Different Biological Characteristics of Macrophages from BALB/c Mice and Severe Combined Immunodeficient Mice.

BACKGROUND: Our previous research has found that absence of lymphocytes during development may affect innate activity of immune cells. To better understand the effect of lymphocyte defects on macrophages during development, we compared the biological characteristics of peritoneal macrophages (PMa) from BALB/c and severe combined immunodeficient (SCID) mice. METHODS: Cytokines such as TNF-α, IL-6, and IL-10 produced by PMa and mRNA of those cytokines of PMa, phagocytosis of chicken red cells by PMa, and cell surface markers of PMa from BALB/c and SCID mice were tested. LPS was used as the stimulator. RESULTS: Compared with PMa from BALB/c mice, PMa from SCID mice produced more TNF-α, IL-6, and less IL10 after LPS stimulation in vitro and exhibited decreased phagocytosis in vivo or in vitro. The MFI of cell surface markers, including TLR4, MHC-II and CD80/CD86, on PMa increased markedly after LPS injection in vivo, but was not significantly different between the two mouse strains. CONCLUSIONS: Lymphocyte defects during development lead to increased inflammatory potential and decreased phagocytosis of PMa. If BALB/c and SCID mice are used as models to compare differences in infections and inflammation, the different biological characteristics of macrophages should be considered.

Clinical and molecular features of 38 children with chronic granulomatous disease in mainland china.

PURPOSE: Chronic granulomatous disease (CGD) is an inherited disorder, with phagocytes failing to produce antimicrobial superoxide due to deficient NADPH oxidase activity. Mutations in the gene encoding CYBB are responsible for the majority of the CGD cases. To date, there have been no reports on large samples of children with CGD in China. Therefore, in this study, we described the clinical and molecular features of 38 suspected CGD patients from 36 unrelated Chinese families. METHODS: Clinical diagnosis was performed using dihydrorhodamine assays detected by flow cytometry. Molecular analysis was used to identify underlying CGD-causative genes. RESULTS: The mean age of onset in our 38 patients was 3.4 months, while the mean age at diagnosis was 31.7 months. Apart from recurrent pneumonia and abscesses, tuberculosis (TB) and Bacille Calmette-Guerin (BCG) infections were notable features in our cohort. Overall, 17 cases died and patient 1 did not participate in the follow-up period . In total, we identified 29 different CYBB gene mutations in 31 patients. We found NCF1 and CYBA mutations in 3 and 2 patients, respectively. In addition, we identified 31 carriers and prenatally diagnosed 4 CGD and 4 healthy fetuses. CONCLUSIONS: The results of our study demonstrate that children with BCG infections or recurrent TB infections should have immune function screening tests performed. Moreover, newborns with family histories of primary immunodeficiency diseases should avoid of BCG vaccination. Molecular analysis is an important tool for identifying patients, carriers, and high-risk CGD fetuses.

Regulation on expression of toll-like receptors on monocytes after stimulation with the 3-o-C12-HSL molecule from Pseudomonas aeruginosa.

Quorum sensing (QS) is a type of cell-to-cell communication. The Pseudomonas aeruginosa QS molecule N-3-(oxododecanoyl)-L-homoserine lactone (3-o-C12-HSL) has the potential to modulate the immune system of its host. However, the mechanism of that activity is yet to be fully characterized. To be able to understand this activity, we determined whether 3-o-C12-HSL has a direct effect on the immune function and the expression of toll-like receptors (TLRs) in monocytes. Monocytes were cultured with 3-o-C12-HSL at different concentrations (0, 10, 25, 50, and 100 µmol/L) for 12 h; upon exposure to 3-o-C12-HSL, IL-12 production in monocytes was inhibited, monocyte proliferation was blocked, TLR2- and 4-mRNA expressions were reduced, and TLR5-mRNA expression was increased in a dose-dependent manner. Strikingly, 3-o-C12-HSL was able to significantly induce mRNA changes in the monocytes even at the lowest concentration (10 µmol/L, P < 0.05). Interestingly, though TLR2- and 4-protein levels were reduced, TLR5 protein expression was not changed. These findings provide a new perspective toward understanding the persistence of chronic inflammation in P. aeruginosa infections. They also suggest that TLR2, 4, and 5 may not share the same signaling pathways during monocyte activation.

