Mamdoh Al-Ameri

Prostaglandin E2 inhibits mast cell-dependent bronchoconstriction in human small airways through the E prostanoid subtype 2 receptor.

BACKGROUND Inhaled prostaglandin (PG) E2 might inhibit asthmatic responses, but the mechanisms involved remain undefined. OBJECTIVE We sought to characterize the direct and indirect effects of PGE2 on human small airways with particular reference to the receptors mediating the responses. METHODS Contraction and relaxation were studied in isolated human bronchi with an inner diameter of 1 mm or less. RESULTS Low concentrations of PGE2 (0.01-1 μmol/L) relaxed the bronchi precontracted by histamine. The bronchodilator response was inhibited by the E prostanoid (EP) subtype 4 receptor antagonist ONO-AE3-208 but unaffected by the EP2 receptor antagonist PF-04418948. Higher concentrations of PGE2 (10-100 μmol/L) contracted the small airways. However, the TP receptor agonists U-46,619, PGF2α, and PGD2 were more potent than PGE2. Moreover, the bronchoconstrictor responses to PGE2 and all other tested prostanoids, including the EP1/EP3 receptor agonist 17-phenyl trinor PGE2 and the partial FP receptor agonist AL-8810, were uniformly abolished by the TP receptor antagonist SQ-29,548. In the presence of TP and EP4 antagonists, PGE2 inhibited the mast cell-mediated bronchoconstriction resulting from anti-IgE challenge. Measurement of the release of histamine and cysteinyl leukotrienes documented that this bronchoprotective action of PGE2 was mediated by the EP2 receptor, unrelated to bronchodilation, and increased with time of exposure. CONCLUSION The pharmacology of PGE2 in isolated human small airways was different from its profile in animal models. This first demonstration of powerful EP2 receptor-mediated inhibition of IgE-dependent contractions in human airways introduces a new selective target for the treatment of asthma. This EP2 control of mast cell-mediated bronchoconstriction is presumably exaggerated in patients with aspirin-exacerbated respiratory disease.

Bitter taste receptor agonists mediate relaxation of human and rodent vascular smooth muscle.

Taste-sensing type 2 receptors (TAS2Rs) have been implicated in extraoral functions. Airway smooth muscle expresses TAS2Rs and is strongly relaxed by TAS2R agonists. We hypothesised that TAS2R agonists might affect vascular smooth muscle as well. Moreover, the general pharmacological profile of TAS2R agonists, which are used to investigate the functions of TAS2R׳s, are undefined. The aim of this study was to pharmacologically characterise the effects of five prototype TAS2R agonists in vascular smooth muscle. Responses to the TAS2R agonists were investigated in guinea-pig aorta and taenia coli, mouse aorta (wild-type and caveolin-1-/- mice) and human pulmonary arteries. Chloroquine, denatonium, dextromethorphan, noscapine and quinine, agonists for TAS2R3, TAS2R4, TAS2R10 and TAS2R14, induced strong endothelium-independent relaxations (responses between 82-96% of maximal relaxations) in phenylephrine pre-contracted guinea-pig aorta that persisted in the presence of L-type Ca2+ and KCa1.1-channel blockers. Experiments in guinea-pig taenia coli revealed that denatonium and quinine also inhibited relaxations to phenylephrine, indicating antagonism of α-adrenoceptors. Only chloroquine and noscapine mediated relaxations when the guinea pig aorta was pre-contracted by U-46619 or PGF2α. Relaxations to chloroquine and noscapine after U-46619 pre-contractions were however markedly impaired in aortae from caveolin-1-/- mice. Chloroquine and noscapine mediated relaxations of human pulmonary arteries that expressed also mRNA for TAS2R3, TAS2R4, TAS2R10 and TAS2R14, at levels similar to that of the α1A adrenoceptor. Notwithstanding whether TAS2Rs are involved or not, TAS2R agonists have profound effects on vascular smooth muscle. Chloroquine and noscapine are of special interest as their effects cannot be accounted for by conventional pathways.

Human lung natural killer cells are predominantly comprised of highly differentiated hypofunctional CD69−CD56dim cells

