroth dilution methods appear equally acceptable for susceptibility testing of anaerobes against temafloxacin and ciprofloxacin. Most major discrepancies occurred with strains whose MIC was near the breakpoint. These results suggest that method variation will not be a significant factor in the susceptibility testing of anaerobic bacteria against other fluoroquinolone agents.

Enzymatically active Peptostreptococcus magnus: association with site of infection.

Fifty-four strains of Peptostreptococcus magnus (11 were recovered from abdominal infections, 18 were from nonpuerperal breast abscesses, and 21 were from diabetic foot infections; the type strain and three other strains were from the American Type Culture Collection, Rockville, Md.) and the type strain of Peptostreptococcus micros were tested for their ability to produce various enzymes, including catalase, hippurate hydrolase, serine dehydratase, threonine dehydratase, collagenase, gelatinase, alkaline phosphatase, and esterase C4. The data were analyzed by cluster analysis. The results showed that all but one strain could be assigned to either of two distinct, valid clusters. The first cluster of 11 strains was composed of strains that were relatively inactive, having produced one or two of the eight strain-dependent enzymes. The second was a large cluster of strains (n = 43) that were considerably more active, all having produced at least three enzymes; the vast majority of strains (89%) produced four or more enzymes. The unclustered strain produced one enzyme that was different from that produced by the strains in the first cluster. The chi 2 test of homogeneity applied to the clustering solution indicated that greater enzyme activity was significantly associated with the site of infection (P less than 0.001). The more enzymatically active P. magnus strains were recovered significantly more often from nonpuerperal breast abscesses and diabetic foot infections than they were from abdominal infections. These results may provide insight into the nature of certain polymicrobial soft tissue infections and suggest that (i) P. magnus may participate more in nonpuerperal breast and diabetic foot infections than in abdominal infections and that (ii) peptostreptococcal production of proteolytic enzymes may have an important adjunctive effect on the pathogenesis of certain soft tissue infections.

Anaerobic pathogenesis: collagenase production by Peptostreptococcus magnus and its relationship to site of infection.

Fifty isolates of Peptostreptococcus magnus from intraabdominal sepsis, nonpuerperal breast abscess, and diabetic foot infections were examined for collagenase activity using bovine type I collagen. Collagenase production was detected in a higher percentage of strains from nonpuerperal breast and diabetic foot specimens (P less than .001). This enzyme may be responsible for P. magnus playing a more central role in the pathogenesis of nonpuerperal breast abscess and diabetic foot disease than in intraabdominal sepsis.

Streptococcus milleri group (Streptococcus anginosus): recovery from intra-abdominal and soft tissue sites.

One hundred thirty-three Streptococcus milleri group (S. anginosus) isolates were recovered from 487 surgical patients. The streptococci were recovered from 33 percent of intra-abdominal infection cultures (84/257). 22 percent of samples from penetrating visceral trauma (19/86), 52 percent of perirectal abscess specimens (13/25), 13 percent of nonpuerperal breast abscess cultures (8/60), and 15 percent of diabetic foot lesions (9/59). Ninety-eight percent of the S. milleri (131/133) were recovered as companion flora in polymicrobial cultures. The organisms were highly susceptible to the beta-lactam antibiotics. The precise pathogenic role of the S. milleri group (S. anginosus) is unknown. However, intrinsic virulence may be expressed in patients with severe infection or other predisposing factors.