5b). These data suggest that demethylation of this CpG island of the Foxp3 promoter region correlates with Foxp3 expression. The methylation status of this region was evaluated in Foxp3− T cells that were activated for 72 hr in the presence of TGF-β alone, simvastatin alone, and the combination of TGF-β/simvastatin (Fig. 5c). After 72 hr, 48% and 42% of the CpGs of dimethylsulphoxide-treated or simvastatin-treated cells were methylated, respectively. However, this region in TGF-β-treated cells was less methylated (26%) than in dimethylsulphoxide-treated

or simvastatin-treated cells and the lowest level of methylation (16%) was observed in the cells treated with simvastatin/TGF-β. Seventy-two hours after activation, the extent of demethylation correlated well with the level of Foxp3 expression learn more detected by FACS analysis (bottom boxes in Fig. 5c). These results suggest that the synergistic action of simvastatin on TGF-β-mediated induction of Foxp3 may be mediated by co-operative control of methylation of the Foxp3 promoter. To directly examine the effects of simvastatin on TGF-β-mediated signal transduction, we measured phosphorylation of Smad3. Significant phosphorylation of

Smad3 was observed 24 hr after activation of cells cultured in the presence of GW-572016 manufacturer TGF-β, but not simvastatin alone, and the levels of Smad3 phosphorylation were not modulated when the cultures were stimulated with both TGF-β and simvastatin (Fig. 6a). In addition, the total amount of Smad4 was comparable in all treatment groups. The lack of an effect of Buspirone HCl simvastatin on Smad3 phosphorylation is consistent with its late time of action and raised the possibility that simvastatin might block steps in the negative-feedback regulation of TGF-β signalling. Smad6 and Smad7 are the major inhibitory Smad proteins in the negative feedback regulation

of the TGF-β signalling pathway. In contrast to Smad3 phosphorylation, we could not detect Smad7 by Western blot analysis 24 hr after T-cell activation and only low levels of Smad6 were observed. The levels of Smad6/7 increased after 48 hr and were maximal at 72 hr after activation (Fig. 6b). Importantly, when combined with TGF-β, simvastatin markedly inhibited the induction of Smad6 at 48 and 72 hr, and completely blocked Smad7 induction at both 48 and 72 hr. Simvastatin alone also decreased levels of Smad6 and completely blocked Smad7 expression at 72 hr. As TGF-β has been reported to play an important role in Foxp3+ Treg homeostasis,16 we also examined the expression of Smad6/7 in nTregs that were activated under conditions similar to those used in our iTreg induction cultures. Foxp3− and Foxp3+ CD4+ T cells were FACS-sorted from Foxp3gfp mice and activated with anti-CD3/CD28 and IL-2 in the absence or presence of TGF-β for 72 hr (Fig. 6c).

[21, 22] This leads to haematogenous dissemination of the organism to target organs, while ischaemic necrosis

of the infected tissue can prevent leucocyte and antifungal agent penetration to the foci of infection.[23] R. oryzae was used as a model system in understanding the basis of fungal pathogenicity. Sequencing the genome of a pathogenic R. oryzae strain there was evidence that the entire genome had been duplicated and retained two copies of three extremely sophisticated systems involved in energy generation and utilisation. This gene duplication has led to the development of gene families related to fungal virulence, fungal cell wall synthesis enzymes and signal transduction, which may contribute to the invasive nature of R. oryzae.[24] The important clinical observations that patients with diabetic ketoacidosis as well as patients receiving dialysis and selleck treated with iron chelator deferoxamine are characteristically susceptible to mucormycosis highlights the central role of host iron in the pathogenesis mTOR inhibitor of mucormycosis.[23] As proof of principle in vitro studies have shown that Rhizopus spp. can accumulate many-fold greater amounts of iron supplied by deferoxamine than A. fumigatus.[25] Deferoxamine per se is not the pathogenetic factor for infection but Rhizopus spp.

