Biliary transporter gene mutations in severe intrahepatic cholestasis of pregnancy: Diagnostic and management implications.

BACKGROUND AND AIMS: Clinical syndromes associated with biallelic mutations of bile acid (BA) transporters usually present in childhood. Subtle mutations may underlie intrahepatic cholestasis of pregnancy (ICP) and oral contraceptive steroid (OCS) induced cholestasis. In five women with identified genetic mutations of such transporters, with eight observed pregnancies complicated by ICP, we examined relationships between transporter mutations, clinical phenotypes, and treatment outcomes. METHODS: Gene mutation analysis for BA transporter deficiencies was performed using Next Generation/Sanger sequencing, with analysis for gene deletions/duplications. RESULTS: Intrahepatic cholestasis of pregnancy was early-onset (9-32 weeks gestation) and severe (peak BA 74-370 µmol/L), with premature delivery (28+1 -370 weeks gestation) in 7/8 pregnancies, in utero passage of meconium in 4/8, but overall good perinatal outcomes, with no stillbirths. There was generally no response to ursodeoxycholic acid and variable responses to rifampicin and chelation therapies; naso-biliary drainage appeared effective in 2/2 episodes persisting post-partum in each of the two sisters. Episodic jaundice occurring spontaneously or provoked by non-specific infections, and OCS-induced cholestasis, had previously occurred in 3/5 women. Two cases showed biallelic heterozygosity for several ABCB11 mutations, one was homozygous for an ABCB4 mutation and a fourth case was heterozygous for another ABCB4 mutation. CONCLUSIONS: Early-onset or recurrent ICP, especially with previous spontaneous or OCS-induced episodes of cholestasis and/or familial cholestasis, may be attributable to transporter mutations, including biallelic mutations of one or more transporters. Response to standard therapies for ICP is often incomplete; BA sequestering therapy or naso-biliary drainage may be effective. Optimized management can produce good outcomes despite premature birth and evidence of fetal compromise.

A novel prognostic model for transplant-free survival in primary sclerosing cholangitis.

OBJECTIVE: Most prognostic models for primary sclerosing cholangitis (PSC) are based on patients referred to tertiary care and may not be applicable for the majority of patients with PSC. The aim of this study was to construct and externally validate a novel, broadly applicable prognostic model for transplant-free survival in PSC, based on a large, predominantly population-based cohort using readily available variables. DESIGN: The derivation cohort consisted of 692 patients with PSC from the Netherlands, the validation cohort of 264 patients with PSC from the UK. Retrospectively, clinical and biochemical variables were collected. We derived the prognostic index from a multivariable Cox regression model in which predictors were selected and parameters were estimated using the least absolute shrinkage and selection operator. The composite end point of PSC-related death and liver transplantation was used. To quantify the models' predictive value, we calculated the C-statistic as discrimination index and established its calibration accuracy by comparing predicted curves with Kaplan-Meier estimates. RESULTS: The final model included the variables: PSC subtype, age at PSC diagnosis, albumin, platelets, aspartate aminotransferase, alkaline phosphatase and bilirubin. The C-statistic was 0.68 (95% CI 0.51 to 0.85). Calibration was satisfactory. The model was robust in the sense that the C-statistic did not change when prediction was based on biochemical variables collected at follow-up. CONCLUSION: The Amsterdam-Oxford model for PSC showed adequate performance in estimating PSC-related death and/or liver transplant in a predominantly population-based setting. The transplant-free survival probability can be recalculated when updated biochemical values are available.

Validation of the prognostic value of histologic scoring systems in primary sclerosing cholangitis: An international cohort study.

