Neutrophil autophagy and extracellular DNA traps contribute to airway inflammation in severe asthma.

BACKGROUND: Autophagy and neutrophil extracellular DNA traps (NETs) are implicated in asthma; however, their roles in asthma pathogenesis have not been elucidated. OBJECTIVES: We compared autophagy and NET production levels from peripheral blood neutrophils (PBNs) of patients with severe asthma (SA) and non-severe asthma (NSA). Additionally, we investigated the inflammatory effects of NETs on human airway epithelial cells (AECs) and peripheral blood eosinophils (PBEs). METHODS: Peripheral blood neutrophils from patients with SA (n = 30) and NSA (n = 38) were treated with interleukin (IL)-8 (100 ng/mL). Autophagy (light chain 3-II expression) and NET production levels were evaluated by Western blot, immunofluorescence microscopy, and PicoGreen assay. The effects of NETs on AECs were assessed by investigating cell death, cell detachment, expression of occludin and claudin-1, and IL-8 production; the effects of NETs on PBEs were examined by investigating the activation and release of eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN). RESULTS: Untreated and IL-8-treated PBNs from the SA group produced higher autophagy and NET levels compared with those from the NSA group (P < 0.01). IL-8 increased autophagy and NET levels in PBNs from the SA group, but not from the NSA group. NET levels were correlated with autophagy levels in PBNs (P < 0.001). IL-8-induced NET production levels negatively were correlated with FEV1/FVC (r = -0.700, P = 0.016). NETs induced cell death, detachment, degradation of occludin and claudin-1, and IL-8 production from AECs. Higher levels of NET-induced ECP and EDN were released from PBEs in SA compared with NSA groups. CONCLUSIONS AND CLINICAL RELEVANCE: Neutrophil autophagy and NETs could enhance asthma severity by damaging airway epithelium and triggering inflammatory responses of AECs and PBEs. Modulating neutrophil autophagy and NET production may be a new target therapy for SA.

Eosinophil activation and novel mediators in the aspirin-induced nasal response in AERD.

BACKGROUND: Eosinophil activation is the key feature of upper and lower airway inflammation in aspirin-exacerbated respiratory disease (AERD). OBJECTIVE: To investigate the mechanism of eosinophil activation and identify novel inflammatory mediators using proteomics. METHODS: Thirty-two asthmatic subjects were enrolled: 18 AERD patients who showed positive responses to the lysine-aspirin nasal provocation test (L-ASA NPT) and 14 aspirin-tolerant asthma (ATA) patients who showed negative responses to the L-ASA NPT (control group). Nasal lavage fluid (NLF) was collected before (baseline), at 10, 30 and 60 min (early response), and at 3 h (late response) after the L-ASA NPT. Eosinophil cationic protein (ECP) and cysteinyl leucotriene (CysLT) levels were measured using an ImmunoCAP system and ELISA respectively. To identify proteins involved in AERD, comparative proteomics was applied using NLFs collected before and after L-ASA NPTs in AERD patients. The clinical relevance of identified novel proteins was evaluated by ELISA using NLFs from the AERD and ATA groups. RESULTS: Eosinophil cationic protein and CysLT levels both increased significantly during the early response in AERD. ECP levels increased until the late response in AERD, while CysLT levels were not significantly increased during the late response. Proteomic analysis showed up-regulation of apolipoprotein A1 (ApoA1), α2-macroglobulin (α2M) and ceruloplasmin (CP), with significant increases in NLF of AERD patients, which was significantly higher in AERD patients with chronic rhinosinusitis. Significant correlations were noted between ECP and CysLT, ApoA1, α2M and CP levels during the early response in AERD patients. CONCLUSION: Eosinophil activation occurred in early and late responses after L-ASA NPT in upper airway mucosa of AERD patients, where ApoA1, α2M and CP as well as CysLT may be involved in eosinophilic inflammation.

Functional variability of the adenosine A3 receptor (ADORA3) gene polymorphism in aspirin-induced urticaria.

BACKGROUND: To improve understanding of aspirin hypersensitivity, this study focused on adenosine as a noncyclooxygenase target molecule of aspirin. Adenosine may affect the release of histamine from cutaneous mast cells through a mechanism mediated by the adenosine A3 receptor. OBJECTIVES: To investigate the genetic contribution of adenosine A3 receptor gene (ADORA3) polymorphisms in the pathogenesis of aspirin-induced urticaria (AIU) in a case-control association study in a Korean population. METHODS: A case-control association study was performed in 385 patients with AIU and 213 normal controls from a Korean population. The functional variability of genetic polymorphisms in the ADORA3 gene was analysed in in vitro studies that included a luciferase reporter assay and an electrophoretic mobility shift assay (EMSA), and ex vivo studies that included real-time polymerase chain reaction for mRNA expression in peripheral blood mononuclear cells and a histamine release assay. RESULTS: A significant association of ADORA3 promoter polymorphism at -1050G/T was found with the phenotype of AIU. Patients with AIU showed higher frequency of the haplotype, ht1 (T(-1050) C(-564) ), compared with normal healthy controls. Moreover, ht1 (TC) was found to be a high-transcript haplotype by the luciferase activity assay, and a -564C allele-specific DNA binding protein was found by EMSA. Increased basophil histamine release was noted in subjects who had the high-transcript haplotype, ht1 (TC). CONCLUSION: These results suggest that the high-transcript haplotype, ht1 (TC), of the ADORA3 gene may contribute to the development of cutaneous hyper-reactivity to aspirin, leading to the clinical presentation of AIU.

