Streptococcus sanguinis-induced cytokine release from platelets.

BACKGROUND: There is increasing evidence that both chronic and acute infections play a role in the development and progression of atherothrombotic disorders. One potential mechanism is the direct activation of platelets by bacteria. A wide range of bacterial species activate platelets through heterogeneous mechanisms. The oral micro-organism S. sanguinis stimulates platelet aggregation in vitro in a strain-dependent manner, although there are no reports of associated cytokine production. OBJECTIVE: The aim of the present study was to determine whether platelet activation by S. sanguinis involved the release of pro-inflammatory and immune modulating factors, and whether activation was enhanced by epinephrine. METHODS AND RESULTS: Four strains of S. sanguinis and one of S. gordonii stimulated the release of RANTES, PF4, sCD40L and PDGF-AB, whereas only one S. sanguinis strain caused the release of sCD62p. Epinephrine enhanced S. sanguinis-induced platelet aggregation and phosphorylation of phospholipase Cγ2 and Erk, but inhibited RANTES, PF4, sCD40L and PDGF-AB release. Wortmannin inhibited S. sanguinis-induced aggregation and release; however, only aggregation was partially reversed by epinephrine. CONCLUSIONS: The present study demonstrates that platelets respond to S. sanguinis with both prothrombotic and pro-inflammatory/immune-modulating responses. Epinephrine, potentially released in response to infection and/or stress, can significantly enhance the prothrombotic response, thereby providing a putative link between bacteraemia and acute coronary events during stress. In contrast, epinephrine inhibited the pro-inflammatory/immune-modulating response by an undetermined mechanism.

Treadmill Velocity Best Predicts 5000-m Run Performance.

In this study, we aimed to investigate physiological determinants of endurance performance that best predict 5000-m average run velocity before and after endurance training. Thirty-nine previously untrained participants completed a 5000-m run; a constant velocity test (measuring running economy); and an incremental treadmill test to determine maximal oxygen uptake, final treadmill velocity, and velocity and oxygen uptake at lactate threshold, before and after six weeks of endurance training. Maximal oxygen uptake, final treadmill velocity, and velocity and oxygen uptake at threshold all increased significantly after training (p < 0.05). Average velocity for 5000 m increased significantly (p < 0.05). Running economy was not significantly altered. Correlation analysis revealed final treadmill velocity was most strongly related to 5000-m performance, in both untrained and trained states (r = 0.89, 0.83). Lactate threshold velocity (r = 0.73, 0.76), maximal oxygen uptake (r = 0.55, 0.51) and oxygen uptake at threshold (r = 0.45, 0.45) also showed significant correlations. In contrast, running economy was not significantly related to performance. These results demonstrate that final treadmill velocity in an VO2max test is the single best predictor of 5000-m performance in untrained and trained states. Furthermore, stepwise regression analysis showed that only velocity at lactate threshold significantly improved the accuracy of prediction provided by final treadmill velocity alone.

Loss of neuronatin expression is associated with promoter hypermethylation in pituitary adenoma.

The imprinted gene, neuronatin (NNAT), is one of the most abundant transcripts in the pituitary and is thought to be involved in the development and maturation of this gland. In a recent whole-genome approach, exploiting a pituitary tumour cell line, we identified hypermethylation associated loss of NNAT. In this report, we determined the expression pattern of NNAT in individual cell types of the normal gland and within each of the different pituitary adenoma subtypes. In addition, we determined associations between expression and CpG island methylation and used colony forming efficiency assays (CFE) to gain further insight into the tumour-suppressor function of this gene. Immunohistochemical (IHC) co-localization studies of normal pituitaries showed that each of the hormone secreting cells (GH, PRL, ACTH, FSH and TSH) expressed NNAT. However, 33 out of 47 adenomas comprising, 11 somatotrophinomas, 10 prolactinomas, 12 corticotrophinomas and 14 non-functioning tumours, irrespective of subtype failed to express either NNAT transcript or protein as determined by quantitative real-time RT-PCR and IHC respectively. In normal pituitaries and adenomas that expressed NNAT the promoter-associated CpG island showed characteristics of an imprinted gene where approximately 50% of molecules were densely methylated. However, in the majority of adenomas that showed loss or significantly reduced expression of NNAT, relative to normal pituitaries, the gene-associated CpG island showed significantly increased methylation. Induced expression of NNAT in transfected AtT-20 cells significantly reduced CFE. Collectively, these findings point to an important role for NNAT in the pituitary and perhaps tumour development in this gland.

The relationship between the systemic inflammatory response, tumour proliferative activity, T-lymphocytic and macrophage infiltration, microvessel density and survival in patients with primary operable breast cancer.