Histologic scoring systems specific for primary sclerosing cholangitis (PSC) are not validated. We recently determined the applicability and prognostic value of three histological scoring systems in a single PSC cohort. The aim of this study was to validate their prognostic use and reproducibility across a multicenter PSC cohort. Liver biopsies from PSC patients were collected from seven European institutions. Histologic scoring was performed using the Nakanuma, Ishak, and Ludwig scoring systems. Biopsies were independently scored by six liver pathologists for interobserver agreement. The prognostic value of clinical, biochemical, and all three histologic scoring systems on predicting composite endpoints 1 (PSC-related death and liver transplantation), 2 (liver transplantation), and 3 (liver-related events), was assessed using univariable and multivariable Cox proportional hazards modeling. A total of 119 PSC patients were identified, and the median follow-up was 142 months. During follow-up, 31 patients died (20 PSC-related deaths), 31 patients underwent liver transplantation, and 35 patients experienced one or more liver-related events. All three staging systems were independent predictors of endpoints 2 and 3 (Nakanuma system: hazard ratio [HR], 3.16 [95% confidence interval (CI), 1.49-6.68] for endpoint 2 and HR, 2.05 [95% CI, 1.17-3.57] for endpoint 3; Ishak system: HR, 1.55 [95% CI, 1.10-2.18] for endpoint 2 and HR, 1.43 [95% CI, 1.10-1.85] for endpoint 3; Ludwig system: HR, 2.62 [95% CI, 1.19-5.80] for endpoint 2 and HR, 2.06 [95% CI, 1.09-3.89] for endpoint 3). Only the Nakanuma staging system was independently associated with endpoint 1: HR, 2.14 (95% CI, 1.22-3.77). Interobserver agreement was moderate for Nakanuma stage (κ = 0.56) and substantial for Nakanuma component fibrosis (κ = 0.67), Ishak stage (κ = 0.64), and Ludwig stage (κ = 0.62). CONCLUSION: We confirm the independent prognostic value and demonstrate for the first time the reproducibility of staging disease progression in PSC using the Nakanuma, Ishak, and Ludwig staging systems. The Nakanuma staging system-incorporating features of chronic biliary disease-again showed the strongest predictive value. (Hepatology 2017;65:907-919).

The UK-PBC risk scores: Derivation and validation of a scoring system for long-term prediction of end-stage liver disease in primary biliary cholangitis.

UNLABELLED: The biochemical response to ursodeoxycholic acid (UDCA)--so-called "treatment response"--strongly predicts long-term outcome in primary biliary cholangitis (PBC). Several long-term prognostic models based solely on the treatment response have been developed that are widely used to risk stratify PBC patients and guide their management. However, they do not take other prognostic variables into account, such as the stage of the liver disease. We sought to improve existing long-term prognostic models of PBC using data from the UK-PBC Research Cohort. We performed Cox's proportional hazards regression analysis of diverse explanatory variables in a derivation cohort of 1,916 UDCA-treated participants. We used nonautomatic backward selection to derive the best-fitting Cox model, from which we derived a multivariable fractional polynomial model. We combined linear predictors and baseline survivor functions in equations to score the risk of a liver transplant or liver-related death occurring within 5, 10, or 15 years. We validated these risk scores in an independent cohort of 1,249 UDCA-treated participants. The best-fitting model consisted of the baseline albumin and platelet count, as well as the bilirubin, transaminases, and alkaline phosphatase, after 12 months of UDCA. In the validation cohort, the 5-, 10-, and 15-year risk scores were highly accurate (areas under the curve: >0.90). CONCLUSIONS: The prognosis of PBC patients can be accurately evaluated using the UK-PBC risk scores. They may be used to identify high-risk patients for closer monitoring and second-line therapies, as well as low-risk patients who could potentially be followed up in primary care.

New Therapeutic Strategies for Primary Sclerosing Cholangitis.

Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease, which in the majority of patients progresses to liver transplantation or death. To date, no medical treatment has been proven to be of benefit, although ursodeoxycholic acid is widely used. The etiopathogenesis of PSC is unclear, although it is associated with inflammatory bowel disease. Various hypotheses have been suggested, which have led to different therapeutic strategies. Recent studies have suggested that the microbiome may play a role in PSC, raising the possibility of efficacy of antibiotics and fecal microbiota transplantation. Gut-homing T cells may be important in the pathogenesis of PSC, and several agents are in development, targeting various receptors, integrins, and ligands on this pathway, including VAP-1, MAdCAM-1, α4ß7, and CCR9. Nuclear receptor agonists such as obeticholic acid and fibrates hold promise, as do other therapies that alter bile acid composition such as norUDCA. Antifibrotic agents such as Loxl2 inhibitors are also being assessed. In conclusion, it is likely that an effective drug therapy for PSC will become available over the next decade.

