Can a modified thrombolysis in myocardial infarction risk score outperform the original for risk stratifying emergency department patients with chest pain?

BACKGROUND: The thrombolysis in myocardial infarction (TIMI) risk score has been shown to risk stratify patients with suspected acute coronary syndromes (ACS) effectively in the emergency department (ED) but cannot be used to guide patient disposition. We aimed to evaluate whether modifying the TIMI risk score to give greater weighting to ischaemic ECG changes and troponin elevations would enhance its risk stratification and thus potentially facilitate safe patient discharge after 12-h troponin testing. METHODS: A prospective diagnostic cohort study was performed within the ED at Manchester Royal Infirmary, a university-affiliated teaching hospital with an annual ED census of approximately 145,000 patients. 804 patients who had presented to the ED with suspected cardiac chest pain were recruited. All patients underwent 12-h troponin T testing and were followed up by telephone and chart review after 30 days for the composite primary outcome of death, acute myocardial infarction (AMI) or urgent coronary revascularisation. RESULTS: The modified TIMI risk score outperformed the original (area under the receiver operator characteristic curve 0.87 versus 0.77, p<0.001). Using a cut-off of more than 2 points the score had a sensitivity of 96.4% for the prediction of 30-day events. The specificity of the score was only 51.0%, suggesting that in practice over 40% of patients would be ineligible for discharge even after troponin testing. CONCLUSIONS: Modifications to the TIMI risk score can improve its performance in the risk stratification of patients presenting to the ED with chest pain. However, a lack of specificity may still limit its use for guiding patient disposition after troponin testing.

Early changes in the haemostatic and procoagulant systems after chemotherapy for breast cancer.

Venous thromboembolism (VTE) following breast cancer chemotherapy is common. Chemotherapy-induced alterations in markers of haemostasis occur during chemotherapy. It is unclear how rapidly this occurs, whether this is upregulated in patients developing VTE and whether changes predict for VTE. Markers of haemostasis, functional clotting assays and vascular endothelial growth factor were measured before chemotherapy and at 24 h, 4 days, 8 days and 3 months following commencement of chemotherapy in early and advanced breast cancer patients and in age- and sex-matched controls. Duplex ultrasound imaging was performed after 1 month or if symptomatic. Of 123 patients, 9.8% developed VTE within 3 months. Activated partial thromboplastin time (APTT), prothrombin time (PT), D-dimer, fibrinogen, platelet count, VEGF and fibrinogen were increased in cancer. Fibrinogen, D-dimer, VEGF and tissue factor were increased, at baseline, in patients subsequently developing VTE. D-dimer of less than 500 ng ml(-1) has a negative predictive value of 97%. Activated partial thromboplastin time, PT and thrombin-antithrombin showed significantly different trends, as early as within 24 h, in response to chemotherapy in patients subsequently developing VTE. Markers of coagulation and procoagulants are increased, before chemotherapy, in patients who subsequently develop VTE. A group of patients at minimal risk of VTE can be identified, allowing targeted thrombopropylaxis to the higher risk group.

Angiogenic characteristics of circulating and tumoural thrombospondin-1 in breast cancer.

In cancer models, thrombospondin-1 (TSP-1) has been shown to inhibit angiogenesis or promote metastasis by increasing adhesion of malignant cells to endothelium. To determine the role of TSP-1 in breast cancer and breast cancer angiogenesis, we have measured TSP-1 in plasma and tumour cytosols and compared levels to established clinicopathological prognostic parameters and intratumoural microvessel density. TSP-1 was measured, by radioimmunoassay, in plasma (pTSP-1) and tumour cytosols (cTSP-1) of women with early breast cancer (EBC) (n=71). pTSP-1 in EBC was compared to pTSP-1 levels in women with advanced breast cancer (ABC) (n=66), normal controls (n=77) and was correlated with prognostic features and microvessel density (MVD) (measured by CD31 immunostaining). cTSP-1 levels were compared to prognostic features and microvessel density. pTSP-1 in women with EBC (median 484, IQR 344-877 ng/ml) and ABC (median 588, IQR 430-952 ng/ml) were elevated when compared to normal controls (median 21, IQR 175-247) (p<0.001). Women with lymph node metastases (n=35) had higher levels of TSP-1 (median 799 ng/ml, IQR 455-943) than women who were node negative (median 343 ng/ml, IQR 267-514) (n=36) (p<0.05). Levels of pTSP-1 in EBC correlated with MVD (R=0.39, p<0.05). Levels of TSP-1 in tumour cytosols of women with EBC (median 1714, IQR 893-5283 ng/ml) correlated with microvessel density (R=0.46, p<0.01). Circulating levels of TSP-1 appear to be a marker of breast cancer aggressiveness and in breast cancer may have a pro-angiogenic rather than anti-angiogenic role.

