Neutrophil-activating Protein Research Articles

Ethanol-mediated alterations in oligodendrocyte differentiation in the developing brain

IntroductionAlterations of white matter integrity and subsequent white matter structural deficits are consistent findings in Fetal Alcohol Syndrome (FAS), but knowledge regarding the molecular mechanisms underlying these abnormalities is incomplete. Experimental rodent models of FAS have shown dysregulation of cytokine expression leading to apoptosis of oligodendrocyte precursor cells (OPCs) and altered oligodendrocyte (OL) differentiation, but whether this is representative of human FAS pathogenesis has not been determined. MethodsFetal brain tissue (12.2–21.4 weeks gestation) from subjects undergoing elective termination of pregnancy was collected according to an IRB-approved protocol. Ethanol (EtOH) exposure status was classified based on a detailed face-to-face questionnaire adapted from the National Institute on Alcohol Abuse and Alcoholism Prenatal Alcohol and Sudden Infant Death Syndrome and Stillbirth (PASS) study. Twenty EtOH-exposed fetuses were compared with 20 gestational age matched controls. Cytokine and OPC marker mRNA expression was quantified by Real-Time Polymerase chain reaction (qRT-PCR). Patterns of protein expression of OPC markers and active Capase-3 were studied by Fluorescence Activated Cell Sorting (FACS). ResultsEtOH exposure was associated with reduced markers of cell viability, OPC differentiation, and OL maturation, while early OL differentiation markers were unchanged or increased. Expression of mRNAs for proteins specific to more mature forms of OL lineage (platelet-derived growth factor α (PDGFRα) and myelin basic protein (MBP) was lower in the EtOH group than in controls. Expression of the multifunctional growth and differentiation-promoting growth factor IGF-1, which is essential for normal development, also was reduced. Reductions were not observed for markers of early stages of OL differentiation, including Nuclear transcription factor NK-2 homeobox locus 2 (Nkx2.2). Expression of mRNAs for the proinflammatory cytokine, tumor necrosis factor-α (TNFα), and several proinflammatory chemokines was higher in the EtOH group compared to controls, including: Growth regulated protein alpha/chemokine (C-X-C motif) ligand 1 (GRO-α/CXCL1), Interleukin 8/chemokine (C-X-C motif) ligand 8 (IL8/CXCL8), Chemokine (C-X-C motif) ligand 6/Granulocyte chemotactic protein 2 (CXCL16/GCP2), epithelial-derived neutrophil-activating protein 78/chemokine (C-X-C motif) ligand 5 (ENA-78/CXCL5), monocyte chemoattractant protein-1 (MCP-1). EtOH exposure also was associated with an increase in the proportion of cells expressing markers of early stage OPCs, such as A2B5 and NG2. Finally, apoptosis (measured by caspase-3 activation) was increased substantially in the EtOH group compared to controls. ConclusionPrenatal EtOH exposure is associated with excessive OL apoptosis and/or delayed OL maturation in human fetal brain. This is accompanied by markedly dysregulated expression of several chemokines and cytokines, in a pattern predictive of increased OL cytotoxicity and reduced OL differentiation. These findings are consistent with findings in animal models of FAS.

HP-NAP ameliorates OXA-induced atopic dermatitis symptoms in mice

Background Atopic dermatitis (AD) is a chronic skin inflammatory disease characterized by disequilibrium between Th1/Th2 lymphocytes. Helicobacter pylori neutrophil-activating protein (HP-NAP) has been reported that it has the potential immunomodulatory effect able to regulate the Th1/Th2 balance. Objective This study aimed to investigate the therapeutic effect of HP-NAP in AD mice model. Methods The model of AD was built with oxazolone (OXA) in BALB/c mice, then HP-NAP was used to treat AD by intraperitoneal injection. Ear thickness was measured by a digital thickness gauge. The ears tissues were collected and subjected to hematoxylin-eosin (H&E) and toluidine blue (TB) staining. The mRNA expression levels of inflammatory cytokines (IL-1β, IL-5, IL-6, and TNF-α) in ear tissue were measured using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The secretion of IgE, IL-4, and IFN-γ was measured by enzyme-linked immunosorbent assay (ELISA). Results Treatment with HP-NAP successfully alleviated the symptoms of AD, such as erythema, horny substance, and swelling. The infiltration of lymphocytes and mast cells were significantly reduced following HP-NAP therapy. The secretion of IgE and IL-4 was significantly attenuated following treatment with HP-NAP. Additionally, HP-NAP observably downregulated inflammatory cytokine expression (e.g. IL-1β, IL-5, IL-6, and TNF-α) in ear tissues. Conclusions and clinical relevance Taken together, our results showed that HP-NAP possessed the potential to be a novel immunomodulatory candidate drug against AD.

