Asthma-related hospitalizations after implementing SABA-free asthma management with a maintenance and anti-inflammatory reliever regimen.

Overreliance on short-acting ß2-agonists (SABA) has been a common feature of asthma management globally for at least 30 years. However, given the evidence against the long-term use of SABA, including potentially increased risk of exacerbations, emergency room visits, overall healthcare resource utilization, and mortality, the latest Global Initiative for Asthma report no longer recommends SABA only therapy. Since 2014, we implemented an ICS-containing reliever strategy at our asthma center at the G Baigorria Hospital in Argentina; we only administered budesonide/formoterol via a single inhaler device across the spectrum of asthma severity and completely eliminated the use of SABA therapy. In this article, we compare hospitalization data from our center, previously reported in the EAGLE study (when inhaled corticosteroids plus as-needed SABA was administered) for the years 1999 and 2004 with data from 2017 to 2018 (when budesonide/formoterol in a single inhaler device was administered as maintenance and/or anti-inflammatory reliever therapy [MART/AIR] without any SABA) from our center, to assess the impact of two distinct asthma management strategies on asthma-related hospitalizations. MART/AIR regimens in our SABA-free center reduced asthma hospitalizations from 9 (1999 and 2004) to 1 (2017 and 2018) (Fisher's exact test, p = 0.031; odds ratio = 0.11; 95% confidence interval [CI] = 0.013-0.98); the hospitalization rate was reduced by 92% (1.47% in 1999 and 2004 to 0.12% in 2017 and 2018). Our data provide preliminary real-world evidence that MART/AIR with budesonide/formoterol simultaneously with SABA elimination across asthma severities is an effective asthma management strategy for reducing asthma-related hospitalizations.

Growth factor effects on apoptosis of rat gastric enterochromaffin-like cells.

Enterochromaffin-like (ECL) cells are histamine-containing endocrine cells in the gastric epithelium that show increased density during chronic atrophic gastritis. The current study determined cell number and apoptosis of isolated rat ECL cells in response to several growth factors. Isolated ECL cells from fundic mucosa (enrichment >90%) were grown in serum-free medium over 2-5 days. Cell number was determined by mitochondrial formazan production; apoptosis was measured by Tdt-mediated dUTP nick end labeling reaction and DNA fragmentation-based enzyme-linked immunosorbent assay. Immunocytochemistry and RT-PCR demonstrated the presence of epidermal growth factor receptor, neuronal growth factor receptor (type 1), and fibroblast growth factor (FGF) receptor (type 1). Gastrin (EC50, approximately 2 pM), transforming growth factor-alpha (TGF alpha; 10-30 ng/ml), and basic FGF (bFGF; 1-10 ng/ml) increased the total number of cultured ECL cells. bFGF augmented the gastrin (1 pM)-induced response. Beta-neuronal growth factor (10 ng/ml) and bFGF (2 ng/ml) decreased the programed death of ECL cells. Interleukin-1beta (100 pg/ml, 24 h) stimulated apoptosis 2- to 3-fold in ECL cells, and simultaneous incubation with TGF alpha (20 ng/ml) or bFGF (2 ng/ml) significantly inhibited this effect. ECL cells express specific receptors for gastrin, epidermal growth factor, neuronal growth factor, and FGF. bFGF prolonged ECL cell survival by inhibiting spontaneous apoptosis. Our data further indicate that TGF alpha and bFGF increase ECL cell number by inhibiting cytokine-induced programed cell death.

Gastrin induces expression and promoter activity of the vesicular monoamine transporter subtype 2.

