Circadian variations of platelet reactivity on clopidogrel in patients treated with elective percutaneous coronary intervention.

Evidence assessing potential diurnal variations of platelet reactivity in patients on clopidogrel treated with elective percutaneous coronary intervention (PCI) for chronic coronary syndrome (CCS) are currently lacking. We prospectively enrolled 15 patients affected by stable coronary artery disease (CAD) previously treated with elective PCI and on clopidogrel for at least 8 days (administered at 8 a.m.). A significant heterogeneity in diurnal levels of ADP-dependent platelet aggregation was found (p = 0.0004), with a peak of platelet reactivity occurring at the 6 a.m. assessment, which resulted significantly increased compared to the afternoon (6 p.m.) evaluation (255 ± 66 vs 184 ± 67, p = 0.002). In addition, at the early-morning evaluation a considerably high proportion of patients with high platelet reactivity (53.3%) were observed. In conclusion, clopidogrel-induced platelet inhibition in patients with CCS after elective PCI follows a circadian rhythm, thus suggesting that a consistent and durable antiplatelet inhibition is often failed with standard clopidogrel administration at morning.

Proteomic analysis of protein purified derivative of Mycobacterium bovis.

BACKGROUND: Tuberculin skin test based on in vivo intradermal inoculation of purified protein derivative from Mycobacterium bovis (bPPD) is the diagnostic test for the control and surveillance of bovine tuberculosis (bTB). METHODS: Proteomic analysis was performed on different bPPD preparations from M. bovis, strain AN5. Proteins were precipitated from bPPD solutions by TCA precipitation. The proteome of bPPD preparations was investigated by bottom-up proteomics, which consisted in protein digestion and nano-LC-MS/MS analysis. Mass spectrometry analysis was performed on a Q-exactive hybrid quadrupole-Orbitrap mass spectrometer coupled online to an Easy nano-LC1000 system. RESULTS: Three hundred and fifty-six proteins were identified and quantified by at least 2 peptides (99% confidence per peptide). One hundred and ninety-eight proteins, which had not been previously described, were detected; furthermore, the proteomic profile shared 80 proteins with previous proteomes from bPPDs from the United Kingdom and Brazil and 139 protein components from bPPD from Korea. Locus name of M. bovis (Mb) with orthologs from M. tuberculosis H37Rv, comparative gene and protein length, molecular mass, functional categories, gene name and function of each protein were reported. Ninety-two T cell mycobacterial antigens responsible for delayed-type hypersensitivity were detected, fifty-two of which were not previously reported in any bPPD proteome. Data are available via ProteomeXchange with identifier PXD005920. CONCLUSIONS: This study represents the highest proteome coverage of bPPD preparations to date. Since proteins perform cellular functions essential to health and/or disease, obtaining knowledge of their presence and variance is of great importance in understanding disease states and for advancing translational studies. Therefore, to better understand Mycobacterium tuberculosis complex biology during infection, survival, and persistence, the reproducible evaluation of the proteins that catalyze and control these processes is critically important. More active and more specific tuberculins would be desirable. Indeed, many antigens contained within bPPD are currently responsible for the cross-reactivity resulting in false-positive results as they are shared between non-tuberculous and tuberculous mycobacteria.

Influence of diffusion on the stability of equilibria in a reaction-diffusion system modeling cholera dynamic.

A reaction-diffusion system modeling cholera epidemic in a non-homogeneously mixed population is introduced. The interaction between population and toxigenic Vibrio cholerae concentration in contaminated water has been taken into account. The existence of biologically meaningful equilibria is investigated together with their linear and nonlinear stability. Using the data collected during the Haiti cholera epidemic, a numerical simulation is performed.

PBMCs are additional sites of productive infection of bovine papillomavirus type 2.

Bovine papillomavirus type 2 (BPV-2) is an oncogenic virus infecting both epithelial and mesenchymal cells. Its life cycle, similar to other papillomaviruses (PVs), appears to be linked to epithelial differentiation. Human and bovine PVs have been known to reside in a latent, episomal form in PBMCs; therefore, it is believed that blood cells, like all mesenchymal cells, function as non-permissive carriers. Here, for the first time in veterinary and comparative medicine, the BPV-2 E5 oncoprotein and the major structural L1 capsid protein, known to be expressed only in productive infections, were shown to occur in defined subsets of PBMCs. E5 oncoprotein was detected in sorted T- and B-cells as well as in monocytes by flow cytometry and Western blot analysis. However, CD4(+) and CD8(+) lymphocytes appeared to be the main circulating targets of the virus, thus possibly representing the most important reservoir of active BPV-2 in blood. L1 protein was identified by flow cytometry in a population of blood cells recognized as lymphocytes by morphological scatter properties. Western blot analysis was performed on lysates obtained from the sorted subpopulations of PBMCs and detected L1 protein in CD4(+) and CD8(+) cells only. Thus, this study showed that CD4(+) and CD8(+) lymphocytes are permissive for BPV-2 and are new, hitherto unknown sites of productive PV infection. In light of these observations, the life cycle of PVs needs to be revisited to gain novel insights into the epidemiology of BPV infection and the pathogenesis of related diseases.