utilise deferoxamine as a siderophore to supply previously unavailable iron to the fungus.[26] However, not all Mucorales have the same susceptibility to iron chelators.[19] Host defences are modulated by a number of factors as evidenced from in vitro and preclinical data but only

from few case reports.[27] Such factors are cytokines and pharmacological agents including certain antifungal drugs. We will herein review relevant in vitro and in vivo studies and scant clinical data. An overview of immune response and its regulations against Mucorales is shown in Fig. 1. Adjunctive cytokine Selleck Lonafarnib treatment for patients with mucormycosis has long ago attracted scientific interest as a means to improve outcome through neutrophil recovery and restoration of host immune responses. Cytokines studied so far include the hematopoietic growth factors, granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), as well as IFN-γ. These cytokines have been shown to stimulate proliferation and differentiation of myeloid progenitor cells to neutrophils (G-CSF, GM-CSF) or monocytes and eosinophils (GM-CSF), to up-regulate chemotaxis, phagocytosis and respiratory burst of phagocytic cells (neutrophils, monocytes, macrophages) (G-CSF, GM-CSF, IFN-γ) and to regulate/enhance protective T-helper type 1 (Th1) responses (IFN-γ).

These cells could, in turn, recruit neutrophils. Because livers of ALD patients, particularly those with AH, are infiltrated by IL-17+ cells [20], and because Th-17 cells play a role in neutrophil recruitment and express

CCR2 [22], we correlated CCL2 liver expression with IL-17+ cell infiltrates. We found that CCL2 liver expression was correlated with numbers of IL-17+ cells. Furthermore, IL-17+ cell infiltrates were correlated strongly with neutrophil infiltrates and with IL-8 liver expression. These results suggest that CCL2 plays a role in the pathogenesis of ALD by recruitment of Th17 cells which, in turn, would recruit neutrophils via an IL-8 effect. Gefitinib However, IL-17+ cell infiltrates may, in part, reflect neutrophil infiltrates. Indeed, we have shown previously, using confocal microscopy, that among liver-infiltrating IL-17+, T lymphocytes and neutrophils were represented most frequently [20]. As each AH episode is thought to be profibrogenic [4], we speculate that CCL2 secreted during the AH inflammatory burden

could enhance the fibrogenesis process. However, we found no difference in liver CCL2 expression between ALD patients with and without cirrhosis; nevertheless, this result should be viewed with caution, as non-cirrhotic patients in our cohort were scarce. We found no correlation between CCL2 liver expression and hepatic steatosis in our patient cohort, whereas CCL2 was involved in hepatic lipid metabolism in an experimental model of alcoholic liver disease YAP-TEAD Inhibitor 1 [16]. This relationship between CCL2 liver expression and steatosis may be present in the beginning of ALD, next but not in severe disease such as cirrhosis. Patients with the G-allele for −2518 A > G CCL2 polymorphism were present more frequently in the severely ill AH group than in other ALD patients. Moreover, among AH patients, the G-allele was more frequent in the severe form of the disease. It was shown previously

that the presence of the −2518 G-allele resulted in significantly greater CCL2 secretion than that found in patients with the A/A homozygous genotype in response to a given inflammatory stimulus [23], and this polymorphism has been implicated in numerous inflammatory diseases, including hepatitis C, acute pancreatitis, Crohn’s disease and, more recently, spontaneous bacterial peritonitis [24,25,28,29]. However, we did not find higher CCL2 plasma levels or liver expression in G-allele carriers in our cohort of patients (data not shown). It is possible that G-allele carriers are more likely to develop a severe form of AH, but that the levels of CCL2 at the time of alcoholic hepatitis are the same as in G-non-carriers. Our finding suggests that G-allele carriers are more likely to develop a severe form of AH than patients without the G-allele when exposed to alcohol.

