Disease Development Is Accompanied by Changes in Bacterial Protein Abundance and Functions in a Refined Model of Dextran Sulfate Sodium (DSS)-Induced Colitis.

Using the acute dextran sulfate sodium (DSS)-induced colitis model, studies have demonstrated that intestinal inflammation is accompanied by major changes in the composition of the intestinal microbiota. Only little is known about the microbial changes and more importantly their functional impact in the chronic DSS colitis model. We used a refined model of chronic DSS-induced colitis that reflects typical symptoms of the human disease without detrimental weight loss usually observed in DSS models. We sampled cecum and colon content as well as colon mucus from healthy and diseased mouse cohorts ( n = 12) and applied 16S rRNA gene sequencing and metaproteomics. An increase of Prevotella sp. in both colon content and mucus was observed. Functional differences were observed between sample types demonstrating the importance of separately sampling lumen content and mucus. The abundance of Desulfovibrio, a sulfate-reducing bacterium, was positively associated with the carbon metabolism. Lachnoclostridium was positively correlated to both vitamin B6 and tryptophan metabolism. In summary, functional changes in the distal colon caused by DSS treatment were more pronounced in the mucus-associated microbiota than in the microbiota present in the distal colon content.

The immunome of soy bean allergy: Comprehensive identification and characterization of epitopes.

BACKGROUND: The precise mapping of multiple antibody epitopes recognized by patients' sera allows a more detailed and differentiated understanding of immunological diseases. It may lead to the development of novel therapies and diagnostic tools. OBJECTIVE: Mapping soy bean specific epitopes relevant for soy bean allergy patients and persons sensitized to soy bean, and analysis of their IgE/IgG binding spectrum. METHODS: Identification of epitopes using sera, applying an optimized peptide phage display library followed by next-generation sequencing, specially designed in silico data analysis and subsequent peptide microarray analysis. RESULTS: We were able to identify more than 400 potential epitope motifs in soy bean proteins. More than 60% of them have not yet been described as potential epitopes. Eighty-three peptides, representing the 42 most frequently found epitope candidates, were validated by microarray analysis using 50 sera from people who have been tested positive in skin prick test (SPT). Of these peptides, 56 were bound by antibodies, 55 by serum IgE, 43 by serum IgG and 30 by both. Person-specific epitope patterns were found for each individual and protein. CONCLUSIONS: For individuals with clinical symptoms, epitope resolved analyses reveal a high prevalence of IgE binding to a few soy bean specific epitopes. Evaluation of individual immune profiles of patients with soy bean sensitization allows the identification of peptides that do facilitate studying individual IgE/IgG epitope binding patterns. This enables discrimination of sensitization from disease, such assay test has the potential to replace SPT assays.

Drimia indica: A Plant Used in Traditional Medicine and Its Potential for Clinical Uses.

Drimia indica (Roxb.) Jessop (Asparagaceae) is a reputed Ayurvedic medicine for a number of therapeutic benefits, including for cardiac diseases, indigestion, asthma, dropsy, rheumatism, leprosy, and skin ailments. The present work aimed to critically and extensively review its traditional uses, phytochemistry, pharmacology, toxicology, and taxonomy together with the mechanisms of action of selected extracts of D. indica. A systematic literature survey from scientific databases such as PubMed, Scopus, and Web of Science as well as from some textbooks and classical texts was conducted. The plant, mainly its bulb, contains various bioactive constituents, such as alkylresorcinols, bufadienolides, phytosterols, and flavonoids. Various scientific studies have proven that the plant has anthelmintic, anticancer, antidiabetic, antimicrobial, antioxidant, and wound healing activities. The present work concludes that D. indica has the potential to treat various diseases, mainly microbial infections. This review also suggests that bufadienolides, flavonoids, and steroids might be responsible for its bioactive potential.

Immunogenic potential of a Salmonella Typhimurium live vaccine for pigs against monophasic Salmonella Typhimurium DT 193.