Serum vascular endothelial growth factor in breast cancer.

Many studies have reported elevated serum concentrations of vascular endothelial growth factor (VEGF) in patients with cancer and claimed that the measurement of circulating VEGF is a surrogate marker of angiogenesis and/or metastasis. To determine the value of VEGF measurement in the diagnosis and prognosis of breast cancer, we measured levels in women with and without breast cancer. Platelet-depleted plasma VEGF levels were measured in premenopausal women at four-day intervals across the menstrual cycle, postmenopausal women and postmenopausal women who had undergone hysterectomy. Platelet-depleted plasma VEGF was also measured in pre- and postmenopausal women with early breast cancer (EBC) and levels compared with intratumoral levels, clinicopathological prognostic parameters and microvessel density. Levels of VEGF were determined using ELISA and immuno-histochemistry. Microvessel density was determined by immunohistochemical CD34 staining. Plasma VEGF in premenopausal women remained stable across the menstrual cycle except for a peak between days 8 and 12. VEGF levels in postmenopausal women were higher than in premenopausal women unless postmenopausal women had undergone hysterectomy. Amongst premenopausal women, levels of VEGF were high in 22 EBC patients when compared to normal premenopausal controls. No correlation was found between plasma and intratumoral VEGF, clinicopathological prognostic parameters or tumour microvessel density. The origin of circulating VEGF differs between pre- and postmenopausal women. Its measurement is unlikely to provide clinically useful diagnostic and prognostic information in women with early and advanced breast cancer.

The feasibility of well-logging measurements of arsenic levels using neutron-activation analysis.

Arsenic is an extremely toxic metal, which poses a significant problem in many mining environments. Arsenic contamination is also a major problem in ground and surface waters. A feasibility study was conducted to determine if neutron-activation analysis is a practical method of measuring in situ arsenic levels. The response of hypothetical well-logging tools to arsenic was simulated using a readily available Monte Carlo simulation code (MCNP). Simulations were made for probes with both hyperpure germanium (HPGe) and bismuth germanate (BGO) detectors using accelerator and isotopic neutron sources. Both sources produce similar results; however, the BGO detector is much more susceptible to spectral interference than the HPGe detector. Spectral interference from copper can preclude low-level arsenic measurements when using the BGO detector. Results show that a borehole probe could be built that would measure arsenic concentrations of 100 ppm by weight to an uncertainty of 50 ppm in about 15 min.

New Insights Into the Role of Ubiquitylation of Proteins.

Posttranslational modification of proteins by the addition of ubiquitin and related modifiers has an essential role in cellular processes such as protein degradation and subcellular localization. This impacts on the study of cell and developmental biology in diseases such as cancer, and on the study of protein folding and stability in Alzheimer's disease and other diseases of protein aggregation and misfolding. Recently, there have been many additions to the ubiquitylation literature that have challenged, revised, and expanded our understanding and future directions of this process. Here we present a comprehensive overview of the classical textbook description of protein ubiquitylation and then review the recent literature that has challenged and revised the canonical models of protein ubiquitylation. We discuss the roles of noncanonical ubiquitylation at sites other than lysine residues, unconventional ubiquitylation of mixed and branched polyubiquitin chains, and highlight the role of other structural and posttranslational modifications in ubiquitylation that have been identified in the recent literature. By highlighting the assumptions that have been challenged and revised in the field of protein ubiquitylation, we hope to stimulate further study and questions about this ubiquitous protein modification.

Alteration in platelet function in patients with early breast cancer.

The aim of this study was to examine our hypothesis that platelets of patients with breast cancer were functionally altered compared to healthy controls. The results have shown that the platelets from women with early breast cancer released significantly more vascular endothelial growth factor (VEGF) when stimulated with thrombin, tissue factor, clotting, or over a period of time. Similarly, release of thrombospondin (TSP) with thrombin and tissue factor was higher, but failed to reach a significant level. Thus, the observed differences in platelet response support our hypothesis, but warrant further work to determine the reason underlying the observed difference and potential clinical relevance of our findings.

Clinical and molecular aspects of nephropathic cystinosis.

