Preparation and evaluation of antigen/N-trimethylaminoethylmethacrylate chitosan conjugates for nasal immunization

The frequent outbreak of respiratory infectious diseases such as influenza and pulmonary tuberculosis calls for new immunization strategies with high effectiveness. Nasal immunization is one of the most potential methods to prevent the diseases infected through the respiratory tract. In this study, we designed a water-soluble system based on antigen/N-trimethylaminoethylmethacrylate chitosan conjugates for nasal immunization. N-trimethylaminoethylmethacrylate chitosan (TMC) was synthesized by free radical polymerization of chitosan and N-trimethylaminoethylmethacrylate chloride and identified by ¹H NMR and FT-IR. Thiolated ovalbumin (OVA) was covalently conjugated to maleimide modified TMC with high conjugation efficiency. OVA conjugated TMC (OVA–TMC) significantly increased uptake of OVA by Raw 264.7 cells, which was 2.38 times higher than that of OVA/TMC physical mixture (OVA + TMC) at 4 h. After nasal administration, OVA–TMC showed higher transport efficiency to superficial and deep cervical lymph nodes than OVA + TMC or OVA alone. Balb/C mice were intranasally given with OVA–TMC three times at 2-week internals to evaluate the immunological effect. The serum IgG, IgG1 and IgG2a levels of the OVA–TMC group were 17.9–87.9 times higher than that of the OVA + TMC group and comparable to that of the intramuscular group. The secretory IgA levels in nasal wash and saliva of the OVA–TMC group were 5.2–7.1 times higher than that of the OVA + TMC group while the secretory IgA levels of the intramuscular alum-precipitated OVA group were not increased. After immunofluorescence staining of nasal cavity, IgA antibody secreting cells were mainly observed in the lamina propria regions and glands of nasal mucosa. OVA–TMC showed little toxicity to the nasal epithelia or cilia of rats after nasal administration for three consecutive days. These results demonstrated that antigen conjugated TMC can induce both systemic and mucosal immune responses after nasal administration and may serve as a convenient, safe and effective vaccine for preventing respiratory infectious diseases.     

Asian sand dust enhances ovalbumin-induced eosinophil recruitment in the alveoli and airway of mice

Asian sand dust (ASD) containing sulfate (SO4(2-)) reportedly causes adverse respiratory health effects but there is no experimental study showing the effect of ASD toward allergic respiratory diseases. The effects of ASD and ASD plus SO4(2-) toward allergic lung inflammation induced by ovalbumin (OVA) were investigated in this study. ICR mice were administered intratracheally with saline; ASD alone (sample from Shapotou desert); and ASD plus SO4(2-) (ASD-SO4); OVA+ASD; OVA+ASD-SO4. ASD or ASD-SO4 alone caused mild nutrophilic inflammation in the bronchi and alveoli. ASD and ASD-SO4 increased pro-inflammatory mediators, such as Keratinocyte chemoattractant (KC) and macrophage inflammatory protein (MIP)-1 alpha, in bronchoalveolar lavage fluids (BALF). ASD and ASD-SO4 enhanced eosinophil recruitment induced by OVA in the alveoli and in the submucosa of the airway, which has a goblet cell proliferation in the bronchial epithelium. However, a further increase of eosinophils by addition of SO4(2-) was not observed. The two sand dusts synergistically increased interleukin-5 (IL-5) and monocyte chemotactic protein-1 (MCP-1), which were associated with OVA, in BALF. However, the increased levels of IL-5 were lower in the OVA+ASD-SO4 group than in the OVA+ASD group. ASD caused the adjuvant effects to specific-IgG1 production by OVA, but not to specific-IgE. These results suggest that the enhancement of eosinophil recruitment in the lung is mediated by synergistically increased IL-5 and MCP-1. IgG1 antibodies may play an important role in the enhancement of allergic reaction caused by OVA and sand dust. However, extra sulfate may not contribute to an increase of eosinophils.     