Primary sclerosing cholangitis: a clinical update.

INTRODUCTION: Primary sclerosing cholangitis (PSC) is a progressive cholestatic disorder that ultimately can lead to cirrhosis, liver failure, malignancy and death. It is strongly associated with inflammatory bowel disease (IBD), and though a rare disease, its incidence is increasing. There are no proven medical therapies for PSC. SOURCES OF DATA: Ovid Medline was utilised to search for articles with keywords 'sclerosing cholangitis' and 'cholangiocarcinoma' and containing titles 'primary sclerosing cholangitis', and references of these papers were cross-referenced for further relevant manuscripts. AREAS OF AGREEMENT: PSC is a rare disease, and there is a strong association with risk loci within the major histocompatibility complex and other genes common to other autoimmune diseases. PSC is a premalignant condition, associated with higher rates of hepatobiliary and colorectal cancer in patients with ulcerative colitis (UC). AREAS OF CONTROVERSY: The pathogenesis is unclear, and competing theories exist surrounding toxic bile acids, enhanced homing of particular T cells from the gut to the liver and increased passage of toxins to the liver through a permeable bowel wall. It is unclear whether the higher rate of colonic cancer in PSC/UC occurs in PSC/Crohn's disease. Ursodeoxycholic acid therapy reduces liver enzymes but has not been shown to improve survival. It may reduce the prevalence of bowel cancer. GROWING POINTS: Recent genetic studies have revealed new risk loci, pointing to the importance of the immune system and its interaction with the biome. AREAS TIMELY FOR DEVELOPING RESEARCH: On the basis of the genetic studies discussed earlier, novel agents are being developed and trialled in the treatment of PSC.

Primary sclerosing cholangitis.

Primary sclerosing cholangitis (PSC) is a chronic, cholestatic liver disease caused by diffuse inflammation and fibrosis that can involve the entire biliary tree. It is a progressive disorder which can ultimately lead to biliary cirrhosis, portal hypertension and hepatic failure. PSC is a complex genetic disorder with male predominance. Environmental predisposing factors include non-smoking. It is closely associated with inflammatory bowel disease (IBD), particularly ulcerative colitis, which occurs in about two thirds of PSC cases. Recent studies have suggested that PSC-IBD is a separate disease entity from IBD alone with distinctive genetic and phenotypic characteristics. Most PSC patients are asymptomatic at presentation; clinical symptoms include fatigue, jaundice, weight loss, right upper quadrant pain and pruritis. Serum biochemical tests indicate cholestasis, and diagnosis is usually established by cholangiography. In symptomatic patients, median survival from presentation to death or liver transplantation is about 12 years. It is a premalignant condition, and the majority of deaths are from malignancy, particularly cholangiocarcinoma or colonic cancer. PSC has no curative treatment. Medical treatment with ursodeoxycholic acid may slow progression of the disease and reduce colonic dysplasia, though trials lack statistical significance. Liver transplantation is the only option in young patients with PSC and advanced liver disease.

Collectives of diagnostic biomarkers identify high-risk subpopulations of hematuria patients: exploiting heterogeneity in large-scale biomarker data.

