A Follow-up Study And Mechanisms Discussion Of RhINF-γ To Prevent Infections In Chronic Granulomatous Disease

Objective1. To describe the clinical features of chronic granulomatous disease (CGD) in children.To analyze the clinical effect of combined treatment program (combination of rhIFN-γ and TMP-SMX) and the clinical efficacy and safety of rhIFN-γ in CGD.2. To detect the percentage of TH17cells (IL-17+/CD4+T cells) and concentration of IL-17、IL-21、IL-23、IFN-y produced by lymphocytes in patients of chronic granulomatous disease (CGD). To provide clinical basis for molecular mechanisms research of excessive inflammatory response and rhIFN-γ treatment.Methods1. The follow-up study of clinical features, including the site of infection,the pathogen of infection,noninfectious inflammatory response,and drug treatment,etc, was performed in CGD patients diagnosed in children s hospital of Fudan University from January2004to December2011. The incidence of infections calculated as per patient-year was compared before and after treatments between patients who received combined treatment program or separated TMP-SMX treatment and the clinical efficacy and safety of rhIFN-y treatment was analyzed.2. Peripheral blood samples was collected from CGD patients and healthy controls, and stimulated with PMA、ionomycin and BFA for4hours. Flowcytometry was used to detect the percentage of TH17cells. PBMCs stimulated with PMA and ionomycin were prepared by centrifugation using Ficoll-Paque, ELISA was used to detect the concentration of cytokines of IL-17、IL-21、IL-23、IFN-γ in PBMC supernatants.CGD patients were divided into several groups including infected and non-infected group, rhIFN-γ used group and unused group, a comparative analysis of TH17cells and the cytokine levels was performed.Results1. A total of29cases was recruited into the study.The mean age of onset and diagnosis was2.5months and2.4years respectively.Total observation period was82.8years while mean duration of per follow-up patient was2.9years.①The total incidence of infections was1.8per patient-year, and the incidence of severe infections and non-severe infections,was0.48and0.80per patient-year. Lung infections and lymphadenitis were the most frequent manifestations both prior to diagnosis (49and26) and during follow-up (26and37).Mycobacteria species was the leading cause of infection(34/61) and death (3/4) in our cohort.②Combined treatment program was used in21cases while separated TMP-SMX treatment was performed in8cases because of drug adverse reactions or self-withdrawal. After combined treatment,the total incidence of infections and the incidence of severe infections were significantly lower than those prior to treatment(total incidence:1.986vs1.098, P=0.002; severe infection:1.290vs0.452, P=0.001), and it was the most marked to the lung (0.758vs0.275, P=0.004).. No significant difference of the incidence of infections was observed in patients receiving separated TMP SMZ treatment. The total incidence of infections and the incidence of non-severe infections in combined treatment program were significant lower than those in separated TMP-SMX treatment (Total infection:1.098vs1.823, P=0.015; non-severe infection:0.646vs1.248, P=0.047), while there was no significant difference in severe infections(0.452vs0.576, P=0.254).③14of29(48%) cases reported adverse event after using rhIFN-γ,the commonest was fever (12/29).There was no life-threatening rhIFN-y-related adverse events reported in our study2. In the second part of our study,a total of32cases was recruited the mean age of blood samples was2.58years.16healthy controls were collected.①In CGD group, the percentage of Th17cells and the concentrations of Th17-derived cytokines including IL-17、IL-21、IL-23were significantly higher than those in healthy control group (p<0.05), while the level of IFN-y did not significantly differ from that of healthy control group (p=0.586).②There was no significant difference in the level of Th17cells and cytokines of IL-17, IL-21, IL-23, IFN-γbetween infection group (n=10) and non-infected group (22cases)(p>0.05). In both groups, the level of Th17cells and Th17-derived cytokines of IL-17、IL-21、IL-23was significantly higher than that in healthy control group (p<0.05), but no significant difference was detected in the level of IFN-γ between patients and healthy controls (p=0.544).③There was no significant difference in the level of Th17cells and the cytokines of IL-17, IL-21, IL-23, IFN-γ between rhIFN-γ used group (n=12) and unused group (20cases)(p>0.05). In both groups, the level of Th17cells and Th17-derived cytokines of IL-17、IL-21、IL-23was significantly higher than that in healthy control group (p<0.05), and the level of IFN-γ did not significantly differ from that of healthy control group (p=0.282).Conclusions1. Lung infections and lymphadenitis are the most frequent manifestations in our cohort, andMycobacteria species is the first cause of infection and death.combined treatment program significantly reduced the incidence of infectionsand the use of rhIFN-y in patients with CGD appeared to be effective and safe.2. Th17cells and Th17-derived cytokines of IL-17、IL-21、IL-23most probably play an important role in excessive inflammatory response and tissue granulomas formation in CGD patients. The level of IFN-y do not increase after infection,and this may be associated with impaired resistance to pathogen infections. Conventional therapeutic and preventive dose of rhIFN-γ can not restrain differentiation and proliferation of Th17cells.