The microfluidic chip was fabricated by MEMS process.

The combination of the traditional LF test strip with capillary-driven gold-coated substrate results in the enhancement of sensitivity as well as the reduction of cost for SERS-based immunodiagnostic techniques. In this work, a calibration curve was obtained to detect the concentration of abrin in the range from 0.1 ng/mL to 1 μg/mL, which is superior to the traditional LF test strip for the same purpose in respect of both sensitivity and quantitation [21]. What is critically important is the Selleckchem TSA HDAC operability of our design strategy, that is, the performance of traditional LF test strips is improved without excessive increase in complication and cost of fabrication. In addition, this SERS-based microfluidic chip can be further developed and applied to other on-demand and point-of-care detection for a substance of interest. Acknowledgements This work is supported by the National Key Basic Research Program (973 Project) (No. 2011CB933101), National Natural Scientific Fund (Nos. 81225010, 81327002, and 31100717), 863 project of China (2012AA022703), Shanghai Science and Technology Fund (Nos. GNS-1480 solubility dmso 13NM1401500

and 11nm0504200), and Shanghai Jiao Tong University Innovation Fund for Postgraduates (No. AE340011). References 1. Felder E, Mossbrugger I, Lange Selleck PKC412 M, Wolfel R: Simultaneous detection of ricin and abrin DNA by real-time PCR (qPCR). Toxins 2012, 4:633–642.CrossRef 2. Dickers KJ, Bradberry Avelestat (AZD9668) SM, Rice P, Griffiths GD, Vale JA: Abrin poisoning. Toxicol Rev 2003, 22:137–142.CrossRef 3. Jang DH, Hoffman RS, Nelson LS: Attempted suicide, by mail order: Abrus precatorius. J Med Toxicol 2010, 6:427–430.CrossRef 4. Balali-Mood

M, Moshiri M, Etemad L: Medical aspects of bio-terrorism. Toxicon 2013, 69:131–142.CrossRef 5. Yang D-P, Chen S, Huang P, Wang X, Jiang W, Pandoli O, Cui D: Bacteria-template synthesized silver microspheres with hollow and porous structures as excellent SERS substrate. Green Chem 2010, 12:2038–2042.CrossRef 6. Lin CC, Yang YM, Chen YF, Yang TS, Chang HC: A new protein A assay based on Raman reporter labeled immunogold nanoparticles. Biosens Bioelectron 2008, 24:178–183.CrossRef 7. Nie S, Emory SR: Probing single molecules and single nanoparticles by surface-enhanced Raman scattering. Science 1997, 275:1102–1106.CrossRef 8. Porter MD, Lipert RJ, Siperko LM, Wang G, Narayanan R: SERS as a bioassay platform: fundamentals, design, and applications. Chem Soc Rev 2008, 37:1001–1011.CrossRef 9. Grubisha DS, Lipert RJ, Park HY, Driskell J, Porter MD: Femtomolar detection of prostate-specific antigen: an immunoassay based on surface-enhanced Raman scattering and immunogold labels. Anal Chem 2003, 75:5936–5943.CrossRef 10. Zong S, Wang Z, Chen H, Hu G, Liu M, Chen P, Cui Y: Colorimetry and SERS dual-mode detection of telomerase activity: combining rapid screening with high sensitivity.

Mutations in this gene lead to inactivity of the CFTR protein and/or reduced expression of the protein at the cytoplasmic membrane [2]. Improper functioning of the CFTR results in the production of viscous mucus and in a defective innate immunity [2, 3]. The reduced functionality of the mucociliary system and the ongoing inflammation result in an increased sensitivity of the CF airways

to infection by bacterial pathogens, of which Pseudomonas aeruginosa and Staphylococcus aureus are the most important. Chronic lung infection with P. aeruginosa is a major cause of morbidity and mortality among the CF patients [4]. It is now well-established that early aggressive antibiotic treatment of new infection with P. aeruginosa is successful in postponing chronic infection. Hence, it is important to detect new selleck kinase inhibitor infection with P. aeruginosa as early as possible so that eradication treatment can be started as soon as possible [5–7]. Currently, routine detection and identification of P. aeruginosa in respiratory samples is done by conventional methods such as culture and biochemical characteristics. Misidentification can occur due to the variable phenotypic characteristics of this species [8]. Moreover, the sensitivity of culture might be limited, especially when compared VEGFR inhibitor to DNA amplification based techniques. Thus far, however,

