Effect of influenza A virus on leukocyte histamine release

Viral respiratory infections provoke asthma in many patients. In the following study we examined the effect of an in vitro incubation of influenza A on leukocyte histamine release. After incubation with a live influenza A (H3N2) virus, calcium ionophore A23187 (0.5, 1.0, and 1.5 microgram/ml)-induced leukocyte histamine release (HR) was enhanced (p less than 0.05). This effect was also found with heat- or ether-inactivated virus. Similarly, influenza A-exposed leukocytes had augmented leukocyte HR during subsequent incubation with ragweed AgE. Incubation of the leukocyte suspension with interferon (800 IU/ml) for 24 hr was also associated with enhanced HR to ragweed AgE. In contrast, interferon did not alter the calcium ionophore A23187 HR. Therefore, although interferon may mediate the enhanced leukocyte HR when ragweed AgE is the inciting stimulus, it does not change HR to the calcium ionophore.     

The coronary endothelium-derived hyperpolarizing factor (EDHF) stimulates multiple signalling pathways and proliferation in vascular cells

In the present study we determined whether the endothelium-derived hyperpolarizing factor (EDHF), in addition to its acute effects on vascular tone, activates intracellular signalling pathways other than those associated with Ca2+-dependent K+ channels. EDHF was generated by rhythmic distension of porcine coronary arteries under conditions of combined nitric oxide (NO) synthase/cyclo-oxygenase blockade, and the EDHF-containing luminal incubate was applied to cultured human coronary endothelial or smooth muscle cells. In both cell types, the luminal incubate activated tyrosine kinases, the mitogen-activated protein (MAP) kinases, extracellular signal regulated kinases 1 and 2 (Erk1/2) and p38, as well as protein kinase B/Akt. The constituent responsible for Erk1/2 phosphorylation was identified as a cytochrome P450 (CYP) metabolite, as Erk1/2 activation was attenuated by pretreating the EDHF donor with the CYP 2C inhibitor sulfaphenazole as well as by CYP 2C antisense oligonucleotides. Erk1/2 phosphorylation in detector cells was also observed following the transfer of supernatant from cultured endothelial cells treated with the CYP inducer beta-naphthoflavone. The CYP 2C product 11,12-epoxyeicosatrienoic acid (11,12-EET) also activated tyrosine kinases, Erk1/2 and p38 MAP kinase. Overexpression of CYP 2C8 in native porcine coronary artery endothelial cells resulted in an increase in endothelial 11,12-EET production and Erk1/2 phosphorylation compared to that detected in untreated cells or cells transfected with an antisense CYP 2C8. Endothelial cell number was unaffected by transfection with LacZ or CYP 2C8 antisense but was significantly enhanced in cells overexpressing CYP 2C8. These observations indicate that EDHF/11,12-EET is not simply a vasodilator and that its continuous release under pulsatile conditions in vivo may affect vascular cell signalling and proliferation.     

Alternaria IgG precipitins and adverse reactions.

Late reactions consisting of fever, malaise, and swelling at the site, 4 to 6 hr after injections of Alternaria extract occurred in several patients receiving immunotherapy with Alternaria. These patients had in common serum IgG precipitins and exquisite leukocyte histamine release sensitivity to Alternaria. Such precipitins were 3 times more frequent in patients receiving Alternaria immunotherapy than a control group of patients receiving immunotherapy with other antigens. A prospective study revealed that 5 of 23 Alternaria-sensitive persons had precipitins before immunotherapy and another 6 developed precipitins during therapy. Only one of the 23 experienced a late Alternaria reaction. Thus, precipitins to Alternaria are common and do not seem to be the basis for the late reactions we observed. The finding of precipitins does not contraindicate immunotherapy.     

Grafting of fibroblasts isolated from the synovial membrane of rheumatoid arthritis (RA) patients induces chronic arthritis in SCID mice-A novel model for studying the arthritogenic role of RA fibroblasts in vivo

The objective of this study was to verify whether isolated rheumatoid arthritis (RA) synovial fibroblasts induce chronic arthritis in SCID mice, in analogy to whole tissue pieces. Fibroblasts were isolated from the synovial membrane of four RA patients (or controls) by out-growth and repeated-passage culture. Following flow-cytometry characterization, 2x10(6)cells were transferred into the left knee joint of SCID mice. The development of arthritis was assessed by joint swelling and histological changes. Human and murine cytokines were measured in vitro in co-cultures (or Transwelltrade mark systems) of human and murine cells. Purified RA synovial fibroblasts, but not healthy synovial or skin fibroblasts, induced hu/mu arthritis within 6 weeks. In-vitro secretion of murine and human interleukin(IL)-6, as well as murine tumour necrosis factor (TNF)-alpha, indicated cross-activation between murine macrophages and human RA fibroblasts. Soluble-factor mechanisms proved more effective than cell-contact mechanisms. Purified RA fibroblasts can, alone, induce hu/mu SCID arthritis. The cytokine profile suggests that xenogeneic interaction between human fibroblasts and murine macrophages may determine the sequence of events leading to hu/mu arthritis.     

