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Abstract The aim of this study was to analyze the expression of mast cell markers toluidine blue, c-kit, and tryptase and presence of mononuclear inflammatory cells in oral lichen planus (OLP) and oral lichenoid lesions related to dental amalgam. Nineteen specimens of OLP, OLLC, and healthy oral mucosa were selected. Mononuclear inflammatory cells were analyzed. Histochemical and immunohistochemical analyses were performed using toluidine blue, anti-c-kit and anti-tryptase reagents, and the results were quantified in areas A and B of connective tissue. Mast cells of all OLP and OLLC samples were positive for toluidine blue, c-kit, and tryptase. The density of toluidine blue+, c-kit+ and tryptase+ mast cells was higher in tissue with OLP and OLLC compared with healthy controls (p < 0.05). No difference was noted in mast cells density between OLP and OLLC (p > 0.05). The density of tryptase+ mast cells was higher in the subepithelial region (area A) than the region below it (Area B) in OLLC (p = 0.047). The mononuclear inflammatory cell density was higher in OLLC compared to OLP, but without statistical significance (p > 0.05). A positive statistical correlation was found between mononuclear immune cells and density of c-kit+ and tryptase+ mast cells in OLP (r = 0.943 and r = 0.886, respectively). Our data demonstrate that the etiopathogenesis process of OLP and OLLC modulates the expansion and degranulation of mast cells; mast cells density, however, was similar between OLP and OLLC. The distribution of mast cells appears to vary along the lamina propria.
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Abstract: Inflammatory periapical lesions are characterized by infiltration of different immune cell types, the functions of which depend on an effective vascular network. This study aimed to evaluate the mast cells density (MCD) in inflamatory odontogenic cysts capsules concerning microvascular density (MVD), microvascular area (MVA), and microvascular perimeter (MVP), and correlate such findings with the type of lesion, intensity of the inflammatory infiltrate, and thickness of the epithelial lining. Twenty inflamatory dentigerous cysts (IDCs), twenty radicular cysts (RCs), and twenty residual radicular cysts (RRCs) were submitted to immunohistochemical analysis using anti-tryptase and anti-CD34 antibodies. RCs exhibited the highest MCD, MVD, MVA, and MVP indexes (p = < 0.001, p = 0.008, p = 0.003 and p = < 0.001, respectively), and lesions with inflammatory infiltrate grade III showed the highest MVD (p = 0.044). Considering epithelial thickness, a higher MVP index was identified in lesions with hyperplastic epithelium (p = 0.018). In IDCs, RCs, and RRCs, a strong positive correlation was observed between MVA and MVP (r = 0.950 and p = < 0.001; r = 0.914 and p = < 0.001; r = 0.713 and p = < 0.001, respectively). In IDCs, a moderate correlation was observed between MCD and both MVA and MVP (r = 0.660 and p = 0.002; r = 0.634 and p = 0.003, respectively). These results suggest that tryptase-positive mast cells might play an important role in the angiogenic activity of IDCs, while RCs had the highest indexes. Our findings also confirmed that the intensity of the inflammatory infiltrate and epithelial thickness influence angiogenesis.
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Humans , Odontogenic Cysts , Radicular Cyst , Epithelium , Tryptases , Mast CellsABSTRACT
Anaphylactic shock (AS) is a systemic and life-threatening type I hypersensitivity reaction and is often encountered at an autopsy. However, postmortem diagnosis of AS can be difficult due to non-specific autopsy findings. Clinically, the analysis of serum mast cell tryptase (MCT) is well known as a useful ancillary test for the diagnosis of AS. However, in order to apply this test to forensic autopsy, it is necessary to confirm its usefulness due to postmortem changes. We carried out serum MCT analysis in 299 autopsy cases including nine AS cases at National Forensic Service from January 2013 to May 2015 and analyzed the difference according to the cause of death and degree of postmortem change. As a result, the MCT level in AS was significantly increased compared to others, and the appropriate cutoff value for postmortem diagnosis of AS was 63.0 µg/L (sensitivity 88.9%, specificity 98.6%). Conclusively, serum MCT analysis is a useful test for postmortem diagnosis of AS and seems to be more appropriate for screening rather than confirmation.
