Mast Cell-deficient Mice And Bacterial Rhinosinusitis

Mast cell deficient (Ws/Ws) rats and wild-type controls(+/+) rats will be chosen to studythe role of mast cells in the pathogenesis of nasal allergy. Ws/Ws rats will be sensitized toovalbumin followed by development of bacterial rhinosinusitis model through intranasalinoculation of selected bacteria. Inflammatory cell infiltration, mast cell degranulation as wellas bacterial-epithelial cell interactions will be quantitatively measured by a 3-D methods. At thesame time, mitochondria and autophagosomes in the nasal epithelia will be studied throughelectron microscopy. In addition, epithelial function will be measured by short-circuit current(Isc)on the nasal epithelia and and by transepithelial flux of horserdish peroxidase (HRP).Micro-osmotic pump will be used for chemical interference. We believe that allergicinflammatory mucosal disease in the upper respiratory tract may be initiated firstly and mainlyby mast cell, instead of the current conception of Th1/Th2 imbalance. Mast cell-dependentallergic reaction is a key factor for inducing the bacteria rhinosinusitis.PartⅠ.Establishment of a mast cell-deficient mice Model of AcuteBacterial Rhinosinusitis by Allergic RhinitisObjective using to examine mast cell function in the nasal cavity,to explore the infectivecourse of bacterial rhinosinusitis based on allergic rhinitis in homozygous mutants Wadsm/m andheterozygote Wadsm/+(The mutation localized at W/c-kit)and the difference between themafter intranasal streptococcus pneumoniae inoculation. Methods Respectively 40 mice ofWadsm/m and Wadsm/+ were randomly divided averagely into 4 groups: A,B (OVA+NS) C,D(OVA+S.p.),E,F(PBS+ S.p.) and G,H(PBS+NS).①Group A,B,C and D were sensitizedto OVA(10％)by intraperitoneal injection on days 0 through 8, and exposed to OVA(6%)intranasally on days 9 through 16, as to induce allergic inflammation. PBS was used in group Cand D in the same way.②Subsequently, group C,D,E and F were inoculated S.p.(1.2×109CFU/ml)intranasally on day 12, and NS was used in other groups .After 2, 5, 10 and14 days, Blood was gathered from the orbital venous sinus after anesthesia. nasal lavage cuhureswere obtained and then the mice were killed. On the 6th day after inoculation, mice were killed.The heads were embedded with paraffin and serial sections were made and stained withhematoxlin-eosin and toluidine blue for histological analysis. The number of polymorphounclearneutrophils and eosinophils per square millimeter of sinus mucosa (PMN/mm2 and Eos/ mm2)were calculated by the use of computer aided microscope. ed. The heads were embedded with paraffin and serial sections were made for histological analysis. The percentage of sinus cavityoccupied by neutrophil cluster (% cluster) and the number of polymorphonuclear leukocytes persquare millimeter of sinus mucosa (PMN/ mm2) were calculated by the use of a computer aidedmicroscope in conjunction with a reconstruction and image analysis system. Results % Clusterand PMN/ mm2 in infected mice appeared to peak on five days,which were significantlyheavier than those in controls( P < 0. 05) . The infection in simple infection decreased by twoweeks. But in complex model of allergy and infection contrast to simple infection, the infectionin wadm/m had not been controlled and Streptococcus pneumoniae were still seen in the nasallavage cuhure by two weeks. Conclusion:AR models were successfully established from wadm/+mice . It was found that mast cell-deficient mice didn`t exhibited .Acute bacterial rhinosinusitisin wads are successfully induced by intranasal inoculation of streptococcus pneumoniae. Thisbacterial infection in the group of simple infection controlled completely and rapidly. Our datademonstrate that an ongoing local allergic response augments bacterial infection in these mice.Incontrast failed to control rhinosinusitis and had a tendency to chronic inflammation, suggestingthat mast cell was important for clearance of bacteria from rhinosinus and mast cell-deficientWads was a convenient tool for investigation of the pathogenesis of experimental rhinosinusitis.Mast cell-deficient mice has been developed for study of a variety of pathological,immunological, and physiological processes in the respiratory tract ,beacause this is a veryconvenient site for experimental manipulations.PartⅡ.Establishment of a Mouse Model of Acute Bacterial Rhinosinusitis by AllergicRhinitisObjective To develop a animal model of bacterial rhinosinusitis in mice with allergicrhinitis, and explore whether ongoing allergic rhinitis enhances the acute sinus infection andinflammation associated with Streptococcus pneumoniae. Methods 40 mice of C57BL6/J wererandomly divided averagely into 4 groups: A (OVA+S.p.), B(OVA+NS), C(PBS+ S.p.) andD(PBS+NS).