The contribution of gut bacterial metabolites in the human immune signaling pathway of non-communicable diseases.

The interaction disorder between gut microbiota and its host has been documented in different non-communicable diseases (NCDs) such as metabolic syndrome, neurodegenerative disease, and autoimmune disease. The majority of these altered interactions arise through metabolic cross-talk between gut microbiota and host immune system, inducing a low-grade chronic inflammation that characterizes all NCDs. In this review, we discuss the contribution of bacterial metabolites to immune signaling pathways involved in NCDs. We then review recent advances that aid to rationally design microbial therapeutics. A deeper understanding of these intersections between host and gut microbiota metabolism using metabolomics-based system biology platform promises to reveal the fundamental mechanisms that drive metabolic predispositions to disease and suggest new avenues to use microbial therapeutic opportunities for NCDs treatment and prevention. Abbreviations: NCDs: non-communicable disease, IBD: inflammatory bowel disease, IL: interleukin, T2D: type 2 diabetes, SCFAs: short-chain fatty acids, HDAC: histone deacetylases, GPCR: G-protein coupled receptors, 5-HT: 5-hydroxytryptamine receptor signaling, DCs: dendritic cells, IECs: intestinal epithelial cells, T-reg: T regulatory cell, NF-κB: nuclear factor κB, TNF-α: tumor necrosis factor alpha, Th: T helper cell, CNS: central nervous system, ECs: enterochromaffin cells, NSAIDs: non-steroidal anti-inflammatory drugs, AhR: aryl hydrocarbon receptor, IDO: indoleamine 2,3-dioxygenase, QUIN: quinolinic acid, PC: phosphatidylcholine, TMA: trimethylamine, TMAO: trimethylamine N-oxide, CVD: cardiovascular disease, NASH: nonalcoholic steatohepatitis, BAs: bile acids, FXR: farnesoid X receptor, CDCA: chenodeoxycholic acid, DCA: deoxycholic acid, LCA: lithocholic acid, UDCA: ursodeoxycholic acid, CB: cannabinoid receptor, COBRA: constraint-based reconstruction and analysis.

Unaccounted risk of cardiovascular disease: the role of the microbiome in lipid metabolism.

PURPOSE OF REVIEW: Not all of the risk of cardiovascular disease can be explained by diet and genetics, and the human microbiome, which lies at the interface of these two factors, may help explain some of the unaccounted risk. This review examines some of the well established links between the microbiome and cardiovascular health, and proposes relatively unexplored associations. RECENT FINDINGS: Byproducts of microbial metabolism are associated with health and disease: Trimethylamine N oxide is associated with atherosclerosis; whereas short-chain fatty acids are associated with decreased inflammation and increased energy expenditure. More broadly, a large number of association studies have been conducted to explore the connections between bacterial taxa and metabolic syndrome. In contrast, the relationship between the microbiome and triglycerides levels remains poorly understood. SUMMARY: We suggest that deeper understanding of the molecular mechanisms that drive linkages between the microbiome and disease can be determined by replacing 16S rRNA gene sequencing with shotgun metagenomic sequencing or other functional approaches. Furthermore, to ensure translatability and reproducibility of research findings, a combination of multiple different complementary '-omic' approaches should be employed.

Gene cloning, expression and immune adjuvant properties of the recombinant fusion peptide Tα1-BLP on avian influenza inactivate virus vaccine.

Thymosin α1 (Tα1) and bursin-like peptide (BLP) are both immunopotentiators. In order to investigate adjuvant of thymosin α1-bursin-like peptide (Tα1-BLP), we cloned the gene of Tα1-BLP and provided evidence that the gene of Tα1-BLP in a recombinant prokaryotic expression plasmid was successfully expressed in E. coli BL21. To evaluate the immune adjuvant properties of Tα1-BLP, chickens were immunized with Tα1-BLP combined with H9N2 avian influenza whole-inactivated virus (WIV). The titers of HI antibody, antigen-specific antibodies, AIV-neutralizing antibodies, levels of Th1-type cytokines (IFN-γ) and Th2-type cytokines (IL-4) and lymphocyte proliferation responses were determined. What's more, the viral loads and pathologic changes of lung tissue were observed by virus challenge experiment and HE staining to evaluate the immune protection of chickens. We found that Tα1-BLP enhanced HI antibody and antigen-specific IgG antibodies titers, increased the level of AIV-neutralizing antibodies, induced the secretion of Th1- and Th2-type cytokines, and promoted the proliferation of T and B lymphocyte, Furthermore, virus challenge experiment and HE staining confirmed that Tα1-BLP contributed to inhibition replication of the virus from chicken lungs and protected the lungs from damage. Altogether, this study suggested that Tα1-BLP is a novel adjuvant suitable for H9N2 avian influenza vaccine.

