Common genetic variants do not predict recurrent events in coronary heart disease patients.

BACKGROUND: It is unclear whether genetic variants identified from single nucleotide polymorphisms (SNPs) strongly associated with coronary heart disease (CHD) in genome-wide association studies (GWAS), or a genetic risk score (GRS) derived from them, can help stratify risk of recurrent events in patients with CHD. METHODS: Study subjects were enrolled at the close-out of the LIPID randomised controlled trial of pravastatin vs placebo. Entry to the trial had required a history of acute coronary syndrome 3-36 months previously, and patients were in the trial for a mean of 36 months. Patients who consented to a blood sample were genotyped with a custom designed array chip with SNPs chosen from known CHD-associated loci identified in previous GWAS. We evaluated outcomes in these patients over the following 10 years. RESULTS: Over the 10-year follow-up of the cohort of 4932 patients, 1558 deaths, 898 cardiovascular deaths, 727 CHD deaths and 375 cancer deaths occurred. There were no significant associations between individual SNPs and outcomes before or after adjustment for confounding variables and for multiple testing. A previously validated 27 SNP GRS derived from SNPs with the strongest associations with CHD also did not show any independent association with recurrent major cardiovascular events. CONCLUSIONS: Genetic variants based on individual single nucleotide polymorphisms strongly associated with coronary heart disease in genome wide association studies or an abbreviated genetic risk score derived from them did not help risk profiling in this well-characterised cohort with 10-year follow-up. Other approaches will be needed to incorporate genetic profiling into clinically relevant stratification of long-term risk of recurrent events in CHD patients.

Influence of a triclosan toothpaste on periodontopathic bacteria and periodontitis progression in cardiovascular patients: a randomized controlled trial.

BACKGROUND AND OBJECTIVE: Triclosan/copolymer toothpaste is effective in controlling plaque and gingivitis and in slowing the progression of periodontitis. This study describes its influence on microbiological and clinical outcomes, over a 5-year period, in patients with established cardiovascular disease (CVD). MATERIAL AND METHODS: Four-hundred and thirty-eight patients were recruited from the Cardiovascular Unit at The Prince Charles Hospital, Brisbane, Australia, and randomized to triclosan or placebo groups. Six sites per tooth were examined annually for probing pocket depth and loss of attachment. These outcomes were analysed, using generalized linear modelling, in 381 patients who had measurements from consecutive examinations. Concurrent load of the periodontal pathogens Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Tannerella forsythia and Porphyromonas gingivalis was determined, using quantitative real-time PCR, in 437 patients with baseline plaque samples. Group comparisons were expressed as geometric means. The chi-square test was used to test for differences between the two groups of patients with regard to the proportion of patients with different numbers of bacterial species. RESULTS: There was no difference in general health or periodontal status between the groups at baseline. There was a significant reduction in the number of interproximal sites showing loss of attachment between examinations, by 21% on average (p < 0.01), in the triclosan group compared with the placebo group. The prevalence of patients with F. nucleatum and A. actinomycetemcomitans was high and remained relatively constant throughout the 5 years of the study. In contrast, the prevalence of T. forsythia and P. gingivalis showed more variability; however, there was no significant difference between the groups, at any time point, in the prevalence of any organism. A significant difference in the geometric means for P. gingivalis (p = 0.01) was seen at years 1 and 4, and for F. nucleatum (p = 0.01) and in the total bacterial load (p = 0.03) at year 2; however, these differences were not statistically significant following a Bonferroni correction for multiple comparisons. There was no difference between the groups in the geometric means for each organism at year 5. CONCLUSION: Within the limitations of the study, these data suggest that the use of triclosan/copolymer toothpaste significantly slowed the progression of periodontitis in patients with CVD but that it had little influence on key subgingival periodontopathic bacteria in these patients over the 5 years of the study.

The eponymous Dr James Marion Sims MD, LLD (1813-1883).

