Persistent systemic monocyte and neutrophil activation in neonatal encephalopathy.

AIM: Circulating immune cell activation is associated with worse outcome in adult and animal models of brain injury. Our aim was to profile the systemic inflammatory response over the first week of life in infants at risk of neonatal encephalopathy (NE) and correlate early neutrophil and monocyte endotoxin and activation responses with outcome. METHODS: Prospective observational study in a tertiary referral university hospital including 22 infants requiring resuscitation at birth who had serial (five time points) neutrophil and monocyte CD11b (marker of cell adhesion), intracellular reactive oxygen intermediates (ROI; cell activation) and Toll-like receptor (TLR; endotoxin recognition) before and after endotoxin stimulation ex vivo compared to neonatal controls. RESULTS: All neonates requiring resuscitation at delivery (n = 122 samples) had higher neutrophil and monocyte CD11b and TLR-4 expression compared with adults and neonatal controls. Neonates with abnormal neuroimaging and/or severe NE had increased CD11b, ROI and TLR-4. Increased polymorphonuclear leukocytes TLR-4 expression was associated with increased mortality in infants with NE. CONCLUSION: Innate immune dysregulation in the first week of life is associated with severity of outcome in neonatal brain injury in this cohort and may be amenable to immunomodulation.

Persistent systemic monocyte and neutrophil activation in neonatal encephalopathy.

AIM: Circulating immune cell activation is associated with worse outcome in adult and animal models of brain injury. Our aim was to profile the systemic inflammatory response over the first week of life in infants at risk of neonatal encephalopathy and correlate early neutrophil and monocyte endotoxin and activation responses with outcome. METHODS: Prospective observational study in a tertiary referral university hospital including 22 infants requiring resuscitation at birth who had serial (five time points) neutrophil and monocyte CD11b (marker of cell adhesion) (intracellular Reactive oxygen intermediates) ROI (cell activation), and Toll-like receptor (endotoxin recognition) before and after endotoxin stimulation ex vivo compared to neonatal controls. RESULTS: All neonates requiring resuscitation at delivery (n = 122 samples) had higher neutrophil and monocyte CD11b and TLR-4 expressions compared with adults and neonatal controls. Neonates with abnormal neuroimaging and/or severe neonatal encephalopathy had increased CD11b, ROI and TLR-4. Increased PMN TLR-4 expression was associated with increased mortality in infants with neonatal encephalopathy (NE). CONCLUSION: Innate immune dysregulation in the first week of life is associated with severity of outcome in neonatal brain injury in this cohort and may be amenable to immunomodulation.

Predicting prostate cancer: analysing the clinical efficacy of prostate cancer risk calculators in a referral population.

BACKGROUND: The decision to proceed to biopsy for the diagnosis of prostate cancer in clinical practice is a difficult one. Prostate cancer risk calculators allow for a systematic approach to the use of patient information to predict a patient's likelihood of prostate cancer. AIMS: In this paper, we validate the two leading prostate cancer risk calculators, the prostate cancer prevention trial (PCPT) and the European Randomized Study of Screening for Prostate Cancer (ERSPC) in an Irish population. METHODS: Data were collected for 337 men referred to one tertiary referral center in Ireland. Calibration analysis, ROC analysis and decision curve analysis were undertaken to ascertain the performance of the PCPT and the ERSPC risk calculators in this cohort. RESULTS: Of 337 consecutive biopsies, cancer was subsequently diagnosed in 146 men (43 %), 98 (67 %) of which were high grade. The AUC for the PCPT and ERSPC risk calculators were 0.68 and 0.66, respectively for the prediction of prostate cancer. Each calculator was sufficiently calibrated in this cohort. Decision curve analysis demonstrated a net benefit via the use of the PCPT and ERSPC risk calculators in the diagnosis of prostate cancer. CONCLUSIONS: The PCPT and ERSPC risk calculators achieve a statistically significant prediction of prostate cancer in this Irish population. This study provides external validation for these calculators, and therefore these tools can be used to aid in clinical decision making.

Prostate cancer risk assessment tools in an unscreened population.