Modulation of Distinct Asthmatic Phenotypes in Mice by Dose-Dependent Inhalation of Microbial Products

Background: Humans with asthma display considerable heterogeneity with regard to T helper (Th) 2–associated eosinophilic and Th17-associated neutrophilic inflammation, but the impact of the environment on these different forms of asthma is poorly understood.Objective: We studied the nature and longevity of asthma-like responses triggered by inhalation of allergen together with environmentally relevant doses of inhaled lipopolysaccharide (LPS).Methods: Ovalbumin (OVA) was instilled into the airways of mice together with a wide range of LPS doses. Following a single OVA challenge, or multiple challenges, animals were assessed for pulmonary cytokine production, airway inflammation, and airway hyperresponsiveness (AHR).Results: Mice instilled with OVA together with very low doses (≤ 10^–3 μg) of LPS displayed modest amounts of Th2 cytokines, with associated airway eosinophilia and AHR after a single challenge, and these responses were sustained after multiple OVA challenges. When the higher but still environmentally relevant dose of 10^–1 μg LPS was used, mice initially displayed similar Th2 responses, as well as Th17-associated neutrophilia. After multiple OVA challenges, however, the 10^–1 μg LPS animals also accumulated large numbers of allergen-specific T regulatory (Treg) cells with high levels of inducible co-stimulatory molecule (ICOS). As a result, asthma-like features in these mice were shorter-lived than in mice sensitized using lower doses of LPS.Conclusions: The nature and longevity of Th2, Th17, and Treg immune responses to inhaled allergen are dependent on the quantity of LPS inhaled at the time of allergic sensitization. These findings might account in part for the heterogeneity of inflammatory infiltrates seen in lungs of asthmatics.Citation: Whitehead GS, Thomas SY, Cook DN. 2014. Modulation of distinct asthmatic phenotypes in mice by dose-dependent inhalation of microbial products. Environ Health Perspect 122:34–42; http://dx.doi.org/10.1289/ehp.1307280     

Patients’ views on dyspepsia and acid suppressant drug therapy in general practice

Objectives: To do an inventory on the opinion of long-term acid suppressant drug (ASD) users on their condition and drug treatment, and from that angle to reflect on possibilities to reduce usage and costs of long-term acid suppressant drug therapy. Methods: In the year 2001, in seven general practices, patients who chronically used ASDs without proper indication were identified. A postal questionnaire based on the literature was sent to these patients (n=498). Results: 318 (64%) returned questionnaires were fit for analysis. Patients experienced dyspepsia as unpleasant and restraining, and were extremely positive about ASDs without differences between H2-receptor antagonists (H2RAs) and proton pump inhibitors (PPIs). ASD use was very compliant. Patients smoked and drank less than the normal population. They did not continue unhealthy habits simply because ASDs enable them to. Most patients were not motivated to stop their ASDs.

Conclusion: Dyspeptic complaints should not be underestimated. Improving lifestyle is not likely to generate much effect on ASD use. Due to a lack of motivation, discontinuing long-term ASD use may not be an efficient way to reduce the use and costs of ASDs. Patient-centred possibilities are prescribing H2RAs instead of PPIs as a first step, as they are perceived by patients to be equally effective, and encouraging patients to self-regulate drug treatment through on-demand regimes.     

Long-term air pollution exposure and lung function in 15 year-old adolescents living in an urban and rural area in Germany: The GINIplus and LISAplus cohorts

