Checkerboard Analysis Research Articles

Phototargeting human periodontal pathogens in vivo

The effects of blue light at 455 nm were investigated on the bacterial composition of human dental plaque in vivo. Eleven subjects who refrained from brushing for 3 days before and during phototherapy participated in the study. Light with a power density of 70 mW/cm(2) was applied to the buccal surfaces of premolar and molar teeth on one side of the mouth twice daily for 2 min over a period of 4 days. Dental plaque was harvested at baseline and again at the end of 4 days from eight posterior teeth on both the exposed side and unexposed sides of the mouth. Microbiological changes were monitored by checkerboard DNA probe analysis of 40 periodontal bacteria. The proportions of black-pigmented species Porphyromonas gingivalis and Prevotella intermedia were significantly reduced on the exposed side from their original proportions by 25 and 56 %, respectively, while no change was observed to the unexposed side. Five other species showed the greatest proportional reduction of the light-exposed side relative to the unexposed side. These species were Streptococcus intermedius, Fusobacterium nucleatum ss. vincentii, Fusobacterium nucleatum ss. polymorphum, Fusobacterium periodonticum, and Capnocytophaga sputigena. At the same time, the percentage of gingival areas scored as being red decreased on the side exposed to light from 48 to 42 %, whereas the percentage scored as red increased on the unexposed side from 53 to 56 %. No adverse effects were found or reported in this study. The present study proposes a new method to modify the ecosystem in dental plaque by phototherapy and introduces a new avenue of prophylactic treatment for periodontal diseases.

Bacterial markers vs. clinical markers to predict progression of chronic periodontitis: a 2‐yr prospective observational study

The aim of this study was to identify sites at risk for future progression, during 2 yr of maintenance, in patients with chronic periodontitis (CP), based on longitudinal clinical and microbiological monitoring. At baseline (2003), clinical and microbiological features were recorded in 50 patients with CP. Two microbial samples were obtained from each patient (one from a clinically healthy site and one from a periodontitis site) and these were analyzed using DNA-DNA hybridization involving 25 bacterial species. After non-surgical periodontal therapy, clinical and microbiological re-examinations were performed at the same or similar sites at 2 yr (2006) and 4 yr (2008) of maintenance. Plaque, bleeding on probing (BoP), and the number of sites with periodontitis (≥4 mm) and severe periodontitis (≥6 mm) all showed a significant decrease at 2 and 4 yr of maintenance after non-surgical intervention. Checkerboard analysis revealed that various bacteria with a high colonization score (≥3) corroborated the clinical findings of pathology at 2003, 2006, and 2008. Different clusters of bacteria, not just the 'red complex', were able to predict progression of chronic periodontitis during 2 yr of maintenance (2006-2008). Therefore, quantified bacterial markers (reflecting bacterial load) and the clinical markers BoP and periodontal probing depth show comparable prediction of future disease condition.

Interaction between caspofungin or voriconazole and cefoperazone-sulbactam or piperacillin-tazobactam byin vitroandin vivomethods

Immunosuppressive patients are at risk of fungal and bacterial infections. Therefore, these patients receive prophylactic, preemptive, empirical or target antifungal and concomitant antibiotic therapy. To this end, caspofungin (CAS) or voriconazole (VRC) antifungals and cefoperazone-sulbactam (CPZ/SAM) or piperacillin-tazobactam (PIP/TAZ) antibiotics may be used. Here, we aimed to investigate the interaction between these antifungals and antibiotics by in vitro and in vivo methods. The interaction was tested by chequerboard analysis and fractional inhibitory concentration index (FICI). It was also tested in a neutropenic mice-invasive candidiasis model and evaluated by fungal burden in kidney tissue of infected animals from the first day to the fifth day of treatment with 24 h intervals. A synergism was detected between CAS and CPZ/SAM (FICI = 0.1) and PIP/TAZ (FICI = 0.3). Fungal burden in tissues of drug-treated mice was reduced compared with controls in a time-dependent manner. In comparison with CAS-alone treated group, there were 1.32 log10 reductions of fungal burden in CAS + CPZ/SAM (p = 0.002) and in CAS + PIP/TAZ group (p = 0.14). The same interactions were not found with VRC and antibiotics. CPZ/SAM had stronger synergistic interaction with CAS than PIP/TAZ. The mechanism of synergism is not well understood. This is most likely due to an increase in the anticandidal effect of CAS plus antibiotics.

