ING2 regulates the onset of replicative senescence by induction of p300-dependent p53 acetylation.

ING2 is a candidate tumor suppressor gene that can activate p53 by enhancing its acetylation. Here, we demonstrate that ING2 is also involved in p53-mediated replicative senescence. ING2 protein expression increased in late-passage human primary cells, and it colocalizes with serine 15-phosphorylated p53. ING2 and p53 also complexed with the histone acetyltransferase p300. ING2 enhanced the interaction between p53 and p300 and acted as a cofactor for p300-mediated p53 acetylation. The level of ING2 expression directly modulated the onset of replicative senescence. While overexpression of ING2 induced senescence in young fibroblasts in a p53-dependent manner, expression of ING2 small interfering RNA delayed the onset of senescence. Hence, ING2 can act as a cofactor of p300 for p53 acetylation and thereby plays a positive regulatory role during p53-mediated replicative senescence.

Clinical Utility of CINtec PLUS Triage in Equivocal Cervical Cytology and Human Papillomavirus Primary Screening.

OBJECTIVES: While cervical cytology is accepted triage for human papillomavirus (HPV)-positive women, the efficiency of cervical screening could be improved by exploiting disease markers with higher specificity. METHODS: CINtec PLUS triage alone and combined with HPV 16/18 genotyping was performed on ThinPrep samples from HPV-positive women. Clinical performance and the potential to reduce or expedite colposcopy referrals were evaluated. RESULTS: The 2-year sensitivity and specificity for cervical intraepithelial neoplasia grade 3 or worse (CIN3+) were 90% and 42%, respectively. Specificity was improved over HPV testing in equivocal cytology and could cut the referral rate by about 40%. When combined with HPV 16/18 genotyping, CINtec PLUS triage of the 12 other high-risk HPV genotypes generally demonstrated better sensitivity for CIN3+ than separate triage of non-type-specific HPV-positive women. This strategy could reduce colposcopy referrals by 31%. CONCLUSIONS: These findings highlight the potential of CINtec PLUS to improve management pathways in HPV-positive women. CINtec PLUS cytology represents a sensitive and efficient triage in HPV-positive women. The clinical performance of the dual-stain was most notable in women younger than 25 years and could potentially improve management pathways.

Stability of human papillomavirus (HPV) in cervical ThinPrep specimens previously lysed with glacial acetic acid: effect on cobas 4800 HPV test performance.

BACKGROUND: The authors previously demonstrated that lysing samples with glacial acetic acid (GAA) before human papillomavirus (HPV) testing does not adversely affect HPV detection with the cobas 4800 HPV Test. However, the long-term impact of GAA on HPV DNA was not explored in that study, and inherent cell loss with the lysing protocol used also was observed. The current study considered the long-term effects of GAA treatment of cervical ThinPrep samples on HPV detection with the cobas 4800 HPV Test. They also modified the manufacturer's lysing procedure for ThinPrep specimens to help prevent cell loss. METHODS: Seventy-eight ThinPrep samples, including previously lysed, archived specimens, were split; then, 1 part was treated with GAA according to the manufacturer's protocol or using a modified protocol, and the other part was left untreated. All samples were tested for HPV using the cobas 4800 HPV Test. The HPV-positive/HPV-negative status of tested samples was used to determine the level of agreement between treated and untreated fractions. Performance of the modified lysing procedure was assessed relative to the manufacturer's protocol. RESULTS: Positive HPV status produced 97% agreement between the GAA-treated/untreated fractions. Concordance between the GAA-treated/untreated fractions did not differ between the 2 lysing methods; however, the average percentage increases in ß-globin and HPV cycle threshold values after lysing were less discernible for the modified lysing method. CONCLUSIONS: GAA treatment of cervical ThinPrep specimens does not have long-term adverse effects on HPV detection with the cobas 4800 HPV Test. The manufacturer's GAA lysing procedure for ThinPrep specimens can be reliably modified for specimens that may subsequently undergo HPV testing.

Human papillomavirus testing in young women: clinical outcomes of human papillomavirus triage in a UK cervical screening program.