Dr James Marion Sims was born in 1813 in Lancaster County, South Carolina. It was while pioneering numerous surgical procedures in Alabama that in 1849 he achieved the outstanding landmark in medical history of successfully, and consistently, repairing vesicovaginal fistulae. Sims soon developed a reputation as a fine surgeon, with new operations and techniques, using novel surgical instruments and his innovative approaches frequently published. Moving to New York City in 1853, he further established hospitals devoted entirely to women's health. Sims was controversial, with flamboyant descriptions of self-confident success, yet they were tempered with sober reflection of failure and loss. Today we remain with the Sims speculum and Sims position, eponymous tributes to his accomplishments as the 'Father of Gynaecology'.

Cardiopulmonary exercise testing prior to myeloablative allo-SCT: a feasibility study.

The feasibility of symptom-limited cardiopulmonary exercise testing (CPET) prior to allo-SCT was assessed in addition to the prognostic value of CPET-derived measures. CPET was performed prospectively on 21 patients with hematologic malignancies, with assessments of peak (for example, peak oxygen consumption, VO2peak) and submaximal (for example, ventilatory threshold (VT)) measures of cardiopulmonary function. No serious adverse events were observed during CPET procedures, with 95% of patients achieving criteria for a peak test. Mean VO2peak was 24.7±6.4 mL kg(-1 )min(-1) (range: 10.9-35.5), equivalent to 29%±17% below that of age-matched healthy controls. All patients proceeded with the conditioning regimen followed by allo-SCT. Median follow-up was 25 months. During this period, 11 (52.4%) patients died (n=6, relapsed disease; n=5, non-relapse mortality (NRM)); 9 patients (43%) developed pulmonary toxicity. In univariate analyses, both peak and submaximal markers of cardiopulmonary function were predictors of OS, pulmonary toxicity and NRM. For OS, the HR for VO2peak and VT were 0.89 (95% CI, 0.8-0.99, P=0.04) and 0.84 (95% CI, 0.71-0.98, P=0.03), respectively. In conclusion, CPET is safe and feasible prior to allo-SCT. Patients have marked impairments in cardiopulmonary function prior to allo-SCT. CPET-derived metrics may complement conventional measures to improve risk stratification.

Perforation as a rare presentation of gastric gastrointestinal stromal tumours: a case report and review of the literature.

INTRODUCTION: Gastrointestinal stromal tumours (GISTs) are the most common connective tissue neoplasms of the gastrointestinal tract, the most common clinical presentation of which is with abdominal pain or gastrointestinal bleeding. METHODS: We describe a case of a perforated gastric GIST as well as reviewing the relevant published literature. RESULTS: A 51-year-old woman presented to the acute assessment unit with a 1-day history of severe epigastric pain on a background of longstanding reflux symptoms. Radiological investigation demonstrated a perforated mass in the gastric antrum and the patient subsequently underwent an emergency distal gastrectomy. She recovered well postoperatively and was discharged home. Her condition remains stable six months following surgery. Histological analysis revealed the perforated lesion to be a GIST. A PubMed search suggests that this is the first English report to describe a perforated gastric GIST. Six further published reports (written in English or with an English abstract) describing the presentation of small bowel GISTs with perforation are reviewed. CONCLUSIONS: We present the first English report of a perforated gastric GIST. More common presentations include abdominal pain and gastrointestinal bleeding. Although rare, GISTs should be considered in the differential diagnoses of perforated gastrointestinal masses.

No evidence of triclosan-resistant bacteria following long-term use of triclosan-containing toothpaste.

