0 mg/dL) levels were high, although other IgG subtypes were normal. Serum immunofixation did not demonstrate M protein, and the level of serum soluble IL-2 receptor was normal. The serum levels of κ (20.6 mg/dL) and λ (18.5 mg/dL) free light chains and the κ/ λ ratio (1.11) were also normal. The patient also did not have any donor specific antigens. A contrast-enhanced CT scan revealed a non-enhanced mass at the renal hilum

and some contrast defect areas in the renal cortex and diffuse marked enlargement of the graft, although no lymph node swelling was observed (Fig. 2A). An MRI also showed a hilum mass lesion with high intensity on T2-weighted images PLX-4720 concentration and low intensity on diffusion-weighted images. A PET-CT scan only detected a light integrated

mass of the hilum. Based on these findings, the patient was suspected of having IgG4-RKD. As the renal function of the patient was stable at that time, a no-treatment follow-up strategy was considered appropriate. However, her renal function deteriorated gradually and the serum Selleckchem BIBW2992 IgG4 level remained high (>400 mg/dL). In November 2012, the patient’s serum creatinine level had increased to 1.56 mg/dL. A biopsy was therefore carried out that showed almost the same findings as the biopsy 2 years after transplantation, although some severe fibrotic lesions and infiltration of IgG4-positive plasma cells were observed directly under the renal capsule. Because of the deterioration in renal function, the methylprednisolone dose was increased to 16 mg/day. Three months after this increase in steroid dose, the hilum mass disappeared on a CT scan (Fig. 2B), but cytomegalovirus antigenmia, JC virus viruria and viraemia screening became positive. An over-immunosuppression state was therefore suspected, and the methylprednisolone dose was decreased to 8 mg/day and mycophenolate mofetil changed to mizoribine. Tenofovir purchase Five months after the initial increase in steroid

dose, a follow-up biopsy in May 2013 showed that plasma cell infiltration in the renal interstitium had decreased markedly, although focal and segmental severe interstitial fibrosis and tubular atrophy with IgG4-positive plasma cells were observed (Fig. 3). Serum IgG4 levels decreased immediately after the increase in steroid dose and remained at <100 mg/dL thereafter. The patient's serum creatinine level also remained stable at around 1.6 mg/dL. The clinical course of the patient is shown in Figure 4. IgG4-RKD usually manifests as plasma cell-rich tubulointerstitial nephritis (TIN), although its clinicopathological features are not well described. Raissan et al. showed that most patients with overt IgG4-RKD had acute or progressive chronic renal failure, involvement of other organ systems, radiographic abnormalities such as small peripheral low-attenuation cortical nodules or diffuse marked enlargement of the kidneys, and elevated IgG4 serum levels (>135 mg/dL).

24; MgSO4, 1.3; CaCl2, 2.4; NaHCO3, 26; and glucose, 10. The tissues are transported to our laboratory under these conditions

within 45 min after removal. The second step is preparing the brain slices for physiological experiments (Fig. 1 middle). Brain slices 500 μm thick are obtained from the transported brain tissue using a microslicer in our laboratory. Several fresh slices, usually 2–3, are prepared from each brain block. For histological evaluation, residual tissue from the brain block is embedded in optimal cutting temperature compound, and then slices 7 μm thick are prepared using a cryostat (Fig. 3). The sections are stained quickly with HE. Histological features are then compared with the translucent image of the fresh slices. The prepared slices are incubated in ACSF at 29–30°C for more than 1 h to allow recovery from any see more damage due to the slicing procedure. The third step is evaluation of the neural activity of the slices. After incubation, each slice is transferred to a submerged recording chamber and perfused AZD6738 continuously with oxygenated ACSF at a flow rate of 1 mL/min. Translucent images taken in infrared light (λ = 930 ± 10 nm) are obtained with a cooled charge-coupled device camera system attached to an inverted epifluorescence microscope to identify the histological architecture. By comparing the microscopic features on the HE sections obtained at the previous

