Function of proprotein convertase subtilisin/kexin type 9 and its role in central nervous system diseases: An update on clinical evidence.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) has attracted lots of attention in preventing the clearance of plasma low-density lipoprotein cholesterol (LDL-C). PCSK9 inhibitors are developed to primarily reduce the cardiovascular risk by lowering LDL-C level. Recently, a number of pleiotropic extrahepatic functions of PCSK9 beyond the regulation of cholesterol metabolism, particularly its effects on central nervous system (CNS) diseases have been increasingly identified. Emerging clinical evidence have revealed that PCSK9 may play a significant role in neurocognition, depression, Alzheimer's disease, and stroke. The focus of this review is to elucidate the functions of PCSK9 and highlight the effects of PCSK9 in CNS diseases, with the aim of identifying the potential risks that may arise from low PCSK9 level (variant or inhibitor) in the clinical practice.

S100A4 exerts robust mucosal adjuvant activity for co-administered antigens in mice.

The lack of clinically applicable mucosal adjuvants is a major hurdle in designing effective mucosal vaccines. We hereby report that the calcium-binding protein S100A4, which regulates a wide range of biological functions, is a potent mucosal adjuvant in mice for co-administered antigens, including the SARS-CoV-2 spike protein, with comparable or even superior efficacy as cholera toxin but without causing any adverse reactions. Intranasal immunization with recombinant S100A4 elicited antigen-specific antibody and pulmonary cytotoxic T cell responses, and these responses were remarkably sustained for longer than 6 months. As a self-protein, S100A4 did not stimulate antibody responses against itself, a quality desired of adjuvants. S100A4 prolonged nasal residence of intranasally delivered antigens and promoted migration of antigen-presenting cells. S100A4-pulsed dendritic cells potently activated cognate T cells. Furthermore, S100A4 induced strong germinal center responses revealed by both microscopy and mass spectrometry, a novel label-free technique for measuring germinal center activity. Importantly, S100A4 did not induce olfactory bulb inflammation after nasal delivery, which is often a safety concern for nasal vaccination. In conclusion, S100A4 may be a promising adjuvant in formulating mucosal vaccines, including vaccines against pathogens that infect via the respiratory tract, such as SARS-CoV-2.

[Spatiotemporal Variations in Fine Particulate Matter and the Impact of Air Quality Control in Zhengzhou].

