Based on thorough studies of many groups using different techniques, the current view on iNKT-TCR/CD1d interaction is that the CDR2α, which discriminates type 1 and type 2

AV14 genes, is not at all, or only very weakly, involved in CD1d-restricted antigen recognition Bioactive Compound Library chemical structure [30]. Whether this holds true for the rat still needs to be shown, especially since own preliminary data obtained with α-GalCer-CD1d dimers and iNKT-TCR transductants suggest that rat AV14 family members may indeed differ in their CD1d/antigen-binding properties. Our data on the F344 iNKT-TCR repertoire are fully consistent with the data from Matsuura and colleagues who used molecular biology methods (RT-PCR and analysis of cDNA libraries) to make predictions on frequencies and organ-specific distribution of rat iNKT cells, as well as on the proportion of canonical iNKT-TCR rearrangements within AV14-containing TCRs [9]. Nevertheless, we could not confirm the proposed organ specificity of AV14 gene usage. It was

not clear that Matsuura and colleagues analyzed several individual animals. Therefore, it is possible that their different results were due to variability between individual animals. Indeed, we found the proposed dominance of type 2 AV14 in spleen and a nearly equal distribution find more of type 1 and type 2 in IHLs, but only in one of four F344 rats (Supporting Information Table 2, animal 2). The impossibility to detect iNKT cells in LEW rats is of particular interest since iNKT cells have been linked to autoimmunity in humans and mouse models and the LEW strain is widely used as model for organ-specific

autoimmune diseases such as experimentally Morin Hydrate induced encephalomyelitis, uveitis, and others. Importantly, the induction of these diseases is not successful in F344 rats [24-26]. Therefore, the clear differences in iNKT-cell frequencies between LEW and F344 rats (and probably between other inbred strains as well) offer the opportunity to map loci controlling the different frequencies and link them (or not) with known disease-associated loci, for example, controlling autoimmunity [24-26]. Moreover, the role of iNKT cells in the development of spontaneous type 1 autoimmune diabetes is not clear [1]. Thus, an obvious candidate for the analysis of iNKT cells are BB inbred rats as they are, apart from NOD mice, the only animal model available for this disease. The observed similarities in the frequencies and phenotype of F344 rat iNKT cells compared with those in the human already suggest that certain rat strains might result in valuable models to study iNKT cells in disease. Indeed, the rather simple mode of in vitro expansion is of special interest, since it opens the possibility of expanding and manipulating iNKT cells in vitro and testing the functional properties of the cells after adoptive transfer.

Furthermore, the leak point pressure of cell-implanted rabbits is significantly higher than that of cell-free injected controls. We conclude that implantation BYL719 chemical structure of autologous bone marrow-derived cells could be an effective treatment for human post-surgical ISD-related urinary incontinence. We have been vigorously investigating regenerative medicine of the lower urinary tract based on tissue engineering and/or stem cell therapy techniques, both in vitro and in vivo.1–9 Tissue engineering strives to form functional tissues by using cells, scaffolds, and growth factors. Stem cell therapy

strives to restore functional structures by injections of adult somatic stem cells into damaged tissues. In this review, we show that implantation of autologous bone marrow-derived cells into injured urethral sphincters leads to the recovery of continence due to the replacement, enhancement, and/or reconstruction of the striated and smooth muscle layers. We group urinary incontinence into two

major categories: (i) stress urinary incontinence and (ii) post-surgical urinary incontinence associated with intrinsic sphincter deficiency (ISD). Stress urinary incontinence is an involuntary leakage of urine that occurs during physical activity, such as coughing, sneezing, or lifting heavy Alectinib research buy objects, and is the most common type.10,11 The majority of stress urinary incontinence cases is related to urethral hypermobility, which results from the loss of bladder neck support.12,13 Urethral hypermobility-related stress urinary incontinence can be improved by surgical therapies to lift the bladder and urethra.14,15 In contrast, post-surgical ISD-related urinary incontinence can occur as a result of radical prostatectomy16,17 or bladder neck surgery.18 It is characterized by severely decreased urethral closure pressure due to malfunction of the closure mechanism and results in intractable urinary incontinence.19 Under these circumstances, improvement of urinary continence requires increased urethral closure pressure. Injection of a bulking agent, such

as collagen, into the periurethral tissue has been widely accepted;20–23 however, the long-term benefits are not satisfactory because the continence rate sharply decreases with time.24,25 Surgical implantation of a device, such as an artificial urinary Decitabine mw sphincter, has also been accepted as a treatment for this type of incontinence.26,27 However, this modality is not popular because the procedure is not covered by insurance. Additionally there are side-effects, such as inflammation and abscesses.28 Thus post-operative ISD-related urinary incontinence has few effective treatments. For that reason, we have vigorously investigated novel treatments that have proved to be effective in an experimental model of ISD-related urinary incontinence and have the potential to be effective in humans.

