Extracellular truncations of h beta c, the common signaling subunit for interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-5, lead to ligand-independent activation

The hypothesis that extracellular truncation of the common receptor subunit for interleukin-3 (IL-3), granulocyte-macrophage colony- stimulating factor, and IL-5 (h beta c) can lead to ligand-independent activation was tested by infecting factor-dependent hematopoietic cell lines with retroviruses encoding truncated forms of h beta c. A truncation, resembling that in v-Mpl, and retaining 45 h beta c-derived extracellular residues, led to constitutive activation in the murine myeloid cell line, FDC-P1. However, infection of cells with retrovirus encoding a more severely truncated receptor, retaining only 7 h beta c- derived extracellular residues, did not confer factor independence on these cells. These experiments show that truncation activates the receptor and define a 37-amino acid segment of h beta c (H395-A431) which contains two motifs conserved throughout the cytokine receptor superfamily (consensus Y/H XX R/Q VR and WSXWS), as essential for factor-independent signaling. The mechanism of activation was also investigated in less severe truncations. A receptor that retains the entire membrane-proximal domain (domain 4) also conferred factor independent growth on FDC-P1 cells; however, a retrovirus encoding a truncated form of h beta c having two intact membrane proximal domains did not have this ability, suggesting that domain 3 may have an inhibitory role in h beta c. The ability of these receptors to confer factor independence was cell specific as demonstrated by their inability to confer factor-independent growth when introduced into the murine IL-3-dependent pro-B cell line BaF-B03. These results are consistent with a model in which activation requires unmasking of an interactive receptor surface in domain 4 and association with a myeloid- specific receptor or accessory component. We suggest that in the absence of ligand intramolecular interactions prevent inappropriate signaling.     

Book reviews

Myointima formation or intimal hyperplasia is a major undesirable problem at the anastomotic ends of narrow bore arterial autografts and in other arterial wall injuries, which often leads to late restenosis and thrombosis and whose pathogenesis is still not understood. Platelets are suspected to intervene at some stages of its development, together with endothelial and muscle cells, the extracellular matrix and, most probably, adhesion receptors. To ascertain whether and at what stage beta 3 integrins are involved, a rat arterial autograft model was used, together with monoclonal antibody P37, which is directed to the sequence 101-109 of the beta 3 subunit of the human platelet fibrinogen receptor integrin alpha IIb beta 3 and inhibits platelet aggregation in vitro and acute thrombosis in vivo . Three groups of animals were used: group I underwent an arterial autograft of a 5-mm segment of the right common iliac artery; group II received, intravenously, a single dose 0.8 mg kg of P37 at 15 min before the graft implantation; and group III was treated as group II but a similar dose of antibody was additionally given on day 14 after the operation. Animals in each group were sacrificed on days 7, 14, 21, 30 and 50 after the operation, and the grafts were removed for light and electron microscopy observation and further time-dependent morphometric analysis. By day 14, group I autografts already showed intimal hyperplasia and secretory smooth muscle cells, while group II and II autografts presented only some degenerative changes in the medial layer, with no signs of hyperplasia. Intimal hyperplasia was observed on day 21 in group II and on day 30 in group III, although less pronounced than in the corresponding controls. However, by day 50, the three groups had the same thickness of myointima. The immunohistochemical determination of metalloproteases suggests no role for these enzymes in the immunoinhibition of myointima formation. We conclude that P37 inhibits the onset of the intimal hyperplasia in the arterial autografts and that this onset in treated animals seems to be related to the decay of the circulating antibody. Further work is required to decide whether a higher or longer presence of circulating P37 can definitively prevent the development of intimal hyperplasia, as well as to ascertain which cells and which beta 3 integrin receptors intervene.     

Sexual assault and harassment,perceived vulnerability,and association with alcohol use in a student population: a cross-sectional survey

BackgroundThe prevalence of sexual assault and harassment at universities is unclear because of under-reporting. Furthermore, student perceptions of vulnerability to sexual assault and how these relate to alcohol consumption are unknown. The present study assesses the prevalence of sexual assault and harassment in a student sample, and how this relates to issues of vulnerability and alcohol use.MethodsParticipants were recruited via a weekly email of notices that is sent to all students attending a town-based Scottish university of about 7200 students. The study was approved by the university ethics committee, and all participants gave informed consent before participating. The online survey was available in November, 2013, and included validated scales examining personal experience of sexual harassment and assault, perception of own and peers' vulnerability to sexual assault, and the Alcohol Use Disorder Identification Test (AUDIT). Data were analysed with χ² and t tests.FindingsThe survey was completed by 135 female and 40 male students, aged 17–56 years (mean 21·9, SD 5·4), 84 (48%) of whom were in their first year of study. There was no significant difference in the proportion of male students (22·5%) and female students (37·0%) who reported having experienced some form of sexual harassment or assault at the university. Participants perceived themselves as significantly less vulnerable than same-sex peers (t(df 160)=6·10, 95% CI ?1·09 to ?0·55; p<0·0001) and judged peers to significantly underestimate their own vulnerability (t(df 159)=4·86, 0·40 to 0·95; p<0·0001). Controlling for sex, AUDIT did not predict judgments of own vulnerability (β=0·02, 95% CI ?0·03 to 0·06, p=0·49), but hazardous drinkers were more likely to report experiences of sexual harassment than non-hazardous drinkers (χ²(df 2)=20·98, p<0·0001).InterpretationPrevalence rates for experiencing sexual harassment or assault were high, and participants underestimated their own vulnerability compared with peers. Although hazardous drinkers were more likely to report experiencing sexual harassment or assault than non-hazardous drinkers, this finding was not shown in their judged vulnerability, again suggesting a misperception. The role of alcohol and vulnerability misperceptions should be considered in future interventions. The study was limited by oversampling female first-year students, thereby producing a non-representative sample.FundingNone.     