3); from these findings, we consider that neutrophil infiltration in LPR may be responsible for the induction of chronic inflammation in local tissue that needs further

experiments to confirm. In summary, the present study reveals that IL-9+IL-10+ T cells are involved in intestinal LPR. Activation of IL-9+IL-10+ T cells promotes the infiltration of Mϕs and neutrophils click here in local tissue. The finding that IL-9+IL-10+ T cells play an important role in the pathogenesis of LPR implies that this subset of T cells may be a novel therapeutic target in the treatment of chronic allergic diseases. This study was supported by grants from the Canadian Institutes of Health Research (CIHR; #191063, #220058), Natural Sciences and Engineering Research Council of Canada and the Natural Science Foundation of China. Dr P. Yang holds a New Investigator Award (CIHR; #177843).

Dr P. C. Yang holds a New Investigator Award from CIHR. Author contributions: Z.Q.L., C.H.S., X.C., L.F.A., W.J.M., L.C. and Y.D. were involved in experiment performance, data collection and reviewing the paper. S.H.H. and P.C.Y. are principle investigators and were involved in project design, data analysis and paper writing. None to declare. “
“The contribution of myeloid-derived suppressor cells (MDSC) in patients suffering from early or recurrent miscarriage is unknown. MDSC are implicated in modulation of T-cell response in healthy pregnancies; however, the role of MDSC in patients suffering BMN 673 datasheet from miscarriage has not been studied. We hypothesized that MDSC play major role in inducing maternal–fetal tolerance and this tolerance is compromised

in patients suffering from miscarriage. MDSC level was assessed by flow cytometry and immunostaining in blood and endometrial decidua, respectively. Activation of T cells was determined by DAPT order MTT proliferation and IL-2 ELISA assays. The miscarriage patients harbor reduced level of functionally suppressive MDSC in blood and endometrium as compared to healthy control women with successful pregnancies. These results suggest MDSC regulate maternal tolerance in healthy pregnancies and that drug inducing MDSC could have therapeutic implication in the miscarriage patients. “
“Intestinal intraepithelial lymphocytes carrying the γδ TCR (γδ iIEL) are involved in the maintenance of epithelial integrity. γδ iIEL have an activated phenotype, characterized by CD69 expression and increased cell size compared with systemic T lymphocytes. As an additional activation marker, the majority of γδ iIEL express the CD8αα homodimer. However, our knowledge about cognate ligands for most γδ TCR remains fragmentary and recent advances show that γδ T cells including iIEL may be directly activated by cytokines or through NK-receptors, TLR and other pattern recognition receptors.

From each animal, three flat sheets of unstripped ileum free of Peyer’s patches were placed in

Teflon holders and mounted in Ussing chambers within 5 min after being cut off from blood supply. Both sides of the sample (exposed area 0·2 cm2) were in contact with 1·6 mL Krebs–Ringer solution, stirred and gassed with humidified 95% O2 + 5% CO2 at 37°C. The transepithelial potential difference Vte check details (mV) was continuously monitored with Calomel electrodes connected to the chambers with Krebs–Ringer-agar bridges. Transepithelial electrical resistance R (Ω/cm2) was calculated from the voltage deflections induced by bipolar current pulses of 10 μA (every 30 s) applied through platinum wires. The potential and resistance data were stored on a PC using custom software (Natural Simstrument, Amsterdam, the Netherlands). During off-line data analysis, corrections were made for resistance of the solution and for potential differences between Calomel electrodes, measured both just before and immediately

after each experiment. The equivalent short-circuit HSP inhibitor current Isc (μA/cm2) was calculated from the continuously monitored values of R and Vte. Reported values for the parameters Vte, R and Isc were obtained at the end of a 15- to 20-min equilibration period. Generally, these values were stable during the subsequent 1- or 2-h experiment. At the end of the experiment, the secretory capacity of the tissue segments was tested by measuring their response (Vte and Isc) to application of the secretagogue carbachol in the serosal compartment (10−4 M). In the Ussing chamber experiments, the measured transepithelial potential

