| Objective:The dynamic model of brucellosis was constructed to reflect the epidemic rule from the perspective of the transmission mechanism of the disease,and the severity of the disease transmission in the six areas with high incidence of brucellosis in xinjiang was estimated.To analyze the clinical characteristics of patients with acute and chronic brucellosis,study the expression of IL-21,IFN-γ,IL-4,IL-6,IL-10,Follicular Helper T Cells(Tfh)and B cells in peripheral blood and the titer of antibody.Combining with the model of Brucella infection in mice,the regulation of IL-21 on IL-6,IL-10,Tfh cells,B cells and germinal center was further analyzed.The correlation of IL-21,Tfh cells,B cells and antibody titers was analyzed,To explore the mechanism of immune regulation of Tfh cells and their secreted cytokine IL-21 in brucellosis.Methods:1)The incidence of brucellosis in Xinjiang from 2008 to 2017 was analyzed,and the fever maps and trend maps of the incidence of brucellosis in various prefectures were drawn.Constructing a Brucellosis Dynamics Model Using Data from Six High-incidence Areas of Brucellosis in Xinjiang(Changji Hui Autonomous Prefecture,Bortala Mongolian Autonomous Prefecture,Bayingolin Mongolian Autonomous Prefecture,Ili Kazakh Autonomous Prefecture,Tacheng Region,and Altay Region)And calculate the severity of disease transmission in six areas with high incidence of brucellosis in xinjiang was estimated.The contour maps of immunity rate and killing rate of positive cattle and sheep were drawn,and the strategies to reduce the incidence of brucellosis were put forward;2)Ninety-eight patients with brucellosis were selected from the First Affiliated Hospital of Xinjiang Medical University and the First Affiliated Hospital of Shihezi University Medical College from January 2017 to December 2018.Among them,50 were in acute stage and48 were in chronic stage.At the same time,40 healthy controls matched by age and sex were collected.After admission,all patients collected basic data and improved routine blood biochemical and ultrasound tests.The expression levels of IL-21,IFN-gamma,IL-4,IL-6 and IL-10 were detected by Cytometric Bead Array(CBA)method.Peripheral blood mononuclear cells(PBMCs)were isolated from peripheral blood of acute and chronic brucellosis patients and healthy controls by density gradient centrifugation.Flow Cytometry(FCM)was used to detect the proportion of Tfh and B cells in peripheral blood.The antibody titer of each group was determined by Serum tube Agglutination Test(SAT).The correlation between Tfh cells,IL-21 and B cells expression and antibody titer were analyzed in acute and chronic brucellosis patients.A retrospective analysis was made of21 patients with brucellosis and thrombocytopenia diagnosed in the First Affiliated Hospital of Xinjiang Medical University from January 2012 to December 2018.Statistical demographic characteristics,clinical features,laboratory examinations and other clinical data,treatment methods,prognosis;3)BALB/C mice were infected with 16M Brucella virulent strain by intraperitoneal injection,or combined with recombinant mouse IL-21.Recombinant Mouse IL-21 Protein(r IL-21)or recombinant mouse IL-21 receptor(r IL-21R Fc)stimulated BALB/C mice.The spleens of BALB/C mice were cultured and identified at the 4th week after challenge to verify the success of challenge.The peripheral blood of mice was taken from the 2nd,4th,8th and 12th weeks after challenge to prepare serum.The expression levels of IL-21,IL-6 and IL-10 were detected by CBA method,the titers of antibodies were determined by SAT,and the expression levels of Tfh and B cells in peripheral blood of mice were detected by FCM.The liver,spleen and lung tissues of rats were observed by morphology and HE staining.The expression of Brucella and Peanut agglutinin(PNA)was analyzed by immunohistochemical staining.To analyze the immunoregulatory effect of Tfh cells on B cells and the effect on antibody secretion by secreting IL-21.Results:1)According to the incidence trend of 14 prefectures,since 2008,the incidence rate of each prefecture has risen sharply.Most of the prefectures reached the peak around 2015(Kizilesu and Hotan continued to rise).Then the disease has been controlled to a certain extent,and began to show a downward trend,and the epidemic has been alleviated to a certain extent.The established dynamic model of brucellosis can reflect the transmission of brucellosis in six regions.According to the model,the basic regeneration number of six high-incidence areas of brucellosis in Xinjiang has reached more than 4.Among them,the most serious area of brucellosis transmission in Xinjiang is Altay,and the basic regeneration number has reached 8.47(95%CI 7.82-9.35),followed by 7.84(95%CI 7.12-8.60)in Bayinguoling Mongolian Autonomous Prefecture,6.70(95%CI 6.12-7.36)in Bortala Mongolian Autonomous Prefecture,6.33(95%CI 5.77-6.93)in Tacheng Prefecture,5.73(95%CI 5.12-6.21)in Changji Hui Autonomous Prefecture,and 4.85(95%CI 4.49-5.29)in Ili Kazakh Autonomous Prefecture.2)The epidemiological history of 98 patients with acute and chronic brucellosis was investigated.91.8%(90/98)of