In addition to standard microbiological culture, we examined explanted suture and tissue specimens using CM to determine whether bacterial Talazoparib purchase biofilms were present. Specimens were prepared as described previously (Kathju et al., 2009a, b). Briefly, suture

and tissue recovered at surgery were placed in Hanks balanced salt solution (HBSS) and placed on wet ice, directly after removal. After rinsing in the HBSS (to remove unattached bacteria) and blotting on sterile paper, specimens were mounted on the bottom of a 35-mm Petri plate on partially solidified agar (Kathju et al., 2009a, b). Specimens were stained for viability assessment using Molecular Probes BacLight Live/Dead kit (Molecular Probes, Eugene, OR). The BacLight kit consists of two nucleic acid stains, Syto9 (green), which enters all bacteria, and propidium iodide (red), which can only enter bacteria with porous cell walls. Once inside the bacteria, the propidium iodide suppresses the Syto9 fluorescence so that live bacteria appear green, whereas dead or damaged cells appear red. In some cases, bacteria stain with both dyes

and appear yellow – these have been interpreted as live, but nonculturable. The nuclei of human cells also take up these nucleic acid stains, but rapidly turn red. They are readily distinguished from bacteria on the basis of size and morphology. In addition, these stains have been used to stain extracellular bacterial DNA Phosphoprotein phosphatase (eDNA), which is commonly found in the EPS and appears as a diffuse staining click here surrounding the bacterial cells (Böckelmann et al., 2006; Thomas et al.,

2008). Fully hydrated specimens were then imaged by CM using a Leica DM RXE upright microscope attached to a TCS SP2 AOBS confocal system (Leica Microsystems, Exton, PA) using either a × 20 air objective or a × 63 long working distance water immersion objective. Live (green) and ‘dead’ (red) bacteria were imaged using 488 and 594 nm lasers; the suture and xenograft were imaged using reflected CM (blue) and bright-field microscopy (gray). Examination of one of the pieces of explanted Surgisis xenograft by CM showed heterogeneously distributed patches of live and dead bacteria and evidence of associated eDNA attached to the xenograft material (Fig. 2a). These organisms had a primarily coccal appearance, consistent with the solitary finding by culture of staphylococci. Interestingly, only one of the four specimens yielded a positive culture result, illustrating the inherent difficulty in detecting biofilm infections and making the case for multiple specimens to be sent for clinical culture, as well as the utility of using independent culture-free methods. Biofilms are commonly patchy on surfaces and this heterogeneity might partly explain the inconsistency in culture data. Examination of explanted suture material also showed evidence of attached and viable biofilm bacteria (Fig. 2c and d).

When the animals were deeply anaesthetized blood was obtained by cardiac puncture of the right ventricle. Bronchoalveolar lavage (BAL) was performed by instilling 0·25 ml PBS through the tracheal cannula, followed by gentle aspiration and repeated with 0·2 ml PBS. Finally, one femur was cut at the epiphysis and the BM cells were flushed with 2 ml PBS. Bronchoalveolar lavage fluid and bone marrow.  Samples of BALF and BM were centrifuged at 300 g for 10 min at 4°. The BAL supernatant

was saved for eotaxin-2 measurement and stored at − 80° until analysis. The cells were resuspended with 0·03% BSA in PBS. The total cell numbers in BAL and BM were determined using standard haematological procedures. Cytospins PXD101 of BAL and BM were prepared and stained with May–Grünwald–Giemsa for differential cell counts by counting 300–500 cells using a light microscope (Zeiss Axioplan 2; Carl Zeiss, Jena, Germany). The cells were identified using standard morphological criteria, and BM mature and immature eosinophils were determined by nuclear morphology, PARP inhibitor cell size and cytoplasmic granulation.23 Lung tissue cells.  The pulmonary circulation was perfused with ice-cold PBS and lungs were removed from the thoracic cavity. The lung tissue was thinly sliced and suspended