BACKGROUND: In the United Kingdom, human papillomavirus (HPV) testing is used to triage women with borderline cytology or mild dyskaryosis; however, in young women, the value of triage is limited by the high HPV prevalence rate. The current study examined the impact of HPV triage on colposcopy referral, colposcopy procedures, and patient outcome in a cervical screening population that included women aged < 25 years. METHODS: Women aged 18 to 65 years attending for cervical screening in Northern Ireland were tested for HPV if their cytology result demonstrated borderline cytology or mild dyskaryosis. Of the 866 women eligible for HPV triage, those who tested negative for HPV were returned to routine screening and women who tested positive were referred to colposcopy. RESULTS: HPV prevalence was 82.07% in women aged < 25 years and 54.69% in women aged ≥ 25 years. Colposcopy referrals increased by 42.67%. The odds of undergoing a large loop excision of the transformation zone (LLETZ) compared with punch biopsy increased by 0.056 per year above the age of 31 years. LLETZ performed in women aged ≥ 25 years and those aged < 25 years yielded rates of cervical intraepithelial neoplasia of type 2 or higher (≥ CIN2) of 57.04% and 80.00%, respectively. The positive predictive value of HPV triage for detecting ≥ CIN2 was 29.92% in women aged < 25 years and 27.51% in the older age group. CONCLUSIONS: HPV triage substantially increased colposcopy referrals. The positive predictive value of a positive HPV test to detect ≥ CIN2 was not affected by age. LLETZ performed in women aged < 25 years yielded higher rates of ≥ CIN2 compared with the older age group.

Targeted neuronal nitric oxide synthase transgene delivery into stellate neurons reverses impaired intracellular calcium transients in prehypertensive rats.

Hypertension is associated with the early onset of cardiac sympathetic hyperresponsiveness and enhanced intracellular Ca(2+) concentration [Ca(2+)](i) in sympathetic neurons from both prehypertensive and hypertensive, spontaneously hypertensive rats (SHRs). Oxidative stress is a hallmark of hypertension, therefore, we tested the hypothesis that the inhibitory action of the nitric oxide-cGMP pathway on [Ca(2+)](i) transients is impaired in cardiac sympathetic neurons from the SHR. Stellate ganglia were isolated from young prehypertensive SHRs and age-matched normotensive Wistar-Kyoto rats. [Ca(2+)](i) was measured by ratiometric fluorescence imaging. Neurons from the prehypertensive SHR ganglia had a significantly higher depolarization evoked [Ca(2+)](i) transient that was also associated with decreased expression of neuronal nitric oxide synthase (nNOS), ß1 subunit of soluble guanylate cyclase and cGMP when compared with the Wistar-Kyoto rat ganglia. Soluble guanylate cyclase inhibition or nNOS inhibition increased [Ca(2+)](i) in the Wistar-Kyoto rats but had no effect in SHR neurons. A nitric oxide donor decreased [Ca(2+)](i) in both sets of neurons, although this was markedly less in the SHR. A novel noradrenergic cell specific vector (Ad.PRSx8-nNOS/Cherry) or its control vector (Ad.PRSx8-Cherry) was expressed in sympathetic neurons. In the SHR, Ad.PRSx8-nNOS/Cherry-treated neurons had a significantly reduced peak [Ca(2+)](i) transient that was associated with increased tissue levels of nNOS protein and cGMP concentration compared with gene transfer of Ad.PRSx8-Cherry alone. nNOS inhibition significantly increased [Ca(2+)](i) after Ad.PRSx8-nNOS/Cherry expression. We conclude that artificial upregulation of stellate sympathetic nNOS via targeted gene transfer can directly attenuate intracellular Ca(2+) and may provide a novel method for decreasing enhanced cardiac sympathetic neurotransmission.

Immunization against GAD induces antibody binding to GAD-independent antigens and brainstem GABAergic neuronal loss.

Stiff person syndrome (SPS) is a highly-disabling neurological disorder of the CNS characterized by progressive muscular rigidity and spasms. In approximately 60-80% of patients there are autoantibodies to glutamic acid decarboxylase (GAD), the enzyme that synthesizes gamma-amino butyric acid (GABA), the predominant inhibitory neurotransmitter of the CNS. Although GAD is intracellular, it is thought that autoimmunity to GAD65 may play a role in the development of SPS. To test this hypothesis, we immunized mice, that expressed enhanced green fluorescent protein (EGFP) under the GAD65 promoter, with either GAD65 (n = 13) or phosphate buffered saline (PBS) (n = 13). Immunization with GAD65 resulted in autoantibodies that immunoprecipitated GAD, bound to CNS tissue in a highly characteristic pattern, and surprisingly bound not only to GAD intracellularly but also to the surface of cerebellar neurons in culture. Moreover, immunization resulted in immunoglobulin diffusion into the brainstem, and a partial loss of GAD-EGFP expressing cells in the brainstem. Although immunization with GAD65 did not produce any behavioral abnormality in the mice, the induction of neuronal-surface antibodies and the trend towards loss of GABAergic neurons in the brainstem, supports a role for humoral autoimmunity in the pathogenesis of SPS and suggests that the mechanisms may involve spread to antigens expressed on the surface of these neurons.

