miR-19b was expressed differently between quiescent and activated HSCs, using comparative analysis of microRNA (miRNA) expression. As is well known, comparative analysis is the gold standard approach for detecting dys-regulated miRNAs. This same approach has been used on HSC in 4 other studies related to the topic.2-5 The profiles of dys-regulated miRNAs in activated HSCs are summarized in Table 1. The same protocol was executed with the following steps in these studies: step 1, quiescent HSCs were isolated from normal rat liver; step 2, activated HSCs were acquired by culturing quiescent HSCs in vitro for 10 or 14 days until activated; step 3, the different miRNA expression Pirfenidone patterns of activated and quiescent

HSCs were analyzed by comparative analysis. However, there was an interesting phenomenon shown in Table 1, which was that the profiles of dys-expressed miRNAs in activated HSCs varied greatly across the studies. The issue remains why the same protocol for detecting

dys-regulated miRNAs in activated HSCs resulted in such different miRNA profiles. Shao-Long Chen M.D.*, Ming-Hua Zheng M.D.*, Tao Yang M.D.*, Mei Song Doxorubicin solubility dmso M.D.*, Yong-Ping Chen M.D.*, * Department of Infection and Liver Diseases, Liver Research Center,The First Affiliated Hospital of Wenzhou Medical College, Wenzhou, China. “
“Bariatric surgery is an increasingly popular approach for effecting significant weight reduction in obese patients with comorbidities, including hepatic steatosis. Here we report a novel case of advanced nonalcoholic steatohepatitis (NASH) fibrosis

with portal hypertension after duodenal switch bariatric surgery, resolving histopathologically with partial reversal of the malabsorptive procedure.1 BPD/DS, biliopancreatic diversion with duodenal switch; CPT, Childs-Pugh-Turcotte; DM, diabetes mellitus; JIB, jejunoileal bypass; MELD, model for endstage liver disease; NASH, nonalcoholic steatohepatitis. A 50-year-old male with a history of morbid obesity and no alcohol prior to presentation presented to the Hepatology Clinic with cirrhosis secondary to NASH. Three years prior, the patient underwent biliopancreatic 上海皓元 diversion with duodenal switch (BPD/DS). Two years postsurgery he had lost 188 pounds with resolution of hypertension and diabetes mellitus (DM). At presentation the patient was noted to have extensive bridging fibrosis on percutaneous liver biopsy with trichrome staining (Fig. 1) complicated by portal hypertensive ascites and mild hepatic encephalopathy. Computed tomography (CT) of the abdomen noted diminished size with nodular contour of the liver and moderate ascites. His initial model for endstage liver disease (MELD) was 19 (international normalized ratio [INR] 1.50, total bilirubin 1.9 mg/dL, creatinine 1.8 mg/dL; weight = 193 lbs; body mass index [BMI] = 29.3; albumin = 3.4 gm/dL [after albumin infusions]) and he was Childs-Pugh-Turcotte (CPT) Class B.

miR-19b was expressed differently between quiescent and activated HSCs, using comparative analysis of microRNA (miRNA) expression. As is well known, comparative analysis is the gold standard approach for detecting dys-regulated miRNAs. This same approach has been used on HSC in 4 other studies related to the topic.2-5 The profiles of dys-regulated miRNAs in activated HSCs are summarized in Table 1. The same protocol was executed with the following steps in these studies: step 1, quiescent HSCs were isolated from normal rat liver; step 2, activated HSCs were acquired by culturing quiescent HSCs in vitro for 10 or 14 days until activated; step 3, the different miRNA expression HM781-36B in vivo patterns of activated and quiescent

HSCs were analyzed by comparative analysis. However, there was an interesting phenomenon shown in Table 1, which was that the profiles of dys-expressed miRNAs in activated HSCs varied greatly across the studies. The issue remains why the same protocol for detecting

dys-regulated miRNAs in activated HSCs resulted in such different miRNA profiles. Shao-Long Chen M.D.*, Ming-Hua Zheng M.D.*, Tao Yang M.D.*, Mei Song ATM/ATR inhibitor M.D.*, Yong-Ping Chen M.D.*, * Department of Infection and Liver Diseases, Liver Research Center,The First Affiliated Hospital of Wenzhou Medical College, Wenzhou, China. “
“Bariatric surgery is an increasingly popular approach for effecting significant weight reduction in obese patients with comorbidities, including hepatic steatosis. Here we report a novel case of advanced nonalcoholic steatohepatitis (NASH) fibrosis