The antiasthma effect of neonatal BCG vaccination does not depend on the Th17/Th1 but IL-17/IFN-γ balance in a BALB/c mouse asthma model.

OBJECTIVE: This study aimed to determine whether the protective effects of the Mycobacterium bovis Bacillus Calmette-Guérin (BCG) vaccination on allergic asthma are associated with the T helper (Th) 17/Th1 balance in a murine asthma model. METHODS: BALB/c neonates were vaccinated with BCG on the first day after birth, sensitized with ovalbumin, and then challenged with allergen. The resulting airway inflammation and responsiveness were measured. The levels of IL-17 and interferon (IFN)-γ in BALF and ratio of Th17/Th1 were investigated. RESULTS: We found that although BCG neonatal vaccination inhibited airway hyperresponsiveness and inflammation following allergen challenge in a BALB/c mouse asthma model, reduced levels of Th2 cytokines were not observed. However, BCG neonatal vaccination reduced IL-17 production and increased IFN-γ production in both the bronchoalveolar lavage fluid and the lung lymphocytes in asthmatic mice. CONCLUSION: The antiasthma effects of neonatal BCG vaccination reversed the IL-17/IFN-γ imbalance in a murine asthma model but did not depend on modifying the Th17/Th1 balance.

Molecular diagnosis of severe combined immunodeficiency--identification of IL2RG, JAK3, IL7R, DCLRE1C, RAG1, and RAG2 mutations in a cohort of Chinese and Southeast Asian children.

Severe combined immunodeficiencies (SCID) are a group of rare inherited disorders with profound defects in T cell and B cell immunity. From 2005 to 2010, our unit performed testing for IL2RG, JAK3, IL7R, RAG1, RAG2, DCLRE1C, LIG4, AK2, and ZAP70 mutations in 42 Chinese and Southeast Asian infants with SCID adopting a candidate gene approach, based on patient's gender, immune phenotype, and inheritance pattern. Mutations were identified in 26 patients, including IL2RG (n = 19), IL7R (n = 2), JAK3 (n = 2), RAG1 (n = 1), RAG2 (n = 1), and DCLRE1C (n = 1). Among 12 patients who underwent hematopoietic stem cell transplantation, eight patients survived. Complications and morbidities during transplant period were significant, especially disseminated bacillus Calmette-Guérin disease which was often difficult to control. This is the first cohort study on SCID in the Chinese and Southeast Asian population, based on a multi-centered collaborative research network. The foremost issue is service provision for early detection, diagnosis, management, and definitive treatment for patients with SCID. National management guidelines for SCID should be established, and research into an efficient platform for genetic diagnosis is needed.

Clinical characteristics and molecular analysis of three Chinese children with Omenn syndrome.

Omenn syndrome (OS) is a rare autosomal recessive genetic disorder and presents symptoms of severe combined immunodeficiency characterized by erythrodermia, eosinophilia, hepatosplenomegaly, lymphadenopathy, and elevated serum IgE levels. OS has been found to be caused by mutations in RAG1 or RAG2 gene that result in partial V(D)J recombination activity. No study on OS has been reported in Chinese children so far. In this study, the genotype and phenotypes of three infants with OS from three unrelated Chinese families were investigated. All the three children had most of the characteristics of OS except normal serum IgE level. Compound heterozygosity mutations in RAG1 gene (1983 G>A; 2444 C>T and 2219 C>T; 3127 C>G) were identified in two cases, and a homozygous deletion mutation with a premature stop codon was found at residue 2302 of RAG1 gene (2302delT, I729X) in the remaining case, including three novel mutations (2302delT, I729X; 2219 C>T, R699W; and 3127 C>G, Y1001X). Spectratyping analysis of T-cell receptor ß-chain variable region (TCRVß) gene rearrangement was performed in case 1 and case 2. All the 25 TCRVß subfamilies presented monoclonal or oligoclonal peaks in case 1 and 11 TCRVß subfamilies were very weak or even absent in case 2. This was the first report about OS in Chinese children. Molecular genetic testing represents an important tool for early confirmed diagnosis and may allow accurate carrier detection and prenatal diagnosis.

