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1.  Jack Edwards 
BMJ : British Medical Journal  2003;327(7418):813.
PMCID: PMC214177
2.  Electrokinetic and Hemostatic Profiles of Nonwoven Cellulosic/Synthetic Fiber Blends with Unbleached Cotton 
Greige cotton contains waxes and pectin on the outer surface of the fiber that are removed when bleached, but these components present potential wound dressing functionality. Cotton nonwovens blended with hydrophobic and hydrophilic fibers including viscose, polyester, and polypropylene were assessed for clotting activity with thromboelastography (TEG) and thrombin production. Clotting was evaluated based on TEG measurements: R (time to initiation of clot formation), K (time from end of R to a 20 mm clot), α (rate of clot formation according to the angle tangent to the curve as K is reached), and MA (clot strength). TEG values correlate to material surface polarity as measured with electrokinetic parameters (ζplateau, Δζ and swell ratio). The material surface polarity (ζplateau) varied from −22 to −61 mV. K values and thrombin concentrations were found to be inversely proportional to ζplateau with an increase in material hydrophobicity. An increase in the swell ratios of the materials correlated with decreased K values suggesting that clotting rates following fibrin formation increase with increasing material surface area due to swelling. Clot strength (MA) also increased with material hydrophobicity. Structure/function implications from the observed clotting physiology induced by the materials are discussed.
PMCID: PMC4285407  PMID: 25459983
nonwoven cotton; hemostasis; thromboelastography; electrokinetic properties; accelerated clotting; contact angle
3.  The in situ local immune response, tumour senescence and proliferation in colorectal cancer 
British Journal of Cancer  2013;109(8):2207-2216.
Immune cell infiltrates are important determinants of colorectal cancer (CRC) outcome. Their presence may be driven by tumour or host-specific factors. From previous studies in mice, senescence, a state of cell cycle arrest, may moderate tumour progression through upregulation of antitumour immune responses. The relationships between senescence and immune infiltrates have not previously been studied in humans. We explore whether a marker of senescence (p16ink4a) in combination with low level expression of a proliferation marker (ki-67) relate to T cell infiltrates in CRC, and whether p16ink4a, Ki-67 and immune infiltrates have similar prognostic value.
Immunostaining of p16inka and Ki-67 was performed within a CRC tissue microarray. Nuclear p16inka and Ki-67 were categorised as high/low. T-cell markers, CD3, CD45RO, CD8 and FOXP3 were scored separately as high/low grade in three areas of the tumour: the invasive margin (IM), tumour stroma and cancer cell nests (CCNs).
Two hundred and thirty stage I–III cancers were studied. High nuclear p16ink4a was expressed in 63% and high proliferation (Ki-67 >15%) in 61%. p16ink4a expression was associated with reduced CD45RO+ cells at the IM (P<0.05) and within the stroma (P<0.05) and reduced CD8+ cells at the IM (P<0.01). A low Ki-67 proliferative index was associated with reduced density of CD3+ cells in CCNs (P<0.01), reduced CD45RO+ cells at the IM (P<0.05) and within the CCNs (P<0.001), reduced FOXP3+ cells at the IM (P<0.001), within the stroma (P=0.001) and within CCNs (P<0.001) and reduced CD8+ cells at the IM (P<0.05) and within the CCNs (P<0.05). Tumours with both a low proliferative index and expression of p16ink4a demonstrated similar consistent relationships with reduced densities of T-cell infiltrates. On multivariate analysis, TNM stage (P<0.001), low CD3 cells at the IM (P=0.014), low CD8 cells at the IM (P=0.037), low proliferation (Ki-67; P=0.013) and low senescence (p16ink4a; P=0.002) were independently associated with poorer cancer survival.
Senescence, proliferation and immune cell infiltrates are independent prognostic factors in CRC. Although related to survival, p16ink4a-associated senescence is not associated with an upregulation of antitumour T-cell responses.
PMCID: PMC3798960  PMID: 24022192
immune cells; senescence; proliferation; colorectal cancer; p16ink4a; CDKN2A
4.  The relationship between lymphocyte subsets and clinico-pathological determinants of survival in patients with primary operable invasive ductal breast cancer 
British Journal of Cancer  2013;109(6):1676-1684.
The importance of lymphocyte subtypes in determining outcome in primary operable ductal invasive breast cancer remains unclear. The aim of present study was to examine the relationship between tumour lymphocyte subsets infiltrate and standard clinico-pathological factors and survival in patients with primary operable invasive ductal breast cancer.
The analysis of the inflammatory cell infiltrate, including lymphocyte subtypes, was undertaken using immunohistochemical techniques and visual quantitative and semi-quantitative techniques in 338 patients with ductal breast cancer.