OBJECTIVE: To compare the prostate cancer prevention trial risk calculator (PCPT-RC) and European randomized study of screening for prostate cancer risk calculator (ERSPC-RC) in a unique unscreened population from the West of Ireland. PATIENTS AND METHODS: Data was prospectively recorded for all 556 consecutive men who underwent prostate biopsy at our institution as part of the Rapid Access Prostate Assessment Clinic program in Ireland. The estimated probabilities of detecting prostate cancer and high-grade disease were calculated using the PCPT and ERSPC risk calculators. For each calculator the discriminative ability, calibration and clinical utility was assessed. RESULTS: Prostate cancer was detected in 49% and high-grade prostate cancer in 34% of men. Receiver operating characteristic curve analysis demonstrated that the PCPT-RCs outperformed the ERSPC-RCs for the prediction of prostate cancer areas underneath the ROC curve (AUC 0.628 vs. 0.588, p = 0.0034) and for the prediction of high-grade prostate cancer (AUC 0.792 vs. 0.690, p = 0.0029). Both risk calculators generally over-predicted the risk of prostate cancer and high-grade disease across a wide range of predicted probabilities. Decision curve analysis suggested greater net benefit using the PCPT-RCs in this population. CONCLUSIONS: Multivariable nomograms can further aid patient counselling for early prostate cancer detection. In unscreened men from Western Ireland, the PCPT-RCs provided better discrimination for overall prostate cancer and high-grade disease compared to the ERSPC-RC. However, both tools overpredicted the risk of cancer detection on biopsy, and it is possible that a different set of predictive variables may be more useful in this population.

Neonatal brain injury and systemic inflammation: modulation by activated protein C ex vivo.

Infection and inflammation can be antecedents of neonatal encephalopathy (NE) and increase the risk of neurological sequelae. Activated protein C (APC) has anti-coagulant and anti-inflammatory effects and provides neuroprotection in brain and spinal cord injury. We examined neutrophil and monocyte responses to lipopolysaccharide (LPS) in infants with NE compared with healthy adult and neonatal controls, and also studied the effect of APC. Whole blood was incubated with LPS and APC and Toll-like receptor (TLR)-4 (LPS recognition), CD11b expression (activation) and intracellular reactive oxygen intermediate (ROI; function) release from neutrophils and monocytes was examined by flow cytometry serially from days 1 to 7. We found a significant increase in neutrophil ROI in infants with NE on day 3 following LPS compared to neonatal controls and this augmented response was reduced significantly by APC. Neutrophil and monocyte CD11b expression was increased significantly on day 1 in infants with NE compared to neonatal controls. LPS-induced neutrophil TLR-4 expression was increased significantly in infants with NE on days 3 and 7 and was reduced by APC. LPS-induced monocyte TLR-4 was increased significantly in infants with NE on day 7. Neutrophil and monocyte activation and production of ROIs may mediate tissue damage in infants with NE. APC modified LPS responses in infants with NE. APC may reduce the inflammatory responses in NE and may ameliorate multi-organ dysfunction. Further study of the immunomodulatory effects of protein C may be warranted using mutant forms with decreased bleeding potential.

Bacterial lipopolysaccharide promotes profibrotic activation of intestinal fibroblasts.

BACKGROUND: Fibroblasts play a critical role in intestinal wound healing. Lipopolysaccharide (LPS) is a cell wall component of commensal gut bacteria. The effects of LPS on intestinal fibroblast activation were characterized. METHODS: Expression of the LPS receptor, toll-like receptor (TLR) 4, was assessed in cultured primary human intestinal fibroblasts using flow cytometry and confocal microscopy. Fibroblasts were treated with LPS and/or transforming growth factor (TGF) beta1. Nuclear factor kappaB (NFkappaB) pathway activation was assessed by inhibitory kappaBalpha (IkappaBalpha) degradation and NFkappaB promoter activity. Fibroblast contractility was measured using a fibroblast-populated collagen lattice. Smad-7, a negative regulator of TGF-beta1 signalling, and connective tissue growth factor (CTGF) expression were assessed using reverse transcriptase-polymerase chain reaction and western blot. The NFkappaB pathway was inhibited by IkappaBalpha transfection. RESULTS: TLR-4 was present on the surface of intestinal fibroblasts. LPS treatment of fibroblasts induced IkappaBalpha degradation, enhanced NFkappaB promoter activity and increased collagen contraction. Pretreatment with LPS (before TGF-beta1) significantly increased CTGF production relative to treatment with TGF-beta1 alone. LPS reduced whereas TGF-beta1 increased smad-7 expression. Transfection with an IkappaBalpha plasmid enhanced basal smad-7 expression. CONCLUSION: Intestinal fibroblasts express TLR-4 and respond to LPS by activating NFkappaB and inducing collagen contraction. LPS acts in concert with TGF-beta1 to induce CTGF. LPS reduces the expression of the TGF-beta1 inhibitor, smad-7.

