cAMP and PMA enhance the effects of IGF-I in the proliferation of endometrial adenocarcinoma cell line HEC-1-A by acting at the G 1 phase of the cell cycle

The present study was undertaken to determine whether endometrial cancer cell line HEC-1-A differ from nontransformed cells, in that the cAMP and protein kinase C pathways may enhance IGF-I effects in mitogenesis by acting at the G 1 phase of the cell cycle instead of G 0 . Immunofluorescence staining of HEC-1-A cells using the proliferating cell nuclear antigen (PCNA) monoclonal antibody and flow cytometric analysis determined that HEC-1-A cells do not enter the G 0 phase of the cell cycle when incubated in a serum-free medium. Approximately 51% of the cells were in G 1 , 12% were in S and 37% in G 2 phase of the cell cycle prior to treatment. Forskolin and phorbol-12-myristate 13-acetate (PMA) were used to stimulate cAMP production and protein kinase C activity, respectively. IGF-I, forskolin and PMA each increased ( P <0.01) [ 3 H]-thymidine incorporation in a dose and time dependent manner. The interaction of forskolin and PMA with IGF-I was then determined. Cells preincubated with forskolin or PMA followed by incubation with IFG-I incorporated significantly more ( P <0.01) [ 3 H]-thymidine into DNA than controls or any treatment alone. It is concluded that forskolin and, to a lesser extent, PMA exert their effect at the G 1 phase of the cycle to enhance IGF-I effects in cell proliferation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75013/1/j.1365-2184.1995.tb00061.x.pd

Insulin resistance in type 1 diabetes: what is ‘double diabetes’ and what are the risks?

In this review, we explore the concept of ‘double diabetes’, a combination of type 1 diabetes with features of insulin resistance and type 2 diabetes. After considering whether double diabetes is a useful concept, we discuss potential mechanisms of increased insulin resistance in type 1 diabetes before examining the extent to which double diabetes might increase the risk of cardiovascular disease (CVD). We then go on to consider the proposal that weight gain from intensive insulin regimens may be associated with increased CV risk factors in some patients with type 1 diabetes, and explore the complex relationships between weight gain, insulin resistance, glycaemic control and CV outcome. Important comparisons and contrasts between type 1 diabetes and type 2 diabetes are highlighted in terms of hepatic fat, fat partitioning and lipid profile, and how these may differ between type 1 diabetic patients with and without double diabetes. In so doing, we hope this work will stimulate much-needed research in this area and an improvement in clinical practice

Reliability of Quantitative Real-Time PCR for Bacterial Detection in Cystic Fibrosis Airway Specimens

The cystic fibrosis (CF) airway microbiome is complex; polymicrobial infections are common, and the presence of fastidious bacteria including anaerobes make culture-based diagnosis challenging. Quantitative real-time PCR (qPCR) offers a culture-independent method for bacterial quantification that may improve diagnosis of CF airway infections; however, the reliability of qPCR applied to CF airway specimens is unknown. We sought to determine the reliability of nine specific bacterial qPCR assays (total bacteria, three typical CF pathogens, and five anaerobes) applied to CF airway specimens. Airway and salivary specimens from clinically stable pediatric CF subjects were collected. Quantitative PCR assay repeatability was determined using triplicate reactions. Split-sample measurements were performed to measure variability introduced by DNA extraction. Results from qPCR were compared to standard microbial culture for Pseudomonas aeruginosa, Staphylococcus aureus, and Haemophilus influenzae, common pathogens in CF. We obtained 84 sputa, 47 oropharyngeal and 27 salivary specimens from 16 pediatric subjects with CF. Quantitative PCR detected bacterial DNA in over 97% of specimens. All qPCR assays were highly reproducible at quantities ≥102 rRNA gene copies/reaction with coefficient of variation less than 20% for over 99% of samples. There was also excellent agreement between samples processed in duplicate. Anaerobic bacteria were highly prevalent and were detected in mean quantities similar to that of typical CF pathogens. Compared to a composite gold standard, qPCR and culture had variable sensitivities for detection of P. aeruginosa, S. aureus and H. influenzae from CF airway samples. By reliably quantifying fastidious airway bacteria, qPCR may improve our understanding of polymicrobial CF lung infections, progression of lung disease and ultimately improve antimicrobial treatments