RPMI-1640 (Sigma-Aldrich) complemented with 10% fetal calf serum (FCS), collagenase (5·25 mg/ml) and DNAse (3 mg/ml; Roche). After 90 min incubation in a shaking water bath (37°), any remaining intact tissue was disrupted by repeated passage through a wide-bore Pasteur pipette and filtered through a 40-μm nylon mesh (BD Biosciences, Erembodegem, Belgium). The parenchyma lung cells were diluted in Percoll (density 1·03 g/ml; Amersham Bioscience, Uppsala, Sweden) and layered on a discontinuous gradient,

centrifuged at 400 g for 20 min. The cells in the top layer, mainly macrophages, dead cells and debris, were discarded. Cells at the Percoll interfaces were collected and washed in PBS complemented with 10% FCS. Total cell numbers were determined using standard haematological procedures. Antibodies.  Fluorescein isothiocyanate (FITC) -labelled anti-mouse CD34 (clone RAM 34; BD Bioscience), phycoerythrin (PE) or FITC-labelled anti-mouse CCR3 (clone 83101; R&D systems, Neratinib solubility dmso Abington, UK), biotinylated anti-mouse stem cell antigen-1 (Sca-1)/Ly6 (clone 177228; R&D Systems) followed by peridinin chlorophyll protein (PerCP) -labelled streptavidin, PE-labelled anti-mouse IL-5Rα (Clone 558488; BD Bioscience), PercP-labelled anti-mouse CD45 (clone 557235; BD Bioscience), FITC-labelled BrdU (BD Bioscience) and rabbit anti-mouse major basic protein (MBP) polyclonal antibody in combination with goat anti-rabbit PE or with biotinylated swine anti-rabbit followed by streptavidin-FITC were used. Animals were sensitized and exposed to OVA or PBS as described above.

78 Most studies on the location of effector/memory T cells in non-lymphoid tissues have focused on entry (homing) or proliferation and survival as determining factors of lymphocyte content in a given tissue. Recent findings have shown that exit from the tissue is an CHIR-99021 concentration active process controlled by chemotactic mechanisms.

The chemokine receptor CCR7 was shown to be required for T-cell exit from inflamed peripheral tissue.79,80 Another chemotactic agent, sphingosin-1-phosphate (S1P), and its receptors are required for the exit from lymph nodes, a finding emerging from studies with the drug FTY 720, which displays immunosuppressive effects. Both CCR7 and S1P receptors are modulated in the course of T-cell activation, and thereby might cause the transient retention of recently activated T cells in the lymph node.81 When CCR7 is knocked out, the number of T cells retained in an inflamed tissue doubles, confirming its importance for continuous circulation.82 For technical reasons, quantification of exit rates for specific subsets of cells and specific tissues is

more difficult. However, a variety of data are available from early studies in which cannulation of the thoracic duct or even single lymph nodes was applied, Selleckchem Bortezomib which provided clear evidence not only that naïve cells entering a lymph node via the high endothelium pass through the tissue within half a day and exit it, but also that large numbers of effector/memory cells attracted to an inflamed tissue, or generated by local proliferation, exit the tissue via the efferent lymph.83 It is conceivable that

the process of emigration also underlies a variety of regulatory influences; T-cell activation upon antigen encounter within the tissue may be one factor, but an influence of inflammation-generated mediators such as prostaglandins has also been described.84 The directional movement towards a chemical acetylcholine compound plays a major role in the recruitment as well as the egress of T cells from the site of an immune response. Leucocytes are able to integrate signals from multiple chemoattractants in their migration.85 In fact, cells migrating away from a local chemoattractant source actually chemotax towards distant attractants. The ability to navigate through chemoattractant arrays may be sufficient to explain entry and egress of T cells during an immune response. However, recent evidence supports the existence of both chemoattractants and chemorepellents that guide the directed movement of leucocytes into and out of tissues. Chemorepulsion is defined as the migration away from peak concentrations of a chemokine and was initially studied in the context of axonal guidance, where the same molecule may act as a chemoattractive or chemorepulsive cue depending on the receptor expressed on the cell surface.