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Objective To investigate the role of mast cells in Staphylococcus aureus enterotoxin B (SEB)-induced atopic dermatitis (AD)-like skin inflammation in BALB/c mice.Methods A total of 24 BALB/c mice were randomly and equally divided into 4 groups to be topically treated with ovalbumin (OVA group),SEB (SEB group),OVA + SEB (OVA + SEB group) and sodium chloride physiological solution (control group) respectively,so as to establish mouse models of epicutaneously induced AD-like skin inflammation.The AD-like skin lesions were evaluated by clinical observation and eczema area and severity index (EASI).Biopsy specimens were obtained from lesional skin of mice and then subjected to toluidine blue staining and immunohistochemical staining to count the mast cells,observe the morphology and distribution of mast cells,and calculate the percentage of degranulated mast cells.Results After 7-week treatment,the OVA group,SEB group and OVA + SEB group all showed severer local skin inflammation,higher EASI scores and denser infiltration of inflammatory cells compared with the control group.Moreover,the OVA + SEB group showed significantly severer local skin inflammation,skin lesions and degree of infiltration of inflammatory cells compared with the OVA group and SEB group (all P ＜ 0.05).The number of mast cells in the dermis of AD-like skin lesions per high-power field (× 400) was significantly higher in the OVA group (median [quartile range]:10.625 [3.675]),SEB group (11.000 [4.163]) and OVA + SEB group (13.875 [8.813]) than that in the control group (5.925 [2.088],all P ＜ 0.05).The SEB group (71.083％ ± 14.519％) and OVA + SEB group (58.767％ ±.16.978％) both showed significantly higher percentage of degranulated mast cells compared with the OVA group (24.050％ ± 11.161％,both P ＜ 0.05) and control group (23.617％ ± 8.132％,both P ＜ 0.05).Bivariate correlation analysis showed that the number of mast cells in the skin lesions was positively linearly correlated with the EASI scores (P ＜ 0.05).Conclusions Epicutaneous application of SEB can induce AD-like skin lesions in mice,and can exacerbate the severity of OVA-induced AD-like skin lesions.Mast cell proliferation,activation/degranulation and tryptase release may participate in the inflammation.
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Objective To evaluate the role of tryptase in intestinal ischemia-reperfusion (Ⅰ/R) injury in rats and the relationship with inflammatory responses,lipid peroxidation and cell apoptosis.Methods Thirty clean-grade healthy male Sprague-Dawley rats,aged 2-3 months,weighing 150-270 g,were divided into 3 groups (n =10 each) using a random number table method:sham operation group (Sham group),intestinal Ⅰ/R group (Ⅱ/R group) and tryptase inhibitor group (PRTM group).Intestinal Ⅰ/R was produced by occlusion of the superior mesenteric artery for 75 min followed by 4-h reperfusion in Ⅱ/R and PRTM groups.The superior mesenteric artery was only exposed but not occluded in Sham group.Protamine 2.5 mg/kg was injected via the caudal vein at 5 min of before reperfusion in PRTM group.The rats were sacrificed at the end of reperfusion,and the small intestinal tissues 1 cm in length 5 cm away from the terminal ileum were removed for examination of the pathological changes (with a light microscope) and for determination of intestinal mucosal mast cell (IMMC) count and expression of tryptase in mast cells (by immunohistochemical SP staining),malondialdehyde (MDA) content (using thiobarbituric acid assay),superoxide dismutase (SOD) activity (by xanthine oxidase method),expression of caspase-3 (by Western blot) and contents of tumor necrosis factor alpha (TNF-ot) and interleukin-6 (IL-6) (by enzyme-linked immunosorbent assay).The degree of intestinal tissue damage was graded using Chiu's scoring system.Results Compared with Sham group,the pathological changes were significantly accentuated,Chiu's score was increased,the expression of tryptase was up-regulated,the IMMC count and contents of TNF-α,IL-6 and MDA were increased,the activity of SOD was decreased,and the expression of caspase-3 was up-regulated in Ⅱ/R and PRTM groups (P＜0.05).Compared with Ⅱ/R group,the pathological changes were significantly attenuated,Chiu's score was decreased,the expression of tryptase was down-regulated,the IMMC count and contents of TNF-α,IL-6 and MDA were decreased,the activity of SOD was increased,and the expression of caspase-3 was down-regulated in PRTM group (P＜0.05).Conclusion Tryptase is involved in the process of intestinal Ⅰ/R injury though promoting inflammatory responses,lipid peroxidation and cell apoptosis in rats.
