Preparation and immunogenicity evaluation of C-HapS-P6 fusion protein vaccine against nontypeable Haemophilus influenzae in mice.

Nontypeable Haemophilus influenzae (NTHi) is the dominant pathogen in several infectious diseases. Currently the use of antibiotics is the main intervention to prevent NTHi infections, however with the emergence of drug resistant strains, it has compromised the treatment of respiratory infections with antibiotics. Therefore there is an urgent need to develop a safe and effective vaccine to prevent NTHi infections. We investigate the potential of C-HapS-P6 fusion protein as a vaccine for treating NTHi in murine models. PGEX-6P2/C-HapS-P6 fusion gene was constructed using overlap extension polymerase chain reaction. The recombined plasmid was transformed into Escherichia coli for protein expression. The mice were subjected to intraperitoneal immunization using purified antigens. Immunoglobulin (Ig) G in serum samples and IgA in nasal and lung lavage fluids were analyzed using enzyme-linked immunosorbent assay. Cytokine release and proliferation capacity of splenic lymphocytes in response to antigens were measured in vitro. The protective effect of the C-HapS-P6 protein against NTHi infection was evaluated by NTHi count and histological examination. The data showed that the C-HapS-P6 fusion protein increased significantly the levels of serum IgG and nasal and lung IgA, and promoted the release of interleukin (IL)-2, interferon-ϒ, IL-4, IL-5, and IL-17 and the proliferation of splenic lymphocytes compared with C-HapS or P6 protein treatment alone. Moreover, C-HapS-P6 effectively reduced the NTHi colonization in the nasopharynx and lungs of mice. In conclusion, our results demonstrated that the C-HapS-P6 fusion protein vaccine can significantly enhance humoral and cell immune responses and effectively prevent against NTHi infection in the respiratory tract in murine models.

Characteristics of Haemophilus influenzae carriage among healthy children in China: A meta-analysis.

BACKGROUND: Haemophilus influenzae (Hi) commonly causes invasive and noninvasive bacterial infections. Nationwide investigation on the carriage characteristics of H influenzae in healthy children in China is lacking. We reviewed the prevalence of H influenzae infections in this population. METHODS: PubMed, CNKI, Wanfang, VIP, and CBM databases were electronically searched to collect cross-sectional studies on the prevalence of Hi among healthy children in China from inception to November 2021. Two reviewers independently screened the literature, extracted the data, and assessed the risk of bias in the included studies. Meta-analysis was performed using Stata 14.0. RESULTS: A total of 28 studies involving 14,301 children were included, among whom there were 2878 children with Hi. The pooled carriage rate of Hi was 0.21 (95% CI: 0.17-0.25). Subgroup analysis indicated no significant sex- or age-related differences. The proportion of Hi in winter (29%) was higher than that in other seasons. Results indicated significant differences among the provinces, with carriage proportions ranging from 0.11 to 0.60. The proportion of nontypeable H influenzae (NTHi) was higher than that of the capsular type. The proportion of Hib in the capsular type (2%) was higher than that in other serotypes. CONCLUSIONS: The carriage rate of Hi in healthy children in China was 21% with no sex-related age differences. The proportion of Hi in winter was high, and the proportions of Hi in different regions were significantly different. NTHi was the predominant serotype detected in children.

Mucosal immunity of mannose-modified chitosan microspheres loaded with the nontyepable Haemophilus influenzae outer membrane protein P6 in BALB/c mice.