To study the spatiotemporal variations in fine particulate matter (PM2.5) and the impact of air quality management in autumn and winter in Zhengzhou, five sites were selected to collect PM2.5 samples from the autumn of 2017 to the winter of 2018, and the characteristics of the chemical components were analyzed. The positive matrix factorization (PMF) model was also applied to identify the sources of PM2.5, and the effect of air quality control was evaluated to provide support for air quality control in autumn and winter in the next stage. The PM2.5 concentrations in the four seasons in Zhengzhou were ranked as winter > autumn > spring > summer. The PM2.5 concentration at Zhengzhou University (ZZU) was the highest (8.7% higher than the average concentration), and the PM2.5 concentrations at the other sites were slightly lower than the average concentration. The concentration of water-soluble ions (WSIs) was low in spring and summer and high in autumn and winter. The average proportions of SO42-, NO3-, and NH4+ in the nine WSIs were as high as 22.5%, 43.6%, and 23.4%, respectively. The proportion of Cl- in winter was higher than that in the other seasons owing to coal combustion (6.7% and 6.6% in 2017 and 2018, respectively). Owing to wind and sand, the proportions of Ca2+ and Mg2+ in spring were the highest (4.4% and 0.4%, respectively), and those at the Jiancezhan (JCZ) and ZZU sites were higher than those at the other sites. K+, as a marker of biomass burning, had a higher proportion in spring, autumn, and winter. The proportion of K+ in the spring of 2018 was 1.9%, those in the autumn and winter of 2017 were 1.6% and 2.1%, respectively, and those in the autumn and winter of 2018 were 1.3% and 1.8%, respectively. JCZ, Hangkonggang (HKG), and Xinmi (XM) had higher proportions of NO3-, and the proportions of SO42- were lower. Secondary organic carbon (SOC) pollution was serious in autumn and winter, and the concentration accounted for more than half of the organic carbon (OC). In 2018, the SOC/OC at the JCZ and ZZU sites decreased compared with that in 2017, but that at the other three sites increased significantly, thereby indicating that different air pollutant emissions in these regions had different performances in response to control policies. The chemical composition reconstruction results showed that the proportion of sulfate was highest in summer (25.0%), the contribution of nitrate was higher in autumn (23.1% and 25.1% for 2017 and 2018, respectively) and winter (20.6% and 23.0% for 2017 and 2018, respectively), the proportion of crustal material was higher in spring (18.2%), and the contribution of secondary organic aerosol (SOA) was the highest in winter (14.1% and 20.5% for 2017 and 2018, respectively). SOA had higher contributions at the JCZ and HKG sites (16.9% and 16.4%, respectively), and ZZU was affected more by primary organic aerosol (14.3%) and crustal materials (12.1%). The PMF results showed that secondary inorganic salts (37.5%), SOA (15.4%), traffic (14.9%), industry (4.8%), coal combustion (16.0%), fugitive dust (6.5%), and biomass burning (2.8%) were the main pollution sources of PM2.5 in Zhengzhou. SOA and coal combustion contributed more in winter and fugitive dust contributed more in spring, followed by autumn. Biomass burning contributed more in spring and autumn. The urban sites JCZ and ZZU and the characteristic site HKG near the airport were more affected by traffic sources (16.9%, 16.2%, and 16.0%, respectively) than the other sites. The impact of biomass burning on the non-urban sites XM and HKG was slightly larger (both 2.7%), and the contribution of coal combustion to the suburban site XM was higher (16.8%). Owing to the construction around ZZU, the loading of fugitive dust at ZZU was higher than that at other sites. Comparing the results of the two-year autumn and winter, the contribution of SOA, traffic, and industry increased in the autumn and winter of 2018, whereas the contribution of secondary inorganic salts, coal combustion, and biomass burning decreased and the contribution of fugitive dust in winter also decreased. The results showed that the control strategies in autumn and winter had significant effects on the primary sources, including fugitive dust, coal combustion, and industry, and SOA precursor volatile organic compounds should be targeted for further pollution control.

Evaluation of the association between extravascular lung water and prognosis of sepsis: A protocol of systematic review.

BACKGROUND: The purpose of this study is to explore the association between extravascular lung water (EVLW) and prognosis of sepsis (PS). METHODS: We will carry out comprehensive literature search in electronic databases (PUBMED/MEDLINE, EMBASE, CENTRAL, WorldSciNet, PsycINFO, Allied and Complementary Medicine Database, CBM, and CNKI) and additional sources. All electronic databases will be searched from their initial to the present without language restrictions. Case-controlled studies reporting the association between EVLW and PS will be evaluated for inclusion. Outcomes of interest will include mortality rate, extravascular lung water index, pulmonary vascular permeability index, blood lactate clearance, oxygenation index, blood gas analysis, PaO2/FiO2, cardiac output index, global end diastolic volume index, intrathoracic blood volume index, systemic resistance index, acute physiology and chronic health scoring system II, and infection-related organ failure scoring system. Study quality will be evaluated using Newcastle-Ottawa Tool, and statistical analysis will be performed utilizing RevMan 5.4 software. RESULTS: This study will summarize the most recent evidence to investigate the association between EVLW and PS. CONCLUSIONS: The results of this study will provide an exhaustive view of the association between EVLW and PS. STUDY REGISTRATION OSF: osf.io/vhnxw.

Mzb1 protects against myocardial infarction injury in mice via modulating mitochondrial function and alleviating inflammation.