BACKGROUND: Ineffective risk stratification can delay diagnosis of serious disease in patients with hematuria. We applied a systems biology approach to analyze clinical, demographic and biomarker measurements (n = 29) collected from 157 hematuric patients: 80 urothelial cancer (UC) and 77 controls with confounding pathologies. METHODS: On the basis of biomarkers, we conducted agglomerative hierarchical clustering to identify patient and biomarker clusters. We then explored the relationship between the patient clusters and clinical characteristics using Chi-square analyses. We determined classification errors and areas under the receiver operating curve of Random Forest Classifiers (RFC) for patient subpopulations using the biomarker clusters to reduce the dimensionality of the data. RESULTS: Agglomerative clustering identified five patient clusters and seven biomarker clusters. Final diagnoses categories were non-randomly distributed across the five patient clusters. In addition, two of the patient clusters were enriched with patients with 'low cancer-risk' characteristics. The biomarkers which contributed to the diagnostic classifiers for these two patient clusters were similar. In contrast, three of the patient clusters were significantly enriched with patients harboring 'high cancer-risk" characteristics including proteinuria, aggressive pathological stage and grade, and malignant cytology. Patients in these three clusters included controls, that is, patients with other serious disease and patients with cancers other than UC. Biomarkers which contributed to the diagnostic classifiers for the largest 'high cancer- risk' cluster were different than those contributing to the classifiers for the 'low cancer-risk' clusters. Biomarkers which contributed to subpopulations that were split according to smoking status, gender and medication were different. CONCLUSIONS: The systems biology approach applied in this study allowed the hematuric patients to cluster naturally on the basis of the heterogeneity within their biomarker data, into five distinct risk subpopulations. Our findings highlight an approach with the promise to unlock the potential of biomarkers. This will be especially valuable in the field of diagnostic bladder cancer where biomarkers are urgently required. Clinicians could interpret risk classification scores in the context of clinical parameters at the time of triage. This could reduce cystoscopies and enable priority diagnosis of aggressive diseases, leading to improved patient outcomes at reduced costs.

Performance of the Glasgow-Blatchford score in predicting clinical outcomes and intervention in hospitalized patients with upper GI bleeding.

BACKGROUND: Data regarding the utility of the Glasgow-Blatchford bleeding score (GBS) in hospitalized patients with upper GI hemorrhage are limited. OBJECTIVE: To evaluate the performance of the GBS in predicting clinical outcomes and the need for interventions in patients with upper GI hemorrhage. DESIGN: Prospective observational study. SETTING: Single, tertiary-care endoscopic center. PATIENTS: Between July 2010 and July 2012, 888 consecutive hospitalized patients managed for upper GI hemorrhage were entered into the study. INTERVENTION: GBS and Rockall scores. MAIN OUTCOME MEASUREMENTS: GBS and Rockall scores were prospectively calculated. The performance of these scores to predict the need for interventions and outcomes was assessed by using a receiver operating characteristic curve. RESULTS: Endoscopy was performed in 708 patients (80%). A total of 286 patients (40.3%) required endoscopic therapy, and 29 patients (3.8%) underwent surgery. GBS and post-endoscopy Rockall scores (post-E RS) were superior to pre-endoscopy Rockall scores in predicting the need for endoscopic therapy (area under the curve [AUC] 0.76 vs 0.76 vs 0.66, respectively) and rebleeding (AUC 0.71 vs 0.64 vs 0.57). The GBS was superior to Rockall scores in predicting the need for blood transfusion (AUC 0.81 vs 0.70 vs 0.68) and surgery (AUC 0.71 vs 0.64 vs 0.51). Patients with GBS scores ≤ 3 did not require intervention. LIMITATIONS: Subjective decision making as to need for endoscopic therapy and blood transfusion. CONCLUSION: Compared with post-E RS, the GBS was superior in predicting the need for blood transfusion and surgery in hospitalized patients with upper GI hemorrhage and was equivalent in predicting the need for endoscopic therapy, rebleeding, and death. There are potential cutoff GBS scores that allow risk stratification for upper GI hemorrhage, which warrant further evaluation.

Defining the spatial distribution of extracellular adenosine revealed a myeloid-dependent immunosuppressive microenvironment in pancreatic ductal adenocarcinoma.