Nephropathic cystinosis, an autosomal recessively inherited lysosomal storage disease, results from impaired transport of the disulfide amino acid cystine out of cellular lysosomes. The consequent accumulation and crystallization of cystine destroys tissues, causing growth retardation in infancy, renal failure at 10 years of age, and a variety of other complications. Early oral therapy with the cystine-depleting agent cysteamine prevents renal deterioration and enhances growth. Although the lysosomal cystine carrier has been extensively studied, its molecular structure remains unknown. The lysosomal cystine transporter gene has been mapped by linkage analysis to human chromosome 17p between polymorphic microsatellite markers D17S1583 and D17S1584. Pertinent recombination events and homozygosity by descent has verified that the cystinosis gene lies in the 3.6 cM genetic interval between these two markers. The cystinosis region has been substantially reduced in size by the observation of recombination events in cystinosis patients between markers D17S1828 and D17S2167. According to radiation hybrid analysis, these two markers are separated by 10.2 cR8000 (centirad using 8000 rad radiation hybrids). Estimates of the physical size of this interval range from 187 to 510 kb. Four yeast artificial chromosomes have been identified which form a contig covering the original cystinosis region. Two P1 clones together may span the new, smaller interval, meaning that the cystinosis gene would lie on one of them. Current efforts are being directed toward using these P1 clones to isolate candidate cDNAs by a variety of methods. The ultimate cloning of the cystinosis gene will reveal how functional lysosomal porters are synthesized, targeted, processed, and integrated into the lysosomal membrane.

Investigation of proposed mechanisms of chemotherapy-induced venous thromboembolism: endothelial cell activation and procoagulant release due to apoptosis.

UNLABELLED: Venous thromboembolism (VTE) during chemotherapy is common, with 7% mortality in metastatic breast cancer (MBC). In a prospective cohort study of patients with breast cancer, we investigated whether vascular endothelial cell activation (VECA), and whether apoptosis, is the cause of chemotherapy-induced VTE. METHODS: Serum markers of VECA, E-selectin (E-sel), vascular cell adhesion molecule 1 (VCAM-1) and d-dimer (fibrin degradation and hypercoagulability marker) were measured prechemotherapy and at 1, 4, and 8 days following chemotherapy. Clinical deep vein thrombosis (DVT) or pulmonary embolism and occult DVT detected by duplex ultrasound imaging were recorded as VTE-positive (VTE+). In patients with MBC, hypercoagulable response to chemotherapy was compared between patients with and without cancer progression. Development of VTE and cancer progression was assessed 3 months following starting chemotherapy. RESULTS: Of the 134 patients, 10 (7.5%) developed VTE (6 [17%] of 36 MBC receiving palliation, 0 of 11 receiving neoadjuvant to downsize tumor, and 4 [5%] of 87 early breast cancer receiving adjuvant chemotherapy, P = .06). Levels of E-sel and VCAM-1 decreased in response to chemotherapy (P < .001) in both VTE+ and patients not developing VTE (VTE-). However, decrease in VECA markers was similar in VTE+ and VTE- patients, implying this is not the cause of VTE. In patients with MBC following chemotherapy, d-dimer (geometric mean) increased by 36% in the 21 patients with MBC responding to chemotherapy but steadily decreased by 11% in the 15 who progressed (day 4, P < .01), implying patients with tumor response (apoptosis) had an early hypercoagulable response. CONCLUSIONS: During chemotherapy for breast cancer, VECA is induced; however, this is not the primary mechanism for VTE. Chemotherapy-induced apoptosis may enhance hypercoagulability and initiate VTE.

Effects of continuous intravenous infusion of an ovine placental extract enriched in placental lactogen on plasma hormones, metabolites and metabolite biokinetics in non-pregnant sheep.

Continuous intravenous infusions of saline or of a placental extract containing ovine placental lactogen were given to three non-pregnant, non-lactating ewes over periods of 36 h, 1 week apart. During saline infusion no placental lactogen was detected in jugular vein plasma. but infusion of the placental extract raised the placental lactogen concentration from undetectable to 40-50 micrograms/l, similar to concentrations in ewes with one fetus on day 90 of pregnancy. By comparison with the saline control period, infusion of the placental extract consistently increased both plasma concentrations and irreversible loss of nonesterified fatty acids. Plasma concentrations of glucose and urea, but not irreversible loss of these metabolites, were consistently increased. Although the placental extract was not subjected to extensive purification, it was enriched in placental lactogen and contained no detectable contamination with insulin, prolactin or growth hormone. The results are suggestive of a role for placental lactogen in modifying metabolism and acting during pregnancy to provide nutrients for fetal metabolism.