step with the translucent image of the fresh slice, the area in which to place the stimulating electrode is determined. This procedure is especially effective for examining neocortical lesions,

including focal cortical dysplasia, because otherwise correct orientation of the fresh slices would be difficult to achieve in such cases. The slice is then stimulated electrically and the spatiotemporal activity evaluated in terms of flavoprotein fluorescence imaging every 100–300 ms. Details of the theoretical background of flavoprotein fluorescence imaging have been described previously.[11] Under the experimental conditions employed, responses represented by changes in signal intensity of about 0.5–3% are usually observed. The images obtained are usually averaged eight times to improve their quality; however, a response can be observed even in a single trial (Fig. 4). The fourth step is morphological and molecular biological Liothyronine Sodium examination to validate the physiological findings (Fig. 1 right). For this purpose, we use a block of brain tissue corresponding to the mirror surface of each of the slices employed for the physiological examination (Fig. 3). These blocks are fixed with 4% paraformaldehyde and embedded in paraffin. This approach allows us to observe microscopic alterations within the blocks. On the other hand, the fresh slice used for optical imaging can also be used for molecular biological study,[6] since the flavoprotein fluorescence method requires no exogenous dye or fixative.

Cells were washed after 48 hr and lysed for 30 min at 4° in radioimmunoprecipitation assay buffer [1% Triton X-100 (v/v), 0·5% sodium deoxycholate (w/v), 0·1% SDS] containing protease inhibitor cocktail (Sigma-Aldrich). Cell debris was spun down at 15 600 g

for 15 min. Precipitates were removed and aliquots of cell lysates were diluted in SDS sample buffer, boiled at 100° for 3 min, spun down, and applied to precast 10% acrylamide Tris–glycine gels at 40 g protein/lane and run at 150 V for 1 hr. Samples were transferred to nitrocellulose membrane (BioRad) at 100 V for 1 hr. Membranes were probed using rabbit anti-mouse Arg I polyclonal antibody (Santa Cruz Biotechnology, click here Santa Cruz, CA) and rabbit anti-mouse iNOS (NOS2) polyclonal antibody

(BD Biosciences) at a 1 : 500 and 1 : 2000 dilutions, respectively, followed by peroxidase-conjugated anti-rabbit antibody (Sigma-Aldrich) at a 1 : 1000 dilution. Bands were visualized using a chemiluminescence reaction. Splenocytes were prepared from naive mice, and enriched for CD90.2+ cells (90% by FACS analysis) using anti-FITC-coated magnetic beads (MACS; Miltenyi Biotec, Bergisch Gladbach, Germany) after incubation with FITC-conjugated anti-CD90.2 mAb. Peritoneal cells were enriched for F4/80+ Mφs (85% by FACS analysis) using anti-FITC-coated Selleckchem BAY 73-4506 magnetic beads (MACS; Miltenyi Biotec) after incubation with FITC-conjugated anti-F4/80 mAb. The T-cell proliferative response was evaluated after co-culturing CD90.2+ spleen cells (2 × 105 cells/200 μl/well) with F4/80+ peritoneal cells in flat-bottom microwell tissue 4��8C culture