only one group has compared both approaches in a long term study for early detection of P. aeruginosa Dimethyl sulfoxide from CF patients [9]. In this national study, we followed CF patients during periods between 1 to 15 months and we compared the sensitivity of conventional culture

techniques with qPCR for the detection of P. aeruginosa in the respiratory samples from CF patients, not chronically infected by P. aeruginosa. Methods Patients and sampling From January 2008 until May 2009, sputum, nasopharyngeal or throat swab samples were routinely collected from 397 CF patients attending all but one of Belgian CF-centres, i.e. Ghent University Hospital (UZG, Ghent), Universitair Ziekenhuis Brussel (UZB, Brussels), St Luc University Hospital (UCL, Brussels), Queen Fabiola Children’s University Hospital and Erasme University Hospital (ULB, Brussels), Antwerp University Hospital (UZA, Antwerp), CF Center Liege (CHC – CHR, Liege). Patients were seen every three months and sputum or nasopharyngeal aspirate/throat swab samples were cultured at every visit. Nasopharyngeal aspirates/throat swab samples were collected in case the patients could not expectorate. All 397 included patients, (median age: 14 years, range: 1-53 years), were considered as P. aeruginosa free and not chronically infected according to the criteria used by the different Belgian CF centers, i.e., the check details European Consensus criteria [10] or those defined by Lee et al. [11].

Sciatic neurectomy resulted in a non-site-specific increase in osteocyte sclerostin expression in both cortical and trabecular bone. This upregulation was not observed following sham sciatic neurectomy. The uniform SC79 supplier increase in sclerostin expression with sciatic neurectomy-induced disuse contrasts with

the regional effects seen with loading, probably because the effect of disuse induced by sciatic neurectomy is a uniform reduction in mechanical strain [40]. Our data, in 19-week-old female mice, are not perfectly consistent with those of others using tail suspension, in 6-week-old male mice, where unloading was associated with an increase in the expression of the sost gene but not the sclerostin protein [6]. Potential reasons for this discrepancy include the possibility that tail suspension permits continued muscle activity which, even in the absence of ground reaction forces, may engender significant changes in bone strain. Nevertheless, mice lacking the

sost gene showed resistance to bone loss induced by tail suspension in both cortical and trabecular regions [8]. The relevance of the present short-term experiment in mice to the human condition must selleck chemicals take into account a number of differences in the two situations including the pattern of their normal bone modeling and remodeling. buy BTSA1 However, the implication of this study for our understanding of the potential role of sclerostin in loading and disuse-related control of bone (re)modeling is probably transferable. Indeed, in agreement with our experimental data, immobilization-induced bone loss in stroke patients is associated with a Palbociclib ic50 state of “hypersclerostinemia” [41]. The circulating sclerostin levels in humans negatively correlate with the circulating PTH levels [42] and osteocytic Sost suppression is likely to mediate the effects of intermittent PTH [43, 44] which synergistically enhances

loading-related osteogenesis in mice [45]. Sclerostin-neutralizing drugs [12, 13] therefore have great potential to provide an effective anabolic treatment for the prevention of fragility fractures in humans. In conclusion, the present data from both cortical and cancellous bone in adult female mice suggest a substantial regulation of osteocyte sclerostin production by bone’s mechanical environment. Exposure to loading is generally associated with downregulation and disuse with upregulation. However, osteocyte sclerostin status appears to be less closely related to the magnitude of local loading-related strain, as determined by surface-bonded strain gauges and by FE analysis, than to the subsequent increase in new bone formation. Further studies are required to elucidate the mechanistic association between changes in osteocytic sclerostin expression and local new bone formation.

At the indicated times about 105 cells were collected by centrifugation (5,000 × g for 10 min). At least 1 mL of the cell-free culture fluid was saved, buy Panobinostat air-saturated and stored on ice until use. The cell selleck compound pellet was resuspended in a small volume of the corresponding culture fluid. Propidium iodide (5 mM, dissolved in phosphate-buffered saline) was added to 20 μL of this cell suspension to stain dead cells (red fluorescence), and the suspension

was immediately transferred onto a coverslip and incubated in the dark for 20 min to allow cells to adhere. All coverslips were pretreated with poly L-lysine (0.05 g*L-1) to fix the cells on the surface. Subsequently, cells were washed twice with the corresponding air-saturated culture fluid directly