The polymorphism of IL-17 G-152A was associated with childhood asthma and bacterial colonization of the hypopharynx in bronchiolitis.

OBJECTIVE: Interleukin (IL)-17 plays an important role in the pathogenesis of asthma. We investigated the association between single-nucleotide polymorphism (SNP) of IL-17 (rs2275913, IL-17 G-152A) and asthma-related traits. Its effect on IL-17 production was also attractive. METHODS: One hundred and sixty eight childhood asthmatic patients, 144 bronchiolitis patients, and 205 healthy controls were recruited in this study. SNP rs2275913 was genotyped by polymerase chain reaction-restriction fragment length polymorphism. Peripheral blood mononuclear cells (PBMCs) from parts of healthy controls with different genotype were isolated and cultured with phytohaemagglutinin (PHA) for detection of IL-17 in the supernatants. RESULTS: SNP rs2275913 was associated with asthma (P = 0.03) in genotype frequency test. Children with homozygous A were 2.29 times more likely to have asthma than others (95% confidence interval 1.39-3.78, P = 0.001). The strength of associations was moderately higher by allergy comorbidity. Furthermore, SNP rs2275913 A allele was associated with abnormal lung function and serum total IgE in asthmatics, although the production of IL-17 by PHA-induced PBMC seemed to be not different among individuals with different genotypes. The distribution of SNP rs2275913 in bronchiolitis was marginally statistically different with controls and demonstrated a tendency close to that in asthma. Higher Streptococcus pneumoniae and Moraxella catarrhalis detection rates were shown in bronchiolitis patients with homozygous A allele than those with other genotypes (20.8% vs. 3.7%, P < 0.01 and 20.8% vs. 6.2%, P = 0.03). CONCLUSION: The preliminary results demonstrate that IL-17 SNP rs2275913 was associated with several asthma-related traits and confers genetic susceptibility to childhood asthma. It may be used to develop markers to assess the risk of asthma, especially in the bronchiolitis population. It may be a potential bridge to connect the bacterial colonization and the onset of asthma.

Clinical characteristics and genotype-phenotype correlation in 62 patients with X-linked agammaglobulinemia.

INTRODUCTION: X-linked agammagobulinemia (XLA) is a primary immunodeficiency disorder caused by Bruton's tyrosine kinase (Btk) gene mutation. Recent studies suggested genotype-phenotype correlation in XLA, but a definitive association remains controversial. PATIENTS AND METHODS: We examined the relationship between specific Btk gene mutations and severity of clinical presentation in 62 patients with XLA. Disease severity was assessed by the age of disease onset and the presence of severe infections, while mutations were classified into severe and mild based on structural and functional consequence by bioinformatics analysis. RESULTS: Fifty-six Btk mutations were identified in 62 patients from 57 kindreds. Variation in phenotypes was observed, and there was a tendency of association between genotype and age of disease onset as well as occurrence of severe infections. CONCLUSION: A critical analysis of the circumstances upon presentation also revealed that under-recognition of recurrent infections and relevant family history are important hurdles to timely diagnosis of XLA.

Genetic variability of respiratory syncytial viruses (RSV) prevalent in Southwestern China from 2006 to 2009: emergence of subgroup B and A RSV as dominant strains.