The majority (91%) of patients had high grade inflammatory cell infiltrate. The median follow-up of the survivors was 164 months. During this period, 65 died of their cancer. On univariate analysis, tumour inflammatory cell infiltrate, macrophages infiltrate (P<0.05), lymphocytic infiltrate (P<0.001) and CD8+ T-lymphocytic infiltrate (P<0.01) were associated with improved cancer-specific survival, whereas neutrophil (P<0.05) and CD138+ B-lymphocytic infiltrate (P<0.001) were associated with poorer cancer-specific survival. On multivariate analysis, tumour lymphocytic infiltrate (P<0.001), macrophage infiltrate (P<0.05), CD8+ T-lymphocytic infiltrate (P<0.01) and CD138+ B-lymphocytic infiltrate (P<0.001) were independently associated with cancer survival. When the significant inflammatory cell types were included with tumour-based factors in multivariate analysis only tumour size (Hazard ratios (HR): 2.55, 95% confidence interval (CI): 1.53–4.27, P<0.001), Ki-67 index (HR: 2.08, 95% CI: 1.08–4.00, P<0.05), lymphovascular invasion (HR: 4.40, 95% CI: 2.07–9.35, P<0.001), macrophage infiltrate (HR: 0.49, 95% CI: 0.33–0.73, P<0.001), lymphocytic infiltrate (HR: 0.11, 95% CI: 0.05–0.23, P<0.001), CD8+ T-lymphocytic infiltrate (HR: 0.57, 95% CI: 0.38–0.87, P<0.001) and CD138+ B-lymphocytic infiltrate (HR: 2.86, 95% CI: 1.79–4.56, P<0.001) were independently associated with cancer survival.
The majority of patients with invasive ductal breast cancer had high-grade inflammatory cell infiltrate. In these patients, inflammatory cells including macrophage and lymphocytic infiltrate, and subsets CD8+ T-lymphocytic infiltrate and CD138+ B-lymphocytic infiltrate had superior prognostic value, compared with hormone status and lymph node involvement in patients with primary operable invasive ductal breast cancer.
PMCID: PMC3777002  PMID: 23982600
breast cancer; lymphocytes subsets; survival
5.  Diabetes regulates mitochondrial biogenesis and fission in neurons 
Diabetologia  2009;53(1):160-169.
Normal mitochondrial (Mt) activity is a critical component of neuronal metabolism and function. Disruption of Mt activity by altered Mt fission and fusion is the root cause of both neurodegenerative disorders and Charcot-Marie-Tooth Type 2A inherited neuropathy. The current study addressed the role of Mt fission in the pathogenesis of diabetic neuropathy (DN).
Mt biogenesis and fission were assayed in both in vivo and in vitro models of DN. Gene, protein, mitochondrial DNA and ultrastructural analyses were used to assess Mt biogenesis and fission.
Our data reveal increased Mt biogenesis in dorsal root ganglion (DRG) neurons from diabetic compared to non-diabetic mice. An essential step in Mt biogenesis is Mt fission, regulated by the Mt fission protein Drp1. Evaluation of in vivo diabetic neurons indicated small, fragmented Mt, suggesting increased fission. In vitro studies reveal short-term hyperglycemic exposure increased expression of Drp1. The influence of hyperglycemia-mediated Mt fission on cellular viability was evaluated by knockdown of Drp1. Knockdown of Drp1 resulted in decreased susceptibility to hyperglycemic damage.
We propose that: 1) Mt undergo biogenesis in response to hyperglycemia, but the increased biogenesis is insufficient to accommodate the metabolic load; 2) hyperglycemia causes an excess of Mt fission, creating small, damaged mitochondria; and 3) reduction of aberrant Mt fission increases neuronal survival and indicates an important role for the fission-fusion equilibrium in the pathogenesis of DN.
PMCID: PMC4011390  PMID: 19847394
Diabetic Neuropathy; Mitochondrial Biogenesis; Mitochondrial Fission
6.  Androgen receptor phosphorylation at serine 515 by Cdk1 predicts biochemical relapse in prostate cancer patients 
British Journal of Cancer  2012;108(1):139-148.
Prostate cancer cell growth is dependent upon androgen receptor (AR) activation, which is regulated by specific kinases. The aim of the current study is to establish if AR phosphorylation by Cdk1 or ERK1/2 is of prognostic significance.
Scansite 2.0 was utilised to predict which AR sites are phosphorylated by Cdk1 and ERK1/2. Immunohistochemistry for these sites was then performed on 90 hormone-naive prostate cancer specimens. The interaction between Cdk1/ERK1/2 and AR phosphorylation was investigated in vitro using LNCaP cells.
Phosphorylation of AR at serine 515 (pARS515) and PSA at diagnosis were independently associated with decreased time to biochemical relapse. Cdk1 and pCdk1161, but not ERK1/2, correlated with pARS515. High expression of pARS515 in patients with a PSA at diagnosis of ⩽20 ng ml−1 was associated with shorter time to biochemical relapse (P=0.019). This translated into a reduction in disease-specific survival (10-year survival, 38.1% vs 100%, P<0.001). In vitro studies demonstrated that treatment with Roscovitine (a Cdk inhibitor) caused a reduction in pCdk1161 expression, pARS515expression and cellular proliferation.
In prostate cancer patients with PSA at diagnosis of ⩽20 ng ml−1, phosphorylation of AR at serine 515 by Cdk1 may be an independent prognostic marker.
PMCID: PMC3553508  PMID: 23321516
androgen receptor; biomarker; phosphorylation; prostate cancer
7.  Upper airway collapsibility, dilator muscle activation and resistance in sleep apnoea 
The European respiratory journal  2007;30(2):10.1183/09031936.00063406.