Evaluation of the inflammatory markers CCL8, CXCL5, and LIF in patients with anastomotic leakage after colorectal cancer surgery

PurposeAnastomotic leakage constitutes a dreaded complication after colorectal surgery, leading to increased morbidity and mortality as well as prolonged hospitalization. Most leakages become clinically apparent about 8 days after surgery; however, early detection is quintessential to reduce complications and to improve patients’ outcome. We therefore investigated the significance of specific protein expression profiles as putative biomarkers, indicating anastomotic leakage.MethodsIn this single-center prospective cohort study serum and peritoneal fluid samples—from routinely intraoperatively inserted drainages—of colorectal cancer patients were collected 3 days after colorectal resection. Twenty patients without anastomotic leakage and 18 patients with an anastomotic leakage and without other complications were included. Protein expression of seven inflammatory markers in serum and peritoneal fluid was assessed by multiplex ELISA and correlated with patients’ clinical data.ResultsMonocyte chemoattractant protein 2 (CCL8/MCP-2), leukemia-inhibiting factor (LIF), and epithelial-derived neutrophil-activating protein (CXCL5/ENA-78) were significantly elevated in peritoneal fluid but not in serum samples from patients subsequently developing anastomotic leakage after colorectal surgery. No expressional differences could be found between grade B and grade C anastomotic leakages.ConclusionMeasurement 3 days after surgery revealed altered protein expression patterns of the inflammatory markers CCL8/MCP2, LIF, and CXCL5/ENA-78 in peritoneal fluid from patients developing anastomotic leakage after colorectal surgery. Further studies with a larger patient cohort with inclusion of different variables are needed to evaluate their potential as predictive biomarkers for anastomotic leakage.

Parenteral immunization with a cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) adjuvanted Helicobacter pylori vaccine induces protective immunity against H. pylori infection in mice

ABSTRACT Under the trend of antibiotic resistance of H. pylori leading to the decrease of eradication rate, the development of a vaccine is the best choice to fight against H. pylori. In this study, we attempted to reduce the amounts of required antigens by using three different parenteral routes of immunization and an adjuvant cGAMP (cyclic guanosine monophosphate-adenosine monophosphate) to enhance the immunogenicity of the vaccine candidate. The immune protection and post-challenge immune responses were assessed and compared in mice immunized with recombinant Helicobacter pylori urease A, urease B, and neutrophil-activating protein adjuvanted with cGAMP. The gastric mucosal colonization by H. pylori was significantly reduced in mice immunized by intranasal and, to a less degree, subcutaneous route, but not by intramuscular route. All immunized mice, regardless of the route of immunization, displayed significant, but comparable, increases in antigen-specific serum IgG and mucosal IgA responses 5 weeks post-challenge. The magnitude of the vaccine-induced protection appeared to be associated with the level of antigen-specific Th1 and particularly Th17 responses, as IL-17 responses were only detected in intranasally immunized mice. Taken together, we explored and confirmed the possibility of using a novel adjuvant (cGAMP) to induce significant protective immunity with 10% of oral vaccine antigen dosage through parenteral immunization, especially intranasal immunization. This may provide an alternative approach to oral immunization for the development of effective H. pylori vaccines.

Oral immunization of mice with a multivalent therapeutic subunit vaccine protects against Helicobacter pylori infection