Intrafamilial variability in Hurler syndrome and Sanfilippo syndrome type A: implications for evaluation of new therapies.

Intrafamilial variability has not been reported previously in Hurler syndrome or Sanfilippo syndrome type A. We describe two families in which sibs with comparable deficiencies of alpha-iduronidase (Hurler) or sulfamidase (Sanfilippo type A) activities in vitro nonetheless have divergence in clinical severity and disease progression. These cases underscore the need for caution in counseling as well as the limitations of using sibs as controls in evaluating the outcome of treatment.

Protection against experimental salmonellosis in mice and sheep by immunisation with aromatic-dependent Salmonella typhimurium.

Mice immunised by the oral or intraperitoneal route with a live aromatic-dependent strain of Salmonella typhimurium exhibited significantly less protection against oral challenge with 50 LD50 of an ovine isolate of S. typhimurium (12313) than when a bovine isolate with the same O antigens and phage-type as strain 12313 was used as the challenge organism. When challenged with 10 LD50, however, protection against both strains was significantly better than that obtained when mice were vaccinated with killed vaccines (heat-killed, acetone-killed or irradiated) even when the antigenic mass of the killed vaccine was increased by up to 500-fold in an attempt to compensate for the expected limited multiplication of the mutant organism. Sheep immunised with the live mutant strain by either the intramuscular or oral route were protected against oral challenge with the virulent ovine isolate of S. typhimurium; unimmunised sheep died of acute enteritis within 7 days, although there was no evidence of systemic invasion by the challenge organism. After challenge, immunised animals ate more food than the unimmunised controls and suffered only transient, mild diarrhoea. Serum antibody titres against O and H antigens measured by direct or antiglobulin tests were significantly higher in sheep immunised by the intramuscular route than in those immunised orally. Sheep in both immunised groups developed skin swellings within 30 min after intradermal inoculation with purified homologous lipopolysaccharide indicating development of immediate-type hypersensitivity, but only those immunised by the intramuscular route showed significant indurated skin swellings characteristic of delayed-type hypersensitivity 48 and 72 h post-inoculation.(ABSTRACT TRUNCATED AT 250 WORDS)

Management of symptomatic lymphocele via percutaneous drainage and sclerotherapy with tetracycline.

Lymph collections following pelvic surgery are usually asymptomatic, but may occasionally produce symptoms requiring invasive therapy. We have successfully obliterated a large lymphocele by employing percutaneous drainage and instilling tetracycline.

Pelvic lymphadenectomy for staging clinically localized prostate cancer. Indications, complications, and results in 217 cases.

We reviewed the findings of 217 consecutive pelvic lymphadenectomies performed in patients with clinically localized prostatic carcinoma focusing particular attention on the importance of completely removing the hypogastric lymph nodes and on the operative complications associated with a more extensive dissection. Metastatic disease was identified in the lymph nodes of 127 patients (58.6%). The hypogastric nodes were involved in two thirds of the patients with lymph node metastases, and in 29 percent the hypogastric nodes were the only site of metastasis. No increased operative morbidity was documented as a result of extending the level of the pelvic lymphadenectomy to include the lower hypogastric nodes. We conclude that although the lower hypogastric lymph nodes have not been routinely included in most pelvic lymphadenectomies, their removal is important in detecting metastases.

The sensitivity of the assessment process in screening mammography.

A follow-up of 3143 women from four screening centres, who were assessed after screening mammography, showed that 371 had breast cancer diagnosed at that assessment and 62 developed a breast cancer in the following 6 years. Of these, 32 were judged to be false-negatives at assessment, with 23 of the 32 at the site originally suspected. The results show that the assessment process is not infallible and that the scale of the problem only becomes clear when an exhaustive search is made for missed cancers.

Tay-Sachs disease as a model for screening inborn errors.

In the absence of treatments for most inborn errors of metabolism, the goal of both geneticists and health care providers has been the prevention of disease through identification of at-risk couples. When the enzyme deficiency responsible for a disorder is known, heterozygotes can frequently be identified by enzyme assay. The presence or absence of specific mutations in the genes coding for these enzymes may be determined directly if the gene of interest has been identified and characterized. Because the inherited metabolic disorders are rare, these approaches are useful only for individuals with a family history of a specific disease or for populations in which the gene frequency for a specific disease is increased. Tay-Sachs disease is a fatal, autosomal recessive, metabolic disease caused by deficient activity of the lysosomal enzyme Hex A. Although it is rare in the general population, in which the heterozygote frequency is approximately 1/167, it is elevated in a few populations, including the Ashkenazi Jewish community, in which the heterozygote frequency is 1/30. The ability to detect TSD heterozygotes reliably and to diagnose TSD prenatally using a simple and rapid enzyme assay has made prevention of this disorder possible through education and carrier screening. The identification of specific TSD mutations at the DNA level enables laboratories to provide more accurate screening and diagnosis in some families. The success of TSD screening in the Ashkenazi Jewish population has made it the prototype for screening among the inborn errors of metabolism. The TSD example becomes increasingly relevant as heterozygote detection becomes possible for other genetic disorders that are increased in well-defined populations. Cystic fibrosis is such a disease in the caucasian population.