T lymphocytes and B lymphocytes specific for other antigens are not activated in the current model. CD4+ regulatory T lymphocytes.  Innate (or natural) regulatory T lymphocytes (iTregs), representing

CD4+CD25+ T lymphocytes, LY2109761 molecular weight are modelled as a distinct population of thymic-derived cells, distinguished from the aforementioned aTregs by not requiring further differentiation to express regulatory activity [52]. Once activated via presentation of autoantigen on MHC class II molecules (MHCII antigen), regulatory T lymphocytes exhibit both cell contact-mediated and cytokine-mediated immunosuppressive activity [46,53,54]. CD8+ T lymphocytes.  CD8+ T lymphocytes in the model are initially activated by MHCI-antigen in the PLN, with help provided Selleck FDA-approved Drug Library by activated CD4+ T lymphocytes [55–58]. Acquired cytotoxic effector activity includes both cell contact- and cytokine-mediated mechanisms [59,60]. B lymphocytes.  B lymphocytes in the model interact with DCs, natural killer (NK) cells and T lymphocytes. They differentiate (in the PLN), present antigen to CD4+ and CD8+ T lymphocytes and produce cytokines and autoantibodies [61–63]. Autoantibodies form immune complexes, influencing antigen uptake

[26,64]. NK cells.  On the recommendation of the scientific advisory board, NK cells were included in the model based on a high degree of scientific interest and investigation [65–68]. Because the data characterizing NK cells in type 1 diabetes and their relative role in disease are sparse relative to other cell types, the use of the NK cell module is optional (i.e. it can be omitted from the virtual mouse simulations). Inclusion of the NK cell module may be used to explore specific hypotheses on the role of NK cells in disease. learn more Activation of NK cells in the model is mediated by DCs and B lymphocytes and is regulated further by cytokines and co-stimulatory molecules [69–74]. Effector activities include cytokine synthesis and killing of immature

DCs and β cells [75,76]. Blood glucose.  The level of blood glucose in the model is regulated by insulin-dependent and insulin-independent mechanisms, based on deviations of insulin and glucose from their basal levels [77,78]. Dietary glucose intake is assumed to be constant and implicitly accounted for in the basal glucose level. Gut and gut-associated lymphoid tissue.  The gut and gut-associated lymphoid tissue (GALT) were built to investigate the role of local immune activity on the efficacy of oral insulin therapy. The gut tissue in the model is simplified to include only DCs. The GALT includes all the biological components present in the modelled PLN. Following the design phase, the components of the model were represented mathematically. As illustrated in Fig.

While CF patients were exclusively colonised with either S. prolificans or FDA-approved Drug Library in vitro P. boydii, patients with other severe underlying diseases were colonised or infected with several (in total: six species) Scedosporium species. Remarkable is that CF patients, who were monitored over up to almost 5 years, were exclusively

colonised with a single Scedosporium species. Due to the limited amount of data, we cannot yet see species-dependent clinical prevalence or a correlation with underlying diseases. At the moment, VOR is the only licensed antifungal agent for the treatment of Scedosporium infections in Europe; all other antifungals are used off-label. MIC/MEC breakpoints for Scedosporium species have not yet been determined. Published studies of susceptibility profiles of Scedosporium species taking the latest taxonomical changes into account are lacking, since the separation

of P. apiosperma from P. boydii was published only in 2010.5 These two sibling species were found to have very similar susceptibility profiles, being most susceptible to MICA and VOR. Our results show that in general, MICA had reasonable in vitro activity against all Pseudallescheria/Scedosporium species except S. prolificans (Table 2). Monotherapy using VOR has frequently been reported to be tolerated by patients MG132 and was successful in treatment of S. apiospermum infections.25–28 MICA exerts antifungal activity via inhibition of (1,3)-β-d-glucan synthase,29 and therefore may enhance in combination therapy the fungicidal effect of other antifungal compounds targeting different cellular elements. Interleukin-2 receptor In vitro synergistic effects of azoles combined with echinocandins were reported by Cuenca–Estrellas et al.1 Other studies demonstrated a profound synergistic effect of azole-terbinafin combinations.3–32 Therefore, in addition to terbinafin, MICA should be also taken into consideration as possible combination therapy option for Scedosporium infections, preferably in combination