To determine the effects of IL-32 over-expression on the expression of PARP, p21, cyclin E and cyclin A related to apoptosis and the cell cycle, we conducted Western blot analysis, demonstrating that the protein

levels of p21 and cleaved-PARP were increased in the IL-32γ-transfected cells compared with the mock-control cells. However, Selleck cancer metabolism inhibitor the expressions of cyclin E and cyclin A were reduced in the IL-32-over-expressing SiHa and CaSki cells (Fig. 5c). These results suggested that IL-32 over-expression inhibits cancer development in cervical cancer cells, via down-regulation of the expressions of E7 and COX-2. In this study, we evaluated the feedback inhibition mechanism of IL-32 pro-inflammatory or cancer pathways in response to the high-risk E7 oncogene in cervical cancer cells. Recently, IL-32 has been associated with the regulation of inflammatory response during infection with the influenza A virus and with the regulation of HIV production.19,20 Expression of IL-32 has been detected in cervical cancer tissues, and IL-32 has been shown to be markedly induced by HPV-16 E7 in a variety of cervical cancer cells.

When IL-32 expression was investigated according to the groups with regard to the FIGO stage IB and IIA–IIIB, there was a statistically significant (χ2 test) IL-32 expression frequency in the stage IIA–IIIB (71%) compared with stage IB (31%) disease (P = 0·014) Cytoskeletal Signaling inhibitor (Table 1). However, IL-32 expression was not correlated with survival of the patients (P = 0·79 and P = 0·90 in stage IB and IIA–IIIB, respectively). Extensive studies using clinical samples are needed to investigate the discrepancy between advanced stage and survival of the patients. Additionally, COX-2 was over-expressed by HPV-16 E7 as reported previously.22,24 The COX-2 induced by HPV-16 oncoproteins has been reported to induce

immortality, the inhibition of apoptosis,33 strong invasion ability,34 angiogenesis35 and suppression of the immune response36 in cervical cancer cells, via a number of mechanisms. The levels of COX-2-derived PGE2 were reduced in the culture media from the NS398-treated SiHa and CaSki cells. The levels of COX-2-derived PGE2 were reduced in the culture media from the NS398-treated SiHa and CaSki Molecular motor cells. Compared with the intracellular expression levels of IL-32, significant secretion of IL-32 was not detected in the supernatants of COX-2 over-expressing and NS398-treated SiHa and CaSki cells using a sandwich IL-32 ELISA.30 Although IL-32 is considered to be mainly intracellular,12,26 one may envisage that some is secreted and triggers pro-inflammation in neighbour cells. It is well known that high-risk HPV-16 expresses E6 and E7 proteins from a single polycitronic mRNA.37 An siRNA targeting HPV-16 E7 region degrades either E6, or truncated E6 (E6*) and E7 mRNAs and simultaneously results in knock-down of both E6 and E7 expression.

Beside the ability to secrete cytokines and express cytotoxic machinery, another critical element for T-cell-mediated immune protection is their ability to proliferate and survive after activation. We observed that after T-cell receptor stimulation in vitro CD45RA+ CD27+ and CD45RA− CD27+ CD4+ T-cell populations expanded more than CD45RA− CD27− and CD45RA+ CD27− subsets

during culture (Fig. 4a,b; see Supplementary Information, Fig. S3a). To understand the extent to which increased cell death, rather than reduced proliferation, contributes to the decline find more of the CD45RA+ CD27− population after in vitro stimulation, we measured the rate of cell death by monitoring Annexin V staining and PI incorporation after activation (Fig. 4c,d). The analysis of early apoptotic (Annexin V+ PI−) and late apoptotic/necrotic (Annexin V+ PI+) cells in the different subsets at day 3 after activation showed that CD4+ CD45RA+