Clinical antibacterial effectiveness of the self‐adjusting file system

To evaluate in vivo the antibacterial effectiveness of the self-adjusting file (SAF) using molecular methods. Root canals from single-rooted teeth with apical periodontitis were instrumented using the SAF system under continuous irrigation with 2.5% NaOCl. DNA extracts from samples taken before and after instrumentation were subjected to quantitative analysis of total bacteria counts and levels of streptococci by quantitative real-time polymerase chain reaction (qPCR). The reverse-capture checkerboard assay was also used to identify 28 bacterial taxa before (S1) and after (S2) SAF instrumentation. SAF was also compared with a conventional hand nickel-titanium instrumentation technique for total bacterial reduction. Data from qPCR were analysed statistically within groups using the Wilcoxon matched pairs test and between groups using the Mann-Whitney U-test and the Fisher's exact test, with significance level set at P < 0.05. Self-adjusting file significantly reduced the total bacterial counts from a mean number of 1.96 × 10(7) cells to 1.34 × 10(4) cells (P < 0.001). Quantitatively, the 99.9% reduction in total bacterial counts associated with the SAF system was significantly superior to the 95.1% reduction obtained by hand instrumentation (P < 0.001). Qualitatively, SAF resulted in significantly more cases with negative PCR results for bacteria (54.5%) than hand instrumentation (4.5%) (P < 0.001). The SAF system succeeded in significantly reducing the streptococcal levels, but four cases still harboured these bacteria in S2. Checkerboard analysis revealed that not only streptococci but also some anaerobic and even as-yet-uncultivated bacteria may resist the effects of chemomechanical procedures. The SAF instrumentation system was highly effective in reducing bacterial populations from infected root canals and performed significantly better than hand instrumentation. However, because half of the samples still had detectable bacteria after preparation with SAF, supplementary disinfection is still required to maximize bacterial elimination.

In Vitro Activity of Doripenem in Combination with Various Antimicrobials Against Multidrug-Resistant Acinetobacter baumannii: Possible Options for the Treatment of Complicated Infection

The aim of this study was to evaluate the in vitro activity of doripenem (DOR) alone and in combination with a variety of commonly used anti-Acinetobacter chemotherapeutic agents against 22 primary multidrug-resistant (MDR) Acinetobacter baumannii isolates (including 17 isolates that were resistant to DOR) from Intensive Care Unit patients. Antibiotic interactions were evaluated using the chequerboard method and the time-kill assay. Considering all antimicrobials in combination with DOR, chequerboard analysis showed synergy in 13 A. baumannii strains (54.2%). Seven strains (29.2%) showed ≥2 synergistic interactions. DOR showed synergy in combination with tigecycline (TIG) (eight strains), colistin (COL) (eight strains), amikacin (AMK) (four strains), ampicillin/sulbactam (two strains), and rifampicin (one strain). Remarkably, synergistic effects were detected only in DOR nonsusceptible strains. Time-kill assays confirmed synergy in eight isolates (giving 10 synergistic interactions) for DOR in combination with TIG (n=4), COL (n=5), and AMK (n=1). No antagonistic interactions were observed with both methods. This study demonstrates the in vitro synergistic activity of DOR in combination with TIG, COL, and AMK against DOR-resistant A. baumannii strains, opening the way to in vivo assessment of novel combination therapies for treatment of infections caused by MDR A. baumannii.

Optimization of acquisition setup for cross‐hole: GPR full‐waveform inversion using checkerboard analysis

ABSTRACTTomographic inversions of cross‐hole ground‐penetrating radar provide images of electromagnetic properties of the shallow subsurface and are used in a wide range of applications. Whereas the resolutions of ray‐based methods like first‐arrival traveltime and first‐cycle amplitude tomography are limited to the scale of the first Fresnel zone, full‐waveform inversions incorporate precise forward modelling using the full recorded signal for a solution of Maxwell’s equation, which results in sub‐wavelength resolutions. In practice, the method can be time‐consuming in data acquisition and expensive in computational costs. To overcome these expenses, a semi‐reciprocal acquisition setup with a reduced number of transmitters and an interchange of transmitter and receiver boreholes instead of a one‐sided equidistant setup in either borehole yielded promising results. Here, this optimized, semi‐reciprocal acquisition setup is compared to a dense, equidistant, one‐sided acquisition setup measured at the field site Krauthausen, Germany. The full‐waveform inversion results are evaluated using the checkerboard test as a capable resolution analysis tool to determine resolvabili‐ties. We introduced also a new method of time‐zero correction by a cross‐correlation of a zero‐offset profile with corresponding horizontal traces of each multi‐offset gather. The obtained experimental results from Krauthausen combined with the checkerboard analysis indicate the main three‐permittivity layers that correspond with different porosities. Also fine‐layered structures within these main layers were reliably imaged. We conclude that the use of the semi‐reciprocal setup is optimum for acquisition speed, inversion speed and obtained permittivity inversion results. Our results indicate that conductivity results are better for denser transmitter‐receiver setups.