Myocardial infarction (MI) leads to the loss of cardiomyocytes, left ventricle dilation and cardiac dysfunction, eventually developing into heart failure. Mzb1 (Marginal zone B and B1 cell specific protein 1) is a B-cell-specific and endoplasmic reticulum-localized protein. Mzb1 is an inflammation-associated factor that participates a series of inflammatory processes, including chronic periodontitis and several cancers. In this study we investigated the role of Mzb1 in experimental models of MI. MI was induced in mice by ligation of the left descending anterior coronary artery, and in neonatal mouse ventricular cardiomyocytes (NMVCs) by H2O2 treatment in vitro. We showed that Mzb1 expression was markedly reduced in the border zone of the infarct myocardium of MI mice and in H2O2-treated NMVCs. In H2O2-treated cardiomyocytes, knockdown of Mzb1 decreased mitochondrial membrane potential, impaired mitochondrial function and promoted apoptosis. On contrary, overexpression of Mzb1 improved mitochondrial membrane potential, ATP levels and mitochondrial oxygen consumption rate (OCR), and inhibited apoptosis. Direct injection of lentiviral vector carrying Len-Mzb1 into the myocardial tissue significantly improved cardiac function and alleviated apoptosis in MI mice. We showed that Mzb1 overexpression significantly decreased the levels of Bax/Bcl-2 and cytochrome c and improved mitochondrial function in MI mice via activating the AMPK-PGC1α pathway. In addition, we demonstrated that Mzb1 recruited the macrophages and alleviated inflammation in MI mice. We conclude that Mzb1 is a crucial regulator of cardiomyocytes after MI by improving mitochondrial function and reducing inflammatory signaling pathways, implying a promising therapeutic target in ischemic cardiomyopathy.

Association between microRNA 25 expression in serum and lung cancer: A protocol for systematic review and meta-analysis.

BACKGROUND: This study aims to identify the association between microRNA 25 (mRNA 25) expression in serum and lung cancer (LC). METHODS: This planned study will cover all eligible case-controlled studies that report association between mRNA 25 expression in serum and LC. It will include published studies from inception to the present in Cochrane Library, PUBMED, EMBASE, Web of Science, Allied and Complementary Medicine Database, VIP database, and China National Knowledge Infrastructure regardless language and geographical location. We will also search other sources, such as conference abstracts and reference lists of related known studies and experts in the domain consulted to avoid missing potential studies. Two contributors will independently examine and select studies, collect all necessary data, and judge study quality for all included studies. We will perform statistical analysis using RevMan V.5.3 software and Stata V.12.0 software. RESULTS: This study will summarize current evidence to present first systematic review of research on the association between mRNA 25 expression in serum and LC. CONCLUSION: This study will present comprehensive evidence to determine whether mRNA 25 expression in serum is associated with LC, and will provide helpful evidence for the future studies. SYSTEMATIC REVIEW REGISTRATION: INPLASY202040056.

LINC00174 is an oncogenic lncRNA of hepatocellular carcinoma and regulates miR-320/S100A10 axis.

Hepatocellular carcinoma (HCC) is one of the deadliest cancers. Multiple long non-coding RNAs (lncRNAs) are recently identified as crucial oncogenic factors or tumour suppressors. In this study, we explored the effects of LINC00174 on the progression of HCC. Expression levels of LINC00174 and microRNA-320 (miR-320) in HCC tissue samples were measured using quantitative real-time polymerase chain reaction (qRT-PCR). The association between pathological indices and LINC00174 was also analysed. Human HCC cell lines Hep3B and Huh7 were used as cell models. CCK-8 and bromodeoxyuridine (BrdU) assays were used to assess the effect of LINC00174 on HCC cell line proliferation. Flow cytometry was used to study the effect of LINC00174 on HCC apoptosis. Transwell assay was conducted to detect the effect of LINC00174 on migration and invasion. Furthermore, luciferase reporter assay and RNA immunoprecipitation (RIP) assay were used to confirm the binding relationship between miR-320 and LINC00174. Additionally, western blot was used to detect the regulatory function of LINC00174 on oncogene S100 calcium binding protein A10 (S100A10). We demonstrated that LINC00174 expression in HCC clinical samples was significantly increased and this was correlated with higher T stage. Its overexpression remarkably accelerated proliferation and metastasis of HCC cells while reduced apoptosis. Accordingly, knockdown of it suppressed the malignant phenotypes of HCC cells. Overexpression of LINC00174 significantly reduced the expression of miR-320 by sponging it, in turn enhanced the expression of S100A10. In conclusion, LINC00174 is a sponge of tumour suppressor miR-320, enhances the expression of S100A10 indirectly and functions as an oncogenic lncRNA in HCC. SIGNIFICANCE OF THE STUDY: LINC00174 is a novel lncRNA, whose function is rarely investigated. It is reported that it is oncogenic in colorectal cancer, while its role in HCC remains unclear. Herein, we report that LINC00174 is significantly up-regulated in HCC tissues and promotes the malignant phenotypes. We demonstrate that LINC00174 functions as a sponge for miR-320, increases the expression level of oncogene S100A10 in HCC. This study helps clarify the mechanism of HCC tumorigenesis and progression, and uncover the role of LINC00174 in human disease.