on the coverslip to remove non-adherent cells. Phase contrast and fluorescence images were taken at room temperature using a customized inverted Leica DMI 6000 B microscope, an oil-immersion objective and a high-sensitivity iXON CCD camera (Andor). Fluorescence microscopy was performed using the bandpass filters BP546/12 (red) and BP470/40 (green) and the emission filters 605/75 AMN-107 datasheet (red) and 525/50 (green). Luminescent cells were identified by bioluminescence microscopy without any filter in a Pecon flow chamber to ensure sufficient oxygen supply [3]. The exposure time for imaging of luminescent cells with the cooled (-80°C) CCD camera was set to 240 s. Phase-contrast, bioluminescence and/or fluorescence images were obtained from the same fields of view. Single cell analysis Images were analyzed using ImageJ 1.37c (National Institute of Health http://​rsb.​info.​nih.​gov/​ij). A screen depicting the contours of the cells was created from the phase contrast image using the self-programmed PlugIn CellEvaluator (Prof.

Dr. J. Rädler, LMU Munich). This screen was superimposed on the background-corrected fluorescence and bioluminescence images. Intensities were determined for each cell and normalized by cell size. The correlation coefficient r is defined as the covariance of two variables (here fluorescence and luminescence) divided by the product of their standard deviations. A value of |r| = 1 indicates 100% correlation. The p-value is a measure of the probability that the correlation is due to chance. Time-lapse histograms were Glycogen branching enzyme generated using Matplotlib (http://​matplotlib.​sourceforge.​net). Acknowledgments This work was financially supported by the Deutsche Forschungsgemeinschaft (Exc114/1) and (Ju270/9-1) and the BMBF (ChemBiofilm). We are indebted to Joachim Rädler for access to the PlugIn CellEvaluator and to Judith Mergerle and Georg Fritz for instruction in its use. We are grateful to Kolja Prothmann for assistance in preparing the illustrations using Matplotlib and to Laure Plener for helpful discussions during the preparation of the manuscript. References 1. Chai Y, Chu F, Kolter R, Losick R: Bistability and biofilm formation in Bacillus subtilis. Mol Microbiol 2008, 67:254–263.

Beyond the data presented herein, no data are currently available to determine whether pre-exposure to environmental stresses might affect bacterial uptake or intracellular killing by amoeba. Other C. jejuni/amoeba studies were performed using bacteria grown in optimal culture conditions (temperature, media and atmospheric conditions) which C59 wnt ic50 are not adapted to stressful conditions [24–28], or simply probe the ability of C. jejuni to sustain stressful conditions during or after interactions

with amoeba [33]. Stress-induced bacterial adaptation to enhance the bacteria’s ability to survive a subsequent interaction with amoeba, and amoeba-mediated enhanced bacterial resistance to stress are complementary mechanisms that are important for the survival of C. jejuni in the environment. STAT inhibitor Our data showed that low nutrient and osmotic stresses were the strongest factors which significantly affected the survival of C. jejuni (Figure  1, decreased survival in pure cultures without amoeba) and the transcription of three virulence-associated genes (Figure  2), and also reduced the uptake of the bacterium by A. castellanii (Figure  3). Our findings are consistent with previous studies that reported that starvation strongly affected C. jejuni invasion in Caco-2 and macrophages [6,

58]. In contrast, our data showed that heat and oxidative stresses did not affect the uptake of C. jejuni by amoebae. These findings differ from previous studies that reported that pre-exposure of C. jejuni to oxidative stress increased the invasion of C. jejuni in intestinal cells [45, 47], and that heat stress reduced the invasion of C. jejuni in Caco-2 and macrophages. These discrepancies are likely due to cell line-specific mechanisms of uptake Cyclooxygenase (COX) and killing, Go6983 order variations in the nature and abundance of appropriate eukaryotic receptors [59], and differences in the experimental set up used to apply the heat stress as indicated above. Correlation

between the effects of stress on transcription of virulence-associated genes and on uptake by amoeba Previous studies have shown that ciaB, htrA, and dnaJ play important roles in the invasion of C. jejuni[11, 34, 35, 38, 39, 55], but most of these studies involve epithelial cells which have little to no phagocytic abilities. The effect of ciaB, htrA and dnaJ on interaction with amoeba in which entry is based on phagocytosis remained to be established. Our working hypothesis was that transcriptional effects triggered on virulence-associated genes by pre-exposure to stress may affect subsequent interactions with amoeba, even if they did not affect bacterial viability. Therefore, we examined whether down- or up- regulation of virulence-related genes correlated with decreased or increased bacterial uptake and/or intra-amoeba survival, with the understanding that correlation does not imply direct causality.