IntroductionThe impact of outdoor air pollution exposure on long-term lung development and potential periods of increased lung susceptibility remain unknown. This study assessed associations between early-life and current residential exposure to air pollution and lung function at 15-years of age in two German birth cohorts.MethodsFifteen year-old participants living in an urban and rural area in Germany underwent spirometry before and after bronchodilation (N = 2266). Annual average (long-term) exposure to nitrogen dioxide (NO₂), particles with aerodynamic diameters less than 2.5 μg/m³ (PM_2.5) mass and less than 10 μg/m³ (PM₁₀) mass, PM_2.5 absorbance and ozone were estimated to each participant's birth-, 10- and 15-year home address using land-use regression and kriging (ozone only) modelling. Associations between lung function variables and long-term pollutant concentrations were assessed using linear regression models adjusted for host and environmental covariates and recent short-term air pollution exposures.ResultsLong-term air pollution concentrations assessed to the birth-, 10- and 15-year home addresses were not associated with lung function variables, before and after bronchodilation, in the complete or study area specific populations. However, several lung function variables were negatively associated with long-term NO₂ concentrations among asthmatics. For example, NO₂ estimated to the 15-year home address was associated with the ratio of forced expiratory volume in one second to forced vital capacity (FEV₁/FVC) and the mean flow rate between 25% and 75% of FVC (−3.5%, 95% confidence interval [−6.0, −1.0] and −297.4 ml/s [−592.6, −2.1] per 5.9 μg/m³ increase in NO₂, respectively). Nearly all effect estimates for the associations between the short-term PM_2.5 mass, PM₁₀ mass and ozone concentrations and the lung function variables were negative in the complete population.ConclusionsEarly-life and current long-term air pollution exposures and lung function at the age of 15 years were not associated in the complete study population. Asthmatics may represent a vulnerable group.     

Methionine Restriction Prevents Lipopolysaccharide-Induced Acute Lung Injury via Modulating CSE/H2S Pathway

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) result in high mortality, whereas effective treatments are limited. Methionine restriction (MR) has been reported to offer various benefits against multiple pathological processes of organ injuries. However, it remains unknown whether MR has any potential therapeutic value for ALI/ARDS. The current study was set to investigate the therapeutic potential of MR on lipopolysaccharide (LPS)-induced ALI and its underlying mechanisms. We found that MR attenuated LPS-induced pulmonary edema, hemorrhage, atelectasis, and alveolar epithelial cell injuries in mice. MR upregulated cystathionine-gamma-lyase (CSE) expression and enhanced the production of hydrogen sulfide (H₂S). MR also inhibited the activation of Toll-like receptors 4 (TLR4)/NF-κB/NOD-like receptor protein 3 (NLRP3), then reduced IL-1β, IL-6, and TNF-α release and immune cell infiltration. Moreover, the protective effects of MR on LPS-induced ALI were abrogated by inhibiting CSE, whereas exogenous H₂S treatment alone mimicked the protective effects of MR in Cse^−/− mice after LPS administration. In conclusion, our findings showed that MR attenuated LPS-induced lung injury through CSE and H₂S modulation. This work suggests that developing MR towards clinical use for ALI/ARDS patients may be a valuable strategy.     

Enhancement of antigen-specific CD4+ and CD8+ T cell responses using a self-assembled biologic nanolipoprotein particle vaccine

To address the need for vaccine platforms that induce robust cell-mediated immunity, we investigated the potential of utilizing self-assembling biologic nanolipoprotein particles (NLPs) as an antigen and adjuvant delivery system to induce antigen-specific murine T cell responses. We utilized OT-I and OT-II TCR-transgenic mice to investigate the effects of NLP-mediated delivery of the model antigen ovalbumin (OVA) on T cell activation. Delivery of OVA with the TLR4 agonist monophosphoryl lipid A (MPLA) in the context of NLPs significantly enhanced the activation of both CD4⁺ and CD8⁺ T cells in vitro compared to co-administration of free OVA and MPLA. Upon intranasal immunization of mice harboring TCR-transgenic cells, NLPs enhanced the adjuvant effects of MPLA and the in vivo delivery of OVA, leading to significantly increased expansion of CD4⁺ and CD8⁺ T cells in lung-draining lymph nodes. Therefore, NLPs are a promising vaccine platform for inducing T cell responses following intranasal administration.     