BACKGROUND: The prognosis for patients with pancreatic ductal adenocarcinoma (PDAC) remains extremely poor. It has been suggested that the adenosine pathway contributes to the ability of PDAC to evade the immune system and hence, its resistance to immuno-oncology therapies (IOT), by generating extracellular adenosine (eAdo). METHODS: Using genetically engineered allograft models of PDAC in syngeneic mice with defined and different immune infiltration and response to IOT and autochthonous tumors in KPC mice we investigated the impact of the adenosine pathway on the PDAC tumor microenvironment (TME). Flow cytometry and imaging mass cytometry (IMC) were used to characterize the subpopulation frequency and spatial distribution of tumor-infiltrating immune cells. Mass spectrometry imaging (MSI) was used to visualize adenosine compartmentalization in the PDAC tumors. RNA sequencing was used to evaluate the influence of the adenosine pathway on the shaping of the immune milieu and correlate our findings to published data sets in human PDAC. RESULTS: We demonstrated high expression of adenosine pathway components in tumor-infiltrating immune cells (particularly myeloid populations) in the murine models. MSI demonstrated that extracellular adenosine distribution is heterogeneous in tumors, with high concentrations in peri-necrotic, hypoxic regions, associated with rich myeloid infiltration, demonstrated using IMC. Protumorigenic M2 macrophages express high levels of the Adora2a receptor; particularly in the IOT resistant model. Blocking the in vivo formation and function of eAdo (Adoi), using a combination of anti-CD73 antibody and an Adora2a inhibitor slowed tumor growth and reduced metastatic burden. Additionally, blocking the adenosine pathway improved the efficacy of combinations of cytotoxic agents or immunotherapy. Adoi remodeled the TME, by reducing the infiltration of M2 macrophages and regulatory T cells. RNA sequencing analysis showed that genes related to immune modulation, hypoxia and tumor stroma were downregulated following Adoi and a specific adenosine signature derived from this is associated with a poorer prognosis in patients with PDAC. CONCLUSIONS: The formation of eAdo promotes the development of the immunosuppressive TME in PDAC, contributing to its resistance to conventional and novel therapies. Therefore, inhibition of the adenosine pathway may represent a strategy to modulate the PDAC immune milieu and improve therapy response in patients with PDAC.

The impact of biomarkers in multivariate algorithms for bladder cancer diagnosis in patients with hematuria.

BACKGROUND: We appraised 23 biomarkers previously associated with urothelial cancer in a case-control study. Our aim was to determine whether single biomarkers and/or multivariate algorithms significantly improved on the predictive power of an algorithm based on demographics for prediction of urothelial cancer in patients presenting with hematuria. METHODS: Twenty-two biomarkers in urine and carcinoembryonic antigen (CEA) in serum were evaluated using enzyme-linked immunosorbent assays (ELISAs) and biochip array technology in 2 patient cohorts: 80 patients with urothelial cancer, and 77 controls with confounding pathologies. We used Forward Wald binary logistic regression analyses to create algorithms based on demographic variables designated prior predicted probability (PPP) and multivariate algorithms, which included PPP as a single variable. Areas under the curve (AUC) were determined after receiver-operator characteristic (ROC) analysis for single biomarkers and algorithms. RESULTS: After univariate analysis, 9 biomarkers were differentially expressed (t test; P < .05). CEA AUC 0.74; bladder tumor antigen (BTA) AUC 0.74; and nuclear matrix protein (NMP22) 0.79. PPP included age and smoking years; AUC 0.76. An algorithm including PPP, NMP22, and epidermal growth factor (EGF) significantly improved AUC to 0.90 when compared with PPP. The algorithm including PPP, BTA, CEA, and thrombomodulin (TM) increased AUC to 0.86. Sensitivities = 91%, 91%; and specificities = 80%, 71%, respectively, for the algorithms. CONCLUSIONS: Addition of biomarkers representing diverse carcinogenic pathways can significantly impact on the ROC statistic based on demographics. Benign prostate hyperplasia was a significant confounding pathology and identification of nonmuscle invasive urothelial cancer remains a challenge.

An early analysis of the cost-effectiveness of a diagnostic classifier for risk stratification of haematuria patients (DCRSHP) compared to flexible cystoscopy in the diagnosis of bladder cancer.