Respiratory syncytial virus (RSV) is the most commonly identified viral agent in young children with acute respiratory tract infections (ARIs) and often causes repeated infections throughout life. This study investigated the genetic variability of the attachment (G) protein gene among RSV strains prevalent in southwestern China. Reverse transcription-PCR (RT-PCR) for a fragment of the RSV G gene was performed with nasopharyngeal aspirates (NPAs) collected from children with ARIs hospitalized in Chongqing Children's Hospital, Chongqing, China. A total of 1,387 NPA specimens were collected from April 2006 to March 2009, and 439 (31.7%) were positive for RSV. During the study period, subgroup A and B viruses accounted for 79.5% (349/439) and 19.8% (87/439) of the total positive samples, respectively. Both subgroup A and B viruses were identified in three samples. Subgroup A viruses were predominant during two epidemic seasons (2006 to 2008), while subgroup B strains prevailed during the 2008-2009 epidemic season. Phylogenetic analyses showed that all 30 group A strains could be clustered into one genotype, genotype GA2, and 30 group B strains could be clustered into three genotypes, genotypes GB1, GB3, and BA, among which 17 genotype BA strains were detected from 23 group B strains selected during the 2008-2009 epidemic season. The G gene of genotype BA was predicted to encode proteins of five different lengths. These data suggest that group A RSV likely predominated in southwestern China and that a new genotype of subgroup B with a 60-nucleotide insertion, named BA-like virus, became the dominant genotype in the 2008-2009 epidemic season.

Analysis of clinical and molecular characteristics of Wiskott-Aldrich syndrome in 24 patients from 23 unrelated Chinese families.

The clinical data of 24 children with Wiskott-Aldrich syndrome (WAS) from 23 unrelated Chinese families were reviewed in the present study. WAS protein (WASP) expression in peripheral blood mononuclear cells was examined by flow cytometry (FCM); WASP gene was amplified by PCR and directly sequenced to analyze mutations in the WASP gene in patients and their female relatives. FCM analysis of 21 patients showed that 18 cases were WASP-negative, and three had partially WASP expression. WASP gene analysis revealed mutations in 23 patients, including five missense mutations, four nonsense mutations, four deletion mutations, three insertion mutations, six splice site mutations, and one complex mutation, among which, 20 unique mutations were detected, including seven novel mutations (168 C>A, 747-748del T, 793-797del C, 1185 ins C, Dup 1251-1267, 1277 insA and 1266 C>G; 1267-1269del C). Five WAS children underwent stem cell transplantation. After 2 months of transplantation, WASP expression was restored to normal in all five patients whereas one patient died of cytomegalovirus-induced interstitial lung disease. WASP gene analysis can make a definite diagnosis of WAS and identify mutation carriers, beneficial for timely treatment and genetic counseling for children with WAS.

Nebulized hypertonic saline/salbutamol solution treatment in hospitalized children with mild to moderate bronchiolitis.

BACKGROUND: The objective of this study was to determine the efficacy and safety of nebulized 3% hypertonic saline solution and salbutamol in the treatment of mild to moderate bronchiolitis. METHODS: In a randomized controlled trial, 93 infants with mild to moderate bronchiolitis were divided into two groups. The infants received inhalation of 2.5 mg (0.5 mL) salbutamol dissolved in either 4.0 mL normal (0.9%) saline (control group, n= 43) or 4.0 mL hypertonic (3%) saline (treatment group, n= 50). The therapy was repeated three times daily until discharge. Cough, wheezing, pulmonary physical signs, and the length of hospital stay were recorded. RESULTS: Wheezing remission time was 3.8 + or - 1.1 days in the control group and 2.7 + or - 0.9 days in the treatment group (P < 0.01). Cough remission time was 6.3 + or - 0.9 days in the control group and 5.3 + or - 0.8 days in the treatment group (P < 0.01). The moist crackles disappeared at 5.4 + or - 0.8 days in the treatment group versus 6.2 + or - 0.9 days in the control group (P < 0.01). Furthermore, the average length of hospital stay decreased from 7.4 + or - 1.5 days in the control group to 6.0 + or - 1.2 days in the treatment group (P < 0.01). No obvious adverse effects were observed. CONCLUSIONS: Inhalation of nebulized 3% hypertonic saline solution and salbutamol is a safe and effective therapy for patients with mild to moderate bronchiolitis.

Clinical and molecular characteristics of 35 Chinese children with Wiskott-Aldrich syndrome.

