Involvement of calyculin A inhibitable protein phosphatases in the cyclic AMP signal transduction pathway of mouse corticotroph tumour (AtT20) cells

1. The role of non-calcineurin protein phosphatases in the cyclic AMP signal transduction pathway was examined in mouse pituitary corticotroph tumour (AtT20) cells. 2. Blockers of protein phosphatases, calyculin A and okadaic acid, were applied in AtT20 cells depleted of rapidly mobilizable pools of intracellular calcium and activated by various cyclic AMP generating agonists. Inhibitors of cyclic nucleotide phosphodiesterases were present throughout. The accumulation of cyclic AMP was monitored by radioimmunoassay, phosphodiesterase activity in cell homogenates was measured by radiometric assay. 3. Neither calyculin A nor okadaic acid altered basal cyclic AMP levels but cyclic AMP formation induced by 41 amino acid residue corticotrophin releasing-factor (CRF) was strongly inhibited (up to 80%). 1-Norokadaone was inactive. Similar data were also obtained when isoprenaline or pituitary adenylate cyclase activating peptide1-38 were used as agonists. 4. Pertussis toxin did not modify the inhibition of CRF-induced cyclic AMP production by calyculin A. 5. Pretreatment with calyculin A completely prevented the stimulation of cyclic AMP formation by cholera toxin even in the presence of 0.5 mM isobutylmethylxanthine (IBMX) and 0.1 mM rolipram. Cholera toxin mediated ADP-ribosylation of the 45K and 52K molecular weight G,, isoforms in membranes from calyculin A-pretreated cells was enhanced to 150-200% when compared with controls. 6. Cholera toxin-induced cyclic AMP was reduced by calyculin A within 10 min when calyculin A was applied after a 90 min pretreatment with cholera toxin. Under these conditions the effect of calyculin A could be blocked by the combination of 0.5 mM IBMX and 0.1 mM rolipram, but not by 0.5 mM IBMX alone. 7. Phosphodiesterase activity in AtT20 cell homogenates showed a significant, 2.7 fold increase after treatment with calyculin A. In control cells phosphodiesterase activity was blocked by 80% in the presence of IBMX (0.5 mM), or IBMX plus rolipram (0.1 mM). In calyculin A-treated cells phosphodiesterase activity was also strongly inhibited by IBMX, but because of the stimulating effect of calyculin A, the activity remaining was still 55% of that found in control homogenates. This activity was reduced to 5% of control by using IBMX and rolipram in combination. Assay of phosphodiesterase in Ca2+ free conditions showed that calyculin A markedly increases the activity of rolipram sensitive (type 4) phosphodiesterase. 8. Taken together, blockers of protein phosphatases (PPases) impaired signal transduction through Gs-mediated pathways and activated cyclic AMP degrading phosphodiesterase(s), indicating that PPases 1 and/or 2A are essential for agonist-mediated regulation of cyclic AMP levels in AtT20 cells, and are thus important in maintaining the secretory phenotype of the cells

A hepatitis C outbreak preceded the HIV outbreak among persons who inject drugs in Athens, Greece: Insights from a mathematical modelling study

People who inject drugs (PWID) comprise one of the major transmission risk groups for human immunodeficiency virus (HIV) and hepatitis C virus (HCV). In 2011, Athens experienced a large HIV outbreak among PWID. Significant public health interventions were implemented in response to the HIV outbreak. The aims of this study were to estimate the indirect effects of the HIV interventions on HCV infection and to evaluate the concept of the association between HCV and HIV infections in the case of Athens. A dynamic, stochastic, individual-based model was developed to simulate HCV transmission among PWID. We calibrated the model to reproduce the observed HCV prevalence among PWID in Greece. Two years prior to the HIV outbreak, an undetected HCV outbreak has occurred. In 2009, the incidence of HCV infection increased from 640 (495, 842) cases in 2008 to 1260 (1060, 1500). The mean time from initiation of injecting drug use to HCV acquisition decreased from 29 months in 2008 to 13 months in 2009. After HIV interventions, HCV incidence declined by 64.8% in 2012, compared to 2009. The averted HCV incidence cases attributed to the HIV-implemented interventions were 2200 (1950, 2480), during 2012-2015. The cumulative number incident HCV cases in Athens during 2002-2015 was about 9900 (7800, 12 100). Our results highlight that before the 2011 HIV outbreak in Athens, an HCV outbreak occurred in 2009. Prevention measures for HIV that took place in the Athens metropolitan area in 2012 reduced significantly the incidence of HCV. © 2019 John Wiley & Sons Lt

Factors associated with HCV test uptake in heroin users entering substitution treatment in Greece