BACKGROUND AND OBJECTIVE: There is a paucity of data in relation to the possible emergence of triclosan (TCS)-resistant bacteria following long-term exposure to TCS toothpaste. Therefore, this study investigated whether long-term continuous exposure to TCS in toothpaste selects for TCS-resistant bacteria within the oral biofilm. MATERIAL AND METHODS: Dental plaque samples were collected from 40 individuals during year 5 of a randomised controlled trial. Participants had been randomly assigned to use TCS (3000 µg/mL TCS) (n = 18) or placebo toothpaste (n = 22). Diluted plaque samples were plated on to Wilkins-Chalgren agar plates containing 5% (v/v) laked sheep red blood cells and TCS (concentrations ranging from 25 to 150 µg/mL) and incubated at 37 °C under microaerophilic and anaerobic conditions for 2-10 d. Selected bacterial isolates were identified by partial 16S rDNA sequencing and TCS minimum inhibitory concentration (MIC) determined for each isolate. RESULTS: At 3000 µg/mL TCS no growth was observed under microaerophilic or anaerobic conditions in either group. The MICs of TCS for all isolates ranged from 125 to 1000 µg/mL in both groups. Species common to both groups had similar MICs. Veillonella parvula and Campylobacter gracilis were the most frequent isolates from both groups, with similar MICs in both groups. CONCLUSION: The use of TCS-containing toothpaste did not appear to lead to an increase in MIC of TCS of oral bacterial isolates.

Improved periodontal health and cardiovascular risk.

BACKGROUND: Previous studies have demonstrated variable effects on systemic inflammatory and immune responses following improved periodontal health. This study examined changes in serum levels of the inflammatory mediators IL-1ß, IL-6, TNF-α and sICAM-1, and antibodies to Porphyromonas gingivalis, human heat shock protein (hHSP) 60 and P. gingivalis GroEL following improvement in periodontal health in high cardiovascular (CV) risk and low CV-risk patients. METHODS: Patients retrospectively selected from a longitudinal study, had undergone yearly periodontal examinations and peripheral blood collections. They had demonstrated a quantifiable improvement in periodontal health (>60% reduction in number of sites with probing depth ≥ 4 mm from the baseline visit) and could be classified as either high CV-risk (≥ 6 classical risk factors, n = 13) or low CV-risk (≤ 1 classical risk factor, n = 14). Serum levels of the cytokines and antibodies were measured using ELISA. RESULTS: For sICAM-1 and anti-P. gingivalis GroEL and anti-hHSP60 antibodies, most patients recorded decreased levels. Reductions in serum sICAM-1 levels were more notable in low CV-risk patients (p = 0.006); and reductions in levels of anti-P. gingivalis GroEL and anti-hHSP60 antibodies (p = 0.001 and 0.009 respectively) were more notable in high CV-risk patients. CONCLUSIONS: This study found that subsequent to improved periodontal health, the anti-HSP (HSP60 and GroEL) antibody response was reduced, particularly for high CV-risk patients. sICAM-1 levels were also lowered, more so for low CV-risk patients.

High antibody levels to P. gingivalis in cardiovascular disease.

Recent evidence suggests that strain variation in the serum IgG response to Porphyromonas gingivalis occurs in periodontal disease and cardiovascular disease (CVD). This study aimed to test the hypothesis that different P. gingivalis strains would elicit different levels of IgG, depending on a patient's cardiovascular (CV) and periodontal health. For CVD patients, serum antibody levels increased significantly with increasing numbers of deep pockets for all strains of P. gingivalis, except W50 (p < 0.001). We used a two-way analysis of variance to examine differences in antibody responses across several CV and periodontal groups simultaneously. There was a significant interaction effect (p < 0.05) between periodontal status and CV status for antibody levels to ATCC33277, UQD605, and Su63. This study shows variation in strain type with respect to serum IgG response in several CV and periodontal categories, providing further support for the role of the immune response to P. gingivalis in the relationship between periodontal disease and CVD.

Decreased cell proliferation in the dentate gyrus does not associate with development of anhedonic-like symptoms in rats.