them had a clear epidemiological history.The clinical manifestations of patients in acute and chronic stages were different.The main clinical manifestations of patients in acute stage were fever,fatigue,muscle and joint pain,poor appetite and sweating,while those in chronic stage were different.The main clinical manifestations were joint pain and fatigue.All patients were screened positive by Tiger Red Plate Agglutination Test.The titers of antibodies in test tube agglutination test varied from 1∶100 to 1∶800 in acute stage to 1∶50 to 1∶400 in chronic stage.Detection of IFN-γexpression level:The expression of IFN-γin patients with acute brucellosis was significantly higher than that in patients with chronic brucellosis and healthy controls,and there were significant differences among groups(P<0.05).Detection of IL-4 expression level:The expression of IL-4 in chronic Brucellosis patients was higher than that in acute Brucellosis patients,and the expression level of IL-4 in two groups was higher than that in healthy controls.There was a statistical difference between the two groups(P<0.05).Detection of IL-21expression level:The expression of IL-21 in patients with acute brucellosis was significantly higher than that in patients with chronic brucellosis and healthy controls,and there were significant differences among groups(P<0.05).The level of IL-6 expression in patients with acute stage was higher than that in patients with chronic stage(P<0.05),and in patients with chronic stage was higher than that in healthy controls,but there was no significant difference between the two groups(P>0.05).The expression of IL-10 in chronic phase was higher than that in acute phase(P<0.05),and in acute phase was higher than that in healthy control,but there was no statistical difference(P>0.05).CD3~+CD4~+T cell expression level:patients in acute phase were lower than those in healthy control group.There was statistical difference between the two groups(P<0.05).Patients in acute phase were lower than those in chronic phase.There was statistical difference between the two groups(P<0.05).Patients in chronic phase were slightly lower than those in healthy control group.There was no statistical difference between the two groups(P>0.05).The expression level of CD3~+CD8~+T cells in patients with acute stage was higher than that in patients with chronic stage(P<0.05),while that in patients with chronic stage was higher than that in healthy controls(P>0.05).The expression level of Tfh cells(CD3~+CD4~+CXCR5~+cells)in patients with acute phase was higher than that in patients with chronic phase,and that in patients with chronic phase was higher than that in healthy controls.There were significant differences among groups(P<0.05).The expression of B cells(CD3~-CD19~+cells)in peripheral blood of patients with acute and chronic brucellosis was higher in patients with acute stage than in patients with chronic stage,and higher in patients with chronic stage than in healthy controls.There was a significant difference between groups(P<0.05).The results showed that the titer of antibody was positively correlated with the level of IL-21,the titer of antibody was positively correlated with the level of Tfh cells,and the titer of antibody was positively correlated with the level of B cells.From January 2012 to December 2018,21 patients diagnosed with brucellosis complicated with thrombocytopenia were admitted to the first affiliated hospital of xinjiang medical university.All 21 patients were male.Uygur 3 cases,Han 14 cases,Kazakh 4 cases.Farmers and herdsmen were the main group.Eleven patients had close contact with cattle and sheep in their homes.Five patients were engaged in slaughtering and wool processing industries.Five patients had unexplained infection.All patients had no history of thrombocytopenia in the past;Brucella growth in blood culture was observed in 12 patients,and serological tube agglutination test was positive in16 patients with titers ranging from 1∶200 to 1∶800.The severity of thrombocytopenia in brucella patients was not correlated with antibody titer.Sixteen patients were given rifampicin capsule combined with doxycycline tablets for symptomatic treatment;three patients were given baifule tablets combined with doxycycline for anti-Brucella treatment;and two patients were given baifule tablets combined with rifampicin for anti-Brucella treatment.Three patients(<10×10~9/L)were treated with glucocorticoid,gamma globulin and platelet transfusion.After 6 months of follow-up,platelets returned to normal in 19patients and slightly lower in 2 patients;3)At the 2nd,4th,8th and 12th week after Brucella infection,the serum of mice in each group was taken for Tiger Red plate test and in vitro agglutination test.The results showed that Tiger Red Plate test A,B and C were all positive in the experimental group,and the titer of test tube agglutination test was 1∶50 in the fourth week,1∶200 to 1∶400 in the eighth week and 1∶400 in the twelfth week.The titer of the test tube agglutination test in the control group could not be measured.Four weeks after the challenge,Brucella was isolated from