Therefore, syk−/− DT40 B-cell mutants were reconstituted with a OneStrep-tagged version of human Syk and left untreated or stimulated through their BCR for six different time points. Cellular lysates were incubated with a streptactin affinity column and obtained proteins were size-separated by 1-D PAGE. Following in-gel digestion of Syk with endoproteinase trypsin, resulting see more phosphopeptide products were enriched by TiO2 microcolumns and identified by liquid chromatography (LC)-coupled tandem mass spectrometry (MS/MS) on an orbitrap mass

spectrometer. As shown in Fig. 1, we detected a total of 32 phosphoacceptor sites, 15 of which were on tyrosine, 11 on serine and six on threonine (see Supporting Information data 1 for annotated MS/MS spectra). Our analysis confirmed

all previously published phosphorylation GSK1120212 supplier events and revealed 19 novel acceptor sites. Notably, almost half of the Syk phosphosites mapped to interdomain B (see Fig. 1) previously implicated in the control of Syk functions 2, 3. Our data show that Syk is extensively modified by phosphorylation on a large number of acceptor sites, which might act individually or in concert to regulate Syk function. To monitor the phosphorylation kinetics of individual acceptor sites we used a quantitative SILAC-based mass-spectrometric approach 29–31. DT40 cells expressing OneStrep-tagged Syk were metabolically labeled in SILAC medium containing arginine and lysine residues with incorporated light or heavy isotopes of carbon and nitrogen.

Different combinations yielded three types of SILAC media. Using 12C6,14N2-Lys and 12C6,14N4-Arg resulted in “light medium” while the combination of 13C6,15N2-Lys and 13C6,15N4-Arg Sitaxentan yielded “heavy medium”. “Intermediate medium” was obtained by using 2D4,12C6,14N2-Lys and 13C6,14N4-Arg. Cells cultured in “light medium” were left untreated and those cultured in “intermediate” or “heavy medium” were BCR-stimulated for different time points. This setup had important consequences. Proteins or peptides derived from the differentially labeled cells can be distinguished in the mass spectrometer by virtue of their distinct absolute molecular masses and hence can unambiguously be assigned to one of the three stimulation conditions. Proteins were purified from the three cell cultures via streptactin affinity chromatography, pooled at a 1:1:1 ratio and separated by 1-D PAGE. The gel slice containing the three pools of Syk was excised and subjected to trypsin digestion. TiO2-enriched phosphopeptides were analyzed by LC-MS/MS and individually quantified using MaxQuant software 32. This strategy allowed an unbiased relative quantification of Syk phosphorylation in resting and stimulated cells. Altogether, we monitored the phosphorylation kinetics of 16 phosphosites, which we grouped into three categories (Fig. 2).

These findings suggest the following pathophysiological process: the astrocytes are affected at an early phase in NMO, CA are expelled from the astrocytes and phagocytized by macrophages finally leading to clearance. A phagocytized figure and subsequent loss of CA can be a histological hallmark of astrocytic injury of NMO. “
“K. R. Sherwood, M. W. Head, R. Walker, C. Smith, J. W. Ironside and J. K. Fazakerley (2011) Neuropathology Atezolizumab order and Applied Neurobiology37, 633–642 RNA integrity in post mortem human variant Creutzfeldt–Jakob disease (vCJD) and control brain tissue Aims: To determine premortem

and post mortem factors affecting quality and yield of RNA isolated from the unique archived brain material in the UK National Creutzfeldt–Jakob Disease Surveillance find more Unit Brain and Tissue Bank and to compare this to control brain tissue with no neurological disease. Methods: In parallel and in replicate, RNA was prepared from the frontal parasagittal or subfrontal cortex of samples dissected from half brains (frozen intact) or from brain samples snap frozen or placed in RNALater. A total of 350 RNA samples from 78 human autopsy cases, 21 variant Creutzfeldt–Jakob disease, 26 other neurological diseases and 31 non-neurological diseases were studied. Results: There was no difference in the quality or yield of RNA isolated from variant