The calibre of the upper airway is thought to be dependant upon its passive anatomy/collapsibility and the activation of pharyngeal dilator muscles. During awake periods, the more collapsible upper airway in obstructive sleep apnoea (OSA) increases the dilator muscle activity through a negative-pressure reflex.
A direct correlation between the critical closing pressure (Pcrit), as a measure of anatomy/collapsability and electromyogram (EMG) activity of genioglossus EMG (GG-EMG) and tensor palatini EMG (TP-EMG), was hypothesised. The relationship between these indices and pharyngeal resistance (Rphar) was also examined.
The study involved eight males with a mean age of 48 (interquartile range 46–52) yrs with OSA, and an apnoea/hypopnoea index of 75 (65–101)·hr−1 on two nights breathing normally and on nasal continuous positive airway pressure (nCPAP). The Pcrit was measured during nonrapid eye movement sleep on nCPAP using brief, incremental reductions in mask pressure. GG-EMG and TP-EMG were measured breath-by-breath, awake, during sleep onset and on nCPAP. Rphar was measured using airway pressures and flow.
Wakeful GG-EMG, early sleep TP-EMG and the sleep decrement in TP-EMG were directly related to Pcrit. Muscle activation was negatively correlated with Rphar for TP-EMG awake and GGEMG early in sleep.
In conclusion these results confirm that dilator muscle activation is directly related to airway narrowing and reduces resistance across patients with obstructive sleep apnoea.
PMCID: PMC3817291  PMID: 17459896
Airway mechanics; airways resistance; muscle function; obstructive sleep apnoea; pharynx
8.  Regulation of cell survival by sphingosine-1-phosphate receptor S1P1 via reciprocal ERK-dependent suppression of Bim and PI-3-kinase/protein kinase C-mediated upregulation of Mcl-1 
Cell Death & Disease  2013;4(11):e927-.
Although the ability of bioactive lipid sphingosine-1-phosphate (S1P) to positively regulate anti-apoptotic/pro-survival responses by binding to S1P1 is well known, the molecular mechanisms remain unclear. Here we demonstrate that expression of S1P1 renders CCL39 lung fibroblasts resistant to apoptosis following growth factor withdrawal. Resistance to apoptosis was associated with attenuated accumulation of pro-apoptotic BH3-only protein Bim. However, although blockade of extracellular signal-regulated kinase (ERK) activation could reverse S1P1-mediated suppression of Bim accumulation, inhibition of caspase-3 cleavage was unaffected. Instead S1P1-mediated inhibition of caspase-3 cleavage was reversed by inhibition of phosphatidylinositol-3-kinase (PI3K) and protein kinase C (PKC), which had no effect on S1P1 regulation of Bim. However, S1P1 suppression of caspase-3 was associated with increased expression of anti-apoptotic protein Mcl-1, the expression of which was also reduced by inhibition of PI3K and PKC. A role for the induction of Mcl-1 in regulating endogenous S1P receptor-dependent pro-survival responses in human umbilical vein endothelial cells was confirmed using S1P receptor agonist FTY720-phosphate (FTY720P). FTY720P induced a transient accumulation of Mcl-1 that was associated with a delayed onset of caspase-3 cleavage following growth factor withdrawal, whereas Mcl-1 knockdown was sufficient to enhance caspase-3 cleavage even in the presence of FTY720P. Consistent with a pro-survival role of S1P1 in disease, analysis of tissue microarrays from ER+ breast cancer patients revealed a significant correlation between S1P1 expression and tumour cell survival. In these tumours, S1P1 expression and cancer cell survival were correlated with increased activation of ERK, but not the PI3K/PKB pathway. In summary, pro-survival/anti-apoptotic signalling from S1P1 is intimately linked to its ability to promote the accumulation of pro-survival protein Mcl-1 and downregulation of pro-apoptotic BH3-only protein Bim via distinct signalling pathways. However, the functional importance of each pathway is dependent on the specific cellular context.
PMCID: PMC3847331  PMID: 24263101
sphingosine-1-phosphate; S1P1; apoptosis; Mcl-1; Bim; breast cancer
9.  The Accuracy of Magnetic Resonance Imaging in Radical Prostatectomy 
Current Urology  2013;7(2):62-64.
The aim of this study was to examine the accuracy of standard magnetic resonance imaging (MRI) in the localised staging of prostate cancer in those who had undergone radical prostatectomy.
Patients and Methods
The cohort consisted of 110 patients who had undergone MRI for staging of prostate cancer and subsequently underwent radical prostatectomy. T stage was analysed both on MRI and from the specimen following radical surgery.
Of the patients 57% of patients had their disease up-staged following radical surgery from preoperative MRI findings. Of those patients who had their disease up-staged following surgery, nearly 50% of patients had gone from organ confined disease at time of MRI to extra-prostatic involvement from the surgical specimen.
We have reported that MRI has a wide range of accuracy. Given developments in MRI technologies further work should be pursued to help in the staging of this disease for which decision to treat is difficult.
PMCID: PMC4017751  PMID: 24917760
Magnetic resonance imaging; Prostate cancer; Prostatectomy
10.  NFκB signalling is upregulated in a subset of castrate-resistant prostate cancer patients and correlates with disease progression 
British Journal of Cancer  2012;107(9):1554-1563.
