Nucleotide oligomerization domain 1 is a dominant pathway for NOS2 induction in vascular smooth muscle cells: comparison with Toll-like receptor 4 responses in macrophages

Background and purpose:

Gram-negative bacteria contain ligands for Toll-like receptor (TLR) 4 and nucleotide oligomerization domain (NOD) 1 receptors. Lipopolysaccharide (LPS) activates TLR4, while peptidoglycan products activate NOD1. Activation of NOD1 by the specific agonist FK565 results in a profound vascular dysfunction and experimental shock in vivo.

Experimental approach:

Here, we have analysed a number of pharmacological inhibitors to characterize the role of key signalling pathways in the induction of NOS2 following TLR4 or NOD1 activation.

Key results:

Vascular smooth muscle (VSM) cells expressed NOD1 mRNA and protein, and, after challenge with Escherichia coli or FK565, NOS2 protein and activity were induced. Macrophages had negligible levels of NOD1 and were unaffected by FK565, but responded to E. coli and LPS by releasing increased NO and expression of NOS2 protein. Classic pharmacological inhibitors for NF-κB (SC-514) and mitogen-activated protein kinase (SB203580, PD98059) signalling pathways inhibited responses in both cell types regardless of agonist. While TLR4-mediated responses in macrophages were specifically inhibited by the pan-caspase inhibitor z-VAD-fmk and the PKC inhibitor Gö6976, NOD1-mediated responses in VSM cells were inhibited by the Rip2 inhibitor PP2.

Conclusions and implications:

Our findings suggest a selective role for NOD1 in VSM cells, and highlight NOD1 as a potential novel therapeutic target for the treatment of vascular inflammation.     

Activation of submucosal 5-HT3 receptors elicits a somatostatin-dependent inhibition of ion secretion in rat colon

Background and purpose:

5-Hydroxytryptamine (5-HT) is a key regulator of the gastrointestinal system and we have shown that submucosal neuronal 5-HT₃ receptors exerted a novel inhibitory effect on colonic ion transport. The aim of the present study was to investigate the precise mechanism(s) underlying this inhibitory effect.

Experimental approach:

Mucosa/submucosa or mucosa-only preparations from rat distal colon were mounted in Ussing chambers for measurement of short-circuit current (I_sc) as an indicator of ion secretion. Somatostatin release was determined with radioimmunoassay. Intracellular cAMP content was measured with enzyme-linked immunoadsorbent assay (elisa). Immunohistochemical techniques were used to study the expression of 5-HT₃ receptors, somatostatin and somatostatin receptors in colonic tissue.

Key results:

In rat distal colonic mucosa/submucosa preparations, pretreatment with 5-HT₃ receptor antagonists enhanced 5-HT-induced increases in I_sc. However, in mucosa-only preparations without retained neural elements, pretreatment with 5-HT₃ receptor antagonists inhibited 5-HT-induced ΔI_sc. Pretreatment with a somatostatin-2 (sst₂) receptor antagonist in mucosa/submucosa preparations augmented 5-HT-induced ΔI_sc. Combination of sst₂ and 5-HT₃ receptor antagonists did not cause further enhancement of 5-HT-induced ΔI_sc. Moreover, both sst₂ and 5-HT₃ receptor antagonists enhanced 5-HT-induced increase in intracellular cAMP concentration in the mucosa/submucosa preparations. 5-HT released somatostatin from rat colonic mucosa/submucosa preparations, an effect prevented by pretreatment with 5-HT₃ receptor antagonists. Immunohistochemical staining demonstrated the presence of 5-HT₃ receptors on submucosal somatostatin neurons and of sst₂ receptors on colonic mucosa.

Conclusion and implications:

Activation of neuronal 5-HT₃ receptors in the submucosal plexus of rat colon suppressed 5-HT-induced ion secretion by releasing somatostatin from submucosal neurons.     