CD27− T cells are significantly more prone to cell death than all other subsets. A time–course of Annexin V staining and PI incorporation showed that by day 15 CD4+ CD45RA+ CD27− T cells are almost completely dead when all other subsets are still present in culture (see Supplementary Information, Fig. S3c). To explore the possibility that pro-survival pathways are defective in CD45RA+ CD27− CD4+ T cells, which makes them susceptible to apoptosis, we investigated the expression of the anti-apoptotic protein Bcl-2, measured by intracellular staining of CD4+ T-cell subsets directly Venetoclax ex vivo (Fig. 5a).30 We found that Bcl-2 expression is significantly

lower in CD45RA+ CD27− CD4+ T cells compared with all the other subsets (P < 0·0001). A critical role in promoting cell survival is also ascribed to Akt, which operates by blocking the function of pro-apoptotic proteins and processes.28,31 Akt is phosphorylated at two sites – serine 473 and threonine Leukotriene-A4 hydrolase 308. We previously showed that there is defective phosphorylation of Akt(ser473) but not Akt(thr308) in highly differentiated CD8+ T cells.28,31 We now show that there is a decrease in pAkt(ser473) from CD45RA+ CD27+ (naive), CD45RA− CD27+, CD45RA− CD27− and CD45RA+ CD27− subsets, respectively (Fig. 5b). Therefore CD45RA+ CD27− CD4+ T cells have potent effector function but have decreased capacity for survival after activation, associated with decreased Bcl-2 expression and Akt(ser473) phosphorylation. Previous studies have shown that within CD8+ T cells cytokines such as IL-15 that drive homeostatic proliferation also induce the generation of CD45RA+ CD27− CD8+ T cells.21,32,33 Although the presence CD4+ CD45RA+ CD27− T cells has been described previously26 the mechanism by which they are induced is not known. We showed previously that IL-7 can induce the proliferation of CD4+ CD45RA+ (naive) T cells without inducing CD45RO expression,34 which was subsequently supported by other studies.

2A). Confirming results obtained on total NK cells, expression of KIR2DL1 but not of KIR3DL1 increased on NKG2C+ cells (Fig. 2C and D). Interestingly, a small but statistically significant increase in KIR2DL1 on NKG2C+ was detected also in CMV-seronegative donors; however, this increase was much smaller than that seen in this website CMV-seropositive donors (Fig. 2C). To discriminate between expression of KIR2DL2/S2 and KIR2DL3, we next cultured PBMCs from donors carrying

the genes for all three receptors. Co-staining of a KIR2DL3 specific Ab with an Ab recognizing KIR2DL2/S2/L3 allowed us to distinguish between expression of KIR2DL2/S2 and KIR2DL3 (Fig. 3A). In five CMV-seropositive donors, strong expansion of KIR2DL3-expressing NK cells was documented, while co-culture with

CMV-infected fibroblasts had no impact on the expression of KIR2DL2/S2 (Fig. 3B and C). To address whether the increased expression of KIR-expressing cells represents true expansion, we determined cell number weekly during the 21-day co-culture with MRC-5 in the presence or absence of CMV. The APO866 price NK-cell number contracted during the first week, followed by an expansion of NK cells exclusively in seropositive donors in the presence of CMV (Supporting Information Fig. 2). Staining for the proliferation marker Ki-67 corroborated these results: infection of MRC-5 with CMV led to a massive up-regulation of Ki-67 on NK cells if these stemmed from CMV-seropositive donors (Fig. 2B). Interestingly, when the KIR repertoire was assessed on Ki-67+ cells, we noted expansion of KIR2DL1/Ki-67 double positive but not of KIR3DL1/Ki-67 double positive cells after co-culture with CMV-infected MRC-5 (Fig. 2E and F). We next aimed