62–5.05) 1.74 (0.61–4.99)  Stratified by age categoryb   ≤70 years 13.5 12.5 1.10 (0.61–1.98) 1.16 (0.64–2.09)   >70 years 13.4 6.5 2.14 (1.07–4.30) 2.22 (1.11–4.47) OP osteoporosis prophylaxis drugs, HR hazard ratio, CI confidence PLX4032 mw interval, DDDs defined daily dosage prednisone equivalents aAdjusted for age categories (≤70, >70) and use of hydrocortisone in the 6 months before baseline bAdjusted for hydrocortisone use in

the 6 months before baseline Discussion This randomised controlled trial showed that active identification of GIOP-eligible patients by community pharmacists did not significantly increase the prescribing rate of bisphosphonates in the total study population. However, subgroup analyses showed that there was a significant increase in the primary endpoint in males and in the elderly (>70 years). Similar results

were seen for the composite endpoint of any prophylactic osteoporosis drug (bisphosphonate, calcium, or selleck chemicals llc vitamin D). To the best of our knowledge, this is the first randomised controlled trial where pharmacists identified GIOP-eligible patients and subsequently contacted the prescriber, without further training of the patient or the physician [22]. The only previously conducted pharmacy-based randomised controlled trial that aimed to increase GIOP found an increased prescribing EPZ015938 clinical trial rate of calcium but not of bisphosphonates [19]. This trial was conducted at 15 community pharmacies (intervention 70 patients, control 26 patients). The pharmacists received training for GIOP, identified eligible patients, gave them education for GIOP and contacted the prescriber when necessary. However, pharmacists in both the intervention and control groups received training about GIOP and the importance of bone mineral density (BMD) testing which may have diluted the results. medroxyprogesterone Another randomised controlled trial has shown a twofold increase (28 patients (22 %) intervention group vs. 14 patients (11 %) control group; relative risk 2.1, 95 % CI 1.1–3.7) in the composite endpoint of BMD testing or incident osteoporosis treatment with a community pharmacist screening programme [21]. In contrast to the present study, all patients and pharmacists received education about osteoporosis.

Other attempts to increase GIOP mostly included educational interventions directed at physicians (general practitioners or rheumatologists) but were often without or with modest results [16–18]. The lack of an overall intervention effect was accompanied by a low number of bisphosphonate-treated patients [14, 17]. It should be noted that the study population did not include patients who already received a prescription for a bisphosphonate in the 6 months prior to baseline. Chitre et al. (2008) similarly excluded these patients and found comparable incident treatment rates for osteoporosis prophylaxis. In addition, our study population included patients who received a bisphosphonate more than 6 months before baseline (10.8 % in the intervention group, 12.

In contrast, a more recent study found that CheA could bind to the receptors independent of CheW and that CheW only strengthened the interaction [86]. An in vivo localization study found that truncated CheA constructs could bind to receptor clusters independently of CheW, whereas full-length click here CheA required CheW for this [87]. Only Htr group 1 matches the expected composition of prokaryotic

taxis signaling complexes (receptor-transducer, CheW, CheA, CheY, [19, 73]). Considering that binding of a CheW domain protein is mandatory for CheA activity [88–93], our findings indicate that only the receptors from group 1 were active under the tested conditions. At least for Htr11 (Car, the cytoplasmic arginine receptor, [42]), the only receptor with known signal that was assigned to a group other than group 1, this would make sense. Hbt.salinarum degrades arginine to ornithine coupled with the production of ATP [94]. This substrate-level phosphorylation allows the cells to grow in the absence

of light and oxygen, making taxis towards arginine crucial under these conditions. Under the aerobic conditions used in our experiments, the cells can produce energy by oxidative phosphorylation. Arginine is indeed metabolized under aerobic conditions and is depleted rapidly from the medium, but it can be resynthesized from ornithine [95]. Consequently, the cells have no need for arginine uptake and arginine taxis could be switched off. Two novel interactors P505-15 ic50 of CheA Two proteins were identified as novel interaction partners of CheA (Figures 3 and 5). The first is PurNH (OE1620R) which is annotated as a phosphoribosylglycinamide formyltransferase (EC 2.1.2.2) / phosphoribosylaminoimidazolecarboxamide formyltransferase (EC 2.1.2.3). Thus it carries out two essential enzymatic activities in purine metabolism. PurNH was fished by CheA, CheW1 and CheY (Figure 5).