Poly-(lactic-co-glycolic-acid)-based particulate vaccines: Particle uptake by dendritic cells is a key parameter for immune activation

Poly(lactic-co-glycolic acid) (PLGA) particles have been extensively studied as biodegradable delivery system to improve the potency and safety of protein-based vaccines. In this study we analyzed how the size of PLGA particles, and hence their ability to be engulfed by dendritic cells (DC), affects the type and magnitude of the immune response in comparison to sustained release from a local depot. PLGA microparticles (MP, volume mean diameter ≈ 112 μm) and nanoparticles (NP, Z-average diameter ≈ 350 nm) co-encapsulating ovalbumin (OVA) and poly(I:C), with comparable antigen (Ag) release characteristics, were prepared and characterized. The immunogenicity of these two distinct particulate vaccines was evaluated in vitro and in vivo. NP were efficiently taken up by DC and greatly facilitated MHC I Ag presentation in vitro, whereas DC cultured in the presence of MP failed to internalize significant amounts of Ag and hardly showed MHC I Ag presentation. Vaccination of mice with NP resulted in significantly better priming of Ag-specific CD8⁺ T cells compared to MP and OVA emulsified with incomplete Freund's adjuvant (IFA). Moreover, NP induced a balanced T_H1/T_H2-type antibody response, compared to vaccinations with IFA which stimulated a predominant T_H2-type response, whereas MP failed to increase antibody titers. In conclusion, we postulate that particle internalization is of crucial importance and therefore particulate vaccines should be formulated in the nano- but not micro-size range to achieve efficient uptake, significant MHC class I cross-presentation and effective T and B cell responses.     

Toll-like receptor 1 and 10 polymorphisms,Helicobacter pylori susceptibility and risk of gastric lesions in a high-risk Chinese population

Genetic polymorphisms of Toll-like receptor (TLR) 1 and 10 may influence Helicobacter pylori (H. pylori) susceptibility. To evaluate associations between TLR1 and 10 polymorphisms, H. pylori infection, and precancerous gastric lesions, a population-based study was conducted in a high-risk Chinese population.Three single-nucleotide polymorphisms, TLR1 rs4833095, TLR10 rs10004195, and TLR10 rs4129009 were genotyped by TaqMan SNP genotyping assay in 2553 participants with diverse gastric lesions. The status of H. pylori infection was determined by ¹³C-urea breath test.TLR1 rs4833095 T and TLR10 rs10004195 T alleles were the minor alleles and showed in linkage disequilibrium (D′ = 0.98, r² = 0.73) in the Chinese population. A decreased risk of H. pylori infection was observed in subjects with TLR1 rs4833095 CT genotype [adjusted odds ratio (OR) = 0.80; 95% confidence interval (CI): 0.66–0.96] or T allele (OR = 0.82; 95%CI: 0.69–0.99). Moreover, subjects carrying TLR1 rs4833095 TT genotype were associated with reduced risks of chronic atrophic gastritis (CAG, OR = 0.66; 95%CI: 0.45–0.97) and intestinal metaplasia (IM, OR = 0.57; 95%CI: 0.36–0.90). The risk of CAG was also decreased in subjects carrying TLR10 rs10004195 T allele (OR = 0.75; 95%CI: 0.57–0.99). Furthermore, haplotype analysis indicated that haplotype TT of rs4833095 and rs10004195 had a protective effect on H. pylori infection (OR = 0.83; 95%CI: 0.72–0.96) or precancerous gastric lesions (OR = 0.78; 95%CI: 0.64–0.96 for CAG, and OR = 0.74; 95%CI: 0.57–0.96 for IM).These findings suggest that TLR1 rs4833095 and TLR10 rs10004195 may play crucial roles in H. pylori susceptibility and gastric pathogenesis.     