with VOR. In contrast to other fungi, such as Aspergillus fumigatus and Candida albicans, the molecular epidemiology of Scedosporium/Pseudallescheria has received far less attention in medical literature. The few studies having addressed this are reviewed by Harun et al.33 More importantly, since the latest taxonomical change in 2010, no studies have addressed the molecular epidemiology of these fungi in patients; so, the true value of several previous studies cannot be ascertained. In 2003, AFLP was shown to be a powerful method for identifying closely related Canidida species, including differentiation between the sibling species C. albicans and C. dubliniensis.34 A similar observation was made for filamentous fungi exemplified on A. fumigatus and its sibling species Neosartorya fisheri, by Klaassen and Osherov.35 In addition, AFLP analysis can provide high resolution fingerprints for intraspecific discrimination.

3B), pointing once again toward MAPK dephosphorylation as the molecular event that is targeted by zinc in IL-2- signaling. Our results suggest that zinc release after stimulation with IL-2 conserves ERK phosphorylation by inhibiting phosphatases, and hereby free zinc acts as a permissive signal. Zinc also inhibits protein tyrosine phosphatases, preserving signaling by the insulin and EGF receptors 28–30. The IL-2R itself, as well as JAK1 and 3 and STAT5, are activated by tyrosine phosphorylation 10. However, no activation of the STAT5-pathway by zinc was found in our experiments (Fig. 2A), indicating that zinc in IL-2R signaling primarily acts on phosphatases

that dephosphorylate ERK. Here,

intracellular localization of zinc signals PD-0332991 datasheet might be relevant, and should be investigated in more detail, as tyrosine phosphorylation of the IL-2R and JAK occurs at the plasma membrane, whereas MAPK are present in cytosol and nucleus. Alternatively, the binding constants for some protein tyrosine phosphatases are in the low nanomolar concentration range 28, and future experiments should compare these values to the susceptibility of DUSPs and PP2A to zinc inhibition. Notably, there are seven DUSP known to dephosphorylate ERK 31, whereas PP2A also dephosphorylates MEK1/2 in addition to ERK 13. Because zinc had an effect on MEK and ERK in Fig. 2F, it seems likely that PP2A is among the molecular targets of zinc in T cells. Nevertheless, ERK dephosphorylation is completely inhibited by zinc, indicating that all other

ERK dephosphorylating LBH589 price enzymes are also susceptible to inhibition by zinc. When the expression of genes specifically triggered by the different pathways was analyzed by PCR (Fig. 3A and B; Supporting Information Fig. 4), i.e. CIS for STAT5 32 and c-fos for ERK 13, corresponding results to the Western blot analysis were found. STAT5-dependent CIS expression was not influenced by chelation or imitation of zinc signals, whereas c-fos induction was significantly decreased by TPEN. ERK signals are involved in proliferation and cellular survival in response to IL-2 10. Hence, we investigated the role of zinc signals in these events. Cells were labeled with (5)6-Carboxyfluorescein diacetate (CFDA) Interleukin-2 receptor to measure proliferation and with propidium iodide to detect cytotoxicity, and analyzed by flow cytometry after growing for 24 h in the presence of various concentrations of TPEN. Concentrations of up to 3 μM TPEN did not lead to a significant reduction of viability, but IL-2-dependent proliferation of CTLL-2 was significantly reduced at TPEN concentrations of 2 μM and above (Fig. 3C), indicating a preferential requirement of zinc signals for IL-2-induced proliferation at concentrations that were not cytotoxic. TPEN can chelate several other metal ions in addition to zinc 33.

