Representative images were taken for each condition, using the same exposure time for each filter, to allow comparison of fluorescence intensity between different fields and conditions. Primary cultures of cortical neurons were obtained from C57BL/6 mice, at day 16 of gestation. After dissociation

and centrifugation of the dissected cortices, the tissue was resuspended in Neurobasal medium (Invitrogen, San Diego, CA), supplemented with 2% (v/v) B27 supplement (Invitrogen) and 100 U/ml penicillin/streptomycin (Invitrogen). Cells were plated at a density of 500 000 cells/well in six-well multi-well plates previously coated with poly-l-lysine. Characterization of the embryonic neuronal cultures confirmed the presence of 95% neurons, as determined by GFAP and NeuN-immunostaining. Primary cultures were kept at 37° in a humidified atmosphere Gefitinib mouse containing 5% CO2. After 8 days in culture, neurons were incubated for 24 hr with a 1 : 1 mixture of Neurobasal medium (500 μl) and conditioned medium (500 μl). The conditioned medium was obtained from untreated N9 cells, N9 cells exposed to LPS PKC412 chemical structure (0·1 μg/ml) for 24 hr, and N9 cells transfected with anti-miR-155 or control oligonucleotides 24 hr before exposure to LPS. In parallel experiments neurons were incubated

with LPS (0·1 μg/ml) for 24 hr. Cell viability of primary neuronal cultures was determined by a modified Alamar Blue assay. This assay measures the redox capacity of neurons and allows the determination of cell viability without the detachment

of the cells, so cell integrity is maintained. Briefly, 1 ml Neurobasal medium supplemented with 10% (v/v) of Alamar Blue dye was added to each well following the 24-hr incubation period with conditioned medium or LPS. After 3 hr of incubation at 37°, 150 μl supernatant was collected from each well and transferred to 96-well plates. The optical density of the supernatant was aminophylline measured at 570 and 600 nm in a microplate reader and cell viability was calculated as a percentage of control cells, using the formula: (A570–A600) of treated cells × 100/(A570–A600) of control cells. All data are presented as mean ± standard deviation (SD) and are the result of three independent experiments, each performed at least in triplicate. One-way analysis of variance combined with Tukey post-hoc test was used for multiple comparisons in cell culture experiments. Statistical differences are presented at probability levels of P < 0·05 (*), P < 0·01 (**) and P < 0·001 (***). Calculations were performed with standard statistical software (GraphPad Prism 5, GraphPad Software, La Jolla, USA). Since miR-155 has been described as being up-regulated in various cells of myeloid origin upon their activation and as contributing to the modulation of the immune response mediated by these cells, we first investigated the expression of this miRNA in mouse N9 microglia cells and primary microglia cultures employing qRT-PCR.

4–8 Therefore, it is thought that FFA might play learn more a role in the pathogenesis of the tubulointerstitial damage in various kidney diseases Free fatty acids loaded into the human proximal tubules are bound to the 14 kDa renal liver-type fatty acid binding protein (L-FABP) and transported to mitochondria or peroxisomes, where they are metabolized by β-oxidization.9 Expression of the L-FABP gene is induced by FFA, and

this protein may regulate the metabolism of FFA and be a key regulator of FFA homeostasis in the cytoplasm.10 Moreover, L-FABP has a high affinity and capacity to bind long-chain fatty acid oxidation products, and may be an effective endogenous antioxidant.11 However, until now, renal L-FABP had not been investigated under pathological conditions of the kidney. Recent development of the method for measuring urinary human L-FABP (hL-FABP), using

a two-step sandwich enzyme linked immunosorbent assay (ELISA) procedure (CMIC, Tokyo, Japan),12 and the establishment of a transgenic (Tg) mouse model harbouring the hL-FABP chromosomal gene have enabled deeper insights into this protein.13 This review is mainly focused on the pathophysiological roles and dynamics of hL-FABP as revealed by Tg animal models of kidney disease. Deterioration of kidney disease is determined to a large extent by the degree of tubulointerstitial buy Y-27632 changes rather than by the extent of histological changes in the glomeruli.3 Therefore, a tubular marker that accurately reflects TCL the tubulointerstitial damage may be an excellent biomarker for early detection or prediction of kidney disease. Although the importance and necessity of measuring clinical parameters in serum or urine of the patients with CKD are emphasized, there are few clinical markers