Salivary cytokines and levels in denture stomatitis: An exploratory case-control study

We aimed to evaluate the microbial and inflammatory characteristics associated with Denture Stomatitis (DS) analyzing: l) Levels of salivary cytokines and cultivable C. albicans; 2) DNA-DNA checkerboard on biofilm associated with mucosal tissue-bearing denture surfaces, 3) Serum C-reactive protein (CRP) levels. Thirty-two subjects were enrolled in the study with control (n = 17) and DS types II and III (n = 15) subjects. Samples were collected from unstimulated whole saliva, serum and swabs from denture surfaces. Salivary levels of inflammatory mediators and CRP were measured by multiplex. Samples from denture and mucosal surfaces were analyzed by DNA-DNA checkerboard. Saliva from DS subjects showed increase in IL-8 (p = 0.04) and IL-1β (p = 0.04) with trend for increase in IL-1β, TNFα and IL-6 levels. C. albicans higher counts in DS saliva (p = 0.03) showed association with elevated levels of IL-8 (p = 0.03) and IL-1α (p = 0.01). CRP levels were not different among groups (p = 0.74). DNA-DNA checkerboard analyses indicated typical periodontal pathogens below the detection threshold of 104 organisms on both denture and inflamed mucosal surfaces. The data suggest that DS is associated with elevation of salivary IL1 and IL-8 together with increased C. albicans. There was no evidence of systemic inflammation as measured by serum C-reactive protein levels.

Clinical and microbiological characteristics of peri‐implantitis cases: a retrospective multicentre study

The aim of this study was to follow patient cases retrospectively in a longitudinal manner from the time of implant placement to the time they were diagnosed with peri-implant disease, and to identify associated clinical and microbiological features of peri-implant disease. A total of 281 patient cases were chosen from the archives of the Oral Microbiological Diagnostic Laboratory, Gothenburg, Sweden, based on bacterial samples taken from diseased implants. A form was designed and filled in separately for each case including data on patient, implant and disease profile. Most cases were severe peri-implantitis cases (91.4%). In 41.3% of the patients, peri-implantitis was developed early, already after having implants in function less than 4 years. The type of implant surface was significantly associated with the time in years implants were in function, before disease was developed (P < 0.05). The microbiological results by both culture and checkerboard analysis, although failed to fully correspond to the severity of the disease in terms of magnitude, proved to show that peri-implantitis is a polymicrobial anaerobic infection with increased number of AGNB (aerobic Gram-negative bacilli) in 18.6% of the patients. Peri-implantitis is a biological complication of implants in function that poses a threat to their long-term survival. It may develop earlier around implants with rough surfaces and it may represent a true infection. Microbiological sampling methods should be improved and uniformed so as to fully unveil the microbiological profile of the disease.

Microbiota of Dentinal Caries as Assessed by Reverse-Capture Checkerboard Analysis

Background/Aims: This study aimed to identify the microbiota of different layers of dentinal caries by using a culture-independent molecular biology approach. Methods: DNA was extracted from samples taken from 3 distinct layers (superficial, middle and deep) of advanced occlusal caries and analyzed for the presence and relative levels of 28 oral bacterial species/phylotypes using a reverse-capture checkerboard hybridization assay. Results: The mean number of target taxa per layer was 7.7 (±3.96) in the superficial, 7 (±3.4) in the middle, and 6.3 (±3.04) in the deep layer. No statistical significance was observed for these differences (p = 0.36). Overall, the most prevalent taxa in the 3 layers were Atopobium genomospecies C1 (72.5%), Fusobacterium nucleatum (69%), Lactobacillus casei (68%), Veillonella species (55%) and Lactobacillus fermentum (52%). No differences were found in the prevalence rates of the most frequent target species in the 3 layers. The most prevalent taxa found at levels above 10⁵ in the advanced front line of deep-dentin caries were Atopobium genomospecies C1, F. nucleatum, Streptococcus mutans, Streptococcus species and Veillonella species. Conclusion: The present results revealed that the prevalences of several established or candidate caries pathogens do not differ significantly in the different zones of dentinal caries lesions. The finding that some as-yet-uncharacterized species and novel species were found in high frequencies join other molecular studies to include them in the set of candidate caries pathogens.