Immunogenicity and protective efficacy of clade 2.3.2.1c and clade 2.3.4.4c H5Nx avian influenza antigen bank vaccines in mice,Korea

• The immunogenicity and protective efficacy of inactivated clade 2.3.2.1c (rgKA435) and clade 2.3.4.4c (rgES2) H5Nx vaccines, which are representatives of an avian influenza antigen bank in Korea, were examined in mice. Mice were vaccinated twice and then challenged with homologous virus. Hemagglutinin inhibition and serum neutralizing antibody titers in the rgES2-vaccinated group were higher (4.4 ± 1.7 and 10.8 ± 2.3 log₂, respectively) than those in the rgKA435-vaccinated group (2.8 ± 1.1 and 2.5 ± 0.9 log₂, respectively). rgES2 conferred 100% protection, with no morbidity, no severe body weight loss, and no virus replication in any of the tissues tested. By contrast, 80% of mice in the rgKA435 group survived. One mouse in this group died at 10 dpi. Virus titers in the lung and turbinate were 10^2.5–3.5 TCID₅₀/0.1 ml at 3–7 dpi and 10^1.5 TCID₅₀/0.1 ml at 3–5 dpi, respectively. In particular, the viral titer in the turbinate from the rgKA435 group at 3 dpi was significantly lower than that in the equivalent control group (p < 0.05). The data suggest that both of these antigen bank vaccines are promising candidates for further evaluation in humans.

     

Acute ethanol administration inhibits Toll-like receptor 4 signaling pathway in rat intestinal epithelia

Excess alcohol intake, as in binge drinking, increases susceptibility to microbial pathogens. Alcohol impairs macrophage function by suppression of the Toll-like receptor 4 (TLR4) pathway. This study investigated the effects of acute ethanol intake on the TLR4 pathway in rat intestinal epithelia, which usually encounters luminal antigens at first and participates in the development of intestinal immunity. Twenty Wistar rats were randomly assigned to an ethanol group given ethanol as a 25% (v/v) solution in water at 7.5 g/kg, or a control group given saline, by oral gavage daily for 3 days. The epithelial histology and ultrastructure, the intestinal microflora, peripheral and portal venous plasma lipopolysaccharide (LPS) levels, and somatostatin (SST) levels in the peripheral plasma and small intestine were evaluated. Somatostatin receptor 2 (SSTR2), TLR4, TANK binding kinase-1 (TBK1), activated nuclear factor-κB (NF-κB), interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) in the intestinal mucosa were assayed. LPS responsiveness with or without SST pretreatment was assayed in vitro by quantification of TLR4, TBK1, activated NF-κB, IFN-γ and TNF-α in isolated intestinal epithelia. Mucosal damage was observed in the ethanol group by light and electron microscopy. Escherichia coli cultures were unchanged in rat intestine of the ethanol group compared with controls, but lactobacilli cultures were reduced (p < 0.05). LPS levels increased in peripheral and portal venous plasma (p < 0.05), but mucosal TLR4, TBK1, nuclear NF-κB, IFN-γ and TNF-α were unchanged in the ethanol group. LPS treatment in vitro up-regulated the level of TLR4, TBK1 and nuclear NF-κB as well as the production of IFN-γ and TNF-α in isolated intestinal epithelia in the control (p < 0.05), but not the ethanol group. The stimulatory effects of LPS on intestinal epithelia isolated from the control group were significantly inhibited by SST pretreatment (p < 0.05). The peripheral plasma and intestinal levels of SST and the mucosal expression of SSTR2 in the ethanol group were significantly higher than in the control group (p < 0.05). These findings suggest the hyposensitivity of intestinal epithelial TLR4 to LPS induced by acute alcohol abuse probably through ethanol per se and ethanol-enhanced intestinal mucosal SST pathway may be a novel mechanism for increased susceptibility to intestinal pathogens.     