ObjectivesPeople who inject drugs (PWID) represent the main risk group for hepatitis C virus (HCV) infection in most middle and high-income countries. Testing PWID is considered as an important prevention measure. Identification of PWID characteristics associated with HCV testing may contribute to strategies targeting the containment of the HCV and HIV epidemics in Greece. MethodsAnonymous behavioural data from 2747 heroin users were collected upon entry in 38 opioid substitution treatment (OST) clinics in Greece during the period 2013-2015. HCV test uptake was the dependent variable while covariates included sociodemographic and addiction-related variables, mostly derived from the EMCDDA treatment demand indicator protocol. ResultsAmong 2299 cases with complete data on HCV testing, 83.5% reported any HCV testing uptake, with 61.2% reporting a recent test (<12months). In the multivariate analyses, any previous HCV testing uptake was associated with age 25years, past drug treatment attempt, injecting or sniffing the primary substance, injection history 5years, and syringe sharing earlier than the past 12months. Past HCV test uptake was higher among those reporting full-time employment and 2-4years injecting histories, and lower among residents of Athens. Recent testing was positively associated with female gender and polysubstance use. ConclusionAny previous HCV testing uptake is high among PWID entering OST in Greece and is associated with older age, longer injecting histories and past drug-related treatment attempts. Efforts to prevent and mitigate the ongoing HCV test epidemic among PWID in Greece should combine treatment with scaling up of screening, targeting especially those younger than 25years and at the beginning of their hazardous use

Downgrading BIRADS 3 to BIRADS 2 category using a computer-aided microcalcification analysis and risk assessment system for early breast cancer

This paper explores the potential of a computer-aided diagnosis system to discriminate the real benign microcalcifications among a specific subset of 109 patients with BIRADS 3 mammograms who had undergone biopsy, thus making it possible to downgrade them to BIRADS 2 category. The system detected and quantified critical features of microcalcifications and classified them on a risk percentage scale for malignancy. The system successfully detected all cancers. Nevertheless, it suggested biopsy for 11/15 atypical lesions. Finally, the system characterized as definitely benign (BIRADS 2) 29/88 benign lesions, previously assigned to BIRADS 3, and thus achieved a reduction of 33% in unnecessary biopsies. © 2010 Elsevier Ltd

Involvement of calyculin A inhibitable protein phosphatases in the cyclic AMP signal transduction pathway of mouse corticotroph tumour (AtT20) cells

1. The role of non-calcineurin protein phosphatases in the cyclic AMP signal transduction pathway was examined in mouse pituitary corticotroph tumour (AtT20) cells. 2. Blockers of protein phosphatases, calyculin A and okadaic acid, were applied in AtT20 cells depleted of rapidly mobilizable pools of intracellular calcium and activated by various cyclic AMP generating agonists. Inhibitors of cyclic nucleotide phosphodiesterases were present throughout. The accumulation of cyclic AMP was monitored by radioimmunoassay, phosphodiesterase activity in cell homogenates was measured by radiometric assay. 3. Neither calyculin A nor okadaic acid altered basal cyclic AMP levels but cyclic AMP formation induced by 41 amino acid residue corticotrophin releasing-factor (CRF) was strongly inhibited (up to 80%). 1-Norokadaone was inactive. Similar data were also obtained when isoprenaline or pituitary adenylate cyclase activating peptide(1–38) were used as agonists. 4. Pertussis toxin did not modify the inhibition of CRF-induced cyclic AMP production by calyculin A. 5. Pretreatment with calyculin A completely prevented the stimulation of cyclic AMP formation by cholera toxin even in the presence of 0.5 mM isobutylmethylxanthine (IBMX) and 0.1 mM rolipram. Cholera toxin mediated ADP-ribosylation of the 45K and 52K molecular weight G(sα) isoforms in membranes from calyculin A-pretreated cells was enhanced to 150–200% when compared with controls. 6. Cholera toxin-induced cyclic AMP was reduced by calyculin A within 10 min when calyculin A was applied after a 90 min pretreatment with cholera toxin. Under these conditions the effect of calyculin A could be blocked by the combination of 0.5 mM IBMX and 0.1 mM rolipram, but not by 0.5 mM IBMX alone. 7. Phosphodiesterase activity in AtT20 cell homogenates showed a significant, 2.7 fold increase after treatment with calyculin A. In control cells phosphodiesterase activity was blocked by 80% in the presence of IBMX (0.5 mM), or IBMX plus rolipram (0.1 mM). In calyculin A-treated cells phosphodiesterase activity was also strongly inhibited by IBMX, but because of the stimulating effect of calyculin A, the activity remaining was still 55% of that found in control homogenates. This activity was reduced to 5% of control by using IBMX and rolipram in combination. Assay of phosphodiesterase in Ca(2+) free conditions showed that calyculin A markedly increases the activity of rolipram sensitive (type 4) phosphodiesterase. 8. Taken together, blockers of protein phosphatases (PPases) impaired signal transduction through Gs-mediated pathways and activated cyclic AMP degrading phosphodiesterase(s), indicating that PPases 1 and/or 2A are essential for agonist-mediated regulation of cyclic AMP levels in AtT20 cells, and are thus important in maintaining the secretory phenotype of the cells