Increased Act1/IL-17R expression in Hirschsprung’s disease

Purpose

Hirschsprung’s disease-associated enterocolitis (HAEC) is the most common cause of morbidity and mortality in Hirschsprung’s disease (HSCR). Altered intestinal epithelial barrier function is implicated in the pathogenesis of HAEC. IL-17 is a proinflammatory cytokine that plays a crucial role in host defense against microbial organisms in the development of inflammatory diseases. Act1 is an essential adaptor molecule required for the IL-17-mediated inflammatory responses via interaction with IL-17 receptor (IL-17R). We designed this study to investigate the hypothesis that Act1/Il-17R expression is upregulated in HSCR.

Methods

We investigated Act1 and IL17R expression in ganglionic andaganglionic bowel of HD patients (n = 10) and controls (n = 10). qPCR, Western blotting and confocal immunofluorescence were performed.

Main results

qPCR and Western blot analysis revealed that Act1 and IL17R are strongly expressed in the aganglionic and ganglionic colon of patients with HSCR. Act1 and IL17R expression was significantly increased in HSCR specimens compared to controls (p < 0.05). Confocal microscopy revealed a markedly increased expression of Act1 and IL17R in the colonic epithelium of patients with HSCR compared to controls.

Conclusion

To our knowledge, we report, for the first time, the expression of Act1 in the human colon. The increased expression of Act1 and Il-17 in the aganglionic and ganglionic bowel in HSCR may result in IL-17-mediated increased inflammatory response leading to the development of HAEC

     

Structure-Based Design of Type II Inhibitors Applied to Maternal Embryonic Leucine Zipper Kinase

相似文献   

The requirement for platelets in allergen-induced late asthmatic airway obstruction. Eosinophil infiltration and heightened airway responsiveness in allergic rabbits

In these studies, we have used an allergic rabbit model to investigate the role of platelets in the late asthmatic response (LAR) by depleting platelets with a guinea pig antirabbit platelet antiserum (APAS). Allergen exposure of immunized rabbits pretreated with normal guinea pig serum (NGPS) to serve as a control resulted in an early- and late-phase obstructive airway response that persisted for 6 h. When the immunized animals were pretreated with APAS, the magnitude of the LAR in terms of dynamic compliance was reduced by 86.2% (p less than 0.03), but there was no difference in the early response curve. Allergen challenge of animals treated with NGPS resulted in an increased bronchial responsiveness to inhaled histamine: PD50 Cdyn geometric mean +/- SEM before, 2.36 mg/ml (3.43-1.64); after, 0.60 mg/ml (0.67-0.54) (p less than 0.01). PD50 RL before, 1.78 mg/ml (2.4-1.32); after, 0.58 mg/ml (0.81-0.47) (p less than 0.05). In contrast, when animals were treated with APAS, there was a significant inhibition of allergen-induced airway hyperresponsiveness to inhaled histamine: PD50 Cdyn geometric mean +/- SEM before, 1.42 mg/ml (2.06-0.98); after, 1.10 mg/ml (1.41-0.86) (p less than 0.4). PD50 RL before, 1.62 mg/ml (2.22-1.39); after, 1.05 mg/ml (1.35-0.82) (p greater than 0.4). Analysis of bronchoalveolar lavage fluid revealed an increase in the number of neutrophils and eosinophils after allergen exposure in control animals (p less than 0.01). However, in animals rendered thrombocytopenic, the number of eosinophils, but not neutrophils, was significantly reduced (p less than 0.03).(ABSTRACT TRUNCATED AT 250 WORDS)     

An inhibitory monoclonal antibody to factor X that blocks prothrombin activation but not prothrombinase enzyme assembly