Most interestingly, in vitro experiments revealed that FcεRI-aggregation and allergen challenge profoundly down-regulate the capability of PDCs to release IFN-α/β upon subsequent stimulation with cytosine–guanine dinucleotide (CpG) motifs [5]. Data showing lower production of IFN-α by human blood DCs from allergic individuals after TLR-9 stimulation [26], as well as down-regulation of FcεRI expression on PDCs after TLR-9 activation and reduced TLR-9 expression after FcεRI cross-linking

[27], indicate that a direct counter-regulation and interaction of FcεRI/TLR-9 mediated mechanisms might be responsible for this effect. This implies that the amount of FcεRI expressed on the surface of PDCs, together with the strength and frequency of signals mediated via FcεRI attenuate X-396 mouse the capacity of PDCs to defend the organism against invading microbial and, in particular, viral antigens. Furthermore, increased IL-10 production of PDCs after FcεRI aggregation observed in vitro might enhance endogenously, together with the Th2-dominated micromilieu in the skin, PDC apoptosis and reduction of the number of PDCs recruited from the blood

and detectable in epidermal AD lesions [5,16]. Taken together, a close cross-talk of FcεRI with TLR-9 and reduced capability of PDCs to release IFN INCB024360 cell line in response to TLR stimulation by viral antigens after FcεRI activation/allergen challenge, together with the relatively lower number of epidermal PDCs in AD compared to other inflammatory skin diseases such as allergic contact dermatitis or psoriasis, might explain in part the increased susceptibility of AD patients to viral infections of the skin observable, for example, by the manifestation of eczema herpeticum, a severe HSV infection spreading over large body areas in AD patients in vivo[28]. Although the oral mucosal epithelium is exposed to high numbers of bacterial products and allergens derived from food, chronic allergic inflammatory reactions are observed less frequently at this

site [4]. This is in contrast to other mucosal surfaces such as the nasal and bronchial mucosa, where local chronic allergic and inflammatory reactions occur often. Most probably, DCs play a major role as both activators and silencers of allergic immune responses within the immunological network of mucosal surfaces. In this context, PJ34 HCl it has been reported that different DC subpopulations reside within the oral and nasal mucosa in humans. The predominant DC population within the oral epithelium consists mainly of classical Birbeck granules containing CD207pos/CD1apos LCs, while significant numbers of PDCs were detected in nasal mucosal epithelium [29]. The myeloid CD1apos DC subpopulation within oral and nasal mucosal epithelium differs further in the expression of various lectins, such as CD206 and CD209, which are expressed only by nasal DCs (nDCs) (Table 1) [29].

GAD-alum-treated patients displayed higher frequencies of in-vitro GAD65-induced CD4+CD25+CD127+ as well as CD4+CD25hiCD127lo and CD4+FoxP3+ cells compared to placebo. Moreover, GAD65 stimulation induced a population of CD4hi cells consisting mainly of CD25+CD127+, which was specific of GAD-alum-treated patients (16 of 25 versus one of 25 in placebo). Assessment of suppressive function in expanded regulatory T cells revealed no difference between GAD-alum- and placebo-treated individuals. Regulatory T cell frequency did not correlate with C-peptide secretion throughout the study. In conclusion, GAD-alum

Copanlisib molecular weight treatment induced both GAD65-reactive CD25+CD127+ and CD25hiCD127lo cells, but no difference in regulatory T cell function 4 years after GAD-alum treatment. Type 1 diabetes (T1D) is a consequence of an autoimmune reaction towards insulin-producing β cells of the pancreas. Immunomodulatory approaches to prevent or treat T1D have been developed and tested, with variable results [1-4]. Autoantigens may be used to induce immunologic tolerance as an alternative to immunosuppression [5]. Glutamic acid decarboxylase 65 (GAD65) is one of the main antigens to which patients with T1D mount a destructive

immune response, INCB024360 and autoantibodies directed against GAD65 (GADA) and T cells specific for GAD65 epitopes are common in T1D patients [6-8]. We have shown previously preservation of residual insulin secretion by GAD-alum treatment

in a Phase II clinical trial in children with recent-onset T1D [3]. In addition, trial participants treated with GAD-alum up-regulated CD4+CD25hiforkhead box P3+ (FoxP3+) cells in response to GAD65 stimulation in vitro clonidine and had a predominant T helper type 2 (Th2) immune response [9, 10]. Preservation of C-peptide secretion was still detectable after 4 years in patients with <6 months T1D duration at baseline in the same trial [11], and the residual C-peptide secretion was accompanied by sustained high levels of GADA, increased memory T cell frequencies and T cell activation upon in-vitro GAD65 stimulation [12]. Recently, additional Phases II and III clinical trials of GAD-alum have been conducted both in Europe and the United States, neither finding an effect on preservation of insulin secretion [13, 14]. The present Phase II trial included patients with a T1D duration of <18 months, whereas the European Phase III trial included patients with a duration of <3 months, which may contribute to the discrepancy in outcome. Self-tolerance is maintained physiologically by regulatory T cells (Treg) in the periphery [15], and defects in Treg function have been hypothesized to be involved in the pathogenesis of autoimmune disease [16].