to characterize factors PLEK2 influencing the expansion of KIR-expressing NK cells. HLA-C1 group Ags are the ligand for KIR2DL2/S2/L3, while HLA-C2 group Ags are the ligands to KIR2DL1 [17]. If CMV-seropositive donors were stratified according to their KIR ligand status, an expansion of KIR2D-expressing NK cells occurred only in the presence of the cognate KIR ligand: KIR2DL1 expanded only in donors carrying a C2 ligand (Fig. 4A and B), whereas KIR2DL2/S2/L3 NK cells expanded exclusively in the presence of the cognate group C1 ligand (Fig. 4C and D). While no ligand has been identified for the activating KIR receptor KIR3DS1 [18], genetic association studies have suggested an epistatic interaction of KIR3DS1 with HLA-Bw4 in HIV infection [19]. Analysis of Bw4-status in conjunction with KIR3DS1 expression in our population showed that expansion of KIR3DS1 occurred irrespective of the presence of Bw4 (day 21 KIRDS1 expression in CMV-exposed versus CMV nonexposed cells in seropositive donors: mean 23 versus 8% in Bw4-negative, and 31 versus 11% in Bw4-positive donors, p < 0.05 for both comparisons).

Treg cells were also separated for further analysis of multiple genes important in their function with the use of real-time RT-PCR. We did not observe any difference in Treg percentages between study and control group but there was lower expression of some molecules including transforming growth factor-β and interleukin-12 family members in Treg cells separated from children with MS compared to the healthy subjects. Our study is the first to report significant disturbances in some gene expression in T regulatory cells separated from

children with MS. The results should be useful for further research in this field, including immunotherapeutic see more interventions. More than 20 years ago, Reaven has postulated the link between insulin resistance, hypertension and dyslipidemia with an increased risk of cardiovascular diseases in adults [1].

Since https://www.selleckchem.com/products/Erlotinib-Hydrochloride.html that time, the metabolic syndrome (MS) has been defined as a cluster of risk factors including abdominal obesity, dyslipidaemia, glucose intolerance and hypertension that increase the risk for coronary heart disease. The three current definitions of MS in adults use similar components, but threshold values for those components are different, this is why Reaven disputes their clinical utility [2]. However, because of epidemic of childhood obesity in the last decades, there is increasing interest in identifying children who are at risk for developing cardiovascular diseases in adulthood. The latest definition of MS in children presented by International Diabetes Farnesyltransferase Federation (IDF) considers the abdominal obesity as essential for the diagnosis; other components (two or more are required) include elevated triglycerides, low HDL cholesterol, high blood pressure and elevated blood glucose [3]. Immunological and

molecular aspects of obesity and MS have been recently intensively investigated (review e.g. in [4]). Many studies suggest that low-grade systemic inflammation plays a role in the pathology of MS (discussed in [5]). Cytokines and chemokines produced by T cells are crucial immune mediators in many pathophysiological obesity-related conditions including atherosclerosis [6, 7]. Recent research in this field concerns T regulatory cells [8]. In the last two decades, there have been tremendous advances in explication of molecular processes which control immune response. One of the most important players in this phenomenon seems to be the small subpopulation of T lymphocytes called T regulatory cells (Tregs). These cells are regarded as the primary mediators of peripheral tolerance and play a pivotal role in the pathogenesis of autoimmune and immunosuppressive diseases. The lack of Treg number and/or function leads to the appearance of autoimmune diseases like thyroiditis, gastritis, insulitis, glomerulonephritis, polyarthritis and others [9].