Analysis of TLR4 (Asp299Gly and Thr399Ile) gene polymorphisms and mRNA level in patients with dengue infection: A case-control study

BackgroundDengue is a systemic viral infection that spreads to humans by the bite of infected Aedes mosquitoes. The secreted NS1 protein of dengue virus activates macrophages and human PBMCs via TLR4 and induce the release of pro-inflammatory cytokines which is responsible for the pathogenesis of disease. Mutations in TLR4 gene have been associated with the increased susceptibility to many viral, bacterial and parasitic diseases.ObjectiveTo study the impact of TLR4 Asp299Gly (rs4986790) and Thr399Ile (rs4986791) gene polymorphisms with susceptibility to dengue infection.MethodsA total of 120 dengue infected (57; DHF/DSS and 63; DF) and 200 healthy controls were included in the study. TLR4 Asp299Gly and Thr399Ile gene polymorphisms was studied by PCR-RFLP. Expression of TLR4 mRNA was evaluated by rRT-PCR.ResultsIndividuals with heterozygous genotype for TLR4 Asp299Gly and Thr399Ile polymorphisms had increased susceptibility to dengue infection (OR-1.70, 95% CI = 1.01–2.86 P = 0.042 and OR-2.17, 95% CI = 1.10–4.28, P = 0.024, respectively). The frequency of Gly and Ile alleles were higher in dengue patients as compared to controls (OR-1.67, 95% CI = 1.05–2.64, P = 0.029 and OR-2.20, 95% CI = 1.19–4.07, P = 0.011, respectively). IIe/Gly haplotype was associated with the risk of the disease when compared with controls (OR = 3.15, 95% CI = 1.09–9.09, P = 0.035). The mRNA expression was higher in DF when compared with DHF/DSS and controls (P = 0.040 and 0.009, respectively).ConclusionA higher expression of TLR4 mRNA was associated with DF. The TLR4 Asp299Gly and Thr399Ile gene polymorphisms were associated with the susceptibility of dengue infection probably by altering the immune response.     

Parenting aggravation and autism spectrum disorders: 2007 National Survey of Children's Health

Background

Studies suggest autism spectrum disorders (ASDs) are associated with high parenting stress and aggravation. Research on specific risk factors is needed.

Objective/Hypotheses

To assess aggravation level among parents of children with and without ASDs.

Methods

The sample of 73,030 children aged 4 to 17 years in the 2007 National Survey of Children’s Health and their parent respondents were divided into mutually exclusive groups based on child ASD status and other special health care needs. Adjusted prevalence ratios (aPR) for associations between a high Aggravation in Parenting scale score and various risk factors were computed from multivariable models.

Results

High-aggravation percentages were comparable for parents of children with a current ASD (36.6%), ASD reported previously but not currently (35.2%), and another (non-ASD) developmental problem (31.2%) but were significantly lower for parents of children with other special health care needs (6.5%) and no special health care needs (5.1%). Within the current-ASD group, high aggravation was associated with young child age (aPR = 1.8 [1.2-2.6]), lack of health insurance (aPR = 1.5 [1.0-2.4]), lack of a medical home (aPR = 2.2 [1.4-3.5]), recent child mental health treatment (aPR = 2.1 [1.5-3.0]), lack of parenting emotional support (aPR = 1.5 [1.1-2.1]), and ASD severity (aPR = 1.4 [1.0-1.6]). Some of these same factors were associated with aggravation in the non-ASD groups. However, the medical home finding was specific to the ASD groups.

Conclusions

Parenting a child with ASD is associated with high aggravation; however, there is variability within health care and social support subgroups. Strategies to strengthen medical home components for children with ASDs should be considered.     