Nontypeable Haemophilus influenzae (NTHi) is a common opportunistic pathogen that colonizes the nasopharynx. NTHi infections result in enormous global morbidity in two clinical settings: otitis media in children and acute exacerbation of chronic obstructive pulmonary disease (COPD) in adults. Thus, there is an urgent need to design and develop effective vaccines to prevent morbidity and reduce antibiotic use. The NTHi outer membrane protein P6, a potential vaccine candidate, is highly conserved and effectively induces protective immunity. Here, to enhance mucosal immune responses, P6-loaded mannose-modified chitosan (MC) microspheres (P6-MCMs) were developed for mucosal delivery. MC (18.75%) was synthesized by the reductive amination reaction method using sodium cyanoborohydride (NaBH3CN), and P6-MCMs with an average size of 590.4±16.2 nm were successfully prepared via the tripolyphosphate (TPP) ionotropic gelation process. After intranasal immunization with P6-MCMs, evaluation of humoral immune responses indicated that P6-MCMs enhance both systemic and mucosal immune responses. Evaluation of cellular immune responses indicated that P6-MCMs enhance cellular immunity and trigger a mixed Th1/Th2-type immune response. Importantly, P6-MCMs also trigger a Th17-type immune response. They are effective in promoting lymphocyte proliferation and differentiation without toxicity in vitro. The results also demonstrate that P6-MCMs can effectively induce MHC class I- and II-restricted cross-presentation, promoting CD4+-mediated Th immune responses and CD8+-mediated cytotoxic T lymphocyte (CTL) immune responses. Evaluation of protective immunity indicated that immunization with P6-MCMs can reduce inflammation in the nasal mucosa and the lung and prevent NTHi infection. In conclusion, MCMs are a promising adjuvant-delivery system for vaccines against NTHi.

Oxidation mechanism of T91 steel in liquid lead-bismuth eutectic: with consideration of internal oxidation.

Clarification of the microscopic events that occur during oxidation is of great importance for understanding and consequently controlling the oxidation process. In this study the oxidation product formed on T91 ferritic/martensitic steel in oxygen saturated liquid lead-bismuth eutectic (LBE) at 823 K was characterized at the nanoscale using focused-ion beam and transmission electron microscope. An internal oxidation zone (IOZ) under the duplex oxide scale has been confirmed and characterized systematically. Through the microscopic characterization of the IOZ and the inner oxide layer, the micron-scale and nano-scale diffusion of Cr during the oxidation in LBE has been determined for the first time. The micron-scale diffusion of Cr ensures the continuous advancement of IOZ and inner oxide layer, and nano-scale diffusion of Cr gives rise to the typical appearance of the IOZ. Finally, a refined oxidation mechanism including the internal oxidation and the transformation of IOZ to inner oxide layer is proposed based on the discussion. The proposed oxidation mechanism succeeds in bridging the gap between the existing models and experimental observations.

[DNA methylation in thyroid carcinoma].

Cancer has become clear that not merely gene variations but also epigenetic modifications may contribute to it. Epigenetic changes refer to stable alterations in gene expression with unrelated to changes in the underlying genetic sequence,resulting in heritable. DNA methylation is one of the common epigenetic changes. It control the gene expression through changing DNA conformation and stability, chromatin structer, DNA-protein interaction. The reversal of dysregulated DNA methylation has emerged as a potential strategy for the treatment of thyroid carcinoma. The artical will provide an overview of how DNA methylation contribute to thyroid carcinoma dissemination,invasion and metastasis and we will summarize the latest epigenetic therapies for thyroid carcinoma.

[Progress in diagnosis and treatment of children allergic rhinitis].

Children allergic rhinitis, referred to as children allergic rhinitis (AR), is a kind of non-infectious inflammation of the nasal mucosa mediated by IgE with the main symtoms of paroxysmal sneezing, rhinorrhoea, nasal itching and nasal obstruction when the susceptible individuals contact the allergen. It is a high reaction disease of the respiratory mucosa common with childhood, which has serious implications to the Children's quality of life, study, rest and growth. The global sampling survey reveals that the morbidity is about 14%, of which 10% in our country and there is an upward trend year by year. At present, drug therapy is still one of the most important methods for children AR. Definite diagnosis, standardized drug therapy and the development of new specific immune therapy make children AR in a good control . This review updates the diagnosis and treatment for children AR, referring to the newest guide by WHO about allergic rhinitis and its impact on asthma (ARIA).

[Preparation and characterization of mouse monoclonal antibody against outer membrane protein P6 of Haemophilus influenzae].