The significance of the inter-relationship between tumour and host local/systemic inflammatory responses in primary operable invasive breast cancer is limited. The inter-relationship between the systemic inflammatory response (pre-operative white cell count, C-reactive protein and albumin concentrations), standard clinicopathological factors, tumour T-lymphocytic (CD4+ and CD8+) and macrophage (CD68+) infiltration, proliferative (Ki-67) index and microvessel density (CD34+) was examined using immunohistochemistry and slide-counting techniques, and their prognostic values were examined in 168 patients with potentially curative resection of early-stage invasive breast cancer. Increased tumour grade and proliferative activity were associated with greater tumour T-lymphocyte (P<0.05) and macrophage (P<0.05) infiltration and microvessel density (P<0.01). The median follow-up of survivors was 72 months. During this period, 31 patients died; 18 died of their cancer. On univariate analysis, increased lymph-node involvement (P<0.01), negative hormonal receptor (P<0.10), lower albumin concentrations (P<0.01), increased tumour proliferation (P<0.05), increased tumour microvessel density (P<0.05), the extent of locoregional control (P<0.0001) and limited systemic treatment (P

The relationship between tumour proliferative activity, the systemic inflammatory response and survival in patients undergoing curative resection for colorectal cancer.

BACKGROUND: The aim of the present study was to examine the relationship between Ki-67, C-reactive protein and cancer-specific survival in patients undergoing resection for colorectal cancer. METHOD: One hundred and forty-seven patients undergoing potentially curative resection for colorectal cancer had preoperative C-reactive protein concentrations and tumour Ki-67 labelling index measured. RESULTS: On univariate analysis, age (P < 0.001), Dukes stage (P < 0.001), C-reactive protein (P < 0.001) and expression of Ki-67 (< 0.01) were associated with poorer cancer-specific survival. Ki-67 labelling index and C-reactive protein were correlated (r(s) = 0.172, P = 0.037). On multivariate analysis, age (HR 1.96, 95% CI 1.26-3.04, P = 0.003), Dukes stage (HR 4.38, 95% CI 2.11-9.09, P < 0.001) and C-reactive protein (HR 4.09, 95% CI 2.04-8.24, P < 0.001) retained significance. CONCLUSION: Increased tumour proliferation is associated with a systemic inflammatory response and poor cancer-specific survival in patients undergoing potentially curative surgery for colorectal cancer.

TNM staging of midgut and hindgut (neuro) endocrine tumors: a consensus proposal including a grading system.

Criteria for the staging and grading of neuroendocrine tumors (NETs) of midgut and hindgut origin were established at the second Consensus Conference in Frascati (Rome) organized by the European Neuroendocrine Tumor Society (ENETS). The proposed tumor-node-metastasis (TNM) classifications are based on the recently published ENETS Guidelines for the Diagnosis and Treatment of gastroenteropancreatic NETs and follow our previous proposal for foregut tumors. The new TNM classifications for NETs of the ileum, appendix, colon, and rectum, and the grading system were designed, discussed, and consensually approved by all conference participants. These proposals need to be validated and are meant to help clinicians in the stratification, treatment and follow-up of patients.

The relationship between the systemic inflammatory response, tumour proliferative activity, T-lymphocytic infiltration and COX-2 expression and survival in patients with transitional cell carcinoma of the urinary bladder.

The relationship between the systemic inflammatory response, tumour proliferative activity, T-lymphocytic infiltration, and COX-2 expression and survival was examined in patients with transitional cell carcinoma of the urinary bladder (n=103). Sixty-one patients had superficial disease and 42 patients had invasive disease. Cancer-specific survival was shorter in those patients with invasive compared with superficial bladder cancer (P<0.001). On univariate analysis, stratified by stage, increased Ki-67 labelling index (P<0.05), increased COX-2 expression (P<0.05), C-reactive protein (P<0.05) and adjuvant therapy (P<0.01) were associated with poorer cancer-specific survival. On multivariate analysis of these significant factors, stratified by stage, only C-reactive protein (HR 2.89, 95% CI 1.42-5.91, P=0.004) and adjuvant therapy (HR 0.29, 95% CI 0.14-0.62, P=0.001) were independently associated with poorer cancer-specific survival. These results would suggest that tumour-based factors such as grade, COX-2 expression or T-lymphocytic infiltration are subordinate to systemic factors such as C-reactive protein in determining survival in patients with transitional cell carcinoma of the urinary bladder.

TNM staging of foregut (neuro)endocrine tumors: a consensus proposal including a grading system.