BACKGROUND: Wiskott-Aldrich syndrome (WAS) is a rare primary immunodeficiency disease, with an incidence of 4/1,000,000 live male births. In China, an estimated number of 35 babies with WAS are born each year, but likely many remain undiagnosed. OBJECTIVES: The objectives of study were to review the clinical and molecular characteristics of a cohort of Chinese children with WAS and to describe the long-term outcome of those who underwent hematopoietic stem cell transplant (HSCT). MATERIALS AND METHOD: Records of 35 patients diagnosed with WAS during 1991-2008 were reviewed. Genetic diagnosis was established by direct gene sequencing. RESULTS: All patients had classical WAS phenotype. WASP mutations were identified in 33 patients from 29 families. Nine patients underwent HSCT at a mean age of 22.1 months (match-unrelated donor, n = 5; mismatched related donor, n = 2; matched-sibling donor, n = 2). Post-transplant immune hemolytic anemia and thrombocytopenia occurred in three patients with complete resolution. All patients survived without significant long-term complications and had full platelet, T and B lymphocyte recovery within 2 years post-transplant. CONCLUSION: In the past decade, there has been significant improvement in clinical and genetic diagnosis of WAS in Chinese. We demonstrated excellent long-term survival in patients who underwent HSCT. Early workup for transplant should be advocated for children with classical WAS before they suffer from major disease complications and morbidities.

Lipopolysaccharide induces IL-6 production in respiratory syncytial virus-infected airway epithelial cells through the toll-like receptor 4 signaling pathway.

Respiratory syncytial virus (RSV) is a leading cause of bronchiolitis in young children. Microbial agents such as endotoxin and RSV are implicated in airway inflammation during the development of reactive airway disease (RAD) later in childhood. Toll-like receptors (TLRs) are involved in an inflammation cascade through pathogen-associated molecular pattern recognition including lipopolysaccharide (LPS) and viral components. In this study, we investigated the expression of TLRs and cytokine-chemokine production profiles of RSV-infected epithelial cells. In live-RSV infected human tracheal epithelial cell line (9HTEo), TLRs 1-10 mRNA levels were up-regulated in a time-dependent manner compared with ultraviolet (UV)-inactivated RSV. RSV was shown to alter TLR4 membrane and cytosolic location in epithelial cells. Stimulating RSV-infected epithelial cells with TLR4 agonist LPS increased synthesis of IL-6, IL-8, and reduced regulated on activation, normal T cell expressed and secreted (RANTES) production. TLR4 neutralizing antibody HTA125 and TLR4-targeting RNA interference experiments revealed that TLR4 signaling pathway played a predominant role in mediating LPS-induced-IL-6 production of RSV infected epithelial cells. Altogether, our studies indicated that TLR4 play a critical role in leading LPS mediated-IL-6 response in RSV infected-epithelial cells and might be an important factor influencing the cytokine-chemokine profile of epithelial cells interacting with virus and endotoxin, which is correlated with phenotypes of RSV diseases.

Mannose-binding lectin polymorphisms and recurrent respiratory tract infection in Chinese children.

UNLABELLED: In order to establish the reference value of mannose-binding lectin (MBL) serum level in children and to investigate the correlation between the polymorphisms of MBL2 gene and serum MBL level in healthy Chinese of Han ethnic group and in children of Chinese Han ethnic group with recurrent respiratory tract infections (RRTI), the concentration of oligomerized MBL was measured by enzyme-linked immunosorbent assay, and MBL2 gene polymorphisms were analyzed by restriction fragment length polymorphism of polymerase chain reaction and polymerase chain reaction-sequence specific primer. The median MBL levels in the 470 normal children were 2536 ng/ml, and the P(2.5)-P(97.5) was 161-5,070 ng/ml. Our research showed that two promoter polymorphisms at -550, -221 of start codon and coding variants at codon 54 of MBL2 gene affected the protein level significantly and the most frequent genotype in Hans is HYPA/HYPA. Our results also showed that serum MBL level was significantly lower in recurrent respiratory tract infections patients compared with healthy controls (Z, -3.04, P = 0.002). The frequency of the promoter LXP haplotype and the B allele was significantly higher in RRTI patients than in controls (chi (2) 4.05, P < 0.05; OR 1.63, 95%CI 1.01 approximately 2.62; chi (2) 4.27, P < 0.05; OR 1.94, 95%CI 1.02 approximately 3.68). CONCLUSION: We have established that the reference value of serum MBL level in Chinese aged between 0 and 6 years (161-5,070 ng/ml), and we found that LXP and the B are risk factors for RRTI.