Use of an improved quantitative polymerase chain reaction assay to determine differences in human rhinovirus viral loads in different populations

Human rhinoviruses (HRV) frequently cause acute respiratory infections and chronic respiratory disease exacerbations. However, testing is not generally offered. We developed a modified HRV quantitative polymerase chain reaction (qPCR) assay to assess viral loads in the community and hospital patients. The assay had a lower limit of detection of 2 log₁₀ viral copies/mL and displayed linearity over 5 log₁₀ viral copies, with a lower limit of quantitation of 4 log₁₀ viral copies/mL. Mean viral loads (95% confidence interval) for hospitalized children, university students, and institutionalized elderly, were 7.08 log₁₀ viral copies/mL (6.7–7.5), 6.87 log₁₀ viral copies/mL (6.5–7.2), and 7.09 log₁₀ viral copies/mL (6.9–7.3), respectively (P = 0.67). Serial specimens of 14 university students showed a decrease of mean viral loads from 6.36 log₁₀ viral copies/mL on day 1 to 2.32 log₁₀ viral copies/mL 7 days past symptom onset (P < 0.001). Using an HRV qPCR, we showed that viral loads did not differ between the community and hospitalized populations and significantly decreased following symptoms onset in healthy individuals.     

Comparison of the release of microRNAs and extracellular vesicles from platelets in response to different agonists

On activation platelets release microRNAs and extracellular vesicles (EV) into circulation. The release of EV from platelets has been shown to be dependent on the agonist; in this study, we investigated whether the microRNA profile or EV released from platelets was also agonist specific.

Washed platelets from healthy subjects were maximally stimulated with agonists specific for the receptors for collagen (Glycoprotein VI (GPVI)), thrombin (PAR1/PAR4), or ADP (P2Y1/P2Y12) with/without inhibiting secondary mediators, using aspirin to block cyclooxygenase-1 and apyrase to remove ADP. The released microRNAs were profiled using TaqMan microRNA microarray cards. Platelet-derived EV (pdEV) were characterized by size (Nanoparticle Tracking Analysis, NTA), for procoagulant activity (Annexin-V binding and support of thrombin generation), and for the EV markers CD63 and HSP70.

Platelet activation triggered the release of 57–79 different microRNAs, dependent upon agonist, with a core of 46 microRNAs observed with all agonists. There was a high level of correlation between agonists (r² > 0.98; p < 0.0001 for all), and with the microRNA content of the parent platelets (r² > 0.98; p < 0.0001). The 46 microRNAs seen in all samples are predicted to have significant effects on the translation of proteins involved in endocytosis, cell cycle control, and differentiation. MiR-223-3p was the most abundant in all samples and has previously been implicated in myeloid lineage development and demonstrated to have anti-inflammatory effects. Stimulation through GPVI produced a pdEV population with significantly more procoagulant activity than the other agonists. Apyrase significantly reduced microRNA and pdEV release, while aspirin had little effect.

These data suggest that all tested agonists trigger the release of a similar microRNA profile while the procoagulant activity of the pdEV was agonist dependent. ADP was shown to play an important role in the release of both microRNAs and pdEV.     

Patient perspectives on de‐simplifying their single‐tablet co‐formulated antiretroviral therapy for societal cost savings

Objectives

The incremental costs of expanding antiretroviral (ARV) drug treatment to all HIV‐infected patients are substantial, so cost‐saving initiatives are important. Our objectives were to determine the acceptability and financial impact of de‐simplifying (i.e. switching) more expensive single‐tablet formulations (STFs) to less expensive generic‐based multi‐tablet components. We determined physician and patient perceptions and acceptance of STF de‐simplification within the context of a publicly funded ARV budget.

Methods

Programme costs were calculated for patients on ARVs followed at the Southern Alberta Clinic, Canada during 2016 (Cdn$). We focused on patients receiving Triumeq® and determined the savings if patients de‐simplified to eligible generic co‐formulations. We surveyed all prescribing physicians and a convenience sample of patients taking Triumeq® to see if, for budgetary purposes, they felt that de‐simplification would be acceptable.

