Analysis of prenatal diagnosis for seven high-risk fetuses with Wiskott-Aldrich syndrome / 中华儿科杂志

<p><b>OBJECTIVE</b>To investigate the value of gene analysis of amniotic fluid exfoliated cells and WASP detection from cord blood in prenatal diagnosis of high-risk fetus with Wiskott-Aldrich syndrome.</p><p><b>METHOD</b>Seven patients with Wiskott-Aldrich syndrome were diagnosed by gene analysis and WASP detected by flow cytometry from 2008 to 2010. After detailed inquiry for medical history and gene analysis of related family members, seven pedigree trees were drawn, including 15 carriers of abnormal genes. From 2008 to 2011, seven samples of amniotic cell gotten by amniocentesis were collected from seven high-risk pregnant women with abnormal gene during 18 to 20 gestational weeks. WASP gene was amplified by polymerase chain reaction (PCR) from DNA of amniotic cell gotten and sequencing was performed directly on the PCR products forward and reversely. Embryo blood sample was collected from one high-risk fetus by needle puncture of umbilical blood vessel and WASP expression was detected by flow cytometry. Karyotyping was performed in amniotic cell gotten cultivated by orthotopic slice and G band staining. Gene analysis of WASP, WASP expression detected by flow cytometry and evaluation of immune function were reexamined in high-risk fetus after delivery.</p><p><b>RESULT</b>Amniocentesis and culture of amniotic cell succeeded in all the seven fetuses. Gene analysis and karyotyping showed that one male fetus and four female fetuses were normal and two female fetuses were carriers. WASP expression detected from embryo blood sample of the patient was normal. After delivery, the result of gene analysis, WASP detection and evaluation of immune function was the same as that of prenatal diagnosis.</p><p><b>CONCLUSION</b>Karyotyping, gene analysis and WASP detection of cord blood can provide reliable service of prenatal diagnosis for high-risk pregnant women with Wiskott-Aldrich syndrome.</p>

Analysis of viral etiology of severe pneumonia in infants and young children in Chongqing area / 中华儿科杂志

<p><b>OBJECTIVE</b>To investigate the prevalence of viral infections and putative association of viral infection with illness severity in young children with severe lower respiratory tract infection (LRTI) in Chongqing.</p><p><b>METHOD</b>Respiratory secretion specimens were collected from 119 hospitalized patients with severe pneumonia from December 2006 to March 2008.After being processed, the samples were detected for respiratory viruses including respiratory syncytial virus (RSV), adenovirus (ADV), human metapneumovirus (hMPV), human bocavirus (HBoV), parainfluenza virus 1, 2, 3 (PIV 1, 2, 3), influenza virus A and B (IVA and IVB) either by PCR or RT-PCR. Clinical data were analyzed along with virological data by using appropriate statistical methods.</p><p><b>RESULT</b>Viral pathogens were identified in specimens of 86 (72.3%) cases, among which RSV was detected in 49 (41.2%) patients. More than one virus was detected in 23 individual (26.7%) samples, of which 19 were dual positive for RSV and another virus. Bacterial cultures were performed for 69 patients. Both bacterial and viral pathogens were identified in 53 (76.8%) patients. Bacterial and viral coinfection was demonstrated in samples from 41 (59.4%) cases.</p><p><b>CONCLUSION</b>Viral pathogens are the main etiology of severe pneumonia in young children in Chongqing area during the study period. RSV was the most frequent viral pathogens, followed by ADV and hMPV. Coinfection with respiratory common viruses was relatively common, though co-infection with viruses did not appear to aggravate the patients' condition.</p>

Analysis of mutation in heavy chain-micro (microHC) gene in a Chinese patient with congenital agammaglobulinemia / 中华儿科杂志

<p><b>OBJECTIVE</b>Mutation in the heavy chain micro (microHC) gene causes a rare type of autosomal recessive agammaglobulinemia. Here we report the molecular and clinical characterization of a compound heterozygous mutation in the microHC gene in a patient with autosomal recessive agammaglobulinemia firstly from China.</p><p><b>METHOD</b>A one-year and ten-month-old male patient and his parents were enrolled in this study. No mutation was found in BTK gene. The microHC gene of the patient and his parents were amplified by polymerase chain reaction (PCR) from genomic DNA. Reverse transcription polymerase chain reaction (RT-PCR) was used to amplify the microHC transcripts. Sequencing was performed directly on the PCR products bidirectionally.</p><p><b>RESULTS</b>Since 8 months of age, the patient had had recurrent fever and persistent cough. He suffered an acute right hemiplegia at 11 months of age and swelling and pain of left hip joint and right knee joint at one year and eight months of age. Cerebrospinal fluid routine examination showed that total cell count was 18 x 10(6)/L [normal range (0 - 15) x 10(6)/L], leukocyte count 7 x 10(6)/L [(0 - 15) x 10(6)/L] and biochemical examination showed protein 0.14 g/L (0.15 - 0.45 g/L), glucose 4.68 mmol/L (2.44 - 4.44 mmol/L) and chloride 116.3 mmol/L (120 - 132 mmol/L). Mycobacterium bovis was identified negative by cerebrospinal fluid smear examination. No obvious abnormity was detected on skull CT examination. Hydrothorax examination showed that total cell count was 848 x 10(6)/L, leukocyte count 785 x 10(6)/L and protein 30.8 g/L (< 30 g/L). Poliovirus isolation from stool sample of the patient was negative. The serum immunoglobulin (Ig) profile was IgG 181 mg/L (normal range, 5.09 - 10.09 g/L); IgM 28.8 mg/dl (400 - 1260 mg/dl) and IgA 22 mg/dl (310 - 670 mg/dl), IgE 4.6 U/ml (normal range < 150 U/ml). There were no B-cells but normal percentage of T-cells (67%) and NK cells (32%) were present in the peripheral blood. The patient had a compound heterozygous mutation in the microHC gene:on one allele, there was an alternative splice site mutation in exon 4 (1956 G > A), for which the patient's father was a carrier. Whereas on the other allele, an insert mutation between 170 and 175 in exon 1 with a premature stop codon (170 - 175 insert C, L11fs60X) was identified, for which the patient's mother was a carrier. The insert mutation in exon 1 of microHC gene was firstly reported. To detect the effect of the splice site mutation in exon 4, microHC cDNA of the patient was amplified by semi-nested PCR. The PCR products were purified and sequenced directly. A 136 bp of intron 4 was found in the transcripts and only the secreted isoform with a missense substitution is present in the patient, while synthesis of the membrane isoform is completely abolished.</p><p><b>CONCLUSION</b>This is the first case with autosomal recessive agammaglobulinemia with compound heterozygous mutation in the microHC gene reported from China. A novel mutation in exon 1 was found.</p>