Susceptibility to mycobacterial infections in children with X-linked chronic granulomatous disease: a review of 17 patients living in a region endemic for tuberculosis.

BACKGROUND: Chronic granulomatous disease (CGD) is a rare disorder of phagocytic oxidative bursts leading to recurrent pyogenic infections. Affected individuals are most prone to infections caused by staphylococci, Salmonella, Candida, and Aspergillus, but previously we observed a high incidence of Mycobacterium tuberculosis infection in Chinese children with CGD. OBJECTIVE: To determine the spectrum of infections in patients with X-linked CGD, with an emphasis on mycobacterial infections, and to review all CYBB gene mutations identified in our center. RESULTS: From 1988 to 2005, 17 Chinese male children were diagnosed to have X-linked CGD. Fifteen mutations were identified, including 3 splice site defects (IVS1-1G>C, 266G>A, IVS3-1G>A), 5 missense mutations (591T>C, 627T>A, 949T>A, 1039T>A, 1512G>C), 3 nonsense mutations (882C>T, 1451C>A, 1569G>T), 1 insertion (756_757insA), and 3 deletions (660_662delTTC, 727delT, 1341delT). Eight of these were novel mutations. Recurrent pneumonia, lymphadenitis, and bacterial skin abscess were the commonest types of infection. Seven patients had tuberculosis (TB). Seven patients had prolonged scarring or abscess formation at the Calmette-Guérin bacillus (BCG) injection site, and 1 had disseminated BCG infection. Three patients had pulmonary aspergillosis. Four patients underwent hemopoietic stem cell transplantation, but 2 died of complications. CONCLUSIONS: Patients with CGD are susceptible to TB and BCG complications. Our observation suggests that oxidative burst is probably important in host defense against mycobacterial infections. Because interferon-gamma is the key cytokine involved in mycobacterial immunity, there may be a stronger indication for its use in CGD patients living in areas endemic for TB.

High seroprevalence of human metapneumovirus infection in children in Chongqing, China.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: hMPV is a common and significant respiratory pathogen in Chinese children. Almost all individuals are exposed to hMPV by age 6 years.

[Toll-like receptor 4 expression and function of respiratory syncytial virus-infected airway epithelial cells].

OBJECTIVE: To observe the epithelial Toll like receptor (TLR)4 expression changes and the signaling pathway function after respiratory syncytial virus (RSV) infection and to explore the mechanisms of RSV-induced airway inflammation. METHODS: 9HTEo-human tracheal epithelial cell line was infected by RSV (MOI = 10), and TLR1-10 mRNA were detected by RT-PCR assay at 3 h post RSV infection. TLR4 mRNA was detected by real time Q-PCR assay at 3 h, 6 h and 9 h post RSV infection, and TLR4 protein expression and cell apoptosis were determined by flow cytometry at 24 h post RSV infection. IL-8 in supernatant was detected by ELISA after RSV-infected cells exposed to lipopolysaccharide (LPS). A normal control group and a RSV infection group were set up for the RT-PCR and flow cytometry experiments, and the data were analyzed by paired t test using GraphPad 4.0 software. A normal group, a RSV group and a UV-inactivated RSV group were set up for the real time Q-PCR, experiments, and the data were analyzed by Kruskal-Wallis test. The ELISA experiments were divided into 4 groups including a normal control, a RSV, a LPS stimulation, and a RSV plus LPS co-stimulation groups, and the data were analyzed by One-way ANOVA test. RESULTS: (1) TLR2-10 mRNA level was significantly up-regulated (t value of TLR2-10: 3.49 -14.47, P < 0.05), especially TLR-2, 6 enhanced expression, compared with the normal epithelial cells. Real time Q-PCR assay showed that TLR4 mRNA started to increase at 3hr (Kruskal-Wallis test value = 8.82, P < 0.05, n = 6) and significantly elevated at 9 hour (Kruskal-Wallis test value = 6.62, P < 0.05, n = 6). UV inactivated-RSV had no effect on the TLR4 mRNA level. (2) Flow cytometry showed that membrane TLR4 mean fluorescence intensity (MFI) increased (RSV: 1.27 +/- 0.48, normal: 0.97 +/- 0.25; t = 2.39, P > 0.05, n = 10) while cytoplasmic TLR4 MFI simultaneously decreased (RSV: 3.08 +/- 1.38, normal: 3.36 +/- 1.31, t = 2.92, P = 0.225, n = 10). Percentage of membrane TLR4-positive cells was higher in RSV infected population [RSV: (11.99 +/- 7.74)%, normal: (1.16 +/- 0.47)%, Mann-Whitney t value = 0.001, P < 0.01, n = 8], most (93.32 +/- 1.7)% of which were Annexin V positive. IL-8 was significantly induced in the RSV plus LPS costimulation group compared with RSV group (F = 59.29, P < 0.01, n = 3). CONCLUSIONS: RSV induced epithelial TLR4 up-regulation, localization changes from cytoplasm to membrane, IL-8 secretion through TLR4 signaling pathway and epithelial cell apoptosis in membrane TLR4 positive population. These results indicate TLR4 is involved in RSV-induced acute or chronic epithelial-dependent inflammation, which might contribute to acute or chronic airway inflammation.