BACKGROUND: Monophasic Salmonella Typhimurium (mSTM) strains account for up to 8.6% of all human Salmonellosis cases. They have an increasing prevalence during recent years and several human cases with hospitalisation were reported. These strains are often isolated from pigs and pork - one primary source of human infection. A Salmonella Typhimurium (STM) live vaccine has been proven successful in controlling of STM infections in pigs for many years. The aim of this study was to test the immunogenicity of the vaccine in weaners during oral challenge with a virulent mSTM strain and to examine the kinetics of STM-specific IgA, IgM and IgG antibodies induced by vaccination and infection. RESULTS: Despite clinical signs being present in both groups, the vaccination led to a significant reduction of diarrhoea, overall clinical symptoms and a milder elevation of the body temperature. Necropsy revealed fewer pathological lesions in the gastrointestinal tract of vaccinated compared to control animals. Moreover, in the ileal and caecal mucosa and in the ileocaecal lymph nodes the challenge strain burden was significantly reduced by vaccination. Significant differences in the antibody responses of both groups were present during the vaccination period and after infection. In vaccinated animals Salmonella-specific IgA and IgG antibody levels increased significantly after vaccination and were even more pronounced in response to challenge. In contrast, similarly low levels of IgM antibodies were detected during the vaccination period in both vaccinated and non-vaccinated animals. However, after challenge IgM antibody levels increased significantly in control pigs while neither IgA nor IgG antibodies were detectable. CONCLUSION: The data demonstrate that mSTM can evoke clinical signs in weaners. Due to the vaccination their incidence and magnitude were significantly milder. Vaccination also led to a significantly reduced challenge strain burden in the intestine and the lymph nodes which is comparable to previous studies using the same vaccine in a challenge with biphasic STM. Therefore, it is concluded that this vaccine induces immunity against monophasic and biphasic STM strains. Furthermore, the results of antibody profiles in response to vaccination and infection provide additional evidence for humoral immune mechanisms triggered during Salmonella infection or vaccination.

Attenuation of graft-versus-host-disease in NOD scid IL-2Rγ(-/-) (NSG) mice by ex vivo modulation of human CD4(+) T cells.

NOD.Cg-Prkdc(scid) IL-2rg(tm1Wjl) /SzJ (NSG) mice are a valuable tool for studying Graft-versus-Host-Disease (GvHD) induced by human immune cells. We used a model of acute GvHD by transfer of human peripheral blood mononuclear cells (PBMCs) into NSG mice. The severity of GvHD was reflected by weight loss and was associated with engraftment of human cells and the expansion of leukocytes, particularly granulocytes and monocytes. Pre-treatment of PBMCs with the anti-human CD4 antibody MAX.16H5 IgG1 or IgG4 attenuated GvHD. The transplantation of 2 × 10(7) PBMCs without anti-human CD4 pre-treatment induced a severe GvHD (0% survival). In animals receiving 2 × 10(7) PBMCs pre-incubated with MAX.16H5 IgG1 or IgG4, GvHD development was reduced and survival was increased. Immune reconstitution was measured by flow cytometry and confirmed for human leukocytes (CD45), CD3(+) /CD8(+) cytotoxic T cells and CD3(+) /CD4(+) T helper cells. Human B cells (CD19) and monocytes (CD14) could not be detected. Histopathological analysis (TUNEL assay) of the gut of recipient animals showed significantly less apoptotic crypt cells in animals receiving a MAX.16H5 IgG1 pre-incubated graft. These findings indicate that pre-incubation of an allogeneic graft with an anti-human CD4 antibody may decrease the frequency and severity of GvHD after hematopoietic stem cell transplantation (HSCT) and the need of conventional immunosuppressive drugs. Moreover, this approach most probably provides a safer HSCT that must be confirmed in appropriate clinical trials in the future. © 2016 International Society for Advancement of Cytometry.

Preclinical good laboratory practice-compliant safety study to evaluate biodistribution and tumorigenicity of a cartilage advanced therapy medicinal product (ATMP).