A compound heterozygosity mutation in the interleukin-7 receptor-alpha gene resulted in severe combined immunodeficiency in a Chinese patient / 中华儿科杂志

<p><b>OBJECTIVE</b>Mutation in the interleukin-7 receptor-alpha (IL-7R alpha) chain causes a rare type of severe combined immunodeficiency (SCID) with presence of NK cells in the peripheral blood. Here we report the molecular and clinical characterization of a compound heterozygosity mutation in the interleukin-7 receptor-alpha gene that resulted in SCID in a patient firstly from China.</p><p><b>METHOD</b>A 5 month-old male patient and his parents were enrolled in this study. Since 15 days of age, the patient had had recurrent fever, persistent cough and diarrhea. He was in poor general condition with pyorrhea and ulceration of the BCG scar. His brother died of severe infection at 4 months of age. He was initially diagnosed as SCID according to clinical manifestation and immunological analysis. A panel of SCID candidate genes including IL-2RG, RAG1/RAG2 and IL-7R alpha of patient and his parents were amplified by polymerase chain reaction (PCR) from genomic DNA. Reverse transcription polymerase chain reaction (RT-PCR) was used to amplify the IL-7R alpha transcripts. Sequencing was performed directly on the PCR products forward and reversely.</p><p><b>RESULT</b>The serum immunoglobulin (Ig) profile was IgG 6867 mg/L (normal range, 3050 - 8870 mg/L); IgM 206 mg/L and IgA 249 mg/L, IgE 2.3 IU/ml (normal range < 150 IU/ml). The patient was treated with IVIG previously. There were no T-cells but increased percentage of B-cells (58%) and NK cells (42%) in the peripheral blood was found. Needle biopsies from enlarged axillary lymph node was identified positive for Mycobacterium bovis under microscope and by culture. The patient had a compound heterozygosity mutation in the IL-7R alpha gene:on one allele, there was a splice-junction mutation in intron 4 (intron 4(+1)G > A), for which his father was a carrier; whereas on the other allele, a nonsense mutation at position 638 in exon 5 with a premature stop codon (638 C > T, R206X) was identified, for which his mother was a carrier. The splice-junction mutation in intron 4 of IL-7R alpha was firstly reported. The IL-7R alpha mRNA expression of the patient was remarkably reduced whereas the parents had relatively normal IL-7R alpha mRNA expression. IL-7R alpha cDNA of the patient was amplified by nested PCR. The PCR products were purified, cloned with a TA Cloning Kit and sequenced directly. A 64 bp deletion was found in exon 4 of IL-7R alpha. No mutation was found in IL-2RG and RAG1/RAG2 of the patient and his parents.</p><p><b>CONCLUSION</b>This is the first case with a compound heterozygosity mutation in the IL-7R receptor alpha gene and T-B+NK+ phenotype from China. Intron 4(+1)G > A was a novel mutation.</p>

A novel missense mutation of FOXP3 causes immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome in a Chinese child / 中华儿科杂志

<p><b>OBJECTIVE</b>To investigate variation of FOXP3 and it's expression in male children presented with severe and early-onset enteropathy, rash with or without insulin-dependent diabetes mellitus (IDDM).</p><p><b>METHODS</b>Four male children presented with severe and early-onset enteropathy, rash, with or without IDDM were subjected to detection of FOXP3 expression on the PBMC by flow cytometry and FOXP3 gene analysis. The maternal gene analysis was subsequently performed once the variant FOXP3 gene was found. All 11 exons and splice sites within FOXP3 gene were amplified by polymerase chain reaction (PCR) from genomic DNA. Reverse transcription polymerase chain reaction was used to amplify the FOXP3 transcripts. Sequence analysis was performed directly on the bulk PCR products forwardly and reversely. The candidate mutation site was compared with that of 100 healthy controls to exclude polymorphism. Flow cytometry was used to determine FOXP3 expression on CD4+CD25+ T cells and the frequency of Tregs in CD4+ T cells.</p><p><b>RESULTS</b>One of the 4 patients showed a G13128A genetic variation in exon 11, which resulted in a Met370Ile substitution. No sequence variations were found at the same site in any of 100 healthy controls, indicating that the Met370Ile substitution is not a polymorphism but a novel missense mutation. The patient's mother was identified as a carrier for this mutation. There was no reduced frequency of Tregs in the peripheral blood of the patient and FOXP3 protein expression is normal as compared with controls.</p><p><b>CONCLUSION</b>A novel missense mutation of FOXP3 which causes IPEX phenotype was identified in a Chinese child according to immunologic screening and gene sequencing. Infants with early-onset IDDM and persistent diarrhea should be suspected as IPEX, FOXP3 gene analysis will be a reliable diagnostic approach to IPEX.</p>

Successful treatment of a patient with Wiskott-Aldrich syndrome using hematopoietic stem cell transplantation--case report and literature review / 中华儿科杂志