BACKGROUND: Urothelial bladder cancer (UBC) is the 5th most common cancer in Western societies. The most common symptom of UBC is haematuria. Cystoscopy the gold standard for UBC detection, allows direct observation of the bladder, but is expensive, invasive, and uncomfortable. This study examines whether an alternative new urine-based diagnostic test, the DCRSHP, is cost-effective as a triage diagnostic tool compared to flexible cystoscopy in the diagnosis of UBC in haematuria patients. METHODS: A model-based cost-utility analysis using cost per quality adjusted life year and life year gained, parameterised with secondary data sources. RESULTS: If the DCRSHP is targeted at haematuria patients at lower risk of having bladder cancer e.g. younger patients, non-smokers, then it can be priced as high as £620, and be both effective and cost-effective. Sensitivity analysis found that DCRSHP is approximately 80% likely to be cost-effective across all willingness to pay values (for a QALY) and prevalence estimates. CONCLUSION: This analysis shows the potential for a non-invasive test to be added to the diagnostic pathway for haematuria patients suspected of having UBC. If the DCRSHP is applied targeting haematuria patients at low risk of UBC, then it has the potential to be both effective and cost-effective.

Unique and Common Features of Innate-Like Human Vδ2+ γδT Cells and Mucosal-Associated Invariant T Cells.

Mucosal-associated invariant T (MAIT) cells are innate-like T cells abundant in humans that can be activated in a TCR-independent manner by inflammatory and antiviral cytokines. In humans, the capacity for TCR-independent activation is functionally linked to a transcriptional program that can be identified by the expression of the C-type lectin receptor, CD161. In addition to MAIT cells, it has been demonstrated that a subset of γδT cells expresses CD161 and can be activated by TCR-independent cytokine stimulation. In this study, we sought to clarify the nature of cytokine-responsive human γδT cells. We could link CD161 expression on Vδ2+ versus Vδ1+ γδT cells to the observation that Vδ2+ γδT cells, but not Vδ1+ γδT cells, robustly produced IFN-γ upon stimulation with a variety of cytokine combinations. Interestingly, both CD161+ and CD161- Vδ2+ γδT cells responded to these stimuli, with increased functionality within the CD161+ subset. This innate-like responsiveness corresponded to high expression of PLZF and IL-18Rα, analogous to MAIT cells. Vδ2+ γδT cells in human duodenum and liver maintained a CD161+ IL-18Rα+ phenotype and produced IFN-γ in response to IL-12 and IL-18 stimulation. In contrast to MAIT cells, we could not detect IL-17A production but observed higher steady-state expression of Granzyme B by Vδ2+ γδT cells. Finally, we investigated the frequency and functionality of γδT cells in the context of chronic hepatitis C virus infection, as MAIT cells are reduced in frequency in this disease. By contrast, Vδ2+ γδT cells were maintained in frequency and displayed unimpaired IFN-γ production in response to cytokine stimulation. In sum, human Vδ2+ γδT cells are a functionally distinct population of cytokine-responsive innate-like T cells that is abundant in blood and tissues with similarities to human MAIT cells.

Are Dominant Strictures in Primary Sclerosing Cholangitis a Risk Factor for Cholangiocarcinoma?

PURPOSE OF REVIEW: Cholangiocarcinoma is a devastating, unpredictable complication of large duct primary sclerosing cholangitis (PSC), which occurs in 5-15% of patients. The aim of this review is to discuss whether dominant strictures (DS) occurring in the larger bile ducts in PSC are a risk factor for the development of cholangiocarcinoma. RECENT FINDINGS: The development of DS is related to specific genetic polymorphisms affecting the innate immune system and the microbiome. In a recent study, the mean survival of PSC patients with DS was much worse (13.7 years) than for those without a DS (23 years). Survival difference was related to a 26% risk of cholangiocarcinoma, which developed only in those with DS. Half of the patients with cholangiocarcinoma presented within 4 months of the diagnosis of PSC. In another study, the risk of developing cholangiocarcinoma was directly related to the presence of underlying IBD, although this remains controversial. Efforts are being made towards surveying for cholangiocarcinoma including magnetic resonance imaging, endoscopic surveillance and serum tumour markers, but so far, an effective surveillance strategy has not been identified. DS should be treated endoscopically in the setting of symptoms, and there is limited evidence to suggest this may impact protectively on progression to cholangiocarcinoma. SUMMARY: It is established that the presence of symptomatic DS occurring in the larger bile ducts in PSC can be the first presentation of cholangiocarcinoma. There is an increasing body of evidence that even when proven to be benign, dominant biliary strictures predispose to the future development of cholangiocarcinoma. Regular surveillance should be targeted at this selected high-risk group of PSC patients.