BACKGROUND: The clinical development of advanced therapy medicinal products (ATMPs), a new class of drugs, requires initial safety studies that deviate from standard non-clinical safety protocols. The study provides a strategy to address the safety aspects of biodistribution and tumorigenicity of ATMPs under good laboratory practice (GLP) conditions avoiding cell product manipulation. Moreover, the strategy was applied on a human ATMP for cartilage repair. METHODS: The testing strategy addresses biodistribution and tumorigenicity using a multi-step analysis without any cell manipulation to exclude changes of test item characteristics. As a safeguard measurement for meeting regulatory expectations, the project design and goals were discussed continuously with the regulatory authority using a staggered scientific advice concept. Subsequently, the strategy was applied to co.don chondrosphere® (huChon spheroid), a tissue-engineered matrix-free ATMP of human normal chondrocytes. In both the biodistribution and tumorigenicity studies, huChon spheroids were implanted subcutaneously into 40 immunodeficient mice. Biodistribution was studied 1 month after implantation. A skin disc containing the huChon spheroid, two surrounding skin rings and selected organs were analyzed by validated, gender-specific, highly-sensitive triplex qPCR and by immunohistochemistry (IHC). RESULTS: No human DNA was detected in distant skin rings and analyzed organs. IHC revealed no direct or indirect indications of cell migration. Tumorigenicity was assessed 6 months after huChon spheroid implantation by palpation, macroscopic inspection, histology and IHC. No mice from the huChon spheroid group developed a tumor at the implantation site. In two mice, benign tumors were detected that were negative for HLA-ABC, suggesting that they were of spontaneous murine origin. CONCLUSIONS: In summary, the presented strategy using a multi-step analysis was confirmed to be suitable for safety studies of ATMPs.

[Strategic considerations on the design and choice of animal models for non-clinical investigations of cell-based medicinal products]. / Strategische Betrachtungen zur Konzeption und Wahl von Tiermodellen bei nicht-klinischen Prüfungen von zellbasierten Therapeutika.

For the development of medicinal products animal models are still indispensable to demonstrate efficacy and safety prior to first use in humans. Advanced therapy medicinal products (ATMP), which include cell-based medicinal products (CBMP), differ in their pharmacology and toxicology compared to conventional pharmaceuticals, and thus, require an adapted regime for non-clinical development. Developers are, therefore, challenged to develop particular individual concepts and to reconcile these with regulatory agencies. Guidelines issued by the European Medicines Agency (EMA), the U.S. Food and Drug Administration (FDA) and other sources can provide direction.The published approaches for non-clinical testing of efficacy document that homologous animal models where the therapeutic effect is investigated in a disease-relevant animal model utilizing cells derived from the same species are commonly used. The challenge is that the selected model should reflect the human disease in all critical features and that the cells should be comparable to the investigated human medicinal product in terms of quality and biological activity. This is not achievable in all cases. In these cases, alternative methods may provide supplemental information. To demonstrate the scientific proof-of-concept (PoC), small animal models such as mice or rats are preferred. During the subsequent product development phase, large animal models (i.e. sheep, minipigs, dogs) must be considered, as they may better reflect the anatomical or physiological situation in humans. In addition to efficacy, those models may also be suitable to prove some safety aspects of ATMP (e.g. regarding dose finding, local tolerance, or undesired interactions and effects of the administered cells in the target tissue). In contrast, for evaluation of the two prominent endpoints for characterizing the safety of ATMP (i.e. biodistribution, tumorigenicity) heterologous small animal models, especially immunodeficient mouse strains, are favourable due to their tolerance to the human cell therapy product. The execution of non-clinical studies under the principles of good laboratory practice (GLP) increases the acceptance of the results by authorities and the scientific community.

DSS treatment does not affect murine colonic microbiota in absence of the host.

The prevalence of inflammatory bowel disease (IBD) is rising globally; however, its etiology is still not fully understood. Patient genetics, immune system, and intestinal microbiota are considered critical factors contributing to IBD. Preclinical animal models are crucial to better understand the importance of individual contributing factors. Among these, the dextran sodium sulfate (DSS) colitis model is the most widely used. DSS treatment induces gut inflammation and dysbiosis. However, its exact mode of action remains unclear. To determine whether DSS treatment induces pathogenic changes in the microbiota, we investigated the microbiota-modulating effects of DSS on murine microbiota in vitro. For this purpose, we cultured murine microbiota from the colon in six replicate continuous bioreactors. Three bioreactors were supplemented with 1% DSS and compared with the remaining PBS-treated control bioreactors by means of microbiota taxonomy and functionality. Using metaproteomics, we did not identify significant changes in microbial taxonomy, either at the phylum or genus levels. No differences in the metabolic pathways were observed. Furthermore, the global metabolome and targeted short-chain fatty acid (SCFA) quantification did not reveal any DSS-related changes. DSS had negligible effects on microbial functionality and taxonomy in vitro in the absence of the host environment. Our results underline that the DSS colitis mouse model is a suitable model to study host-microbiota interactions, which may help to understand how intestinal inflammation modulates the microbiota at the taxonomic and functional levels.