with portal hypertension after duodenal switch bariatric surgery, resolving histopathologically with partial reversal of the malabsorptive procedure.1 BPD/DS, biliopancreatic diversion with duodenal switch; CPT, Childs-Pugh-Turcotte; DM, diabetes mellitus; JIB, jejunoileal bypass; MELD, model for endstage liver disease; NASH, nonalcoholic steatohepatitis. A 50-year-old male with a history of morbid obesity and no alcohol prior to presentation presented to the Hepatology Clinic with cirrhosis secondary to NASH. Three years prior, the patient underwent biliopancreatic MCE公司 diversion with duodenal switch (BPD/DS). Two years postsurgery he had lost 188 pounds with resolution of hypertension and diabetes mellitus (DM). At presentation the patient was noted to have extensive bridging fibrosis on percutaneous liver biopsy with trichrome staining (Fig. 1) complicated by portal hypertensive ascites and mild hepatic encephalopathy. Computed tomography (CT) of the abdomen noted diminished size with nodular contour of the liver and moderate ascites. His initial model for endstage liver disease (MELD) was 19 (international normalized ratio [INR] 1.50, total bilirubin 1.9 mg/dL, creatinine 1.8 mg/dL; weight = 193 lbs; body mass index [BMI] = 29.3; albumin = 3.4 gm/dL [after albumin infusions]) and he was Childs-Pugh-Turcotte (CPT) Class B.

Interestingly, based on data from 39 patients with haemophilia A, we found

variable levels of endogenous thrombin potential (from <10% to approximately 58%) in patients with <1% FVIII in plasma [17]. In the field of haemophilia not complicated by the presence of inhibitors, the potential applications of the TGA include: assessment of the coagulation profile in patients with similar residual levels of FVIII/FIX activities but different bleeding phenotypes; monitoring of treatment regimens such as prophylaxis (e.g. correlation with trough factor levels and/or incidence of breakthrough bleeds). Phenotype characterization is an interesting issue for both haemophilia A and B, selleck kinase inhibitor particularly as wide variability exists in the clinical expression of the disease. To investigate this aspect, we evaluated adult patients with severe haemophilia with no history Roscovitine of inhibitors and treated exclusively on-demand in a single centre, case-control study [18]. Cases included patients classified as mild bleeders (≤2 spontaneous bleeding episodes/year and an annual factor consumption lower than 500 IU kg−1); controls were patients with more than two spontaneous bleeds per year and a factor consumption >500 IU kg−1year−1 (a subgroup

of controls had a markedly severe bleeding tendency: 25 or more bleeding episodes per year and an annual factor consumption >2000 IU kg−1). Based on the clinical characteristics of cases and controls, we found that patients with severe haemophilia B were significantly more represented among mild bleeders than controls (32% vs. 8%, P = 0.03). Moreover, cases with their first bleed at a significantly older age, had significantly fewer bleeds/year,

medchemexpress lower factor use and better orthopaedic and Pettersson scores than controls (severe bleeders) [18]. Mild bleeders also had significantly higher thrombin generation (expressed as endogenous thrombin potential in platelet-rich plasma) than controls (severe bleeders). According to univariate analysis, haemophilia B was a variable associated with a mild bleeding tendency as well as higher thrombin generation in patients with this tendency; higher levels of factor antigens were also associated with a mild bleeding tendency. After adjusting for other variables, the only significant factor was the type of factor mutation, meaning that less severe gene defects were associated with a mild bleeding tendency. The overall conclusion from this study is that the TGA may be used to detect the coagulation phenotype, but the role of mutation is related to the presence/absence of antigen in plasma which may be related to the thrombin generation that we observed in the plasma of our patients, warranting further research.

The urease B subunit was recently shown to lead to Th17

responses in the mouse model of H. pylori infection [35]. When recombinant urease B was incubated directly with mouse splenic lymphocytes, IL-17-producing cells were increased, and when macrophages were incubated with recombinant urease B, IL-6 and IL-23 were produced to support Th17 development. H. pylori LPS has been shown to induce weaker immune responses than LPS from other bacteria. Particularly, LPS from H. pylori did not induce strong IL-1β, IL-6, or IL-8 responses [36] as other bacterial LPS does. H. pylori LPS was also shown Selleckchem Tanespimycin to induce little NF-κB activation through TLR-4, but was shown in this study to induce IL-12 and IL-18 responses, which are thought to be pro-inflammatory. This is in contrast to another study that showed a lack of IL-12 and IL-2 induction by lymphocytes incubated with H. pylori LPS, which was accompanied by decreased cytotoxic AZD3965 order activity by lymphocytes incubated with H. pylori LPS compared to that of E. coli [37]. The beginning of 2011 was marked by a promising publication in the field of H. pylori vaccine development made by Moss et al. [38]. They used a computational method to predict novel T-cell epitopes. The multi-epitope vaccine was administered intranasally or intramuscularly to H. pylori-infected