Neutrophil immunosurveillance for heart transplant rejection: a prospective study.

Immunologic surveillance for rejection detection in human heart transplantation offers many potential advantages. To date, investigative efforts have focused primarily on the acquired immune system, particularly the lymphocyte. Little attention has been given to aspects of innate immune function. We have previously reported that perioperative neutrophil adhesion molecule expression is associated with early rejection episodes after human cardiac transplantation. Herein we have investigated the utility of neutrophil immunosurveillance in human heart transplant recipients at later time points. We recruited patients more than 3 months after transplantation. Neutrophil assessment was performed simultaneously with an endomyocardial biopsy that showed rejection. No significant relationship was seen between neutrophil maturity (P = .622; n = 34), adhesion marker expression (P = .567; n = 34), respiratory burst (P = .604; n = 34), or apoptosis rates (P = .662; n = 34) and contemporary rejection status at >3 months after transplantation. However, interesting relationships were noted between neutrophil adhesion markers at this late stage and historical rejection status. Higher levels of the adhesion protein CD11b observed at this late stage were significantly associated with a history of higher rejection grades in the first postoperative biopsy (Spearman rank coefficient 0.359; R = 0.304; P = .005; n = 62). Other aspects of neutrophil function and persistence were not significantly associated with rejection history. This finding, combined with the previously reported findings, supports a role for an individual phenotype in neutrophil function in early rejection episodes after transplantation.

Endoglin negatively regulates transforming growth factor beta1-induced profibrotic responses in intestinal fibroblasts.

BACKGROUND: Fibroblasts isolated from strictures in Crohn's disease (CD) exhibit reduced responsiveness to stimulation with transforming growth factor (TGF) beta1. TGF-beta1, acting through the smad pathway, is critical to fibroblast-mediated intestinal fibrosis. The membrane glycoprotein, endoglin, is a negative regulator of TGF-beta1. METHODS: Intestinal fibroblasts were cultured from seromuscular biopsies of patients undergoing intestinal resection for CD strictures or from control patients. Endoglin expression was assessed using confocal microscopy, flow cytometry and western blot. The effect of small interfering (si) RNA-mediated knockdown and plasmid-mediated overexpression of endoglin on fibroblast responsiveness to TGF-beta1 was assessed by examining smad phosphorylation, smad binding element (SBE) promoter activity, connective tissue growth factor (CTGF) expression and ability to contract collagen. RESULTS: Crohn's stricture fibroblasts expressed increased constitutive cell-surface and whole-cell endoglin relative to control cells. Endoglin co-localized with filamentous actin. Fibroblasts treated with siRNA directed against endoglin exhibited enhanced TGF-beta1-mediated smad-3 phosphorylation, and collagen contraction. Cells transfected with an endoglin plasmid did not respond to TGF-beta1 by exhibiting SBE promoter activity or producing CTGF. CONCLUSION: Fibroblasts from strictures in CD express increased constitutive endoglin. Endoglin is a negative regulator of TGF-beta1 signalling in the intestinal fibroblast, modulating smad-3 phosphorylation, SBE promoter activity, CTGF production and collagen contraction.

External validation of the 2007 and 2001 Partin tables in Irish prostate cancer patients.