NeoCheck: a prescription-screening tool to optimise pharmacotherapy for hospitalised neonates.

AIMS OF THE STUDY: To develop a screening tool to optimise neonatal drug prescription, which is often based on low-quality evidence. METHODS: Neonatal pharmacotherapy recommendations were identified by literature review and synthesised into NeoCheck tool statements. In a two-round modified Delphi process, experts from Swiss neonatal intensive care units (NICUs) rated their agreement with individual statements using a five-point Likert scale (5 = totally agree). Statements with >65% scores ≥4 in round 1 and >75% scores ≥4 in round 2 were selected. RESULTS: We identified 1375 clinical guidelines via literature review. After synthesis, 158 statements were submitted to 23 experts (1 clinical pharmacist, 22 neonatologists; 65% with >10 years neonatology practice) from 10 Swiss NICUs. Nineteen items did not reach the agreement threshold and were eliminated in the second Delphi round. The final NeoCheck tool comprises 141 statements in 11 medical domains concerning 49 neonatal diseases. Most (79%) statements concern all neonates, 13% concern preterm (<37 weeks gestational age) infants and 3% concern very preterm (<32 weeks gestational age) infants CONCLUSIONS: NeoCheck is the first prescription-screening tool developed to optimise neonatal pharmacotherapy. In a future prospective study, its effect on NICU prescription optimisation and the quality of care will be assessed.

Intracranial Hemorrhage and Autoimmune Thrombocytopenia in a Neonate: A Rare "Unpredictable" Event.

Neonatal thrombocytopenia is a rare complication of maternal autoimmune thrombocytopenia, and no maternal predictors of its gravity and potential complications have been identified. Neonatal cerebral hemorrhage, a feared event in the setting of autoimmune thrombocytopenia, is fortunately uncommon, but it can occur in utero or in the perinatal period, with potentially serious consequences. The authors report the case of a boy born to a mother affected by autoimmune thrombocytopenia, who presented with severe thrombocytopenia at birth and developed intracranial hemorrhage despite mild maternal thrombocytopenia at delivery and a prompt preventive treatment of the newborn. Platelet count should be tested at birth in all babies born from mothers with autoimmune thrombocytopenia, irrespective of maternal platelets counts during pregnancy or at delivery, and should be closely monitored during the first days of life. Systematic early and serial cranial ultrasound might be advocated in the setting of neonatal thrombocytopenia.

A novel GBE1 mutation and features of polyglucosan bodies autophagy in adult polyglucosan body disease.

We report the clinical, neuro-imaging, pathological and biochemical features of an Italian family in which two siblings have the Adult Polyglucosan Body Disease (APBD). APBD is a rare autosomal recessive disorder characterized by a gradually progressive involvement of both the central and peripheral nervous systems caused by the deficiency of the glycogen branching enzyme (GBE1). The two affected siblings, a 64-year-old man and his 67-year-old sister who had complained of urinary urgency and sporadic incontinence and also progressive gait difficulty for 6 and 7 years respectively, had severely impaired deep sensations on direct examination and a moderately severe symmetrical, axonal sensory-motor neuropathy on electrophysiological testing. GBE1 activity was below 25% of the normal rate in leukocytes and sural nerves. The siblings were homozygous for the novel GBE1 mutation p.N541D. All other members of the pedigree are heterozygous and manifest no symptoms, even in the very elderly. The affected siblings showed polyglucosan bodies (PBs) included within non-myelinating Schwann cells and within lymphocyte vesicles, which were positive for the autophagy markers P62 and LC3-II at immunofluorescence microscopy.

Evaluation of the GEM®PCL Plus point-of-care device for neonatal coagulation assessment: an observational study on cord blood.