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Objective To investigate the role of Staphylococcus aureus enterotoxin B (SEB) in non-IgE mediated activation of mast cells (MCs) by in vitro co-culture of laboratory of allergic disease 2 (LAD2) cells and SEB.Methods The LAD2 cells were incubated with SEB at different concentrations of 0.01,0.1,1,10 and 100 μg/ml,A23187 positive control and negative control separately for 30 minutes.Then,effects of SEB on the morphology of MCs were observed by using a light microscope,and culture supernatants of the above incubation systems were collected.The concentration of tryptase released from MCs was analyzed by enzymatic activity assay,and the level of histamine was detected by enzyme-linked immunosorbent assay (ELISA).Results After 30-minute co-culture of LAD2 cells and SEB,MCs showed larger size,obscure boundaries,increased number of protuberances on the cell surface and decreased refractivity,with a radial burr fin-like appearance.After 30-minute co-culture of LAD2 cells and SEB at different concentrations of 0.01,0.1,1,10 and 100 μg/ml,the concentrations of tryptase in the culture supematants were 4.116 ± 0.651,5.344 ± 0.874,3.806 ± 0.459,1.309 ± 0.247,0.310 ± 0.199 ng/ml respectively.Additionally,the tryptase levels were significantly higher in the 0.01-,0.1-,1-μg/ml SEB groups than in the negative control group(1.538 ± 0.490,all P ＜ 0.05),and gradually decreased along with the increase of SEB concentrations.The histamine levels in the 0.01-,0.1-,1-,10-and 100-μg/ml SEB groups were 242.409 ± 63.915,522.491 ± 73.466,550.926 ± 84.466,334.397 ± 33.640,226.527 ± 5.678 ng/ml respectively.In the 0.01-,0.1-,1-μg/ml SEB groups,the levels of histamine released from MCs were gradually increased along with the increase of SEB concentrations,and were significantly higher than those in the negative control group (146.436 ± 3.100,all P ＜ 0.05).However,with the continued increase of SEB concentrations,the histamine levels gradually decreased.Conclusion SEB can directly activate MCs by a non-IgE mediated mechanism,followed by morphologic changes of MCs and release of tryptase and histamine.
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OBJECTIVES@#To explore the value of mast cell tryptase and brain natriuretic peptide（BNP） in the differential diagnostic of sudden death due to hypersensitivity and coronary atherosclerotic heart disease.@*METHODS@#Totally 30 myocardial samples were collected from the autopsy cases in the Department of Forensic Pathology, Shanxi Medical University during 2010-2015. All samples were divided into three groups： death of craniocerebral injury group, sudden death of hypersensitivity group and sudden death of coronary atherosclerotic heart disease group, 10 cases in each group. Mast cell tryptase and BNP in myocardium were detected by immunofluorescence staining and Western Blotting.@*RESULTS@#Immunofluorescence staining showed that the positive staining mast cell tryptase appeared in myocardium of sudden death of hypersensitivity group and coronary atherosclerotic heart disease group. Among the three groups, the expression of mast cell tryptase showed significantly differences through pairwise comparison （P<0.05）; The expression level of BNP in sudden death of coronary atherosclerotic heart disease group were significantly higher than the sudden death of hypersensitivity group and death of craniocerebral injury group （P<0.05）. The difference of the expression level of BNP between the sudden death of hypersensitivity group and the death of craniocerebral injury group had no statistical significance （P>0.05）.@*CONCLUSIONS@#The combined detection of the mast cell tryptase and BNP in myocardium is expected to provide help for the forensic differential diagnosis of sudden death due to hypersensitivity and coronary atherosclerotic heart disease.