Neuronal surface and glutamic acid decarboxylase autoantibodies in Nonparaneoplastic stiff person syndrome.

IMPORTANCE: High titers of autoantibodies to glutamic acid decarboxylase (GAD) are well documented in association with stiff person syndrome (SPS). Glutamic acid decarboxylase is the rate-limiting enzyme in the synthesis of γ-aminobutyric acid (GABA), and impaired function of GABAergic neurons has been implicated in the pathogenesis of SPS. Autoantibodies to GAD might be the causative agent or a disease marker. OBJECTIVE: To investigate the characteristics and potential pathogenicity of GAD autoantibodies in patients with SPS and related disorders. DESIGN: Retrospective cohort study and laboratory investigation. SETTING: Weatherall Institute of Molecular Medicine, University of Oxford. PARTICIPANTS: Twenty-five patients with SPS and related conditions identified from the Neuroimmunology Service. EXPOSURES: Neurological examination, serological characterization and experimental studies. MAIN OUTCOMES AND MEASURES: Characterization of serum GAD antibodies from patients with SPS and evidence for potential pathogenicity. RESULTS: We detected GAD autoantibodies at a very high titer (median, 7500 U/mL) in 19 patients (76%), including all 12 patients with classic SPS. The GAD autoantibodies were high affinity (antibody dissociation constant, 0.06-0.78 nmol) and predominantly IgG1 subclass. The patients' autoantibodies co-localized with GAD on immunohistochemistry and in permeabilized cultured cerebellar GABAergic neurons, as expected, but they also bound to the cell surface of unpermeabilized GABAergic neurons. Adsorption of the highest titer (700 000 U/mL) serum with recombinant GAD indicated that these neuronal surface antibodies were not directed against GAD itself. Although intraperitoneal injection of IgG purified from the 2 available GAD autoantibody­ositive purified IgG preparations did not produce clinical or pathological evidence of disease, SPS and control IgG were detected in specific regions of the mouse central nervous system, particularly around the lateral and fourth ventricles. CONCLUSIONS AND RELEVANCE: Autoantibodies to GAD are associated with antibodies that bind to the surface of GABAergic neurons and that could be pathogenic. Moreover, in mice, human IgG from the periphery gained access to relevant areas in the hippocampus and brainstem. Identification of the target of the non-GAD antibodies and peripheral and intrathecal transfer protocols, combined with adsorption studies, should be used to demonstrate the role of the non-GAD IgG in SPS.

Continuity of care for women with breast cancer: a survey of the views and experiences of patients, carers and health care professionals.

PURPOSE OF THE RESEARCH: The need for continuity of care in the management of women diagnosed with breast cancer is important yet challenging. The aim was twofold; to explore 1) the concept of continuity of care from the perspectives of women diagnosed with breast cancer, their carers and their health care professionals (HCPs), 2) actual experiences amongst these three groups relating to continuity of care. METHODS AND SAMPLE: A three-phase cross-sectional survey was conducted involving eight focus group interviews with women who had been diagnosed with breast cancer (n = 47) and structured questionnaires to both carers (n = 33) and HCPs (n = 44). Data were analysed according to three categories of continuity of care; relational, informational and managerial. KEY RESULTS: According to the patients: 'Continuity of Care' is the continuous care over time; it involves the relationship between the HCP and the patient. It is not just a follow-up review. The relationship is built on trust, loyalty and constancy. The perception is that the providers of care know you, know your case history and your future care is agreed on.' Across all phases of the study the three categories of continuity of care were identified but there were differences as to the weight different groups placed on them. Continuity of care was reported to be achieved for the majority of the respondents across all three samples however deficiencies in the service were identified. CONCLUSION: The results provide an opportunity to improve service; recommendations have been made and steps to implementation taken.

The relationship between socioeconomic deprivation and metabolic/cardiovascular risk factors in a cohort of patients with type 2 diabetes mellitus.