Efficacy response of inhaled beclomethasone dipropionate in asthma is proportional to dose and is improved by formulation with a new propellant

BACKGROUND: This study tested the hypothesis that there would be improved asthma control with increasing doses of beclomethasone dipropionate (BDP) formulated in hydrofluoroalkane-134a (HFA-BDP) and the standard chlorofluorocarbon propellants (CFC-BDP). Because HFA-BDP has improved lung deposition compared with CFC-BDP, this study also tested the hypothesis that HFA-BDP would provide more effective control of asthma than CFC-BDP. METHODS: In this multicenter, randomized, parallel-group blinded study, asthmatic subjects who had deterioration in asthma control after discontinuation of inhaled corticosteroids were randomized to receive one of 6 possible treatments: 100 microg/d, 400 microg/d, or 800 microg/d of HFA-BDP or 100 microg/d, 400 microg/d, or 800 microg/d of CFC-BDP for 6 weeks. Changes in spirometry, daytime asthma symptom and nighttime asthma-related sleep disturbance scores, morning and evening peak expiratory flows, and daily use of inhaled beta-agonist for symptom control on diary cards were assessed over 6 weeks of treatment. RESULTS: Three hundred twenty-three patients were randomized to the 6 treatment groups, which had similar demographics and baseline lung function. There were significantly larger changes from baseline at week 6 in FEV(1) percent predicted with increasing doses of both HFA-BDP and CFC-BDP. The FEV(1) percent predicted dose-response curve for HFA-BDP was shifted to the left compared with the dose-response curve for CFC-BDP. By using the Finney bioassay method, it was calculated that 2.6 times as much CFC-BDP would be required to achieve the same improvement in FEV(1) percent predicted as HFA-BDP (95% confidence interval, 1.1-11.6). All treatment groups except the 100 microg/d CFC-BDP group tolerated study drug well. Ten (17%) of 59 patients in this group reported an acute asthma episode, increased asthma symptoms (6 of the 8 reports of increased asthma symptoms were classified as severe), or both, and 8 patients withdrew from the study (3 for adverse events related to asthma). CONCLUSIONS: Increasing doses of inhaled corticosteroids lead to improved lung function and asthma control. Moreover, the reformulation of BDP in HFA enables effective asthma control at much lower doses than CFC-BDP.     

Analysis of 4-nitroquinoline-1-oxide induced mutations at the hprt locus in mammalian cells: possible involvement of preferential DNA repair

Mutation spectra induced by 4-nitroquinoline 1-oxide (4NQO)at the hprt locus for both normal (AA8) and 4NQO-sensitive (UV5)Chinese hamster ovary cells were determined to investigate theeffect of DNA repair on the nature of induced mutations. TheUV5 cell line is three times more sensitive to 4NQO than theAA8 parental cell line. In UV5 cells, the dGuo-N2-AQO adduct,which is considered to be the most toxic and mutagenic adductin Escherichia coli, is poorly repaired. The molecular natureof 30hprt mutants isolated from AA8 and 20 isolated from UV5cells was determined by sequence analysis of in vitro amplifiedhprtcDNA. Both similarities and differences emerged. In bothcell lines we found that (i) 4NQO is basically a base substitutionmutagen acting almost exclusively at G residues and (ii) G transversionsare prevalent over G transitions in both cell lines, independentlyfrom the ability to repair dGuo-N2-AQO. A high proportion (13/25)of splice mutations was observed in AA8 cells, statisticallydifferent (P < 0.04, Fisher‘s exact test) from theincidence of splice mutants in UV5 cells (4/20). In AA8 mutants,all but two of the point mutations were due to lesions localizedon the non-transcribed strand, suggesting preferential repairof the transcribed strand. Compared with AA8, the proportionof mutants due to lesions present on the transcribed strandwas higher in UV5 cells, as expected if a preferential repairmechanism was impaired in the sensitive cell line. Our dataare consistent with the molecular defect in DNA repair recentlycharacterized in UV5. ³To whom correspondence should be addressed     

L-arginine-dependent nitric oxide formation and nitrite release in bone marrow-derived macrophages stimulated with bacterial lipopeptide and lipopolysaccharide.