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Humans , Male , Anaphylaxis , Autopsy , Blotting, Western , Case-Control Studies , Coronary Artery Disease/complications , Death, Sudden, Cardiac/etiology , Diagnosis, Differential , Fluorescent Antibody Technique , Forensic Pathology , Myocardial Infarction , Myocardium/metabolism , Natriuretic Peptide, Brain/metabolism , Tryptases/metabolismABSTRACT
Objective To observe the expression of clasmatoblast-secreted tryptase in patients with diabetic cardiomyopathy (DCM) and its relationship with fasting blood glucose. Methods Patients with DCM (DCM group, n=32) or ischemic cardiomyopathy (ICM group, n=26) and age-matched healthy volunteers (healthy control group, n=20) were recruited from Changzheng Hospital of Second Military Medical University between August 2012 and March 2013. The clinical characteristics of the participants were collected and the biochemical indices including fasting bloodglucose were detected. ELISA detection reagent kit was used for detecting serum tryptase level in each group. Pearson analysis was used to analyze the correlation between plasma tryptase level and fasting glucose. Results The general clinical characteristics and the fasting blood glucose were not significantly different between the three groups. The plasma tryptase levels in DCM group and ICM group were significantly higher than that in the healthy control group ([7. 19 ± 0. 62]µg/L, [6. 81 ± 0. 94]µg/L vs [2. 37 ± 0. 56] µg/ L, P<0. 01). DCM group had higher serum tryptase level than ICM group, but with no significant difference. Pearson analysis showed that the plasma tryptase level was positively correlated with fasting blood glucose level (r= 0. 637, P< 0. 01). Conclusion The plasma tryptase level is increased in patients with DCM, and t is positively correlated with the fasting blood glucose in all the participants of three groups.
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Objective To explore the value of mast cell tryptase and brain natriuretic peptide(BNP)in the differential diagnostic of sudden death due to hypersensitivity and coronary atherosclerotic heart dis-ease.Methods Totally 30 myocardial samples were collected from the autopsy cases in the Department of Forensic Pathology, Shanxi Medical University during 2010—2015. All samples were divided into three groups:death of craniocerebral injury group, sudden death of hypersensitivity group and sudden death of coronary atherosclerotic heart disease group, 10 cases in each group. Mast cell tryptase and BNP in myocardium were detected by immunofluorescence staining and Western Blotting.Results Immunofluo-rescence staining showed that the positive staining mast cell tryptase appeared in myocardium of sudden death of hypersensitivity group and coronary atherosclerotic heart disease group. Among the three groups, the expression of mast cell tryptase showed significantly differences through pairwise comparison(P0.05).Conclusion The combined detection of the mast cell tryptase and BNP in myocardium is expected to provide help for the forensic differential diagnosis of sudden death due to hypersensitivity and coronary atherosclerotic heart disease.