INTRODUCTION: External validation of predictive prostate cancer nomograms is of critical importance within distinct geographical locations, prior to their institution into routine clinical practice. We performed external validation of the 2007 and 2001 Partin tables in a cohort of Irish prostate cancer patients. PATIENTS AND METHODS: Men enrolled in the Irish Prostate Cancer Research Consortium (n = 175) and who had undergone radical prostatectomy between 2004 and 2008 were used to externally validate the 2007 and 2001 Partin tables. A comparative analysis of the clinical and pathological parameters of the Irish and Partin patient cohorts was performed. The reported receiver operating characteristic (ROC) curve derived area under the curve (AUC) values were used to assess for variations in predictive accuracy. Statistical analyses were calculated with R software. RESULTS: AUC values assigned to the differentiation of extra-prostatic extension and seminal vesicle invasion using the 2007 tables are 22 and 3%, respectively. The 2007 Partin tables showed superior accuracy for all parameters, excluding seminal vesicle invasion. CONCLUSION: Cumulatively the Partin tables showed poor discriminate ability for prediction of post-radical prostatectomy pathological outcomes in Irish men, necessitating caution in their clinical utilisation.

Novel predictive tools for Irish radical prostatectomy pathological outcomes: development and validation.

AIMS: We developed and validated prostate cancer predictive models for Irish patients, allowing individualised predictions of radical prostatectomy pathological outcomes. METHODS: Retrospective review of the Irish Prostate Cancer Research Consortium database from 2003 to 2008 was performed. Two predictive models were formulated: a replica of the Partin tables (n = 169) and a look-up table based on PSA and biopsy Gleason Score (n = 253). Clinico-pathological parameters were compared to the Partin data set. Internal validation was performed. RESULTS: In total, 70% of patients were at clinical stage T1c. 5.8% had a PSA less than 4.1 ng/ml, whereas 25% of the Partin patients had a PSA in this range. Maximal predictive accuracy was seen for seminal vesicle invasion (area under the curve = 72%). Prediction of extra-prostatic extension and lymph node involvement was only equivalent to that of a chance phenomenon. CONCLUSIONS: Our current results do not support the introduction of the formulated predictive models into routine clinical practice.

Carbonic anhydrase IX expression in prostate cancer.

Tumour hypoxia is associated with over 70% of solid tumours including prostate and colorectal cancer. Hypoxia promotes tumour progression and resistance to treatment. Carbonic anhydrase IX (CA IX) is an endogenous marker of hypoxia. It is expressed in lung and renal cell carcinomas and is associated with a poor prognosis. CA IX has an important role in maintaining pH levels in the highly metabolically active cancer cell. The expression of CA IX in prostate cancer has not previously been investigated. Immunohistochemistry was used to examine CA IX expression in 59 patients, using tissue microarrays (TMAs) and full sections of BPH, surrounding stroma and prostate adenocarcinoma. Cores reviewed included 189 BPH, 130 Gleason grade 3, 93 Gleason grade 4, 40 Gleason grade 5. CA IX expression in colorectal cancer and HIF 1alpha in prostate cancer acted as positive controls. There was only occasional cell staining for CA IX expression. Although prostate cancer is a hypoxic tumour it does not express CA IX. This implies it relies on alternative pathways for maintaining pH balance in cancer. These studies would indicate that CA IX is not a suitable marker of hypoxia in prostate cancer.

Simvastatin impairs smad-3 phosphorylation and modulates transforming growth factor beta1-mediated activation of intestinal fibroblasts.

BACKGROUND: Transforming growth factor (TGF) beta1, acting through the smad pathway, is critical to fibroblast-mediated intestinal fibrosis. Simvastatin exhibits antifibrotic properties. This study assessed the effects of simvastatin on TGF-beta1-mediated intestinal fibroblast activation. METHODS: Human intestinal fibroblasts were activated with TGF-beta1 with or without simvastatin or the cholesterol pathway intermediates farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP). Collagen-Ialpha2 expression was assessed by reverse transcriptase-polymerase chain reaction. Connective tissue growth factor (CTGF) and smad phosphorylation were evaluated by western blot, and plasminogen activator inhibitor (PAI) 1 activity by enzyme-linked immunosorbent assay. Fibroblast filamentous (F)-actin accumulation was assessed by confocal microscopy and contraction by a fibroblast-populated collagen lattice (FPCL) model. RESULTS: TGF-beta1 treatment of fibroblasts induced smad-2/3 phosphorylation, CTGF and collagen-Ialpha2 production, F-actin bundling, FPCL contraction and PAI-1 activation. Pretreatment with simvastatin inhibited the induction of CTGF and collagen-Ialpha2, PAI-1 activation, F-actin bundling and FPCL contraction. The inhibitory effect of simvastatin on PAI-1 activation was reversed by GGPP and FPP. Simvastatin pretreatment inhibited TGF-beta1-mediated phosphorylation of smad-3. CONCLUSION: Simvastatin abrogates TGF-beta1-mediated intestinal fibroblast activation by inhibition of smad-3 phosphorylation. These findings offer a mechanism for the antifibrotic effects of simvastatin and a therapeutic entry point in the treatment of intestinal fibrosis.