Synthesis and biological activity of novel N-cycloalkyl-(cycloalkylaryl)-2-[(3-R-2-oxo-2H-[1,2,4]triazino[2,3-c]quinazoline-6-yl)thio]acetamides

In this paper the novel N-cycloalkyl-(cycloalkylaryl)-2-[(3-R-2-oxo-2H-[1,2,4]triazino[2,3-c]quinazoline-6-yl)thio]acetamides synthesis by aminolysis of activated by thionyl chloride or carbonyldiimidazole [(3-R-2-oxo-2H-[1,2,4]triazino[2,3-c]quinazolin-6-yl)-thio]acetic acids and alkylation of the 3-R-6-thio-6,7-dihydro-2H-[1,2,4]triazino[2,3-c]quinazoline-2-ones potassium salts with N-cycloalkyl-(cycloalkylaryl)-2-chloroacetamides are proposed. The structures of compounds are determined by ¹H, ¹³C NMR, LC-MS and EI-MS analysis. The in vitro anticancer, antibacterial activity and Photobacterium leiognathi Sh1 bioluminescence inhibition of synthesized compounds were revealed. SAR results were discussed. Compound 4.10 was found to be the most anticancer active one, selectively influenced on the non-small cell lung and CNS cancer cell lines, especially on the HOP-92 (log GI₅₀ = −6.01) and U251 (log GI₅₀ = −6.00).     

A TLR2 agonist is a more effective adjuvant for a Chlamydia major outer membrane protein vaccine than ligands to other TLR and NOD receptors

Chlamydia trachomatis (Ct) is the most common sexually transmitted bacterial pathogen in the World and there is an urgent need for a vaccine to prevent these infections. To determine what type of adjuvant can better enhance the immunogenicity of a Chlamydia vaccine, we formulated the recombinant major outer membrane protein (Ct-rMOMP) with several ligands for Toll-like receptors (TLR) and the nucleotide-binding oligomerization domain (NOD) including Pam₂CSK₄ (TLR2/TLR6), Poly (I:C) (TLR3), monophosphoryl lipid A (TLR4), flagellin (TLR5), imiquimod R837 (TLR7), imidazoquinoline R848 (TRL7/8), CpG-1826 (TLR9), M-Tri-_DAP (NOD1/NOD2) and muramyldipeptide (NOD2). Groups of female BALB/c mice were immunized intramuscularly (i.m.) three times with the Ct-rMOMP and each one of those adjuvants. Four weeks after the last immunization the mice were challenged intranasally (i.n.) with 10⁴C. trachomatis mouse pneumonitis (MoPn) inclusion forming units (IFU). As negative antigen control, mice were immunized with the Neisseria gonorrhoeae recombinant porin B (Ng-rPorB) and the same adjuvants. As a positive vaccine control, mice were inoculated i.n. with 10⁴ IFU of MoPn. The humoral and cell mediated immune responses were determined the day before the challenge. Following the challenge the mice were weighed daily and, at 10 days post-challenge (p.c.), they were euthanized, their lungs weighted and the number of IFU in the lungs counted. As determined by the IgG2a/IgG1 ratio in the sera, mice immunized with Ct-rMOMP + Pam₂CSK₄ showed a strong Th2 biased humoral immune response. Furthermore, these mice developed a robust cellular immune response with high Chlamydia-specific T cell proliferation and levels of IFN-γ production. In addition, based on changes in body weight, weight of the lungs and number of IFU recovered from the lungs, the mice immunized with Ct-rMOMP + Pam₂CSK₄, were better protected against the i.n. challenge than any group of mice immunized with Ct-rMOMP and the other adjuvants. In conclusion, Pam₂CSK₄ should be evaluated as a candidate adjuvant for a C. trachomatis vaccine.     

Fusicoccin Counteracts the Toxic Effect of Cadmium on the Growth of Maize Coleoptile Segments