mice, followed by a boost with the peptides themselves formulated in liposomes with CpG oligonucleotides and heat-labile enterotoxin. The vaccine induced a broad immune response, as determined MCE公司 by IFN-γ production, and led to a sterilizing immunity 32 weeks after challenge in 5 of 19 mice. Another promising vector platform for the

expression of H. pylori antigens was published in the beginning of 2011 by Iankov, et al. [39]. They produced a measles virus (MV) vaccine strain encoding the H. pylori neutrophil-activating protein (NAP). Nine months post vaccination, all animals immunized with MV strains expressing the secretory NAP antigen developed a strong humoral immunity against NAP within 2-4 weeks. By using IFN-γ ELISpot assay, they also confirmed effective NAP-specific cell-mediated immunity. Their experiments importantly demonstrated that immunization with a live replication competent vaccine expressing H. pylori molecules (NAP or potentially CagA, VacA, etc.) induced not only robust antibody production but also distinctive cell-mediated response against H. pylori antigens. Improved efficacy of vaccines may be achieved in new trials of vaccine formulations that include multiple antigens and use methods to optimize cellular immunity. An approach made by Chen et al. [40] used a H. pylori oipA gene-encoded construct co-delivered by IL-2 gene-encoded construct and B subunit heat-labile toxin of Escherichia coli gene-encoded construct.

Approximately 34% of the surveys were conducted before 1990; approximately 38% were between 1990 and 1999, and 28% were in 2000 or later. Overall, approximately 32% of the survey population was male and 44% was female; sex was not reported for 24% of the total sample. The results selleckchem of this systematic review reveal the limited availability of HBsAg seroprevalence data around the globe. Although at least one usable survey was found for all countries except Guyana and Macedonia, five or fewer surveys meeting inclusion criteria were found for one third of the countries. The median number of surveys was nine per country (range, 0-376), and more than

half of the total surveys were from 11 countries. For 50 countries, no surveys in emigrants were found. Availability of data differed substantially by region; 1,066 usable surveys were found for Asia, but only 58 for Central America. Surveys used in each country-specific meta-analysis are available at http://www.plan-a.com. HBsAg seroprevalence rates reported for most countries varied significantly from survey to survey. This variation was observed in surveys among emigrants and among in-country populations and was

expected, given that surveys were carried out in different populations at different times. For example, rates in India ranged from 0.25% among pregnant women attending antenatal clinics in Calcutta during 2002-2004 to 11.4% among rural adults in Western Maharashta click here in 1992.17, 18 Rates in China ranged from 0.7% in a 1999 survey of young children

in Taipei City to 39% in adult males in Massago, Taiwan, in 1996.19, 20 Country-specific RE pooled prevalence rates calculated by combining all available studies for each of the 102 countries are shown in Table 3 (no weighting by study quality was included). Countries with the highest pooled HBsAg rates were Sudan (18.6%), Liberia (16.5%), Guinea (16.3%), Eritrea (15.5%), and Zimbabwe (13.9%). Weighted average CHB rates for the FB in the United States by world region of origin were calculated by using the country-specific RE pooled prevalence rates and the number of FB in the United States from each country in the region. FB persons who migrated from Africa had the highest average CHB rate (10.3%), followed by FB from Asia (7.27%), Oceania (4.78%), and the 上海皓元 Caribbean (4.52%). The weighted average CHB prevalence rate from the RE meta-analyses for all FB living in the United States was 3.45% (95% confidence interval [CI]: 2.72-4.19). CIs for the country-specific RE pooled CHB rates were broad (Table 3). Cochran’s Q test and I2 statistic performed for each country-specific meta-analysis supported heterogeneity among the surveys for the majority of countries (Supporting Table 5). The I2 statistic was 55% or higher (indicating significant heterogeneity) for all except three meta-analyses.14, 15 Q tests were significant (P <0.