①Group A and B were sensitized to OVA(10％)by intraperitoneal injectionon days 0 through 8, and exposed to OVA(6%) intranasally on days 9 through 16, as to induceallergic inflammation. PBS was used in group C and D in the same way.②Subsequently, groupA and C were inoculated S.p.(1.2×109CFU/ml)intranasally on day 12, and NS was used ingroup B and D. On the 6th day after inoculation, mice were killed. Blood was gathered from theorbital venous sinus after anesthesia. The heads were embedded with paraffin and serialsections were made and stained with hematoxlin-eosin and toluidine blue for histologicalanalysis. The number of polymorphounclear neutrophils and eosinophils per square millimeterof sinus mucosa (PMN/mm2 and Eos/ mm2) were calculated by the use of computer aided microscope. Results AR models were successfully established on 9 from group A and 8 fromgroup B. Histological examination of the sinus of group A and B revealed significant mucosaledema and dilated venules. The symptoms were mild in group C, and no symptom wasobserved in group D. PMN/mm2 in group A(139.29±26.51)mm2, were significantly heavierthan group B(70.67±16.74)mm2, C(63.04±14.73)mm2 and D(40.24±14.05)mm2 respectively(P＜0.01); Eos/ mm2 and serous IL-5 level in group A(134.63±25.49)mm2,(48.21±13.94)pg/ml and B(116.21±25.17)mm2,(40.83±7.78)pg/ml, were higher than group C(16.71±2.68)mm2,(23.89±8.65)pg/ml(P＜0.05)and D(13.39±4.95)mm2,(24.58±6.50)pg/ml(P＜0.05). Conclusion Our data demonstrate that an ongoing local allergic response augmentsbacterial infection in these mice, and allergic sensitization alone does not augment the sinusinfection without pathogenic bacterium.Pa r tⅢ.Sur f a c t ant Pr o t e in D Expr e s s i on in Chr oni c Rhino s inus i t i s andImmune ResponsesObjectives Surfactant-associated proteins (SP) D are in the family of collectin proteinsthat play an integral part in the innate defense system. SP-D expression and function are alteredin a variety of inflammatory and infectious diseases of the lungs, such as asthma, allergies, andcystic fibrosis. Our studies are the first to identify the presence of these proteins in the nasalcavity. The objective of this study was to immunolocalize SP-D in human sinonasaltissue.Methods Respectively 40 mice of Wadsm/m and Wadsm/+ were randomly dividedaveragely into 4 groups: A,B (OVA+NS) C,D(OVA+S.p.),E,F(PBS+ S.p.) and G,H(PBS+NS).①Group A,B,C and D were sensitized to OVA(10％)by intraperitoneal injection on days0 through 8, and exposed to OVA(6%) intranasally on days 9 through 16, as to induce allergicinflammation. PBS was used in group C and D in the same way.②Subsequently, group C,D,Eand F were inoculated S.p.(1.2×109CFU/ml)intranasally on day 12, and NS was used in othergroups . After 2, 5, 10 and 14 days, The heads were embedded with paraffin and serial sectionswere made for histological analysis. nasal mucosal biopsies were performed in patients withvarious forms of chronic hyperplastic rhinosinusitis with nasal polyposis and nondiseasedmucosa from patients undergoing (n = 10) Immunolocalization of surfactant proteins wasperformed with antibodies SP-D using immunoperoxidase staining technique. Isotype-negativecontrols were performed on all specimens.RESULTS: Analyses of mucosal biopsy specimensfrom nasal tissue reveals staining within respiratory and intermediate (metaplastic)-type surfaceepithelium. In addition, staining was intense in the submucosal ductal epithelium of theseromucinous glands. These properties appear to be consistent regardless of disease state andlocation within the sinuses.CONCLUSION: These are secreted proteins that are intricately involved in innate immunity in the lungs. Their secretion in the upper airway indicates thatfuture studies may allow manipulation of these proteins and development of novel treatments forsinonasal pathology.