to predict and monitor the progression of CKD. Urinary protein is widely accepted to help physicians predict the risk of disease progression and the risk of dialysis for individual patients.14,15 However, in patients with nephrosclerosis, renal dysfunction deteriorates in spite of the low levels of urinary protein levels. In order to clarify the clinical relevance of urinary excretion of hL-FABP, urinary hL-FABP levels in 120 nondiabetic adult patients were measured.12 As a result, urinary hL-FABP was shown to reflect the progression rate of kidney disease, as determined by significantly higher hL-FABP levels in patients with deteriorating renal function as opposed to low levels in those with stable renal function. Moreover, in order to confirm the clinical usefulness of urinary hL-FABP as a maker for the monitoring of CKD, a multicenter trial was carried out.16 In this study, urinary hL-FABP was demonstrated to be more sensitive than urinary protein in predicting the progression of CKD.

Ecstasy and related compounds release neuroactive compounds including serotonin, dopamine

and noradrenaline as well as blocking neuronal re-uptake of these compounds. This leads to the elevated mood state as well as alterations in thermoregulation and autonomic dysfunction. This is also associated with enhanced release of arginine vasopressin, cortisol and adrenocorticotrophin.2 N-benzylpiperazine has gained popularity as a rave drug for producing sensation of euphoria, energy and desire to socialize and is not subject to the controlled drug restrictions that outlaw ecstasy.3 Seliciclib molecular weight While piperazine-based hallucinogens or stimulants are not currently used therapeutically, they are misused. Party pills containing BZP have many names on the market (e.g. A2, Nemesis, Frenzy, Charge Herbal, Black Pepper Extract, LBH589 research buy Herbal Ecstasy, Good Stuff, Legal X).4 BZP has been called a ‘natural’ product by some retailers, describing it as a ‘pepper extract’ or ‘herbal high’, when in fact the drug is entirely synthetic and has not been found to occur naturally. Piperazine derivatives were first synthesized in the 1950s as antihelminthic agents, but because of their lack of efficacy and significant side-effects they

were withdrawn from the market. In the 1970s and 1980s several studies showed that BZP had a stimulant, amphetamine-like effect, and in the 1990s the drug became popular for as recreational drug. In 2002, it was made illegal in USA and banned in most parts of Europe and Australia soon afterwards.5 In New Zealand, the sale of BZP and the other listed piperazines became illegal as of April 2008. The sale of BZP is legal in the UK and Canada and in general is sold as a legal alternative

to Ecstasy.1 The prevalence of party pill usage in the USA and the UK is increasing; exact numbers are unknown but in New Zealand in 2007 it was so widely used that an estimated 5 million pills were sold.6 Serious toxicity can occur even at a usual standard dose and are similar to methylenedioxymethamphetamine (MDMA, ‘ecstasy’) effects. In general, tablets and capsules contain 70–1000 mg BZP. Some products contain BZP in combination with TFMPP (3-Trifluoromethylphenylpiperazine) generally in a ratio of 2:1. An ingestion of 50–100 mg of BZP in an adult is unlikely to cause Mephenoxalone serious toxicity. Doses over 250 mg of a piperazine-based designer drug would be likely to cause moderate toxicity, such as anxiety, agitation, hypertension, tachycardia, palpitations, gastrointestinal upset and headache. Seizures, tremor, hallucinations, fever, chest pain and jaw clenching may accompany this. An increase of the dose to 500 mg can cause these effects to be prolonged and fatal.4,7 Apparent drug–drug synergism and adverse behavioural effects (e.g. seizures) are associated with high-dose administration of BZP especially in combination with TFMPP.

WANG BO, WISE ANDREA F, HUUSKES BROOKE M, RICARDO SHARON D Monash University Introduction: MicroRNA (miR), including miR-let7, is highly effective at reducing

renal fibrosis and reversing progression of disease in rodent models. However, the advancement of miR therapies is hampered by difficulties in delivering miR in a robust and sustainable manner. Thus, it is imperative to develop an efficient delivery method for targeting miR to injured kidneys to exert their anti-fibrotic function. Mesenchymal stem cells (MSC) have demonstrated a strong safety profile in both completed and numerous ongoing clinical trials. The ability of MSC to transfer molecules and organelles suggests their potential usefulness as delivery vehicle for therapeutic miR treatment that is an innovative approach. Methods: C57BL6/J mice underwent 40 mins selleck chemicals of unilateral ischemia/reperfusion