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BACKGROUND/AIMS: Previous studies have revealed that mast cells (MCs) may activate the protease-activated receptors and release of neuropeptides involved in the pathogenesis of irritable bowel syndrome (IBS). The levels of protease-activated receptor 2 (PAR-2) and tryptase can contribute to understanding the pathogenesis of IBS. METHODS: Colonoscopic biopsies were performed of 38 subjects (20 with IBS-diarrhea [IBS-D], eight with IBS-constipation [IBS-C], and 10 healthy volunteers). The mRNA and protein levels of tryptase and PAR-2 were assessed by real-time PCR and Western blot. The levels of vasoactive intestinal peptide (VIP), substance P (SP), and calcitonin gene-related peptide (CGRP) were measured by immunohistochemistry, and MCs were counted by toluidine blue staining. RESULTS: Significant increases in the mRNA expression of tryptase (p<0.05, IBS-D, IBS-C vs control) and PAR-2 (p<0.05, IBS-D, IBS-C vs control) and in the tryptase protein level (p<0.05, IBS-D, IBS-C vs control) were detected in IBS. Elevations of MCs, CGRP, VIP and SP (p<0.05, IBS-D vs control) were observed for IBS-D only. CONCLUSIONS: Tryptase levels may upregulate the function of PAR-2, resulting in the release of neuropeptide and they were correlated with clinical symptoms associated with IBS.
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Biopsy , Blotting, Western , Calcitonin Gene-Related Peptide , Immunohistochemistry , Inflammation , Irritable Bowel Syndrome , Mast Cells , Neuropeptides , Real-Time Polymerase Chain Reaction , Receptor, PAR-2 , Receptors, Proteinase-Activated , RNA, Messenger , Substance P , Tolonium Chloride , Tryptases , Vasoactive Intestinal PeptideABSTRACT
Objective To explore the changes of serum IgE and tryptase caused by anaphylactic shock rats and discuss the relation to PMI and preservative environm ent of corpse and specim en. Methods Rats were used for establishing anaphylactic shock m odels and random ly divided into room tem perature group, refrigeration group, frozen group, manual hem olysis group, specim en preservation group. And the control group was also established. The blood sam ples were collected after rats were sacrificed. The de-gree of hem olysis was graded according to the color of the upper layer of the serum . The mass concen-tration of IgE and tryptase in each group was detected by ELISA. Results The levels of serum IgE and tryptase in anaphylactic shock dead rats were higher than that of the control group. Room tem perature and frozen m ade obviously differences on the levels of serum IgE and tryptase with various PMI. The levels of serum IgE and tryptase in refrigeration group show ed relatively stable. The levels of serum tryptase and IgE were elevated with differently increasing hem olysis. The levels of serum IgE and tryptase show ed no obvious changes during the specim en kept under different tem perature conditions for 25 days. Conclusion Serum IgE and tryptase obviously increased in anaphylactic shock rats. H ow ever, the levels were influenced by PMI and environm ental tem perature, especially under the conditions of room tem perature and frozen.
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BACKGROUND: Mastocytosis is a clonal disorder characterized by the accumulation of abnormal mast cells in the skin and/or in extracutaneous organs. OBJECTIVES: To present all cases of mastocytosis seen in the Porto Hospital Center and evaluate the performance of World Health Organization diagnostic criteria for systemic disease. METHODS: The cases of twenty-four adult patients with mastocytosis were reviewed. Their clinical and laboratorial characteristics were assessed, and the properties of the criteria used to diagnose systemic mastocytosis were evaluated. RESULTS: The age of disease onset ranged from 2 to 75 years. Twenty-three patients had cutaneous involvement and 75% were referred by dermatologists. Urticaria pigmentosa was the most common manifestation of the disease. One patient with severe systemic mast cell mediator-related symptoms showed the activating V560G KIT mutation. The bone marrow was examined in 79% of patients, and mast cell immunophenotyping was performed in 67% of the participants. Systemic disease was detected in 84% of cases, and 81% of the sample had elevated serum tryptase levels. All the diagnostic criteria for systemic mastocytosis had high specificity and positive predictive value. Bone marrow biopsy had the lowest sensitivity, negative predictive value and efficiency, while the highest such values were observed for mast cell immunophenotyping. Patients were treated with regimens including antihistamines, sodium cromoglycate, alpha-interferon, hydroxyurea and phototherapy. CONCLUSIONS: Cutaneous involvement is often seen in adult mastocytosis patients, with most individuals presenting with indolent systemic disease. Although serum tryptase levels are a good indicator of mast cell burden, bone marrow biopsy should also be performed in patients with normal serum tryptase, with flow cytometry being the most adequate method to diagnose systemic disease. .