Cognitive performance in adults with post-COVID syndrome: Results from a German case-control study.

Numerous studies on post-COVID syndrome (PCS) describe persisting symptoms of cognitive impairment. Previous studies, however, often investigated small samples or did not assess covariates possibly linked to cognitive performance. We aimed to describe 1) global and domain-specific cognitive performance in adults with PCS, controls with previous SARS-CoV-2 infection and healthy controls, 2) associations of sociodemographics, depressive symptoms, anxiety, fatigue, somatic symptoms and stress with cognitive performance and subjective cognitive decline (SCD), using data of the LIFE-Long-COVID-Study from Leipzig, Germany. Group differences in cognitive performance and associations with sociodemographic and neuropsychiatric covariates were assessed using multivariable regression analyses. Our study included n = 561 adults (Mage: 48.8, SD: 12.7; % female: 70.6). Adults with PCS (n = 410) performed worse in tests on episodic memory (b = -1.07, 95 % CI: -1.66, -0.48) and visuospatial abilities (b = -3.92, 95 % CI: -6.01, -1.83) compared to healthy controls (n = 64). No impairments were detected for executive function, verbal fluency, and global cognitive performance. Odds of SCD were not higher in PCS. A previous SARS-CoV-2 infection without PCS (n = 87) was not linked to cognitive impairment. Higher age and higher levels of stress and fatigue were linked to worse performance in several cognitive domains. Routine administration of tests for episodic memory and visuospatial abilities might aid in the identification of individuals at risk for cognitive impairment when reporting symptoms of PCS. Low numbers of participants with severe COVID-19 infections possibly limit generalizability of our findings.

Near intron pairs and the metazoan tree.

Gene structure data can substantially advance our understanding of metazoan evolution and deliver an independent approach to resolve conflicts among existing hypotheses. Here, we used changes of spliceosomal intron positions as novel phylogenetic marker to reconstruct the animal tree. This kind of data is inferred from orthologous genes containing mutually exclusive introns at pairs of sequence positions in close proximity, so-called near intron pairs (NIPs). NIP data were collected for 48 species and utilized as binary genome-level characters in maximum parsimony (MP) analyses to reconstruct deep metazoan phylogeny. All groupings that were obtained with more than 80% bootstrap support are consistent with currently supported phylogenetic hypotheses. This includes monophyletic Chordata, Vertebrata, Nematoda, Platyhelminthes and Trochozoa. Several other clades such as Deuterostomia, Protostomia, Arthropoda, Ecdysozoa, Spiralia, and Eumetazoa, however, failed to be recovered due to a few problematic taxa such as the mite Ixodesand the warty comb jelly Mnemiopsis. The corresponding unexpected branchings can be explained by the paucity of synapomorphic changes of intron positions shared between some genomes, by the sensitivity of MP analyses to long-branch attraction (LBA), and by the very unequal evolutionary rates of intron loss and intron gain during evolution of the different subclades of metazoans. In addition, we obtained an assemblage of Cnidaria, Porifera, and Placozoa as sister group of Bilateria+Ctenophora with medium support, a disputable, but remarkable result. We conclude that NIPs can be used as phylogenetic characters also within a broader phylogenetic context, given that they have emerged regularly during evolution irrespective of the large variation of intron density across metazoan genomes.

Transcriptomic signatures reveal a shift towards an anti-inflammatory gene expression profile but also the induction of type I and type II interferon signaling networks through aryl hydrocarbon receptor activation in murine macrophages.