Helicobacter pylori is a human class I carcinogen and no effective prophylactic or therapeutic H. pylori vaccine has yet been marketed. H. pylori can escape the host immune response, but the precise immune protection mechanisms in humans remain unknown. In this study, we developed a multivalent, subunit H. pylori vaccine candidate by formulating three commonly used H. pylori antigens, neutrophil-activating protein (NAP), urease subunit A (UreA) and subunit B (UreB) with the mucosal adjuvant, a double-mutant heat-labile toxin (dmLT) from Escherichia coli, and evaluated its immunogenicity and therapeutic efficacy in a mouse model of H. pylori infection. We found that oral immunization of H. pylori-infected mice significantly reduced gastric bacterial colonization at both 2 and 8 weeks after immunization. The reduction in bacterial burdens was accompanied with significantly increased serum antigen-specific IgG responses and mucosal IgA responses. Moreover, oral immunization also induced Th1/Th17 immune responses, which may play a synergistic role with the specific antibodies in the elimination of H. pylori. Thus, our vaccine candidate appears able to overcome the immune evasion mechanism of H. pylori, restore the suppression of Th2 immune responses with the induction of a strong humoral immune response. These results lay the foundation for the development of an optimized oral therapeutic H. pylori vaccine with increased immunogenicity of UreA and UreB, as well as providing long-term immunity.

Cloning and expression of Helicobacter pylori Neutrophil-activating protein fused to the 6xHis tag in Escherichia coli

Helicobacter pylori (Hp) has been strongly implicated as a causative factor in peptic ulcer disease, and in significantly increasing the risk of developing gastric cancer. Commercial vaccines nowadays have shown less efficiency on preventing the disease caused by Hp. Therefore, this study was performed to clone nap gene from Hp and over-express the encoded protein (NAP) in E. coli cells. NAP (Neutrophil-activating protein) has been considered as a potential antigen of Hp, can be used to produce peptic ulcer prevention vaccine. The obtained results showed that E. coli OmniMAX and E. coli BL21 (DE3) strains carrying recombinant plasmid pET11a-nap were cloned successfully and remained stable in medium supplemented with ampicillin. Over-expression of NAP protein fused with 6xHis in E. coli was demonstrated via SDS-PAGE and Western blot. The result revealed that the highest target protein production level (29.73%) was obtained in the medium added 0.5 mM IPTG at 37 oC after 6 hours of induction. This is the preliminary research for over-production and purification of Hp-NAP protein that may be used in further applications.

Oral administration of recombinant Bacillus subtilis spores expressing Helicobacter pylori neutrophil-activating protein suppresses peanut allergy via up-regulation of Tregs.

Helicobacter pylori neutrophil-activating protein (NAP) is an immune modulator with anti-Th2 inflammation activity that can be used to prevent IgE-mediated allergic reactions. Cholera toxin B (CTB) is a mucosal adjuvant that can induce antigen tolerance. Bacillus subtilis spores are an ideal vehicle for the oral delivery of heterologous antigens. We investigated the therapeutic effect of recombinant NAP Bsubtilis spores on peanut allergies in a mouse model. Female C3H/HeJ mice were sensitized and challenged with peanut extract by oral administration. Before challenge, recombinant NAP and CTB-NAP (CNAP) spores were orally administered to sensitized mice for 4weeks. Faecal peanut-specific IgA and serum-specific IgE, IgG1, and IgG2a levels were measured, and the intestinal microbiota was analysed. Mice were intraperitoneally injected with anti-CD25 antibodies for regulatory T cell (Treg) depletion to evaluate the efficacy of Tregs in preventing peanut allergy. After challenge, anaphylactic reactions, plasma histamine, Tregs, and splenocyte interleukin (IL)-10, IL-4, IL-5 and interferon-γ (IFN-γ) levels were evaluated. After 4weeks of recombinant spore treatment, faecal IgA levels and serum IgG2a levels were increased, while serum IgG1 and IgE levels were reduced. Intestinal microbiota analysis revealed that CNAP spores increased the taxonomic abundance of Firmicutes at the phylum level and Clostridia at the class level. After challenge, the administration of NAP or CNAP spores to mice was found to ameliorate anaphylactic reactions and decrease plasma histamine levels. Administration of NAP or CNAP spores also enhanced IL-10 and IFN-γ secretion, and suppressed IL-4 and IL-5 secretion. The protective effect of CNAP spores was more pronounced than that of NAP spores; this therapeutic effect was lost after Treg depletion. Recombinant NAP spores successfully suppressed Th2 inflammation via the up-regulation of Tregs; this may serve as a novel therapeutic approach for treating food allergies.

Draft genome sequence of multidrug-resistant β-lactamase-producing Bacillus cereus S66 isolated from China.

