The prognostic utility of the transcription factor SRF in docetaxel-resistant prostate cancer: in-vitro discovery and in-vivo validation.

BACKGROUND: Docetaxel based therapy is one of the first line chemotherapeutic agents for the treatment of metastatic castrate-resistant prostate cancer. However, one of the major obstacles in the treatment of these patients is docetaxel-resistance. Defining the mechanisms of resistance so as to inform subsequent treatment options and combinations represents a challenge for clinicians and scientists. Previous work by our group has shown complex changes in pro and anti-apoptotic proteins in the development of resistance to docetaxel. Targeting these changes individually does not significantly impact on the resistant phenotype but understanding the central signalling pathways and transcription factors (TFs) which control these could represent a more appropriate therapeutic targeting approach. METHODS: Using a number of docetaxel-resistant sublines of PC-3 cells, we have undertaken a transcriptomic analysis by expression microarray using the Affymetrix Human Gene 1.0 ST Array and in conjunction with bioinformatic analyses undertook to predict dysregulated TFs in docetaxel resistant prostate cancer. The clinical significance of this prediction was ascertained by performing immunohistochemical (IHC) analysis of an identified TF (SRF) in the metastatic sites from men who died of advanced CRPC. Investigation of the functional role of SRF was examined by manipulating SRF using SiRNA in a docetaxel-resistant PC-3 cell line model. RESULTS: The transcription factors identified include serum response factor (SRF), nuclear factor kappa-B (NFκB), heat shock factor protein 1 (HSF1), testicular receptor 2 & 4 (TR2 &4), vitamin-D and retinoid x receptor (VDR-RXR) and oestrogen-receptor 1 (ESR1), which are predicted to be responsible for the differential gene expression observed in docetaxel-resistance. IHC analysis to quantify nuclear expression of the identified TF SRF correlates with both survival from date of bone metastasis (p = 0.003), survival from androgen independence (p = 0.00002), and overall survival from prostate cancer (p = 0.0044). Functional knockdown of SRF by siRNA demonstrated a reversal of apoptotic resistance to docetaxel treatment in the docetaxel-resistant PC-3 cell line model. CONCLUSIONS: Our results suggest that SRF could aid in treatment stratification of prostate cancer, and may also represent a therapeutic target in the treatment of men afflicted with advanced prostate cancer.

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.

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.

Cyclodextrin mediated delivery of NF-κB and SRF siRNA reduces the invasion potential of prostate cancer cells in vitro.

Prostate cancer is the most common cancer in men of the western world. To date, no effective treatment exists for metastatic prostate cancer and consequently, there is an urgent need to develop new and improved therapeutics. In recent years, the therapeutic potential of RNA interference (RNAi) has been extensively explored in a wide range of diseases including prostate cancer using numerous gene delivery vectors. The aims of this study were to investigate the ability of a non-viral modified cyclodextrin (CD) vector to deliver siRNA to the highly metastatic PC-3 prostate cancer cell line, to quantify the resulting knockdown of the two target genes (RelA and SRF) and to study the effects of the silencing on metastasis. Data from a Matrigel in vitro invasion assay indicated that the silencing of the target genes achieved by the CD vector resulted in significant reductions (P=0.0001) in the metastatic potential of these cells. As the silencing of these target genes was shown not to have a negative impact on cell viability, we hypothesise that the mechanism of invasion inhibition is due, in part, to the significant reduction observed (P⩽0.0001) in the level of pro-inflammatory cytokine, MMP9, which is known to be implicated in the metastasis of prostate cancer.

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.

Physical activity and its mechanistic effects on prostate cancer.

Beneficial effects of physical activity have been illustrated in numerous aspects of health. With the increasing incidence of prostate cancer and changes in physical activity of men, understanding the link between the two has important implications for changing this cancer burden. Both positive and negative associations between physical activity and prostate cancer have been previously demonstrated in observational epidemiological studies. Elucidating the biological mechanisms would lead to a better understanding of how physical activity influences the progression of prostate cancer. This review was undertaken to: (1) identify evidence in literature that demonstrates the effects of physical activity on skeletal muscle secretomes, (2) indicate the plausible signaling pathways these proteins might activate, and (3) identify evidence in literature that demonstrates the roles of the signaling pathways in prostate cancer progression and regression. We also discuss proposed biological mechanisms and signaling pathways by which physical activity may prevent the development and progression of prostate cancer. We discuss proteins involved in the normal and aberrant growth and development of the prostate gland that may be affected by physical activity. We further identify future directions for research, including a better understanding of the biological mechanisms, the need to standardize physical activity and identify mechanistic end points of physical activity that can then be correlated with outcomes.

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.

The impact of high-dose statin therapy on transendothelial neutrophil migration and serum cholesterol levels in healthy male volunteers.

RATIONALE: Cardiac surgery presents a risk to all major organs due to activation of the systemic inflammatory response. Patients referred for cardiac surgery are typically older, usually have comorbid conditions, and are thus at higher risk of postoperative multiorgan dysfunction. Patients demonstrating evidence of organ dysfunction require intensive postoperative management. Any means to predict and reduce the inflammatory response mounted postcardiac surgery could translate into a clinical benefit for the patient and reduce the length of stay in intensive care. OBJECTIVE: Statins are commonly used to prevent primary and secondary cardiovascular disease through their cholesterol-lowering effects. However, they have been shown to have anti-inflammatory properties, which may help reduce postoperative mortality and morbidity for patients undergoing cardiac surgery. The purpose of this study was to analyze the in vivo effects of high-dose atorvastatin (statin) on ex vivo neutrophil migration in healthy volunteers. METHODS: Thirteen healthy male volunteers consented and were placed on high-dose (40 mg) statin therapy for 2 weeks. At week 0 and week 2, full blood count, liver function, serum cholesterol and creatine kinase were assessed, as was neutrophil migration. RESULTS: Neutrophil migration of healthy volunteers was significantly reduced after 2 weeks of high-dose statin therapy (p = 0.002), as was serum cholesterol (p <0.001). There was no change in liver function during statin treatment. CONCLUSION: Statins have an established role as cholesterol-lowering agents, and this study demonstrates that they also potentially have an anti-inflammatory effect in healthy male volunteers.

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.

Detecting soluble clusterin in in-vitro and in-vivo models of prostate cancer.

PSA, the only relevant marker for prostate cancer, has a low predictive value; moreover its low threshold leads to unnecessary biopsies with associated complications. Identification of prognostic factors is an important goal in prostate cancer. In the search for new markers, clusterin, has some potential as it is closely linked with cancer progression and resistance to apoptosis. We looked at the expression of secreted clusterin (sCLU) in prostate cells to determine correlations with progression and drug resistance. The plasmatic expression of sCLU was also investigated in order to use it as a potential marker for prostate cancer. sCLU expression was studied using Western blotting on cultured prostate cells, PWR-1E, PC3 and PC3 Docetaxel resistant cells in the cytosol and culture medium. An inhouse ELISA test was developed to determine sCLU expression in culture media and plasma samples. A patient cohort was identified from the Prostate Cancer Research Consortium Bio-Resource and plasmatic expression of sCLU was studied using western blotting and the inhouse ELISA test. Only the fully processed form of sCLU was identified in the medium of cells with increased expression associated with increased progression of disease and resistance to docetaxel. Plasmatic expression of sCLU was significantly higher in the plasma of patients with high grade prostate cancer with extracapsular extension than in the plasma of prostate cancer patients without extracapsular extension. Plasmatic sCLU may be an effective prognostic marker of prostate cancer and needs to be tested in a multimarker approach.

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.

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.

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.

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.