Cell line models suggest that activation of NFκB is associated with progression of prostate cancer. This pathway may be a therapeutic target if these observations translate to clinical specimens.
Immunohistochemistry measured NFκBp65 (p65), NFκBp65 nuclear localisation signal (NLS), NFκBp65 phosphorylated at ser 276 (p65ser276), NFκBp65 phosphorylated at ser 536 (p65ser536), IκBα phosphorylated at ser 32/36 (pIκBαser32/36) and MMP-9 protein expression in 61 matched hormone naive prostate cancer (HNPC) and castrate-resistant prostate cancer (CRPC) tumours. Animal and cell models were used to investigate the role of NFκB inhibition in prostate carcinogenesis.
In HNPC tumours, NLS expression significantly associated with a shorter time to disease recurrence and disease-specific death. In CRPC tumours p65, pIκBαser32/36 and MMP-9 expression significantly associated with shorter time to death from disease recurrence and shorter disease-specific death. MMP-9 and pIκBαser32/36 expression significantly associated with metastases at recurrence and were independent of Gleason sum and prostate-specific antigen at recurrence. Expression of phosphorylated Akt was associated with increased p65 activation in mouse models and inhibition of NFκB in LNCaP cells significantly reduced cellular proliferation and induced apoptosis.
These results provide further evidence that the NFκB pathway could be exploited as a target for CRPC.
PMCID: PMC3493754  PMID: 23093296
MMP-9; pIκBα; NFκB; hormone naïve prostate cancer
11.  Persistence of the 2009 Pandemic Influenza A (H1N1) Virus on N95 Respirators 
In the United States, the 2009 pandemic influenza A (H1N1) virus (pH1N1) infected almost 20% of the population and caused >200,000 hospitalizations and >10,000 deaths from April 2009 to April 2010. On 24 April 2009, the CDC posted interim guidance on infection control measures in health care settings explicitly for pH1N1 and recommended using filtering face respirators (FFRs) when in close contact with a suspected- or confirmed-to-be-infected individual, particularly when performing aerosol-generating procedures. The persistence and infectivity of pH1N1 were evaluated on FFRs, specifically N95 respirators, under various conditions of absolute humidity (AH) (4.1 × 105 mPa, 6.5 × 105 mPa, and 14.6 × 105 mPa), sample matrices (2% fetal bovine serum [FBS], 5 mg/ml mucin, and viral medium), and times (4, 12, 24, 48, 72, and 144 h). pH1N1 was distributed onto N95 coupons (3.8 to 4.2 cm2) and extracted by a vortex-centrifugation-filtration process, and the ability of the remaining virus to replicate was quantified using an enzyme-linked immunosorbent assay (ELISA) to determine the log10 concentration of the infectious virus per coupon. Overall, pH1N1 remained infectious for 6 days, with an approximately 1-log10 loss of virus concentrations over this time period. Time and AH both affected virus survival. We found significantly higher (P ≤ 0.01) reductions in virus concentrations at time points beyond 24 to 72 h (−0.52-log10 reduction) and 144 h (−0.74) at AHs of 6.5 × 105 mPa (−0.53) and 14.6 × 105 mPa (−0.47). This research supports discarding respirators after close contact with a person with suspected or confirmed influenza infection due to the virus's demonstrated ability to persist and remain infectious.
PMCID: PMC3623216  PMID: 23335770
12.  Expression and prognostic significance of Src family members in renal clear cell carcinoma 
British Journal of Cancer  2012;107(5):856-863.
The aim of this study was to determine whether Src family kinases (SFK) are expressed in renal cell cancer and to assess their prognostic significance.
mRNA expression levels were investigated for the 8 SFK members by quantitative real-time PCR in 19 clear cell cancer tissue samples. Immunohistochemical staining was utilised to assess expression of Src kinase, dephosphorylated Src kinase at Y530 (SrcY530), phosphorylated Src at Y419 (SrcY419) and the downstream focal adhesion kinase (FAK) marker at the Y861 site (FAK Y861) in a cohort of 57 clear cell renal cancer specimens. Expression was assessed using the weighted histoscore method.
Src, Lyn, Hck, Fgr and Fyn were the most highly expressed in renal cancer. All members were more highly expressed in T2 disease, and furthermore expression levels between T2 and T3 disease showed a significant decrease for Lck, Lyn, Fyn, Blk and Yes (P=0.032). Assessment of membrane, cytoplasm and nuclear expression of Src kinase, SrcY530 and SrcY419 were not significantly associated with cancer-specific survival. High expression of cytoplasmic FAK Y861 was associated with decreased cancer-specific survival (P=0.001). On multivariate analysis, cytoplasmic FAK Y861 was independently associated with cancer-specific survival (hazard ratio 3.35, 95% CI 1.40–7.98, P=0.006).
We have reported that all SFK members are expressed in renal cell carcinoma. The SFK members had their highest levels of expression before the disease no longer being organ confined. We hypothesise that these SFK members are upregulated before the cancer spreading out-with the organ and given that Src itself is not associated with cancer-specific survival but the presence of FAK Y861, a downstream marker for SFK member activity is associated with decreased cancer-specific survival, we hypothesise that another SFK member is associated with decreased cancer-specific survival in renal cell cancer.