(IR) injury and were injected with GFP+/luciferase+ MSCs or PBS and imaged from 0–7 days using whole body bioluminescence imaging for cell tracing. miR-let7c modified MSCs were generated and characterised and miR expression assayed with Taqman microRNA assay. The miR-let7c-MSCs were co-cultured with NRK52E, a kidney proximal tubular cell line, using a Transwell system with/without TGF-β1 for 72 hours, and the expression of fibrotic genes assessed using qPCR. Results: Following IR, MSCs homed to the injured kidney where Metformin price they remained for up to 3 days. miR-let7c was successfully engineered and expressed in MSCs. The modified miR-let7c-MSCs maintained a normal karyotype and proliferative ability, but importantly

produced miR-let7c into the exogenous environment through exosome delivery. MSC-delivered miR-let7c was endocytosed into NRK52E cells, confirmed by the up-regulation of miR-let7c expression and fluorescent microscopy. After 3 days co-culturing, the miR-let7c-MSCs strongly inhibited the up-regulation of TGF-β type I receptor (TGBR1), a specific target of miR-let7c, and reduced a-smooth muscle actin and collagen mRNA expression, when NRK52E cells were treated with TGF-β1. Conclusion: MSCs home to the injured kidney in mice with IR injury. In vitro studies show that miR-let7c produced from modified MSC can be endocytosed into kidney epithelial cells leading to the inhibition of fibrotic genes and TGBR1 induced by TGF-β1. This data will pave the way for the application of miR, or siRNA, as an innovative Florfenicol RNAi therapeutic strategy for renal disease therapy, but may also offer promise for other degenerative chronic disorders. YAMANAKA SHUICHIRO1,2, YOKOTE SHINYA1, KATSUOKA YUICHI2, IZUHARA LUNA2, OGURA MAKOTO1, YOKOO TAKASHI1 1Department of Internal Medicine, Division of Nephrology and Hypertension; 2Division of Regenerative Medicine Introduction: We have previously demonstrated that mesenchymal stem cells (MSCs) differentiate into functional kidney cells capable of urine and erythropoietin production, indicating that MSCs may be used for kidney regeneration.

For all subsequent statistical analyses, IL-8, TNFα and IL-10 concentrations present in un-stimulated cultures were subtracted to give stimulus-specific cytokine levels for each

individual. The ratio of IL-10: TNFα was calculated from stimulus-specific cytokine levels. As cytokine concentrations, IL-10: TNFα ratios, smp0–3 h RP: m0–3 h RP cytokine ratios, and leucocyte percentages did not meet parametric assumptions, the Mann–Whitney U-test and Kruskal–Wallis tests were used to compare between two independent groups and K independent groups, respectively. The Wilcoxon signed-rank Maraviroc clinical trial test was used for paired comparison of periodate-treated and mock-treated WB culture cytokine production. This study comprised a total of 47 individuals from the Diokhor Tack community aged 6–53 years old, of whom 13 were not infected, 14 infected with S. mansoni only and 20 co-infected with S. mansoni and S. haematobium (Table 1). Only two participants

in the co-infected group were also positive for soil-transmitted nematode eggs. S. mansoni infection intensity did not significantly differ according to gender (F1,30: 1·433, P = 0·241), age group (F1,30: 1·397, P = 0·246) or between infected and co-infected groups (F1,30: 2·380, P = 0·133). S. haematobium infection intensity also did not significantly differ between males and females (F1, 17: 0·240, P = 0·631) Cell Cycle inhibitor or between age groups (F3,17: 2·501, P = 0·132) in the co-infected group. To investigate innate/early immune responses to 0–3 h RP, IL-8, TNFα and IL-10 were quantified in http://www.selleck.co.jp/products/AG-014699.html whole-blood supernatants 24 h post-stimulation. Levels of all three cytokines were significantly higher in 0–3 h RP-stimulated cultures than in un-stimulated cultures (IL-8 Z: −5·968, P < 0·001; TNFα Z: −5·905, P < 0·001; IL-10 Z: −5·968, P < 0·001) with

all 47 participants mounting a detectable cytokine response to 0–3 h RP. Participants also produced higher levels of IL-8, TNFα and IL-10 in response to zymosan than in un-stimulated control cultures (IL-8 Z: −5·968, P < 0·001; TNFα Z: −5·841, P < 0·001; IL-10 Z: −5·905, P < 0·001). Interestingly, stimulus-specific IL-8 and IL-10 levels were higher in response to 0–3 h RP than to an equivalent concentration of zymosan in paired cultures (Wilcoxon signed-rank test, IL-8 Z: −5·661, P < 0·001 and IL-10 Z: −4·370, P < 0·001), whilst TNFα levels were higher in response to zymosan than to 0–3 h RP (Wilcoxon signed-rank test, Z: −4·529, P < 0·001). There was no significant correlation between levels of any of the 0–3 h RP-specific cytokines, and schistosome infection intensity and levels did not differ between age groups (data not shown).