The effects of cadmium (Cd; 0.1–1000 μM) and fusicoccin (FC) on growth, Cd²⁺ content, and membrane potential (E _m) in maize coleoptile segments were studied. In addition, the E _m changes and accumulation of Cd and calcium (Ca) in coleoptile segments treated with Cd²⁺ combined with 1 μM FC or 30 mM tetraethylammonium (TEA) chloride (K⁺-channel blocker) were also determined. In this study, the effects of Ca²⁺-channel blockers [lanthanum (La) and verapamil (Ver)] on growth and content of Cd²⁺ and Ca²⁺ in coleoptile segments were also investigated. It was found that Cd at high concentrations (100 and 1000 μM) significantly inhibited endogenous growth of coleoptile segments and simultaneously measured proton extrusion. FC combined with Cd²⁺ counteracted the toxic effect of Cd²⁺ on endogenous growth and significantly decreased Cd²⁺ content (not the case for Cd²⁺ at the highest concentration) in coleoptile segments. Addition of Cd to the control medium caused depolarization of E _m, the extent of which was dependent on Cd concentration and time of treatment with Cd²⁺. Hyperpolarization of E _m induced by FC was suppressed in the presence of Cd²⁺ at 1000 μM but not Cd²⁺ at 100 μM. It was also found that treatment of maize coleoptile segments with 30 mM TEA chloride caused hyperpolarization of E _m and decreased Cd²⁺ content in coleoptile segments, suggesting that, in the same way as for FC, accumulation of Cd²⁺ was dependent on plasma membrane (PM) hyperpolarization. Similar to FC, TEA chloride also decreased Ca²⁺ content in coleoptile segments. La and Ver combined with Cd²⁺ (100 μM) significantly decreased Cd content in maize coleoptile segments, but only La completely abolished the toxic effect of Cd²⁺ on endogenous growth and growth in the presence of FC. Taken together, these results suggest that the mechanism by which FC counteracts the toxic effect of Cd²⁺ (except at 1000 μM Cd²⁺) on the growth of maize coleoptile segments involves both stimulation of PM H⁺-ATPase activity by FC as well as Cd²⁺-permeable, voltage-dependent Ca channels, which are blocked by FC and TEA chloride-induced PM hyperpolarization.     

Episodic Trihalomethane Species and Levels in Tap Water at a Start of Operation of Advanced Treatment in Osaka Prefectural Water Supplies

Empirical equations for explaining trihalomethane (THM) distribution were established based on data on tap water in Osaka City before and after starting the advanced treatment and were represented by the following equations for bromodichloromethane (S₁), dibromochloromethane (S₂) and bromoform distribution factors (S₃) as a function of chloroform distribution factor (S₀), respectively: S₁ = −0.97S₀² + 0.75S₀ + 0.19 (regression coefficient: r = 0.71), S₂ = 0.13S₀²−0.76S₀ + 0.51 (r = 0.98) and S₃ = 0.83S₀²−0.99S₀ + 0.30 (r = 0.97). Further, the derived equations were verified by THMs data of Osaka Prefecture and its surrounding cities (except for Osaka City), receiving a combination of advanced treated water and conventionally treated different source water.     

Adjuvant-active aqueous extracts from Artemisia rupestris L. improve immune responses through TLR4 signaling pathway

Activating innate immunity by an adjuvant is required in vaccine development. The study aims to investigate adjuvant effects of aqueous extracts of Artemisia rupestris L. (AEAR) in vivo and in vitro. ICR mice were subcutaneously administered with antigen and AEAR at various doses to evaluate their immune responses of antibodies, dendritic cells (DCs), regulatory T cells (Treg), splenic lymphocyte, and cytokine. The evaluation results showed that AEAR could largely increase titers of antigen-specific antibodies (IgG, IgG₁, and IgG_2a) and T cell proliferation. AEAR also increased expression of IFN-γ in CD8⁺T cells as well as IL-4 and INF-γ expression in CD4⁺T cells. Expression levels of MHC-II, CD40, CD80, and CD86 on DCs were significantly elevated, whereas the Treg frequency was significantly decreased. AEAR (200 μg) showed remarkable adjuvant activity. Furthermore, AEAR enhanced MHC-II, CD40, CD80, and CD86 expression as well as the yields of TNF-α and IL-12 on DCs through toll-like receptor4 (TLR4) in vitro. Those results indicated that AEAR could serve as an efficacious immune stimulator for vaccines because it significantly enhanced specific immune responses by promoting DCs maturation and reduced Treg through TLR4 signaling pathway.