CRISPR/Cas9 mediated gene knockout reveals a more important role of PBP1 than PBP2 in the perception of female sex pheromone components in Spodoptera litura.

Three different pheromone binding proteins (PBPs) can typically be found in the sensilla lymph of noctuid moth antennae, but their relative contributions in perception of the sex pheromone is rarely verified in vivo. Previously, we demonstrated that SlitPBP3 plays a minor role in the sex pheromone detection in Spodoptera litura using the CRISPR/Cas9 system. In the present study, the roles of two other SlitPBPs (SlitPBP1 and SlitPBP2) are further verified using the same system. First, by co-injection of Cas9 mRNA/sgRNA into newly laid eggs, a high rate of target mutagenesis was induced, 51.5% for SlitPBP1 and 46.8% for SlitPBP2 as determined by restriction enzyme assay. Then, the homozygous SlitPBP1 and SlitPBP2 knockout lines were obtained by cross-breeding. Finally, using homozygous knockout male moths, we performed electrophysiological (EAG recording) and behavioral analyses. Results showed that knockout of either SlitPBP1 or SlitPBP2 in males decreased EAG response to each of the 3 sex pheromone components (Z9,E11-14:Ac, Z9,E12-14:Ac and Z9-14:Ac) by 53%, 60% and 63% (for SlitPBP1 knockout) and 40%, 43% and 46% (for SlitPBP2 knockout), respectively. These decreases in EAG responses were similar among 3 pheromone components, but were more pronounced in SlitPBP1 knockout males than in SlitPBP2 knockout males. Consistently, behavioral assays with the major component (Z9,E11-14:Ac) showed that SlitPBP1 knockout males responded in much lower percentages than SlitPBP2 knockout males in terms of orientation to the pheromone, along with reduction in close range behaviors such as hairpencil display and mating attempt. Taken together, this study provides direct functional evidence for the roles of SlitPBP1 and SlitPBP2, as well as their relative importance (SlitPBP1 > SlitPBP2) in the sex pheromone perception. This information is valuable in understanding mechanisms of sex pheromone perception and may facilitate the development of PBP-targeted pest control techniques.

Expression Profile and Functional Characterization Suggesting the Involvement of Three Chemosensory Proteins in Perception of Host Plant Volatiles in Chilo suppressalis (Lepidoptera: Pyralidae).

The high sensitivity of the olfactory system is essential for feeding and oviposition in moth insects, and some chemosensory proteins (CSPs) are thought to play roles in this system by binding and carrying hydrophobic odorants across the aqueous sensillar lymph. In this study, to identify the olfactory CSPs from a repertoire of 21 CSP members in the notorious rice pest Chilo suppressalis (Walker) (Lepidoptera: Pyralidae), tissue expression patterns were firstly examined by quantitative real-time polymerase chain reaction (qPCR). It showed that CSP2 was antennae specific and seven more CSPs (CSP1, 3, 4, 6, 15, 16, and 17) were antennae biased in expression, suggesting their olfactory roles; while other CSPs were multiple-tissue expressed and non-antennae biased, suggesting other functions for these genes. To further determine the ligand binding specificity, three putative olfactory genes (CSP1-3) were expressed in Escherichia coli cells, and binding affinity of these three recombinant CSP proteins were measured for 35 plant volatiles by the ligand binding assays. CSP1 and CSP2 exhibited high binding affinities (Ki ≤ 10.00 µM) for four (2-tridecanone, benzaldehyde, laurinaldehyde and 2-pentadecanone) and two (2-heptanol and (+)-cedrol) host plant volatiles, respectively; the three CSPs also showed moderate binding affinity (Ki = 10.01-20.00 µM) for 16 plant volatiles. Our study suggests that the three CSPs play essential roles in the perception of host plant volatiles, providing bases for the elucidation of olfactory mechanisms in this important pyralid pest.

Evaluation of the diagnostic performance of panfungal polymerase chain reaction assay in invasive fungal diseases.