WT/AngII mice were also treated with either tissue factor antibody, antithrombin III, heparin, hirudin, or murine APC. TF immunoblockade or hirudin treatment did not prevent the AngII-induced acceleration of thrombosis. While antithrombin III treatment prevented the acceleration in both thrombus onset and flow cessation, heparin

only improved the time for blood flow cessation. Neither HSP inhibitor WT mice treated with murine APC nor EPCR-TgN were protected against AngII-induced thrombus development. A similar lack of protection was noted in PAI-1deficient mice. These findings implicate a role for thrombin generation pathway in the accelerated thrombosis induced by AngII and suggest that an impaired protein C pathway and increased PAI-1 do not MG-132 mw make a significant contribution to this model of microvascular thrombosis. “
“Please cite this paper as: Frantz, Engelberger, Liaudet, Mazzolai, Waeber and Feihl (2012). Desensitization of Thermal Hyperemia in the Skin is Reproducible. Microcirculation 19(1), 78–85. Objective:  Local heating increases skin blood flow SkBF (thermal hyperemia). In a previous study, we reported that a first local thermal stimulus could attenuate

the hyperemic response to a second one applied later on the same skin spot, a phenomenon that we termed desensitization. However, other studies found no evidence for desensitization in similar conditions. The aim of the present work was to test whether it was related to differences in instrumentation. Methods:  Twenty-eight healthy young males were studied. Two pairs of heating chambers, one custom-made (our study) and one commercial (other groups), were affixed to forearm skin. SkBF was measured with single-point laser-Doppler flowmetry (LDF) (780 nm) in one pair, and

laser-Doppler imaging (LDI) (633 nm) in the other. A temperature step from 34 to 41°C, was applied for 30 minutes and repeated after two hours. Results:  During the Bcl-w second thermal challenge, the plateau SkBF was lower than during the first thermal and was observed with each of the four combinations of SkBF measurement techniques and heating equipment (p < 0.05 for all conditions, range −9% to −16% of the initial value). Conclusion:  Desensitization of thermal hyperemia is not specific to peculiar operating conditions. In nonglabrous human skin, a local rise in temperature is a powerful stimulus for local vasodilation, mediated by neurogenic reflexes and locally released substances [12,13,15,16]. The mechanisms implicated in this so-called thermal hyperemia remain incompletely defined. In contrast with thermoregulatory skin vasodilation, it is not mediated by central reflexes because it is unaffected by regional nerve block [17] and is preserved in grafted skin [5].

coli O78. Further experiments are needed to determine the optimal timing and route of inoculation.

Furthermore, it will be necessary to test other serovars and challenge routes to confirm that the protection conferred by AESN1331 is efficacious under field conditions, where various serovars of APEC can infect birds. APEC strains that cause respiratory infection and septicemia have also been implicated in cellulitis; MAPK inhibitor thus, immunization with AESN1331 may reduce condemnation and downgrading of carcasses resulting from APEC. Clearly, an inexpensive and effective vaccine against APEC infection in broilers would have a significant economic impact on the industry. We are grateful to Professor Chihiro Sasakawa, Institute for Medical Science, University of Tokyo, for the gift of E. coli SM10λpir and pCVD442 and for advice regarding suicide vector selection technique; to Dr. Tetsuo Nunoya and Dr. Akira Iwata for reviewing the manuscript;

and to Dr. Kunio Doi for excellent support. We also thank Yuji Kuroyama, Tamotsu Sato, Kouji Toriumi and our staff for technical assistance. None of the authors of this paper has any financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper. IWR-1 mouse “
“Antibodies are well known for their role in humoral immunity, and their prominent involvement in the protection afforded by successful vaccines against infections. A less appreciated function