The need for standards in the management of patients with endocrine tumors of the digestive system prompted the European Neuroendocrine Tumor Society (ENETS) to organize a first Consensus Conference, which was held in Frascati (Rome) and was based on the recently published ENETS guidelines on the diagnosis and treatment of digestive neuroendocrine tumors (NET). Here, we report the tumor-node-metastasis proposal for foregut NETs of the stomach, duodenum, and pancreas that was designed, discussed, and consensually approved at this conference. In addition, we report the proposal for a working formulation for the grading of digestive NETs based on mitotic count and Ki-67 index. This proposal, which needs to be validated, is meant to help clinicians in the stratification, treatment, and follow-up of patients.

Best practice no 183. Examination of parathyroid gland specimens.

The pathological examination of parathyroid glands is an essential component of the evaluation of hyperparathyroidism. Traditionally, this has involved intraoperative frozen sections during bilateral surgical exploration of the neck, to confirm removal of parathyroid tissue. With recent developments in imaging, some diseased glands can be localised preoperatively, enabling removal by minimally invasive, targetted surgery, with or without additional non-histological intraoperative procedures to confirm the removal of all hyperfunctioning parathyroid tissue. This article reviews these developments and describes the ideal approach to reporting parathyroid specimens.

The relationship between tumour T-lymphocyte infiltration, the systemic inflammatory response and survival in patients undergoing curative resection for colorectal cancer.

There is increasing evidence that both local and systemic inflammatory responses play an important role in the progression of a variety of common solid tumours. The aim of the present study was to examine the relationship between tumour T-lymphocyte subset infiltration, the systemic inflammatory response and cancer-specific survival in patients with colorectal cancer. In all, 147 patients undergoing potentially curative resection for colorectal cancer were studied. Circulating concentrations of C-reactive protein were measured prior to surgery. CD4+ and CD8+ T-lymphocyte infiltration of the tumour was assessed using immunohistochemistry and a point counting technique. When patients were grouped according to the percentage tumour volume of CD4+ T-lymphocytes, there was no difference in terms of age, sex, tumour site, stage and tumour characteristics. However, there was an inverse relationship between percentage tumour CD4+ T-lymphocytes and C-reactive protein (P<0.01). On univariate analysis, both C-reactive protein concentrations (P<0.001) and percentage tumour volume of CD4+ (P<0.05) T-lymphocytes were associated with cancer-specific survival. The results of the present study show that low tumour CD4+ T-lymphocyte infiltration is associated with elevated C-reactive protein concentrations and both predict poor cancer-specific survival.

The relationship between T-lymphocyte subset infiltration and survival in patients with prostate cancer.

The relationship between tumour stage, T-lymphocyte subset infiltration and survival was examined in patients with prostate cancer (n=80). On multivariate analysis PSA (HR 2.47, 95% CI 1.27-4.83, P=0.008) and CD4+ T-lymphocyte count (HR 2.29, 95% CI 1.25-4.22, P=0.008) had independent significance. Increased CD4+ T-lymphocyte infiltration within the tumour was stage independent and associated with poor outcome in patients with prostate cancer.

Evaluation of a cumulative prognostic score based on the systemic inflammatory response in patients undergoing potentially curative surgery for colorectal cancer.

The value of combining Dukes' stage and C-reactive protein to form a cumulative prognostic score was assessed in 147 patients undergoing potentially curative resection for colorectal cancer. The cancer-specific survival rates at 3 years for patients with a cumulative prognostic score of 0, 1 and 2 were 100, 77 and 40%, respectively (HR 4.76, 2.78-8.15, P<0.001).

Tumour T-lymphocyte subset infiltration and tumour recurrence following curative resection for colorectal cancer.

AIM: The relationship of tumour T-lymphocytic subset infiltration and recurrence in patients undergoing potentially curative resection for colorectal cancer has not been clearly defined. METHODS: Tumour sections from patients who had undergone potentially curative resection for colorectal cancer were stained and counted for CD4+ and CD8+ T-lymphocytes. RESULTS: Twenty-three patients developed recurrence during the follow-up period. Patients were grouped according to whether or not they developed recurrence. The groups were similar in age, sex, site of tumour, Dukes stage and the numbers of patients receiving adjuvant therapy. The total percentage volume of labelled CD4+ T-lymphocytes in the tumour was significantly lower in the patients who recurred (p<0.05). CONCLUSIONS: The results of the present pilot study suggest that a reduction in tumour T-lymphocyte infiltration, in particular CD4+ T-lymphocyte infiltration, is associated with recurrence in patients following potentially curative resection for colorectal cancer.

The relationship between T-lymphocyte infiltration, stage, tumour grade and survival in patients undergoing curative surgery for renal cell cancer.

The present study examined the relationship between tumour stage, grade, T-lymphocyte subset infiltration and survival in patients who had undergone potentially curative surgery for renal clear-cell cancer (n=73). Intratumoural CD4+ T-lymphocyte infiltrate was associated with poor cancer-specific survival, independent of grade, in this cohort.