Timely diagnosis of invasive fungal diseases (IFDs) is important, as delays in treatment initiation are associated with increased mortality rates. However, early diagnosis of IFDs in immunocompromised patients remains difficult. The conventional diagnostic methods currently used for IFDs are not sufficiently effective. Molecular tests, such as polymerase chain reaction (PCR)-based assays, have great potential to improve the early diagnosis of IFDs due to their sensitivity and specificity. In the present study, the diagnostic performance of panfungal PCR assays in IFD patients who received bone marrow transplantation was evaluated. The results suggested that panfungal PCR assay offered a quick and convenient guide for clinical decision-making by identifying higher numbers of fungal species in comparison with the conventional blood culture method. Furthermore, panfungal PCR assay exhibited a sensitivity of 93% and a specificity of 71% in the diagnosis of IFD patients based on the EORTC/MSG criteria. Thus, the present study concluded that the reported PCR-based method was effective and sensitive in early IFD diagnosis and should be integrated into clinical decision-making for the treatment of IFDs in the future.

Deficiency in Calcium-Binding Protein S100A4 Impairs the Adjuvant Action of Cholera Toxin.

The calcium-binding protein S100A4 has been described to promote pathological inflammation in experimental autoimmune and inflammatory disorders and in allergy and to contribute to antigen presentation and antibody response after parenteral immunization with an alum-adjuvanted antigen. In this study, we extend these findings by demonstrating that mice lacking S100A4 have a defective humoral and cellular immune response to mucosal (sublingual) immunization with a model protein antigen [ovalbumin (OVA)] given together with the strong mucosal adjuvant cholera toxin (CT), and that this impairment is due to defective adjuvant-stimulated antigen presentation by antigen-presenting cells. In comparison to wild-type (WT) mice, mice genetically lacking S100A4 had reduced humoral and cellular immune responses after immunization with OVA plus CT, including a complete lack of detectable germinal center reaction. Further, when stimulated in vitro with OVA plus CT, S100A4-/- dendritic cells (DCs) showed impaired responses in several CT-stimulated immune regulatory molecules including the co-stimulatory molecule CD86, inflammasome-associated caspase-1 and IL-1ß. Coculture of OVA-specific OT-II T cells with S100A4-/- DCs that had been pulse incubated with OVA plus CT resulted in impaired OT-II T cell proliferation and reduced production of Th1, Th2, and Th17 cytokines compared to similar cocultures with WT DCs. In accordance with these findings, transfection of WT DCs with S100A4-targeting small interfering RNA (siRNA) but not mock-siRNA resulted in significant reductions in the expression of caspase-1 and IL-1ß as well as CD86 in response to CT. Importantly, also engraftment of WT DCs into S100A4-/- mice effectively restored the immune response to immunization in the recipients. In conclusion, our results demonstrate that deficiency in S100A4 has a strong impact on the development of both humoral and cellular immunity after mucosal immunization using CT as adjuvant.

CRISPR/Cas9 mediated BLOS2 knockout resulting in disappearance of yellow strips and white spots on the larval integument in Spodoptera litura.

The custom-design bacterial CRISPR/Cas9 system has been recently used in some insects, indicating a powerful technique for studies on gene function and transgenic insects. However, its use in lepidopteran pests is scarce. Here, we reported a CRISPR/Cas9 system mediated mutagenesis of biogenesis of lysosome-related organelles complex1, subunit 2 (BLOS2) gene in a noctuid pest Spodoptera litura. A fragment of SlitBLOS2 was identified by analyzing a S. litura transcriptome database by local basic BLAST, and the full length cDNA was acquired by RACE strategy. To clarify the function of SlitBLOS2, CRISPR/Cas9 based target mutagenesis of SlitBLOS2 was achieved, displaying a mosaic translucent integument in 62.3-70.6% larvae of G0 generation. Further PCR-based genotype analysis demonstrated various mutations occurred at the SlitBLOS2 specific target site. A homozygote mutant individual was obtained in G1 generation, in which the yellow strips and white spots on the larval integument completely disappeared. Our study clearly demonstrates the function of SlitBLOS2 in the integument coloration, and thus provides a useful marker gene for genome editing based gene functional study and pest control strategy in S. litura as well as other lepidopteran pests.