Bacillus cereus is associated with a variety of human diseases such as haematological malignancies, tetanus, gas gangrene, food poisoning and pseudomembranous colitis. Our team found and isolated a strain, named B. cereus S66, from radiation-contaminated soil in Xinjiang Uygur Autonomous Region. In this study, the drug resistance and toxicity of B. cereus S66 was systematically studied and analysed. Genomic DNA of B. cereus S66 was sequenced using an Illumina HiSeq 2000 sequencing system. The generated reads were assembled using SOAPdenovo, and GapCloser was used to fill in the gaps. The draft genome was annotated, and virulence genes and drug resistance genes were identified by comparison with multiclass databases. The genome of B. cereus strain S66 is 5 628 370 bp with a GC content of 34.95% and contains various genes related to toxicity and antimicrobial resistance. The number of toxicity genes identified was 496, some of which are associated with gastrointestinal infection, eye infection and neutrophil-activating protein NapA. Antimicrobial resistance-related genes comprised 3.23% of the total genes and conferred resistance to lincomycin, tetracycline, fosfomycin, aminoglycosides, linezolid, chloramphenicol, rifampicin, macrolides, daunorubicin and mupirocin. Three different β-lactamases were also found in isolate S66. The genome sequence of B. cereus S66 provides a valuable reference to further research on multidrug resistance characteristics and pathogenic system analysis of Bacillus sp. in the clinic.

Toll-like receptor agonist rMBP-NAP enhances antitumor cytokines production and CTL activity of peripheral blood mononuclear cells from patients with lung cancer.

Toll-like receptor (TLR) agonists are known for their ability to inhibit tumor progression via enhancing antitumor cytokines production and cytotoxic T lymphocyte (CTL) activity. Recombinant Helicobacter pylori neutrophil-activating protein fused with maltose-binding protein (rMBP-NAP) has been reported as a novel TLR agonist for antitumor treatment in murine models. The present study aimed to determine the potential and efficacy of the rMBP-NAP for antitumor treatment prior to further clinical trials. The rMBP-NAP was expressed and purified for subsequent experiments. Peripheral blood mononuclear cells (PBMCs) from health donors and patients with lung cancer (LC) were incubated with PBS and 0.2 mg/ml rMBP-NAP. Antitumor cytokines production was assayed using ELISA and reverse transcription-quantitative polymerase chain reaction analysis. The cytolytic activity of PBMCs and the number of Interferon-γ (IFN-γ)-secreting cells were assayed using lactate dehydrogenase and Enzyme-linked ImmunoSpot assays, respectively. The results from the present study revealed that the expression of IFN-γ, interleukin (IL)-2, tumor necrosis factor-α and IL-12 of PBMCs from patients with LC and healthy donors were significantly increased following treatment with rMBP-NAP (P<0.05). Additionally, rMBP-NAP significantly upregulated the number of IFN-γ-secreting cells in PBMCs and prominently increased the cytotoxic activity of PBMCs (P<0.05). Furthermore, the expression of TLR2 was significantly enhanced following rMBP-NAP stimulation (P<0.05), which indicated that rMBP-NAP may serve an antitumor role via TLR2 signaling pathways. Overall, these results demonstrated that rMBP-NAP possesses the potential to be a novel immunomodulatory candidate drug and requires further evaluation in clinical trials.

Efficacy, immunogenicity, and safety of a parenteral vaccine against Helicobacter pylori in healthy volunteers challenged with a Cag-positive strain: a randomised, placebo-controlled phase 1/2 study