(Vte) and equivalent short-circuit current (Isc) are indicative of the basal epithelial secretion, while the increase in these parameters (dVte and dIsc) in response to the secretagogue carbachol reflects the maximal secretory capacity. Paracellular mucosal-to-serosal permeability was determined using NaFl 17-DMAG (Alvespimycin) HCl as a model molecule (25). After the equilibration period, NaFl was added to the mucosal compartment (0·01 g/L) and 200-μL serosal samples were taken every 7·5 min and replaced by Krebs–Ringer. The concentration of NaFl was determined using a fluorimeter (Polarstar Galaxy fluorescence multi-well plate reader; BMG LabTech GmbH, Jena, Germany), with 485 nm and 530 nm as excitation and emission wavelengths respectively. Steady-state NaFl-flux was quantified and expressed as ng/cm2/h. For each animal, average values of electrophysiological parameters and NaFl-flux were calculated from simultaneous measurements of three ileal samples. Statistical analyses were performed using SPSS v.12·0 software (SPSS Inc., Chicago, IL, USA).

The suppressive function correlated with reduced proliferation of myelin-specific T cells in vivo after intravenous GA treatment. In contrast, subcutaneous treatment with GA PLX3397 price inhibited the pro-inflammatory IFNγ-producing T cell phenotype rather than suppressing T cell proliferation. These data indicate that (1) GA engages directly with circulating monocytes to induce type II monocyte suppressor function; and (2) the therapeutic efficacy of GA may be expanded by employing different routes of GA administration to engage alternative

mechanisms of suppression of autoreactive T cells in MS. Multiple sclerosis (MS) is an autoimmune disease where the central nervous system (CNS) is attacked by the host immune system [1]. Experimental autoimmune encephalomyelitis (EAE) is an animal model

of MS that is induced by immunization with myelin oligodendrocyte glycoprotein peptides (MOG35–55) or other myelin components [2]. The pathogenesis of both MS and EAE is initiated by myelin-specific CD4 T cells whereby both TH1 and TH17 cells contribute to pathogenic processes [3–5]. In this context, activated CD4 T cells infiltrate the tissue of the CNS and generate a local inflammatory environment resulting in the recruitment of the monocyte, macrophage and CD8 T cell populations that are responsible for the damage to CNS tissue [3, 6]. Glatiramer acetate (GA) is a randomly associated selleck products copolymer comprised O-methylated flavonoid of l-alanine, l-tyrosine, l-glutamic acid and l-lysine

in a defined molar ratio [7]. Although previous studies have shown that GA relieves clinical symptoms in patients with MS and suppresses EAE in mice, the mechanism of action is not yet fully understood. It has been shown that T cell phenotype skewing from TH1 to TH2 [8, 9], decreased TH17 inflammation [10] and antigen-specific expansion of Foxp3+ T regulatory cells (Treg) [11] can contribute to disease suppression. In addition, increased lymphocyte apoptosis, enhanced neuronal repair and T cell receptor (TCR) antagonism to myelin components are also associated with GA treatment [12–14]. It is therefore likely that GA treatment does not depend on a single mechanism, but alters the dysregulated immune system in multiple ways to suppress autoimmunity. It has been recently reported that blood monocytes from naïve mice exhibit the ability to suppress T cell function and that this suppressor function is lost upon induction of EAE [15]. These findings identify monocytes as a potential therapeutic target for controlling autoimmunity. In vitro studies have shown that GA can alter the activation state and cytokine pattern of a variety of different antigen-presenting cells (APCs) [16–19]. In fact, monocytes from GA-treated patients and mice produce elevated levels of anti-inflammatory factors [11, 20]. Furthermore, subcutaneous GA treatment has been shown to induce type II suppressor monocyte in a model of EAE [11].