Passive immunisation of neonatal lambs via colostrum and milk of ewes previously immunised with live attenuated Salmonella typhimurium protects neonatal lambs from experimental salmonellosis.

Lambs sucking non-immunised ewes or ewes immunised 4-5 weeks before lambing with live attenuated, aromatic-dependent (aroA) Salmonella typhimurium (strain CS 332) were challenged orally at either 2, 4 or 7 days of age with virulent S. typhimurium (strain CS 94) at doses ranging from 10(9) to 10(13) colony forming units. No lambs displayed signs of clinical salmonellosis and all survived challenge but those sucking immunised ewes had organisms of the challenge strain in their faeces for much shorter periods of time than lambs of the control ewes. High titres of specific antibodies were measured in colostrum and milk of immunised ewes in comparison with very low titres measured in samples from control ewes; these differences were reflected by the titres of antibodies in the sera of corresponding lambs. At 2 days after lambing, the major antibody isotype in the colostrum of immunised ewes and sera of their lambs was IgM whereas at 7 days IgG1 was the predominant isotype. While it was clear that vaccination of pregnant ewes with the live attenuated vaccination conferred protection against experimentally-induced salmonellosis in their lambs, considerable protection was observed in control lambs in spite of there being very low titres of antibodies in the mammary secretion of their dams. The latter observation could be related to the presence of contain non-antibody potent bactericidal factors previously described in colostrum and milk.

Plasma concentrations of tumour dimeric pyruvate kinase are increased in patients with chronic cardiac failure.

BACKGROUND: The aim of the study was to assess the effect of chronic cardiac failure (CCF) on circulating plasma concentrations of tumour metabolic marker dimeric pyruvate kinase type M2 (M2-PK). METHODS: Fifty patients with clinically stable CCF were studied. Patients with a history of past or ongoing malignancy were excluded. Dimeric M2-PK was measured by enzyme-linked immunosorbent assay in EDTA plasma. RESULTS: Dimeric M2-PK concentration increased significantly (P = 0.005) with increasing clinical severity of CCF as assessed by the New York Heart Association classification grade. CONCLUSIONS: Chronic cardiac failure results in an increased circulating plasma concentration of M2-PK, probably related to increased glycolytic flux. The physiological significance of the results is at present unclear but may relate to maintaining a balance between energy production and the supply of glycolytic intermediates to maintain synthetic processes. The results of this investigation will also have significance in the clinical interpretation of M2-PK concentrations.

Development of a method to measure plasma and whole blood choline by liquid chromatography tandem mass spectrometry.

BACKGROUND: Current gold standard markers for myocardial damage are troponins I and T, which are both sensitive and specific for the detection of myocardial infarction, but require up to 6 h to become reliably elevated in serum. Investigation into markers with potential to identify patients with early ischaemic changes is therefore intense. Choline is reported to be prognostic in patients presenting with acute coronary syndromes via its release from ischaemic cell membranes. METHODS: Liquid chromatography tandem mass spectrometry was used to develop a method to quantitate choline in plasma and blood. The method involves addition of a deuterated internal standard to an aliquot of plasma or blood followed by organic solvent addition, which precipitates the proteins in the sample. Preparation was carried out directly into a 96-deep-well plate. Chromatography of choline used a strong cation exchange column and separation used a Waters Atlantis dC18 analytical column positioned directly before the mass spectrometer source, allowing on-line preanalytical clean up of the sample. RESULTS: The lower limit of quantitation was 0.38 micromol/L, linearity was observed up to 754 micromol/L, with a working concentration range of 0.38-224 micromol/L, inter- and intra-assay coefficients of variation were <6% and <4%, respectively. Samples were stable throughout five freeze-thaw cycles and recovery was between 94% and 114%. CONCLUSIONS: The assay was successfully validated in accordance with FDA guidelines and is suitable for quantitation of choline in research and clinical settings.