Creutzfeldt–Jakob disease, other neurological disease and non-neurological disease brains. RNA preparations from archived frozen half brains or snap frozen autopsy samples were generally of poor quality (RNA integrity number < 5). There was a highly significant negative correlation

between the number of times frozen half brains had been sampled and the quality of RNA. Samples stored in RNALater provided higher-quality RNA (RNA integrity number > 5). Age at death, gender, post mortem interval and freezer storage time had no effect on RNA quality. Conclusion: Reasonable-quality Fludarabine datasheet RNA can be isolated from samples dissected from archived frozen human half brains but repeated sampling results in RNA degradation. Better-quality RNA is obtained from samples placed in RNALater than from snap frozen samples. The quality and yield of RNA are not affected by age at death, gender, post mortem interval of >6 h or freezer storage time. “
“K.-Y. Ryu, N. Fujiki, M. Kazantzis, J. C. Garza, D. M. Bouley, A. Stahl, X.-Y. Lu, S. Nishino and R. R. Kopito (2010) Neuropathology and Applied Neurobiology36, 285–299 Loss of polyubiquitin gene Ubb leads to metabolic and sleep abnormalities in mice Aims: Ubiquitin performs essential roles in a myriad of signalling pathways required for cellular function and survival. Recently, we reported that disruption of the stress-inducible ubiquitin-encoding gene Ubb reduces ubiquitin content in the hypothalamus and leads to adult-onset obesity coupled with a loss of arcuate nucleus neurones and disrupted energy homeostasis in mice.

Junctional ectopic tachycardia may be a spectrum of injury to the AV node in which

partial injury may be associated with increased automaticity and more complete injury with AVB. Although to date there has been no report documenting an association between congenital find more junctional ectopic tachycardia in the absence of AVB and maternal autoantibodies, given the lack of symptoms among otherwise healthy women with infants who have complete AVB and/or maternal autoimmune-mediated cardiomyopathy, prospective serological evaluation of the mothers of affected infants should be considered. A spectrum of structural heart disease has been reported among foetuses and infants with maternal autoimmune-mediated cardiovascular disease. In children with maternal autoimmune-mediated Vemurafenib mouse congenital AVB, structural congenital heart disease has been reported in 16–42% [22, 38]. These lesions have included persistent ductus arteriosus most of which have required intervention, and atrial and ventricular septal defects. Of greater interest, semilunar and atrioventricular valve abnormalities have also been described in association with AVB, including

stenosis, regurgitation and dysplasia without functional changes (Fig. 4) [22, 38, 54]. Inflammation and fibrosis as well as haemodynamic changes could potentially contribute to the evolution of at least some of these lesions, as suggested in one case of acute chordal rupture with moderate mitral insufficiency in 7-week old infant with echocardiographic evidence of EFE involving left ventricular papillary muscles and chordae [54]. The incidence of structural and even functional heart disease among infants of mothers

with autoantibodies in the absence of AVB is still not certain. In one study that assessed structural abnormalities in a series of 165 pregnancies with autoimmune disease and anti-Ro antibodies, four offspring had structural heart disease suggesting a potential incidence of 2.8%, which represents an increase over that of the general FER population [51]. Maternal autoimmune-mediated pathology could be aetiological in the evolution of other forms of congenital heart disease, as suggested in a recent case of prenatally diagnosed hypoplastic left heart syndrome [55]. Further prospective longitudinal investigations of pregnancies (and offspring) in women with anti-Ro and anti-La antibodies with and without autoimmune disease are necessary at this time to determine the true incidence of congenital structural, functional and rhythm-related cardiovascular disease associated with maternal autoantibodies.

The temperature programme was a 5-min denaturing step at 94 °C, 35 amplification cycles (94 °C for 30 s, 58 °C for 30 s, and 72 °C for 30 s), and a final extension step of 72 °C for 10 min. After amplification, 5-μL samples of the PCR products were separated on a 1.5% agarose

gel and stained with ethidium bromide. Images were recorded and analysed using an EDAS 290 system (Kodak, NY), with band density measurements expressed in pixels. The integrated density value (IDV) was determined based on the number BIBW2992 of registered pixels minus background: IDV=Σ(each pixel value minus background). The IDV of each band expressed in nanograms was obtained by comparison with the 300-bp band (equivalent to 80 ng μL−1) of the GeneRuler molecular weight marker (Fermentas Life Sciences, MD). To compare the values obtained from the different study groups with the basal values, a one-sample t-test was performed using the statistica 8 (2007) software for Windows. P<0.05 was considered significant. Fragments of tissue from one mouse of each group (NI-MG, ISSI-MG, CI-MG, and NbI-MG) were obtained and fixed in phosphate-buffered saline with 10% formaldehyde Ensartinib for 24 h. They were then washed in Tris-HCl buffer (0.1 M, pH 7.2), longitudinally cut, and decalcified in a 10% EDTA aqueous solution for 15 days. The tissue was embedded in paraffin, and five sections of 5 μm were hydrated and antigenically reactivated in a citrate buffer (0.01 M citric acid,