IL6 −174 Genotype Associated with Aggregatibacter actinomycetemcomitans in Indians

Genetic factors have recently been associated with presence of Aggregatibacter actinomycetemcomitans subgingivally in populations living in industrialized countries. The aim of this study was to analyse associations between Interleukin-6 (IL6) single nucleotide polymorphisms and presence and levels of A. actinomycetemcomitans and other subgingival microbes in a rural Indian population. A total of 251 individuals from a rural village in India with a periodontal phenotype ranging from healthy to severe periodontitis were included. Checkerboard DNA-DNA analysis was performed to detect 40 periodontal taxa in subgingival plaque samples. Genomic DNA was extracted to genotype five polymorphisms in the IL6 promoter region. The IL6-74 GG genotype was associated with high (above median) counts of A. actinomycetemcomitans (both in all subjects and in periodontally healthy only) and with presence and counts of Capnocytophaga sputigena. Differences in detection of several other bacteria were noted between periodontitis and healthy subjects. These findings support the influence of genetic factors on the subgingival microbiota.

Apical Root Canal Microbiota as Determined by Reverse-capture Checkerboard Analysis of Cryogenically Ground Root Samples from Teeth with Apical Periodontitis

Introduction Bacteria located in the apical root canal system potentially participate in the pathogenesis of apical periodontitis. Detection and identification of apical bacteria can be compromised because of limitations in conventional sampling and identification procedures. This study identified several bacterial taxa in the apical and middle/coronal segments of primarily infected root canal system by using pulverized root segments and a culture-independent molecular method. Methods Seventeen extracted teeth with attached apical periodontitis lesions were sectioned to obtain 2 root fragments (apical and middle/coronal segments). Root fragments were cryogenically ground, and DNA was extracted from samples. After multiple displacement amplification, DNA from samples was used as template in a reverse-capture checkerboard hybridization assay targeting 28 bacterial taxa. Results Bacterial DNA was detected in all samples. The most prevalent taxa in the apical root canal system were Olsenella uli (76.5%), Prevotella baroniae (71%), Porphyromonas endodontalis (65%), Fusobacterium nucleatum (53%), and Tannerella forsythia (47%). O. uli, P. endodontalis, and Propionibacterium acnes were as frequently detected in apical samples as they were in middle/coronal samples. P. baroniae, T. forsythia, and F. nucleatum were found more frequently in the apical part of the canal as compared with matched coronal segments. Streptococcus species were more prevalent in middle/coronal samples. The median and mean of shared bacterial taxa between matched apical and middle/coronal segments were 27% and 41%, respectively. Conclusions Several candidate endodontic pathogens were very prevalent in the apical root canal system. The apical microbiota was usually complex and differed in species composition when compared with the microbiota of middle/coronal samples from the same tooth.

The human male reproductive tract antimicrobial peptides of the HE2 family exhibit potent synergy with standard antibiotics

Reproductive tract infections pose a serious threat to health and fertility. Due to the emergence of antibiotic resistant pathogens, antimicrobial proteins and peptides of the reproductive tract are extensively characterized in recent years toward developing newer strategies to treat genital tract infections. Pathogen growth inhibition using a combination of naturally occurring male reproductive tract antimicrobial peptides and commonly used antibiotics has not been reported. Checker board analyses were carried out to determine the nature of interaction (synergistic, additive and antagonistic) between HE2alpha and HE2beta2 peptides and the commonly used antibiotics. Using Escherichia coli as the target organism, the minimal inhibitory concentration and fractional inhibitory concentration indices were determined. We demonstrate for the first time that the human male reproductive tract antimicrobial peptides HE2alpha and HE2beta2 act synergistically with the commonly used antibiotics to inhibit E. coli growth. A combination of HE2alpha and HE2beta2 peptides resulted in an additive effect. Interestingly, the synergistic effects of HE2 peptides were highest with doxycycline and ciprofloxacin, antibiotics generally used to treat epididymitis. Results of this study demonstrate the potential of endogenous HE2 peptides to be pharmacologically important in designing novel strategies to treat reproductive tract infections.