Background: In contrast to the extensive knowledge about human natural killer (NK) cells in peripheral blood, relatively little is known about NK cells in the human lung. Knowledge about the composition, differentiation, and function of human lung NK cells is critical to better understand their role in diseases affecting the lung, including asthma, chronic obstructive pulmonary disease, infections, and cancer. Objective: We sought to analyze and compare the phenotypic and functional characteristics of NK cells in the human lung and peripheral blood at the single‐cell level. Methods: NK cells in human lung tissue and matched peripheral blood from 132 subjects were analyzed by using 16‐color flow cytometry and confocal microscopy. Results: CD56dimCD16+ NK cells made up the vast majority of NK cells in human lungs, had a more differentiated phenotype, and more frequently expressed educating killer cell immunoglobulin‐like receptors compared with NK cells in peripheral blood. Despite this, human lung NK cells were hyporesponsive toward target cell stimulation, even after priming with IFN‐&agr;. Furthermore, we detected a small subset of NK cells expressing CD69, a marker of tissue residency. These CD69+ NK cells in the lung consisted predominantly of immature CD56brightCD16− NK cells and less differentiated CD56dimCD16+ NK cells. Conclusion: Here, we characterize the major NK cell populations in the human lung. Our data suggest a model in which the majority of NK cells in the human lung dynamically move between blood and the lung rather than residing in the lung as bona fide tissue‐resident CD69+ NK cells. Graphical abstract Figure. No Caption available.

Intracellular Ca2+-handling differs markedly between intact human muscle fibers and myotubes

BackgroundIn skeletal muscle, intracellular Ca2+ is an important regulator of contraction as well as gene expression and metabolic processes. Because of the difficulties to obtain intact human muscle fibers, human myotubes have been extensively employed for studies of Ca2+-dependent processes in human adult muscle. Despite this, it is unknown whether the Ca2+-handling properties of myotubes adequately represent those of adult muscle fibers.MethodsTo enable a comparison of the Ca2+-handling properties of human muscle fibers and myotubes, we developed a model of dissected intact single muscle fibers obtained from human intercostal muscle biopsies. The intracellular Ca2+-handling of human muscle fibers was compared with that of myotubes generated by the differentiation of primary human myoblasts obtained from vastus lateralis muscle biopsies.ResultsThe intact single muscle fibers all demonstrated strictly regulated cytosolic free [Ca2+] ([Ca2+]i) transients and force production upon electrical stimulation. In contrast, despite a more mature Ca2+-handling in myotubes than in myoblasts, myotubes lacked fundamental aspects of adult Ca2+-handling and did not contract. These functional differences were explained by discrepancies in the quantity and localization of Ca2+-handling proteins, as well as ultrastructural differences between muscle fibers and myotubes.ConclusionsIntact single muscle fibers that display strictly regulated [Ca2+]i transients and force production upon electrical stimulation can be obtained from human intercostal muscle biopsies. In contrast, human myotubes lack important aspects of adult Ca2+-handling and are thus an inappropriate model for human adult muscle when studying Ca2+-dependent processes, such as gene expression and metabolic processes.

Surgery for pulmonary metastases from colorectal cancer: survival and prognostic factors

Background This study aimed to describe overall survival following pulmonary metastasectomy for colorectal cancer (CRC) in Sweden, and to assess the discrimination of a recently proposed risk prediction model. Methods Individual-level data of 756 patients who underwent resection of pulmonary metastases from CRC between 2009 and 2015 were obtained from ThoR, a Swedish national quality register for thoracic surgery. We classified patients into three risk categories according to the number of preoperative risk factors [age, disease-free interval (DFI), presence of extrathoracic lesions, number of pulmonary metastases] established in a prior study. We estimated the hazard ratios (HRs) and 95% confidence interval (CI) by Cox regression and the restricted mean survival time difference as group contrast measures. Results During a median follow-up time of 2.9 years, 35% (268/756) patients died. At 5 years, overall survival was 56% (95% CI: 51-60%). In a Cox regression model with risk category as the only independent variable, the HR for all-cause mortality was 1.94 (95% CI: 1.38-2.72, P<0.001) and 4.35 (95% CI: 2.49-7.62, P<0.001) in the moderate- (n=558) and high-risk categories (n=32), respectively, versus the low-risk category (n=166). At 5 years, the differences in restricted mean survival time were 6 months (P<0.001) and 1.5 years (P<0.001) in the moderate- and high-risk categories, respectively, versus the low-risk category. Conclusions Five-year survival after surgery for pulmonary metastases from CRC in Sweden was similar or higher compared with contemporary reports. A prognostic model, initially developed in Japanese patients, had excellent discrimination in an external validation cohort of Swedish patients.

Video-assisted thoracoscopic versus open thoracotomy lobectomy: a Swedish nationwide cohort study

