Diagnostic Value of Immunological Biomarkers in Children with Asthmatic Bronchitis and Asthma.

Background and Objectives: This study aimed to investigate the diagnostic value of immunological biomarkers in children with asthmatic bronchitis and asthma and to develop a machine learning (ML) model for rapid differential diagnosis of these two diseases. Materials and Methods: Immunological biomarkers in peripheral blood were detected using flow cytometry and immunoturbidimetry. The importance of characteristic variables was ranked and screened using random forest and extra trees algorithms. Models were constructed and tested using the Scikit-learn ML library. K-fold cross-validation and Brier scores were used to evaluate and screen models. Results: Children with asthmatic bronchitis and asthma exhibit distinct degrees of immune dysregulation characterized by divergent patterns of humoral and cellular immune responses. CD8+ T cells and B cells were more dominant in differentiating the two diseases among many immunological biomarkers. Random forest showed a comprehensive high performance compared with other models in learning and training the dataset of immunological biomarkers. Conclusions: This study developed a prediction model for early differential diagnosis of asthmatic bronchitis and asthma using immunological biomarkers. Evaluation of the immune status of patients may provide additional clinical information for those children transforming from asthmatic bronchitis to asthma under recurrent attacks.

A Cohort of 169 Chronic Granulomatous Disease Patients Exposed to BCG Vaccination: a Retrospective Study from a Single Center in Shanghai, China (2004-2017).

PURPOSE: Clinical diagnosis and treatment for chronic granulomatous disease (CGD) have advanced greatly in recent years. However, CGD patients in China have unique clinical features and infection spectrums, which are challenging to their caretakers. Here, we summarized the clinical characteristics, genetic features, treatment, and prognosis of CGD in a single center in Shanghai. METHODS: One hundred sixty-nine CGD patients were recruited between January 2004 and May 2017 based on clinical diagnosis. Electronic medical charts were reviewed to collect clinical data. RESULTS: Among the 169 patients recruited, CYBB mutations were identified in 150 cases, whereas CYBA mutations were identified in 7 cases, NCF1 in 5, and NCF2 in 7. The medium age at onset was 1 month (interquartile range 1-3). The medium age at diagnosis was 8 months (interquartile range 3-19). The most common infection sites were the lung (95.9%), lymph node (58.5%), skin (45.4%), intestinal (43.1%), and perianal (38.5%). Bacillus Calmette-Guérin (BCG) infections were common (59.2%). In addition, other non-infectious complications were also common, including anemia (55.4%) and impaired liver functions (34.6%). Thirty-one patients received stem cell transplantation. By the end of this study, 83/131 patients survived. CONCLUSIONS: Similar to other non-consanguineous populations, X-linked CGD accounted for the majority of the cases in China. However, BCG infections were a clinical challenge unique to China. In addition, severe infections were the major cause of death and the overall mortality was still high in China.

Mutation of the BTK gene and clinical feature of X-linked agammaglobulinemia in mainland China.

INTRODUCTION: X-Linked agammaglobulinemia is a prototypical humoral immunodeficiency with the mutation of the Bruton's tyrosine kinase gene. METHODS: We investigated the gene mutation and clinical features of 30 Chinese X-linked agammaglobulinemia (XLA) patients from 27 families. There were 26 mutations, including 11 novel and 15 recurrent mutations, distributing over the entire gene. The nucleotide and amino acid aberration, 1129C>T(H333Y) and 1196T>A(I355N), in SH2 have not been reported before. Five (I355N, W124R, R520X, I590F, G594E) of the 24 mutations not detected in the mothers receiving gene analysis were determined to be de novo. Two mutations occurred within intronic splice-site sequences (intron5(-2)A>G, intron17(-2)A>T). RESULTS AND DISCUSSION: There are eight mutations in the PH domain, two mutations in the SH3 domain, three mutations in the SH2 domain, one mutation in the TH domain, and other 16 mutations in the TK domain. The mutations of protein domain is most common in TK (53%) domain and then in PH(8%) domain. Missense and nonsense mutations were found equal in 46% of the detected mutations. All of the patients are alive, but one died of liver cancer. Clinical features and serum Igs levels range variedly and were not correlated with genotypes. Our results demonstrated molecular genetic characteristics of XLA in mainland China.

Clinical characteristics and immunogenetics of BCGosis/BCGitis in Chinese children: a 6 year follow-up study.