plates at different T-cell/ Mφ ratios (2 : 1, 5 : 1, 10 : 1 and 20 : 1) in the presence of 2.5 μg/ml concanavalin A (Con A; Sigma-Aldrich). The presence of naive Mφs increased proliferation of CD90.2+ T cells and the most effective Mφ-to-T-cell ratio was 1 : 10 (data not shown). The PD-1/PD-Ls pathway blockade on the proliferative response was evaluated after co-culturing CD90.2+ spleen cells (2 × 105 cells/200 μl/well) with infected or non-infected F4/80+ peritoneal cells in flat-bottom microwell tissue culture plates treated with 5 μg/ml isotype control, anti-PD-1, anti-PD-L1 or anti-PD-L2 in the presence of 2.5 μg/ml Con A (Sigma-Aldrich). Cultures were maintained at 37° in a humidified 5% CO2 atmosphere for 3 days and 0·5 μCi/well [methyl-3H] thymidine (Amersham, Chicago, IL) was added for the last 18 hr of culture. Cells were collected with a cell harvester (Cambridge Technology, Watertown, MA) and processed for standard liquid scintillation counting using a counter from Beckman Instruments (Fullerton, CA). Values are represented as counts per minute from triplicate wells. The T. cruzi-infected and non-infected peritoneal cells were obtained and single cell suspensions were prepared in RPMI-1640 supplemented as above.

Methods: A retrospective evaluation of 42 patients has been performed. The study population consisted of 24 males (57.1%) and 18 females (42.9%), ranging in age from 25 to 81 years (mean, 62.6 years). The primary location of the tumor was the mandibular alveolar crest (18 cases), retromolar trigon (9), selleck chemicals floor of the mouth (8), cheek (5), and oral commissure (2). For reconstruction a single free flap technique was used eight times; a double free flap technique, seven times; free and locoregional flap association, 25 times; and a single locoregional flap and two associated locoregional flaps, one time each.

Postoperative follow-up ranged from 12 to 144 months. Final results were evaluated with regards to deglutition, speech, oral competence, and esthetic outcome. Results: When free bone-containing flaps or two free flaps technique were used, the functional results were better (normal diet, 67%–71%; good oral competence, 100%–71%; good or intelligible speech, 100%–86%).

When free and locoregional flap association was chosen, the esthetic results were best (excellent, 76%; acceptable 24%; poor 0%). The worst results were obtained with the use of a single free soft tissue flap and with the use of single or double locoregional flap technique. Conclusion: Bone reconstruction of the lateral mandible is indicated whenever possible. EPZ015666 supplier In elderly or poor prognosis patients acceptable results can be achieved with free soft tissue flaps techniques. When the defect involves different structures of the oral cavity, the best results from are provided by the association of two free flaps. Finally, the association of free and locoregional flaps is a good option for external coverage reconstruction. © 2010 Wiley-Liss, Inc. Microsurgery 30:517–525, 2010. “
“The main advantage of deep inferior epigastric perforator (DIEP) flap breast reconstruction is muscle preservation. Perforating vessels, however, display anatomic variability and intraoperative decisions must balance flap perfusion with muscle or nerve sacrifice. Studies that aggregate DIEP flap reconstruction may not accurately reflect the degree of rectus preservation.

At Beth Israel Deaconess Medical Center from 2004–2009, 446 DIEP flaps were performed for breast reconstruction. Flaps were divided into three categories: DIEP-1, no muscle or nerve sacrifice (126 flaps); DIEP-2, segmental nerve sacrifice and minimal muscle sacrifice (244 flaps); DIEP-3, perforator harvest from both the medial and lateral row, segmental nerve sacrifice and central muscle sacrifice (76 flaps). Although the rate of abdominal bulge was similar among groups, fat necrosis was significantly higher in DIEP-1 when compared with DIEP-3 flaps (19.8% vs. 9.2%, P = 0.049). We describe a DIEP flap classification system and operative techniques to minimize muscle and nerve sacrifice. © 2010 Wiley-Liss, Inc. Microsurgery, 2010.