Performance Evaluation of the New Roche Cobas AmpliPrep/Cobas TaqMan HIV-1 Test Version 2.0 for Quantification of Human Immunodeficiency Virus Type 1 RNA

Despite FDA approval and CE marking of commercial tests, manufacturer-independent testing of the technical aspects of newly developed tests is important. To evaluate the analytical performance and explore the clinical applicability of the new Roche COBAS AmpliPrep COBAS TaqMan HIV-1 test, version 2.0 (CAP/CTM v2.0), platform comparison was performed with the Roche CAP/CTM test, version 2.0, the COBAS Amplicor HIV-1 Monitor Test, version 1.5 (CAP/CA v1.5), the COBAS AmpliPrep COBAS TaqMan HIV-1 Test (CAP/CTM v1.0), and the Abbott m2000 RealTime HIV-1 assay on panels and diagnostic samples. Specificity was tested for HIV-2 samples. Furthermore, samples from HIV-1-seropositive individuals with CAP/CA v1.5-measured viral loads below 50 HIV-1 RNA copies per ml (cp/ml) and replicates of HIV-1-seronegative plasma were tested in a checkerboard analysis. CAP/CTM v2.0 is HIV-1 specific, with broad genotype inclusivity and no serious underquantification of viral load relative to the other assays used. Low viral loads below the threshold of quantification for CAP/CA v1.5 are observed with CAP/CTM v2.0. A CAP/CTM v2.0-measured viral load of >50 copies/ml in these samples correlated with therapy failure. In conclusion, CAP/CTM v2.0 is an accurate and reliable test for HIV-1 viral load measurement relative to the other assays used with respect to specificity, sensitivity, and genotype inclusivity.

Comparison of antifungal efficacies of moxifloxacin, liposomal amphotericin B, and combination treatment in experimental Candida albicans endophthalmitis in rabbits

The goal of this study was to compare in vitro and in vivo efficacy of moxifloxacin and liposomal amphotericin B (Amp-B) monotherapies and combination treatment against Candida albicans in an exogenous endophthalmitis model in rabbit eyes. Microplate dilution tests and checkerboard analysis were performed to detect in vitro efficacies. Endophthalmitis was induced by intravitreal injection of C. albicans in 40 rabbit eyes with simultaneous intravitreal drug injection according to prophylactic treatment groups. Group 1 (control group) received 0.1 mL of balanced salt solution, group 2 (moxi group) 100 microg moxifloxacin/0.1 mL, group 3 (Amp-B group) 10 microg liposomal Amp-B/0.1 mL, and group 4 (combi group) both 100 microg moxifloxacin/0.1 mL [DOSAGE ERROR CORRECTED] and 10 microg liposomal Amp-B/0.05 mL intravitreally. Clinical examination, quantitative analysis of microorganisms, and histopathologic examination were performed as in vivo studies. The minimum inhibitory concentration of liposomal Amp-B against C. albicans was found to be 1 microg/mL. Moxifloxacin showed no inhibition of in vitro C. albicans growth. The minimum inhibitory concentration values of liposomal Amp-B for C. albicans were reduced two- to eightfold with increasing concentrations of moxifloxacin in vitro. In vivo, there was no C. albicans growth in the combi group (zero of eight eyes), whereas three eyes (37.5%) showed growth in the Amp-B group. Vitreous inflammation, retinal detachment, focal retinal necrosis, and outer nuclear layer loss were found to be lower in the moxi group compared with the control group. Ganglion cell and inner nuclear layer loss was observed in all eyes (100%) in both the moxi and combi groups, whereas only in 25% (two of eight eyes) in the Amp-B group. Moxifloxacin strongly augments the efficacy of liposomal Amp-B against C. albicans in vitro, although it has no in vitro antifungal activity when used alone. It is interesting that we found a synergistic effect for in vitro tests but failed to demonstrate it in vivo. When 100 microg moxifloxacin/0.1 mL is given intravitreally, it has some toxic effects that are limited to the inner retinal layers.