Introduction: The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that regulates a broad range of target genes involved in the xenobiotic response, cell cycle control and circadian rhythm. AhR is constitutively expressed in macrophages (Mϕ), acting as key regulator of cytokine production. While proinflammatory cytokines, i.e., IL-1ß, IL-6, IL-12, are suppressed through AhR activation, anti-inflammatory IL-10 is induced. However, the underlying mechanisms of those effects and the importance of the specific ligand structure are not yet completely understood. Methods: Therefore, we have compared the global gene expression pattern in activated murine bone marrow-derived macrophages (BMMs) subsequently to exposure with either benzo[a]pyrene (BaP) or indole-3-carbinol (I3C), representing high-affinity vs. low-affinity AhR ligands, respectively, by means of mRNA sequencing. AhR dependency of observed effects was proved using BMMs from AhR-knockout (Ahr-/-) mice. Results and discussion: In total, more than 1,000 differentially expressed genes (DEGs) could be mapped, covering a plethora of AhR-modulated effects on basal cellular processes, i.e., transcription and translation, but also immune functions, i.e., antigen presentation, cytokine production, and phagocytosis. Among DEGs were genes that are already known to be regulated by AhR, i.e., Irf1, Ido2, and Cd84. However, we identified DEGs not yet described to be AhR-regulated in Mϕ so far, i.e., Slpi, Il12rb1, and Il21r. All six genes likely contribute to shifting the Mϕ phenotype from proinflammatory to anti-inflammatory. The majority of DEGs induced through BaP were not affected through I3C exposure, probably due to higher AhR affinity of BaP in comparison to I3C. Mapping of known aryl hydrocarbon response element (AHRE) sequence motifs in identified DEGs revealed more than 200 genes not possessing any AHRE, and therefore being not eligible for canonical regulation. Bioinformatic approaches modeled a central role of type I and type II interferons in the regulation of those genes. Additionally, RT-qPCR and ELISA confirmed a AhR-dependent expressional induction and AhR-dependent secretion of IFN-γ in response to BaP exposure, suggesting an auto- or paracrine activation pathway of Mϕ.

Thy-1 (CD90) regulates the extravasation of leukocytes during inflammation.

Human Thy-1 (CD90) has been shown to mediate adhesion of inflammatory cells to activated microvascular endothelial cells via interaction with Mac-1 (CD11b/CD18) in vitro. Since there are no data showing the physiological relevance of Thy-1 for the recruitment of inflammatory cells in vivo, different inflammation models were investigated in Thy-1-deficient mice and littermate controls. In thioglycollate-induced peritonitis, the number of neutrophils and monocytes was significantly diminished in Thy-1-deficient mice. During acute lung inflammation, the extravasation of eosinophils and monocytes into the lung was significantly reduced in Thy-1-deficient mice. Moreover, during chronic lung inflammation, the influx of eosinophils and monocytes was also strongly decreased. These effects were independent of Thy-1 expression on T cells, as shown by the transplantation of WT BM into the Thy-1-deficient mice. In spite of the strong Thy-1 expression on T cells in the chimeric mice, the extravasation of the inflammatory cells in these mice was significantly diminished, compared to control mice. Finally, the altered number and composition of infiltrating leukocytes in Thy-1-deficient mice modified the chemokine/cytokine and protease expression at the site of inflammation. In conclusion, Thy-1 is involved in the control of inflammatory cell recruitment and, thus, also in conditioning the inflammatory microenvironment.

Evaluation of the tolerability and immunogenicity of ultraviolet C-irradiated autologous platelets in a dog model.

BACKGROUND: The THERAFLEX ultraviolet (UV) platelets (PLTs) pathogen reduction system for PLT concentrates (PCs) operates using ultraviolet C (UVC) light at a wavelength of 254 nm. UVC treatment can potentially alter proteins, which may affect drug tolerance in humans and influence the immunogenicity of blood products. This preclinical study in beagle dogs was designed to evaluate the safety pharmacology of UVC-irradiated PCs after intravenous administration and to determine whether they are capable of eliciting humoral responses to PLTs and plasma proteins. STUDY DESIGN AND METHODS: Six beagle dogs each were transfused once every other week for 10 weeks with UVC-irradiated or nonirradiated PCs. All PCs were autologous canine single-donor products prepared from whole blood. Safety pharmacology variables were regularly assessed. The impact of UVC irradiation on PLT and plasma proteomes was analyzed by one- and two-dimensional gel electrophoresis. Serum samples were tested for UVC-induced antibodies by Western blot and flow cytometry. RESULTS: Dogs transfused with UVC-irradiated PCs showed no signs of local or systemic intolerance. Few but significant changes in PLT protein integrity were observed after UVC irradiation. Even after repeated administration of UVC-irradiated PCs, no antibodies against UVC-exposed plasma or PLT proteins were detected. CONCLUSIONS: Repeated transfusions of autologous UVC-treated PCs were well tolerated in all dogs studied. UVC irradiation did not cause significant plasma or PLT protein modifications capable of inducing specific antibody responses in the dogs. High-resolution proteomics combined with antibody analysis introduces a comprehensive and sensitive method for screening of protein modifications and antibodies specific for pathogen reduction treatment.