Loss of ACTH expression in cultured human corticotroph macroadenoma cells is consistent with loss of the POMC gene signal sequence.

The proopiomelanocortin (POMC) gene is highly expressed in the pituitary gland where the resulting mRNA of 1200 base pairs (bp) gives rise to a full-length protein sequence. In peripheral tissues however both shorter and longer POMC variants have been described, these include for example placental tissue which contain 800 (truncated at the 5' end) and 1500 as well as the 1200 bp transcripts. The importance of the 800 bp transcript is unclear as the lack of a signal sequence renders the molecule to be non-functional. This transcript has not been previously demonstrated in the pituitary gland. In this report we show evidence of a 5' truncated POMC gene in human pituitary corticotroph macroadenoma cells (JE) maintained in primary culture for >1 year. The original tumour tissue and the derived cells during early passage (up to passage 4-5) immunostained for ACTH and in situ hybridisation confirmed the presence of the POMC gene in the cultured cells. These cells also secreted 15-40 pg/10(5) cells/24 h ACTH. In addition, as expected RT-PCR demonstrated the presence of all three POMC gene exons and is thus indicative of a full-length POMC gene. In late culture passages (passages 8-15) JE cells ceased to express ACTH and cell growth became very slow due presumably to cells reaching their Hayflick limit. ACTH immunostaining in these cells was undetectable and ACTH secretion was also at the detection limits of the assay and no greater than 10 pg/10(5) cells/24 h. ACTH precursor molecules were also undetectable. RT-PCR for the POMC gene in these late passage cells showed that only exon 3 was detectable, in contrast to early passage cells where all three exons were present. In summary we isolated in culture, human pituitary cells that possessed initially all three exons of the POMC gene and immunostained for ACTH. On further passaging these cells showed a loss of exons 1 and 2 in the POMC gene and a loss of ACTH immunostaining and secretion. We would like to suggest that the loss of ACTH peptide expression in these late passage cells is in part due to the loss of the POMC signal sequence. An alternative explanation for our findings is that there were originally two populations of corticotrophs in the cultures, one of which possessed the full-length POMC gene and the other only the 5' truncated POMC transcript and it is these latter cells which survived in culture. In either scenario this is the first report of the 5' truncated POMC gene occurring in pituitary cells.

Incorporation of map kinases into the platelet cytoskeleton.

Erk1 (p44) and erk2 (p42) mitogen-activated protein (MAP) kinases are activated in agonist-stimulated platelets, although their role(s) in the activation process is unknown. In the present study, erk1, erk2 and the phosphorylated forms of both enzymes became associated with the contractile cytoskeleton in thrombin-stimulated platelets. Enzyme incorporation was accompanied by an increase in MAP kinase activity in the cytoskeleton, which was inhibited by PD98059. Pretreatment of the platelets with the arginine-glycine-aspartic acid-serine (RGDS) polypeptide enhanced both the cytoskeletal association and the enzyme activity, but cytochalasin D had no significant effect. Platelets from a patient with Glanzmann's thrombasthenia lack the alpha(IIb)beta(3) integrin and form only a rudimentary cytoskeleton, however, this cytoskeleton is enriched with both erk1 and erk2. These data suggest either that MAP kinases play a role in cytoskeletal rearrangement or that the cytoskeleton act as a frame to align MAP kinases with substrates in a highly integrated signal transduction pathway.

Aberrant expression of G(1)/S regulators is a frequent event in sporadic pituitary adenomas.

Components of the pRb/p16/cyclin D1/CDK4 pathway are frequent targets in numerous tumour types, including those of pituitary origin. However, previous studies of pituitary tumours have examined individual components of this pathway. Therefore, to determine their overall contribution we have simultaneously examined the immunohistochemical status of pRb, p16 and cyclin D1 and analysed the CDK4 gene for a characterized activating mutation. Of the total pituitary tumour cohort (29 clinically non-functioning adenomas and 16 somatotrophinomas) abnormal expression of either pRb, p16 or cyclin D1 was observed in 36 of 45 (80%) tumours and was significantly (P = 0.005) associated with non-functioning tumours (27/29; 93%) compared with somatotrophinomas (9/16, 56%). Loss of either pRb or p16 expression was mutually exclusive in 23 of 45 (51%) tumours, whilst concomitant loss of pRb and p16 expression was observed in five tumours. Cyclin D1 overexpression was observed in 22 of 45 (49%) tumours, however, there was no significant association between overexpression of cyclin D1 and the expression status of either pRb or p16. In addition, no activating mutations within codon 24 of the CDK4 gene were detected. This study provides evidence for the first time that components of the pRb/p16/cyclin D1/CDK4 pathway, either alone or in combination, are frequently deregulated in human pituitary tumours, suggesting that this pathway may be a useful target in drug or gene therapeutic approaches.