0.01 M sodium citrate) according to the method of Pérez-Torres et al. (2009). Endogenous peroxidase was blocked with aqueous 3% H2O2. Nonspecific antigenic

sites were blocked with 4% bovine serum albumin, fraction V, dissolved in Tris-HCl and 0.01% Triton X-100 for 20 min at room temperature. The blocking solution was decanted, and the primary antibody for TLR2 or TLR4 was added (rabbit and goat polyclonal anti-mouse TLR2 and TLR4 antibodies, respectively; Santa Cruz Biotechnology, CA) in a 1 : 50 dilution in Tris-HCl. After an overnight incubation at 4 °C, the secondary antibody (anti-rabbit for TLR2 (Match 4 Kit, Biocare Medical Co. CA) or anti-goat click here for TLR4 (Goat HRP-Polymer Kit, Biocare Medical Co.) was added, and the tissue was incubated for 60 min in a humid chamber at room temperature. The horseradish peroxidase-coupled complementary polymer (MHR2P for Match4 and Goat HRP-Polymer for Goat Kit, Biocare Medical Co.) for the secondary antibody was added and incubated at room temperature for 30 min. Colour development was assessed after incubation for 5 min with diaminobenzidine (DAB500 Chromogen System, Biocare Medical Co.) at room temperature. Specimens were counterstained with Mayer’s haematoxylin. Finally, the tissue was dehydrated and mounted with resin (Ecomount Mounting Medium, Biocare Medical Co.) for analysis under a light microscope. Negative staining controls were run in parallel for all mouse groups without anti-TLR2 and anti-TLR4 antibodies.

The written consent was obtained from the majority of participants of the study (Innsbruck cohort). Due to the retrospective nature of our study it was not possible to obtain written consent from every patient. The need for patient consent was therefore waived for these cases by the institutional ethic committee.

Frozen tumor tissue material of the Innsbruck patient cohort was collected during resection Acalabrutinib solubility dmso of primary breast carcinoma lesions at the Department of Obstetrics and Gynecology of Innsbruck Medical University in the years 1989–2003. Publicly available microarray expression and clinical data were obtained from the Cancer Genome Atlas (TCGA, data-freeze February 27, 2012) [39] or Gene Expression Omnibus, repository (GEO, studies GSE1456 and GSE3494). Characteristics of Innsbruck and TCGA patient collectives is provided in Supporting Information Table 1, features of GSE1456 and GSE3494 cohorts were published elsewhere [40]. All animals experiments were performed in accordance with the Austrian animal welfare law and animal experiment act (BGBl. I Nr. 114/2012). The experimental protocols were approved by the Committee of Animal Care of the Austrian Federal Ministry find more of Science and Research (BMWF-66.011/0186-II/3b/2011 and BMWF-66.011/0068-II/3b/2013). FVB/N-Tg(MMTVneu)202Mul/J CD45.1+ CD45.2− (MMTVneu Stat1+/+ CD45.1+ CD45.2−) transgenic

mice were purchased from Jackson Laboratory (Bar Harbor, ME). C57BL/6 Stat1−/− CD45.1− CD45.2+ animals (provided by Dr. Thomas Decker) were backcrossed to obtain MMTVneu Stat1−/− and MMTVneu CD45.1+ CD45.2+ mouse strains [4]. Backcrossing was accomplished for five generations using MAX/BAX system (Charles River, Sulzfeld, Germany) and

additionally for two generations without marker assistance to achieve 99.5% FVBN/J genetic background. Genotyping for the neu Tg and Stat1 KO was performed as described in [4], the CD45.1 and CD45.2 status was examined by flow cytometry of tail vein blood leukocytes. Development of mammary tumors was investigated by weekly palpation. All animal experiments were performed with mice bearing age-matched tumors. Tumors, spleens, and lungs were excised, cut into small pieces, and digested at 37°C with Liberase TM (0.15 U/mL; Roche, Mannheim, Germany) and DNaseI Epothilone B (EPO906, Patupilone) (10 μg/mL, Sigma-Aldrich, St. Louis, MO) in RPMI 1640 (PAA, Pasching, Austria) medium. BM cells were flushed with 0.5% FCS 2 mM EDTA in PBS from femora and tibiae. The cell suspensions and blood were subjected to the RBC lysis in ACK buffer (Ammonium chloride potassium buffer, 0.15 M NH4Cl, 10 mM KHCO3, 0.1 mM Na2EDTA) and strained through 70 μm cell strainers (BD Bioscience, Franklin Lakes, NJ) prior to their use for flow cytometry, sorting, or culture. Extracellular staining of single-cell suspensions was described elsewhere [41]. In all extracellular stainings, viable cells are defined as 7AAD− or DAPI−.