Important role for FcγRIIB on B lymphocytes for mucosal antigen-induced tolerance and Foxp3+ regulatory T cells.

FcγRIIB, the only FcγR expressed on B cells, is important in the maintenance of immunological tolerance to self-Ags. In this study, we investigated the role of FcγRIIB in Ag-specific CD4 T cell tolerance induced by mucosally administered Ag (OVA) coupled to cholera toxin B subunit (Ag/CTB) or given alone. We found that sublingual administration of Ag/CTB conjugate or intragastric administration of a >100-fold higher dose of Ag alone efficiently suppressed parenteral immunization-induced Ag-specific T cell proliferation and delayed-type hypersensitivity responses in FcγRIIB-expressing wild-type (WT), but not FcγRIIB(-/-), mice. Such mucosally induced tolerance (oral tolerance) associated with induction of Ag-specific Foxp3(+) regulatory T cells was restored in FcγRIIB(-/-) mice by adoptive transfer of either WT B cells or WT dendritic cells before the mucosal Ag/CTB treatment; it was even more pronounced in µMT mice that received FcγRIIB-overexpressing B cells before treatment. Furthermore, cell transfer in either WT or µMT mice of WT but not FcγRIIB(-/-) B cells pretreated for 1 h in vitro with Ag/CTB conjugate induced Ag-specific immunological tolerance, which was further enhanced by adoptive transfer of WT B cells pretreated with anti-Ag IgG immune complexed Ag/CTB. We conclude that FcγRIIB expression on B cells, in addition to dendritic cells, is important for mucosal induction of Ag-specific immune tolerance.

Uncoupling of natural IgE production and CD23 surface expression levels.

CD23, the low affinity receptor for immunoglobulin E (IgE), has been proposed to play a critical role in the regulation of IgE production, based on altered IgE levels in CD23-deficient mice and transgenic mouse models, as well as in mouse strains with mutations in the CD23 gene, e.g. 129 substrains. Here, we have investigated a mouse line termed LxT1 that expresses reduced CD23 surface levels on B cells, and its influence on natural IgE production. Extensive phenotypic analysis showed that CD23 surface expression was reduced in LxT1 compared to the control, without affecting B cell development in general. This CD23(low) surface level in LxT1 mice is not as a result of reduced CD23 mRNA expression levels or intracellular accumulation, but linked to a recessive locus, a 129-derived region spanning 28 Mb on chromosome 8, which includes the CD23 gene. Sequence analysis confirmed five mutations within the CD23 coding region in LxT1 mice, the same as those present in New Zealand Black (NZB) and 129 mice. However, this CD23(low) phenotype was not observed in all 129 substrains despite carrying these same CD23 mutations in the coding region. Moreover, serum IgE levels in LxT1 mice are as low as those in the C57BL/6 (B6) strain, and much lower than those in 129 substrains. These data indicate that the CD23 surface level and serum IgE level are uncoupled and that neither is directly regulated by the mutations within the CD23 coding region. This study suggests that caution should be taken when interpreting the immunological data derived from mice with different genetic background, especially if the gene of interest is thought to influence CD23 surface expression or serum IgE level.

B lymphocytes treated in vitro with antigen coupled to cholera toxin B subunit induce antigen-specific Foxp3(+) regulatory T cells and protect against experimental autoimmune encephalomyelitis.

The ability of activated B cells to protect against various experimental autoimmune or allergic diseases makes them attractive for use in cell-based therapies. We describe an efficient way to generate B cells with strong suppressive functions by incubating naive B cells with a relevant Ag conjugated to cholera toxin B subunit (CTB). This allows most B cells, irrespective of BCR, to take up and present Ag and induces their expression of latency-associated polypeptide (LAP)/TGF-ß and after adoptive transfer also their production of IL-10. With OVA as model Ag, when naive T cells were cocultured in vitro with B cells pretreated with OVA conjugated to CTB (OVA/CTB) Ag-specific CD4(+) Foxp3 regulatory T (Treg) cells increased >50-fold. These cells effectively suppressed CD25(-)CD4(+) effector T (Teff) cells in secondary cultures. Adoptive transfer of OVA/CTB-treated B cells to mice subsequently immunized with OVA in CFA induced increase in Foxp3 Treg cells together with suppression and depletion of Teff cells. Likewise, adoptive transfer of B cells pulsed with myelin oligodendrocyte glycoprotein peptide(35-55) (MOGp) conjugated to CTB increased the number of Treg cells, suppressed MOGp-specific T cell proliferation and IL-17 and IFN-γ production, and prevented the development of experimental autoimmune encephalomyelitis. Similar effects were seen when B cells were given "therapeutically" to mice with early-stage experimental autoimmune encephalomyelitis. Our results suggest that B cells pulsed in vitro with relevant Ag/CTB conjugates may be used in cell therapy to induce Ag-specific suppression of autoimmune disease.