Depressive disorders have been proposed to be caused by stress-induced down-regulation of hippocampal neurogenesis. Nevertheless, several reports have recently pointed out that, in rodent models of depression, suppression of generation of new hippocampal neurons is not by itself sufficient to induce the development of depression-related symptoms. In the present study, we used the cell proliferation blocker methylazoxymethanol (MAM) and the rat chronic mild stress (CMS) model of depression to challenge the neurogenic theory of depression. In order to achieve a comparable reduction in hippocampal cytogenesis, rats were either chronically treated with MAM for 2 weeks, or subjected to an 8 week regime of chronic mild stress. Consumption of a palatable sucrose solution was monitored once a week to assess the development of anhedonic behavior. Prior to terminal perfusion, the animals were injected with bromodeoxyuridine, a marker of proliferating cells. The number of proliferating cells and total cell number and volume were estimated for the granule cell layer of the ventral hippocampal formation. Unlike CMS, chronic injections with MAM did not induce anhedonia-like symptoms in rats. Both MAM-treated and CMS-exposed groups of rats showed a comparable significant reduction in cell proliferation in the granular cell layer of the ventral hippocampal formation. However, the total cell number was reduced for CMS-exposed rats only while the granule cell layer volume was conserved for both groups. Our results show that suppression of cell proliferation in the hippocampal formation is not an absolute factor for induction of an anhedonia-like state in rats. However, it may still represent an important causal factor for vulnerable subjects.

Inflammation, heat shock proteins and periodontal pathogens in atherosclerosis: an immunohistologic study.

BACKGROUND: Inflammation is a significant component of atherosclerosis lesions. Bacteria, including periodontopathogens, have been demonstrated in atherosclerotic plaques and cross-reactivity of the immune response to bacterial GroEL with human heat shock protein 60 has been suggested as a link between infections and atherosclerosis. METHODS: In this study, the nature of the inflammatory infiltrate and the presence of human heat shock protein 60 and GroEL were examined in 31 carotid endarterectomy specimens. Additionally, monoclonal antibodies were used to detect the presence of six bacteria, including those implicated in periodontal disease. RESULTS: The inflammatory cell infiltrate of the lesions was dominated by CD14(+) macrophages and CD4(+) T cells. Most cells of the infiltrate as well as the endothelium were HLA-DR(+), indicating activation; however, there was an absence of CD25 expression, demonstrating that the activated T cells were not proliferating. Few CD1a(+) and CD83(+) cells were noted. Human heat shock protein 60 expression was evident on endothelial cells and cells with the appearance of smooth muscle cells and lymphocytes. GroEL and bacteria were detected within intimal cells. Chlamydia pneumoniae, Porphyromonas gingivalis, Fusobacterium nucleatum, Tannerella forsythia, Prevotella intermedia, and Actinobacillus actinomycetemcomitans were found in 21%, 52%, 34%, 34%, 41%, and 17% of arteries, respectively. CONCLUSION: These results give evidence for a specific immune response associated with atherosclerosis. Whether bacteria initiate the observed inflammation in atherosclerotic lesions is not clear; however, the present study shows that maintenance of inflammation may be enhanced by the presence of periodontopathic bacteria.

EBV EBNA 2 stimulates CDK9-dependent transcription and RNA polymerase II phosphorylation on serine 5.

EBNA 2 is one of only five viral genes essential for the infection and immortalization of human B cells by the cancer-associated virus Epstein-Barr virus (EBV). EBNA 2 activates cellular and viral transcription and associates with components of the basal transcription apparatus and a number of coactivators. We provide the first evidence to show that the mechanism of transcriptional activation by EBNA 2 also involves phosphorylation of the C-terminal domain (CTD) of RNA polymerase II (pol II). We found that transcriptional activation by EBNA 2 was inhibited by a dominant-negative mutant of the pol II CTD kinase, CDK9, and by low concentrations of the CDK9 inhibitor 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole. Moreover, using chromatin immunoprecipitation assays we demonstrated that EBNA 2 stimulates both pol II recruitment and pol II phosphorylation on serine 5 of the CTD in vivo. These results identify a new step in the transcription cycle that is subject to regulation by a key EBV-encoded transcription factor and highlight CDK9 inhibitors as potential anti-EBV agents.

Estimators of the precision of stereological estimates: an example based on the CA1 pyramidal cell layer of rats.