This study shows that stimulating bone marrow-derived macrophages with either lipopolysaccharide (LPS) or the lipopeptide N-palmitoyl-S-(2,3-bis(palmitoyloxy)-(2RS)-propyl)-(R)- cysteinyl-alanyl-glycine (Pam3Cys-Ala-Gly), a synthetic analogue of the N-terminal part of bacterial lipoprotein, leads to the formation of nitric oxide (NO) and nitrite (NO2-), a stable analogue of NO. NO was detected by applying the chemiluminescence method and by measuring the activity of exogenously added soluble guanylate cyclase (GC), which is strongly and selectively activated by NO. Synthesis of NO and NO2- occurs via activation of the L-arginine and NADPH-dependent enzyme(s) present in the cytosol of bone marrow-derived macrophages. No produced by this non-constitutive L-arginine pathway is thought to be responsible for the cytostatic and killing properties of macrophages (Stuehr & Nathan, 1989). Macrophages stimulated either with LPS or Pam3Cys-Ala-Gly exhibited a 6-hr lag time before engaging in nitrite synthesis, a time at which expression of the NO-forming enzyme had already reached its maximum. The regulation of NO and NO2- synthesis during macrophage development seems to differ from that of cytokine synthesis. Whereas cytokine release varies during a culture period up to 20 days, NO synthesis and expression of the NO-forming enzyme remain unaltered. These studies show that, similar to LPS, Pam3Cys-Ala-Gly is a potent activator of 'the oxidative L-arginine pathway' in bone marrow-derived macrophages. Whether both stimuli use the same signal transfer mechanism to induce this pathway and whether NO synthesized by this pathway is involved in the activation of the enzyme guanylate cyclase in macrophages requires clarification.     

A multicenter study on skin-test reactivity of human volunteers to venom as compared with whole body Hymenoptera antigens

Twenty-four control and 104 stinging insect—allergic patients were tested in this multicenter study, which compared the utility of individual venom and whole body extract (WBE) preparations for diagnosis. This study indicates that venom skin testing is more specific and more reliable than is testing with WBE. There was a high rate of WBE skin-test reactivity among controls. False-positive skin testing with venom was not a problem at concentrations of 1 μg/ml or less. Only one mild and easily treated adverse reaction occurred in this study.     

Response of the ENPP1-Deficient Skeletal Phenotype to Oral Phosphate Supplementation and/or Enzyme Replacement Therapy: Comparative Studies in Humans and Mice

Inactivating mutations in human ecto-nucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1) may result in early-onset osteoporosis (EOOP) in haploinsufficiency and autosomal recessive hypophosphatemic rickets (ARHR2) in homozygous deficiency. ARHR2 patients are frequently treated with phosphate supplementation to ameliorate the rachitic phenotype, but elevating plasma phosphorus concentrations in ARHR2 patients may increase the risk of ectopic calcification without increasing bone mass. To assess the risks and efficacy of conventional ARHR2 therapy, we performed comprehensive evaluations of ARHR2 patients at two academic medical centers and compared their skeletal and renal phenotypes with ENPP1-deficient Enpp1^asj/asj mice on an acceleration diet containing high phosphate treated with recombinant murine Enpp1-Fc. ARHR2 patients treated with conventional therapy demonstrated improvements in rickets, but all adults and one adolescent analyzed continued to exhibit low bone mineral density (BMD). In addition, conventional therapy was associated with the development of medullary nephrocalcinosis in half of the treated patients. Similar to Enpp1^asj/asj mice on normal chow and to patients with mono- and biallelic ENPP1 mutations, 5-week-old Enpp1^asj/asj mice on the high-phosphate diet exhibited lower trabecular bone mass, reduced cortical bone mass, and greater bone fragility. Treating the Enpp1^asj/asj mice with recombinant Enpp1-Fc protein between weeks 2 and 5 normalized trabecular bone mass, normalized or improved bone biomechanical properties, and prevented the development of nephrocalcinosis and renal failure. The data suggest that conventional ARHR2 therapy does not address low BMD inherent in ENPP1 deficiency, and that ENPP1 enzyme replacement may be effective for correcting low bone mass in ARHR2 patients without increasing the risk of nephrocalcinosis. © 2021 American Society for Bone and Mineral Research (ASBMR).