Inhibition of respiratory syncytial virus of subgroups A and B using deoxyribozyme DZ1133 in mice.

Respiratory syncytial virus (RSV) commonly infects the upper and lower respiratory tracts. Currently, there is no effective treatment available. Deoxyribozymes are a potential therapeutic for RSV and their activity is based on the ability to bind and cleave complementary RNA sequences to inhibit protein expression. DZ1133 is a deoxyribozyme that targets the conserved genomic RNA sequence of the RSV nucleocapsid protein and has been shown to significantly inhibit various strains of RSV including subgroups A and B, standard A2 and CH18537 strains, and CQ381513, CQ381170, BJ01 and BJ04 strains. Treatment with DZ1133 decreased viral plaque formation in lungs of RSV-infected BALB/c mice. In addition, viral mRNA expression was reduced, airway inflammation was alleviated, and leukocyte counts were reduced in bronchoalveolar lavage fluid of RSV-infected mice. The antiviral effect of DZ1133 was dose-dependent (0.2-0.8mg) and more efficient than antisense oligonucleotide inhibition of gene expression. However, levels of cytokines TNF-alpha, IFN-gamma, IL-12, and IL-10 induced by RSV infection were not affected by DZ1133 treatment. Our data demonstrate that DZ1133 is a potential therapeutic agent against both subgroups A and B RSV infection in vivo.

Inhibition of respiratory syncytial virus in cultured cells by nucleocapsid gene targeted deoxyribozyme (DNAzyme).

Respiratory syncytial virus (RSV), which presents the primary cause of bronchiolitis and pneumonia among infants and causes significant morbidity and mortality in immunodeficient patients, remains a health problem worldwide. Unfortunately, an effective vaccine is currently unavailable and pharmacologic treatment needs further optimization for RSV disease. Because RSV is a non-segmented negative-strand RNA virus, it may be sensitive to the genome RNA cleaving by DNAzyme, an artificial nucleic acids molecule with high catalytic capability of cleaving complementary RNA molecules. Thus, RSV-targeted DNAzymes potentially present as a therapeutic candidate of RSV diseases. In this study, DNAzymes targeting the RSV genomic RNA or mRNA were designed and synthesized, one of which (DZn1133) did cleave RSV RNA in vitro, inhibit the transcription and expression of F viral gene, reduce the RSV yield by about 7 logs and protect more than 90% RSV-infected Hep-2 cells from a cytopathic effect at 8 microM. Moreover, 10 wild RSV strains isolated from clinic patients including both subgroups A and B were all suppressed by DZn1133 with greater anti-RSV activity than antisense DNA or ribavirin.