Statins alter neutrophil migration by modulating cellular Rho activity--a potential mechanism for statins-mediated pleotropic effects?

The ability of neutrophils to sense and migrate toward damaged tissue is a vital component of the innate immune response. Paradoxically, this same migration serves as the hallmark of a number of inflammatory conditions, including ischemic reperfusion injury, atherosclerosis, arthritis, and Crohn's disease. More recent evidence suggests that neutrophil infiltration into the cardiac allograft following transplantation is a contributing factor in allograft rejection. We have demonstrated previously a positive correlation between the degree of neutrophil migration and subsequent rejection grades in a cohort of cardiac transplant recipients. Intracellular signaling pathways that are intimately involved in neutrophil migration thus offer potential targets of manipulation in the treatment of such conditions. 3-hydroxy-3-methylyglutaryl-coenzyme A reductase inhibitors or statins are emerging as potential anti-inflammatory agents and have a proven survival benefit in the transplant population. Yet, little is known about their ability to modulate neutrophil function and their subsequent mechanism of action. We demonstrate here that pravastatin, simvastatin, and atorvastatin significantly reduce neutrophil transendothelial migration toward the chemoattractant fMLP. This effect is independent of any change in neutrophil adhesion or adhesion molecule expression but is related to the ability of statins to reduce fMLP-induced Rho activity in neutrophils. This was confirmed by the ability of the Rho precursor geranylgeranyl pyrophosphate to rescue the statin-mediated reduction in neutrophil transendothelial migration. Understanding the mechanisms of action of statins in the neutrophil allows for their use in targeting excessive migration in inappropriate inflammatory conditions.

Differential mechanisms of bicalutamide-induced apoptosis in prostate cell lines.

Bicalutamide is a non-steroidal antiandrogen used in the treatment of prostate cancer. Although widely accepted as an androgen receptor antagonist, the mechanism by which it induces apoptosis remains unclear. Defining exact pathways by which bicalutamide induces its apoptotic effects would help to advance its clinical applications. We aimed to (a) examine the apoptotic effects of bicalutamide at 24 h and (b) comment on the role of the caspases and calpains in mediating bicalutamide-induced apoptosis in androgen-dependent and androgen-independent cells. PWR-1E, PC-3 and DU-145 cells were treated with bicalutamide and assessed for apoptosis by flow cytometry at 24 h. DU-145 cells were used to compare differences between two different metastatic receptor-negative cells and to verify apoptotic induction at 48 h. To delineate a specific pathway of action for bicalutamide, PC-3 and PWR-1E cells were pretreated with specific inhibitors of caspase-dependent (zVAD-FMK) and caspase-independent pathways (calpain 2 inhibitor). Bicalutamide induced apoptosis in androgen-dependent PWR-1E cells via a caspase-dependent and calpain-independent mechanism. In androgen-independent PC-3 cells, bicalutamide also induced apoptosis by mechanisms that were partially inhibited by pan-caspase inhibition but were partially calpain dependent. Understanding into how bicalutamide exerts its effects in androgen-independent cells will yield further insights into the treatment of hormone-refractory disease.

Maternal and neonatal lipopolysaccharide and Fas responses are altered by antenatal risk factors for sepsis.