Gastric enterochromaffin-like cells produce histamine in response to the antral hormone gastrin and accumulate the biogenic amine in secretory organelles via vesicular monoamine transporter subtype 2. The putative effects of gastrin on vesicular monoamine transporter subtype 2 expression and promoter activity are poorly understood. In the present study we used highly enriched rat enterochromaffin-like cells (purity, >90%) and rat pheochromocytoma cells stably transfected with a gastrin/cholecystokinin B receptor to investigate the expression and transcriptional regulation of vesicular monoamine transporter subtype 2. Stimulation of vesicular monoamine transporter subtype 2 mRNA and protein expression was observed in isolated enterochromaffin-like cells after 3- to 7-h incubation with gastrin (10(-7) M), forskolin (10(-5) M), or ionomycin (10(-5) M). Deletion analysis of the rat vesicular monoamine transporter subtype 2 promoter defined the minimal promoter sequence necessary for full basal activity as a -121 bp segment upstream of exon 1 containing two Sp1 sites (-97 to -88 bp and -68 to -59 bp) and a cAMP-responsive element (-44 to -35 bp). Gastrin (10(-7) M) stimulated extracellular signal related kinase1/2 phosphorylation, activated Sp1 and cAMP-responsive element-binding protein, and further induced activity of the complete rat vesicular monoamine transporter subtype 2 promoter (-800 bp) in gastrin/cholecystokinin B receptor cells. The -121-bp fragment was able to confer full gastrin responsiveness, and site-directed mutagenesis of the Sp1 and cAMP-responsive element motifs demonstrated their crucial importance for basal and inducible activities. Comparison of promoter activity of histidine decarboxylase, chromogranin A, or vesicular monoamine transporter subtype 2 in transfected cell lines revealed significant differences in basal and gastrin-stimulated activities. Our current study provides the first evidence that gastrin directly stimulates the expression and promoter activity of vesicular monoamine transporter subtype 2. Sp1 and cAMP-responsive element-binding protein recognition motifs located within 121 bp upstream of exon 1 appear to be indispensable for full basal and inducible promoter activities. Diverging effects of gastrin on histidine decarboxylase, chromogranin A, and vesicular monoamine transporter subtype 2 promoter may account for the coordinated synthesis and storage of histamine in this neuroendocrine cell type.

Effect of interleukin-1beta on the ascending and descending reflex in rat small intestine.

Acute inflammation of the intestine is associated with motility changes. We investigated the acute effect of inflammatory mediators such as interleukin-1beta, interleukin-2 and tumor necrosis factor-alpha (TNF-alpha) on electrically stimulated ascending and descending reflex responses of the rat small intestine. Exogenous application of interleukin-1beta caused a concentration-dependent inhibition of the oral contraction (0.1 ng/ml: -22.9+/-3.8%, 10 ng/ml: -57.0+/-7.4%, P < 0.05, n=10) but had no effect on anal relaxation. The interleukin-1 receptor antagonist alone had no significant effect on the reflex response, but prevented the inhibitory effect of interleukin-1beta (10 ng/ml: -3.9+/-11.4%, n=8). Interleukin-2 and TNF-alpha had no significant effect on the oral contractile and the anal inhibitory response (n.s., n=10). Using reverse transcriptase polymerase chain reaction (RT-PCR) the presence of mRNA of the interleukin-1 receptor was demonstrated in the rat small intestine. Preincubation of the preparation with indomethacin (10(-6) M), the histamine H1 receptor antagonist, pyrilamine (10(-8) M), and the histamine H3 receptor antagonist, clobenpropit (10(-8) M), decreased the oral contraction by 60.1+/-7.7%, 42.8+/-6.9% and 44.4+/-14.2% as well as the anal relaxation. These data suggest that acute administration of interleukin-1beta inhibits the ascending and descending contractile reflex pathway and this effect seems not to be mediated by prostaglandins or histamine receptors.

Functional impairment of rat enterochromaffin-like cells by interleukin 1 beta.

BACKGROUND & AIMS: Histamine-producing enterochromaffin-like (ECL) cells play an integrative role in the regulation of acid secretion. Decreased mucosal histamine concentrations and increased levels of interleukin (IL) 1 beta, IL-6, and IL-8 have been detected in the gastric mucosa inflamed with Helicobacter pylori. The aim of this study was to investigate the response of isolated ECL cells to these cytokines. METHODS: Enriched rat gastric ECL cells (85%-95%) were cultured for 2-4 days. RESULTS: Polymerase chain reaction showed IL-1 and IL-6, but not IL-8 receptors, in ECL cell complementary DNA. Positive receptor staining with biotinylated IL-1 beta corresponded to ECL cell enrichment (92%). IL-6 and IL-8 had no effect on histamine secretion. IL-1 beta (2 U/mL) stimulated basal histamine secretion and nitric oxide production within 60 minutes and cyclic guanosine monophosphate production within 20 minutes. Pretreatment for 20 minutes with IL-1 beta (2 U/mL) attenuated gastrin-stimulated histamine secretion by 40%-50%, reversed by the IL-1 receptor antagonist (10 U/ mL). Pretreatment for 20 minutes with IL-1 beta (2 U/mL) completely inhibited gastrin-stimulated (1 nmol/L) histidine decarboxylase activity. IL-1 beta (2 U/mL, 60 minutes) increased lactate dehydrogenase release to 25% of cell content. Cells pretreated with IL-1 beta did not respond to gastrin after a further 48-hour culture and showed decreased histamine content. CONCLUSIONS: ECL cells appear to express IL-1 receptors. IL-1 beta causes sustained functional impairment of ECL cells in vitro.

IL-1beta-induced apoptosis in rat gastric enterochromaffin-like cells is mediated by iNOS, NF-kappaB, and Bax protein.