Use of immunoproteomics to identify immunogenic proteins in a rat model of acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS) is a common and life­threatening clinical syndrome, and seeking biomarkers of ARDS has been an area of continuing research. The present study hypothesized that alterations to certain immunogenic substances occur in injured lungs and are able to specifically bind with corresponding proteins in the blood, and that these proteins may be readily detected. To investigate this hypothesis, a rat model of ARDS was established by cecal ligation and puncture surgery, and an immunoproteomics approach, using serum as the primary antibody in a western blot analysis, was used with the aim of identifying immunogenic proteins in the injured lungs. Ingenuity Pathway Analysis (IPA) was used for bioinformatics analysis, and mass spectrometric analysis was used to identify a total of 38 differentially expressed immunogenic proteins. Bioinformatics analysis revealed that the top canonical pathways in which the identified proteins may be involved were gluconeogenesis I, glycolysis I, choline degradation I, NADH repair and heme degradation. IPA Biomarker Filter analysis with the terms 'acute respiratory distress syndrome/acute lung injury' was used to screen 13 proteins as candidate biomarkers. These proteins were described as antigens, and suggested that paired antibodies may be detected in the plasma of patients at high risk of ARDS. Analysis of these identified proteins may provide novel insights into the potential pathological mechanisms of ARDS.

The effectiveness of choline citrate infusions monitored by lymphocyte transformation test (LTT) in multiple sclerosis. A new approach to the diagnosis and treatment of the disease.

The efficacy of intravenous choline citrate infusions was investigated in 34 patients with multiple sclerosis (MS) by clinical evaluation and by monitoring of lymphocyte proliferation in vitro against fragments of myelin basic protein (MOG-35-55, MBP15-31, PLP 39-15) over a period of 12 weeks. Patients have been diagnosed with MS at least one year before entering the study and suffered from mild relapsing/remitting course to long-term chronic progressive disease. Twenty one patients exhibited positive lymphocyte proliferation to myelin fragments prior to treatment and were therefore selected for further studies. Choline citrate was administered with a dosage of 1200mg/ 2x week for a period of 3 months. This treatment resulted in a significant decrease of lymphocyte proliferation to neural fragments (MOG- 35-55, MBP15-31) in lymphocyte transformation test (LTT). There was no significant SI change of PLP Peptide (PLP 39-15) LTT found after treatment with choline citrate. During the 3 mo observation period, patients remained stable and no side-effects of the treatment were observed. In addition, some patients reported long-lasting improvement (less paresthesia and increase of muscle strength in lower extremities) which was demonstrated up to 3 years later. In one spectacular case a commercial pilot was able to return to duty again after treatment. This pilot was allowed back in to his position as a commercial flying cockpit member and is on duty for more than 4 yrs now.

Screening for mast cell tryptase and serum IgE antibodies in 18 patients with anaphylactic shock during general anaesthesia.

BACKGROUND: In the perioperative setting multiple agents can cause anaphylaxis. Often the reactions are dramatic, and due to their lifethreatening potential it is crucial that the responsible agent is identified in order to avoid future adverse reactions. The aim of the present study was to measure the concentration of serum mast cell tryptase (MCT), to investigate the prevalence of serum IgE antibodies against ammonium groups, choline, morphine, suxamethonium, thiopentone and latex and to perform skin prick tests (SPTs) in 18 patients experiencing an anaphylactic reaction during induction of general anaesthesia. METHODS: Serum samples from 18 patients with an anaphylactic reaction during general anaesthesia were analyzed for MCT and specific IgE against ammonium groups, choline, morphine, suxamethonium, thiopentone and latex. Skin prick tests were performed in 11 out of 18 patients. RESULTS: Ten patients had elevated MCT levels and specific IgE against ammonium ion, morphine and (with the exception of patient nos 3, 9 and 10) suxamethonium. Seven of these patients had positive SPTs to suxamethonium. One of the patients tested positive to latex in addition to suxamethonium. Two patients showed elevated MCT, while specific IgE against the drugs tested was not detected. Three patients tested positive to ammonium ion, morphine and suxamethonium, but negative to MCT. Three patients tested negative to both MCT and specific IgE. CONCLUSIONS: Fifteen out of 18 sera tested positive for MCT and/or specific IgE against neuromuscular blocking drugs (NMBDs). Ten of the 18 patients experienced an IgE-mediated anaphylactic reaction to NMBDs during anaesthesia, verified by detection of specific IgE and elevated levels of MCT.