Four of the nine mutations (9%) that were detected in embB306 indicating resistance to ethambutol were not detected by the DST method, giving the lowest rate of concordance (44.4%) of the PCR with the DST method. One of the greatest concerns of national tuberculosis control programs in several countries

is the emergence and spread of drug resistant and MDR-TB. The actual extent and type of drug-resistant tuberculosis in Jordan is unknown. To determine this, the present study characterized 100 M. tuberculosis strains by PCR that were identified as drug resistant in the reference laboratory for mycobacteria. This is the first investigation involving the molecular characterization of drug resistance of M.

tuberculosis clinical isolates from Jordan. It was initiated as a result of the growing demand for rapid molecular characterization Sirolimus purchase of Mycobacterium PLX3397 nmr strains isolated from patients whose clinical details and history suggested the presence of drug-resistant M. tuberculosis, i.e. previous tuberculosis, recent immigration from or travel to an area with a high prevalence of MDR-TB, failure to respond to therapy, or contact with a known MDR-TB patient (Watterson et al., 1998). In this study, 34 isolates resistant to one or more of the tested drugs were identified. This is comparable to what has been reported in the neighboring countries, with resistance to isoniazid and rifampicin being more common than resistance to ethambutol. The World Health Organization has estimated the prevalence of MDR-TB in several Mediterranean and neighboring fantofarone countries as follows: Bahrain 3.5%, Egypt 5%, Iran 7.1%, Iraq 5.6%, Israel 5.6%, Kuwait 2.4%, Lebanon 2.4%, Oman 1.8%, Qatar 1.1%, Saudi Arabia 3.4%, United Arab Emirates 3.8%, and Yemen 3.2% (WHO, 1997, 2000a, b, 2003). In Jordan, there is very limited documentation of MDR-TB cases. Previous studies reported that over 90% of the M. tuberculosis rifampicin-resistant

clinical isolates harbored mutations in the 81-bp core region of the rpoB516, rpoB526, and rpoB531, the most frequent (70–95%) worldwide (Bártfai et al., 2001; Mokrousov et al., 2003). The discrepancies between the molecular and phenotypic drug resistance reported in this study have been reported by others previously (Baldeviano-Vidalon et al., 2005; Chan et al., 2007; Plinke et al., 2009). These discrepancies are most likely caused by problems with conventional susceptibility testing (Plinke et al., 2009) or by a single base substitution of a silent point mutation. Another possibility is the presence of heterogeneous isolates or mixed populations of resistant and susceptible M. tuberculosis bacilli in the initial sputum specimens with mutant genotypes being recognized by the molecular assay and therefore masking or dominating the susceptible genotypes.

Therefore, the DEC-205 receptor can deliver antigen to DCs for presentation to both CD4+ and CD8+ T cells, and when that is performed in the steady state it leads to deletional tolerance or anergy of the antigen-specific T cells. Targeting steady-state

MG132 immature DCs with antigen-linked anti-DEC-205 antibody, apart from inducing anergy and deletion of cognate T cells [20,35], can also lead to the induction and/or expansion of Tregs[47,82]. Anti-DEC-205/OVA drove short-lived proliferation of OVA-specific CD4+ T cells in vivo and led to the induction of CD25+/CTLA-4+ T cells with regulatory properties which could suppress proliferation and IL-2 production of conventional CD4+ T cells in a dose-dependent manner [82]. This phenomenon was corroborated further in CD4+ and CD8+ T cell-driven hypersensitivity models, where suppression of immune responses could be achieved in vivo by the induction of CD4+CD25+ Tregs by antigen-linked anti-DEC-205. To investigate further whether DCs are able to induce Tregs from truly naive FoxP3- CD4+ T cells, peptide ligands were targeted to DCs through DEC-205 and FoxP3 expression was analysed Selumetinib order at the single-cell level [47]. In this study, which used T cells from Rag2−/− TCR transgenic mice to exclude pre-existing FoxP3+ cells, it was shown that the converted Tregs expressed FoxP3 just as

do natural Tregs. It was also demonstrated that minute antigen doses with suboptimal DC activation were necessary for Treg induction, which was enhanced further by the addition of transforming growth factor (TGF)-β or in the absence of IL-2. Importantly, these FoxP3+ Tregs could be expanded by immunogenic presentation of antigen and also retained their surface phenotype and suppressor activity. Recently, Yamazaki and Steinman reported that CD8+DEC205+ splenic DCs are particularly well equipped to induce FoxP3+ Tregs from FoxP3- precursors [45]. This occurs in the presence Doxorubicin concentration of low doses of