<p><b>OBJECTIVE</b>Wiskott-Aldrich syndrome (WAS) is a primary immunodeficiency diseases. The patients with classical WAS have poor prognosis. The hematopoietic stem cell transplantation is the most effective method to cure WAS at present. In this report, a patient with WAS was cured with HLA identical sibling bone marrow transplantation (BMT).</p><p><b>METHODS</b>Wiskott-Aldrich syndrome protein (WASP) was detected using flow cytometry and WASP were analyzed for the diagnosis. The bone marrow was collected from the elder sister who was the HLA identical sibling donor. A total of 4.38x10(8)/kg mononuclear cell (MNC) and 3.78x10(6)/kg CD34+ cells were collected and transfused into the patient after the conditioning regimen with busulfan/cyclophosphamide. Cyclosporine only was used for graft-versus-host disease prophylaxis. WASP and short tandem repeats (STR) were detected as the evidence of engraftment.</p><p><b>RESULTS</b>The diagnosis was WAS: WASP (-IVS9+2T>C, WASP-negative). The patient received busulfan/cyclophosphamide 9 days before the transplantation. WBC decreased to 0.1x10(9)/L in d+4; The absolute number of neutrophils (ANC) was 0.8x10(9)/L in d+13, and exceeded 1.0x10(9)/L later on. From d(-9)-d+14 the patient was dependent on platelet transfusion. From d+15 the patient's PLT>50x10(9)/L and returned to normal after d+30. In d+9-d+10 mild GVHD (I degree) occurred but subsided after the steroid treatment. From d+50, WASP was detected positive and STR showed full donor DNA chimera. Follow-up for 510 d post-transplant, the patient suffered only from mild cold twice, no eczema, no bleeding occurred. The PLT is normal and no chronic GVHD occurred. The levels of IgG, IgM and IgA of the patient were approximately normal.</p><p><b>CONCLUSION</b>The HLA-identical sibling's BMT seems to be the periorit treatment of choice for the WAS patient.</p>

High seroprevalence of human metapneumovirus infection in children in Chongqing, China / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The human metapneumovirus (hMPV) is a newly discovered respiratory viral pathogen that was first isolated in 2001 in the Netherlands. Its global distribution and long history of infection in humans have been well documented. In this study, we assessed the seropositivity of hMPV IgG antibodies in children in Chongqing, China.</p><p><b>METHODS</b>The specificity of the enzyme linked immunosorbent assay (ELISA) was first validated by using respiratory syncytial virus (RSV) infected, antigen subtracted reference serum and by performing western blotting using anti-hMPV animal serum. This assay was used to determine the presence of the IgG antibody against hMPV and RSV in 325 serum samples obtained from children aged 0 - 6 years.</p><p><b>RESULTS</b>No crossreaction was detected by ELISA between the antibodies to hMPV and RSV. The seropositivity of the anti-hMPV IgG antibody was 74.5% in children aged 0 to 5 months, 64.0% in 6 to 11 months, 72.7% in 12 to 23 months, 87.1% in 24 to 35 months and 90.3% in 3 to 6 years.</p><p><b>CONCLUSIONS</b>hMPV is a common and significant respiratory pathogen in Chinese children. Almost all individuals are exposed to hMPV by age 6 years.</p>

Characterization of human metapneumoviruses isolated in Chongqing, China / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Human metapneumovirus (hMPV) has recently been recognized as a notable respiratory pathogen in children. However, no isolation processes and only a limited understanding of hMPV epidemiology present in Chinese children are documented by far.</p><p><b>METHODS</b>Nasopharyngeal aspirates from 86 hospitalized children with acute respiratory tract infections from December 2004 to July 2005 were inoculated onto Vero-E6 cells for hMPV isolation. Total RNA was extracted from infected cells with a cytopathic effect and then subjected to a RT-PCR amplification of the N and F genes of the hMPV. Nucleotide sequences of amplified F gene products were examined using a variation analysis with the MegAlign program and the phylogenetic tree construction using the neighbor-joining algorithm with a Phylip package.</p><p><b>RESULTS</b>Six strains of hMPV were isolated from the samples during winter, spring and summer. The most common symptoms were coughing and cyanosis, and the diagnoses were bronchiolitis, bronchopneumonia, infantile asthma, or upper respiratory tract infection. All isolates were in the A2 genetic sublineage and shared a high percentage of homology with the F gene in the nucleotide (99.8%-100%) and amino acid (99.3%-100%) sequences.</p><p><b>CONCLUSIONS</b>This report indicates that hMPV is an important viral agent for acute respiratory tract infections present in Chongqing, China. Knowledge of phylogeny and genes will benefit the studies on the treatment and prophylaxis of hMPV infection.</p>

Primary immunodeficiency complicated with Bacillus Calmette-Guerin infection: identification and clinical phenotype of a case of novel interleukin-12Rbeta1 gene mutation / 中华儿科杂志

<p><b>OBJECTIVE</b>Interleukin-12 receptor beta1 (IL-12 Rbeta1) deficiency is a rare primary immunodeficiency (PID) characterized by selective susceptibility to weakly virulent organisms, including Mycobacterium bovis, BCG, non-tuberculous environmental mycobacteria and non-typhoidal salmonellosis. The present study was conducted to identify the mutation type and to analyze clinical phenotype.</p><p><b>METHODS</b>Based on the typical clinical manifestations and immunologic tests in this case, a varieties of PIDs were excluded and IL-12Rbeta1 deficiency was suspected. IL-12Rbeta1 chain expressed on Epstein-Barr virus-transformed lymphoblastoid B cell lines were detected by flow cytometric assay. The IL-12Rbeta1 gene sequences of the patient and her parents were analyzed by PCR-directed sequencing. The IL-12Rbeta1 gene sequences of the patient's younger brother also had been analyzed prenatally and after birth.</p><p><b>RESULTS</b>After inoculating BCG, the patient suffered from multiple BCG infectious lymphadenitis. There was no detectable IL-12Rbeta1 on the Epstein-Barr virus-transformed lymphoblastoid B cell lines from the patient, while only mild expression on the cell line from her mother. Sequencing analysis by using sense and antisense primers separately, a novel IL-12Rbeta1 gene mutation was found in the patient which was homozygous single nucleotide substitution, a nonsense mutation with nucleotide substitution of C to T at position 853 (853C-->T) in exon 9 leading the glutamate at position 285 to the stop codon mutation (Q285X). The parents were carriers of the mutated IL-12Rbeta1 gene. But her younger brother has normal IL-12Rbeta1 gene.</p><p><b>CONCLUSION</b>The novel IL-12Rbeta1 gene mutation is responsible for BCG infection in this case and genetic analysis is useful in carrier detection and prenatal diagnosis is feasible when the mother had a baby with identified IL-12Rbeta1 gene mutation before.</p>