The skin is constantly subjected to environmental insults (microbial, chemical and physical) that may trigger immune responses 20. It has been proposed that the presence of NLRP3 in the skin (keratinocytes and tissue resident dendritic cells) provides a first line of defence by enabling the rapid sensing of invading pathogens, thereby triggering an innate immune response via NLRP3 inflammasome activation 21, 22. Sensitising allergens that penetrate the skin surface induce a delayed type hypersensitivity reaction, called contact hypersensitivity (CHS) 23, 24. Evidence has been presented for the involvement of NOD-like receptors (NLR) as well as IL-1β,

IL-18 and caspase-1 in the mouse CHS

model 25, 26. Recent work has also suggested that IL-18 plays an important role by distinguishing the presence check details of contact allergens from irritants 27 (Table 1). The outcome of skin immune responses with respect to tolerance or immunity is dependent on skin NLRP3 inflammasome activation, and secreted IL-1β and IL-18 may regulate the quality of an allergen-specific PLX4032 cost T-cell response in CHS 25. Furthermore, mice deficient in IL-1β have impaired CHS to trinitrochlorobenzone 28. These discoveries suggest that modulation of the NLRP3 inflammasome may offer a therapeutic strategy to modulate T-cell responses in patients suffering from allergic CHS. Excitingly, manipulation of the NLRP3 inflammasome may also offer a perspective to induce tolerance towards a given contact allergen. Type 2 diabetes (T2D) occurs when beta cells in the pancreas fail to produce sufficient insulin to overcome insulin resistance. Several lines of evidence support the role of IL-1β in the pathogenesis of T2D; expression of the IL-1Ra is reduced in the pancreatic islets of these patients, with IL-1β being produced in response to high glucose concentrations,

leading to decreased cell proliferation and apoptosis 29. Larsen et al. have reported that anakinra treatment results in decreased glycated haemoglobin (HbA1c) levels and increased insulin production in T2D patients 30. An IL-1β antibody, Xoma 052, was shown to restore glycemic control in T2D patients Amobarbital in a double-blind, placebo-controlled, dose-escalation study 31. In this regard, it is also relevant that glyburide, a sulphonylurea drug used to treat T2D, inhibits the NLRP3 inflammasome 32. T2D is a burgeoning global health problem and this advance in understanding the pathogenesis will offer novel therapeutic avenues in the future. Inflammation appears to provide a local environment in which many tumours flourish and IL-1β has a key role in this process 33. Inflammasome-mediated pathogen recognition 34 provides a potential, but as yet unproven, link between infection-induced inflammation and cancer.

In this study, we address the question: find more can Ab targeting the high affinity FCR engineered to express CTL epitopes stimulate high-avidity CTL

responses that are capable of efficient anti-tumor activity? We have previously shown that Ab–DNA vaccines engineered to express CTL epitopes can stimulate high-frequency responses to self and foreign epitopes but it was unclear if these were of high avidity 26. Initially a DNA vaccine incorporating the H-2Kb OVA epitope, SIINFEKL, within a human IgG1 molecule was screened for stimulation of high-avidity CTL responses. The SIINFEKL epitope OVA was grafted into CDRH2 region alongside an I-Ab restricted CD4 helper epitope from Hepatitis B (HepB) surface Ag. C57BL/6 mice immunized with this DNA construct demonstrated high-frequency epitope-specific responses compared to a control irrelevant peptide (p<0.0001) (Fig. 1B). It was next assessed if encoding an epitope within an Ab–DNA vaccine could break tolerance to a self Ag. An epitope from the melanoma Ag tyrosinase related