Prevalence of Murine Helicobacter spp. Infection Is Reduced by Restocking Research Colonies with Helicobacter-Free Mice

Most academic research colonies of mice are endemically infected with enterohepatic Helicobacter spp. (EHS). We evaluated EHS prevalence in surveillance mice before and after a 10-y period of requiring that imported mice be free of EHS by embryo transfer rederivation or purchase from approved vendors. In 2009, composite fecal samples from CD1 surveillance mice representing colony health in 57 rooms located in 6 facilities were evaluated for EHS infection by using PCR assays. Fecal samples were screened with primers designed to detect all known EHS, and positive samples were further assayed by using primers specific for H. hepaticus, H. bilis, H. rodentium, and H. typhlonicus. Most EHS were detected in surveillance mice within the first month of dirty bedding exposure, with prevalence ranging from 0% to 64% as monoinfections or, more commonly, infections with multiple EHS. Compared with 1999 prevalence data, EHS remained endemic in colonies importing the lowest number of EHS-free mice. EHS were absent or the prevalence was greatly reduced in colonies receiving the highest percentage of EHS-free mice. This study demonstrates that the management decision to require exclusive importation of EHS-free mice reduced EHS prevalence on an institutional scale without intensive labor and expense associated with other techniques or interference with research objectives.Abbreviation: EHS, enterohepatic Helicobacter spp.; ET, embryo transfer; Hb, H. bilis; Hh, H. hepaticus; Hm, H. mastomyrinus; Hr, H. rodentium; Ht, H. typhlonicusEnterohepatic Helicobacter spp. (EHS) infections are endemic in the majority of research mouse colonies. In 2007, 84% of mice shipped from academic institutions worldwide for embryo transfer (ET) rederivation at our institution were PCR-positive for EHS. H. hepaticus (Hh) was detected in 64% of the mouse shipments either as a monoinfection or in combination with other EHS including H. bilis (Hb), H. rodentium (Hr), H. typhlonicus (Ht), and H. mastomyrinus (Hm).³⁰ Although EHS generally cause subclinical infection in immunocompetent mice, opportunistic infections have the potential to confound experimental data in mouse models.^9,17,34 Importantly, chronic EHS infection in immunodeficient and select inbred strains of mice can induce liver¹⁰ and lower bowel carcinoma,¹³ typhlocolitis, and rectal prolapse,^16,21,28 and reduce reproductive performance.²⁵ In addition, EHS-induced inflammatory responses may alter host immune responses to unrelated experimental infections (for example, promoting elevated systemic IFNγ responses).^3,20Key challenges to eradication of EHS from rodent colonies are determining infection status, eliminating endemic infections, and instituting management practices that prevent reinfection. EHS are disseminated through fecal–oral transmission within a colony and are transmissible to surveillance mice through dirty-bedding exposure.^1,19,24,32 For routine surveillance, PCR assay of feces or cecal mucosal scrapings for genus-specific Helicobacter 16S rRNA genes is the most efficient means of detecting EHS infection, with speciation (if desired) of positive results by culture, restriction fragment length polymorphism analysis, species-specific PCR, or sequence analysis.³⁴ In 1999, as determined by species-specific PCR assays of cecal scrapings from 59 surveillance mice exposed to dirty bedding from colony mice in 26 rooms representing 4 mouse facilities, EHS were endemic on our campus, with prevalence in surveillance mice of 41% for Hh, 82% for Hr, and 6% for Hb.³² Husbandry practices used to minimize cage-to-cage transmission of EHS included microisolation caging, sanitized forceps to transfer mice, and a cage change order from known Helicobacter-free mice to mice of unknown or known EHS infection status (that is, clean to dirty traffic flow of personnel and equipment).³² Although EHS eradication potentially could be accomplished campus-wide by using labor-intensive antibiotics^7,15 and cross-fostering,^4,29,31 we hypothesized that a more cost-effective approach, without confounding experimental data, would be to restrict importation of mice to EHS-free sources. Vendors were screened to establish that production colonies were SPF for EHS, and a new requirement was instituted for embryo transfer (ET) rederivation of mice obtained from random sources, typically other academic institutions, replacing traditional quarantine practices. This study used PCR data from 1999 and 2009 to evaluate the success of this approach, which was defined as a marked decrease in the prevalence of EHS infection over time.     