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Adult , Aged , Female , Humans , Male , Middle Aged , Young Adult , Mastocytosis, Systemic/diagnosis , World Health Organization , Age Factors , Age of Onset , Biopsy, Needle , Bone Marrow/pathology , Disease Progression , Flow Cytometry , Immunophenotyping , Mutation , Mast Cells/pathology , Portugal , Reproducibility of Results , Sensitivity and Specificity , Time FactorsABSTRACT
Midazolam is a type of anesthetic agent frequently used for conscious sedation during a variety of medical procedures. Anaphylactic reactions to midazolam are rarely reported. However, we observed a case of midazolam hypersensitivity in which emergency measures were required to ensure patient recovery after administration of midazolam as a sedative. The occurrence of the anaphylactic reaction to midazolam was confirmed by elevated serum tryptase levels. The current case report presents a discussion of our findings.
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Humans , Anaphylaxis , Conscious Sedation , Emergencies , Hypersensitivity , Midazolam , TryptasesABSTRACT
Objective To explore theroles of histamine and tryptase in chronic pancreatitis pain development and maintenance. Methods Rat models of chronic pancreatitis were prepared, and the histamine and tryptase contents were examined in the pancreatic specimens of both model group and the control group. The pancreas incubation supernatants of both groups were injected into the plantar surface of the rat hindpaw, and then the pain thresholds were observed at 0. 5, 1, 3, 5, and 7 h after injection. Rats injected with the supernatant of chronic pancreatitis model were also given H1 receptor blocker or protease inhibitor toobserve their treatment for hyperalgesia. Results The histamine and tryptase contents in the pancreas specimenswere increased in chronic pancreatitis model rats. Injection of supernatant from the chronic pancreatitis specimens had a profound hyperalgesia effect within 5 h after injection, and the effect could be partially reduced by H1 receptor blockers and protease inhibitors. Conclusion Our findings suggest that histamine and tryptase are important mediators in the development and maintenance of the chronic pancreatitis pain.
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BACKGROUND: Few studies have evaluated the ultrastructure of the superficial skin nerves in urticaria. OBJECTIVE: The objective of this study was to describe findings in superficial skin nerves in cases of drug-induced acute urticaria. METHODS: Seven patients with drug-induced acute urticaria were included in the study. Skin biopsies were obtained from the urticarial lesion and from the apparently normal skin. The 14 fragments collected were processed for immunogold electron microscopy using single stains for antitryptase and anti-FXIIIa antibodies, as well as double immunogold labeling for both. RESULTS: Some sections showed mast cells in the process of degranulation. Following double immunogold staining, 10 nm (FXIIIa) and 15 nm (Tryptase) gold particles were found together throughout the granules in mast cells, indicating that tryptase and FXIIIa are located inside each one of the granules of these cells. Interestingly, we found strong evidence of the presence of tryptase and factor XIIIa in the superficial skin nerves of these patients, both in cases of urticarial lesions (wheals) and in the apparently normal skin. CONCLUSIONS: Tryptase and FXIIIa are present in the superficial nerves of the skin in drug-induced acute urticaria. This is the first report of tryptase and FXIIIa expression in the superficial skin nerves of patients with urticaria. Tryptase may be participating in neural activation in these patients, while FXIIIa may be present in the nerves to guarantee the functional integrity of structures.