antigen and requires TGF-β expressed by the DEC-205+ DCs themselves. This may explain partially why, in some cases, DC targeting by antigen-linked anti-DEC-205 antibody led to the conversion of conventional CD4+ T cells to CD25+CD4+ Tregs[47,82]. The therapeutic potential of DEC-205-mediated antigen delivery has begun to be explored in mouse models of type 1 diabetes [69,70]. The first such study utilized a CD4+ T cell-driven model in which mice express haemagglutinin (HA) under the control of the rat insulin promoter (INS) and an I-Ed-restricted TCR specific for HA110–120. These mice have a diabetes incidence of 40%. When treated with HA peptide-linked anti-DEC-205 repeatedly from birth until 12–16 weeks of age, diabetes was prevented in most animals.

2A). In this experimental setting, we also observed a significant increase Lumacaftor chemical structure in the expression of the activation marker CD38 on B-cell surface after IFN-β treatment (Supporting Information Fig. 2B). Given that this protein is notoriously type I IFN inducible

[20], this result clearly shows that B lymphocytes are target of the IFN-β therapy confirming previous study by Zula et al. [21] who described a rapid activation of IFN signal transduction pathways in B cells present in unseparated blood from RRMS patients soon after IFN-β injection. In the past, we dissected the regulation of TLR7 in maturing monocyte-derived DCs and observed that its transcription was dependent on the endogenous IFN-β release [22]. Thus, to evaluate whether IFN-β therapy would modulate TLR7 expression in MS patients, we first monitored by real-time RT-PCR TLR7 level of transcription, together with that of TLR9, in MS patients versus HDs. It was of great interest to find that PBMCs obtained from MS patients display a clear defect, as compared with those of HDs, in TLR7 expression that was statistically significant (25 HDs and 45 MS patients analyzed) (Fig. 2A). This difference was not observed in the transcription

of TLR9 gene (Fig. 2B), demonstrating that in MS patients, the defective TLR7 expression is specific. Furthermore, we observed that in PBMCs isolated from the same MS patients HSP cancer following 1 month of IFN-β therapy, the level of TLR7 mRNA was restored to the level observed in HDs, while that of TLR9 was not modulated (Fig. 2A and B). In the attempt to investigate which TLR7-expressing cell types in the peripheral blood might be responsible for this defect in MS patients, B cells and monocytes were purified from both HDs and MS patients at baseline and 1 month after the beginning of IFN-β therapy, since these two leukocyte populations express TLR7. Data on TLR7 expression in B cells isolated from HDs or MS (7 and 13 individuals, respectively) did not mirror the impairment observed in the context of the

mixed cell population of PBMCs (Fig. 2C and D), although a slightly enhanced level of TLR7 transcription in response to IFN-β http://www.selleck.co.jp/products/sorafenib.html occurred also in this experimental setting. As observed in unseparated PBMCs, TLR9 levels of B cells did not differ in HDs and MS patients irrespective of IFN-β treatment. Interestingly, when the expression of TLR7 was analyzed in monocytes of MS patients (13 individuals), a different picture appeared. Indeed, a lower TLR7 mRNA level was highlighted in monocytes from MS patients than that obtained from HD (8 individuals) and, moreover, also a robust induction was observed in response to IFN-β therapy (longitudinal analysis of 5 patients at baseline and 1 month after IFN-β treatment) (Fig. 2E). TLR9 expression was absent in monocytes (data not shown). These data for the first time indicated a defect in TLR7 signaling in monocytes of MS patients.