Catheter ablation of accessory pathways, atrioventricular nodal reentrant tachycardia, and the atrioventricular junction: final results of a prospective, multicenter clinical trial. The Atakr Multicenter Investigators Group

BACKGROUND: The purpose of this study was to evaluate the safety and efficacy of a temperature-controlled radiofrequency catheter ablation system. METHODS AND RESULTS: The patient population included 1050 patients who had undergone ablation of atrioventricular nodal reentrant tachycardia (AVNRT), an accessory pathway (AP), or the atrioventricular junction (AVJ). Ablation was successful in 996 patients. The probability of success was highest among patients who had undergone ablation of the AVJ, lowest in patients who had undergone ablation of an AP, and in between for patients who had undergone ablation of AVNRT. A major complication occurred in 32 patients. Four variables predicted ablation success (AVJ, AVNRT, or left free wall AP ablation and an experienced center). Four factors predicted arrhythmia recurrence (right free wall, posteroseptal, septal, and multiple APs). Two variables predicted development of a complication (structural heart disease and the presence of multiple targets), and 3 variables predicted an increased risk of death (heart disease, lower ejection fraction, and AVJ ablation). CONCLUSIONS: These findings may serve as a guide to clinicians considering therapeutic options in patients who are candidates for ablation.     

Evidence-based dexamethasone dosing in malignant brain tumors: what do we really know?

Purpose

The present study aims to conduct a systematic review of literature reporting on the dose and dosing schedule of dexamethasone (DXM) in relation to clinical outcomes in malignant brain tumor patients, with particular attention to evidence-based practice.

Methods

A systematic search was performed in PubMed, Embase, Web of Science, Cochrane, Academic Search Premier, and PsycINFO to identify studies that reported edema volume reduction, symptomatic relief, adverse events and survival in relation to dexamethasone dose in glioma or brain metastasis (BM) patients.

Results

After screening 1812 studies, fifteen articles were included for qualitative review. Most studies reported a dose of 16 mg, mostly in a schedule of 4 mg four times a day. Due to heterogeneity of studies, it was not possible to perform quantitative meta-analysis. For BMs, best available evidence suggests that higher doses of DXM may give more adverse events, but may not necessarily result in better clinical condition. Some studies suggest that higher DXM doses are associated with shorter survival in the palliative setting. For glioma, DXM may lead to symptomatic improvement, yet no studies directly compare different doses. Results regarding edema reduction and survival in glioma patients are conflicting.

Conclusions

Evidence on the safety and efficacy of different DXM doses in malignant brain tumor patients is scarce and conflicting. Best available evidence suggests that low DXM doses may be noninferior to higher doses in certain circumstances, but more comparative research in this area is direly needed, especially in light of the increasing importance of immunotherapy for brain tumors.

     

Minimum spanning tree analysis of the human connectome

One of the challenges of brain network analysis is to directly compare network organization between subjects, irrespective of the number or strength of connections. In this study, we used minimum spanning tree (MST; a unique, acyclic subnetwork with a fixed number of connections) analysis to characterize the human brain network to create an empirical reference network. Such a reference network could be used as a null model of connections that form the backbone structure of the human brain. We analyzed the MST in three diffusion‐weighted imaging datasets of healthy adults. The MST of the group mean connectivity matrix was used as the empirical null‐model. The MST of individual subjects matched this reference MST for a mean 58%–88% of connections, depending on the analysis pipeline. Hub nodes in the MST matched with previously reported locations of hub regions, including the so‐called rich club nodes (a subset of high‐degree, highly interconnected nodes). Although most brain network studies have focused primarily on cortical connections, cortical–subcortical connections were consistently present in the MST across subjects. Brain network efficiency was higher when these connections were included in the analysis, suggesting that these tracts may be utilized as the major neural communication routes. Finally, we confirmed that MST characteristics index the effects of brain aging. We conclude that the MST provides an elegant and straightforward approach to analyze structural brain networks, and to test network topological features of individual subjects in comparison to empirical null models.     