The value of the dihedral angle determined by C5′ atom of ribose, the neighboring oxygen atom, α phosphorus atom and the bridging oxygen atom varied from −162.25° to 53.63° for the most bent conformers. The dihedral angle determined by C5′-connected ribose oxygen atom, α phosphorus atom, the bridging oxygen and the β phosphorus atom varied from 162.63° to 93.87° for the most bent conformers. It was observed that the lowest energy conformers were characterized by the least linear conformation of ATP. The energy difference between the geometrically extreme structures was 54.25 kcal mol−1, due to the presence of hydrogen bonds Ruxolitinib in vivo stabilizing the ATP molecule. During the molecular dynamics simulation of ATP–enzyme complexes

the ATP conformation became more bent. However, the lowest energy conformers did not result in the binding pose, which would be in accordance with the mutagenesis data (Yamashita et al., 2008), and therefore the compromise conformer was accepted as the final one. The obtained mode of interaction of ATP with the enzyme is consistent with the reported mutagenesis analysis (Yamashita et al., 2008) and literature data concerning the mechanism of ATP hydrolysis by helicases/NTPases (Frick & Lam, 2006; Yamashita et al.,

2008). IDH mutation The binding pocket of JEV NS3 helicase/NTPase is formed by positively charged residues, i.e. Lys200, Arg461 and Arg464 of motifs I, II and VI. The most crucial residue, Lys200, projects into the pocket and recognizes the β-phosphate moiety of ATP. It forms a salt bridge with Asp285 and Glu286, which stabilizes the binding site structure. Arg461 and Arg464 in motif VI constitute an arginine finger and act as sensors recognizing the γ- and α-phosphate of ATP. It was reported that they are critical for conformational switching upon ATP hydrolysis (Ahmadian et al., 1997; Niedenzu et al., 2001; Caruthers & McKay, 2002; Yamashita et al., 2008). As stressed by Yamashita et al. (2008), the conserved water molecule necessary for ATP hydrolysis is coordinated by residues

Glu286, His288 and Gln457. Thr201 directs the molecule of ATP toward interactions with Lys200 and conserved arginines. His288 was reported as essential for RNA unwinding activity (Utama et al., 2000a, b). The side chain conformations Ketotifen of the JEV NS3 helicase/NTPase binding pocket residues were additionally refined in the docking procedure of known JEV NS3 helicase/NTPase inhibitors, 1–2 (Fig. 2), followed by molecular dynamics simulation. In the case of ring-expanded nucleoside 1 (Fig. 3a), the ligand structure is stabilized by two intramolecular hydrogen bonds: one between the C3′ hydroxylic group of the sugar moiety and a nitrogen atom of the imidazole ring, and the other one between one of the keto groups and the sugar ring oxygen atom. The other keto group of the inhibitor is engaged in the network of hydrogen bond with Arg464 and, through the water molecules, with the main chain NH hydrogen atoms of Gly197 and Ser198.

Vascular adhesion protein-1 (VAP-1; AOC3) is the best characterized SRT1720 ic50 ectooxidase in terms of leukocyte traffic 3, 4. It belongs to the primary amine oxidases (also known as semicarbazide-sensitive amine oxidases). VAP-1 is expressed in vascular endothelial, smooth muscle and fat cells, and it catalyzes oxidative deamination of primary amines. Regarding endothelial cells, VAP-1 is expressed in most vessels intracellularly but, apparently, under normal (non-inflammatory) conditions it can be expressed on the luminal surface of only certain types of vessels such as high endothelial venules. In most other vessels such as flat-walled venules, VAP-1 is translocated

from cytoplasmic vesicles to the luminal surface only upon induction of inflammation. During