protein 2 (TRP2) was engineered into a human IgG1 Ab alongside the HepB CD4 epitope. Immunized C57BL/6 mice also demonstrated high-frequency TRP2-specific responses, although these were lower than OVA-specific responses (p<0.0001) (Fig. 1C). The ELISPOT assays in this study use total splenocyte Palbociclib populations and it is possible that other IFN-γ producing cells reside within this population. To address this, CD8+ cells were depleted prior to use in the ELISPOT assay. Depletion of the CD8+ cells eliminates the TRP2-specific response but has no effect upon the HepB helper peptide-specific response (Fig. 1D). To determine if there was any advantage in immunizing with Ab–DNA vaccine as compared to simple peptide immunization, T-cell responses to OVA/HepB

or TRP2/HepB human IgG1 DNA vaccines were compared to vaccination with HepB/OVA or TRP2/HepB 4-Aminobutyrate aminotransferase linked peptides. Mice immunized with peptide show significantly lower frequency responses compared to human IgG1 DNA immunized mice (p<0.0001 and p=0.003, respectively) (Fig. 1e). Functional avidity of CD8 responses has been shown to be important in the induction of anti-tumor immunity. Analysis of the functional avidity revealed that responses induced in human IgG1 DNA immunized mice were over 100-fold higher compared to peptide immunized mice for both OVA and TRP2 epitopes (p<0.0001 and p=0.0009, respectively) (Fig. 2A and B). OVA human IgG1 DNA shows avidity of 1×10−11 M compared to OVA peptide at 1.3×10−9 M. TRP2 human IgG1 DNA demonstrates an average avidity of 6×10−12 M compared to TRP2 peptide at 1.7×10−9 M.

Cell death induction was detected by the addition of propidium iodide (PI; Sigma-Aldrich, St. Louis, MO, USA) at a final concentration of 10 μg/mL and analyzed by flow cytometry. Similar experiments were performed with serum samples previously heated at 56°C for 30 min

to inactivate complement and with both IgG and IgM fractions isolated from the serum of healthy donors HD2 and HD4. We considered a serum sample to be positive when the percentage of dead cells was ≥20% and at least two times the percentage observed for the untreated cells. To determine if the cytotoxic effect of serum samples was mediated by the anti-NeuGcGM3 antibodies, L1210 cells were cultured for 3 days with 10 μmol/L of D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (Matreya, LLC, PA, USA), an inhibitor of glucosylceramide synthetase that affects glycosphingolipids biosynthesis. With this same objective, before cell death induction, serum samples were incubated with Wnt activity 15 μg of NeuGcGM3, previously air dried and sonicated in PBS, in order to block the anti-NeuGcGM3 antibodies. As a control for apoptosis induction, L1210 cells were treated with 10 μM CIGB 300 for 20 min at 37°C [51], an apoptosis inducer kindly provided by Dr. Perea from the Centre of Genetic Engineering and Biotechnology. To determine the nuclear and membrane morphology, after incubation with serum samples during the indicated times,

L1210 cells were dried on microscope slides, fixed with 4% formaldehyde and stained with check details H&E. Apoptotic or oncotic necrotic cells were identified by morphological criteria. Cell death with chromatin condensation, cell shrinkage and formation of apoptotic bodies was regarded as apoptosis. Morphologic criteria such as karyolysis, cell membrane disruption and cellular swelling were used to determine oncotic necrosis [52, 53]. To visualize antibody binding to the cell membrane and incorporation of PI after 30 min of treatment with the sera, cells were washed and blocked with PBS containing 1% FCS, and incubated with FITC-conjugated goat antihuman Igs (IgM + IgG) (Jackson ImmunoResearch Laboratories) for 30 min at room temperature in the dark and with

PI for 10 min at a final concentration of 10 μg/mL. After washing with PBS, cells were immediately visualized on a fluorescence microscope (OLYMPUS BH-2, Tokyo, Japan). The involvement of caspase-3 in induced Acetophenone cell death was studied after 2 or 4 h of incubation of L1210 cells with the serum samples. Next, the cells were stained with FLICA (SR-DEVD-FMK; Immunochemistry Technologies, Bloomington, IN, USA), following the manufacturer’s instructions. The cells were visualized on a fluorescence microscope (OLYMPUS BH-2). Data analyses were performed using Graph-Pad Prism 5.03 Software. Each experiment was repeated at least twice. Unless specified otherwise, data is described as mean ± SD. Mann–Whitney U test was used as a nonparametric test for pair-wise comparisons.