Because of the complex and dynamic structure of the brain, there is perhaps no other organ system in which the application of stereological methods can contribute so much with regard to understanding normal and pathological processes. In order to design the studies in an optimal manner, with regard to the number of individuals, sections, probes, and to be able to critically evaluate the stereological studies made by others, it is important for neuroscientists to have an understanding of the precision or reproducibility of a stereological estimation procedure. This precision or reproducibility is often referred to as the coefficient of error of the estimate, which is a statistical expression for the size of the standard error of the mean of repeated estimates, relative to the mean of the estimates. Like the 'margin of error' associated with public opinion polls, it indicates how much the estimate would vary if it were repeated numerous times. It is difficult and time consuming to empirically derive the coefficient of error of estimates made of features observed in histological preparations. To overcome this obstacle, it is common practice to try to get a feeling for the precision of an estimate by estimating the coefficient of error itself. In this paper, we will compare and discuss the coefficient of error of estimates of volume and cell number made with different numbers of sections and probes in the CA1 pyramidal cell layer of the rat hippocampus. The conclusions drawn from this analysis indicate that, using practically feasible and anatomically sensible sampling schemes, the Gundersen-Jensen coefficient of error estimator or the 'Split-Sample' coefficient of error estimator can provide useful information about the precision of stereological estimates even in highly irregular brain regions and requires little work.

Histopathology and fibrillin-1 distribution in severe early onset Marfan syndrome.

Marfan syndrome (MFS) is an autosomal dominant condition which may involve the cardiovascular, ocular, skeletal, and other systems. Mutations causing MFS are found in the FBN1 gene, encoding fibrillin-1, an extracellular matrix protein involved in microfibril formation. In the most severe cases, mutations are generally found in exons 24-32, and children with these mutations usually die in the first years of life, of cardiopulmonary failure. We present clinical, molecular and histopathological studies on a patient with severe early onset MFS. He has a mutation in exon 25 of FBN1, a G>A transition at nucleotide position 3131 that converts the codon TGC, coding for cysteine at position 1044, to TAC, coding for tyrosine (C1044Y). This has resulted in abnormalities of the extracellular matrix and a severe clinical phenotype, although he has survived to the age of 14 years.

Cross-reactivity of GroEL antibodies with human heat shock protein 60 and quantification of pathogens in atherosclerosis.

BACKGROUND/AIMS: Chronic infections such as those caused by Chlamydia pneumoniae and periodontopathic bacteria such as Porphyromonas gingivalis have been associated with atherosclerosis, possibly due to cross-reactivity of the immune response to bacterial GroEL with human heat shock protein (hHSP) 60. METHODS: We examined the cross-reactivity of anti-GroEL and anti-P. gingivalis antibodies with hHSP60 in atherosclerosis patients and quantified a panel of six pathogens in atheromas. RESULTS: After absorption of plasma samples with hHSP60, there were variable reductions in the levels of anti-GroEL and anti-P. gingivalis antibodies, suggesting that these antibodies cross-reacted with hHSP60. All of the artery specimens were positive for P. gingivalis. Fusobacterium nucleatum, Tannerella forsythia, C. pneumoniae, Helicobacter pylori, and Haemophilus influenzae were found in 84%, 48%, 28%, 4%, and 4% of arteries, respectively. The prevalence of the three periodontopathic microorganisms, P. gingivalis, F. nucleatum and T. forsythia, was significantly higher than that of the remaining three microorganisms. CONCLUSIONS: These results support the hypothesis that in some patients, cross-reactivity of the immune response to bacterial HSPs including those of periodontal pathogens, with arterial endothelial cells expressing hHSP60 may be a possible mechanism for the association between atherosclerosis and periodontal infection.

Immersion autometallographic tracing of zinc ions in Alzheimer beta-amyloid plaques.