Knockdown of PPARβ CDK activation clearly inhibited MAT2A expression as well as transcriptional activity. In culture-activated primary rat HSCs that exhibit an induction of PPARβ,3

silencing this gene inhibited MAT2A expression. This analysis established that PPARβ is a positive regulator of the MAT2A gene during HSC activation. Forced expression of MAT2A in RSG-treated cells reverses the quiescent state of HSCs by lowering the expression of PPARγ and concomitantly inducing PPARβ and the activation marker, α-SMA. These findings imply a reciprocal regulation between PPARs and MAT2A during quiescence and activation. Our previous work has shown that MAT2A knockdown reduces HSC activation, and the overexpression findings support the silencing data.15 Forced expression of MAT2A also lowered C/EBPβ protein levels, but we did not see a significant modulation of MAT2A when C/EBPβ reserves were altered. Hence, in quiescent and activated HSCs, PPARs appear to be the major modulators of MAT2A transcription, and MAT2A deregulates PPARs and other proteins during HSC activation. In conclusion, we have unraveled an important mechanism of transcriptional regulation of the MAT2A gene by two PPAR proteins that occupy the same binding site on the MAT2A promoter. In quiescent HSCs, the PPARγ subtype acts as a negative regulator of MAT2A transcription. During HSC activation, BI 6727 a dramatic reduction in PPARγ expression

and activity releases the inhibitory tone that this transcription factor exerts on MAT2A and allows positive regulators like PPARβ to bind to the MAT2A PPRE and induce the expression of this gene. MAT2A is the only SAM-synthesizing enzyme in HSCs and is a strong determinant of HSC activation and proliferation. Therefore, multiple levels of control of this gene may exist in HSCs apart from those described in this work. Identifying other novel factors that control MAT2A both transcriptionally and posttranscriptionally in HSCs is a subject of future investigation. Our sincere thanks go to Dr. Shelly Lu for expert advice and guidance. Additional Supporting Information may be found in the online version of this article.

“
“Liver fibrosis is a common medchemexpress pathway leading to cirrhosis. Cilostazol, a clinically available oral phosphodiesterase-3 inhibitor, has been shown to have antifibrotic potential in experimental non-alcoholic fatty liver disease. However, the detailed mechanisms of the antifibrotic effect and its efficacy in a different experimental model remain elusive. Male C57BL/6J mice were assigned to five groups: mice fed a normal diet (groups 1 and 2); 0.1% or 0.3% cilostazol-containing diet (groups 3 and 4, respectively); and 0.125% clopidogrel-containing diet (group 5). Two weeks after feeding, groups 2–5 were intraperitoneally administered carbon tetrachloride (CCl4) twice a week for 6 weeks, while group 1 was treated with the vehicle alone.

Knockdown of PPARβ Fulvestrant clearly inhibited MAT2A expression as well as transcriptional activity. In culture-activated primary rat HSCs that exhibit an induction of PPARβ,3

silencing this gene inhibited MAT2A expression. This analysis established that PPARβ is a positive regulator of the MAT2A gene during HSC activation. Forced expression of MAT2A in RSG-treated cells reverses the quiescent state of HSCs by lowering the expression of PPARγ and concomitantly inducing PPARβ and the activation marker, α-SMA. These findings imply a reciprocal regulation between PPARs and MAT2A during quiescence and activation. Our previous work has shown that MAT2A knockdown reduces HSC activation, and the overexpression findings support the silencing data.15 Forced expression of MAT2A also lowered C/EBPβ protein levels, but we did not see a significant modulation of MAT2A when C/EBPβ reserves were altered. Hence, in quiescent and activated HSCs, PPARs appear to be the major modulators of MAT2A transcription, and MAT2A deregulates PPARs and other proteins during HSC activation. In conclusion, we have unraveled an important mechanism of transcriptional regulation of the MAT2A gene by two PPAR proteins that occupy the same binding site on the MAT2A promoter. In quiescent HSCs, the PPARγ subtype acts as a negative regulator of MAT2A transcription. During HSC activation, Alvelestat research buy a dramatic reduction in PPARγ expression

and activity releases the inhibitory tone that this transcription factor exerts on MAT2A and allows positive regulators like PPARβ to bind to the MAT2A PPRE and induce the expression of this gene. MAT2A is the only SAM-synthesizing enzyme in HSCs and is a strong determinant of HSC activation and proliferation. Therefore, multiple levels of control of this gene may exist in HSCs apart from those described in this work. Identifying other novel factors that control MAT2A both transcriptionally and posttranscriptionally in HSCs is a subject of future investigation. Our sincere thanks go to Dr. Shelly Lu for expert advice and guidance. Additional Supporting Information may be found in the online version of this article.