PMCID: PMC3426751  PMID: 22814579
Src kinase; Src; FAK; renal cancer
13.  The relationship between components of tumour inflammatory cell infiltrate and clinicopathological factors and survival in patients with primary operable invasive ductal breast cancer 
British Journal of Cancer  2012;107(5):864-873.
The importance of the components of host local inflammatory response in determining outcome in primary operable ductal invasive breast cancer is not clear. The aim of this study was to examine the relationship between components of the tumour inflammatory cell infiltrate and standard clinicopathological factors including hormone status (oestrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor (HER)-2), Ki-67 and survival in patients with primary operable invasive ductal breast cancer.
Tumour inflammatory cell infiltrate, hormone status (ER, PR and HER-2), Ki-67 and standard clinicopathological factors were determined using routine pathological and immuno-histochemical techniques in 468 patients.
The large majority (94%) of ductal tumours had evidence of inflammatory cell infiltrate. The general inflammatory cell infiltrate was positively associated with high grade (P<0.001), the absence of ER (P<0.001), the absence of PR (P<0.01), the presence of vascular invasion (P<0.05) and high lymphocytic infiltrate, plasma cell infiltrate, other inflammatory cell infiltrate and macrophage infiltrate (all P<0.001). The median follow-up of the survivors was 165 months. During this period, 93 patients died of their cancer. On univariate analysis, stratified for ER status, tumour size (P<0.01), lymph node involvement (P<0.001), tumour plasma cell infiltrate (P<0.001), other inflammatory cell infiltrate (P<0.05) and treatment (P<0.05) were associated with poorer cancer-specific survival whereas lymphocyte infiltrate (P<0.001) was associated with improved cancer-specific survival. On multivariate analysis, stratified for ER status, lymph node involvement (P<0.05) was independently associated with poorer cancer-specific survival whereas increased tumour lymphocyte infiltrate (P<0.001) was independently associated with improved cancer-specific survival.
The results of this study show that, using routine histology, the general inflammatory cell infiltrate was a common feature and was positively associated with high grade, the absence of ER, the absence of PR, the presence of vascular invasion and high-grade infiltration of lymphocytes, plasma cells, other inflammatory cells and macrophages. Also, that within a mature cohort of patients, a high lymphocytic infiltrate was associated with improved survival, independent of clinicopathological characteristics including ER status, in primary operable ductal invasive breast cancer. These results rationalise previous work and provide a sound basis for future studies in this important area of breast cancer research.
PMCID: PMC3426752  PMID: 22878371
primary ductal invasive breast cancer; hormone status; tumour inflammatory cell infiltrate; lymphocytes; plasma cells; survival
14.  Physical and sexual abuse in childhood as predictors of early onset cardiovascular events in women 
Circulation  2012;126(8):920-927.
Although child abuse is widespread and has been associated with cardiovascular disease (CVD) risk factors, its association with CVD events is not established.
Methods and Results
We examined associations of child abuse with CVD events among 66,798 women in the Nurses’ Health Study 2. Proportional hazards models estimated hazard ratios (HR) and 95% confidence intervals (CI) for myocardial infarction (n=262), stroke (n=251), and total CVD (n=513). Severe physical abuse was reported by 9% and forced sex by 11% of participants. Adjusting for age, race, childhood body type, parental education and family CVD history, the HR for CVD events was 0.91 (95% CI: 0.70–1.17) for mild physical abuse, 1.02 (0.82–1.26) for moderate physical abuse, and 1.46 (1.11–1.92) for severe physical abuse compared to none. Compared to women without childhood sexual abuse, the HR was 1.10 (0.88–1.35) for unwanted sexual touching, and 1.56 (1.23–1.99) for forced sex. After adjustment for adult lifestyle and medical risk factors, the HR for severe physical abuse was 1.13 (0.85–1.51) and that for forced sex was 1.25 (0.98–1.60); these intermediates accounted for much of the association of severe child abuse with CVD. Associations were similar for retrospectively and prospectively reported events. Women with abuse were less likely to release medical records. The associations were stronger for unconfirmed self-reported events than endpoints which were corroborated with additional information or medical record review.
Severe child abuse is a prevalent risk for early adult CVD that is partially mediated by preventable risk factors.
PMCID: PMC3649533  PMID: 22787111
myocardial infarction; stroke; women; epidemiology; violence
16.  Expression of sphingosine 1-phosphate receptor 4 and sphingosine kinase 1 is associated with outcome in oestrogen receptor-negative breast cancer 
British Journal of Cancer  2012;106(8):1453-1459.
We previously reported that sphingosine 1-phosphate receptor 4 (S1P4) is expressed and stimulates the ERK-1/2 pathway via a human epidermal growth factor receptor 2 (HER2)-dependent mechanism in oestrogen receptor-negative (ER−) MDA-MB-453 breast cancer cells.
Clinical relevance of S1P4 and sphingosine kinase 1 (SK1, which catalyses the formation of S1P) was assessed in a cohort of 140 ER− breast tumours by immunohistochemistry (IHC) and the weighted histoscore method. Additional evidence for a functional interaction between S1P4 and SK1 and between HER2 and SK1 was obtained using MDA-MB-453 cells.