Older peoples' perceptions of oral health: 'it's just not that simple'

Abstract: Objectives: Little is known about older persons’ perceptions of oral health and oral health care. The purpose of this study was to explore the viewpoint of older adults’ regarding their oral health care practices. Methods: A qualitative interpretive methodology was employed comprising three analytic levels: coding of data into concepts, analysis of concepts into themes, followed by an in‐depth analysis of relationships within concepts and between themes. In‐depth individual interviews were conducted with 19 participants aged 65 to 87 years. Results: Older people’s decision to access oral health care involves complex and personally meaningful strategies. A dental visit surfaces hopes and fears based on past and present experiences. Mouth and teeth are not merely objects of dental care; they represent a person’s social and relational self. Age‐related changes challenge the relational self as represented in societal ideal images of youth and perfection (the perfect smile). This study highlights older peoples’ resilience and determination when faced with the dilemmas in accessing oral health care – it costs, personally as well as financially. Contrary to the assumption that older peoples’ oral health status is related to neglect, rather for many, it is the result of the intersection of their history with technological advances. Conclusions: These findings challenge oral health care practitioners to be sensitive to the contexts affecting their older client’s oral health care status. They do not ‘just go’ to the dentist; they bring with them their past dental experiences and their hopes for the future. It matters how one is treated at this vulnerable time.     

Non‐invasive prenatal determination of fetal sex: translating research into clinical practice

Hill M, Finning K, Martin P, Hogg J, Meaney C, Norbury G, Daniels G, Chitty LS. Non‐invasive prenatal determination of fetal sex: translating research into clinical practice. The effectiveness and clinical utility of non‐invasive prenatal diagnosis (NIPD) for fetal sex determination using cell‐free fetal DNA (cffDNA) was assessed by undertaking a prospective national audit of UK testing. NIPD was performed using real‐time polymerase chain reaction analysis of the DYS14 or SRY gene in cffDNA extracted from maternal plasma. All cases referred for fetal sex determination from 1 April 2006 to 31 March 2009 were ascertained from two laboratories offering the test. Fetal gender determined by NIPD was compared with that based on ultrasound, invasive test or phenotype at birth. Indication and rate of invasive testing was ascertained. In the first year, results were issued in 150/161 pregnancies tested. Of the 135 with outcome data, results were concordant in 130/135 [96.3% (95% CI 91.6–98.8%)]. Reporting criteria were changed and in the subsequent 511 pregnancies the concordancy rate increased to 401/403 [99.5% (95% CI 98.2–99.9%)]. Over the 3 years only 32.9% (174/528) underwent invasive testing. NIPD for fetal sex determination using cffDNA is highly accurate when performed in National Health Service laboratories if stringent reporting criteria are applied. Parents should be advised of the small risk of discordant results and possible need for repeat testing to resolve inconclusive results.     

The combined use of steroids and immune checkpoint inhibitors in brain metastasis patients: a systematic review and meta-analysis

BackgroundImmune checkpoint inhibitors (ICI) have been a breakthrough for selected cancer patients, including those with brain metastases (BMs). Likewise, steroids have been an integral component of symptomatic management of BM patients. However, clinical evidence on the interaction between ICI and steroids in BM patients is conflicting and has not adequately been summarized thus far. Hence, the aim of this study was to perform a systematic literature review and meta-analysis on the association between steroid use and overall survival (OS) in BM patients receiving ICI.MethodsA systematic literature search was performed. Pooled effect estimates were calculated using random-effects models across included studies.ResultsAfter screening 1145 abstracts, 15 observational studies were included. Fourteen studies reported sufficient data for meta-analysis, comprising 1102 BM patients of which 32.1% received steroids. In the steroid group, median OS ranged from 2.9 to 10.2 months. In the nonsteroid group, median OS ranged from 4.9 to 25.1 months. Pooled results demonstrated significantly worse OS (HR = 1.84, 95% CI 1.22-2.77) and systemic progression-free survival (PFS; HR = 2.00, 95% CI 1.37-2.91) in the steroid group. Stratified analysis showed a consistent effect across the melanoma subgroup; not in the lung cancer subgroup. No significant association was shown between steroid use and intracranial PFS (HR = 1.31, 95% CI 0.42-4.07).ConclusionsAdministration of steroids was associated with significantly worse OS and PFS in BM patients receiving ICI. Further research on dose, timing, and duration of steroids is needed to elucidate the cause of this association and optimize outcomes in BM patients receiving ICI.