The Clinical And Molecular Features Of Two Types Of Leukocyte Functional Defect Diseases And Partial Pathogenesis Exploration

PART ONE: CLINICAL AND MOLECULAR FEATURES OF PATIENTS WITH CHRONIC GRANULOMATOUS DISEASEObjective:To discuss the clinical features,laboratory results and gene mutations of 26 patients with chronic granulomatous disease(CGD).Methods:1.Twenty six patients diagnosed with CGD in Children's Hospital of Chongqing Medical University during June 2015 to February 2017 were enrolled into this study.The clinical manifestations and laboratory results of these patients were summarized.2.Flow cytometry was used to analyze the function of respiratory burst of neutrophils in these patients and then Neutrophil oxidative index(NOI= MFI after stimulated/MFI before stimulated)was calculated.3.Genes relevant to CGD(CYBB?CYBA?NCF1?NCF2)were analyzed using PCR technique.Results:1.Basic information: All the 26 patients were male,15 cases(57.7%)came from southwest China.The average age of onset and diagnosis was 2.3 months old and 1.4 years old,respectively.14 cases(53.8%)had a family history of male relatives died in young ages.2.Clinical manifestations: Recurrent fever(26 cases,100%),pneumonia(24 cases,92.3%),diarrhea(13 cases,50%),skin and subcutaneous tissue infections(14 cases,53.8%),hepatosplenomegaly or abnormal liver function(12 cases,46.2%)were the common manifestations of patients in our cohort.Twenty one cases got BCG vaccination and 66.7% of them experienced vaccinated scar fester,lymphadenitis or tuberculosis after BCG vaccination.In addition,3 cases(11.5%)developed hepatapostema and 2 cases(7.7%)suffered recurrent urinary tract infection.3.Laboratory examinations: Markedly elevated leukocyte counts with a predominance of neutrophils and elevated CRP were found.Immunoglobulins were in normal or high level,Ig E was significantly increased in 9 cases(34.6%),and lymphocyte subsets had no significant abnormalities.NBT in 80.8% of the patients were markedly decreased,most of them were 0 after neutrophil stimulated by PMA.Only 50% of the patients found pathogenic bacteria.4.Respiratory burst of neutrophils and gene mutations: NOI in patients was markedly decreased(1.365±0.1018)compared with normal people(>100)whereas NOI in their mothers was in normal or low level.Mutations in CYBB gene were found in 21 patients(80.8%)and only 1 patient(3.8%)had a mutation in CYBA gene.There were 4 patients(15.4%)didn't find mutation gene and 15 XLR-CGD carriers were identified.Conclusion:CGD patients usually suffer severe bacterial and fungal infections since neonatal period.Patients have abnormal reactions after BCG vaccination and have family history(male relatives die in young ages)with markedly increased leukocyte counts and normal or high level of immunoglobulins,abnormal NBT should be suspicious of CGD.Respiratory burst of neutrophils using flow cytometry is a rapid clinical diagnosis method and gene analysis is necessary for CGD patients and carriers.PART TWO: AN EXPLORATION OF RELATED FACTORS OF THE DECREASED MEMORY B CELL IN PATIENTS WITH CHRONIC GRANULOMATOUS DISEASEObjective:To discuss whether there exist decreased memory B cell(MBC)in CGD patients and search for its related factors.To explore the mechanisms of IFN-? in treating recurrent infections of CGD patients.Methods:1.Flow cytometry was used to measure the percentages of MBC,T follicular helper(Tfh)cell,PD-1 on Tfh cell as well as CD40 L on CD4+T cell.2.The percentage of MBC was measured using flow cytometry after healthy children's PBMC co-cultured with different concentration of s CD40L(1?g/ml?1.5?g/ml?2?g/ml)for 24,36 and 48 hours.3.The percentage of MBC was measured using flow cytometry after CGD patients' PBMC co-cultured with different concentration of H2O2(10?mol/L?30?mol/L)or IFN-?(2000U/ml?3000U/ml)for 24 hours.Results:1.The percentage of MBC was markedly decreased in CGD patients(1.267%±0.1416%)compared with healthy controls(2.824%±0.27%),P<0.0001.2.The percentage of CD40 L on CD4+T cell was significant decreased in CGD patients(8.186%±2.736%)compared with healthy controls(46.43%±4.619%),P<0.0001.3.There were no significant differences(P<0.01)of Tfh cells and PD-1 between CGD patients(Tfh 5.625%±0.5005%;PD-1 9.039%±1.984%)and healthy controls(Tfh 6.269%±1.177%;PD-1 18.26%±10.11%).4.With the elongation of culture time,MBC in healthy children was decreased after PBMC co-cultured with s CD40L(P=0.0028).5.There were no significant effects of H2O2 and IFN-? on stimulating MBC in CGD patients,on the contrast,MBC was decreased in a time-dependent manner(P<0.0001).Conclusion:The decreased MBC in CGD patients may have a connection with the decreased CD40 L on the surfaces of CD4+T cell.Although the percentage of Tfh cell was normal,the expression of CD40 L on Tfh cells should be measured to verify the function of Tfh cells in CGD patients is normal or not.IFN-? has a good effect on recurrent infections but it is not by means of stimulating MBC.PART THREE: CLINICAL AND MOLECULAR FEATURES OF THREE PATIENTS WITH LEUKOCYTE ADHESION DEFICIENCY TYPE-1Objective:To explore the clinical features,abnormal expression of CD18 and gene mutations in 3 patients with leukocyte adhesion deficiency type-1(LAD-1).Methods:1.Clinical materials of the 3 patients were summarized and common primary immunodeficiency diseases were excluded.2.Flow cytometry was used to measure the expression of CD18 on leukocytes' surfaces.3.ITGB2 gene mutation analysis was performed using PCR sequencing.Results:1.Clinical manifestations: Omphalitis was the initial symptom in the 3 patients and then they experienced severe and recurrent skin,mucosa and soft tissue infections(pneumonia,otitis media,thrush,gingivitis and loss of deciduous teeth).Case 1 and case 2 had delayed umbilical cord separation and 2 patients(case 1 and case 3)suffered frequently from chronic skin infection and delayed wounds healing(>1 month).One patient(case 1)had family history(relatives died in young ages).2.Laboratory examinations: Markedly increased leukocyte counts with a predominance of neutrophils and anemia were observed.Ig G?Ig A?Ig M were increased,lymphocyte subsets and NBT had no significant abnormalities.3.Flow cytometry of CD18: CD18 on the surfaces of leukocytes were markedly decreased in the 3 patients whereas were normal in their parents(>90%).Case 2 was severe deficiency of CD18(CD18 on lymphocyte,neutrophil and monocyte was 0,0.23%,0 respectively),Case 1 and case 3 had a moderate deficiency of CD18(Case 1: 9.49%,0.04%,0.45%;Case 3: 10.14%,0.67%,2.54%).4.ITGB2 gene analysis: Five mutations were identified(c.167-168 ins GG,c.1884C>A,c.533C>T,c.817G>C,c.1768T>C).Two novel mutations(c.167-168 ins GG,c.1884C>A in case 1)and 5 carriers were confirmed.Conclusion:Children suffering recurrent skin,mucosa and soft tissue infections,omphalitis,delayed umbilical cord separation,poor wounds healing,gingivitis with loss of deciduous teeth,markedly elevated leukocyte counts with a predominance of neutrophils and normal or high level of immunoglobulins should be suspicious of LAD-1.CD18 expression measured by flow cytometry is a rapid clinical diagnosis method and ITGB2 gene analysis is the golden standard for LAD-1 diagnosis.