An easy to perform autometallographic technique (AMG) for capturing zinc ions in Alzheimer plaques is presented. The possibility of visualizing loosely bound or free zinc ions in tissue by immersion autometallography (iZnS(AMG)) is a relatively recent development. The iZnS(AMG) staining is caused by zinc-sulphur nanocrystals created in 1-2 mm thick brain slices that are immersed in a 0.1% sodium sulphide, 3% glutaraldehyde phosphate buffered solution, the NeoTimm Solution (NTS), for 3 days. When the zinc-sulphur nanocrystals are subsequently silver-enhanced by autometallography, the plaques are readily identified as spheres of dark interlacing strands of different sizes, embedded in the pattern of zinc-enriched terminals. The zinc specificity of the iZnS(AMG) technique was tested by immersion of brain slides in the chelator DEDTC prior to the NTS immersion. The iZnS(AMG) detection of zinc ions is easily standardized and can be used in the quantification of plaques with stereological methods. This technique is the first to detect zinc in plaques in the cerebellum of transgenic PS1/APP mice and the first to detect zinc ions in plaques and dystrophic neurites at electron microscopical levels.

Cultures, genes, and neurons in the development of song and singing in brown-headed cowbirds (Molothrus ater).

In brown-headed cowbirds, Molothrus ater, as in many songbird species, vocalizations are fundamental to reproduction. In our studies, experiments utilizing different social housing regimes and geographic comparisons have indicated the social learning of males' vocalizations and associated abilities to use vocalizations effectively during the breeding season. Here, we describe studies indicating roles of cultural and genetic background, and of social influences from females, on male vocal development. These influences can interact with neural regions, including song learning and song control nuclei, but also visual-processing nuclei, in the development of signaling. We argue that a developmental systems approach to the study of vocal behavior provides a structure to organize these different influences and how they may interact with one another over development. A systems approach requires that researchers study the social context in which signals and signalers develop - both the ontogenetic arena in which young animals learn their signals from older animals, and the functional arena in which young and older animals socially interact with one another.

Stereological length estimation using spherical probes.

Lineal structures in biological tissue support a wide variety of physiological functions, including membrane stabilization, vascular perfusion, and cell-to-cell communication. In 1953, Smith and Guttman demonstrated a stereological method to estimate the total length density (Lv) of linear objects based on random intersections with a two-dimensional sampling probe. Several methods have been developed to ensure the required isotropy of object-probe intersections, including isotropic-uniform-random (IUR) sections, vertical-uniform-random (VUR) slices, and isotropic virtual planes. The disadvantages of these methods are the requirements for inconvenient section orientations (IUR, VUR) or complex counting rules at multiple focal planes (isotropic virtual planes). To overcome these limitations we report a convenient and straightforward approach to estimate Lv and total length, L, for linear objects on tissue sections cut at any arbitrary orientation. The approach presented here uses spherical probes that are inherently isotropic, combined with unbiased fractionator sampling, to demonstrate total L estimation for thin nerve fibres in dorsal hippocampus of the mouse brain.

Activation of human immunodeficiency virus transcription in T cells revisited: NF-kappaB p65 stimulates transcriptional elongation.

Human immunodeficiency virus type 1 (HIV-1) is able to establish a persistent latent infection during which the integrated provirus remains transcriptionally silent. Viral transcription is stimulated by NF-kappaB, which is activated following the exposure of infected T cells to antigens or mitogens. Although it is commonly assumed that NF-kappaB stimulates transcriptional initiation alone, we have found using RNase protection assays that, in addition to stimulating initiation, it can also stimulate elongation from the HIV-1 long terminal repeat. When either Jurkat or CCRF/CEM cells were activated by the mitogens phorbol myristate acetate and phytohemagglutinin, elongation, as measured by the proportion of full-length transcripts, increased two- to fourfold, even in the absence of Tat. Transfection of T cells with plasmids carrying the different subunits of NF-kappaB demonstrated that the activation of transcriptional elongation is mediated specifically by the p65 subunit. It seems likely that initiation is activated because of NF-kappaB's ability to disrupt chromatin structures through the recruitment of histone acetyltransferases. To test whether p65 could stimulate elongation under conditions where it did not affect histone acetylation, cells were treated with the histone deacetylase inhibitor trichostatin A. Remarkably, addition of p65 to the trichostatin A-treated cell lines resulted in a dramatic increase in transcription elongation, reaching levels equivalent to those observed in the presence of Tat. We suggest that the activation of elongation by NF-kappaB p65 involves a distinct biochemical mechanism, probably the activation of carboxyl-terminal domain kinases at the promoter.