Differential expression of unconventional myosins in apoptotic and regenerating chick hair cells confirms two regeneration mechanisms.

Hair cells of the inner ear are damaged by intense noise, aging, and aminoglycoside antibiotics. Gentamicin causes oxidative damage to hair cells, inducing apoptosis. In mammals, hair cell loss results in a permanent deficit in hearing and balance. In contrast, avians can regenerate lost hair cells to restore auditory and vestibular function. This study examined the changes of myosin VI and myosin VIIa, two unconventional myosins that are critical for normal hair cell formation and function, during hair cell death and regeneration. During the late stages of apoptosis, damaged hair cells are ejected from the sensory epithelium. There was a 4-5-fold increase in the labeling intensity of both myosins and a redistribution of myosin VI into the stereocilia bundle, concurrent with ejection. Two separate mechanisms were observed during hair cell regeneration. Proliferating supporting cells began DNA synthesis 60 hours after gentamicin treatment and peaked at 72 hours postgentamicin treatment. Some of these mitotically produced cells began to differentiate into hair cells at 108 hours after gentamicin (36 hours after bromodeoxyuridine (BrdU) administration), as demonstrated by the colabeling of myosin VI and BrdU. Myosin VIIa was not expressed in the new hair cells until 120 hours after gentamicin. Moreover, a population of supporting cells expressed myosin VI at 78 hours after gentamicin treatment and myosin VIIa at 90 hours. These cells did not label for BrdU and differentiated far too early to be of mitotic origin, suggesting they arose by direct transdifferentiation of supporting cells into hair cells.

Vitamin D improves endothelial dysfunction and restores myeloid angiogenic cell function via reduced CXCL-10 expression in systemic lupus erythematosus.

Patients with systemic lupus erythematosus (SLE) have accelerated cardiovascular disease and dysfunctional endothelial repair mechanisms. Myeloid angiogenic cells (MACs), derived from circulating monocytes, augment vascular repair by paracrine secretion of pro-angiogenic factors. We observed that SLE MACs are dysfunctional and secrete pro-inflammatory cytokines. We also found that the vitamin D receptor was transiently expressed during MAC differentiation and that in vitro, calcitriol increased differentiation of monocytes into MACs in both SLE and in a model using the prototypic SLE cytokine, interferon-alpha. The active form of vitamin D (calcitriol) restored the SLE MAC phenotype towards that of healthy subjects with reduced IL-6 secretion, and normalised surface marker expression. Calcitriol also augmented the angiogenic capacity of MACs via the down-regulation of CXCL-10. In SLE patients treated with cholecalciferol for 12 weeks, the improvement in endothelial function correlated with increase in serum 25(OH)D concentrations independently of disease activity. We also show that MACs were able to positively modulate eNOS expression in human endothelial cells in vitro, an effect further enhanced by calcitriol treatment of SLE MACs. The results demonstrate that vitamin D can positively modify endothelial repair mechanisms and thus endothelial function in a population with significant cardiovascular risk.

The need to embrace molecular profiling of tumor cells in prostate and bladder cancer.