INTRODUCTION: Point of care devices (POCT) are used for coagulation evaluation in adults. Reduced blood volumes and the direct use of whole blood allow studies when venous puncture is difficult, such as in newborns. Elimination of sample transport is attractive for use in emergencies and intensive care. OBJECTIVE: To prospectively compare neonatal coagulation parameters measured by the GEM®PCL POCT versus a central laboratory. MATERIALS AND METHODS: Prothrombin Time (PT) and activated Partial Thromboplastin Time (aPTT) were performed on whole cord blood (POCT) and plasma (central laboratory) collected from consecutive newborns at Geneva University Hospitals. Agreement was assessed with a Bland & Altman plot and intra-class correlation coefficient (ICC) in 213 newborns cord blood; intra-assay variability (repeatability) was assessed using ICC and coefficient of variation (CV). RESULTS: 189 samples were available for the agreement analysis, 24 were excluded for technical problems. The 95% limits of agreements in the Bland & Altman plot ranged from -5.6 to 11.6 and from -39.6 to 11.6seconds for the PT and aPTT, respectively. The ICC between the two methods was 0.28 (CI 95% 0.06 to 0.47) for PT and 0.20 (CI 95% -0.06 to 0.42) for aPTT. Repeatability (ICC) on the 43 eligible samples was 0.46 (CI 95% 0.19 to 0.67) for PT and 0.52 (CI 95% 0.26 to 0.71) for aPTT. The CV was 10.6% and 12% for PT and aPTT, respectively. CONCLUSIONS: In newborn cord blood, PT and aPTT measurements with the GEM®PCL POCT had poor agreement with the central laboratory and poor repeatability.

Bovine papillomavirus type 2 (BPV-2) E5 oncoprotein binds to the subunit D of the V1-ATPase proton pump in naturally occurring urothelial tumors of the urinary bladder of cattle.

BACKGROUND: Active infection by bovine papillomavirus type 2 (BPV-2) was documented for fifteen urinary bladder tumors in cattle. Two were diagnosed as papillary urothelial neoplasm of low malignant potential (PUNLMP), nine as papillary and four as invasive urothelial cancers. METHODS AND FINDINGS: In all cancer samples, PCR analysis revealed a BPV-2-specific 503 bp DNA fragment. E5 protein, the major oncoprotein of the virus, was shown both by immunoprecipitation and immunohistochemical analysis. E5 was found to bind to the activated (phosphorylated) form of the platelet derived growth factor ß receptor. PDGFßR immunoprecipitation from bladder tumor samples and from normal bladder tissue used as control revealed a protein band which was present in the pull-down from bladder cancer samples only. The protein was identified with mass spectrometry as "V1-ATPase subunit D", a component of the central stalk of the V1-ATPase vacuolar pump. The subunit D was confirmed in this complex by coimmunoprecipitation investigations and it was found to colocalize with the receptor. The subunit D was also shown to be overexpressed by Western blot, RT-PCR and immunofluorescence analyses. Immunoprecipitation and immunofluorescence also revealed that E5 oncoprotein was bound to the subunit D. CONCLUSION: For the first time, a tri-component complex composed of E5/PDGFßR/subunit D has been documented in vivo. Previous in vitro studies have shown that the BPV-2 E5 oncoprotein binds to the proteolipid c ring of the V0-ATPase sector. We suggest that the E5/PDGFßR/subunit D complex may perturb proteostasis, organelle and cytosol homeostasis, which can result in altered protein degradation and in autophagic responses.

Productive infection of bovine papillomavirus type 2 in the urothelial cells of naturally occurring urinary bladder tumors in cattle and water buffaloes.

BACKGROUND: Papillomaviruses (PVs) are highly epitheliotropic as they usually establish productive infections within squamous epithelia of the skin, the anogenital tract and the oral cavity. In this study, early (E) and late (L) protein expression of bovine papillomavirus type 2 (BPV-2) in the urothelium of the urinary bladder is described in cows and water buffaloes suffering from naturally occurring papillomavirus-associated urothelial bladder tumors. METHODS AND FINDINGS: E5 protein, the major oncoprotein of the BPV-2, was detected in all tumors. L1 DNA was amplified by PCR, cloned and sequenced and confirmed to be L1 DNA. The major capsid protein, L1, believed to be only expressed in productive papillomavirus infection was detected by Western blot analysis. Immunohistochemical investigations confirmed the presence of L1 protein both in the cytoplasm and nuclei of cells of the neoplastic urothelium. Finally, the early protein E2, required for viral DNA replication and known to be a pivotal factor for both productive and persistent infection, was detected by Western blot and immunohistochemically. Electron microscopic investigations detected electron dense particles, the shape and size of which are consistent with submicroscopic features of viral particles, in nuclei of neoplastic urothelium. CONCLUSION: This study shows that both active and productive infections by BPV-2 in the urothelium of the bovine and bubaline urinary bladder can occur in vivo.

Bovine papillomavirus type 2 infection and microscopic patterns of urothelial tumors of the urinary bladder in water buffaloes.