of antibodies is their capacity to dampen the autoimmune responses associated with some inflammatory diseases. Nevertheless, this paradoxical activity of antibodies is used to treat patients with autoimmune disease. Preparations of polyclonal serum IgG, which are obtained from pools of thousands of human blood donors and Resveratrol are called intravenous immunoglobulin (IVIg), are commonly used to treat patients suffering from immunothrombocytopenia. Although of important clinical significance the anti-inflammatory function of polyclonal IgG remains poorly understood. Previous studies have primarily addressed its mode of action in a prophylactic setting. However, IVIg is usually applied therapeutically in the clinic. In a study published in this issue of the European Journal of Immunology, Schwab et al. [Eur. J. Immunol. 2014. 44: 1444–1453] focus specifically on the protective effect of IVIg in a therapeutic setting, in four different mouse models of autoantibody-mediated pathology, in order to better approach the condition in human disease and therapy. This Commentary discusses how their findings have key implications for our understanding, and further deciphering, of the mode of action of this therapy. Antibodies were discovered for their protective roles against microbial infections, and are thought to mediate most of the beneficial effects afforded by licensed vaccines [1].

In conclusion, IRE1α appears to mediate early processes in B cell maturation, particularly in connection with VDJ rearrangement [91] [92]. To evaluate the role of IRE1α in plasma cell differentiation, Zhang and collaborators used IRE1Α dominant-negative mutants [91]. B cells

expressing RNAse- or kinase- dominant-negative mutants of IRE1α, or cells lacking the intracytoplasmic tail were unable to secrete immunoglobulins. When these cells were transduced with XBP-1s and stimulated with LPS, immunoglobulin secretion was restored in the RNAse- or kinase- dominant-negative mutants expressing cells. In contrast, the cells lacking the cytoplasmic tail of IRE1α did not restored immunoglobulin secretion when transduced with XBP-1s. Thus, IRE1α cytoplasmic BGB324 datasheet region have another role in addition to its catalytic activity in antibody production, perhaps acting as a scaffold for other proteins [91]. XBP-1 conditional knockout mice (XBP1flox/floxCD19cre/+) were generated to answer the question of whether XBP-1 altered the formation of memory B cells. XBP-1-deficient B cells were able to differentiate into post-GC memory B cells (IgDloB220+CD138−) and preplasma memory B cells (IgDloB220loCD79b+CD138−) in vivo, but no plasma cell was encountered in these mice [93]. Interestingly, XBP1flox/floxCD19cre/+ mice were protected against systemic

lupus erythematosus [59, 93]. Murine splenic B cells and I.29 B cell lymphoma were stimulated with LPS or treated with tunicamycin, followed by chromatin precipitation. XBP-1 was found bound to the ERDJ3 promoter in association with enhanced LY294002 purchase ERDJ3 transcription [94]. ERdj3 is a co-chaperone that associates with BiP/IgH complexes [20]. Furthermore, XBP-1 indirectly regulates IgH expression by controlling transcription of OBF1, which codes for a specific IgH transcriptional co-activator. XBP-1 binds to the OBF1 promoter, possibly through an ACGT/C sequence found in human and mice OBF1 promoters [94]. These are

the first evidences that demonstrate XBP-1 acting directly on target gene promoter during plasma cell differentiation [20, 94]. During the plasmacytic differentiation programme the PERK branch of the UPR and its downstream targets are silenced [91, 95, 96]. Two independent studies provided evidences that the IRE1/XBP-1, but not PERK/eIF2α, DNA ligase axis of UPR was activated in B lymphocytes after LPS treatment [91, 95]. Interestingly, B lymphocyte maturation occurred normally in PERK-deficient animals and their B cells could differentiate into plasma cells and secrete antibodies [95]. A third study showed that under LPS induced differentiation, I.29 μ+ B cell line activated IRE1α and consequently spliced XBP-1 mRNA at early phases. PERK was partially phosphorylated, but the LPS-elicited PERK activation was insufficient to phosphorylate eIF2α and to induce GADD34 and CHOP, downstream events of PERK activation. Curiously, pretreatment of I.

Here, we have extended these observations by showing that in silico predicted HLA-I binding 9mer peptides derived from M. tuberculosis proteins induce T-cell-dependent responses that appear to be HLA-II restricted because they are totally blocked by a pan HLA-II antibody as well as by an anti-HLA-DR antibody. As in our previous study

with vaccinia virus-derived peptides,39 there was a trend of correlation between HLA class II restricted antigenicity and a measured high peptide HLA-I binding affinity, so six of eight antigenic M. tuberculosis peptides bind HLA-I with a KD < 50 nm. However, in accordance with our recent observation on flu epitopes,28 Y-27632 we found that two of the M. tuberculosis peptides with intermediate binding affinities to HLA class I were also capable of stimulating a