PURPOSE: Current treatment strategies for urological cancer are still based on empirical formulae as opposed to treatment tailored for each cancer patient. To individualize treatment, the multiple molecular abnormalities within tumor cell populations needs to be mapped out. The aim of this article is to explain molecular profiling (MP) and its associated techniques so that the process is not purely seen as a research tool but as a future adjunctive measure in patient diagnosis and treatment. EXPERIMENTAL DESIGN: A Medline search of publications relating to MP of prostate and bladder cancer was carried out. A review article was written combining the relevant published literature along with the clinical and scientific experience of both centers. RESULTS: The advent of MP now provides a strategy by which these molecular abnormalities can be assessed. As well as being of diagnostic and prognostic use, these molecular profiles will identify putative molecular abnormalities within tumor cells that may be appropriate for therapeutic modulation. CONCLUSIONS: In prostate and bladder cancer, mapping out the molecular abnormalities could be translated into a valuable tool to help solve difficult issues regarding patient management decisions.

Urothelial cancer gene regulatory networks inferred from large-scale RNAseq, Bead and Oligo gene expression data.

BACKGROUND: Urothelial pathogenesis is a complex process driven by an underlying network of interconnected genes. The identification of novel genomic target regions and gene targets that drive urothelial carcinogenesis is crucial in order to improve our current limited understanding of urothelial cancer (UC) on the molecular level. The inference of genome-wide gene regulatory networks (GRN) from large-scale gene expression data provides a promising approach for a detailed investigation of the underlying network structure associated to urothelial carcinogenesis. METHODS: In our study we inferred and compared three GRNs by the application of the BC3Net inference algorithm to large-scale transitional cell carcinoma gene expression data sets from Illumina RNAseq (179 samples), Illumina Bead arrays (165 samples) and Affymetrix Oligo microarrays (188 samples). We investigated the structural and functional properties of GRNs for the identification of molecular targets associated to urothelial cancer. RESULTS: We found that the urothelial cancer (UC) GRNs show a significant enrichment of subnetworks that are associated with known cancer hallmarks including cell cycle, immune response, signaling, differentiation and translation. Interestingly, the most prominent subnetworks of co-located genes were found on chromosome regions 5q31.3 (RNAseq), 8q24.3 (Oligo) and 1q23.3 (Bead), which all represent known genomic regions frequently deregulated or aberated in urothelial cancer and other cancer types. Furthermore, the identified hub genes of the individual GRNs, e.g., HID1/DMC1 (tumor development), RNF17/TDRD4 (cancer antigen) and CYP4A11 (angiogenesis/ metastasis) are known cancer associated markers. The GRNs were highly dataset specific on the interaction level between individual genes, but showed large similarities on the biological function level represented by subnetworks. Remarkably, the RNAseq UC GRN showed twice the proportion of significant functional subnetworks. Based on our analysis of inferential and experimental networks the Bead UC GRN showed the lowest performance compared to the RNAseq and Oligo UC GRNs. CONCLUSION: To our knowledge, this is the first study investigating genome-scale UC GRNs. RNAseq based gene expression data is the data platform of choice for a GRN inference. Our study offers new avenues for the identification of novel putative diagnostic targets for subsequent studies in bladder tumors.

Restricted differentiation potential of progenitor cell populations obtained from the equine superficial digital flexor tendon (SDFT).

The aim of this study was to characterize stem and progenitor cell populations from the equine superficial digital flexor tendon, an energy-storing tendon with similarities to the human Achilles tendon, which is frequently injured. Using published methods for the isolation of tendon-derived stem/progenitor cells by low-density plating we found that isolated cells possessed clonogenicity but were unable to fully differentiate towards mesenchymal lineages using trilineage differentiation assays. In particular, adipogenic differentiation appeared to be restricted, as assessed by Oil Red O staining of stem/progenitor cells cultured in adipogenic medium. We then assessed whether differential adhesion to fibronectin substrates could be used to isolate a population of cells with broader differentiation potential. However we found little difference in the stem and tenogenic gene expression profile of these cells as compared to tenocytes, although the expression of thrombospondin-4 was significantly reduced in hypoxic conditions. Tendon-derived stem/progenitor cells isolated by differential adhesion to fibronectin had a similar differentiation potential to cells isolated by low density plating, and when grown in either normoxic or hypoxic conditions. In summary, we have found a restricted differentiation potential of cells isolated from the equine superficial digital flexor tendon despite evidence for stem/progenitor-like characteristics.