Gab1, SHP2, and protein kinase A are crucial for the activation of the endothelial NO synthase by fluid shear stress

Fluid shear stress enhances NO production in endothelial cells by a mechanism involving the activation of the phosphatidylinositol 3-kinase and the phosphorylation of the endothelial NO synthase (eNOS). We investigated the role of the scaffolding protein Gab1 and the tyrosine phosphatase SHP2 in this signal transduction cascade in cultured and native endothelial cells. Fluid shear stress elicited the phosphorylation and activation of Akt and eNOS as well as the tyrosine phosphorylation of Gab1 and its association with the p85 subunit of phosphatidylinositol 3-kinase and SHP2. Overexpression of a Gab1 mutant lacking the pleckstrin homology domain abrogated the shear stress-induced phosphorylation of Akt but failed to affect the phosphorylation or activity of eNOS. The latter response, however, was sensitive to a protein kinase A (PKA) inhibitor. Mutation of Gab1 Tyr627 to phenylalanine (YF-Gab1) to prevent the binding of SHP2 completely prevented the shear stress-induced phosphorylation of eNOS, leaving the Akt response intact. A dominant-negative SHP2 mutant prevented the activation of PKA and phosphorylation of eNOS without affecting that of Akt. Moreover, shear stress elicited the formation of a signalosome complex including eNOS, Gab1, SHP2 and the catalytic subunit of PKA. In isolated murine carotid arteries, flow-induced vasodilatation was prevented by a PKA inhibitor as well as by overexpression of either the YF-Gab1 or the dominant-negative SHP2 mutant. Thus, the shear stress-induced activation of eNOS depends on Gab1 and SHP2, which, in turn, regulate the phosphorylation and activity of eNOS by a PKA-dependent but Akt-independent mechanism.     

Sonographic,manometric, and myographic evaluation of the anal sphincters morphology and function

PURPOSE: A direct comparison of anal sphincter physiology (muscle performance and anatomy-muscle thickness) has not yet been undertaken but may be of importance in patients with defecation disorders. METHODS: We evaluated 15 healthy volunteers by means of anorectal manometry to determine pressure functions of the internal and external anal sphincter. Transcutaneous electromyography was recorded to assess the electrical activity of the external anal sphincter. Thickness of the anal sphincter muscles was measured sonographically from within the anal canal with dorsal projection using a 7.5-MHz 360° rectal panorama scanner. RESULTS: It was shown that neither is the muscle thickness of the external anal sphincter during rest (6.26±1.02 mm) or during squeezing (7.40±1.39 mm) correlated to its squeeze pressure (138.8±15.2 mmHg), nor is the diameter of the internal anal sphincter (2.09±0.58 mm) correlated to its resting pressure (61.1±15.2 mmHg). Finally, the thicker the internal anal sphincter muscle, the lower the electrical activity of the external anal sphincter during squeezing (r=0.74,P <0.001). CONCLUSION: It is concluded that both manometry to assess sphincter function and sonography to determine sphincter morphology are important measures in the evaluation of continence function.     

A multimedia consent tool for research participants in the Gambia: a randomized controlled trial

ObjectiveTo assess the effectiveness of a multimedia informed consent tool for adults participating in a clinical trial in the Gambia.MethodsAdults eligible for inclusion in a malaria treatment trial (n = 311) were randomized to receive information needed for informed consent using either a multimedia tool (intervention arm) or a standard procedure (control arm). A computerized, audio questionnaire was used to assess participants’ comprehension of informed consent. This was done immediately after consent had been obtained (at day 0) and at subsequent follow-up visits (days 7, 14, 21 and 28). The acceptability and ease of use of the multimedia tool were assessed in focus groups.FindingsOn day 0, the median comprehension score in the intervention arm was 64% compared with 40% in the control arm (P = 0.042). The difference remained significant at all follow-up visits. Poorer comprehension was independently associated with female sex (odds ratio, OR: 0.29; 95% confidence interval, CI: 0.12–0.70) and residing in Jahaly rather than Basse province (OR: 0.33; 95% CI: 0.13–0.82). There was no significant independent association with educational level. The risk that a participant’s comprehension score would drop to half of the initial value was lower in the intervention arm (hazard ratio 0.22, 95% CI: 0.16–0.31). Overall, 70% (42/60) of focus group participants from the intervention arm found the multimedia tool clear and easy to understand.ConclusionA multimedia informed consent tool significantly improved comprehension and retention of consent information by research participants with low levels of literacy.     

The impact of childhood vaccines on bacterial carriage in the nasopharynx: a longitudinal study

Background

There is increasing evidence that childhood vaccines have effects that extend beyond their target disease. The objective of this study was to assess the effects of routine childhood vaccines on bacterial carriage in the nasopharynx.

Methods

A cohort of children from rural Gambia was recruited at birth and followed up for one year. Nasopharyngeal swabs were taken immediately after birth, every two weeks for the first six months and then every other month. The presence of bacteria in the nasopharynx (Haemophilus influenzae, Streptococcus pneumoniae, Staphylococcus aureus) was compared before and after the administration of DTP-Hib-HepB and measles-yellow fever vaccines.

Results

A total of 1,779 nasopharyngeal swabs were collected from 136 children for whom vaccination data were available. The prevalence of bacterial carriage was high: 82.2% S. pneumoniae, 30.6%, S.aureus, 27.8% H. influenzae. Carriage of H. influenzae (OR = 0.36; 95% CI: 0.13, 0.99) and S. pneumoniae (OR = 0.25; 95% CI: 0.07, 0.90) were significantly reduced after measles-yellow fever vaccination; while DTP-Hib-HepB had no effect on bacterial carriage.