Effect of neonatal BCG vaccination on phenotype and function of splenic dendritic cells of BALB/c mice / 中华儿科杂志

<p><b>OBJECTIVE</b>The impact of dendritic cells (DCs) and regulatory T cells (Tr) on the pathogenesis of asthma have been investigated over the past decades. As the professional antigen presenting cells, DCs not only prime immune response directing Th0 cells toward different T subtypes but also induce immune tolerance. As an immunoregulator, Bacillus Calmette-Guerin (BCG) has potential to be applied in allergic diseases such as asthma for prevention. Previous study showed that neonatal BCG vaccination could induce Th1/Tr1 development in mice in vivo. To further identify the mechanism of neonatal BCG vaccination on T cell subsets differentiation, the present study was designed to investigate the impact of BCG vaccination on splenic DCs development in neonatal mice.</p><p><b>METHODS</b>Neonatal specific pathogen free (SPF) BALB/c mice (2-3 days) were divided into intraperitoneal BCG-treated group, subcutaneous BCG-treated group and control group; simultaneously adult SPF BALB/c mice (6-8 weeks) were divided into intraperitoneal BCG-treated and control group. The BCG-treated mice were inoculated with 1 x 10(5) CFU BCG, the mice in control group were not inoculated with any vaccine. Four weeks post BCG vaccination, spleen cells were isolated. With flow cytometry, subtype and maturity of splenic DCs were analysed. Moreover, cells were further separated into mononuclear cell by Ficoll solution. The mononuclear cells were stimulated by 1 microg/ml lipopolysaccharide (LPS) for 18 or 10(5) CFU /ml BCG for 48 hours at 37 degrees C in a humidified atmosphere containing 5% CO2 and cytokines concentration was detected by ELISA.</p><p><b>RESULTS</b>(1) CD11c(+) CD8alpha(+) and CD11c(+) CD8alpha(-) DCs were found in spleen cells of the BALB/c mice. In comparison with the control group, the percent of CD11c(+) CD8alpha(-) DCs in intraperitoneal BCG group significantly declined (P < 0.01) and that of CD11c(+) CD8alpha(+) DCs significantly increased (P < 0.01), there were no significant difference in DC subtypes between intraperitoneal and subcutaneous BCG-vaccinated mice. In contrast, the percent of CD11c(+) CD8alpha(-)DCs markedly increased (P < 0.01) and that of CD11c(+) CD8alpha(+)DCs noticeably reduced (P < 0.01) in adult BCG-vaccinated mice. The percent of CD11c(+)CD8alpha(-)DC was significantly higher and that of CD11c(+) CD8alpha(+)DC was significantly lower in adult-vaccinated BALB/c mice than that of neonatal-vaccinated ones. (2) The expression of costimulatory molecules CD40 on CD11c(+) CD8alpha(-) DCs and CD86 on CD11c(+) CD8alpha(+) DCs in neonatal BCG-treated BALB/c mice was higher than the controls. There were no significant difference in expression of costimulatory molecules on DC between neonatal BCG-vaccinated mice. Compared with the control group, expression of CD40 and MHC-II molecules on CD11c(+) CD8alpha(-) and CD11c(+) CD8alpha(+)DC was significantly higher and that of CD86 was significantly lower in adult BCG-vaccinated mice. The expression of costimulatory molecules on DC had no significant difference between neonatal and adult BCG vaccinated BALB/c mice. (3) As compared with the control mice, concentration of IL-12p70 induced by LPS and IL-10 induced by BCG in vitro from spleen cells culture supernatant was noticeably elevated (P < 0.05) in neonatal BCG-treated BALB/c mice, but that of IL-6 did not change by LPS or BCG stimulation.</p><p><b>CONCLUSION</b>(1) By up-regulating splenic CD8alpha(+)DCs and inducing IL-12p70 and IL-10 production in BALB/c mice, neonatal BCG vaccination promoted Th1/Tr1 development. (2) The effect of BCG vaccination on DC was different between neonates and adult BALB/c mice.</p>

Analysis of neutralizing activity of sera from 0 to 6 years old children in Chongqing area against human metapneumovirus / 中华儿科杂志

<p><b>OBJECTIVES</b>Human metapneumovirus (hMPV) has been identified as one of the most important viral pathogens for acute respiratory tract infections in children. This study was to observe neutralizing activity to hMPV in sera collected from children without respiratory illnesses aged 0 to 6 years and to investigate the correlation between the level of hMPV IgG antibody and neutralizing activity in the same serum.</p><p><b>METHODS</b>Sera of 0 to 3 years old children were collected from patients hospitalized for surgery without documented respiratory illnesses, and sera of 3 to 6 years old children were remainder of serum specimens taken for health checkup. Total number of serum specimens was 325. Ten serum samples were selected for neutralizing activity detection in every age group and the sum was 50. The neutralizing titer was assessed by microneutralization assay which had been well documented previously.</p><p><b>RESULTS</b>Among 50 serum samples, eight in which hMPV IgG antibody was proved negative by ELISA previously did not show any neutralizing activity. The remaining 42 samples, as expected, showed variable neutralizing activity. The geometric mean neutralizing titer to hMPV for all the samples was 1:129. In the age group of 0-5 months, the geometric mean titer was 1:160. In the age groups of 6-11 months, 12-23 months, 24-35 months, 3-6 years, the geometric mean titers were 1:58.9, 1:114.9, 1:172.8 and 1:160.0, respectively. The level of hMPV antibody and neutralizing activity in the same serum was significantly correlated (r = 0.668).</p><p><b>CONCLUSION</b>Most 0-6 year-old children in Chongqing area have neutralizing antibody in their serum. Whether such neutralizing activity is high enough to prevent infection with different subtypes of hMPV is unknown.</p>