OBJECTIVE: To prepare and identify monoclonal antibody against Haemophilus influenzae(Hi) outer membrane protein P6. METHODS: Recombinant protein P6 as an immunogen was administered intraperitoneally to BALB/c mice. The splenocytes of the mouse were isolated from spleen and hybridized with Sp2/0 myeloma cells. Indirect ELISA was used for screening hybridoma and the number of chromosomes in hybridoma cells was determined by karyotype analysis. The titers and specificity of monoclonal antibodies in their culture supernatant were detected by indirect ELISA. The immunoglobulin class, subclasses and type of the monoclonal antibody were identified with colloidal gold labeled IsoQuick(TM) strips. RESULTS: Two hybridoma cell lines designated α2G3 and γ2C4 were obtained. Karyotype analysis showed that the chromosome numbers of α2G3 and γ2C4 were 103 and 95, respectively. The highest titers of antibodies in their culture supernatant were 1:256 and 1:512, respectively. Both monoclonal antibodies only reacted with standard or clinical isolated strains of Hi, and they both did not react with other bacteria. A2G3 was IgG2b, and γ2C4 was IgM, both of which were kappa light chains. They could recognize different antigen epitope of protein P6. CONCLUSION: Two hybridoma cell lines producing the monoclonal antibodies against protein P6 of Hi outer membrane are obtained.

[Mouse macrophage-derived chemokine as an adjuvant enhances the protective effect of P6 protein vaccine of nontypeable Haemophilus influenzae].

OBJECTIVE: To express P6 protein of nontypeable Haemophilus influenzae (NTHi) in prokaryotic cells and observe the immune effect of macrophage-derived chemokine (MDC) as an adjuvant on NTHi-P6 protein vaccine. METHODS: The encoding sequence of NTHi-P6 protein was inserted into plasmid PGEX-6P2, and the recombinant plasmid PGEX-6P2/P6 was transformed into E.coli XL1-Blue. In the expression system, the P6 protein was induced and expressed. BALB/c mice were randomized into three groups that were respectively inoculated intraperitoneally with P6 protein combined with Freund's adjuvant and MDC, P6 protein combined with Freund's adjuvant and only PBS, 3 times at 0, 14 and 28 days, respectively. Sera were collected from 12 mice of each group 14 days after each injection. The titer of IgG antibodies was detected by ELISA. Splenocytes were isolated from 3 mice of each group for determining the levels of IL-4 and IFN-γ by ELISA. Furthermore, the other 15 mice of each group were challenged with 10×LD50 NTHi ATCC49247 to observe the protective effect of the vaccine. RESULTS: The IgG antibody could be induced by p6 protein combined with Freund's adjuvant and MDC and P6 protein combined with Freund's adjuvant, and the titers were 1:1 140.25 and 1:3 044.38, respectively. The differences in the antibody titers and the levels of IFN-γ between the two groups were significant statistically (P<0.05). There was no difference in IL-4 levels between the two groups (P>0.05). After challenged with 10×LD50 NTHi ATCC49247, the survival rate of mice in P6 protein combined with Freund's adjuvant and MDC group was 80%, and significantly higher than that in the PBS group (P<0.05), but no statistical difference was noted when compared with P6 protein combined with Freund's adjuvant group. CONCLUSION: MDC as an adjuvant could enhance the immune protective effect of NTHi-P6 protein vaccine to an extent.

[Effect of ambroxol on biofilm of Haemophilus influenzae and bactericidal action].

OBJECTIVE: To establish a biofilm model of Haemophilus influenzae and observe the effect of ambroxol on biofilm of Haemophilus influenzae and bactericidal action. METHOD: Thirty strains of Haemophilus influenzae were isolated from adenoids of children with adenoidal hypertrophy. Two strains which could build stronger biofilms was selected in a 96-well plate. The effect of ambroxol on biofilms were determined by crystal violet, and the structure of biofilms were observed by scanning electron microscope (SEM). The numbers of viable bacterial in biofilm after ambroxol treatmented determined by plate culture count. RESULT: Through crystal violet assay, significant difference (P < 0.01) between the two group after treatment was found when ambroxol concentration reached at 0.25 mg/ml and 0.49 mg/ml. The biofilms was destroyed by SEM. Ambroxol had the positive effect on bacterial killing by plate culture count,and the effect was in a dose dependent. CONCLUSION: Ambroxol could destroy the biofilm of Haemophilus influenzae, and had bactericidal function in vitro.