Vascular endothelial growth factor in allergen-induced nasal inflammation.

BACKGROUND: Increased vessel number and permeability are important features of the nasal mucosa in allergic rhinitis (AR), and are mediated in part by the cytokine vascular endothelial growth factor (VEGF). Eosinophils are the major effector cells in the nasal secretions of patients with AR during the responses to allergen challenges. To evaluate the involvement of VEGF in nasal allergic inflammation, we monitored the levels of VEGF, eosinophil cationic protein (ECP), and specific antibodies in the nasal lavage fluids (NLFs) of patients with AR in response to Dermatophagoides pteronyssinus (Dpt). METHODS: Sixty-three subjects with sensitization to Dpt were enrolled: 29 patients with AR (group I) who showed positive responses in a nasal provocation test (NPT) with Dpt; and 34 asymptomatic controls (group II) who showed sensitization to Dpt but negative NPT results. NLF samples were collected at baseline, 10, 30, and 60 min, and at 3, 6, and 24 h during the NPT. The ECP levels in the NLF samples were measured using the ImmunoCAP system. VEGF and Dpt-specific IgE, IgA, and IgG in the NLF samples were detected by ELISA. RESULTS: The eosinophil counts and ECP levels in the samples were significantly increased in group I, but not in group II, during the early and late responses. Although the baseline VEGF level was not significantly different between groups I and II, increased VEGF production was noted in group I after the NPT, especially during the early response. The level of Dpt-specific IgA was significantly increased in group I during the NPT. A relationship was found between the levels of VEGF and ECP or Dpt-specific IgA in the NLF samples collected at 10 min and at 3-6 h (P<0.05, respectively). CONCLUSION: Nasal VEGF secretion in response to allergen exposure may augment eosinophilic inflammation in the nasal mucosa of patients with AR.

Association of TNF-alpha genetic polymorphism with HLA DPB1*0301.

BACKGROUND: We speculated TNF-alpha can be one of candidate gene for aspirin-intolerant asthma (AIA) because TNF-alpha is pro-inflammatory cytokine and known to be increased level in asthmatic airways. In addition, genetic interaction between TNF-alpha and human antigen leucocyte (HLA) DPB1*0301, which is a strong genetic marker for AIA, was examined for its close location within chromosome 6. METHOD: To investigate genetic association of TNF-alpha with an AIA phenotype, three study groups (163 patients with AIA, 197 patients with aspirin-tolerant asthma (ATA), 257 normal control subjects) were enrolled. Single nucleotide polymorphisms (SNPs) were genotyped using a single-base extension method and HLA DPB1 genotyping was determined by high-throughput sequencing method. RESULTS: All five SNPs of TNF-alpha were tested; there were no significant differences in allele and genotype frequencies among the three groups. However, significant association between TNF-alpha-308G>A polymorphism and atopy status was noted (P<0.05). Gene to gene interaction between TNF-alpha-1031T>C (or -863C>A or -857C>A) and HLA DPB1*0301could synergistically increase the susceptibility to AIA with odds ratio (OR) to 7.738 (or OR=8.184 for -863C>A, OR=7.500 for -857C>T, P<0.001, respectively). CONCLUSION: TNF-alpha promoter polymorphism may significantly increase susceptibility to AIA by gene-to-gene interaction with HLA DPB1*0301.

Relationship between neurokinin 2 receptor gene polymorphisms and serum vascular endothelial growth factor levels in patients with toluene diisocyanate-induced asthma.

BACKGROUND: Among the various pathogenic mechanisms of toluene diisocyanate (TDI)-induced asthma, a contribution from neurogenic inflammation has been suggested. OBJECTIVE: To evaluate neurokinin 2 receptor (NK2R) gene polymorphisms in association with the clinical phenotype of TDI-induced asthma, 70 TDI-induced occupational asthma (TDI-OA)patients, 59 asymptomatic exposed controls (AEC), and 93 unexposed healthy controls (NC) were enrolled in the study. METHODS: Two single-nucleotide polymorphisms (SNPs) of NK2R, 7853G>A (Gly231Glu) and 11 424G>A (Arg375His), were genotyped using a single base extension method. The levels of PC20 methacholine, specific IgE and IgG to TDI-human serum albumin conjugate, and serum vascular endothelial growth factor (VEGF), matrix metalloproteinase-9, and TGF-beta1 were compared according to the NK2R genotypes of the subjects with TDI-OA and AEC. RESULTS: No significant differences in allele, genotype, or haplotype frequencies of these two SNPs were noted among the three groups (P>0.05, respectively). Moreover, subjects with the NK2R 7853GG genotype had higher serum VEGF levels than those with GA or AA among the TDI-exposed workers (P=0.040). CONCLUSION: The NK2R 7853GG genotype may contribute to increased serum VEGF levels, which result in airway inflammation after TDI exposure.