In this study, the clinical and immunogenetical features in a cohort of Chinese patients with BCGosis/BCGitis were investigated. For the patients with abnormal immunological functions, Sanger sequencing was used to identify the involved genes. There were 74 confirmed cases of BCGosis/BCGitis during 2007-2012. Classified by infected tissues and organs, no cases only had local infection, 39 patients had a regional infection, 21 patients had a distant infection and 14 patients had a disseminated infection. Thirty-two patients (43.2%) had definitive primary immunodeficiency diseases (PID) and chronic granulomatous disease (CGD) is the most common PID (n = 23, accounted for 71.9% of all PID patients). For CGD patients, based on the anti-tuberculosis treatment, administration of rhIFN-γ resulted in better control of BCGosis/BCGitis. The results indicate that PIDs are associated with susceptibility to BCG disease. For children with BCGosis/BCGitis, immune function evaluation is necessary, and IFN-γ treatment for BCGosis/BCGitis patients with CGD is effective.

[Clinical features of chronic granulomatous disease].

OBJECTIVE: Chronic granulomatous disease (CGD) is a rare primary immunodeficiency of phagocytic oxidative bursts leading to recurrent severe bacterial and fungal infections as well as granuloma formation. There were few reports on the clinical characteristics of this disease in China. The purpose of this study was to evaluate the clinical features of 48 Chinese cases with CGD which were confirmed by clinical features, dihydrorhodamine (DHR) assay and gene mutation analysis. METHOD: The study cohort was the population of CGD patients diagnosed in Children's Hospital of Fudan University from January, 2004, to June, 2011. Cases included in our analysis were restricted to those who had complete data of the clinical symptoms and laboratory tests. The patients were followed up by outpatient visiting and telephone call regularly for 0.5 to 6 years. The history and data of physical examination and treatment of 48 cases were collected and reviewed. RESULT: All the patients were diagnosed by DHR analysis. The age of onset of all the 48 patients were less than 6 months, including 43 male and 5 female. The mean age at diagnosis was 2.42 years; 12 patients were infants under six months, 10 were between 6 and 12 months, 9 were between 1 and 2 years, 5 patients were between 2 and 3 years, 4 were between 4 and 5 years, and 8 were between 6 and 10 years. Recurrent respiratory infection (44/48) and chronic diarrhea (31/48) were the common symptoms in all the patients, and then skin lesion (22/48), including marked reaction at BCG infected site, pustular eruption and infected skin ulcer and urinary tract infection (3/48) were also general symptoms in our study. In addition, lymphadenectasis occurred in 31 cases and 23 of them were considered to be associated with BCG vaccination. The pathogens caused the infection were mycobacteria (52.08%), fungi (43.75%) and pyogenic bacteria. Thirty-seven patients had mutations in CYBB/CYBA/NCF1/NCF2 genes. Recombinant human interferon-gamma (rhIFN-γ) plus sulfamethoxazole were used for the prevention and treatment of infection, the frequency and severity of the disease could be reduced. CONCLUSION: The age at onset and diagnosis of the present group of CGD was younger. Clinical symptoms were associated with recurrent mycobacterial, fungal and pyogenic bacterial infection, which involved respiratory tract, alimentary tract, skin and lymph node. rhIFN-γ partially improved the prognosis of CGD.

[Effects of sodium valproate on neutrophils' oxidative metabolism and oxidant status in children with idiopathic epilepsy].

OBJECTIVE: To evaluate the influence of VPA treatment on neutrophils' oxidative metabolism and oxidant status in epileptic children. METHOD: Twenty-six newly diagnosed epileptic children with idiopathic epilepsy and 30 healthy children were included in the study. The activation rates of neutrophils and stimulation indexes were detected in patients before and 6 months and 12 months after VPA treatment respectively and in all the healthy children by flow cytometry with dihydrorhodamine as fluorochrome. The activities of myeloperoxidase from neutrophils were also detected. Malondialdehyde as an indicator of lipid peroxidation and antioxidant enzymes including superoxide dismutase, catalase, and glutathione peroxidase were measured in plasma respectively. RESULT: The activation rates of neutrophils in patients treated with VPA after 6 and 12 months were (11.50 ± 6.52)% and (14.31 ± 5.76)% respectively, which were significantly higher than the data of control group (5.90 ± 3.77)% and pretreatment level (7.42 ± 3.15)%. The stimulation indexes 6 and 12 months after VPA therapy were (474.88 ± 118.98) and (416.31 ± 110.00) respectively, which were lower than the data of control group (544.83 ± 140.83) and pretreatment level (535.23 ± 111.55). The plasma MPO activities and levels of malondialdehyde in VPA treated patients were also higher while the activities of SOD and CAT were significantly lower than the control and untreated groups. GSH-Px levels did not differ between the groups. Multiple linear regression analysis showed that the time of treatment and the activation rates of neutrophils were indicators which had positive correlation with the levels of plasma MDA and that SOD activities were inversely correlated with MDA levels. CONCLUSION: VPA which is frequently used in childhood epilepsy may activate the neutrophils of patients and cause oxidative stress and prolonged treatment may aggravate it.