Results

Of 1780 patients receiving ARVs, 62% (n = 1038) were on STF; 58% (n = 607) of patients on STF were on Triumeq®. The total annual cost of ARVs was $26 222 760. The cost for Triumeq® was $8 292 600. If every patient on Triumeq® switched to generic abacavir/lamivudine and Tivicay® (dolutegravir), total costs would decrease by $4 325 040. All physicians (n = 13) felt that de‐simplifying could be safely achieved. Forty‐eight per cent of 221 patients surveyed were agreeable to de‐simplifying for altruistic reasons, 27% said no, and 25% said maybe.

Conclusions

De‐simplifying Triumeq® generates large cost savings. Additional savings could be achieved by de‐simplifying other STFs. Both physicians and patients agreed that selective de‐simplification was acceptable; however, it may not be acceptable to every patient. Monitoring the medical and cost impacts of de‐simplification strategies seems warranted.

     

Increased binding of fibrinogen to glycoprotein IIIa-proline33 (HPA-1b, PlA2, Zwb) positive platelets in patients with cardiovascular disease.

AIMS: The GPIIb-IIIa complex on the platelet membrane plays an important part in thrombosis as it is the receptor for fibrinogen. The gene for platelet membrane glyco-protein IIIa has multiple alleles one of which, the GPIIIa-Proline33 (HPA-1b, PlA2, Zwb) allele has been reported in some, but not all studies, to be associated with an increased risk of myocardial infarction. We investigated whether the presence of the Pro33 form of GPIIIa on the platelet membrane is associated with increased fibrinogen binding. METHODS AND RESULTS: Blood samples from 70 patients (54 male) with stable angina of whom 22 (18 male) had a history of previous myocardial infarction, were analysed for the GPIIIa-Leu-Pro33 polymorphism at the genomic level, and for whole blood flow cytometric measurement of platelet fibrinogen binding. The GPIIIa-Pro33 form was present in 20 (28.6%) patients (1 homozygous) representing an allele frequency of 0.85 and 0.15 (GPIIIa-Leu33:Pro33). The incidence of myocardial infarction was higher (40.0%) in patients positive for GPIIIa-Pro33 than in those without (32.0%) but this was not significant (P=0.58). Fibrinogen binding to ADP-stimulated platelets was significantly higher in the GPIIIa-Pro33 positive group at all ADP concentrations (<0.0001; two way ANOVA). There was no association between fibrinogen binding and the level of expression of the GPIIb-IIIa complex, platelet volume or platelet count. Fibrinogen binding in response to thrombin stimulation was not different between the groups (P>0.05). CONCLUSIONS: The increased tendency of platelets from patients with the Pro33 form of GPIIIa may predispose patients with this allele to a higher risk of acute thrombotic events, and argues for selective use of therapeutic agents that inhibit ADP-mediated platelet activation in occlusive vascular disease states.     

The effect of dietary restriction on mitochondrial protein density and flight muscle mitochondrial morphology in Drosophila

Dietary restriction (DR) extends life span in diverse organisms and may do so by attenuating production of mitochondrial reactive oxygen species (ROS). However, measurements of ROS production from isolated mitochondria of organisms subjected to DR have produced inconsistent results. In the fruit fly Drosophila, DR does not reduce production of ROS from isolated mitochondria. In this study, we used Drosophila to test whether DR lowered mitochondrial density. We assessed mitochondrial densities of flies on DR and Control diets using (a) the activities of mitochondrial enzymes and (b) electron microscopy. Both methods showed no overall effect of DR on mitochondrial density; however, mitochondrial enzyme activities and morphology differed significantly between DR and Control flies. We concluded that life-span extension by DR in Drosophila is not mediated through a reduction in mitochondrial density. If DR in Drosophila extends life span by reducing ROS production, then it does so through mechanisms that operate only in vivo.     

A mutation of the common receptor subunit for interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor, and IL-5 that leads to ligand independence and tumorigenicity

The cytokines interleukin-3, interleukin-5, and granulocyte-macrophage colony-stimulating factor bind with high affinity to a receptor complex that contains a ligand-specific alpha-chain and a common beta-chain, h beta c. We report here the isolation of a mutant form of h beta c, from growth factor-independent cells, that arose spontaneously after infection of a murine factor-dependent hematopoietic cell line (FDC-P1) with a retroviral h beta c expression construct. Analysis of this h beta c mutation shows that a small (37 amino acid) duplication of extracellular sequence that includes two conserved sequence motifs is sufficient to confer ligand-independent growth on these cells and lead to tumourigenicity. Because this is a conserved region in the cytokine receptor superfamily, our results suggest that the large family of cytokine receptors has the capacity to become oncogenically active.