These results also suggest that Th17-derived Tregs, inducible Tregs from other T-cell origins, and naturally occurring Tregs may have different stabilities 55. In support of this notion, recent studies have shown epigenetic differences between naturally occurring Tregs and induced Tregs 57. Thus, improved understanding of epigenetic and gene expression profiles in T-cell lineages is essential for studies of T-cell commitment, plasticity and reciprocity under both physiological and pathological conditions. Mounting evidence suggests that human CD4+ Tregs can differentiate into IL-17-producing Th17 cells (IL-17+FOXP3+), and that Th17 cells can express Small molecule library FOXP3 and RORγt

(RORγt+FOXP3+)

24, 25, 52. It has not previously known whether Th17 cells can be differentiated into Tregs. In addition, all these studies were performed with polyclonal CD4+ T cells purified with magnetic beads or FACS sorting, thus the purity and/or potential contamination with other cell populations could directly influence the results. To address these important issues, in the check details present study, we established Th17 clones from TILs containing high percentages of IL-17-producing cells, and we confirmed the purity of these clones by assessing TCR-Vβ expression. We then showed that these Th17 clones could significantly increase Th17+IFN-γ+ and Th17+FOXP3+ double-positive T-cell populations and could differentiate into functional Tregs following multiple rounds of unbiased TCR stimulation. Our studies further confirm the developmental plasticity of human Th17 cells at a clonal level, suggesting that Th17 cells not

only can differentiate into Th1 cells but can also convert to Tregs 21. Notably, these data implicate that Th17 cells may have dual functions, performing regulatory as well effector roles in human diseases including inflammatory disorders and cancers. The commitment of Th17 cells to Th1 and/or Treg lineages may depend on specific physiological and pathological conditions, such as the local proinflammatory cytokine milieu and pathogen- or tumor antigen-mediated stimulation. In support of our concept, recent studies have shown that FOXP3+ PtdIns(3,4)P2 Tregs can acquire an effector cell phenotype expressing T-bet and IFN-γ in the presence of strong inflammatory responses during lethal infection 58. In addition, environmental IDO can regulate the conversion of FOXP3+ Tregs to Th17-like cells in tumor-draining lymph nodes 59. Besides possessing potent suppressive function, our data also showed that these Th17-Treg differentiated T cells secreted moderate amounts of IL-10 and TGF-β1 after stimulation with OKT3 and PBMCs that may amplify their negative regulatory functions, and which is consistent with studies from other groups 14.

[9] A necrotic eschar in maxillary, facial, or sino-orbital mucosal surfaces in an immunocompromised host may be an early RAD001 mw sentinel marker of invasive

mucormycosis. Pleuritic pain in a neutropenic host also may signify an angioinvasive filamentous fungus. Pleuritic pain in a neutropenic or HSCT patient receiving voriconazole prophylaxis has a high probability of being invasive mucormycosis instead of aspergillosis. Diplopia is an early manifestation of sino-orbital mucormycosis in a diabetic patient that usually signifies involvement of the extraocular muscles or their innervating nerves.[10] Hyperglycaemia in diabetic patients may produce blurring of vision, but does not produce diplopia. During sino-orbital mucormycosis, hyphae involving the ethmoid sinus breach the lamina papyracea to invade the medial rectus muscle creating dysconjugate vision. The organism may extend along the emissary veins to the ethmoid sinus to the cavernous sinus and encroach upon the critical cranial nerves involve III, IV, V (1, 2) and VI. Diplopia in a diabetic patient or other compromised host with ethmoidal sinusitis should be assessed aggressively for sino-orbital mucormycosis. Necrotic cutaneous lesions in immunocompromised

patients may also be caused by mucormycosis. The differential diagnosis includes www.selleckchem.com/products/napabucasin.html other angioinvasive pathogens including Aspergillus, Fusarium, Pseudallescheria, Scedosporium species. Pseudomonas aeruginosa and occasionally members of Enterobacteriaceae in the same host also cause ecthyma gangrenosum. The preponderance