Recent advances in immunotoxicity and its impact on human health: causative agents, effects and existing treatments.

BACKGROUND: A strong immune system is a primary requirement to keep the body safe from different ailments and infections. A human knowingly or unknowingly often comes to the exposure of many foreign substances such as pollutants, chemicals, metals and drugs that affect the immune system. Though some of these substances are known to exert immunotoxicity, their precise role as immunotoxicant is still unidentified, hence, the testing of these substances has to be taken into account. The present manuscript aimed to explore the mechanism behind immunotoxicity, biomarkers involved, manifestations, testing of immunotoxicity and its management. METHODS: Relevant literature on immunotoxicity, collected from online scientific databases like Scopus, PubMed and Google Scholar, was rigorously reviewed. RESULTS: Earlier reports showed that immunotoxic effects of chemicals and pharmaceuticals may cause various health problems including allergic reactions, skin disorders, respiratory infections, gastrointestinal problems, and autoimmune disorders. If diagnosed in time, many of these conditions can be managed with the help of existing treatments although the complete treatment is sometimes a big challenge. CONCLUSION: Based on the review of available literature, it can be concluded that immunotoxicity is one of the key factors responsible for several infectious diseases, autoimmune disorders and even cancers. Hence, the requirement of advanced diagnostic tools and established treatments for immunotoxicity is highly recommended.

Mycobacterium tuberculosis Affects Protein and Lipid Content of Circulating Exosomes in Infected Patients Depending on Tuberculosis Disease State.

Tuberculosis (TB), which is caused by the bacterium Mycobacterium tuberculosis (Mtb), is still one of the deadliest infectious diseases. Understanding how the host and pathogen interact in active TB will have a significant impact on global TB control efforts. Exosomes are increasingly recognized as a means of cell-to-cell contact and exchange of soluble mediators. In the case of TB, exosomes are released from the bacillus and infected cells. In the present study, a comprehensive lipidomics and proteomics analysis of size exclusion chromatography-isolated plasma-derived exosomes from patients with TB lymphadenitis (TBL) and treated as well as untreated pulmonary TB (PTB) was performed to elucidate the possibility to utilize exosomes in diagnostics and knowledge building. According to our findings, exosome-derived lipids and proteins originate from both the host and Mtb in the plasma of active TB patients. Exosomes from all patients are mostly composed of sphingomyelins (SM), phosphatidylcholines, phosphatidylinositols, free fatty acids, triacylglycerols (TAG), and cholesterylesters. Relative proportions of, e.g., SMs and TAGs, vary depending on the disease or treatment state and could be linked to Mtb pathogenesis and dormancy. We identified three proteins of Mtb origin: DNA-directed RNA polymerase subunit beta (RpoC), Diacyglycerol O-acyltransferase (Rv2285), and Formate hydrogenase (HycE), the latter of which was discovered to be differently expressed in TBL patients. Furthermore, we discovered that Mtb infection alters the host protein composition of circulating exosomes, significantly affecting a total of 37 proteins. All TB patients had low levels of apolipoproteins, as well as the antibacterial proteins cathelicidin, Scavenger Receptor Cysteine Rich Family Member (SSC5D), and Ficolin 3 (FCN3). When compared to healthy controls, the protein profiles of PTB and TBL were substantially linked, with 14 proteins being co-regulated. However, adhesion proteins (integrins, Intercellular adhesion molecule 2 (ICAM2), CD151, Proteoglycan 4 (PRG4)) were shown to be more prevalent in PTB patients, while immunoglobulins, Complement component 1r (C1R), and Glutamate receptor-interacting protein 1 (GRIP1) were found to be more abundant in TBL patients, respectively. This study could confirm findings from previous reports and uncover novel molecular profiles not previously in focus of TB research. However, we applied a minimally invasive sampling and analysis of circulating exosomes in TB patients. Based on the findings given here, future studies into host-pathogen interactions could pave the way for the development of new vaccines and therapies.