FUNDAMENTOS: Poucos autores têm estudado a ultraestrutura dos nervos superficiais na urticária. OBJETIVO: Descrever os achados nos nervos cutâneos superficiais em casos de urticária aguda induzida por medicamentos. MÉTODOS: Sete pacientes com urticária aguda induzida por medicamentos foram incluídos no estudo. Foram obtidas biopsias da pele da lesão urticariforme e da pele aparentemente normal. Os 14 fragmentos coletados foram processados usando imunomarcação com ouro para anticorpos anti-triptase e anti-FXIIIa separadamente, além da dupla imunomarcação com ambos anticorpos. A seguir as amostras foram submetidas à análise por microscopia imunoeletrônica. RESULTADOS: Alguns cortes demonstraram mastócitos em processo de degranulação. Após a imunomarcação dupla, partículas de ouro de 10 nm (FXIIIa) e partículas de ouro de 15 nm (Triptase) apresentavam-se juntas em grânulos de mastócitos indicando que a triptase e o FXIIIa se localizam dentro de cada um dos grânulos dessas células. Curiosamente, foi encontrada uma forte evidência da presença da triptase e do fator XIIIa nos nervos superficiais dos pacientes avaliados, tanto em lesões urticadas, como na pele aparentemente normal. CONCLUSÕES: A triptase e o FXIIIa estão presentes nos nervos superficiais da pele na urticária aguda medicamentosa. Este é o primeiro relato da expressão de triptase e de FXIIIa nos nervos superficiais na urticária. A triptase poderia estar participando da ativação neural nos pacientes estudados. O FXIIIa poderia estar presente nos nervos, com a finalidade de manter a integridade funcional dessas estruturas.
Subject(s)
Adult , Female , Humans , Middle Aged , Drug Hypersensitivity/pathology , Skin/innervation , Urticaria/pathology , Drug Hypersensitivity/immunology , Factor XIIIa/metabolism , Immunohistochemistry , Microscopy, Immunoelectron , Peripheral Nerves/ultrastructure , Skin/enzymology , Tryptases/metabolism , Urticaria/chemically induced , Urticaria/immunologyABSTRACT
Protease activated receptor-2 (PAR-2) is a G-protein-coupled receptor. Recent studies indicate that PAR-2 is mainly expressed in leukocytes and activated by pancreatin and (or) tryptase, which subsequently induces inflammation through degranulation of leukocytes. Activation of PAR-2 in leukocytes is possibly involved in the pathogenesis of rheumatoid arthritis.
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Objective To observe the variation of tryptase expression in the colonic mucosa of patients with diarrhea-predominant irritable bowel syndrome(IBS-D) and the effects of their colonic mucosa cultured supernatants on mice visceral sensitivity. Methods The IBS-D patients study: of twenty-four patients with IBS-D and 12 healthy volunteers, four mucosal biopsies of each individual were collected at ascending colon under colonoscopy. The quantity of mucosal mast cells were detected by toluidine blue stains, and the tryptase concentration of colonic mucosa cultured supernatants were determined by enzyme-linked immunosorbent assay (ELISA). Animal experiments: fifty-six male six to eight weeks old C57BL/6 mice were divided into 7 groups and then were administrated with healthy volunteer's ascending colonic mucosa-cultured supernatants 100 μl (group A), IBS-D patient' s ascending colonic mucosa-cultured supernatants 100 μl (group B), IBS-D ascending colonic mucosacultured supernatants 100 μl with Fut-175(50 μg/ml, group C), tryptase with different concentration:0.6 ng (group D), 2.2 ng (group E), tryptase 2.2 ng with Fut-175 (group F), and 0.9% sodium chloride as control (group G) respectively. After six hours, the abdominal myoelectric activities (AMAs) were recorded during colorectal balloon distension (CRD), and the expressions of substance P (SP), Calcitonin gene-related peptide (CGRP) in spinal dorsal horn of lumbosaeral region were detected by immunohistochemistry. Results The number of mast cells in the ascending colon of patients with IBS-D [6.33 ± 1.64) / HP] was higher than that in controls [(3.08 ± 0.77 ) / HP], the difference was statistical significant (P = 0.000). The tryptase concentration of ascending colonic mucosa cultured supernatants in IBS-D patients [(0. 072 ± 0. 023) ng/mg] was higher than that in controls [(0.026 ± 0.014) ng/mg], the difference was statistical significant ( P = 0. 000). In animal experiments, when the inside pressure of balloon was 30, 45, 60 mmHg, respectively, the AMAs value in group B [(27.50±5.23), (41.69±5.80), (54. 03±6.13) μV, respectively] and group E [(27. 05±1.66), (40. 66±3.39), (54. 38± 4.68) μV, respectively] were significantly higher than those in group A, group C, group D, group F and group G (P＜0. 05, respectively). The average integrated option densities (IOD) of SP and CGRP in spinal dorsal horn in groupB (121.72 ± 4.67,123.54±4.32)and E (124.69±5.06,126.32±5.09) were higher than in group A, group C, group D,group F and group G (P＜0.05, respectively). Conclusion The quantity of mast cell and the content of tryptase in the ascending colonic mucosa of IBS-D patients increased significantly. And their colonic mucosa cultured supernatants caused the mice visceral hypersensitivity, which indicated that tryptase may involved in the variation of visceral sensitivity in IBS-D patients.