Recently, it was shown that APRIL (a-proliferation-inducing ligand) triggers the differentiation of IgM+ B cells into low-affinity IgA plasma cells within the LP in response to Toll-like receptor (TLR) stimulation of epithelial cells [7]. B cell activating factor (BAFF) belonging to the tumour necrosis factor (TNF) family was also shown to sustain the differentiation of IgM+ CD27+ marginal zone B cells into IgA plasma cells, independently of CD40 [7], in the subepithelial regions of the mucosa. In contrast, the T-dependent production of high-affinity IgA occurs in the germinal centres (GC) of the Peyer’s patches and requires CD40–CD40L

interactions [8]. During a T-dependent response, CSR is promoted by CD40–CD40L interactions

and modulated by various cytokines that target specific CH genes prior Smoothened Agonist in vitro to germline transcription [9]. A panel of cytokines, including TGF-β, interleukin (IL)-10 and others can skew CSR towards IgA. CD40L, BAFF and APRIL trigger the activation of both nuclear factor (NF)-κB1 and NF-κB2 [10]; however, only the NF-κB1 pathway leads to NF-κB p65 activation. The NF-κB subunits (p50, p52, p65, c-Rel, RelA and RelB) function as dimers and have been shown to be both differentially activated [11,12] and also to possess distinct target DNA binding site specificities [13,14] that depend upon dimer composition. The CD40/CD40L interaction activates and phosphorylates the latent cytoplasmic NF-κB/IκB complex. This process is followed by IκB proteolysis and the translocation Selleck BGB324 of NF-κBp50 or p65 into the nucleus, where these NF-κB subunits up-regulate

gene expression by binding κB site-containing gene promoters [15]. NF-κB1 may also affect other independent pathways upon activation of TNF receptor-associated factors, such as Janus kinases (JAK) and signal transducers and activators of transcription (STAT) PI-1840 [16]. Complex interactions exist between NF-κB subunits and STAT3 that can differently modulate B cell responses to pathogens. Phosphorylated p65 dimer can bind to non-phosphorylated STAT3 and this complex can then bind to κB sites, but not on γ-activated sites (GAS–STAT component) [17]. Alternatively, the phosphorylated form of STAT3 can interact with the phosphorylated NF-κB p50. This complex enhances the transcription of GAS-dependent genes [18]. Moreover, phosphorylated STAT3 can form a complex with a non-phosphorylated NF-κB dimer and bind to κB sites [19]. The recruitment and activation of STAT3 can also induce downstream expression of numerous cytokine receptors, including IL-10 receptor (IL-10R). IL-10 participates in many biological responses, including cell proliferation, survival, apoptosis and differentiation [20,21], and is an important factor in the regulation of Ig production.

5C). Taken together, these results indicate that RAR-α mediates the regulation of cytokine production by RA. Next, to determine if RA directly affects NKT cells, the CD1d-expressing NKT-cell line DN32.D3 was stimulated with Con A or α-GalCer in the presence of RA (Supporting Information Fig. 5A). As shown in Fig. 5D and E, the secretion of IFN-γ and IL-4 but not TNF-α was reduced by RA. The mRNA expression was consistent with the quantitation data of the secreted cytokines (Supporting Information Fig. 5B). Because TNF-α production, which is regulated by NFAT, was not reduced by RA, we examined the changes in other signaling

molecules that are activated upon TCR stimulation. learn more As a result,

the phosphorylation of MAPK, especially JNK, was reduced by RA (Fig. 5F). We measured the amount of IκB, as an indicator of NFκB signaling, by western blot, and it was not influenced by RA. Therefore, these data suggest that RA regulates cytokine production in NKT cells directly, the mechanism of which might include a modification of MAPK signaling pathway. In the current study, we demonstrated, for the first SCH772984 in vitro time, how RA regulates NKT cell-mediated diseases and NKT-cell responses in vivo. We showed that RA ameliorated Con A-induced hepatitis but not α-GalCer-induced hepatitis. This distinct role of RA can be explained by the finding that RA differentially regulated the secretion of various pathogenic cytokines from NKT cells, with unaltered NKT-cell activation. Mechanistically, our observations indicate that RA affects NKT cells directly by modulating signaling molecules Progesterone such as RAR-α and MAPK. We first attempted to examine the influence of endogenous RA using vitamin A-deficient mice; however, the results did not correlate with the data obtained from RA-pretreated