A monoclonal antibody (designated alpha BFX-2b) prepared against bovine factor X inhibited factor X activity in human, bovine, porcine, rabbit, and canine plasma. In assays using purified prothrombinase components, factor Xa, factor Va, phospholipid vesicles, and calcium ion with the fluorescent active site thrombin inhibitor dansylarginyl-N-(3-ethyl-1,5- pentanediyl)amide, the antibody inhibited the conversion of prothrombin to thrombin. Antibody alpha BFX-2b also blocked prothrombinase cleavage of the macromolecular substrates prethrombin 1 and prethrombin 2 but did not inhibit factor Xa hydrolysis of the synthetic substrate benzoyl- Ile-Glu-Gly-Arg-p-nitroanilide. The antibody also prevented the inactivation of factor Xa by antithrombin III but did not prevent the inactivation by soybean trypsin inhibitor. Antibody alpha BFX-2b bound factor Xa with a stoichiometry of 1:1 and an apparent dissociation constant of 9.0 x 10(-11) mol/L as estimated from its inhibition of prothrombinase activity. Antibody alpha BFX-2b did not prevent binding of factor Xa to factor Va-phospholipid as measured by using fluorescence polarization or high-pressure liquid gel chromatography with the fluorescent Factor Xa analogue dansyl-glutamyl-glycyl-arginyl- Xa. Immunoblotting of factor X following electrophoresis on sodium dodecyl sulphate-polyacrylamide gels and transfer to nitrocellulose indicated that the antigenic determinant recognized by antibody alpha BFX-2b was found on the heavy chain of factors X and Xa. From these observations it can be concluded that antibody alpha BFX-2b recognizes a highly conserved epitope on the factor X heavy chain that is remote from the topographic sites required for prothrombinase complex assembly and substrate hydrolysis but may be located at or near a portion of the macromolecular substrate binding site.     

Platelet-collagen interaction: inhibition by ristocetin and enhancement by von Willebrand factor-platelet binding

The contribution of von Willebrand factor (vWF)-platelet binding to platelet-collagen interaction was examined in vitro. The binding of vWF to platelets was mediated and regulated by ristocetin. Subthreshold concentrations of ristocetin (less than or equal to 1 mg/mL), insufficient to cause ristocetin-induced platelet aggregation (RIPA), were added to platelet-rich plasma (PRP) prior to the addition of collagen. The collagen-induced platelet aggregation (CIPA) was modified by ristocetin and the degree of alteration was dependent on the ristocetin concentration. Response as a function of ristocetin concentration was designated the Collagen-Platelet Aggregation Response (CoI-PAR). In normal PRP the CoI-PAR was a progressive inhibition followed by decreasing inhibition and then an enhanced response. The enhanced response occurred over a narrow range of ristocetin concentrations (0.8 to 1.0 mg/mL). In the absence of vWF (severe von Willebrand's disease, Type I, vWF less than 1%) the CoI-PAR was a progressive, eventually complete inhibition with no enhanced response (with ristocetin concentrations up to 3.0 mg/mL). With addition of vWF to this PRP an enhanced response was observed at a ristocetin concentration inversely proportional to the vWF level. PRP from a patient with severe Hemophilia A showed a response within the normal range. Subthreshold ristocetin did not cause plasma protein precipitation or platelet release of 3H-serotonin, nor induce micro platelet aggregate formation. Digestion of platelet membrane glycoproteins (GP(s] with chymotrypsin demonstrated that upon removal of GPI, RIPA was absent, CIPA retained and the CoI-PAR was progressive inhibition, with no enhancement. With removal of GPs I, II, and III, RIPA, CIPA, and the CoI-PAR were absent. A dose-response 125I-vWF- platelet binding occurred with increasing ristocetin concentrations which was unchanged by the addition of collagen. These results demonstrated that ristocetin-platelet association inhibited CIPA, and vWF-platelet binding enhanced platelet-collagen adhesion and platelet aggregation. The in vitro-enhanced CIPA represents a vWF-dependent aggregation of sufficient magnitude to overcome the inhibitory effect of ristocetin. These studies demonstrate an influential interaction of ristocetin, vWF, and collagen with the platelet membrane and imply an important hemostatic contribution of vWF-platelet binding in platelet- collagen interaction.