When PurNH was subsequently used as bait, it fished CheA and most of the group 1 Htrs. In all experiments, PurNH showed an interaction and exchange behavior identical to that of CheA (Additional file 4), indicating that it is statically bound to CheA. Figure 5 Interactions of the core signaling proteins CheW1 and CheA and their novel interaction partners PurNH and OE4643R. Plots show Sorafenib clinical trial the association score of the proteins identified in one-step (A-D) or two-step (E-H) bait fishing experiments with CheW1 (A, E), CheA (B, F), PurNH (C, G) and OE4643R (D, H). The dashed line indicates the threshold used in this study for assuming an interaction. The proteins CheA, CheW1, CheW2, PurNH and OE4643R are labeled in the plots when identified with an association score above the threshold. For the underlying data see Additional file 3 and Additional file 4. The second novel interactor is OE4643R, a conserved protein of unknown function. OE4643R belongs to the uncharacterized protein family DUF151 (Pfam, [96]) and the cluster of orthologous buy Elafibranor groups COG1259 (“uncharacterized conserved protein”) [97, 98].

The qualitative and quantitative differences found for GI microbiota affected the level of volatile organic compounds (VOC) and amino acids in faecal and urine samples. A few studies considered the metabolome of faecal or urine samples [10, 22]. The concept of human metabolome encompasses the idea of microbial and metabolic cooperation, and it aims to systematically examine changes in numerous low molecular mass metabolites of biological fluids as the response to different stimuli such as drugs or diseases [31–33]. The

combination of GC-MS/SPME and 1H NMR metabolic profiles together with CAP analysis allowed the identification of specific molecules which significantly A-1210477 changes in T-CD children. The largest level of esters was found for HC, whereas ethyl-acetate and octyl-acetate seemed to be over-synthesized in T-CD children. Overall, esterification reactions at the colon level are considered as the microbial strategy to remove or detoxify acids or alcohols [34]. Median values of aldehydes were the highest in HC compared to T-CD children. Previously, the highest level of alcohols was found in CD children at diagnosis compared to T-CD and HC [10]. In this study, some alcohols such as Epigenetics inhibitor 1-octen-3-ol, ethanol and 1-propanol were higher in T-CD than HC children. Ethanol seems to be an important mediator to develop of non-alcoholic steatohepatitis

(NASH). It was hypothesized that when intestinal Alvocidib manufacturer bacteria synthesize alcohol they may induce endotoxemia [35]. NASH was also associated to occult CD [36]. The present study confirmed the higher level of some short chain fatty acids (SCFA) of HC compared to T-CD children [10, 37]. It was suggested that Lactobacillus and Bifidobacterium modified the metabolism of the large intestine by increasing the synthesis of SCFA [10, 38]. SCFA are some of the most important by-products of anaerobes

in the colon. They represent the main fuel for colonocytes and are involved MG-132 nmr in water and electrolyte absorption by colon mucosa, even under diarrheic conditions [39]. The increase of butyric acid is especially significant since it plays a key role in the regulation of cell proliferation and differentiation of colon epithelial cells. It was also shown that faecal and urine samples of T-CD had an altered level of free amino acids compared to HC children. Indeed, a large number of free amino acids and related compounds were found at the highest level in T-CD children. Another report [22], also showed that serum and urine samples of adult CD patients had altered level of amino acids. Peptides enter enterocytes either after preliminary digestion by brush border peptidases into amino acids or as di- and tri-peptides which are split inside the cell by cytoplasmic peptidases. Non specific inflammatory alterations of the intestinal mucosa (e.g.

jejuni during slaughter can contaminate cooling water, knives and poultry meat in the processing plant [4]. During transmission of C. jejuni from animals, primarily poultry, to humans, this important zoonotic foodborne pathogen encounters various stresses, such as non-growth temperatures, starvation, hypo- and hyper-osmotic stress, and desiccation [5, 6].