the spleen of mice in each experimental group,and the challenge was successful.The changes of il-6,il-10and il-21 were detected at the time points of week 2,week 4,week 8 and week 12 after challenge.The results indicated that,with the extension of time,the expressions of il-6,il-10 and il-21 gradually increased from week 2 and reached the highest level at week 12.After intraperitoneal injection of Recombinant Mouse IL-21 Protein,the expression of IL-10 and IL-21 in experimental group B was up-regulated at different time points,but the change of IL-6 was not obvious.After intraperitoneal injection of Recombinant Mouse IL-21 R Fc,the expression of IL-10 and IL-21 in the experimental group C decreased compared with the A experimental group,and the change of IL-6 was not obvious.suggesting IL-21 promoted the secretion of IL-10,but did not significantly regulate the secretion of IL-6.The expression of Tfh cells and B cells increased gradually with the prolongation of challenge time,and the expression was highest at 12th week after intraperitoneal injection was given.After Recombinant Mouse IL-21 Protein,the expression of Tfh cells and B cells was up-regulated in each time period compared with experimental group A.After intraperitoneal injection of Recombinant Mouse IL-21 R Fc,the expression of Tfh cells and B cells decreased at different time points compared with the experimental group A;IL-21 inhibited the regulation of Tfh cells and B cells.In this study,the level of IL-21,the expression of Tfh cells,the expression of B cells and the antibody titer of the 2nd week,4th week,8th week,and 12th week after infection with Brucella were performed.Correlation analysis showed that with the prolongation of the challenge time,the level of IL-21,the expression of Tfh cells,the expression of B cells showed an upward trend,and the antibody titer increased.The levels of IL-21,the level of Tfh cells,and the level of B cells were positively correlated with antibody titers;IL-21promoted the regulation of Tfh cells and B cells,and the expression of PNA staining by spleen indicated that Tfh cells passed.When IL-21 enhancement,PNA staining positive signal of spleen increased,while after IL-21 antagonism,PNA staining positive signal of spleen decreased.Its secreted IL-21 induces the formation of germinal centers in the mouse spleen.After brucella infection in mice,with the extension of challenge time,liver,spleen and lung showed different degree of pathological changes and positive signals of brucella staining.Conclusion:1)According to the dynamic model analysis of infectious diseases,the six regions with the most serious transmission of brucellosis in xinjiang are altay region,bayingolin Mongolian autonomous prefecture,bortala Mongolian autonomous prefecture,tacheng region,changji hui autonomous prefecture and ili kazakh autonomous prefecture.In this paper,two disease control strategies were proposed to control the epidemic situation of brucellosis in humans by increasing the immunization rate of cattle and sheep or the slaughter rate of cattle and sheep with brucellosis positivity,and the specific quantitative control threshold of six high-incidence areas of brucellosis in xinjiang was calculated;2)In acute stage brucella patients,the expression of pro-inflammatory cytokines ifn-gamma,IL-6 and IL-21 was mainly increased,which mediated the inflammatory response of the host and caused various clinical manifestations.The chronic stage is dominated by the increase of anti-inflammatory cytokines IL-4 and IL-10,which inhibit the further development of inflammatory response,causing the pathogen to be difficult to remove,causing non-specific manifestations throughout the body.CD8+T cells were mainly immunized in patients with acute brucella disease.The number of CD8+T cells in patients with chronic brucella disease decreased,and CD4+T cells were lower than those in normal people,suggesting that brucella may cause chronic persistent infection by inhibiting host immunity.In the acute and chronic course of brucella infection,the expression of Tfh cells,IL-21 and B cells was positively correlated with the titer of antibodies.The Tfh cell-IL-21-B cell-antibody immune regulatory network is involved in the pathogenesis of brucella infection.Antibody titer could not be used as an evaluation index of severity of brucellosis complicated with thrombocytopenia;3)IL-21 can promote the secretion of IL-10 in mice infected with brucella,but has no obvious promotion or inhibition effect on IL-6.In the acute stage of brucella infection in mice".Tfh cell-IL-21-B cell-antibody"jointly promoted the occurrence of infection.Tfh cells induce B cell maturation and antibody production through the secretion of IL-21.Antagonizing the function of IL-21 can reduce the secretion of Tfh cells and B cells as well as the production of antibodies,which may be beneficial to the host to resist the infection of brucella.After antagonizing IL-21,the degree of liver inflammation decreased.Anti-infective drugs combined with il-21 antagonist are expected to be a new treatment for brucellosis. |
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