animals (unpublished observation). We found that RA deficiency affected the activation status of cells in naïve mice by an unknown mechanism. RA signaling is biphasic and has the potential to display opposite effects in various models [20-25]. These controversial findings have not been explained completely, and our observations and future studies may explain this discordance. In this study, to minimize the effect of vitamin A-deficiency on NKT cells, disulfiram was used to pretreat the animals for 3 days to reduce the amount of endogenous RA. Aggravated liver injury was observed in disulfiram-treated mice, demonstrating the regulatory role of endogenous RA in Con A-induced hepatitis (Fig. 1D and E). Disulfiram can induce liver injury by hitherto unknown mechanisms when it is administered to treat alcohol abuse [31, 32]. Our observations suggest that a defect in RA synthesis via disulfiram treatment might cause the liver to become susceptible to inflammation and increase liver injury in patients.

The BCA protein assay (Thermo Fisher) was used to

determine the protein concentration of each of the cleared lysates. A 30 μg sample of each caecum or colon lysate protein was boiled for 5 min in reducing sample buffer containing DTT and resolved by SDS–PAGE, transferred to PVDF membranes and probed with the indicated antibodies. The membranes were exposed to enhanced chemifluorescence substrate (GE Healthcare, Piscataway, NJ), followed by scanning on a Typhoon Trio+ imaging system (GE Healthcare) to obtain a digital image of the probed protein. The bands were then quantified with ImageQuant software selleck chemical (GE Healthcare). Caecum and colon snips obtained from untreated and C. difficile-infected mice were homogenized with a rotor/stator-type homogenizer while immersed in TRIzol RNA reagent (Life Technologies, Grand Island, NY). The TRIzol RNA reagent and the RNeasy Mini kit (Qiagen, Valencia, CA) were used in successive steps to isolate RNA from the caecum and colon samples, each according to its manufacturer’s instructions. An Agilent Bioanalyser (Agilent Technologies, Palo Alto, CA) and a Nanodrop instrument (Thermo Fisher) were used to determine RG-7388 ic50 the quality and concentration of each RNA isolate, respectively.

Complementary DNA (cDNA) was generated from each RNA sample using the RT2 First Strand kit (Qiagen). Expression levels of the genes under study were determined by using two different sets of mouse RT2 Profiler PCR cards (Qiagen), each custom-made to contain eight replicate sets of

48 primer pairs (Table 1). Each well of the replicate sets was loaded with 5 ng of cDNA reaction product. Each card was run on a LightCycler 480 real-time PCR system (Roche). The relative RNA expression levels were inferred from the Ct values. Xbp1 splicing was assessed as previously described.[39] Briefly, the Superscript III RT-PCR kit (Life Technologies) was used to amplify both unspliced and spliced Xbp1 in RNA samples obtained at the end of the experimental period. The primers used in the assay flanked the Xbp1 intron and had the following sequences: upstream: ttgtggttgagaaccagg; downstream: tccatgggaagatgttctgg. Quantitative RT-PCR, including methods for verifying primer efficiency and specificity, were performed as previously described.[40] The Ct value for each gene Dynein of each sample was normalized against the geometric mean of the Gapdh and Hprt for that sample.[41] For the following assays, differences between untreated and C. difficile-infected mice were evaluated for significance by using paired t-tests at P ≤ 0.05: diversity of the bacterial community examined by pyrosequencing; cell numbers obtained by analysing the flow cytometric data; mRNA expression for the UPR genes Gadd34 and Wars obtained by single gene quantitative RT-PCR; and protein expression or phosphorylation assessed by immunoblotting.