oxidative deamination of primary amines, the substrate (the primary amine) is converted into an aldehyde, and ammonium and hydrogen peroxide are released (Fig. 2). The aldehyde products are involved in non-enzymatic formation of advanced glycation end-products, which are aberrantly glycosylated proteins capable of triggering inflammation and vascular malfunction. The hydrogen peroxide, on the other hand, is a powerful redox-signaling molecule at low concentrations. In particular, it can alter cellular responsiveness by inactivating phosphatases Selleckchem Ferroptosis inhibitor within the cells. The role of VAP-1 in leukocyte trafficking has been demonstrated by the use of function-blocking antibodies (which block the binding between leukocytes and endothelium but do not interfere with VAP-1′s enzymatic activity), small molecule enzyme inhibitors and gene-deficient mice 3, 4. Lymphocyte, monocyte and granulocyte binding to vessels in various lymphatic and non-lymphatic tissues has been shown to be inhibited by anti-VAP-1 antibodies in in vitro frozen section assays. In vitro flow chamber assays have revealed that blocking of VAP-1 by Oxalosuccinic acid mAbs or enzyme inhibitors reduces leukocyte rolling and adhesion on and, in particular, transmigration through the treated endothelial monolayer

4, 5. The contribution of VAP-1 in leukocyte extravasation under physiological shear has been confirmed in multiple in vivo assays. In intravital videomicroscopy, inhibition of VAP-1 by mAbs or enzyme inhibitors, results in increased rolling velocity, reduced adhesion and reduced transmigration 4, 6. The same alterations are also seen in VAP-1-deficient mice 7 (Table 1). Finally, inflammatory reactions can be alleviated in multiple in vivo models, such as those for peritonitis, arthritis, hepatitis, autoimmune diabetes, diabetic retinopathy, age-related macular degeneration, ischemia-reperfusion injury, transplant rejection and colitis, by anti-VAP-1 mAbs or enzyme inhibitors 3, 4, 6, 8–12. In malignancies, VAP-1 inhibition results in a decreased influx of immune-suppressing myeloid-derived suppressor cells into the tumors 13.

1), there was little change in splenic F5 T cell numbers compared with dox-fed controls (data not shown). Therefore, these data suggest that basal Bcl2 expression by naïve CD8 T cells in replete F5 hosts does not depend on IL-7 signalling. To further examine whether or not basal Bcl2 expression depends on IL-7 signalling, we examined Bcl2 levels in thymocytes, since both IL-7Rα and Bcl2 expression are dynamically regulated during development. IL-7Rα is expressed in DN thymocytes, required for normal DN survival and expansion 29, but is completely lost in DPs. Following successful

positive selection, CD4 and CD8 single positive (SP) thymocytes re-express IL-7Rα (Fig. 4A). Correlating with IL-7Rα, Bcl2 levels were high in WT DNs, greatly reduced in DPs and expression restored in SPs of WT thymocytes (Fig. 4A), Selleckchem ABT737 consistent with the view that IL-7 signalling is regulating Bcl2 expression in vivo during thymic development. To test whether Bcl2 expression in this developmental context was directly dependent

on IL-7 signalling, we examined thymic development of Il7r−/− and dox-fed F5 TetIL-7R mice. In Il7r−/− mice, although thymus size is approximately 100-fold less than WT 30, the gross thymic phenotype is remarkably normal in terms of the four major subsets defined by CD4 and CD8 expression. Interestingly, regulation of Bcl2 expression during thymic development was virtually identical selleck to that of WT (Fig. 4A).