A Small-Molecule Inhibitor of T. gondii Motility Induces the Posttranslational Modification of Myosin Light Chain-1 and Inhibits Myosin Motor Activity

Toxoplasma gondii is an obligate intracellular parasite that enters cells by a process of active penetration. Host cell penetration and parasite motility are driven by a myosin motor complex consisting of four known proteins: TgMyoA, an unconventional Class XIV myosin; TgMLC1, a myosin light chain; and two membrane-associated proteins, TgGAP45 and TgGAP50. Little is known about how the activity of the myosin motor complex is regulated. Here, we show that treatment of parasites with a recently identified small-molecule inhibitor of invasion and motility results in a rapid and irreversible change in the electrophoretic mobility of TgMLC1. While the precise nature of the TgMLC1 modification has not yet been established, it was mapped to the peptide Val46-Arg59. To determine if the TgMLC1 modification is responsible for the motility defect observed in parasites after compound treatment, the activity of myosin motor complexes from control and compound-treated parasites was compared in an in vitro motility assay. TgMyoA motor complexes containing the modified TgMLC1 showed significantly decreased motor activity compared to control complexes. This change in motor activity likely accounts for the motility defects seen in the parasites after compound treatment and provides the first evidence, in any species, that the mechanical activity of Class XIV myosins can be modulated by posttranslational modifications to their associated light chains

Minimizing the evidence-practice gap – a prospective cohort study incorporating balance training into pulmonary rehabilitation for individuals with chronic obstructive pulmonary disease

Background: We have recently demonstrated the efficacy of balance training in addition to Pulmonary Rehabilitation (PR) at improving measures of balance associated with an increased risk of falls in individuals with Chronic Obstructive Pulmonary Disease (COPD). Few knowledge translation (KT) projects have been conducted in rehabilitation settings. The goal of this study was to translate lessons learnt from efficacy studies of balance training into a sustainable clinical service. Methods: Health care professionals (HCPs) responsible for delivering PR were given an hour of instruction on the principles and practical application of balance training and the researchers offered advice regarding; prescription, progression and practical demonstrations during the first week. Balance training was incorporated three times a week into conventional PR programs. Following the program, HCPs participated in a focus group exploring their experiences of delivering balance training alongside PR. Service users completed satisfaction surveys as well as standardized measures of balance control. At six month follow-up, the sustainability of balance training was explored. Results: HCPs considered the training to be effective at improving balance and the support provided by the researchers was viewed as helpful. HCPs identified a number of strategies to facilitate balance training within PR, including; training twice a week, incorporating an interval training program for everyone enrolled in PR, providing visual aids to training and promoting independence by; providing a set program, considering the environment and initiating a home-based exercise program early. Nineteen service users completed the balance training [ten male mean (SD) age 73 (6) y]. Sixteen patients (84 %) enjoyed balance training and reported that it helped them with everyday activities and 18 (95 %) indicated their wish to continue with it. Scores on balance measures improved following PR that included balance training (all p < 0.05). At six month follow-up balance training is being routinely assessed and delivered as part of standardised PR. Conclusions: Implementing balance training into PR programs, with support and training for HCPs, is feasible, effective and sustainable. Trail registration Clinical Trials ID: NCT02080442 (05/03/2014) Electronic supplementary material The online version of this article (doi:10.1186/s12890-015-0067-2) contains supplementary material, which is available to authorized users

Clinical emergence of neurometastatic merkel cell carcinoma: a surgical case series and literature review