Production and characterization of monoclonal antibodies against L-carnitine: radioimmunologic assays for L-carnitine determination.

Monoclonal antibodies against L-carnitine have been produced and characterized. These antibodies have been found to specifically bind L-carnitine and, with different affinities, other carnitine-related compounds. No binding was observed with choline or acetylcholine. These antibodies have been used to measure L-carnitine in biological samples and serum. Data obtained demonstrate that, in biological samples, by using radiolabelled carnitine, it is possible quickly to detect small amounts of carnitine. The high specificity of the test is clearly demonstrated.

Production and characterization of monoclonal antibodies that specifically bind to phosphatidylcholine.

A series of monoclonal antibodies (mAbs) that react with phosphatidylcholine (PC) were established. All mAbs were highly specific to PC and no cross-reaction with other phospholipids were observed. The results obtained with two typical monoclonal antibodies, JE-1 and JE-8, were described. The analysis using synthetic PC analogs with modified polar head groups showed that the methyl groups on the quaternary nitrogen of the choline moiety were important for the binding. Each mAbs showed distinct acyl chain specificities of the PC molecules, and JE-1 showed considerable reactivity with PC with saturated fatty acids, whereas JE-8 could not react with the PC. Both mAbs bound to PC with unsaturated fatty acids, but showed distinct reactivity profiles. Both mAbs reacted only weakly with water-soluble haptens such as phosphorylcholine and L-alpha-glycerophosphocholine, suggesting that the hydrophobic moiety of the PC molecule is important for the maximum affinity. The interaction between the mAbs and the hydrophobic moieties of PC molecules was further studied by analyzing the effect of the mAbs on the activities of phospholipase A2 and phospholipase C. JE-1 inhibited both enzyme activities, while JE-8 inhibited only the phospholipase C activity, indicating that JE-1 interacts more thoroughly with the hydrophobic region of the PC molecule than JE-8 does.

Naturally occurring IgG-antibody-like substance reacting with quaternary ammonium group and neuromuscular blockers: a common finding in humans and other species?

IgG antibodies reacting with the quaternary ammonium group of choline and alcuronium appeared to be present in the sera of humans, guinea pigs, and rats. The antibodies, particularly those 'against' alcuronium, were detected in every serum by paper immunosorbent test(s). This finding, if substantiated, would be of fundamental importance in view of the wide distribution of choline derivatives within the body and in the environment. It may also be relevant to the histamine-releasing activity of neuromuscular blockers and compounds possessing the quaternary ammonium group.

Immunohistochemical localization of bursin in epithelial cells of the avian bursa of Fabricius.

Antibodies to the avian B-cell-differentiating hormone bursin (lysyl-histidyl-glycine amide) were raised in mice and rabbits by immunizing with bursin conjugates in Freund's adjuvant. Immunohistochemical staining with these bursin-specific antibodies was restricted to follicular and dendritic reticular epithelial cells of the bursa of Fabricius, and was not found in control avian tissues.

Allergy to suxamethonium: persisting abnormalities in skin tests, specific IgE antibodies and leucocyte histamine release.