Intramuscular immunisation with a vaccine composed of three recombinant Helicobacter pylori antigens-vacuolating cytotoxin A (VacA), cytotoxin-associated antigen (CagA), and neutrophil-activating protein (NAP)-prevented infection in animal models and was well tolerated and highly immunogenic in healthy adults. We aimed to assess the efficacy of the vaccine in prevention of a H pylori infection after challenge with a CagA-positive strain (BCM 300) in healthy volunteers. In this randomised phase 1/2, observer-blind, placebo-controlled, single-centre study, healthy non-pregnant adults aged 18-40 years who were confirmed negative for H pylori infection were randomly assigned (3:4) to three intramuscular doses of either placebo or vaccine at 0, 1, and 2 months. Randomisation was via a computer-generated list with study numbers ensuring the correct ratio within a block size of seven. Participants were consecutively assigned in a double-blind manner to existing study numbers of the study protocol. Investigators and participants were blinded to allocation throughout the study. One month after the third immunisation, participants underwent challenge with a CagA-positive H pylori strain, which, for safety reasons, was initially administered in a subset of participants. The primary efficacy outcome was the efficacy of the vaccine as measured by the proportion of participants infected with H pylori 12 weeks after the challenge. At the end of the study, participants infected with H pylori were treated for 14 days with combination therapy consisting of a proton pump inhibitor and two antibiotics twice daily. Safety and immunogenicity were monitored at pre-established visits. This trial is registered with ClinicalTrials.gov, number NCT00736476, and is completed. 63 patients were randomly assigned, 27 to placebo and 36 to the vaccine. 34 participants (19 in the vaccinated group and 15 in the placebo group) underwent infectious challenge, all but one of whom experienced transient mild-to-moderate epigastric symptoms. 12 weeks after infectious challenge, six (32%) of 19 people in the vaccinated group and six (40%) of 15 people in the placebo group remained positive for H pylori. Eradication was successful in everyone who remained infected at 12 weeks. The geometric mean concentrations of antibodies specific to CagA (202 [95% CI 69-588] vs 4·73 [95% CI 1·41-16]; p=0·001), VacA (1469 [838-2577] vs 73 [39-138]; p=0·001), and NAP (208 [139-313] vs 8·01 [5·05-13]; p=0·001) were significantly higher in the vaccine group than in the placebo group 12 weeks after infectious challenge. Compared with placebo, the vaccine did not confer additional protection against H pylori infection after challenge with a CagA-positive strain, despite increased systemic humoral responses to key H pylori antigens. The finding of spontaneous clearance of H pylori infection in more than half the participants in the placebo group is remarkable and suggests important immune protection in the healthy adult population. Novartis Vaccine and Diagnostics.

Production and delivery of Helicobacter pylori NapA in Lactococcus lactis and its protective efficacy and immune modulatory activity

Helicobacter pylori neutrophil-activating protein A subunit (NapA) has been identified as a virulence factor, a protective antigen and a potent immunomodulator. NapA shows unique application potentials for anti-H. pylori vaccines and treatment strategies of certain allergic diseases and carcinomas. However, appropriate production and utilization modes of NapA still remain uncertain to date. This work has established a novel efficient production and utilization mode of NapA by using L. lactis as an expression host and delivery vector, and demonstrated immune protective efficacy and immune modulatory activity of the engineered L. lactis by oral vaccination of mice. It was observed for the first time that H. pylori NapA promotes both polarized Th17 and Th1 responses, which may greatly affect the clinical application of NapA. This report offers a promising anti-H. pylori oral vaccine candidate and a potent mucosal immune modulatory agent. Meanwhile, it uncovers a way to produce and deliver the oral vaccine and immunomodulator by fermentation of food like milk, which might have striking effects on control of H. pylori infection, gastrointestinal cancers, and Th2 bias allergic diseases, including many food allergies.

Enhanced Mutant Screening in One-step PCR-based Multiple Site-directed Plasmid Mutagenesis by Introduction of Silent Restriction Sites for Structural and Functional Study of Proteins

Site-directed mutagenesis (SDM) has been widely used for studying the structure and function of proteins. A one-step polymerase chain reaction (PCR)-based multiple site-directed plasmid mutagenesis method with extended non-overlapping sequence at the 3′ end of the primer increases the PCR amplification efficiency and the capacity of multi-site mutagenesis. Here, we introduced silent restriction sites in the primers used in this PCR-based SDM method by utilizing SDM-Assist software to generate mutants of Helicobacter pylori neutrophil-activating protein (HP-NAP), whose gene has low GC content. The HP-NAP mutants were efficiently generated by this modified mutagenesis method and quickly identified by a simple restriction digest due to the presence of the silent restriction site. This modified PCR-based SDM method with the introduction of a silent restriction site on the primer is efficient for generation and identification of mutations in the gene of interest.

Serum Antibodies against Helicobacter pylori Neutrophil Activating Protein in Carriers of IL-4 C-590T Genetic Polymorphism Amplify the Risk of Gastritis and Gastric Cancer.