Conclusions

Nasopharyngeal bacterial carriage is unaffected by DTP-Hib-HepB vaccination and reduced after measles-yellow fever vaccination.     

Ebola Risk Perception in Germany, 2014

Ebola virus disease (EVD) outbreaks have occurred during the past 5 decades, but none has affected European countries like the 2014 epidemic in West Africa. We used an online questionnaire to investigate risk perceptions in Germany during this epidemic peak. Our questionnaire covered risk perceptions, knowledge about transmission routes, media use, reactions to the outbreak, attitudes toward measures to prevent the spread of EVD and vaccination against EVD, and willingness to volunteer for aid missions. Of 974 participants, 29% indicated that they worried about EVD, 4% correctly stated virus transmission routes, and 75% incorrectly rated airborne transmission and transmission by asymptomatic patients as possible. Many indicated that if a patient were flown to Germany for treatment in a nearby hospital, they would adapt preventive behavior. Although most participants were not worried about EVD at the current stage of the epidemic, misperceptions regarding transmission were common and could trigger inappropriate behavior changes.     

Type A dissection following endovascular thoracic aortic stent-graft repair.

PURPOSE: To describe our experience with endovascular stent-graft repairs in the thoracic aorta focusing on the secondary complication of type A dissection. METHODS: Between January 1996 and April 2004, 73 patients were treated for traumatic thoracic aortic rupture (n=15), type B dissection (n=22), or atherosclerotic descending thoracic aortic aneurysms (TAA, n=36). A retrospective review of the records found 5 (6.8%) patients (3 men; median age 64 years, range 43-87) who experienced a type A dissection at a median 20 days (range 2-124) after thoracic stent-graft repair for 3 type B dissections, 1 TAA, and a late type I endoleak that appeared 28 months after initial stent-graft repair of a traumatic dissection. RESULTS: In 3 patients (2 dissections, 1 endoleak), a tear in the aortic wall at the proximal stent-graft was responsible for a retrograde type A dissection. Underlying disease was the cause of the type A dissection in the 2 other patients (1 dissection, 1 TAA) and was unrelated to the stent-grafts. Three patients underwent open surgery at 3, 26, and 124 days after stent-graft placement; 2 procedures were successful, but the third patient died 3 months later due to multiorgan failure. Two type A dissections were untreated: one patient died from cardiac tamponade 14 days after successful stent-graft exclusion of the type I endoleak; the other patient refused further treatment and survived. The procedure-related mortality following acute retrograde type A dissection was 40%. CONCLUSIONS: Endovascular stent-graft repair of the thoracic aorta is associated with lower morbidity and mortality rates than surgical repair, although potentially lethal complications, acute or delayed, may occur.     

CD4<Superscript>+</Superscript>CD25<Superscript>+</Superscript> Regulatory T Cells Have Divergent Effects on Intestinal Inflammation in IL-10 Gene-Deficient Mice

The regulatory effect of murine CD4⁺CD25⁺ T-cells in vivo appears to be dependent on the secretion of IL-10. The lack of IL-10 in the IL-10 gene-deficient mouse has a profoundly negative effect on the mouse’s regulation of the response to intestinal bacteria, resulting in severe enterocolitis. We investigated the effect of neonatal injection with wild-type CD4⁺CD25⁺ T-cells on the intestinal immune response in IL-10 gene-deficient mice. At the time of analysis, 8–15 weeks later, all mice demonstrated an increased, antigen-stimulated systemic response. However, the intestinal response was divergent with about half of the mice developing an intestinal inflammation with a high injury score, the other half demonstrating a remarkable reduction in injury score with a marked decrease in intestinal IFNγ release. Our data demonstrate that CD4⁺CD25⁺ T-cells can be activated in IL-10 gene-deficient mice and that this stimulation under stringent conditions has the potential to reduce intestinal inflammation.     

Virulence and thrombocyte affectation of two Aspergillus terreus isolates differing in amphotericin B susceptibility

Aspergillus terreus-induced invasive infections exhibit high lethality, partly due to the intrinsic resistance for amphotericin B (AmB). We compared the virulence and pathogenesis of an AmB-resistant isolate of A. terreus (ATR) with that of a rare variant showing enhanced sensitivity for AMB (ATS). The modifications that result in enhanced AmB sensitivity of isolates are not associated with reduced virulence in vivo; instead, the ATS-infected mice died even faster than the ATR-infected animals. Since A. terreus enters the blood stream in most patients and frequently induces thrombosis, we studied a putative correlation between virulence of the two A. terreus isolates and their effect on thrombocytes. Those mice infected with the more virulent ATS isolate had lower thrombocyte numbers and more phosphatidylserine exposure on platelets than ATR-infected mice. In vitro experiments confirmed that ATS and ATR differ in their effect on thrombocytes. Conidia, aleurioconidia and hyphae of ATS were more potent than ATR to trigger thrombocyte stimulation, and thrombocytes adhered better to ATS than to ATR fungal structures. Furthermore, ATS secreted more soluble factors that triggered platelet stimulation than ATR. Thus, it might be suggested that the capacity of a fungal isolate to modulate thrombocyte parameters contributes to its virulence in vivo.