Background The aim of this nationwide observational cohort study was to investigate the early postoperative complications and long-term survival following video-assisted thoracoscopic surgery (VATS) lobectomy compared to open thoracotomy lobectomy for early stage non-small cell lung cancer (NSCLC). Methods We used the Swedish national quality register for general thoracic surgery and included all patients who underwent lobectomy for NSCLC during 2012-2015. We compared postoperative complications and long-term survival in patients who underwent VATS lobectomy at our institution to patients who underwent open lobectomy at the other seven hospitals in Sweden. We used inverse probability of treatment weighting to limit differences in baseline characteristics between the groups and used standardized mean differences to assess balance after weighting. Results We included 1,601 patients who underwent open (n=1,316) or VATS (n=285) lobectomy for NSCLC. The mean age was 67.7 years in both groups and comorbidities were well balanced, but the open thoracotomy group had a higher proportion of patients with more advanced cancer stage. After weighting, all baseline characteristics were well balanced. Most patients (84%) did not have postoperative complications; 83% vs. 86% in the open and VATS group, respectively (P=0.41). The 30- and 90-day mortality was 0.7% vs. 0.3% (P=0.38) and 1.7% vs. 0.3% (P=0.09) in the open thoracotomy and VATS group, respectively. There were significantly more transfusions (5.0% vs. 1.4%, P=0.008) and pneumonia (5.5% vs. 0.6%, P=0.002) in the in the open thoracotomy and VATS group, respectively. The overall survival at 1 and 5 years was 92% vs. 97% and 63% vs. 78% in the open thoracotomy and VATS group, respectively; HR (95% CI): 0.47 (0.33-0.68). Conclusions We found less postoperative complications and better long-term survival following VATS lobectomy compared to open thoracotomy lobectomy for NSCLC. The implementation of a VATS lobectomy program did not compromise patient safety or the oncological efficacy.

An optimized protocol for the isolation and functional analysis of human lung mast cells.

Background: Mast cells are tissue-resident inflammatory cells defined by their high granularity and surface expression of the high-affinity IgE receptor, FcεRI, and CD117/KIT, the receptor for stem cell factor (SCF). There is a considerable heterogeneity among mast cells, both phenotypically and functionally. Human mast cells are generally divided into two main subtypes based on their protease content; the mucosa-associated MCT (tryptase positive and chymase negative mast cell) and the connective tissue associated-residing MCTC (tryptase and chymase positive mast cell). Human lung mast cells exhibit heterogeneity in terms of cellular size, expression of cell surface receptors, and secreted mediators. However, knowledge about human lung mast cell heterogeneity is restricted to studies using immunohistochemistry or purified mast cells. Whereas the former is limited by the number of cellular markers that can be analyzed simultaneously, the latter suffers from issues related to cell yield. Aim: To develop a protocol that enables isolation of human lung mast cells at high yields for analysis of functional properties and detailed analysis using single-cell based analyses of protein (flow cytometry) or RNA (RNA-sequencing) expression. Methods: Mast cells were isolated from human lung tissue by a sequential combination of washing, enzymatic digestion, mechanical disruption, and density centrifugation using Percoll (WEMP). As a comparison, we also isolated mast cells using a conventional enzyme-based protocol. The isolated cells were analyzed by flow cytometry. Results: We observed a significant increase in the yield of total human lung CD45+ immune cells and an even more pronounced increase in the yield of CD117+ mast cells with the WEMP protocol in comparison to the conventional protocols. In contrast, the frequency of the rare lymphocyte subset innate lymphoid cells group 2 (ILC2) did not differ between the two methods. Conclusion: The described WEMP protocol results in a significant increase in the yield of human lung mast cells compared to a conventional protocol. Additionally, the WEMP protocol enables simultaneous isolation of different immune cell populations such as lymphocytes, monocytes, and granulocytes while retaining their surface marker expression that can be used for advanced single-cell analyses including multi-color flow cytometry and RNA-sequencing.

Long-term survival after surgery for pulmonary metastases from colorectal cancer: an observational cohort study

Background Evidence for pulmonary metastasectomy following colorectal cancer (CRC) is scarce. The aim of the study was to investigate long-term survival and identify prognostic factors to aid patient selection. Methods We included all patients who underwent pulmonary resections for CRC metastases between January 01, 2004 and December 31, 2015 in a population-based cohort study. The primary outcome measure was all-cause mortality and was ascertained from Swedish national registers. The Kaplan-Meier estimator was used to calculate cumulative survival. We used Cox regression for estimation of hazard ratios (HR) and 95% confidence intervals (CI) for the association between patient characteristics and survival. Results We included 184 patients. The number of procedures per year increased from 1 in 2004 to 34 in 2015. During a median follow-up time of 3.2 years, 36% (66/184) patients died. Overall survival at 5 years was 60% (95% CI: 50-68%) and was significantly lower compared to an age- and gender-matched Swedish population. Carcinoembryonic antigen (CEA) level was identified as a prognostic factor for mortality in the age and sex-adjusted analysis (HR, 2.46; 95% CI: 1.15-5.26, P=0.020). Conclusions We found a steady increase in the number of pulmonary metastasectomies after CRC during the study period. We identified prethoracotomy CEA level as a prognostic factor for long-term survival, which was consistent with prior reports. The 5-year overall survival rate in our study was 60%, which was high in comparison with prior reports. Although our results indicated that current patient selection criteria were reasonable, definitive evidence of efficacy is pending.