Allergic airway response associated with the intestinal microflora disruption induced by antibiotic therapy / 中华儿科杂志

<p><b>OBJECTIVE</b>Over the past several decades, there has been a significant increase in allergy and asthma in the world, which correlates with alterations in microflora and widespread use of antibiotics. The authors have developed a mouse model of antibiotics-induced microbiota disruption. In that model, mice were challenged by intranasal exposure to Aspergillus fumigatus allergens to explore the relation of allergic airway response and intestinal microflora disruption.</p><p><b>METHODS</b>Sixty female BALB/c mice were divided at random into 6 groups with 10 mice in each. (1) First antibiotic therapy group: the mice were given oral cefoperazone for 7 days, on day 7, mice were inoculated with Candida albicans (10(9)/ml, 50 microl) orally. (2) First control group: the mice were treated as first antibiotic therapy group, but cefoperazone and Candida albicans were replaced by saline. The mice in groups (1) and (2) were sacrificed on day 8, and cecal contents were collected for quantitative analysis of the intestinal bacterial flora. (3) Antibiotic therapy and challenge group: the mice were treated as the first antibiotic therapy group, then challenged (day 9 and 16) by intranasal exposure to Aspergillus fumigatus allergen. (4) Second antibiotic therapy group: the mice were treated as the first antibiotic therapy group, then challenged (day 9 and 16) by intranasal exposure to saline. (5) Challenge group: the mice were treated as the first control group, then challenged (day 9 and 16) by intranasal exposure to Aspergillus fumigatus allergen. (6) Second control group: the mice were treated as the first control group, then challenged (day 9 and 16) by intranasal exposure to saline. The mice in (3) - (6) group were killed for analysis of allergic airway response on day 19.</p><p><b>RESULTS</b>The quantity of Enterobacteriaceae, Enterococcus, Bifidobacterium and Lactobacillus in first antibiotic therapy group was significantly lower than that in the first control group, the quantity of Candida albicans increased in the first antibiotic therapy group as compared with the first control group. Mice intestinal microflora were disrupted with weight reduction and increased moisture in feces. After challenging with Aspergillus fumigatus allergens via intranasal inhalation, the total cell count, eosinophils, lymphocytes and neutrophils increased in BALF, especially in bronchoalveolar lavage fluid (BALF) from the mice in antibiotic therapy and challenge groups. IL-4 level in BALF from antibiotic therapy and challenge group (45.35 +/- 2.36) pg/ml was higher than that in the second control group (35.32 +/- 2.53) pg/ml. The expression of GATA-3 mRNA in the mice lung tissue (0.569 +/- 0.023) was higher than that in the second control group (0.410 +/- 0.020), and the ratios of T-bet/GATA-3 (0.578 +/- 0.021) decreased as compared with that in the second control group (0.804 +/- 0.035). IFN-gamma level in BALF from any group was not significantly different. In the absence of antibiotics, mice exposed to Aspergillus fumigatus allergen did not develop an allergic response in the airways.</p><p><b>CONCLUSIONS</b>The allergic (Th2) immune response can be induced by airway challenge with Aspergillus fumigatus allergen in the mice in which the intestinal microflora disruption resulted from antibiotic therapy, this result suggests that the intestinal microflora disruption resulted from antibiotic therapy is a risk factor for allergy and asthma.</p>

Isolation of human metapneumovirus from children with acute respiratory tract infection in Chongqing, China / 中华儿科杂志

<p><b>OBJECTIVE</b>To isolate and characterize the newly discovered human metapneumovirus (hMPV) in Chongqing, China and elucidate the clinical manifestations of hMPV infection.</p><p><b>METHODS</b>Eighty-six patients hospitalized for acute respiratory tract infection in Children's Hospital, Chongqing University of Medical Sciences from December 2004 to July 2005 were enrolled in the present study. Nasopharyngeal aspirates were collected for screening for common respiratory viruses by direct immunofluorescence assay, including respiratory syncytial virus, influenza virus types A and B, parainfluenza virus types 1, 2, 3 and adenovirus, and for inoculating onto Vero-E6 and LLC-MK2 cells for hMPV isolation. Cultures were maintained in the presence of trypsin and observed for development of cytopathic effect (CPE) for 3 weeks. Presence of hMPV was first indicated by positive CPE and subsequently confirmed by reverse transcription polymerase chain reaction targeting N and F genes. Sequence of amplified F fragments were analyzed and submitted to NCBI GenBank. The clinical findings of hMPV infection were collected and analyzed.</p><p><b>RESULTS</b>Of the collected 86 NPAs, six showed CPE characterized by clustering of infected cells, increased granules and eventuall detachment from cell monolayer and obvious syncytium formation. Successful isolation of hMPVs was confirmed with RT-PCR targeting hMPV F and N genes. Of the six hMPV-positive specimens, two were collected in winter (December, January), two in spring (May) and the other two in autumn (June, July). All the six patients were younger than 2 years of age with disease spectrum of bronchiolitis (2/6), bronchopneumonia (2/6), infantile asthma (1/6) and upper respiratory tract infection (1/6). Clinical findings included fever, cough, wheezing, polypnea, cyanosis and rales. Parainfluenza 3 and adenovirus seemed to beviral pathogens of co-infection.</p><p><b>CONCLUSION</b>Six hMPVs were successfully isolated in the mainland of China for the first time. HMPV appears to be one important viral pathogen for acute lower respiratory tract infections in young children with a detection rate of 7% (6/86) by viral isolation. The virus causes respiratory diseases similar to those caused by respiratory syncytial virus. These findings highlight the need for future investigations to define disease burden of hMPV infection and molecular epidemiology among children in China.</p>