Di-(2-ethylhexyl) phthalate substitutes accelerate human adipogenesis through PPARγ activation and cause oxidative stress and impaired metabolic homeostasis in mature adipocytes.

The obesity pandemic is presumed to be accelerated by endocrine disruptors such as phthalate-plasticizers, which interfere with adipose tissue function. With the restriction of the plasticizer di-(2-ethylhexyl)-phthalate (DEHP), the search for safe substitutes gained importance. Focusing on the master regulator of adipogenesis and adipose tissue functionality, the peroxisome proliferator-activated receptor gamma (PPARγ), we evaluated 20 alternative plasticizers as well as their metabolites for binding to and activation of PPARγ and assessed effects on adipocyte lipid accumulation. Among several compounds that showed interaction with PPARγ, the metabolites MINCH, MHINP, and OH-MPHP of the plasticizers DINCH, DINP, and DPHP exerted the highest adipogenic potential in human adipocytes. These metabolites and their parent plasticizers were further analyzed in human preadipocytes and mature adipocytes using cellular assays and global proteomics. In preadipocytes, the plasticizer metabolites significantly increased lipid accumulation, enhanced leptin and adipsin secretion, and upregulated adipogenesis-associated markers and pathways, in a similar pattern to the PPARγ agonist rosiglitazone. Proteomics of mature adipocytes revealed that both, the plasticizers and their metabolites, induced oxidative stress, disturbed lipid storage, impaired metabolic homeostasis, and led to proinflammatory and insulin resistance promoting adipokine secretion. In conclusion, the plasticizer metabolites enhanced preadipocyte differentiation, at least partly mediated by PPARγ activation and, together with their parent plasticizers, affected the functionality of mature adipocytes similar to reported effects of a high-fat diet. This highlights the need to further investigate the currently used plasticizer alternatives for potential associations with obesity and the metabolic syndrome.

Aryl Hydrocarbon Receptor Activation by Benzo[a]pyrene Prevents Development of Septic Shock and Fatal Outcome in a Mouse Model of Systemic Salmonella enterica Infection.

This study focused on immunomodulatory effects of aryl hydrocarbon receptor (AhR) activation through benzo[a]pyrene (BaP) during systemic bacterial infection. Using a well-established mouse model of systemic Salmonella enterica (S.E.) infection, we studied the influence of BaP on the cellular and humoral immune response and the outcome of disease. BaP exposure significantly reduced mortality, which is mainly caused by septic shock. Surprisingly, the bacterial burden in BaP-exposed surviving mice was significantly higher compared to non-exposed mice. During the early phase of infection (days 1-3 post-infection (p.i.)), the transcription of proinflammatory factors (i.e., IL-12, IFN-γ, TNF-α, IL-1ß, IL-6, IL-18) was induced faster under BaP exposure. Moreover, BaP supported the activity of antigen-presenting cells (i.e., CD64 (FcγRI), MHC II, NO radicals, phagocytosis) at the site of infection. However, early in infection, the anti-inflammatory cytokines IL-10 and IL-22 were also locally and systemically upregulated in BaP-exposed S.E.-infected mice. BaP-exposure resulted in long-term persistence of salmonellae up to day 90 p.i., which was accompanied by significantly elevated S.E.-specific antibody responses (i.e., IgG1, IgG2c). In summary, these data suggest that BaP-induced AhR activation is capable of preventing a fatal outcome of systemic S.E. infection, but may result in long-term bacterial persistence, which, in turn, may support the development of chronic inflammation.

Characterization of murine non-adherent bone marrow cells leading to recovery of endogenous hematopoiesis.