Family meetings are usually a good way to interact with the indigenous patient and the family. Effective communication skills XAV 939 are needed to have effective discussions. Here the clinician needs to actively listen and give time for replies and questions. Patients and families should not feel unduly pressured to choose or embark on a particular pathway of care. It can be helpful to let the caregivers know that this is a medical recommendation and that the physician is, with their assent, primarily

responsible for the decisions. Above all it should be a shared decision making process with the patient’s best interest the primary consideration at all times. It is important to discuss cultural requirements and preferences early in the conservative management Y-27632 ic50 pathway so that the impact of family and kinship relatives can be managed. Family/kinship rules may mean that certain family members of an indigenous person, who in mainstream society would be regarded as distant relatives, may have strong cultural responsibilities to that person. It is imperative therefore to identify early in the planning stages

who is the culturally appropriate person, or persons to be involved in the decision making process so that they can give consent for treatment and discuss goals of care. Where English is not the main language of the person and/or their

family, interactions and family meetings will always need to be held in the presence of a cultural broker (aboriginal liaison officer) and or an interpreter to explain treatment pathways and care issues so that informed choices are made. Informed choices can be only made TCL in an environment where all stakeholders can participate freely. An interpreter or translator can be an invaluable resource in such situations to ensure that information is conveyed and received accurately. The use of a family member as an interpreter may not always be appropriate and the health care team should be sensitive to these issues. Given the remoteness and accessibility issues in the life of indigenous Australians, it may be sometimes difficult to bring the patients to the ‘tertiary services’. In many instances, ‘services’ may have to be taken to the patient. One effective way of doing this is by tele or video case-conferencing with the local clinic, DMO/primary GP, patient and family as well as the renal team in attendance. The range of environmental and social conditions in the remote setting may also necessitate flexible models of care and creative solutions to sourcing equipment and medications etc. Patients in the ‘remote setting’ who have chosen the non-dialysis pathway will have to be supported and cared for at home.

A master transcriptional regulator of human Th9 cells still awaits identification, and even FoxP3, which delineates murine Treg cells, is not exclusively specific for human Treg cells, since it can be upregulated upon polyclonal TCR activation alone [15]. Epigenetics determines the cell-type-specific status of the chromatin landscape. Epigenetic modifications, Selleckchem EPZ6438 especially histone modifications and DNA methylation, have been shown to regulate gene accessibility and thus help establish gene expression programs. Inclusion of epigenetics in defining Th subsets allows for better specification

of these subsets, and in particular, offers an approximation of their degree of flexibility [16, 17]. Nevertheless, recently a new concept emerged

for the specification of Th-cell identity which takes regulatory elements of the genome into consideration. Enhancers are extragenic DNA sequences that mediate the combinatorial recruitment of transcription factors to “enhance” transcription of cognate target genes [18]. They are the accessible part of a cell’s genome and are hypersensitive to digestion by DNaseI. New technologies such as genome-wide microarrays and high-throughput sequencing have contributed to establish enhancer landscapes for certain Th-cell subsets (reviewed in [19]). BMN 673 ic50 Interestingly, Protein kinase N1 several independent studies demonstrated that these enhancer landscapes determine Th-cell identity irrespective of the putative master transcriptional regulators because the enhancer landscapes of Th1, Th17, and Treg cells were not affected following the deletion of Tbet, ROR-γt, and FoxP3, respectively [20-22]. TCR-dependent signals have been shown to generate the initial phase of the enhancer landscape, which is then followed by modification of cytokine signaling in a STAT-dependent manner. For example,

many differentially active enhancers in Th1 and Th2 and Th17 cells have been shown to be STAT4, STAT6, or STAT3 dependent, respectively [20-22]. Master transcriptional regulators therefore rather seem to fine-tune Th-cell functions, while the enhancer landscape sets the tone in response to environmental signals such as microbe-elicited cytokine milieus. The expression of certain chemokine receptors has significantly contributed to the categorization of Th-cell subsets in humans [23]. The circulating immunological T-cell memory compartment is generally divided into effector memory (TEM) and central memory (TCM) subsets. TEM cells circulate to nonlymphoid tissues whereas TCM cells home to secondary lymphoid organs.