Clinical characteristics of 12 persistently wheezing children with human bocavirus infection / 中华儿科杂志

<p><b>OBJECTIVE</b>The impact of human bocavirus (HBoV), a newly identified human parvovirus, on childhood persistent wheezing has not been identified. In this study, the clinical features of infantile persistent wheezing induced by HBoV was analyzed.</p><p><b>METHODS</b>Tracheal aspirates were collected by bronchofibroscope or nasopharyngeal (NP) aspirates from April, 2006 to January, 2007. HBoV DNA in the tracheal aspirates of 33 children with persistent wheezing and in NP aspirates of 6 children with persistent wheezing, who had at least or more than four weeks wheezing. RSV was identified by virus isolation in Hep-2 cells and antigen detetion by direct immunofluorescence assay (DIFA) which was also used for diagnosis of adenovirus, influenza A and B, parainfluenza 1, 2, 3 infection.</p><p><b>RESULTS</b>Of the 39 children with persistent wheezing, 12 cases (31%) were positive for HBoV DNA. Age of HBoV-positive patients ranged from 2 month to 1 year. The results of sequencing of PCR products proved that sequences of HBoV DNA from these 12 samples were exactly identical to the those of HBoV stored in GeneBank (accession numbers DQ000495 and DQ000496). Two cases with HBoV infection were found to be co-infected with RSV. Ten of the 12 HBoV-positive samples were collected during the period from winter to spring (1 in November, 4 in December, 2 in January and 3 in April), the other two HBoV-positive samples were collected during the period from summer to autumn (1 in May and the other in July). Seven of the 12 HBoV DNA-positive patients had fever, 5 of them had high fever. Significantly more patients with HBoV infection had fever as compared to patients with RSV infection. All the HBoV positive patients showed abnormal findings on chest X ray such as interstitial infiltrates, lung infiltration and hyperinflation. Abnormal findings on chest X ray were found in higher proportion of HBoV positive patients as compared with RSV positive patients. And other manifestations such as wheezing, cough and respiratory distress had no significant difference between HBoV and RSV infected patients.</p><p><b>CONCLUSIONS</b>This study further demonstrated that HBoV probably is a common pathogen of lower respiratory infection in children and might particularly be associated with persistent wheezing.</p>

Clinical analysis of 942 cases of Kawasaki disease / 中华儿科杂志

<p><b>OBJECTIVE</b>The study was designed to investigate the clinical characteristics and the effects of therapeutic proposal on Kawasaki disease (KD).</p><p><b>METHODS</b>Clinical features, diagnosis and treatment for totally 942 patients with KD hospitalized during Jan, 2000 to Dec, 2004 were reviewed. Clinical features of typical and incomplete KD were compared. Also, influential factors for KD resistant to intravenous immune globulin (IVIG) therapy were analyzed. Five hundred and ten cases were followed up for analyzing the prognosis of coronary artery lesion (CAL).</p><p><b>RESULTS</b>(1) 774 cases were diagnosed as typical KD, and 168 cases as incomplete KD. The incidence of infants with incomplete KD was higher than that of infants with typical KD (18.5% vs. 10.1%, P < 0.01). As compared with typical KD, the cases of incomplete KD had a long duration of fever before final diagnosis [(7.7 +/- 2.9) d vs. (7.0 +/- 2.4) d, P < 0.01], high hemoglobin level [Hb, (106.6 +/- 13.4) g/L vs. (103.5 +/- 12.3) g/L, P < 0.01], high hematocrit [Hct, (32.0 +/- 4.3)% vs. (31.0 +/- 4.0)%, P < 0.01], and high prevalence of CAL (23.8% vs. 16.8%, P < 0.05), respectively. The occurrence rate and emerging time of clinical manifestations in incomplete KD and in typical KD were presented, respectively: non-exudative conjunctivitis [occurrence rate, 64.9% vs. 93.5%; emerging time, (4.4 +/- 1.4) d vs. (4.0 +/- 1.6) d, respectively (P < 0.05 or P < 0.01)], erythema and cracking of lips [occurrence rate, 50.6% vs. 94.8%; emerging time, (4.9 +/- 1.4) d vs. (4.5 +/- 1.6) d, respectively (P < 0.05 or P < 0.01)], rash [occurrence rate, 35.1% vs. 87.7%; emerging time, (3.9 +/- 1.9) d vs. (3.4 +/- 1.7) d, respectively (P < 0.05 or P < 0.01)], erythema and edema of extremity [occurrence rate, 26.8% vs. 71.4%; emerging time, (6.7 +/- 1.5) d vs. (5.3 +/- 1.7) d, respectively (P < 0.01)], cervical lymphadenopathy [occurrence rate, 34.5% vs. 68.0%; emerging time, (4.3 +/- 2.5) d vs. (3.6 +/- 2.2) d, respectively (P < 0.05 or P < 0.01)], strawberry tongue [occurrence rate, 31.0% vs. 59.8%; emerging time, (5.6 +/- 2.2) d vs. (4.9 +/- 1.8) d, respectively (P < 0.05 or P < 0.01)], membranous desquamation of fingertips [occurrence rate, 34.5% vs. 56.3%; emerging time, (11.7 +/- 3.3) d vs. (10.3 +/- 2.7) d, respectively (P < 0.01)], and desquamation peri-anus [occurrence rate, 42.9% vs. 50.0%; emerging time, (6.7 +/- 2.7) d vs. (6.9 +/- 2.5) d, respectively (P > 0.05)]. Except for peri-anus desquamation, other clinical manifestations in incomplete KD were sporadical as compared to typical KD. (2) Six per cent (51/857) of cases were resistant to the IVIG therapy. As compared to the group responding to IVIG therapy, high prevalence of CAL (31.4% vs. 17.1%, P < 0.05), long fever duration [(10.6 +/- 3.9) d vs. (7.5 +/- 2.3) d, P < 0.01], low Hb level [(99.9 +/- 14.1) g/L vs. (104.3 +/- 12.4) g/L, P < 0.01], low Hct [(30.1 +/- 4.5)% vs. (31.2 +/- 4.0)%, P < 0.05], low platelet [PLT, (256.9 +/- 142.4) x 10(9)/L vs. (309.7 +/- 131.5) x 10(9)/L, P < 0.05], and low albumin level [ALB, (27.8 +/- 8.4) g/L vs. (33.5 +/- 6.7) g/L, P < 0.01] were found in the group resistant to IVIG therapy, respectively. (3) In patients who received IVIG 1 g/kg and 2 g/kg, the recovery rates from CAL were 83.1% and 89.7% (P > 0.05), respectively. The prevalence of CAL in those without CAL in acute and subacute stages was 0.9% and 3.5% (P > 0.05), respectively, during 2 year-follow-up period.</p><p><b>CONCLUSION</b>(1) Infants appeared to have more chances to suffer from incomplete KD. Incomplete KD had high prevalence of CAL. The peri-anus desquamation might be an important clue for early diagnosis of incomplete KD. (2) In acute stage, the influential factors for KD resistance to IVIG therapy included prolonged fever, non-elevated PLT, and persistent decrease in Hb, Hct and ALB levels. (3) Children receiving IVIG 1 g/kg and 2 g/kg had the similar effects on recovery and prevention from CAL within the first two years after KD onset.</p>