Non-adherent bone marrow-derived cells (NA-BMCs) are a mixed cell population that can give rise to multiple mesenchymal phenotypes and that facilitates hematopoietic recovery. We characterized NA-BMCs by flow cytometry, fibroblast colony-forming units (CFU-f), real-time PCR, and in in vivo experiments. In comparison to adherent cells, NA-BMCs expressed high levels of CD11b(+) and CD90(+) within the CD45(+) cell fraction. CFU-f were significantly declining over the cultivation period, but NA-BMCs were still able to form CFU-f after 5 days. Gene expression analysis of allogeneic NA-BMCs compared to bone marrow (BM) indicates that NA-BMCs contain stromal, mesenchymal, endothelial cells and monocytes, but less osteoid, lymphoid, and erythroid cells, and hematopoietic stem cells. Histopathological data and analysis of weight showed an excellent recovery and organ repair of lethally irradiated mice after NA-BMC transplantation with a normal composition of the BM.

Indol-3-Carbinol and Quercetin Ameliorate Chronic DSS-Induced Colitis in C57BL/6 Mice by AhR-Mediated Anti-Inflammatory Mechanisms.

Inflammatory bowel diseases (IBD), such as Crohn's disease and ulcerative colitis, are multifactorial inflammatory disorders of the gastrointestinal tract, characterised by abdominal cramps, bloody diarrhoea, and anaemia. Standard therapies, including corticosteroids or biologicals, often induce severe side effects, or patients may develop resistance to those therapies. Thus, new therapeutic options for IBD are urgently needed. This study investigates the therapeutic efficacy and safety of two plant-derived ligands of the aryl hydrocarbon receptor (AhR), quercetin (Q), and indol-3-carbinol (I3C), using a translationally relevant mouse model of IBD. Q and I3C are administered by gavage to C57BL/6 wild-type or C57BL/6 Ahr-/- mice suffering from chronic colitis, induced by dextran sulphate sodium (DSS). The course of the disease, intestinal histopathological changes, and in-situ immunological phenotype are scored over 25 days. Our results show that both Q and I3C improved significantly clinical symptoms in moderate DSS colitis, which coincides with a significantly reduced histopathological score. Even in severe DSS colitis I3C, neither Q nor the therapy control 6-thioguanine (6-TG) can prevent a fatal outcome. Moreover, treatment with Q or I3C restored in part DSS-induced loss of epithelial integrity by induction of tight-junction proteins and reduced significantly gut inflammation, as demonstrated by colonoscopy, as well as by immunohistochemistry revealing lower numbers of neutrophils and macrophages. Moreover, the number of Th17 cells is significantly reduced, while the number of Treg cells is significantly increased by treatment with Q or I3C, as well as 6-TG. Q- or I3C-induced amelioration of colitis is not observed in Ahr-/- mice suggesting the requirement of AhR ligation and signalling. Based on the results of this study, plant-derived non-toxic AhR agonists can be considered promising therapeutics in IBD therapy in humans. However, they may differ in terms of efficacy; therefore, it is indispensable to study the dose-response relationship of each individual AhR agonist also with regard to potential adverse effects, since they may also exert AhR-independent effects.

Development of Monoclonal Antibodies against Pea Globulins for Multiplex Assays Targeting Legume Proteins.

Legume proteins are widely used as food ingredients, but only some (soybean, lupin, and peanut) must be declared under consumer safety regulations to protect allergy sufferers. It is not yet mandatory to declare pea proteins as allergens even though they are predicted to be allergenic based on cross-reactivity in sensitized people. The processing of legume proteins can modify their allergenic properties and hence the need for specific and precise methods for the detection of all major legume allergens. There are many commercially available tests for known food allergens but not for ingredients that are yet to be classified as allergenic. We therefore generated sets of pea-specific antibodies targeting globulins to be used in a multiplex assay for the simultaneous detection of soybean, lupin, peanut, and pea proteins. We focused on the 7S globulin family, which is the least conserved among the four legumes, allowing the specific detection of proteins from each species. Having confirmed the specificity and sensitivity of the multiplex assay, we evaluated different processing steps for proteins rich in pea globulins to demonstrate the impact of food processing on antibody binding. Our sensitive multiplex assay provides a fast and reliable method for the specific detection of soybean, lupin, peanut, and pea allergens and is therefore ideal for food safety and authenticity testing applications.