Cells were stained with TMRE (Sigma-Aldrich, St Louis, MO, USA) in PBS to a final concentration of 125 nM, and incubated for 30 min at 37°C with 5% CO2 to assess mitochondrial membrane potential (ΔΨm). Total mitochondrial mass and membrane potential were also determined using mitotracker green and red dyes (Invitrogen), respectively, according to manufacturers’ instructions. For in vitro culture experiments, CD8+ T cells were purified >90% by magnetic-activated cell sorting (MACS) using anti-CD8α microbeads PD0332991 clinical trial and LS columns (Miltenyi Biotec, Bergisch Gladbach, Germany) following manufacturer’s instructions. For microarray and Western blot analysis,

CD8+ T cells were purified >98% using the Easysep PE selection kit (StemCell Technologies) using PE-CD8α (eBioscience). Primary naïve CD8+ T cells were cultured in 24-well plates at 1×106 cells/mL at 37°C with 5% CO2 in RPMI-1640 medium (Sigma-Aldrich) supplemented with glutamine, 2-mercaptoethanol and antibiotics (all Sigma-Aldrich). Where used, IL-7 (Peprotech, Rocky Hill, NJ, USA) was supplemented at 50 ng/mL. CD8+ T cells were sorted, check details and total RNA was prepared using the RNEasy mini kit (Qiagen). RNA was quality and quantity controlled for degradation on a BioAnalyzer

2100 (Agilent). Since the starting quantity of RNA for each sample did not exceed  μg, two cycle amplification was performed, as recommended by the manufacturer (Affymetrix). The GC-RMA (Robust Multiarray Analysis) algorithm was applied to the probe level data (CEL files). Quality control and data processing was performed Teicoplanin at the Bloomsbury Centre for Bioinformatics, University College London, using the limma,

gcrma, simpleaffy, annotate, annaffy and affycoretools R packages in Bioconductor. Multiple testing correction was applied for the data using the Benjamini and Hochberg False Discovery Rate. Annotation of each probe set was derived using the NetAffx site (Affymetrix). Microarray data were deposited in ArrayExpress (accession number E-TABM-991). Cell pellets containing 1×106 cells were lysed at 4°C in 1 mL 1% NP40 lysis buffer. Protein lysates were run on 12% SDS-PAGE acrylamide gels and protein content analysed on nitrocellulose membrane with the following antibodies: Mcl1 (Rockland), Bcl2, BclXL, Bok, Bax, total Bad, Bim, Bid, Bak, Puma, pBad (Ser112) (all from Cell Signaling Technology), and Actin (Santa Cruz). Densitometry calculations of proteins were calculated in the ImageJ v1.43 (NIH, Public Domain). The authors thank Biological Services for animal husbandry and technical support, Hugh Brady for providing Bad transgenic mice. This work was supported by the Medical Research Council UK under programme code U117573801. Conflict of interest: The authors declare no financial or commercial conflict of interest. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset.

Future studies to investigate LPS-induced CGRP synthesis in monocytes/macrophages of RAMP1 over-expressing

transgenic mice20 and knockout mice37 should verify this hypothesis. In the present study, we have used exogenous CGRP, peptide CGRP receptor antagonist CGRP8-37 and non-peptide CGRP receptor antagonist BIBN4096BS, HSP inhibitor clinical trial to establish the possible role of CGRP receptor signalling in basal and LPS-induced pro-inflammatory and anti-inflammatory chemokines and cytokines in the RAW 264.7 macrophage cell line. The affinities of αCGRP, CGRP8-37 and BIBN4096BS to bind human CGRP receptors have been well established, with the affinities BIBN4096BS (Ki = 14·4 ± 6·3 pm) > αCGRP (Ki = 31·7 ± 1·6 pm) > CGRP8-37 (Ki = 3·6 ± 0·7 nm), respectively.25 Hence, the physiological concentrations for MG132 both CGRP and BIBN4096BS are within nm range25 whereas for CGRP8-37, it is within the μm range.38 We used the physiological range of concentrations of the antagonists in the current study. The mechanisms underlying the blocking activities of both antagonists on CGRP receptors are rather different. Since CGRP8-37 peptide includes all but the first seven amino acids at the C-terminal