Twenty-one patients, who had previously experienced an anaphylactic reaction to suxamethonium during general anaesthesia, were selected for this study. Initially, skin tests with muscle relaxants were carried out in the twenty-one patients, detection of specific anti-choline IgE in nineteen, and leucocyte histamine release in seventeen. These three tests were then repeated between 1 year and 4 years after the initial evaluation. In the majority of patients, sensitization to the muscle relaxants persisted for more than 1 year after the anaphylactic reaction. Only three patients out of twenty-one (4%) had negative skin tests when retested 1-4 years later. A reduction in leucocyte histamine release was noticed in one of the seventeen retested patients (6%). Modifications of anti-choline IgE were observed in five of nineteen patients (26%). The persistence of sensitization to suxamethonium may result from repeated stimulation by occasional contacts with quaternary ammonium compounds. This study demonstrates the reliability of skin tests, leucocyte histamine release and detection of anti-choline IgE to diagnose allergic reactions to suxamethonium, even when they are performed a long time after the initial anaphylactic reaction.

[Anaphylactic complications due to suxamethonium]. / Accidents anaphylactiques au suxaméthonium.

A study was carried out on 36 patients who had presented with an anaphylactic reaction when they had been received anaesthetic induction agents including suxamethonium. After having been examined, they were assessed with various immunoallergic tests (skin tests, LHL, a search for specific anticholine IgE antibodies). They were compared with a group of 120 control patients with the same age, sex and professional characteristics. This study confirmed the part played by specific IgE antibodies in accidents involving suxamethonium. The specificity of the tests that could be used for the diagnosis was excellent. However, as far as sensitivity of the tests went, skin tests and LHL were more sensitive than the search for specific IgE antibodies. There was no statistical relationship between the limit for skin reactions and the degree of histamine release of the level of anticholine IgE antibody.

Anaphylactic reactions to suxamethonium prevention of mediator release by choline.

IgE antibodies cross-reacting with choline and suxamethonium were found in the sera of 15 of 22 patients with life-threatening sensitivity to suxamethonium, one of the most commonly used muscle relaxants. Furthermore, choline that is one of the metabolites of acetylcholine and a monovalent part of the suxamethonium molecule could act as a hapten inhibitor in vitro and in vivo, blocking specifically the basophil histamine release induced by suxamethonium and the positive skin tests to the drug in allergic patients. These results confirm the IgE dependency of allergic reactions to suxamethonium and suggest a possible way of preventing such reactions with choline.

Detection of IgE antibodies to suxamethonium after anaphylactoid reactions during anaesthesia.

Choline covalently coupled to an insoluble support was used in a radioimmunoassay to detect IgE antibodies to suxamethonium in serum samples from patients who experienced life-threatening anaphylactoid reactions after receiving the drug. Direct binding and inhibition experiments and correlations with clinical findings showed that use of choline in such an assay is relevant to the detection of suxamethonium-reactive antibodies. IgE antibodies were found in 9 of 10 patients who reacted to suxamethonium and whose skin-tests to the drug were positive and in 8 other patients who had reactions to other muscle-relaxant drugs. The positive reactions in the latter group were probably due to cross-reacting antibodies that recognise quaternary ammonium groups on more than one muscle relaxant.

Clonal nature of the immune response to phosphorylcholine (PC). V. Cross-idiotypic specificity among heavy chains of murine anti-PC antibodies and PC-binding myeloma proteins.

Seven mouse myeloma proteins with specificity for phosphorylcholine (PC) were found to share a common antigenic determinant. This group of proteins contained members which differed in genetic origin, heavy chain class, kappa-chain subgroup, individual antigenic determinants and specificity for choline analogues. The cross-idiotypic determinant, VH-PC, was antigenically similar in each of the proteins and was associated with the variable portion of the heavy chain in the region of the antibody combining site. Further studies showed that an indistinguishable determinant was present on IgM anti-PC antibodies isolated from all strains of mice tested regardless of histocompatibility or heavy chain allotype. In view of the finding that this cross-idiotypic determinant was not found on antibodies or myeloma proteins which lacked specificity for PC, the data strongly suggest that a particular heavy chain variable region has been preserved in all mouse antibodies with specificity for PC.

The frequency of phosphorylcholine-specific B cells in conventional and germfree BALB/C mice.