BACKGROUND & AIMS: Enterochromaffin-like (ECL) cells are histamine-containing endocrine cells in the gastric mucosa. Previous studies have shown that the proinflammatory cytokine interleukin (IL)-1beta present during chronic gastritis inhibits histamine synthesis in ECL cells and leads to sustained functional impairment. This study investigated the effects of IL-1beta on ECL cell apoptosis and the related signal-transduction mechanisms. METHODS: ECL cells were isolated by pronase digestion and a combination of elutriation, gradient centrifugation, and 48-hour culture (purity >/=90%). Apoptosis was measured by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling reaction and cell death detection enzyme-linked immunosorbent assay. RESULTS: IL-1beta (100 pg/mL) increased the rate of programmed cell death 2-3 fold in ECL cells after 24 hours of incubation (total of 12%-14%). This effect was completely inhibited by the NF-kappaB inhibitor, proteasome inhibitor I, and the nitric oxide synthase inhibitor (iNOS) N(G)-monomethyl-L-arginine (10(-4) mol/L), but not by the caspase 3 inhibitor, Asp-Glu-Val-Asp-CHO. Western blot analysis, reverse-transcription polymerase chain reaction (PCR), and in situ PCR showed that IL-1beta induced gene expression (after 2-4 hours) and protein synthesis (6-18 hours) of the iNOS isoform in ECL cells. Bax protein expression was increased in response to IL-1beta. In contrast, bcl-2 gene expression was increased in response to basic fibroblast growth factor, which has been shown to counteract IL-1beta- induced apoptosis. CONCLUSIONS: These data suggest that IL-1beta induces programmed cell death in isolated rat ECL cells via activation of NF-kappaB, iNOS, and the Bax protein.

Clinical relevance of the Helicobacter pylori gene for blood-group antigen-binding adhesin.

Infection with Helicobacter pylori is associated with different human gastric diseases. Biochemical studies, in vitro adherence assays, and in vivo animal models revealed that epithelial attachment of H. pylori can be mediated by the blood-group antigen-binding adhesin (BabA) targeting human Lewis(b) surface epitopes. Studies with transgenic mice expressing the Lewis(b) epitope have shown that such attachment can alter disease outcome. In the current study, the presence of the babA2 gene encoding the adhesin was investigated in clinical isolates from a German population by using PCR and reverse transcription-PCR. A positive genotype was correlated to allelic variations in the genes encoding VacA and CagA and also to the prevalence of duodenal ulcer, distal gastric adenocarcinoma, mucosa-associated lymphoid tissue lymphoma, and antral gastritis. The presence of babA2 was significantly associated with duodenal ulcer (P = 0.0002) and adenocarcinoma (P = 0.033). In contrast, type 1 strains (vacAs1- and cagA-positive) were associated with only duodenal ulcer (P = 0.004) but not adenocarcinoma (P = 0.235). Genotype presence of babA2, vacAs1, and cagA ("triple-positive" strains) showed a highly significant correlation to the prevalence of ulcer (P = 0.000002) and adenocarcinoma (P = 0.014) and discriminated significantly better between disease outcome than did the current type 1 classification. These results indicate that the babA2 gene is of high clinical relevance and would be a useful marker to identify patients who are at higher risk for specific H. pylori-related diseases.

The mechanism of histamine secretion from gastric enterochromaffin-like cells.

Enterochromaffin-like (ECL) cells play a pivotal role in the peripheral regulation of gastric acid secretion as they respond to the functionally important gastrointestinal hormones gastrin and somatostatin and neural mediators such as pituitary adenylate cyclase-activating peptide and galanin. Gastrin is the key stimulus of histamine release from ECL cells in vivo and in vitro. Voltage-gated K(+) and Ca(2+) channels have been detected on isolated ECL cells. Exocytosis of histamine following gastrin stimulation and Ca(2+) entry across the plasma membrane is catalyzed by synaptobrevin and synaptosomal-associated protein of 25 kDa, both characterized as a soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein. Histamine release occurs from different cellular pools: preexisting vacuolar histamine immediately released by Ca(2+) entry or newly synthesized histamine following induction of histidine decarboxylase (HDC) by gastrin stimulation. Histamine is synthesized by cytoplasmic HDC and accumulated in secretory vesicles by proton-histamine countertransport via the vesicular monoamine transporter subtype 2 (VMAT-2). The promoter region of HDC contains Ca(2+)-, cAMP-, and protein kinase C-responsive elements. The gene promoter for VMAT-2, however, lacks TATA boxes but contains regulatory elements for the hormones glucagon and somatostatin. Histamine secretion from ECL cells is thereby under a complex regulation of hormonal signals and can be targeted at several steps during the process of exocytosis.