High S1P4 expression is associated with shorter disease-free (P=0.014) and disease-specific survival (P=0.004), and was independent on multivariate analysis. In addition, patients with tumours that contain high and low levels of SK1 and S1P4, respectively, have a significantly shorter disease-free survival (P=0.043) and disease-specific survival (P=0.033) compared with patients whose tumours contain both low S1P4 and SK1 levels. In addition, high tumour expression of SK1 was significantly associated with shorter disease-specific survival (P=0.0001) in patients with HER2-positive tumours. Treatment of MDA-MB-453 cells with the SK1 inhibitor, SKi (2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole) reduced the basal and S1P/S1P4-induced activation of ERK-1/2 and altered HER2 trafficking in these cells.
These findings highlight an important role for S1P4 and SK1 in ER− breast cancer progression.
PMCID: PMC3326679  PMID: 22460268
sphingosine 1-phosphate; sphingosine kinase; oestrogen receptor; HER2; sphingosine 1-phosphate receptor 4; extracellular signal regulated kinase
17.  The interrelationships between Src, Cav-1 and RhoGD12 in transitional cell carcinoma of the bladder 
British Journal of Cancer  2012;106(6):1187-1195.
The aim of this current study was to assess the expression and activity of Src family kinases, focal adhesion kinase (FAK), caveolin (Cav-1) and RhoGD12 in bladder cancer.
Fifty-eight patients with a new diagnosis of bladder cancer undergoing transurethral resection were included. Immunohistochemical staining was utilised to assess expression of c-Src, dephosphorylated (SrcY530), phosphorylated Src (Y419), phosphorylated FAK (FAK Y861), Cav-1 and RhoGD12. Expression was assessed using the weighted histoscore method.
High expression of dephosphorylated Y527, phosphorylated Y416 and phosphorylated FAK Y861 in the membrane were associated with increased cancer-specific survival (P=0. 01, P=0.001, P=0.008, respectively) and expression of Y416 in the membrane was an independent factor on multivariate analysis when combined with known clinical parameters (P=0.008, HR 0.288, 95% CI 0.11–0.72).
These results demonstrate that in contrast to other solid tumours, activation of the Src family members and downstream signalling proteins are associated with a good prognosis in transitional cell carcinoma of the bladder, and activated Src has a positive relationship with RhoGD12.
PMCID: PMC3304420  PMID: 22353809
Src kinase; Cav-1; RhoGD12; bladder cancer; survival
18.  The relationship between tumour necrosis, tumour proliferation, local and systemic inflammation, microvessel density and survival in patients undergoing potentially curative resection of oesophageal adenocarcinoma 
British Journal of Cancer  2012;106(4):702-710.
There is increasing evidence that the local and systemic inflammatory responses are associated with survival in oesophageal cancer. The aim of this study was to examine the relationship between tumour necrosis, tumour proliferation, local and systemic inflammation and microvessel density and survival in patients undergoing potentially curative resection of oesophageal adenocarcinoma.
The interrelationship between tumour necrosis, tumour proliferation, local inflammatory response (Klintrup–Makinen criteria, intra-tumoural CD8+ lymphocyte and macrophage infiltration), systemic inflammatory response (modified Glasgow Prognostic score (mGPS)), and microvessel density was examined in 121 patients undergoing potentially curative resection for oesophageal adenocarcinoma (including type I and II tumours of the gastro-oesophageal junction).
Tumour necrosis was not significantly associated with any tumour measure other than the degree of differentiation. On multivariate analysis, only age (HR 1.93, 95% CI 1.23–3.04, P=0.004), mGPS (HR 2.91, 95% CI 1.51–5.62, P=0.001), positive to total lymph node ratio (HR 2.38, 95% CI 1.60–3.52, P<0.001) and macrophage infiltration (HR 1.49, 95% CI 1.02–2.18, P=0.041) were independently associated with cancer-specific survival in oesophageal adenocarcinoma. Intra-tumoural macrophages were associated with tumour proliferation (P<0.001) and CD8+ lymphocytes infiltration (P<0.01).
The results of this study suggest that tumour necrosis does not link local and systemic inflammatory responses and is not significantly associated with survival. In contrast, tumour macrophage infiltration appears to have a central role in the proliferative activity and the coordination of the inflammatory cell infiltrate and is independently associated with poorer survival in patients with oesophageal adenocarcinoma.
PMCID: PMC3322960  PMID: 22240784
oesophageal cancer; C-reactive protein; tumour inflammatory infiltrate; microvessel; necrosis; survival
19.  Comparison of Visual and automated assessment of Ki-67 proliferative activity and their impact on outcome in primary operable invasive ductal breast cancer 
British Journal of Cancer  2012;106(2):383-388.
Immunohistochemistry of Ki-67 protein is widely used to assess tumour proliferation, and is an established prognostic factor in breast cancer. There is interest in automating the assessment of Ki-67 labelling index (LI) with possible benefits in handling increased workload, with improved accuracy and precision.
Patients and methods:
Visual and automated assessment of Ki-67 LI and survival were examined in patients with primary operable invasive ductal breast cancer. Tissue microarrays (n=379 patients) immunostained for Ki-67 were scored visually and automatically with the Slidepath Tissue IA system.