Background:Gastric cancer arises, mainly, on an inflammatory background. Helicobacter pylori neutrophil activating (HP-NAP) protein functions as a potent pro-inflammatory mediator. Similarly, IL-4 plays a critical role in the inflammation pathway, the levels of which are altered by C to T transition at position -590 in its promoter region. Here, we have aimed to assess the risk of gastritis and gastric cancer in the co-presence of these two inflammation modulating mediators.Methods:Gastritis (n=58) and gastric cancer (n=31) patients were evaluated and compared with H. pylori-positive asymptomatic controls (n=46), for serum antibodies against recombinant HP-NAP and IL-4 C-590T single nucleotide polymorphism using immunoblotting and PCR-RFLP, respectively. Multivariable logistic regression, adjusting for age, gender and ethnicity, was used for data analysis.Results:In terms of susceptibility to gastritis, seropositivity to HP-NAP projected a risk impact of 4.62 fold (OR=4.62, 95% CI=1.50-14.22), which when present in IL-4 -590 T carriers augmented the risk up to 9.7 fold (OR=9.70, 95% CI=2.06-45.69). A similar pattern, but of a stronger magnitude, occurred for the risk of gastric cancer, which was estimated at 9.07 fold (OR=9.07, 95% CI=1.99-42.0) for HP-NAP-seropositive subjects and was drastically amplified (OR=33.64, 95% CI=2.06-548.68), when double-positive (HP-NAP seropositive/IL-4 -590 T carrier) subjects were examined against double negatives (HP-NAP seronegative/IL-4 -590 CC).Conclusion:Our preliminary data indicate that serum antibodies against HP-NAP represent a state of risk, which is further exacerbated in IL-4 -590 T carriers. These biomarkers, if validated in larger prospective studies, can be used to screen for gastric cancer susceptibility

Recombinant protein rMBP-NAP restricts tumor progression by triggering antitumor immunity in mouse metastatic lung cancer.

Recombinant Helicobacter pylori neutrophil-activating protein fused with maltose-binding protein (rMBP-NAP), a potential TLR2 ligand, was reported to possess immunomodulatory effects on in situ tumors in our previous study. In the present work, we attempt to elucidate the effect of rMBP-NAP at the local immune modulation in B16-F10-induced metastatic lung cancer. Our results demonstrated that growth of B16-F10 melanoma metastases in the lung was significantly arrested after rMBP-NAP treatment, along with marked reduction in metastatic lung nodules and significant increase in survival. The treatment induced both local and systemic immune responses, which were associated with higher influx of CD4+/CD8+ T cells and drove toward Th1-like and cytotoxic immune environment. Moreover, rMBP-NAP also showed significant anti-angiogenic activity by reducing vascularization in lung tumor sections. rMBP-NAP could induce antitumor immunity through activating Th1 cells and producing pro-inflammatory cytokines, which are responsible for the effective cytotoxic immune response against cancer progression. Our findings indicate that rMBP-NAP might be a novel antitumor therapeutic strategy.

Oral Immunization with a Multivalent Epitope-Based Vaccine, Based on NAP, Urease, HSP60, and HpaA, Provides Therapeutic Effect on H. pylori Infection in Mongolian gerbils

Epitope-based vaccine is a promising strategy for therapeutic vaccination against Helicobacter pylori (H. pylori) infection. A multivalent subunit vaccine containing various antigens from H. pylori is superior to a univalent subunit vaccine. However, whether a multivalent epitope-based vaccine is superior to a univalent epitope-based vaccine in therapeutic vaccination against H. pylori, remains unclear. In this study, a multivalent epitope-based vaccine named CWAE against H. pylori urease, neutrophil-activating protein (NAP), heat shock protein 60 (HSP60) and H. pylori adhesin A (HpaA) was constructed based on mucosal adjuvant cholera toxin B subunit (CTB), Th1-type adjuvant NAP, multiple copies of selected B and Th cell epitopes (UreA27–53, UreA183–203, HpaA132–141, and HSP60189–203), and also the epitope-rich regions of urease B subunit (UreB158–251 and UreB321–385) predicted by bioinformatics. Immunological properties of CWAE vaccine were characterized in BALB/c mice model. Its therapeutic effect was evaluated in H. pylori-infected Mongolian gerbil model by comparing with a univalent epitope-based vaccine CTB-UE against H. pylori urease that was constructed in our previous studies. Both CWAE and CTB-UE could induce similar levels of specific antibodies against H. pylori urease, and had similar inhibition effect of H. pylori urease activity. However, only CWAE could induce high levels of specific antibodies to NAP, HSP60, HpaA, and also the synthetic peptides epitopes (UreB158–172, UreB181–195, UreB211–225, UreB349–363, HpaA132–141, and HSP60189–203). In addition, oral therapeutic immunization with CWAE significantly reduced the number of H. pylori colonies in the stomach of Mongolian gerbils, compared with oral immunization using CTB-UE or H. pylori urease. The protection of CWAE was associated with higher levels of mixed CD4+ T cell (Th cell) response, IgG, and secretory IgA (sIgA) antibodies to H. pylori. These results indic ate that a multivalent epitope-based vaccine including Th and B cell epitopes from various H. pylori antigens could be a promising candidate against H. pylori infection.