of CGRP, it works as a competitive antagonist to block the binding of full-length CGRP to its receptor. In contrast, the specific affinities of BIBN4096BS depend on its interaction with the RAMP1 subunit of CGRP receptor.39 From the literature, the role of CGRP in the induction of pro-inflammatory and anti-inflammatory chemokines and cytokines is controversial.21–23 In these studies, depending on the cell type and concentration, CGRP exhibits either stimulating or suppressing effect on the production of MCP-1, IL-1β, TNFα, IL-6 and IL-10. Consistently, CGRP receptor signalling in the current study also demonstrates positive or negative effects on basal and LPS-induced release of these inflammatory mediators depending on the concentration of CGRP and CGRP receptor antagonists. Generally speaking, a lower concentration of CGRP seems to facilitate the basal Bcl-w release of MCP-1, TNFα and IL-6 but had no effect on the basal release of IL-1β and IL-10. The facilitating effects were

blocked by a lower concentration of CGRP8-37 (10 nm), suggesting that CGRP receptor mediates the effect. In contrast, a higher concentration of CGRP suppressed basal TNFα release but had no effect on others. Contrary to the effect of CGRP, a higher concentration of the peptide antagonist CGRP8-37 significantly increased the basal release of all chemokines and cytokines examined, but the lower concentration had no effect at all. Non-peptide antagonist BIBN4096BS also manifested the same tendency. However, at higher concentration, it only significantly increased the basal release of MCP-1, IL-6 and IL-10 but had no effect on IL-1β and TNFα. Similar to CGRP8-37, a lower concentration of BIBN4096BS had no effect on the basal release of chemokines and cytokines.

5B). These results were in line with immunohistochemical data showing PD-0332991 mouse that a higher percentage of CD4+ lymphocytes than neutrophils were positive for IL-17 (Fig. 4). Importantly, the IL-17 we detected on cells

could have originated from endogenous or exogenous factors and bound by IL-17 receptors on cell surfaces [21, 22]. To determine if these leukocytes were actively expressing IL-17, the cells were subjected to fixation and permeabilization. The fluorescence intensity of IL-17 staining increased slightly, but with statistical significance, in both CD4+ T cells and Ly-6G+ cells (Fig. 5B and Supporting Information Fig. 5). These resulted indicated that infiltrated lymphocytes and neutrophils express IL-17. Since fungal growth and leukocyte infiltration coordinately contribute to corneal destruction, PD0325901 cost we wondered whether either of these processes was occurring in inoculated nude mice. In inoculate BALB/c mice, pseudohyphae were detected as early

as 6 h postinoculation and abundant by 12 and 24 h postinoculation (Fig. 6A). In striking contrast, few pseudohyphae were detected at these time points in nude mice. Similarly, leukocyte infiltration was already obvious in the corneas of BALB/c mice at 6 h, but few leukocytes were present in nude mice throughout the observation period (Fig. 6A and B). Colony-forming assay showed that the pathogen burdens gradually increased in immunocompetent mice, but decreased in nude mice soon after inoculation (Fig. 6C). Together, these results suggest that nude mice have an innate mechanism that inhibits Candida blastospore transformation

and leukocyte infiltration. In Resveratrol support of the latter, real-time polymerase chain reaction (RT-PCR) assay demonstrated that the expression of chemokines (e.g. CXCL12, CXCL10, CXCL2, CXCL1, and CCL2) including the IL-17 inducer IL-6 was upregulated during the first day of inoculation in BALB/c and nude mice, but their levels were significantly lower in nude mice (Fig. 6D). To determine whether the decreased production of chemokines in nude mice corneas was an intrinsic property of resident corneal cells rather than systemic immune components, cornea buttons were removed following inoculation and placed in overnight culture in vitro. Like the findings above, corneal buttons of nude mice showed decreased chemokine production compared with those of BALB/c mice (Fig. 6E). Corresponding to the fact that IL-17-neutralized mice became insensitive to CaK induction, the inoculated corneas of anti-IL-17-treated mice had reduced production of above chemokines compared with isotype control antibody-treated mice (Supporting Information Fig. 6). Our results indicated that reduced chemokine production is correlated with CaK resistance in nude mice.