Visual and automated Ki-67 LI were in excellent agreement (ICCC=0.96, P<0.001). On univariate analysis, visual (P<0.001) and automated Ki67 LI (P<0.05) were associated with cancer-specific survival in patients with invasive ductal breast cancer overall and in patients who received endocrine therapy (Tamoxifen) (P<0.01 for visual and P<0.05 for automated scoring).
Automated assessment of Ki-67 LI would appear to be comparable to visual Ki-67 LI. However, automated Ki-67 LI assessment was inferior in predicting cancer survival in patients with breast cancer, including patients who received Tamoxifen.
PMCID: PMC3261670  PMID: 22251968
primary invasive breast cancer; visual counting method; automated counting method; survival
20.  Upregulated FGFR1 expression is associated with the transition of hormone-naive to castrate-resistant prostate cancer 
British Journal of Cancer  2011;105(9):1362-1369.
Prostate cancer (PC) represents a global health issue. Treatment for locally advanced and metastatic PC remains unsatisfactory. The androgen receptor (AR) has been validated in having a key role in both naïve and castrate-resistant PC (CRPC). However, the significance of other signalling pathways in CRPC is less well validated.
To gain a better insight into the molecular signalling cascades involved in clinical CRPC, we performed gene expression profiling using the Illumina DASL assay and studied matched hormone-naive (HN) and CR prostate tumours (n=10 pairs). Ingenuity Pathways Analysis (IPA) was used to identify potential networks involved, and further validation was performed in in vitro cell models and clinical tumours.
Expression of 50 genes was significantly different between HN and CRPC. IPA revealed two networks of particular interest, including AR and FGFR1, respectively. FGFR1 expression was confirmed to be significantly upregulated in CRPC (P⩽0.005), and abnormal FGFR1 expression was associated with shorter time to biochemical relapse in HNPC (P=0.006) and less favourable disease-specific survival in CRPC (P=0.018).
For the first time, our gene expression profiling experiment on archival tumour materials has identified upregulated FGFR1 expression to be associated with PC progression to the CR state.
PMCID: PMC3241546  PMID: 21952621
prostate cancer; formalin-fixed, paraffin-embedded samples; gene expression analysis; hormone-naive and castrate-resistant prostate cancer; fibroblast growth factor receptor 1
21.  Mucous Gland Metaplasia in the Esophagus and Gastric Mucosa in Baboons 
Anticancer research  2011;31(6):2187-2190.
Chewing of regurgitated food elicits in baboons life-long gastro-esophageal reflux (GER). The acid reflux transforms the multilayered squamous cell epithelium of the esophagus into columnar-lined mucosa with mucus-producing accessory glands. The function of this mucous gland metaplasia (MGM), which mimics Barrett’s mucosa with MGM in humans, is to buffer the gastric acid entering the esophagus during regurgitation. In a previous study of entire esophagi, the majority of baboons showed MGM. The gastric mucosa was not investigated.
Materials and Methods
Hematoxylin-eosin-stained sections from the esophagus, from the lesser gastric curvature and from the greater gastric curvature were collected separately from 50 adult baboons. The presence of MGM was assessed in each one of these locations.
MGM was demonstrated in 92% (46/50) of blocks from the esophagus, in 98% (49/50) of blocks from the lesser curvature and in 90% (45/50) of those of the greater curvature (fundus).
The majority of the animals had MGM, not only in the esophagus but also in the proximal gastric mucosa. Rationally, MGM in baboons starts in the distal esophagus and proceeds downwards, towards the proximal stomach. The histogenesis of the MGM in Barrett’s mucosa in humans (that is Barrett’s mucosa type 2) remains elusive. Therefore the baboon might be an important animal model for studying the histogenesis of Barrett’s mucosa with MGM in humans, a recognized pre-cancerous lesion.
PMCID: PMC3468912  PMID: 21737639
Esophagus; baboon; gastric reflux; mucus metaplasia
22.  Upregulation of MAPK pathway is associated with survival in castrate-resistant prostate cancer 
British Journal of Cancer  2011;104(12):1920-1928.
Recent evidence has implicated the MAP kinase (MAPK) pathway with the development of castrate-resistant prostate cancer (CRPC). We have previously reported gene amplification of critical members of this pathway with the development of castrate-resistant disease. In addition, we have shown that rising Raf-1 expression, with the development of CRPC, influences time to biochemical relapse. We therefore sought to further analyse the role of both Raf-1 and its downstream target MAPK in the molecular pathogenesis of CRPC.
Protein expression of Raf-1 and MAPK, including their activation status, was analysed using immunohistochemistry in a database of 65 paired tumour specimens obtained before and after the development of CRPC and correlated with other members of the pathway.
Patients whose nuclear expression of MAPK rose with the development of CRPC had a significantly shorter median time to death following biochemical relapse (1.40 vs 3.00 years, P=0.0255) as well as reduced disease-specific survival when compared with those whose expression fell or remained unchanged (1.16 vs 2.62 years, P=0.0005). Significant correlations were observed between protein expression of Raf-1 and MAPK with the type 1 receptor tyrosine kinases, Her2 and epidermal growth factor receptor, as well as the transcription factor AP-1 in CRPC tumours.