strong HLA-II restricted T-cell responses. As the eight antigenic 9mer epitopes appear to be restricted by HLA-II DR molecules (Fig. 1), we tried to predict the binding of all the 157 9mers used in this study to all DR alleles present among the donors using the publicly available MHC-II predictor NetMHCIIpan48 (http://www.cbs.dtu.dk/services). Forty-eight peptides including the two antigenic peptides LEEIGILLL and IVFATAARY were predicted to be either strong binders (SB, predicted KD < 50nm) or weak binders (WB, predicted KD < 500 nm), respectively, to one or more DR alleles present among the donors, (see Supplementary material Tables S1 and Antiinfection Compound Library S2). However, the two donors (no. 19 and 32) who reacted with these two peptides did not express the predicted HLA-DR alleles. We have recently developed a technology for assaying the binding of peptides to recombinant HLA-DR molecules.32 However, only three of the eight antigenic M. tuberculosis peptides showed binding to three of the 14 tested HLA-DR molecules, PtdIns(3,4)P2 but none of these three HLA-DR molecules were expressed by the two peptide-reactive donors. These negative data might reflect the

fact that the number of assayed HLA-DR molecules only represent one-third of the HLA-DR subtypes expressed by the TB peptide immune donors. In addition, the 10-day peptide exposure period might favour low-affinity interactions that might be missed in our biochemical assay. However, so far we have no definitive proof that the eight antigenic 9mer TB peptides discovered in the present study do bind to HLA-DR. It is well established that CD4+ T cells are instrumental in the control of M. tuberculosis infections.6,7,9–11 For this reason, MHC-II restricted epitopes identified in the present study as capable of stimulating CD4+ T-cell responses may be of importance for the development of effective peptide-based vaccines against TB. In addition, it has been shown that CD4+ T cells are required for priming as well as secondary expansion of CD8+ memory T cells.

Each harvested cornea or auricle was homogenized in 0.5 mL buffer (0.1M Tris-HCl pH 8.0, 0.02M EDTA in distilled water) using a Tissue-Tearor (Biospec Products, Bartlesville, OK, USA) at 18 000 rpm for 30 s. The homogenates were aliquoted, and three tenfold dilutions (1:10, 1:100 and 1:1000) were prepared in phosphate-buffered sodium solution (PBS). The dilutions were chosen based on pilot experiments. One hundred microliter aliquots of each diluted sample were spread on 90 mm Sabouraud’s dextrose agar plates in triplicate. The plates were incubated at 37°C for 48 h, and the plates that yielded

clearly isolated fungal colonies were used for counting. The results were later converted to a pathogen load for the whole cornea or ear. The levels of IFN-γ and IL-17A in sera or corneal homogenates CP-690550 order were assayed using Mouse ELISA MAXTM Deluxe Sets for IFN-γ and IL-17A (BioLegend) according

to the protocol supplied by the manufacturer. Standard curves were prepared at the same time and used for calculation of the cytokine concentrations. The readings for the corneal homogenates were then converted Selleck ICG-001 to the total gross amount of cytokine in each cornea. In this study, ELISA was used both for measuring the levels of cytokines of interest and confirmation of the neutralization of cytokines. RT-PCR was performed to evaluate the expression of genes of interest at the mRNA level with ribosomal protein L5 (RPL5) as reference gene (Supporting Information Table 2). In brief, corneas were harvested using a 2 mm diameter trephine into ice-cold TRIzol reagent (Invitrogen, Gaithersburg,

USA). Two infected corneas from two infected animals were pooled into one sample, with the untreated corneas from the same mice used as controls. Total RNA was extracted using isopropanol precipitation, purified with NucleoSpin RNA clean-up columns (Macherey-Nagel, Düren, Germany), and reverse transcribed into cDNA using a PrimeScript RT Reagent Kit (Takara, Shiga, Japan). PCR using Taqman primers was performed in an ABI7500 amplifier (Applied Biosystems, many Foster City, CA, USA). Triplicates were set for each sample, and the cycling condition was as follows: 10 s at 95°C, followed by 45 cycles of amplification for 15 s at 95°C, and 1 min at 60°C. The SDS 7500 software (Applied Biosystems) was used to obtain the fractional cycle number for threshold fluorescence (threshold cycle, Ct) of each reaction. The average of the PCR duplicates was used to calculate the relative Ct of a gene against RPL5 (ΔCt = Ctgene – CtRPL5) for each sample, and the average ΔCt for the three samples in each group was used to calculate the ΔΔCt of the CaK samples against control (ΔΔCt = ΔCtCaK – ΔCtcontrol).