AIM: There is conflicting information on the impact of socioeconomic deprivation on outcomes in diabetes. Our aim was to study the relationship between socioeconomic deprivation and clinical outcome indicators in a patient cohort with type 2 diabetes mellitus [T2DM]. METHODS: We recruited a random sample of 446 patients with T2DM [57% male] stratified by Income Deprivation Measure. Data on patient specific socioeconomic status, educational attainment, behaviour and attitudes were gathered by patient interview. Data analysis was by logistic and linear regression with correction for age, gender and duration of diabetes. RESULTS: The study cohort showed high levels of deprivation with 80% reporting a household annual income of <£20,000 and 69.5% having no formal educational qualification. The cohort was actively managed with high usage of lipid lowering [90.1%], antihypertensive [80.6%] and antiplatelet agents [78%] and correspondingly good control of modifiable risk factors: HbA1c 7.6 [1.4]%, systolic blood pressure 134.2 [20.6], diastolic blood pressure 72.7 [11.2]mmHg, total cholesterol 4.0 [0.9]mmol/L. Socioeconomic disadvantage was strongly related to measures of integration and attitude to diabetes. Despite this there were no significant or consistent relationships between Income Deprivation Measure/self-reported annual household income/educational attainment on metabolic or cardiovascular risk factors. CONCLUSIONS: Successful management of modifiable risk factors can be achieved in a way that is independent of socioeconomic position.

Neuronal nitric oxide synthase gene transfer decreases [Ca2+]i in cardiac sympathetic neurons.

Gene transfer of neuronal nitric oxide synthase (nNOS) can decrease cardiac sympathetic outflow and facilitate parasympathetic neurotransmission. The precise pathway responsible for nitric oxide (NO) mediated inhibition of sympathetic neurotransmission is not known, but may be related to NO-cGMP activation of cGMP-stimulated phosphodiesterase (PDE2) that enhances the breakdown of cAMP to deactivate protein kinase A (PKA), resulting in a decrease in Ca(2+) influx mediated exocytosis of the neurotransmitter. We investigated depolarization evoked Ca(2+) influx in nNOS gene transduced sympathetic neurons from stellate ganglia with a noradrenergic cell specific vector (Ad.PRS-nNOS or empty vector), and examined how nNOS gene transfer affected cAMP and cGMP levels in these neurons. We found that targeting nNOS into these sympathetic neurons reduced amplitudes of voltage activated Ca(2+) transients by 44%. nNOS specific inhibition by N-[(4S)-4-Amino-5-[(2-aminoetyl](amino] pentyl]-N'-nitroguanidine (AAAN) reversed this response. nNOS gene transfer also increased intracellular cGMP (47%) and decreased cAMP (29%). A PDE2 specific inhibitor Bay60-7557 reversed the reduction in cAMP caused by Ad.PRS-nNOS. These results suggest that neuronal NO modulates cGMP and PDE2 to regulate voltage gated intracellular Ca(2+) transients in sympathetic neurons. Therefore, we propose this as a possible key step involved in NO decreasing cardiac sympathetic neurotransmission.

Sequential chemotactic and phagocytic activation of human polymorphonuclear neutrophils.

Human polymorphonuclear neutrophils (PMN) chemotax to a foreign entity. When the chemoattractants' origins are reached, specific receptors bind to the invader's surface, initiating phagocytosis, phagosome formation, and fusion with granule membranes, generating the bactericidal oxidative burst, and releasing lytic enzymes, specific peptides, and proteins. We explored the initial signaling involved in these functions by observing naïve, unprimed PMN in suspension using fluorescent indicators of cytoplasmic signals (Delta[Ca(2+)](i) and DeltapH(i)) and of bactericidal entities (oxidative species and elastase) exposed to N-formyl-methionyl-leucyl-phenylalanine (fMLP) and/or multivalent immune complexes (IC). fMLP and IC each initiate a rapid transient rise in [Ca(2+)](i), mostly from intracellular stores, simultaneously with a drop in pH(i); these are followed by a drop in [Ca(2+)](i) and a rise in pH(i), with the latter being due to a Na(+)/H(+) antiport. The impact of a second stimulation depends on the order in which stimuli are applied, on their dose, and on their nature. Provided that [Ca(2+)](i) is restored, 10(-7) M fMLP, previously shown to elicit maximal Delta[Ca(2+)](i) but no bactericidal functions, did not prevent the cells' responses with Delta[Ca(2+)](i) to a subsequent high dose of fMLP or IC; conversely, cells first exposed to 120 mug/ml IC, previously shown to elicit maximal Delta[Ca(2+)](i) and bactericidal functions, exhibited no subsequent Delta[Ca(2+)](i) or DeltapH(i) to either stimulus. While exposure to 10(-7) M fMLP, which saturates the PMN high-affinity receptor, did not elicit bactericidal release from these naïve unprimed PMN in suspension, 10(-5) M fMLP did, presumably via the low-affinity receptor, using a different Ca(2+) source.