Impaired IFN-γ production after stimulation with bacterial components by natural killer cells from gastric cancer patients.

Gastric adenocarcinoma is a major health problem world-wide, as this is the second most common cause of cancer death in the world. It has been estimated that infection by Helicobacter pylori cause at least half of the gastric cancers. Previously, we have demonstrated that H. pylori antigens directly activate NK cells to secrete IFN-γ. There is also a marked synergistic effect in NK cells stimulated with bacterial lysate and low levels of IL-12, a cytokine which is produced by macrophages and dendritic cells in the H. pylori-infected stomach. The present study was designed to investigate whether NK cells from gastric cancer patients display an altered ability to respond to components from H. pylori and other bacteria. The results show that NK cells from peripheral blood of gastric cancer patients have a severely suppressed ability to produce IFN-γ after stimulation with H. pylori lysate and the synthetic bacterial lipoprotein FSL-1. Furthermore, the synergistic effect of IL-12 and lysate is absent in gastric cancer patients, unless the concentration of IL-12 is increased 10-fold. We also demonstrate that there is a similar lack of IFN-γ production from NK cells isolated from the gastric mucosa of cancer patients. In addition, we propose that the observed suppression is due to tumour-derived TGF-ß and that increased expression of the transcription factor GATA-3 may be responsible for the TGF-ß induced suppression.

B lymphocytes promote expansion of regulatory T cells in oral tolerance: powerful induction by antigen coupled to cholera toxin B subunit.

Mucosal administration of Ag conjugated to cholera toxin B subunit (CTB) can efficiently induce peripheral immunologic tolerance, so-called oral tolerance, associated with development of Foxp3(+)CD25(+)CD4(+) regulatory T (Treg) cells. Using an established sublingual tolerization regimen with Ag(OVA)/CTB conjugate, wherein CTB mediates Ag uptake and presentation by most B lymphocytes irrespective of their Ag specificity, we have assessed the importance of B cells for induction of Ag-specific Treg cells and oral tolerance. We found that Treg cells are reduced in microMT(-/-) B cell-deficient mice compared with wild-type (WT) mice. After sublingual Ag/CTB treatment, Treg cells increased much more in WT than in microMT(-/-) mice; however, adoptive transfer of B cells before treatment normalized Treg cell development and functional oral tolerance. B cells from OVA/CTB-treated mice expressed more IL-10 and less CD86 than control B cells. Adoptive transfer of these cells before parenteral immunization with OVA led to efficient suppression of proliferation and to induction of apoptotic depletion of Ag-specific CD25(-)CD4(+) effector T cells associated with the expansion of Treg cells. However, also OVA/CTB-treated microMT(-/-) mice could suppress the immune response to parenteral immunization with OVA, which was associated with a strong increase in Foxp3(-)CD4(+) T cells expressing LAP/TGF-beta. Our results indicate that mucosal tolerance comprises at least two separate pathways: one being B cell dependent and associated with expansion of Treg cells and Treg-mediated suppression and depletion of effector T cells, and one being B cell independent and associated with development of Foxp3(-)LAP(+)TGF-beta(+) regulatory T cells.

Sublingual immunization induces broad-based systemic and mucosal immune responses in mice.

The potential of sublingual (s.l.) delivery of vaccine was examined in mice. We show the existence of a dense network of dendritic cells (DCs) in the s.l. epithelium and a rapid and transient increase in the frequency of s.l. DCs after topical application of cholera toxin (CT) adjuvant under the tongue. S.l. immunization with ovalbumin and CT induced vigorous systemic and mucosal antibody responses. Such treatment promoted mixed Th1 and Th2 cytokine responses and induced cytotoxic CD8(+) T cells in lung tissues and in systemic lymphoid organs. S.l. immunization was comparable to intranasal immunization and was superior to oral immunization regarding the magnitude and anatomic dissemination of the induced immune responses. S.l. administration of live influenza virus at a dose lethal by the nasal route was well tolerated and did not redirect virus to the olfactory bulb. These features underscore the potential of the s.l. mucosa to serve as an alternative vaccine delivery route.