of cases of cutaneous mucormycosis is associated with direct inoculation rather than haematogenous dissemination.[1] Characteristic hyphal structures are seen on biopsy Dynein and wet mount of tissue. Earlier recognition of sinus and pulmonary lesions by CT scanning is an important advance over conventional sinus and chest radiographs. Early CT findings may reveal pulmonary or sinus lesions before localising symptoms in immunocompromised patients who are at high risk for invasive sino-pulmonary mucormycosis. Among the lesions associated with angioinvasive filamentous fungi are nodules, halo signs, reverse halo signs, cavities, wedge-shaped infiltrates and pleural effusions associated with pleuritic pain.[11] Among these lesions, the reverse halo sign in the neutropenic patient has high predictive value for mucormycosis.[12] Early recognition of risk factors, clinical manifestations and diagnostic imaging findings may increase the probability of an early recognition and lead logically to a definitive diagnosis by culture and biopsy of tissue or the use of novel molecular and antigenic assays.

Three type strains [M. abscessus (ATCC 19977T), M. massiliense (KCTC 19086T= CIP 108297T) and M. bolletii (KCTC 19281T= CIP 108541T)], and 101 M. abscessus-M. chelonae group clinical isolates (M. abscessus, 46; M. massiliense, 49; M. bolletii, two; and M. chelonae, four strains) were used in the present study. In addition to the 85 strains that were used in a previous report (7), 16 strains (Inje collection) were newly included. Mycobacteria were cultivated on Ogawa media or blood agar plates at 37°C under 5% CO2

for 4 days, after which they were subjected to clarithromycin susceptibility testing and sequence analysis. Total DNAs were extracted from cultured colonies using the bead beater-phenol extraction method (17) and used as templates for PCR. The following primer pairs were used: ermF (5′-GAC CGG GGC CTT CTT CGT GAT-3′) and ermR1 (5′-GAC TTC CCC GCA CCG NVP-LDE225 chemical structure ATT CC-3′) for the whole erm(41) (GenBank accession No. CU458896) and primers 19 (5′-GTA GCG AAA TTC CTT GTC GG-3′) and 21 (5′-TTC CCG CTT AGA TGC TTT CAG-3′) for 23S rRNA gene (18). Template DNA (approximately 50 ng) and 20 pmol of each primer were added to a PCR mixture tube (AccuPower PCR PreMix; Bioneer, Daejeon, Korea) that contained 1 unit of Taq DNA polymerase, 250 μM deoxynucleotide triphosphate, 10 mM Tris-HCl (pH 8.3), 10 mM KCl, 1.5 mM MgCl2, and gel loading dye. The final volume was

then adjusted to 20 μl with distilled water, after which the reaction mixture this website was amplified using a model 9700 Thermocycler (Perkin-Elmer Cetus, Norwalk, NJ, USA). The PCR products were purified using QIAEX II gel extraction U0126 cost kits (Qiagen, Hilden, Germany), and were then sequenced directly using forward and reverse primers on an Applied Biosystems automated sequencer (model 377) using BigDye Terminator Cycle Sequencing kits (Applied Biosystems, Warrington, UK). Both strands were sequenced as a cross-check. The resultant 23S rRNA gene and erm(41) sequences were aligned using ClustalW in the MEGA 4.0 (19) and the sequence similarities were analyzed using MegAlign software (DNAStar, Madison, WI, USA) (20). Mycobacterium tuberculosis erm(37) and M. abscessus erm(41) were retrieved from the

GenBank and used to compare with newly determined sequences. The newly determined erm(41) sequences of M. massiliense (accession no. FJ358487 to FJ358490), M. bolletii (accession no. FJ358491), and M. abscessus (accession no. FJ358483 to FJ358486) were deposited in GenBank. M. abscessus (ATCC 19977T), M. massiliense (KCTC 19086T= CIP 108297T), and M. bolletii (KCTC 19281T= CIP 108541T), which are known for their susceptibility to clarithromycin, were used as controls. The MIC of clarithromycin were determined in microtiter plates (21) using the broth dilution method with slight modification as described previously (7). To prepare a stock solution, clarithromycin (Boryung, Seoul, Korea) was solubilized in distilled water with glacial acetic acid (2 μl/ml) (22).