[Bifidobacterium DNA upregulates Th1 type response of umbilical cord blood mononuclear cell].

OBJECTIVE: To study the effect of bifidobacterium genomic DNA on umbilical cord blood mononuclear cell (CBMC), and investigate the immunoregulation of bifidobacterium DNA and explore possible mechanisms by which bifidobacterium acts against allergic reaction. METHODS: Bifidobacterium genomic DNA (bDNA) and human DNA (hDNA) were extracted with phenol/chloroform/isoamyl alcohol and stored at -20 degrees C for later use. Parts of bDNA were completely digested with DNaseI (d-bDNA) at 37 degrees C. CBMCs were isolated with Ficoll from umbilical cord blood and incubated at 37 degrees C in a 5% CO2 humidified incubator. These cells were divided into four groups, control group: without any stimulant; bDNA group: stimulated with 25 microg/ml bDNA; d-bDNA group: stimulated with 25 microg/ml d-bDNA; hDNA group: stimulated with 25 microg/ml hDNA. The cells were stimulated with different stimulants in vitro, at the end of incubation culture supernatant and cells were collected. IL-12 and IL-10 levels in the culture supernatant were measured by enzyme linked immuno sorbent assay (ELISA); cells secreting IL-4 and IFN-gamma were counted by enzyme linked immunospot (ELISPOT) assay; and total RNA was isolated from the cells to assay T-bet and GATA3 mRNA expression levels by reverse transcription polymerase chain reaction (RT-PCR). RESULTS: Six hours after stimulation there was no significant difference in IL-12 level in supernatant among the four groups; 12 hours after stimulation, IL-12 level in supernatant of bDNA treated group was significantly higher than that of each of the other groups, so were the results obtained at 24 hours and 48 hours after stimulation (P < 0.05). No significant difference could be detected in IL-12 level in supernatant among the other 3 groups. On the other hand, 6 hours after stimulation there was no significant difference in IL-10 level in supernatant among the four groups. But 12 and 24 hours after stimulation IL-10 level in supernatant of bDNA treated group was lower than that of each of the other groups, but the difference was not statistically significant. The count of IFN-gamma secreting cells of bDNA treated group was higher than that of the other groups, while IL-4 secteting cells of bDNA treated group were lower than that of the other groups. After bDNA stimulation, nuclear factor T-box expressed in T cells (T-bet) mRNA expression was conspicuously enhanced as compared to the other three groups (P < 0.05). GATA3 mRNA transcription in CBMC had no significant change after bDNA stimulation. CONCLUSION: bDNA could promote secretion of Th1 type cytokine IL-12, while Th2 type cytokine IL-10 level of cell supernatant was decreased. bDNA could stimulate secretion of IFN-gamma by CBMC and inhibit secretion of IL-4. T-bet mRNA expression was highly enhanced after bDNA stimulation. bDNA could upregulate Th1 type response, which may be one of important mechanisms by which bifidobacterium inhibit allergic response.

[Gene diagnosis of X-linked agammaglobulinemia].

OBJECTIVE: X-linked agammaglobulinemia (XLA) is the most common disorder among primary immunodeficiency diseases, which is caused by mutations in the cytoplasmic Bruton's tyrosine kinase (BTK) gene, characterized by lack of mature, circulating B lymphocytes, hypogammaglobulinemia, and recurrent bacterial infections. Mutations in BTK are highly diverse. In this study, genetic analysis was performed on BTK to realize the feature of gene mutation of XLA in Mainland of China. METHODS: Seven patients from 7 different families were enrolled in the analysis. RT-PCR was employed to reverse transcript total RNA and 8 couples of primers were designed for PCR. PCR products were sequenced and the mutation sites were identified. RESULTS: Seven completely different mutations were identified in the 7 patients. All the 7 mutations located at BTK coding region. Three of the 7 mutations were located in pleckstrin homology functional area, 2 mutations located in BTK area, and in other 2 cases at Src homology 2 and Src homology 3 regions, respectively. The mutations in 2 of 7 cases were in exon 18, and the others were in exon 2, 5, 6, 8 and 10, respectively. The types of mutation included 3 missense (L11P, I590F and Y591S), two nonsense (W281X, and Q234X) mutations resulting in premature stop codons. A 10-base pair nucleotides duplicated insertion located between the nucleotide 596 and 597 resulting in frameshift, and a 8 base pair deletion at the nucleotide position 472 resulting in frameshift. Four of the 7 mutations are novel mutation types and have not been reported. Four of 7 mothers were analyzed, 3 of them were carrier and 1 was normal. CONCLUSION: The patients enrolled in this study had classical clinical features of XLA. All the 7 identified mutations located at BTK coding region and 4 of them were novel mutations. Genetic analysis can be used for diagnosis of XLA and distinguish it from other hypogammaglobulinemia.