We conclude that the Her2/Raf-1/MAPK/AP-1 axis may promote the development of CRPC, leading to early relapse, and reduced disease-specific survival. In addition, members of the pathway may act as novel therapeutic and/or diagnostic targets for prostate cancer.
PMCID: PMC3111196  PMID: 21559022
MAP kinase pathway; castrate-resistant prostate cancer; prostate cancer; Her2; AP-1; Raf
23.  Genera of diaporthalean coelomycetes associated with leaf spots of tree hosts 
Four different genera of diaporthalean coelomycetous fungi associated with leaf spots of tree hosts are morphologically treated and phylogenetically compared based on the DNA sequence data of the large subunit nuclear ribosomal DNA gene (LSU) and the internal transcribed spacers and 5.8S rRNA gene of the nrDNA operon. These include two new Australian genera, namely Auratiopycnidiella, proposed for a leaf spotting fungus occurring on Tristaniopsis laurina in New South Wales, and Disculoides, proposed for two species occurring on leaf spots of Eucalyptus leaves in Victoria. Two new species are described in Aurantiosacculus, a hitherto monotypic genus associated with leaf spots of Eucalyptus in Australia, namely A. acutatus on E. viminalis, and A. eucalyptorum on E. globulus, both occurring in Tasmania. Lastly, an epitype specimen is designated for Erythrogloeum hymenaeae, the type species of the genus Erythrogloeum, and causal agent of a prominent leaf spot disease on Hymenaea courbaril in South America. All four genera are shown to be allied to Diaporthales, although only Aurantiosacculus (Cryphonectriaceae) could be resolved to family level, the rest being incertae sedis.
PMCID: PMC3409416  PMID: 23105154
leaf spot disease; molecular phylogeny; systematics
24.  Stress and the risk of multiple sclerosis 
Neurology  2011;76(22):1866-1871.
Several studies have shown that stressful life events are associated with a subsequent significant increase in risk of multiple sclerosis (MS) exacerbations. We wanted to study prospectively whether stress can increase the risk of developing the disease itself.
We studied 2 cohorts of female nurses: the Nurses' Health Study (NHS) (n = 121,700) followed from 1976 and the Nurses' Health Study II (NHS II) (n = 116,671) followed from 1989. The risk of MS after self-report on general stress at home and at work in the NHS in 1982 was studied prospectively using Cox regression. Logistic regression was used to retrospectively estimate the effects of physical and sexual abuse in childhood and adolescence collected in the NHS II 2001. We identified 77 cases of MS in the NHS by 2005 and 292 in the NHS II by 2004. All analyses were adjusted for age, ethnicity, latitude of birth, body mass index at age 18, and smoking.
We found no increased risk of MS associated with severe stress at home in the NHS (hazard ratio 0.85 [95% confidence interval (CI)] 0.32–2.26). No significantly increased risk of MS was found among those who reported severe physical abuse during childhood (odds ratio [OR] 0.68, 95% CI 0.41–1.14) or adolescence (OR 0.77, 95% CI 0.46–1.28) or those having been repeatedly forced into sexual activity in childhood (OR 1.47, 95% CI 0.87–2.48) or adolescence (OR 1.21, 95% CI 0.68–2.17).
These results do not support a major role of stress in the development of the disease, but repeated and more focused measures of stress are needed to firmly exclude stress as a potential risk factor for MS. Neurology® 2011;76:1866–1871
PMCID: PMC3115807  PMID: 21624985
25.  Is there an association with phosphorylation and dephosphorylation of Src kinase at tyrosine 530 and breast cancer patient disease-specific survival 
British Journal of Cancer  2010;103(12):1831-1834.
Recent work has demonstrated that c-Src and fully activated Y419Src expression was associated with poor clinical outcome of breast cancer patients. It is unknown whether different activation stages of c-Src equally influence disease-specific survival of breast cancer patients.
Immunohistochemistry was performed on 165 resected breast cancers using antibodies to phosphorylated and dephosphorylated Src kinase tyrosine site 530. Expression was assessed using the weighted histoscore method.
Majority of phosphorylated and dephosphorylated Y530Src expression was observed in the nucleus and cytoplasm. Only 3.6% of phosphorylated Y530Src (pY530Src) expression was detected in the membrane, compared with 53% with dephosphorylated Y530Src. Nuclear expression of pY530Src correlated negatively with oestrogen receptor (ER) status (χ2 P<0.001), whereas cytoplasmic phosphorylated and dephosphorylated Y530Src expression correlated negatively with membrane c-Src expression (χ2 P=0.008, χ2 P<0.001). On univariate and multivariate analysis, no significant association was noticed between phosphorylated or dephosphorylated Y530Src expression and disease-specific survival at any cellular location.
ER-negative breast cancer patients were more likely to express pY530Src in the nucleus. Breast cancer patients with higher cytoplasmic expression of phosphorylated or dephosphorylated Y530Src were more likely not to express c-Src at the membrane. Phosphorylated and dephosphorylated Y530Src expression is not associated with survival of patients.
PMCID: PMC3008598  PMID: 21063412
Src kinase; breast cancer; immunohistochemistry; disease-specific survival

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