The frequency of B cells specific for phosphorylcholine (PC) in spleens of nonimmune conventionally-reared and germfree adult Balb/c mice was determined by an in vitro splenic focus technique. Analysis of spleen cells from 11 conventionally reared donors revealed an average of 19.1 PC-specific clonal precursor cells/10-6 donor B cells when calculated utilizing a cloning efficiency previously determined for the in vitro splenic focus technique of 4%. This number is one-tenth the frequency determined for 2, 4-dinitrophenyl- and 2,4,6-trintrophenyl-specific B cells; however, both idiotypic and isoelectric analysis reveal that the majority of B cells specific for PC yield monoclonal antibodies which share the TEPC 15 idiotype and contain a homogeneous IgM peak with a pK of 4.60. Thus this single clone appears on the average to be represented by more than 10-3 B cells in conventionally reared nonimmune adults. A similar frequency for precursor cells of this clonotype was found in an analysis of seven germfree adult mice so that the high frequency of B cells of this colontype appears to be independent of previous antigenic stimulation. Among the 11 conventionally reared donors analyzed, frequencies varied from 1.6 to 52/10-6 B cells--an extreme degree of variation when compared to the almost invariant frequencies observed for 2, 4-dinitrophenyl, 2, 4, 6-trinitrophenyl, and fluoroscein. This variation is interpreted as a reflection of the life cycle of a single clone with a possible role for antigen implied by smaller variations observed in grem free mice.

Anti-receptor antibody. I. Isolation and characterization of the immunoglobulin receptor for phosphorylcholine.

We have demonstrated a phosphorylcholine-binding protein in lysates from radioiodinated splenocytes of immunized mice. Immunoprecipitation with anti-receptor antibody of lysates from splenocytes obtained from animals undergoing a primary response or from mice immunized 4 to 6 months earlier with antigen demonstrated that this protein contained H and L chains. Therefore, we have isolated an antigen-specific receptor from the surface of spleen cells from both immunized and "memory" animals. This receptor Ig comprises approximately 10% of total cell surface Ig.

Anti-receptor antibody. II. Induction of long-term unresponsiveness in neonatal mice.

We have examined the ability of anti-receptor antibody (ARA) to induce specific unresponsiveness to the hapten, phosphorylcholine (PC), in neonatal and adult mice. When ARA is given to adult mice, suppression is of short duration. Cells from such mice are responsive in vitro, indicating that suppression in vivo is probably due to blockade of receptors by persisting ARA. ARA given to neonatal mice induces long-term unresponsiveness. The mice apparently have decreased numbers of PC-responsive cells, since cells from such mice are unresponsive both in vitro and in adoptive transfer. Furthermore, cells from neonatally suppressed animals do not suppress the response of normal cells either in vitro or in adoptive transfer, indicating that unresponsiveness is most likely not due to active suppression. We therefore conclude that ARA given to neonates depletes the clone of receptor-bearing cells at the time ARA is given. Clonal depletion may result from antibody-dependent cell mediated cytotoxicity.

Heterogeneity of the BALB/c antiphosphorylcholine antibody response at the precursor cell level.

Immune responsiveness to phosphorylcholine (PC) in BALB/c mice has been characterized by combining (a) usuage of highly sensitive radioimmunoassays for quantitation of antibody, heavy-chain class, and idiotype on a weight basis; (b) isolation of PC-specific B cells in fragment cultures; and (c) stimulation in a carrier-primed environment with the PC hapten coupled to carrier through a tripeptide spacer in order to maximize carrier recognition. The specificity and accuracy of the radioimmunoassays have veen verified by specific inhibition, lack of nonspecific binding, and excellent concordance of values for monoclonal antibody concentration obtained independently for Fab and idiotype content. The latter evidence also serves as strong confirmation of the monoclonality of in vitro monofocal responses as well as the preservation of the idiotype on antibodies of differing immunoglobulin classes. The results indicate that while B cells expressing the TEPC 15 idiotype predominate, other idiotypes may be represented by 2-50% of PC-specific precursors, and monoclonal antibodies even of the TEPC 15 idiotype are produced in both the IgM and IgG1 immunoglobulin classes. These findings are confirmed by the analysis of serum antibodies produced in carrier-primed mice immunized with hapten coupled through a tripeptide spacer, thus re-emphasizint the enhancement of primary responsiveness, particularly IgG1 production, by maximizing carrier recognition. The finding of idiotype diversity in the PC response, as well as diversity of expression in terms of quantity and immunoglobulin class of antibody synthesized by the clonal progeny of B cells within the TEPC 15 clonotype, emphasize the heterogeneity of the B-cell population both in terms of specificity repertoire and the physiological state of cells even within a single clonotype.