The recombinant fusion protein of cholera toxin B and neutrophil-activating protein expressed on Bacillus subtilis spore surface suppresses allergic inflammation in mice.

The neutrophil-activating protein of Helicobacter pylori (HP-NAP) has been identified as a modulator with anti-Th2 inflammation activity, and cholera toxin B (CTB) is a mucosal adjuvant that can also induce antigen tolerance. In this study, we constructed a CTB-NAP fusion protein on the surface of Bacillus subtilis spore and evaluate the efficiency of oral administration of the recombinant CTB-NAP spores in preventing asthma in mice. Oral administration of recombinant CTB or CTB-NAP spores significantly decreased serum ovalbumin (OVA)-specific IgE (p<0.001) and increased fecal IgA (p<0.01) compared to the treatment with non-recombinant spores. Oral administration of recombinant CTB or CTB-NAP spores induced IL-10 and IFN-γ expression and reduced IL-4 levels in bronchoalveolar lavage fluid (BALF). Moreover, CTB and CTB-NAP spores reduced the eosinophils in BALF and inflammatory cell infiltration in the lungs. Furthermore, CD4+CD25+Foxp3+ Tregs in splenocytes were significantly increased in mice treated with recombinant CTB or CTB-NAP spores. The number of CD4+CD25+Foxp3+ Tregs caused by CTB-NAP was higher than that by CTB alone. Our study indicated that B. subtilis spores with surface expression of subunit CTB or CTB-NAP could inhibit OVA-induced allergic inflammation in mice. The attenuated inflammation was attributed to the induction of CD4+CD25+Foxp3+ Tregs and IgA. Moreover, the fusion protein CTB-NAP demonstrated a better efficiency than CTB alone in inhibiting the inflammation.

The protective role of Helicobacter pylori neutrophil-activating protein in childhood asthma

BackgroundHelicobacter pylori quantity and HP-NAP gene expression were evaluated in the faeces of healthy and asthmatic children. MethodsH. pylori DNAs and RNAs were isolated from the stool samples of 92 asthmatic children (AC; 3–8 years) and 88 healthy controls (HC). Quantitative PCR was used to determine the quantity of H. pylori and HP-NAP expression relative to the 16S rRNA (reference gene). Gene expression was analysed using the delta delta-Ct method. ResultsH. pylori DNA was detected in the stool samples of 18 (20.4%) of the 88 HC (p<0.0001, OR=0.79) and none of AC. No meaningful statistical differences were found between individuals with positive and negative family histories for asthma in AC and HC (p>0.05). H. pylori quantity was higher in seven of 18 H. pylori-positive samples, but HP-NAP expression levels were low in four of these seven samples. Based on a multivariate logistic regression analysis of these three variables together, only males displayed a significant difference based on gender differences (p<0.02) and it was determined that, based on the OR value of 0.46 and the 95% CI range of 0.241–0.888, male gender was an independent protective factor in asthma. ConclusionsHP-NAP levels vary to the relative concentrations of bacteria in the stationary or late logarithmic phases. Different napA expression levels may be caused by different endogenous napA gene expression or different environmental conditions.

Differential effects of DEAE negative mode chromatography and gel-filtration chromatography on the charge status of Helicobacter pylori neutrophil-activating protein.