Combined effects of neonatal Bacillus Calmette-Guerin vaccination and respiratory syncytial infection on experimental asthma in mice / 中华儿科杂志

<p><b>OBJECTIVE</b>Neonatal Bacillus Calmette-Guerin (BCG) vaccination could decrease asthma prevalence in human according to "hygiene hypothesis". The authors proposed a hypothesis that effect of BCG vaccination on inhibiting asthma in human might be reversed by respiratory virus infection. The objective of this study was to observe combined effects of neonatal BCG vaccination and respiratory syncytial virus (RSV) infection on experimental asthma in mice.</p><p><b>METHODS</b>Neonatal BALB/c mice were divided into five groups. Control and ovalbumin (OVA) groups were mock-vaccinated at birth and mock-infected at 3 weeks of age. BCG + OVA group was BCG-vaccinated and mock-infected. RSV + OVA group was mock-vaccinated and RSV-infected. BCG + RSV + OVA group was BCG-vaccinated and RSV-infected. Except for control group, all the other groups underwent ovalbumin (OVA) sensitization and challenge. Airway responsiveness to inhaled methacholine was measured and bronchoalveolar lavage (BAL) was performed after the last challege. Cells in BAL fluid (BALF) were counted. Cytokines in BALF and serum OVA-specific IgE were detected by ELISA and inflammatory characteristics of lungs was scored by staining with hematoxylin and eosin.</p><p><b>RESULTS</b>(1) The numbers of total white cells, lymphocytes, monocytes, neutrophils, and eosinophils in the BALF from all OVA-sensitized/challenged groups were significantly greater than those in control (P < 0.01), and BCG + OVA group had significantly lower total white cells, lymphocytes and eosinophils as compared with other OVA-sensitized/challenged groups (P < 0.05 or 0.01). (2) All OVA-sensitized/challenged groups had significantly lower IFNgamma (P < 0.05) and higher IL-4 (P < 0.05) level in BALF as compared with control, but there was no significant difference among all OVA sensitized/challenged groups. There was no significant difference in IL-10 level between all experimental groups. (3) All OVA-sensitized/challenged groups showed significantly higher serum OVA-specific IgE titers than control (P < 0.05 or 0.01), but no significant difference was found among all OVA sensitized/challenged groups. (4) RSV + OVA and BCG + RSV + OVA groups displayed the highest airway resistance and subsequently in order as follows: OVA group, BCG + OVA group and control group in severity of airway hyperreactivity (AHR), but no significant difference was found between RSV + OVA and BCG + RSV + OVA groups. (5) Histological score of peribronchiolitis, perivasculitis, alveolitis, and peribronchial eosinophilia in all OVA-sensitized/challenged groups was significantly higher than that in control. BCG + OVA group had significantly milder peribronchiolitis and peribronchial eosinophilia than the other OVA-sensitized/challenged groups (P < 0.05) and significantly milder alveolitis than OVA and BCG + RSV + OVA groups (P < 0.05).</p><p><b>CONCLUSION</b>Neonatal BCG vaccination decreased asthmatic inflammation and AHR and RSV infection could reverse anti-asthma effect of neonatal BCG vaccination in OVA-sensitized/challenged mouse model.</p>

Effect of Anti-Respiratory Syncytial Virus Activities by RNA Interference and Ribavirin in vitro / 实用儿科临床杂志

Objective To investigate the effect of RNA interference(RNAi) on inhibiting respiratory syncytial virus(RSV) replication through comparing the anti-RSV activities between pshRNA7816 and ribavirin in cell culture system.Methods The recombinated plasmid pshRNA7816 and ribavirin was added to HEp-2 cells.Methyl thiazolyl tetrazolium(MTT) assay was used to detect cytotoxicity of pshRNA7816 and ribavirin on normal HEp-2 cells and protective effects of them on RSV infected HEp-2 cells.The effects of pshRNA7816 and ribavirin on change of cytopathogenic effect(CPE) of HEp-2 cells induced by RSV infection were observed through microscopically.Results pshRNA7816 had not significant toxicity on the growth of HEp-2 cells,but the ribavirin had significant toxicity when the concentration above 1.0 mmol/L.The pshRNA7816 and ribavirin could alleviate the CPE of HEp-2 cells induced by RSV infection,but the pshRNA7816 showed a more potent inhibition than ribavirin.The inhibition rates of pshRNA7816 were significantly higher than the maximum inhibition rate of ribavirin on RSV infection(P