In dox-fed F5 TetIL-7R mice, IL-7Rα is expressed ectopically on DP thymocytes as previously described 24. Analysing cell size of thymocytes from F5 TetIL-7R mice revealed an increase in cell size in both DP and SP subsets (Fig. 4B), confirming that IL-7R signalling was functional in these cells. As is true in WT thymocytes, F5 thymocytes upregulate Bcl2 expression as they mature from DP and SP stages. Significantly, ectopic expression of IL-7Rα on DPs of dox-fed F5 TetIL-7R mice did not result in ectopic expression of Bcl2. Rather, Bcl2 expression between the DPs and SPs of these mice was similar to that observed in F5 control thymocytes (Fig. 4C). Taken together, these data suggest SPTLC1 that basal Bcl2 expression in vivo is not dependent on IL-7 signalling, and that in normal homeostatic conditions, IL-7 must be promoting survival by a mechanism other than simply inducing expression level of Bcl2. Since Bcl2 expression levels could not account for the accelerated apoptosis of IL-7R– F5 T cells, we used microarray analysis to identify IL-7-regulated genes that may be involved in regulating survival of these cells. We compared gene expression between F5 T cells from control, dox-fed F5 TetIL-7R and dox free F5 TetIL-7R mice.

2), and suspended in 150 μL of the same buffer. The suspension was then heated to 50°C, and 150 μL of embedding agarose added from the kit at the same temperature. The suspension was then allowed to solidify in molds. Thereafter, the agarose suspension was incubated at 4°C for 20 min. The

agarose blocks were then incubated overnight at 37°C in 540 μL of lysis buffer I (Bio-Rad) containing 20 μL of lysozyme/lysostaphin solution (lysozyme 25 HDAC inhibitors cancer mg/mL, lysostaphin 2 mg/mL; Bio-Rad) and 20 μL of N-acetylmuramidase solution (N-acetylmuramidase SG 5 mg/mL, Dainippon Pharmaceutical, Osaka, Japan). The agarose blocks were washed once with wash buffer (Bio-Rad) and then incubated overnight at 50°C in 520 μL of proteinase K solution (> 23 U/mL). Then, they were then washed five times with wash buffer (1 hr per wash; Bio-Rad). Before restriction enzyme digestion, the agarose blocks were washed twice (1 hr per wash) with 0.1 × wash buffer, and then balanced for 1 hr in an appropriate restriction enzyme buffer. Restriction enzyme digestion with SmaI (TaKaRa) was performed overnight at 30°C. Restriction enzyme digestion with ApeI (TaKaRa) 5-Fluoracil cost and SacII (TaKaRa)

was performed overnight at 37°C. Electrophoresis was carried out using a CHEF DR III System (Bio-Rad) in 1% PFGE certified agarose (Bio-Rad) with 0.5 × tris/borate/EDTA buffer. The pulse time was 1–12 s, current 6 V/cm, temperature 14°C, and running time 22.5 hr. The agarose gel was stained with ethidium bromide (0.5 μg/mL) and visualized under UV light. The PFGE profiles of the strains were then visually compared. TMC0356 genomic DNA was digested with 11 restriction enzymes (Fig. 1). Banding patterns were obtained by digestion with all restriction enzymes except DraI and RsaI. ApaI, SacII, and SmaI were selected because the bands obtained after digesting the DNA with those enzymes were widely separated (from 24 kb to 290 kb). Ten different macrorestriction Tangeritin patterns were

obtained after digestion of genomic DNA of 15 L. gasseri strains with SmaI and separation by PFGE (Fig. 2). Similar banding patterns were obtained for TMC0356, JCM 1031, and JCM 1131; however, a thick band of 42.9 kb was confirmed for TMC0356 but not for JCM1031 and JCM 1131. No other strain showed a banding pattern similar to that of TMC0356. The genomic DNA profiles of the 15 L. gasseri strains digested with SacII are shown in Figure 3. The banding patterns were similar for TMC0356, JCM1031 and JCM 1131; however, a thick band of 42.9 kb was confirmed for TMC0356 but not for JCM1031, JCM 1131. No other strain showed a banding pattern similar to that of TMC0356. The genomic DNA profiles of the 15 L. gasseri strains digested with Apa I are shown in Figure 4. TMC0356, JCM1031 and JCM 1131 showed identical banding patterns, and hence could not be distinguished. A strain (TMC0356F-100) obtained after subculturing TMC0356 in skim milk 100 times was also analyzed by PFGE.