[Altered behaviour and expression of Fos in rats born in hypergravity and their re-adaptation to the normal gravity].

Changes in behaviour relevant to the vestibular system were studied in Long-Evans rats which were fertilized, born and housed in 2 acceleration of gravity for 4 months and thereafter exposed to 1 acceleration of gravity, and expression of Fos protein in the brain stem was examined. Data from the hypergravity rats were compared respectively with those from the rotation group and the labyrinthectomized group. Static and locomotion modes of the hypergravity rats were changed, tension of extensor was enhanced and the abilities in locomotion equalization and orientation in swimming and air-righting response were reduced. The adaptation process varied with different behaviours. The time for recovery of the ability of orientating in swimming was the longest, taking more than 1 month. The Fos protein expression provides a useful tool for mapping brain functional activities after sensory stimulation, showing a low basal level in normal and labyrinthectomized groups. The hypergravity rats, on the other hand, exhibited more Fos-positive cells in the superior colliculus, inferior colliculus, periaqueductal gray, raphe dorsal nucleus and solitary nucleus. In contrast, the inferior olivary nuclei, locus coeruleus and vestibular nuclei were not strongly labeled. These spatial patterns of Fos expression suggest that a decrease in gravity-inertial force may activate a neural pathway different from the vestibulo-olivar pathways activated by an increase in gravity-inertial force.

[Studies on the anticancer effect of HH07A, a derivative of hainanensine].

The effect of HH07A on the growth of tumor cells in vitro was investigated using the techniques of cell growth curve determination and soft-agar colony-forming assay. The result showed that HH07A inhibited the growth of L1210 cells and HL-60 cells at a concentration of 1.5 micrograms.ml-1 and 4.0 micrograms.ml-1, respectively. Among the cells tested, L1210 cells were shown to be the most sensitive, followed by KB cells and HL-60 cells (with IC50 of 2.29, 4.13 and 4.36 micrograms.ml-1, respectively). Normal mouse granulocyte-macrophage progenitor cells (GM-CFC) were less sensitive to the drug (with IC50 of 11.15 micrograms.ml-1) as compared with the tumor cells. As they were exposed to HH07A 3.5 micrograms.ml-1 for 5 days, HL-60 cells did not show NBT reductive ability. Intraperitoneal injection of HH07A exerted inhibitory effect on the ascitic tumors of L1210 and S180 in mice. Oral or ip administration of HH07A also showed some effect on S180 solid tumors in mice.

[Cytocidal effect of HH07A, a derivative of hainanensine, on L1210 cells in vitro].

The L1210 cells rapidly ceased to proliferate and its mitotic index decreased markedly after being exposed to HH07A 2 micrograms . ml-1 during exponential growth phase. However, 34.6% of the cells were still able to form colonies in soft agar if HH07A was removed after 24 h of incubation (the colony-forming efficiency for control cells was 63.7%) and the cell viability remained at about 94%. The DNA contents in L1210 cells were measured with a flow-cytometer. The results showed that the cell-cycle was blocked at the S phase and the number of cells in G2 + M phase decreased significantly. The time response course for L1210 cells indicated that the inhibitory effect of HH07A on L1210 clonogenic cells was slightly time dependent.

Efficacy of ivermectin for control of microfilaremia recurring after treatment with diethylcarbamazine. II. Immunologic changes following treatment.

We compared the effect of a single dose of ivermectin with that of a standard course of diethylcarbamazine (DEC) on several parameters of the host's antifilarial immune response in 60 patients with bancroftian filariasis enrolled in a double-blind drug trial. All participants had measurable serum levels of antifilarial antibodies and parasite antigens at the onset of the study. Drug-induced clearance of microfilaremia was associated with a temporary increase in HC 11 antigenemia and a decrease in serum levels of antibodies to soluble filarial antigens. Antigenemia progressively declined in patients who remained amicrofilaremic after treatment, but declined and then increased in persons with recurrent microfilaremia. Treatment triggered a sustained increase in serum levels of interleukin-1, tumor necrosis factor, and interleukin-6 in all patients studied. Although ivermectin and DEC are believed to exert their antiparasite activity via different mechanisms, the same pattern of serologic changes was observed in patients treated with either drug.

Characteristic autofluorescence for cancer diagnosis and its origin.

The autofluorescence spectra of human tissues excited with the 365-nm line of pulsed xenon ion laser have been measured. The autofluorescence spectra of cancer tissues usually show characteristic peaks near 630 nm and 690 nm, which do not appear in the spectra of the corresponding normal tissues. This characteristic fluorescence can be taken as a criterion for cancer diagnosis; a consistency of 89% with the traditional biopsy method has been obtained in preliminary clinical application to the diagnosis of buccal carcinoma. Spectroscopic investigations suggest that the porphyrin compounds localized and retained in cancer tissues might be responsible for this characteristic autofluorescence.