Merkel cell carcinoma (MCC) is a rare cutaneous neuroendocrine neoplasm of possible viral origin and is known for its aggressive behavior. The incidence of MCC has increased in the last 15 years. Merkel cell carcinoma has the potential to metastasize, but rarely involves the central nervous system. Herein, we report three consecutive surgical cases of MCC presenting at a single institution within 1 year. We used intracavitary BCNU wafers (Gliadel®) in two cases. Pathological features, including CK20 positivity, consistent with MCC, were present in all cases. We found 33 published cases of MCC with CNS involvement. We suggest that the incidence of neurometastatic MCC may be increasing, parallel to the increasing incidence of primary MCC. We propose a role for intracavitary BCNU wafers in the treatment of intra-axial neurometastatic MCC

Constitutively Activated NLRP3 Inflammasome Causes Inflammation and Abnormal Skeletal Development in Mice

The NLRP3 inflammasome complex is responsible for maturation of the pro-inflammatory cytokine, IL-1β. Mutations in NLRP3 are responsible for the cryopyrinopathies, a spectrum of conditions including neonatal-onset multisystem inflammatory disease (NOMID). While excessive production of IL-1β and systemic inflammation are common to all cryopyrinopathy disorders, skeletal abnormalities, prominently in the knees, and low bone mass are unique features of patients with NOMID. To gain insights into the mechanisms underlying skeletal abnormalities in NOMID, we generated knock-in mice globally expressing the D301N NLRP3 mutation (ortholog of D303N in human NLRP3). NOMID mice exhibit neutrophilia in blood and many tissues, including knee joints, and high levels of serum inflammatory mediators. They also exhibit growth retardation and severe postnatal osteopenia stemming at least in part from abnormally accelerated bone resorption, attended by increased osteoclastogenesis. Histologic analysis of knee joints revealed abnormal growth plates, with loss of chondrocytes and growth arrest in the central region of the epiphyses. Most strikingly, a tissue “spike" was observed in the mid-region of the growth plate in the long bones of all NOMID mice that may be the precursor to more severe deformations analogous to those observed in NOMID patients. These findings provide direct evidence linking a NOMID-associated NLRP3-activating mutation to abnormalities of postnatal skeletal growth and bone remodeling

Pharmacogenomics of Interferon-ß Therapy in Multiple Sclerosis: Baseline IFN Signature Determines Pharmacological Differences between Patients

Multiple sclerosis (MS) is a heterogeneous disease. In order to understand the partial responsiveness to IFNbeta in Relapsing Remitting MS (RRMS) we studied the pharmacological effects of IFNbeta therapy. Large scale gene expression profiling was performed on peripheral blood of 16 RRMS patients at baseline and one month after the start of IFNbeta therapy. Differential gene expression was analyzed by Significance Analysis of Microarrays. Subsequent expression analyses on specific genes were performed after three and six months of treatment. Peripheral blood mononuclear cells (PBMC) were isolated and stimulated in vitro with IFNbeta. Genes of interest were measured and validated by quantitative realtime PCR. An independent group of 30 RRMS patients was used for validation. Pharmacogenomics revealed a marked variation in the pharmacological response to IFNbeta between patients. A total of 126 genes were upregulated in a subset of patients whereas in other patients these genes were downregulated or unchanged after one month of IFNbeta therapy. Most interestingly, we observed that the extent of the pharmacological response correlates negatively with the baseline expression of a specific set of 15 IFN response genes (R = -0.7208; p = 0.0016). The negative correlation was maintained after three (R = -0.7363; p = 0.0027) and six (R = -0.8154; p = 0.0004) months of treatment, as determined by gene expression levels of the most significant correlating gene. Similar results were obtained in an independent group of patients (n = 30; R = -0.4719; p = 0.0085). Moreover, the ex vivo results could be confirmed by in vitro stimulation of purified PBMCs at baseline with IFNbeta indicating that differential responsiveness to IFNbeta is an intrinsic feature of peripheral blood cells at baseline. These data imply that the expression levels of IFN response genes in the peripheral blood of MS patients prior to treatment could serve a role as biomarker for the differential clinical response to IFNbet