“
“Liver fibrosis is a common medchemexpress pathway leading to cirrhosis. Cilostazol, a clinically available oral phosphodiesterase-3 inhibitor, has been shown to have antifibrotic potential in experimental non-alcoholic fatty liver disease. However, the detailed mechanisms of the antifibrotic effect and its efficacy in a different experimental model remain elusive. Male C57BL/6J mice were assigned to five groups: mice fed a normal diet (groups 1 and 2); 0.1% or 0.3% cilostazol-containing diet (groups 3 and 4, respectively); and 0.125% clopidogrel-containing diet (group 5). Two weeks after feeding, groups 2–5 were intraperitoneally administered carbon tetrachloride (CCl4) twice a week for 6 weeks, while group 1 was treated with the vehicle alone.

Research suggests that individuals are more likely to minimize adverse experiences rather than fabricate them.50 In any case, the proportion of migraineurs reporting sexual and physical abuse are nearly identical in this and our earlier clinic-based survey.7 Our findings suggest that childhood maltreatment, particularly emotional abuse, may be risk factors for development of chronic headache, including transformed migraine. Although depression and anxiety are related to childhood maltreatment and to chronic frequency, the association of emotional abuse and chronic migraine/transformed migraine is independent of these psychiatric APO866 ic50 disorders. The

finding that emotional abuse was associated with an earlier age of migraine onset suggests a possible role in migraine pathophysiology. (a)  Conception and Design (a)  Drafting the Manuscript (a)  Final Approval of the Completed Manuscript “
“Photophobia refers to a sensory disturbance provoked by light. However, because it arises distinctly in a broad range of clinical conditions, its definition remains elusive. Many underscore the painful sensory aspects of photophobia, while others emphasize its unpleasant, check details affective qualities. To add further complexity, recent discoveries in photophobia research have raised disparate and potentially

conflicting results. In this installment of an occasional series, we asked clinicians and scientists to give their interpretation of what these discoveries tell us about photophobia in the clinic, and MCE公司 vice versa. “
“This section of Headache annually reviews the status

of recently completed and ongoing major clinical trials involving common headache disorders. The review will focus on multicenter trials of new therapies, as well as novel formulations of previously approved therapeutics. Table 1 summarizes the major therapeutic headache trials that are ongoing at the present time, according to data obtained from both the “ClinicalTrials.Gov” website and from corporate press releases and presentations. 2013 was a year of limited progress in the clinical development of new antimigraine products. Indeed, there were more discontinuations than initiations of clinical development for new chemical entities within the field of migraine. For the fourth year in a row, no new therapeutic agents were approved by the Food and Drug Administration (FDA) for the acute and/or prophylactic treatment of migraine, although a novel patch formulation of sumatriptan did obtain FDA approval (as noted below). Data from only one major clinical efficacy trial of a new chemical entity (ie, BMS-927711) were reported in 2013. Nonetheless, 2013 was a year in which early stage clinical development progress was made with a group of antibodies targeting calcitonin gene-related peptide (CGRP).

A precontrast computed tomography scan showed a diffusely hyperdense liver and a large amount of ascites (Panel A). Transjugular liver biopsy demonstrated cirrhosis with polymorphonuclear infiltrate, Mallory bodies (arrows), and ballooning degeneration (arrowheads) (Panel B). Electron microscopy showed lysosomal myeloid bodies with dense deposits

(asterisks) within hepatocytes (Panel C). On the basis of clinical history and histologic findings, amiodarone-induced liver cirrhosis was diagnosed, and the drug was replaced with another antiarrhythmic agent (propafenone). He was discharged with improved general condition with marked reduction of ascites. Although asymptomatic elevations of aminotransferases have been reported in up to 25% of the patients

treated with long-term amiodarone therapy, symptomatic liver dysfunction has been reported to occur in fewer than 1% of these patients.1, 2 However, amiodarone-induced Temsirolimus purchase hepatitis can progress to cirrhosis, resulting in decompensated hepatic failure, although this rarely happens.1, 2 Amiodarone is one of the cationic amphiphilic drugs (CADs) that can cause phospholipidosis in liver.3 In this case, the findings demonstrating intralysosomal myelin figures and electron-dense material on electron microscopy are compatible with the findings of CAD-induced phospholipidosis.4 However, the functional consequences of phospholipidosis on cellular or tissue function have not been well explained. Rather, liver damage is considered to be caused by amiodarone-induced INCB018424 cell line inhibition of mitochondrial β-oxidation.5 “
“We read with great interest the article by Lange and coworkers.1 It is known that mitochondrial toxicity related to the nucleoside analogues can result in macrovesical hepatic steatosis and lactic acidosis. In vivo, these results were shown as side effects of long-term lamivudine use in patients with human immunodeficiency virus (HIV).2 It was noted that the amount of mitochondrial DNA