Helicobacter pylori neutrophil-activating protein (HP-NAP) is involved in H. pylori-associated gastric inflammation. HP-NAP is also a vaccine candidate, a possible drug target, and a potential diagnostic marker for H. pylori-associated diseases. Previously, we purified recombinant HP-NAP by one-step diethylaminoethyl (DEAE) negative mode chromatography by collecting the unbound fraction at pH 8.0 at 4°C. It remains unclear why HP-NAP does not bind to DEAE resins at the pH above its isoelectric point during the purification. To investigate how pH affects the surface net charge of HP-NAP and its binding to DEAE resins during the purification, recombinant HP-NAP expressed in Escherichia coli was subjected to DEAE negative mode chromatography at pH ranging from 7.0 to 9.0 at 25°C and the surface charge of purified HP-NAP was determined by capillary electrophoresis. A minimal amount of HP-NAP was detected in the elution fraction of DEAE Sepharose resin at pH 8.5, whereas recombinant HP-NAP was detected in the elution fraction of DEAE Sephadex resin only at pH 7.0 and 8.0. The purified recombinant HP-NAP obtained from the unbound fractions was not able to bind to DEAE resins at pH 7.0 to 9.0. In addition, the surface charge of the purified HP-NAP was neutral at pH 7.0 to 8.0 and was either neutral or slightly negative at pH 8.5 and 9.0. However, recombinant HP-NAP purified from gel-filtration chromatography was able to bind to DEAE Sepharose resin at pH 7.0 to 9.0 and DEAE Sephadex resin at pH 7.0. At pH 8.5 and 9.0, only the negatively charged species of HP-NAP were found. Thus, recombinant HP-NAP with different charge status can be differentially purified by DEAE negative mode chromatography and gel-filtration chromatography. Furthermore, the charge distribution on the surface of HP-NAP, the presence of impure proteins, and the overall net charge of the resins all affect the binding of HP-NAP to DEAE resins during the negative purification.

Systemic and mucosal pre-administration of recombinant Helicobacter pylori neutrophil-activating protein prevents ovalbumin-induced allergic asthma in mice.

Previous epidemiologic studies have demonstrated an inverse association between Helicobacter pylori infection and the frequency of allergic asthma. The neutrophil-activating protein (NAP) of H. pylori has been identified as a modulator possessing anti-Th2 inflammation activity. Here, we sought to determine whether systemic or mucosal pre-administration of recombinant H. pylori NAP (rNAP) could prevent ovalbumin (OVA)-induced allergic asthma in mice. Mice were exposed to purified rNAP through intraperitoneal injection or inhalation and then sensitized with OVA. Following a challenge with aerosolized OVA, the bronchoalveolar lavage fluid (BALF) cell count, lung tissue histology, BALF cytokines and serum IgE were evaluated. Both intraperitoneal injection and inhalation of rNAP prior to OVA sensitization significantly reduced eosinophil accumulation and inflammatory infiltration in lung tissue in OVA-induced asthma mice; eosinophils were reduced in the BALF of rNAP-treated mice. In addition, IL-4 and IL-13 levels were lower (P < 0.01), IL-10 and IFN-γ levels were higher (P < 0.01) and IgE serum levels were lower (P < 0.01) in the treated groups compared to the control group. Systemic and mucosal pre-administration of rNAP could suppress the development of OVA-induced asthma in mice; rNAP may be utilized as part of novel strategies for the prevention or treatment of allergic diseases.

One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode.

Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor of Helicobacter pylori (H. pylori). It plays a critical role in H. pylori-induced gastric inflammation by activating several innate leukocytes including neutrophils, monocytes, and mast cells. The immunogenic and immunomodulatory properties of HP-NAP make it a potential diagnostic and vaccine candidate for H. pylori and a new drug candidate for cancer therapy. In order to obtain substantial quantities of purified HP-NAP used for its clinical applications, an efficient method to purify this protein with high yield and purity needs to be established. In this protocol, we have described a method for one-step negative chromatographic purification of recombinant HP-NAP overexpressed in Escherichia coli (E. coli) by using diethylaminoethyl (DEAE) ion-exchange resins (e.g., Sephadex) in batch mode. Recombinant HP-NAP constitutes nearly 70% of the total protein in E. coli and is almost fully recovered in the soluble fraction upon cell lysis at pH 9.0. Under the optimal condition at pH 8.0, the majority of HP-NAP is recovered in the unbound fraction while the endogenous proteins from E. coli are efficiently removed by the resin. This purification method using negative mode batch chromatography with DEAE ion-exchange resins yields functional HP-NAP from E. coli in its native form with high yield and purity. The purified HP-NAP could be further utilized for the prevention, treatment, and prognosis of H. pylori-associated diseases as well as cancer therapy.