Oral tolerance induction with antigen conjugated to cholera toxin B subunit generates both Foxp3+CD25+ and Foxp3-CD25- CD4+ regulatory T cells.

Oral administration of Ag coupled to cholera toxin B subunit (CTB) efficiently induces peripheral immunological tolerance. We investigated the extent to which this oral tolerance is mediated by CD25+CD4+ regulatory T cells (T(reg)). We found that total T(reg), KJ1-26+ T(reg) and CTLA-4+ T(reg) were all increased in Peyer's patches, mesenteric lymph nodes, and, to a lesser extent, in spleen of mice after intragastric administration of OVA/CTB conjugate, which also increased TGF-beta in serum. This could be abolished by co-administering cholera toxin or by treatment with anti-TGF-beta mAb. CD25+ T(reg), but also CD25-CD4+ T cells from OVA/CTB-treated BALB/c or DO11.10 mice efficiently suppressed effector T cell proliferation and IL-2 production in vitro. Following adoptive transfer, both T cell populations also suppressed OVA-specific T cell and delayed-type hypersensitivity responses in vivo. Foxp3 was strongly expressed by CD25+ T(reg) from OVA/CTB-treated mice, and treatment also markedly expanded CD25+Foxp3+ T(reg). Furthermore, in Rag1(-/-) mice that had adoptively received highly purified Foxp3-CD25-CD4+ OT-II T cells OVA/CTB feeding efficiently induced CD25+ T(reg) cells, which expressed Foxp3 more strongly than naturally developing T(reg) and also had stronger ability to suppress effector OT-II T cell proliferation. A remaining CD25- T cell population, which also became suppressive in response to OVA/CTB treatment, did not express Foxp3. Our results demonstrate that oral tolerance induced by CTB-conjugated Ag is associated with increase in TGF-beta and in both the frequency and suppressive capacity of Foxp3+ and CTLA-4+ CD25+ T(reg) together with the generation of both Foxp3+ and Foxp3-CD25- CD4+ T(reg).

Role of arachidonic acid in hyposmotic membrane stretch-induced increase in calcium-activated potassium currents in gastric myocytes.

AIM: To study effects of arachidonic acid (AA) and its metabolites on the hyposmotic membrane stretch-induced increase in calcium-activated potassium currents (I(KCa)) in gastric myocytes. METHODS: Membrane currents were recorded by using a conventional whole cell patch-clamp technique in gastric myocytes isolated with collagenase. RESULTS: Hyposmotic membrane stretch and AA increased both I(K(Ca))) and spontaneous transient outward currents significantly. Exogenous AA could potentiate the hyposmotic membrane stretch-induced increase in I(K(Ca)). The hyposmotic membrane stretch-induced increase in I(K(Ca)) was significantly suppressed by dimethyleicosadienoic acid (100 micromol/L in pipette solution), an inhibitor of phospholipase A2. Nordihydroguaiaretic acid, a lipoxygenase inhibitor, significantly suppressed AA and hyposmotic membrane stretch-induced increases in I(K(Ca)). External calcium-free or gadolinium chloride, a blocker of stretch-activated channels, blocked the AA-induced increase in I(K(Ca)) significantly, but it was not blocked by nicardipine, an L-type calcium channel blocker. Ryanodine, a calcium-induced calcium release agonist, completely blocked the AA-induced increase in I(K(Ca)); however, heparin, a potent inhibitor of inositol triphosphate receptor, did not block the AA-induced increase in I(K(Ca)). CONCLUSION: Hyposmotic membrane stretch may activate phospholipase A2, which hydrolyzes membrane phospholipids to ultimately produce AA; AA as a second messenger mediates Ca(2+) influx, which triggers Ca(2+)-induced Ca(2+) release and elicits activation of I(K(Ca)) in gastric antral circular myocytes of the guinea pig.