The diagnosis of neonatal sepsis is difficult, resulting in unnecessary treatment to minimize morbidity and mortality. We hypothesized that exposure to antenatal risk factors for sepsis alters the perinatal neutrophil phenotype. The study setting was a tertiary referral university-affiliated maternity and neonatal hospital. Neutrophils from adults, normal neonates, neonates with antenatal sepsis risk factors and their respective maternal samples were incubated alone, with agonistic Fas antibody or with lipopolysaccharide (LPS). Surface receptor CD11b expression and the percentage apoptosis (persistent inflammatory response) were assessed using flow cytometry. Both mothers and asymptomatic neonates exposed to maternal sepsis risk factors had increased spontaneous neutrophil apoptosis compared to their respective controls. Infants with sepsis were LPS and Fas hyporesponsive. Maternal neutrophils had a delay in apoptosis in all groups with enhanced LPS and Fas responses associated with neonatal sepsis. CD11b expression was not altered significantly between groups. Maternal neutrophil function is altered in neonatal sepsis and may have a diagnostic role. Neonatal sepsis was associated with LPS hyporesponsiveness, potentially increasing susceptibility to infection.

In silico mining identifies IGFBP3 as a novel target of methylation in prostate cancer.

Promoter hypermethylation is central in deregulating gene expression in cancer. Identification of novel methylation targets in specific cancers provides a basis for their use as biomarkers of disease occurrence and progression. We developed an in silico strategy to globally identify potential targets of promoter hypermethylation in prostate cancer by screening for 5' CpG islands in 631 genes that were reported as downregulated in prostate cancer. A virtual archive of 338 potential targets of methylation was produced. One candidate, IGFBP3, was selected for investigation, along with glutathione-S-transferase pi (GSTP1), a well-known methylation target in prostate cancer. Methylation of IGFBP3 was detected by quantitative methylation-specific PCR in 49/79 primary prostate adenocarcinoma and 7/14 adjacent preinvasive high-grade prostatic intraepithelial neoplasia, but in only 5/37 benign prostatic hyperplasia (P < 0.0001) and in 0/39 histologically normal adjacent prostate tissue, which implies that methylation of IGFBP3 may be involved in the early stages of prostate cancer development. Hypermethylation of IGFBP3 was only detected in samples that also demonstrated methylation of GSTP1 and was also correlated with Gleason score > or =7 (P=0.01), indicating that it has potential as a prognostic marker. In addition, pharmacological demethylation induced strong expression of IGFBP3 in LNCaP prostate cancer cells. Our concept of a methylation candidate gene bank was successful in identifying a novel target of frequent hypermethylation in early-stage prostate cancer. Evaluation of further relevant genes could contribute towards a methylation signature of this disease.

Differential white cell count relationships with human cardiac allograft rejection.

The focus of research in allograft rejection has targeted the lymphocyte, with little attention given to the neutrophil. Recent data indicate that a perioperative neutrophil influx into the cardiac allograft influences early rejection. Factors that influence neutrophil transendothelial migration might offer predictive markers of rejection. We explored the relationship between the number of circulating neutrophils in heart transplant recipients and the development of rejection. Differential white cell counts were obtained prior to transplantation and concurrently with subsequent endomyocardial rejection surveillance biopsies for 53 heart transplant recipients undergoing 410 biopsies. Preoperative differential white cell counts had no relationship with rejection. In the first 3 months after transplantation, no relationship was found between contemporary differential white cell counts and rejection. However, more than 3 months following surgery, rejection grade positively correlated on univariate analysis with neutrophil counts and the usage of cyclosporine, prednisolone, and mycophenolate. There was no relationship with eosinophils or lymphocytes. Multivariate analysis demonstrated a persistent relationship among rejection severity, neutrophil count, and prednisolone usage. A significant positive association of higher steroid usage with higher rejection grades must reflect efforts to treat patients with rejection. The significant association of higher neutrophil counts with higher rejection severity might suggest a pathological contribution to rejection. However, given the neutrophilia response to acute steroid administration, we must conclude that the neutrophil association was related to steroid administration. The absence of a relationship between white cell counts and rejection suggests that functional rather than antiproliferative strategies may offer the greatest therapeutic potential.

Application of proteomic strategies to the identification of urinary biomarkers for prostate cancer: a review.