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OBJECTIVE To compare sensitivity in typeⅠ hypersensitivity between BN and Wistar rats, and to establish a sensitive and reliable determination system for typeⅠ hypersensitivity. METHODS BN and Wistar rats were sc given ovalbumin (OVA) 10, 20 and 40 μg·kg~(-1) every other day for 5 times and normal control group with sc normal saline. The total immunoglobulin E (IgE) levels in serum were determined with ELISA and the specific IgE levels in serum were determined by passive cutaneous anaphylaxis on the 21st day from the 1st injection. The blood pressure, serum histamine and tryptase levels were determined after challenge on the 22nd day. RESULTSTotal IgE, specific IgE, histamine and tryptase levels in serum significantly increased and blood pressure decreased in OVA 10, 20 and 40 μg·kg~(-1) BN rat groups compared with normal control group, while in Wistar rats these symptoms only appeared in OVA 40 μg·kg~(-1) group. CONCLUSION BN rats are more sensitive than Wistar rats in typeⅠ hypersensitivity. The blood pressure, serum total IgE, specific IgE, histamine and tryptase levels can be used as the important indicators in typeⅠ hypersensitivity.
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Mast cells play a central role in the initiation and development of allergic diseases through release of various mediators. Tryptase has been known to be a key mediator in mast cell-mediated inflammatory reactions. In the present study, we investigated whether the transcription of tryptase gene in human mast cells was induced by microphthalmia (mi)-associated transcription factor (MITF). We observed that the human CD34+ progenitor-derived cultured mast cells and human mast cell line HMC-1 expressed strongly the transcripts of tryptase-beta1 and MITF-A, which is a MITF alterative splicing isoform. The transcriptional activity of tryptase gene was specifically higher in HMC-1 cells compared to the tryptase-negative cells. Using mutant constructs of tryptase promoter, we observed that two E-box (CANNTG) motifs including between -817 to -715 and -421 to -202 are able to involve in the transactivation of tryptase gene by MITF-A. In addition, the binding of these motifs-containing oligonucleotides to MITF proteins was detectable by EMGA using the nuclear extracts of HMC-1 cells and anti-MITF mAb. The overexpression of MITF-A elevated tryptase production by HMC-1 cells, while the introduction of specific siRNA against MITF attenuated the expression and enzymatic activity of tryptase. These data suggest that MITF might play a role in regulating the transcription of tryptase gene in human mast cells.
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Protease activated receptor-2(PAR-2)is a G-protein-coupled receptor.Recent studies indicate that PAR-2 is mainly expressed in leukocytes and activated by pancreatin and(or)tryptase,which subsequently induces inflammation through degranulation of leukocytes.Activation of PAR-2 in leukocytes is possibly involved in the pathogenesis of rheumatoid arthritis.