Value of Heparin Blocking Attachment of Respiratory Syncytial Virus to Human Airway Epithelial Cells / 实用儿科临床杂志

Objective To investigate the mechanisms of attachment of respiratory syncytial virus(RSV) to human airway epithelial cells.Methods Attachment of RSV to CFBE was quantitatively assessed by flow cytometry.Various polyclonal and monoclonal antibodies to RSV and heparin were pre-incubated with RSV.The blocking effects of these antibodies and heparin on attachment were evaluated.Results CFBE cells reduced capacity being bound by RSV.All the antibodies used were failed to block attachment of RSV on CFBE,whereas heparin blocked RSV attachment in a dose-response manner and the blokade by heparin was almost complete at the concentration of 0.4 mg/L.Conclusions Flow cytometry provides direct evaluation of attachment without growth of virus.Heparin-like molecules on cell surface of CFBE appears to be involved in attachment between RSV and human epithelial cells.

Construction and effect of the recombinant pshRNA plasmid against respiratory syncystial virus M2-1 gene / 中华儿科杂志

<p><b>OBJECTIVE</b>Respiratory syncystial virus (RSV) is the most common cause of lower respiratory infections in infants worldwide. There is no reliable vaccine or antiviral drug against RSV at present. RNA interference (RNAi) technology is a potent method to degrade expression of the cognate mRNA. In order to inhibit the replication of RSV at gene level, the effects of specific RNAi against M2-1 gene of RSV on inhibition of viral replication in cell culture system was observed in this study.</p><p><b>METHODS</b>RSV M2-1 gene, which plays a key role in RSV transcription, was chosen in this study and was used as target gene and recombinant plasmid pshRNA7816 targeting the mRNA of RSV M2-1 gene coding sequence was constructed. The pshRNA7816 was transfected into Hep2 cells. The effects of the pshRNA7816 on changes of cytopathogenic effect (CPE) of Hep2 cell induced by RSV infection were observed microscopically. Viral plaque forming assay and MTT assay were used to detect the viral titer change and protective function of the pshRNA7816 on RSV infected Hep2 cell.</p><p><b>RESULTS</b>The recombinant RNAi plasmid pshRNA7816 which targets the mRNA of RSV M2-1 gene was successfully constructed. The pshRNA7816 significantly reduced CPE of RSV infected Hep2 cells, reduced the viral titer of RSV in the cells (P < 0.001). The pshRNA7816 raised the survival rate of RSV infected Hep2 cells (P < 0.001). Non-specific pshRNA plasmid did not show anti-RSV effects (P > 0.05).</p><p><b>CONCLUSION</b>The recombinant pshRNA7816 plasmid which targeted the mRNA of RSV M2-1 gene showed a significant and specific anti-RSV effect.</p>

Effects of Bacillus Calmette-Guerin vaccination on immune functional development of splenic T cell in neonatal mice / 中华儿科杂志

<p><b>OBJECTIVE</b>Almost every neonate receives Bacillus Calmette-Guerin (BCG) vaccination in China. The authors' previous study showed that BCG promoted cord blood monocyte-derived dendritic cells maturation and induced high level of interleukin (IL)-10, medium level of interferon (IFN)-gamma, but low level of IL-4 production by cord naive T cells. The experiments in the present study were designed to explore the effects of neonatal BCG vaccination on immune functional development of splenic T cells in mice in vivo.</p><p><b>METHODS</b>Neonatal BALB/c mice were inoculated with BCG intraperiotoneally. Four weeks later, spleen cells of mice were isolated and surface molecular markers of CD4, CD25 and CD44 and intracellular IFN-gamma, IL-10, and IL-4 in CD3(+) T cells were detected by flow cytometry. Furthermore, mRNA expression of transcription factor T-bet, Foxp3 and GATA-3 were analyzed by RT-PCR.</p><p><b>RESULTS</b>The percentage of total CD4(+) T cells decreased [(23.50 +/- 2.59)% vs. (47.38 +/- 10.41)%, P < 0.01] but the percentage of CD25(+) [(24.92 +/- 2.74)% vs. (20.27 +/- 2.85)%, P < 0.05] and CD44(+) [(89.29 +/- 2.56)% vs. (82.98 +/- 5.51)%, P < 0.05] T cells in CD4(+) T cells was higher in BCG-vaccinated mice than that in controls. Meanwhile, the percentage of IFN-gamma positive [(6.52 +/- 2.40)% vs. (3.13 +/- 2.03)%, P < 0.05] and IL-10 positive [(14.81 +/- 3.65)% vs. (10.90 +/- 1.61)%, P < 0.05] but not IL-4 positive [(1.17 +/- 0.46)% vs (1.51 +/- 0.75)%, P > 0.05] cells in CD3(+) T cells of BCG-vaccinated mice was significantly higher than that of non-BCG-vaccinated mice. In comparison with BCG-naive mice, T-bet was significantly high in BCG-vaccinated mice [T-bet/beta-actin 0.44 +/- 0.11 vs. 0.28 +/- 0.06, P < 0.05], but there was no significant difference in GATA-3 [GATA-3/beta-actin 0.46 +/- 0.08 vs. 0.50 +/- 0.10,P > 0.05] and Foxp3 [Foxp3/beta-actin vs. 0.27 +/- 0.11 and 0.30 +/- 0.16, P > 0.05] mRNA expression between the two groups.</p><p><b>CONCLUSION</b>Neonatal BCG vaccination could induce strong Th1 but weak Th2 response as reported previously. Though neonatal BCG vaccination was not capable of inducing CD4(+)CD25(+) regulatory T cell response with Foxp3 expression, it caused increase of IL-10(+) CD3(+) cells which might represent some regulatory T cells producing IL-10.</p>