Comparison between polymerase chain reaction‐based and checkerboard DNA hybridization techniques for microbial assessment of subgingival plaque samples

To compare polymerase chain reaction (PCR) with subsequent reverse hybridization (micro-IDent test) and checkerboard DNA-DNA hybridization for the identification of 13 bacterial species in subgingival plaque samples. Subgingival plaque samples were taken using paper points and curettes from two sites each with pocket depth <4, 4-6 and >6 mm at baseline and 3 months in 25 periodontitis subjects and two sites in 25 periodontally healthy subjects. Samples were analysed for their content of 13 bacterial species using both assays. Similarities for each species between techniques were determined using regression analysis. Differences between health and periodontitis were determined using the Mann-Whitney test. Three hundred and fifty samples were evaluated using both techniques. Regression analysis indicated that 10/13 test species showed significant positive correlations between the counts determined by checkerboard analysis and levels determined by the PCR-based test after adjusting for 13 comparisons. The highest rank correlations of 0.58, 0.49 and 0.46 were seen for Treponema denticola, Fusobacterium nucleatum and Eubacterium nodatum, respectively (p<0.0001). Both tests could distinguish samples from healthy and periodontitis subjects. Detection patterns of 10/13 test species in subgingival plaque samples from periodontitis and healthy subjects were similar using the two molecular techniques.

In vitro activity of tigecycline in combination with various antimicrobials against multidrug resistant Acinetobacter baumannii

BackgroundInfections sustained by multidrug-resistant (MDR) and pan-resistant Acinetobacter baumannii have become a challenging problem in Intensive Care Units. Tigecycline provided new hope for the treatment of MDR A. baumannii infections, but isolates showing reduced susceptibility have emerged in many countries, further limiting the therapeutic options. Empirical combination therapy has become a common practice to treat patients infected with MDR A. baumannii, in spite of the limited microbiological and clinical evidence supporting its efficacy. Here, the in vitro interaction of tigecycline with seven commonly used anti-Acinetobacter drugs has been assessed.MethodsTwenty-two MDR A. baumannii isolates from Intensive Care Unit (ICU) patients and two reference strains for the European clonal lineages I and II (including 3, 15 and 6 isolates that were resistant, intermediate and susceptible to tigecycline, respectively) were tested. Antimicrobial agents were: tigecycline, levofloxacin, piperacillin-tazobactam, amikacin, imipenem, rifampicin, ampicillin-sulbactam, and colistin. MICs were determined by the broth microdilution method. Antibiotic interactions were determined by chequerboard and time-kill assays. Only antibiotic combinations showing synergism or antagonism in both chequerboard and time-kill assays were accepted as authentic synergistic or antagonistic interactions, respectively.ResultsConsidering all antimicrobials in combination with tigecycline, chequerboard analysis showed 5.9% synergy, 85.7% indifference, and 8.3% antagonism. Tigecycline showed synergism with levofloxacin (4 strains; 16.6%), amikacin (2 strains; 8.3%), imipenem (2 strains; 8.3%) and colistin (2 strains; 8.3%). Antagonism was observed for the tigecycline/piperacillin-tazobactam combination (8 strains; 33.3%). Synergism was detected only among tigecycline non-susceptible strains. Time-kill assays confirmed the synergistic interaction between tigecycline and levofloxacin, amikacin, imipenem and colistin for 5 of 7 selected isolates. No antagonism was confirmed by time-kill assays.ConclusionThis study demonstrates the in vitro synergistic activity of tigecycline in combination with colistin, levofloxacin, amikacin and imipenem against five tigecycline non-susceptible A. baumannii strains, opening the way to a more rationale clinical assessment of novel combination therapies to combat infections caused by MDR and pan-resistant A. baumannii.

In vitro antifungal combination effects of micafungin with fluconazole, voriconazole, amphotericin B, and flucytosine against clinical isolates of Candida species

Micafungin (MCFG) is an echinocandin antifungal agent that exhibits potent activity against most species of Candida and Aspergillus. We investigated the in vitro antifungal combination effects of MCFG with four other antifungal agents - fluconazole (FLCZ), voriconazole (VRCZ), amphotericin B, and flucytosine - against clinical isolates of 54 Candida spp. by checkerboard analysis. The synergistic antifungal effects of MCFG-FLCZ and MCFG-VRCZ were 11% and 15%, respectively, and the latter displayed a synergistic activity of 63% against Candida glabrata. Antagonism was not observed in any of the combinations tested.