Microscopic patterns of thirty-four urothelial tumors of the urinary bladder of water buffaloes from the Marmara and Black Sea Regions of Turkey are here described. All the animals grazed on lands rich in bracken fern. Histological diagnosis was assessed using morphological parameters recently suggested for the urinary bladder tumors of cattle. Papillary carcinoma was the most common neoplastic lesion (22/34) observed in this study, and low-grade carcinoma was more common (seventeen cases) than high-grade carcinoma (five cases). Papilloma, papillary urothelial neoplasm of low malignant potential (PUNLMP), and invasive carcinomas were less frequently seen. Carcinoma in situ (CIS) was often detected associated with some papillary and invasive carcinomas. De novo (primary) CIS was rare representing 3% of tumors of this series. A peculiar feature of the most urothelial tumors was the presence in the tumor stroma of immune cells anatomically organized in tertiary lymphoid organs (TLOs). Bovine papillomavirus type-2 (PV-2) E5 oncoprotein was detected by molecular and immunohistochemistry procedures. Early protein, E2, and late protein, L1, were also detected by immunohistochemical studies. Morphological and molecular findings show that BPV-2 infection contributes to the development of urothelial bladder carcinogenesis also in water buffaloes.

Productive infection of bovine papillomavirus type 2 in the placenta of pregnant cows affected with urinary bladder tumors.

Papillomaviruses (PVs) are believed to be highly epitheliotropic as they usually establish productive infections within stratified epithelia. In vitro, various PVs appear to complete their entire life-cycle in different trophoblastic cell lines. In this study, infection by and protein expression of bovine papillomavirus type 2 (BPV-2) in the uterine and chorionic epithelium of the placenta has been described in four cows suffering from naturally occurring papillomavirus-associated urothelial bladder tumors. E5 oncoprotein was detected both by Western blot analysis and immunohistochemically. It appears to be complexed and perfectly co-localized with the activated platelet-derived growth factor ß receptor (PDGFßR) by laser scanning confocal microscopy. The activated PDGFßR might be involved in organogenesis and neo-angiogenesis rather than in cell transformation during pregnancy. The major capsid protein, L1, believed to be only expressed in productive papillomavirus infection has been detected by Western blot analysis. Immunohistochemical investigations confirmed the presence of L1 protein both in the cytoplasm and nuclei of cells of the uterine and chorionic epithelium. Trophoblastic cells appear to be the major target for L1 protein expression. Finally, the early protein E2, required for viral DNA replication and known to be expressed during a productive infection, has been detected by Western blot and immunohistochemically. Electron microscopic investigations detected viral particles in nuclei of uterine and chorionic epithelium. This study shows that both active and productive infections by BPV-2 in the placenta of pregnant cows can occur in vivo.

Incidence of early neonatal mortality and morbidity after late-preterm and term cesarean delivery.

OBJECTIVE: To determine the age-stratified risk of intrapartum and neonatal mortality as well as morbidities of clinical relevance after elective cesarean delivery (ECD). METHODS: This work was a cohort study including 56 549 prospectively recorded late-preterm and term deliveries. We analyzed the effect of cesarean delivery (CD) before the onset of labor on the following multiple neonatal outcomes before hospital discharge, compared with planned vaginal delivery (PVD) and emergency CD: mortality, birth depression, special care admission, and respiratory morbidity. We adjusted for confounders by multivariate analysis and stratified the risk according to gestational age (GA). RESULTS: Mortality and morbidities had a strong GA-related trend with the lowest incidences consistently found between 38 and 40 weeks of gestation independent of delivery mode. Compared with infants delivered via PVD, infants delivered via ECD had significantly higher rates of mortality (adjusted risk ratio [aRR]: 2.1), risk of special care admission (aRR: 1.4), and respiratory morbidity (aRR: 1.8) but not of depression at birth (aRR: 1.1). Compared with emergency CD, newborns delivered via ECD had less depression at birth (aRR: 0.6) and admission to special care (aRR: 0.8), but mortality (aRR: 0.8) and respiratory morbidity (aRR: 1.0) rates were similar. CONCLUSIONS: Gestational age-specific risk estimates are lowest between 38 and 40 weeks and should be included in the informed-consent process. The information should also be used to allow for appropriate preparation with respect to adequate staff and equipment. ECD is consistently associated with increased intrapartum and neonatal mortality, risk of admission, and respiratory morbidity compared with PVD and has no advantage over emergency CD in terms of mortality. Neonatal morbidities are lower after ECD than emergency CD only with term births. Our data provide evidence that ECD should not be performed before term.