In the post-genomic era, genes and proteins are now studied on a more comprehensive scale. Studying disease processes at only the genetic or transcriptomic level will give an incomplete amount of information. A proteomic approach potentially allows for a more global overview of how disease processes affect the proteins present in cells, tissues and organisms. The challenge arises in determining which proteins are affected in specific diseases and establishing which of these changes are unique to a particular disease. Existing and emerging proteomic technologies allow for high throughput analysis of proteins in a variety of sample types. Prostate cancer is a significant male health problem in the Western world. It is widely accepted that more specific prognostic and diagnostic markers of prostate cancer are urgently required. The present paper suggests that urine may be an attractive biofluid in which to pursue the identification of novel biomarkers of prostate cancer. This review introduces some proteomic techniques including mass spectrometry and the newer, quantitative proteomic strategies. It focuses on the potential application of these platforms to novel urinary biomarker identification in prostate malignancy. It also includes a synopsis of the current literature on urinary proteomics.

Expression and regulation of connective tissue growth factor by transforming growth factor beta and tumour necrosis factor alpha in fibroblasts isolated from strictures in patients with Crohn's disease.

BACKGROUND: Connective tissue growth factor (CTGF) stimulates fibroblast proliferation and extracellular matrix production. Fibroblasts may initiate stricture formation in Crohn's disease through overexpression of CTGF. Stricturing that occurs in patients with Crohn's disease after treatment with anti-tumour necrosis factor (TNF) alpha may be due to dysregulation of CTGF homeostasis. The aim of this study was to examine CTGF expression and regulation in fibroblasts isolated from patients with Crohn's disease. METHODS: Fibroblasts were isolated by a primary explant technique from serosal biopsies of strictured segments of bowel in eight patients undergoing resection for Crohn's disease and from normal colon in seven patients having resection for benign or malignant colorectal disease. Cells were stimulated with transforming growth factor (TGF) beta and TNF-alpha. CTGF protein and mRNA expression were measured by western blotting and real-time polymerase chain reaction respectively. RESULTS: Mean(s.d.) CTGF protein expression in strictured Crohn's fibroblasts was higher than that in normal fibroblasts (56.5(9.7) versus 17.0(10.0) respectively; P = 0.011). In normal and strictured Crohn's fibroblasts, culture with TGF-beta increased CTGF protein and mRNA expression. Co-culture of normal fibroblasts with TNF-alpha suppressed TGF-beta-stimulated CTGF expression. CONCLUSION: : Increased expression of CTGF in strictured Crohn's fibroblasts underlies its role in fibrosis. TNF-alpha suppresses fibrosis by downregulating fibroblast CTGF expression, an effect that may be lost following anti-TNF-alpha treatment, thereby promoting stricture formation.

Novel titanocene anti-cancer drugs and their effect on apoptosis and the apoptotic pathway in prostate cancer cells.

Advanced prostate cancer is not curable by current treatment strategies indicating a significant need for new chemotherapeutic options. Highly substituted ansa-titanocene compounds have shown promising cytotoxic activity in a range of cancers. The objectives of this study are to examine the effects of these titanocene compounds on prostate cancer cells. Prostate cell lines were treated with three novel titanocene compounds and compared to titanocene dichloride and cisplatin. Percent apoptosis, viability and cell cycle were assessed using propidium iodide DNA incorporation with flow cytometry. Cytochrome C was assessed by western blotting of mitochondrial and cytoplasmic fractions. Apoptosis Inducing Factor was assessed by confocal microscopy. These novel compounds induced more apoptosis compared to cisplatin in a dose dependent manner. Compound Y had the most significant effect on cell cycle and apoptosis. Despite the release of cytochrome C from the mitochondrial fraction there was no inhibition of apoptosis with the pan caspase inhibitor, ZVAD-FMK. AIF was shown to translocate from the cytosol to the nucleus mediating a caspase independent cell death. Bcl-2 over expressing PC-3 cells, which were resistant to cisplatin induced apoptosis, underwent apoptosis following treatment with all the titanocene compounds. This study demonstrates possible mechanisms by which these novel titanocene compounds can mediate their apoptotic effect in vitro. The fact that they can induce more apoptosis than cisplatin in advanced cancer cell lines would confer an advantage over cisplatin. They represent exciting new agents with future potential for the treatment of advanced prostate cancer.