defects is a predictive marker for the clinical expression of mitochondrial toxicity.2 In case of the severity of the underlying liver dysfunction due to cirrhosis, or acute exacerbation of chronic liver disease, the function of mitochondria in hepatocytes declines immediately. In light of these data, severe mitochondrial MCE公司 dysfunction leading to lactic acidosis can be aggravated by nucleoside analogues in patients with an underlying disease, as discussed above. Moreover, hepatic steatosis may be an additional risk factor for these patients with hyperlactatemia who are using entecavir. Obesity was reported as a possible risk factor for mitochondrial toxicity in patients with HIV who are undergoing highly active antiretroviral therapy.3 So, in spite of the small numbers of patients with lactic acidosis under entecavir, we worry about the effect of body mass index on treatment toxicity. Akif Altınbas MD*, Fuat Ekiz MD*, Osman Yuksel MD, Assoc. Prof.

[13, 14] In addition, the report that SRY (sex determining region Y)-box 17 (Sox17) and pancreatic and duodenal homeobox 1 (Pdx1) are expressed in PBGs in a fashion that is distinct from the adjacent epithelial lining of fetal bile ducts implies a potential role for PBGs as a niche of multipotent stem cells within the extrahepatic bile

duct (EHBD).[8] Here, we sought further Selleckchem KU-60019 insight into the cellular composition of PBGs and their molecular relationships with the epithelium proper of the duct mucosa. Our working hypothesis was that PBGs are populated by mature and undifferentiated cells capable of proliferation in pathological states. To test this hypothesis, we developed a novel whole-mount in situ immunostaining technique that preserves the anatomical integrity of gallbladder Selumetinib supplier and EHBDs in suckling and adult mice. Applying confocal microscopy and three-dimensional (3D) reconstruction, we identified PBGs within the submucosal compartment along the entire length of the ductular system, except the gallbladder. Most notably, we discovered

that PBGs elongate to form complex epithelial networks that course and branch within the walls, expressing cytokeratin (CK)-19, Sox17, and Pdx1 and demonstrating cellular proliferation after viral infection and bile duct ligation (BDL). The gallbladder, cystic duct, and extrahepatic bile ducts of Balb/c mice (Charles River Laboratories Inc., Wilmington, MA) were microdissected en bloc from mice at 3 and 7 days and 2 months of age (N = 7 in each group); this 上海皓元医药股份有限公司 anatomic unit will be referred to as EHBD, unless otherwise specified. EHBDs were fixed in ice-cold 3.7% formalin for 20 minutes, washed in 1× phosphate-buffered saline (PBS) for 10 minutes at room temperature (RT), permeabilized in Dent’s fixative (80% methanol/20% dimethyl sulfoxide) for 15 minutes, rehydrated through a series of methanol dilutions (75%, 50%, then 25% methanol in distilled H2O) for 7 minutes per dilution, washed in 1× PBS for 10 minutes and then in diluent solution (1× PBS with 1% bovine serum albumin and 0.1% Triton X-100) for 1.5 hours, followed by blocking in diluent solution

containing 10% normal donkey serum for an additional 2 hours and incubation with rabbit anti-cytokeratin (CK) antibody (Ab; N1512, undiluted; Dako North America, Carpinteria, CA) overnight at 4oC. EHBDs were then washed in diluent and incubated in donkey anti-rabbit DyLight 488 secondary Ab (711-485-152, diluted 1:333; Jackson Immunoresearch, West Grove, PA) for 5 hours at RT. To complete the assay, EHBDs were washed in diluent and dehydrated in 100% methanol. CK-specific signal using this Ab panel was reproduced using goat anti-mouse CK-19 Ab (33111, diluted at 1:100; Santa Cruz Biotechnology, Santa Cruz, CA). The same protocol was applied to the detection of α-tubulin with the addition of Abs to include mouse anti-α-tubulin Ab (T7451, diluted at 1:333; Sigma-Aldrich, St.