IGF-I secreted by osteoblasts acts as a potent chemotactic factor for osteoblasts

Osteoblast recruitment to the site of future bone formation is essential for skeletal development, bone remodeling and fracture healing. A number of factors associated with bone tissue have been reported to induce directional migration of osteoblasts but the mechanism remains to be clarified. In this study, to explore a major chemotactic factor(s) for osteoblasts, we examined the serum-free medium conditioned by MC3T3-E1 osteoblast-like cells for its ability to induce osteoblast migration. Employing sequential chromatography and tandem mass spectrometry analysis, we purified and identified IGF-I as a potent chemotactic factor from the conditioned medium. IGF-I induced cell migration of both MC3T3-E1 cells and primary mouse osteoblasts, and checkerboard analysis revealed that IGF-I markedly induced directional migration (chemotaxis) of osteoblasts. Neutralization of mouse IGF-I with monoclonal antibodies resulted in delayed osteoblast monolayer wound healing and cellular polarization but addition of human IGF-I reversed these effects. IGF-I also promoted cell spreading on fibronectin in an integrin β1-dependent manner. IGF-I induced Akt and Rac activation and localized accumulation of phosphatidylinositol 3,4,5-triphosphate (PtdIns (3,4,5)P 3) at the membrane in osteoblasts. The phosphatidyl inositol 3 kinase (PI3K) inhibitor LY294002 inhibited IGF-I-induced cell migration and wound healing. Together, the results suggest that IGF-I secreted from osteoblasts in the bone tissue is a potent chemotactic factor that may play a major role in recruitment of osteoblasts during bone formation.

Cleavage of Type II Collagen by Cathepsin K in Human Osteoarthritic Cartilage

Cathepsin K is a cysteine protease of the papain family that cleaves triple-helical type II collagen, the major structural component of the extracellular matrix of articular cartilage. In osteoarthritis (OA), the anabolic/catabolic balance of articular cartilage is disrupted with the excessive cleavage of collagen II by collagenases or matrix metalloproteinases. A polyclonal antibody against a C-terminal neoepitope (C2K) generated in triple-helical type II collagen by the proteolytic action of cathepsin K was prepared and used to develop an enzyme-linked immunosorbent assay to study the generation of this epitope and the effects of its presence in normal adult and osteoarthritic femoral condylar articular cartilage. The generation of the C2K epitope in explant culture and the effect of a specific cathepsin K inhibitor were studied. The neoepitope, which is not generated by the collagenase matrix metalloproteinase-13, increased with age in articular cartilage and was significantly elevated in osteoarthritic cartilage compared with adult nonarthritic cartilage. Moreover, in explants from three of eight OA patients, the generation of the neoepitope in culture was significantly reduced by a specific, nontoxic inhibitor of cathepsin K. These data suggest that cathepsin K is involved in the cleavage of type II collagen in human articular cartilage in certain OA patients and that it may play a role in both OA pathophysiology and the aging process.

CYR61/CCN1 and WISP3/CCN6 are chemoattractive ligands for human multipotent mesenchymal stroma cells

Background:CCN-proteins are known to be involved in development, homeostasis and repair of mesenchymal tissues. Since these processes implicate recruitment of cells with the potential to be committed to various phenotypes, we studied the effect of CYR61/CCN1 and WISP3/CCN6 on migration of human bone marrow derived mesenchymal stroma cells (MSCs) in comparison to in vitro osteogenic differentiated MSCs using a modified Boyden chamber assay.Results:CYR61 and WISP3 were purified as fusion proteins with a C-terminal Fc-tag from baculovirus infected SF21 cells using protein G sepharose columns. CYR61 and WISP3 stimulated cell migration of undifferentiated MSCs in a dose-dependent manner. CYR61 and WISP3 had similar effects on committed osteogenic precursor cells. Checkerboard analysis revealed that CYR61 and WISP3 stimulated true directed cell migration (chemotaxis) of MSCs and committed osteogenic precursors. In MSCs the chemotactic activity of WISP3 but not CYR61 was mediated through integrin ανß5.Conclusion:Our results indicate that CYR61 and WISP3 can function as soluble ligands transmitting chemotactic signals to human MSCs but differ in the involvement of integrin ανß5. This may be relevant for their possible role in connective tissue repair.