Despite the well-known fact that Campylobacter is a fastidious bacterium, human campylobacteriosis cases have significantly increased presumably due to the ability of this pathogen to survive under harsh environmental conditions [7–10] in addition to its low infectious dose (400~800 bacteria) [11]. For example, high genetic diversity of Campylobacter spp. and the ability to transform into a viable-but-non-culturable CUDC-907 mw state may enhance its adaptability to unfavorable growth conditions [7, 8]. Additionally, biofilm formation and stringent response also contribute to the survival of Campylobacter under PRN1371 stress conditions [9, 10]. However, the molecular mechanisms for stress resistance are still largely unknown in Campylobacter. In many bacterial species, alternative sigma factors play an important role in regulation of stress-defense genes

under hostile environmental conditions [12]. Because a sigma factor can coordinate gene transcription in response to environmental stimuli, many bacteria possess multiple alternative sigma factors, some of which are often dedicated to stress responses. For example, RpoS is a sigma factor important for adaptive responses in many Gram-negative pathogens, and RpoS mutations in Escherichia coli, Salmonella, Pseudomonas and Tideglusib in vivo Vibrio significantly impair bacterial ability to resist various stresses, such as starvation,

low pH, oxidative stress, hyperosmolarity, heat and cold [13–17]. In E. coli, RpoS is involved in resistance to high osmolarity in stationary-phase cells and survival in cold-shock by selleck chemicals llc regulating one set of RpoS-dependent genes, including otsA and otsB, which are necessary for synthesis of internal trehalose as an osmoprotectant and important for survival at low temperature [18, 19]. In addition, RpoS controls the acid resistance in E. coli by modulating gadC, a gene involved in the glutamate-dependent low pH-resistance, hdeAB, encoding pH-regulated periplasmic chaperons, and cfa, a gene for cycloporpane fatty acid synthesis [20]. As another stress-response sigma factor, RpoE regulates extracytoplasmic functions related to sensing and responding to bacterial periplasmic and extracellular environmental changes, which contributes to heat- and oxidative stress resistance in many Gram-negative bacteria, including E. coli, Pseudomonas and Salmonella [21, 22]. The RpoE mutation in Salmonella reduces bacterial survival and growth in macrophages by the loss of RpoE-dependent gene expression such as htrA, a gene required for oxidative stress resistance [23, 24].

In the GO-FORWARD study, GLM was shown to be effective in patients who showed lower responses or who were refractory to prior MTX therapy [9, 10]. In the present retrospective analysis, manifestation of effectiveness appeared to be delayed in the bio-switching group compared with the bio-naïve group, suggesting the necessity for longer follow-up when evaluating effectiveness in patients who switch between biological therapies. In a post-hoc

analysis of the effectiveness in relation to the reasons for switching, the effectiveness did not differ significantly according to the reason (data not shown). This suggests that patients undergoing switching will respond to this therapy, regardless of the reasons for switching. This supports findings by Smolen et al. [12] that switching from other this website anti-TNF agents to GLM was effective regardless of the reasons for switching, indicating that GLM can serve as the second anti-TNF agent when patients are switched from another TNF agent. Of the five

anti-TNF agents available, including certolizumab pegol, all have different affinities to TNF-α; therefore, switching from one anti-TNF agent to another is likely to be effective. Expression of antibodies to anti-TNF antibody agents such as infliximab, adalimumab, and certolizumab pegol monotherapy is not uncommon; however, incidences of anti-GLM find more antibodies in the GO-FORWARD [9] and GO-FORTH [13] studies were remarkably low. Because GLM is prepared by the transgenic mouse technique, it is an antibody with high affinity for the antigen [19], which means that formation of unstable PLX4032 cell line proteins or aggregations, which can serve as immunogens,

is unlikely. Studies of GLM (100 mg) monotherapy were conducted in Caucasian and South American countries in GO-FORWARD [9, 10] and in Japan in GO-FORTH [13] and GO-MONO [16], and showed that GLM is an appropriate biological agent for preventing the loss of effectiveness in Caucasian, South American, and Japanese populations receiving long-term RA treatment [9, 10, 13, 16]. As a result of findings from the GO-FORTH [13] and GO-MONO [16] studies, in addition to Sitaxentan the 50-mg dose, GLM (100 mg) every 4 weeks—as monotherapy and in combination with MTX—has been approved in Japan. Further studies at this dose level in larger numbers of patients are necessary. Apart from the usual limitations relating to observational data and retrospective analyses, particularly with regard to selection and enrolment bias, a major limitation of our analysis is the small patient numbers, especially for patients receiving GLM (100 mg) monotherapy. In addition, evaluation of levels of anti-GLM antibodies and the effects of GLM on structural joint damage in this real-life setting would have been useful; however, this was not evaluated in the original study.