Automated workflows for modelling chemical fate, kinetics and toxicity.

Automation is universal in today's society, from operating equipment such as machinery, in factory processes, to self-parking automobile systems. While these examples show the efficiency and effectiveness of automated mechanical processes, automated procedures that support the chemical risk assessment process are still in their infancy. Future human safety assessments will rely increasingly on the use of automated models, such as physiologically based kinetic (PBK) and dynamic models and the virtual cell based assay (VCBA). These biologically-based models will be coupled with chemistry-based prediction models that also automate the generation of key input parameters such as physicochemical properties. The development of automated software tools is an important step in harmonising and expediting the chemical safety assessment process. In this study, we illustrate how the KNIME Analytics Platform can be used to provide a user-friendly graphical interface for these biokinetic models, such as PBK models and VCBA, which simulates the fate of chemicals in vivo within the body and in vitro test systems respectively

The Extracellular Domain of Myelin Oligodendrocyte Glycoprotein Elicits Atypical Experimental Autoimmune Encephalomyelitis in Rat and Species

Atypical models of experimental autoimmune encephalomyelitis (EAE) are advantageous in that the heterogeneity of clinical signs appears more reflective of those in multiple sclerosis (MS). Conversely, models of classical EAE feature stereotypic progression of an ascending flaccid paralysis that is not a characteristic of MS. The study of atypical EAE however has been limited due to the relative lack of suitable models that feature reliable disease incidence and severity, excepting mice deficient in gamma-interferon signaling pathways. In this study, atypical EAE was induced in Lewis rats, and a related approach was effective for induction of an unusual neurologic syndrome in a cynomolgus macaque. Lewis rats were immunized with the rat immunoglobulin variable (IgV)-related extracellular domain of myelin oligodendrocyte glycoprotein (IgV-MOG) in complete Freund’s adjuvant (CFA) followed by one or more injections of rat IgV-MOG in incomplete Freund’s adjuvant (IFA). The resulting disease was marked by torticollis, unilateral rigid paralysis, forelimb weakness, and high titers of anti-MOG antibody against conformational epitopes of MOG, as well as other signs of atypical EAE. A similar strategy elicited a distinct atypical form of EAE in a cynomolgus macaque. By day 36 in the monkey, titers of IgG against conformational epitopes of extracellular MOG were evident, and on day 201, the macaque had an abrupt onset of an unusual form of EAE that included a pronounced arousal-dependent, transient myotonia. The disease persisted for 6–7 weeks and was marked by a gradual, consistent improvement and an eventual full recovery without recurrence. These data indicate that one or more boosters of IgV-MOG in IFA represent a key variable for induction of atypical or unusual forms of EAE in rat and Macaca species. These studies also reveal a close correlation between humoral immunity against conformational epitopes of MOG, extended confluent demyelinating plaques in spinal cord and brainstem, and atypical disease induction

Features of MOG required for recognition by patients with MOG antibody-associated disorders

Antibodies to myelin oligodendrocyte glycoprotein (MOG-Abs) define a distinct disease entity. Here we aimed to understand essential structural features of MOG required for recognition by autoantibodies from patients. We produced the N-terminal part of MOG in a conformationally correct form; this domain was insufficient to identify patients with MOG-Abs by ELISA even after site-directed binding. This was neither due to a lack of lipid embedding nor to a missing putative epitope at the C-terminus, which we confirmed to be an intracellular domain. When MOG was displayed on transfected cells, patients with MOG-Abs recognized full-length MOG much better than its N-terminal part with the first hydrophobic domain (P < 0.0001). Even antibodies affinity-purified with the extracellular part of MOG recognized full-length MOG better than the extracellular part of MOG after transfection. The second hydrophobic domain of MOG enhanced the recognition of the extracellular part of MOG by antibodies from patients as seen with truncated variants of MOG. We confirmed the pivotal role of the second hydrophobic domain by fusing the intracellular part of MOG from the evolutionary distant opossum to the human extracellular part; the chimeric construct restored the antibody binding completely. Further, we found that in contrast to 8-18C5, MOG-Abs from patients bound preferentially as F(ab')(2) rather than Fab. It was previously found that bivalent binding of human IgG1, the prominent isotype of MOG-Abs, requires that its target antigen is displayed at a distance of 13-16 nm. We found that, upon transfection, molecules of MOG did not interact so closely to induce a Forster resonance energy transfer signal, indicating that they are more than 6 nm apart. We propose that the intracellular part of MOG holds the monomers apart at a suitable distance for bivalent binding; this could explain why a cell-based assay is needed to identify MOG-Abs. Our finding that MOG-Abs from most patients require bivalent binding has implications for understanding the pathogenesis of MOG-Ab associated disorders. Since bivalently bound antibodies have been reported to only poorly bind C1q, we speculate that the pathogenicity of MOG-Abs is mostly mediated by other mechanisms than complement activation. Therefore, therapeutic inhibition of complement activation should be less efficient in MOG-Ab associated disorders than in patients with antibodies to aquaporin-4

Persistence of functional memory B cells recognizing SARS-CoV-2 variants despite loss of specific IgG

Although some COVID-19 patients maintain SARS-CoV-2-specific serum immunoglobulin G (IgG) for more than 6 months postinfection, others eventually lose IgG levels. We assessed the persistence of SARS-CoV-2-specific B cells in 17 patients, 5 of whom had lost specific IgGs after 5–8 months. Differentiation of blood-derived B cells in vitro revealed persistent SARS-CoV-2-specific IgG B cells in all patients, whereas IgA B cells were maintained in 11. Antibodies derived from cultured B cells blocked binding of viral receptor-binding domain (RBD) to the cellular receptor ACE-2, had neutralizing activity to authentic virus, and recognized the RBD of the variant of concern Alpha similarly to the wild type, whereas reactivity to Beta and Gamma were decreased. Thus, differentiation of memory B cells could be more sensitive for detecting previous infection than measuring serum antibodies. Understanding the persistence of SARS-CoV-2-specific B cells even in the absence of specific serum IgG will help to promote long-term immunity

HiTSEE KNIME: a visualization tool for hit selection and analysis in high-throughput screening experiments for the KNIME platform

We present HiTSEE (High-Throughput Screening Exploration Environment), a visualization tool for the analysis of large chemical screens used to examine biochemical processes. The tool supports the investigation of structure-activity relationships (SAR analysis) and, through a flexible interaction mechanism, the navigation of large chemical spaces. Our approach is based on the projection of one or a few molecules of interest and the expansion around their neighborhood and allows for the exploration of large chemical libraries without the need to create an all encompassing overview of the whole library. We describe the requirements we collected during our collaboration with biologists and chemists, the design rationale behind the tool, and two case studies on different datasets. The described integration (HiTSEE KNIME) into the KNIME platform allows additional flexibility in adopting our approach to a wide range of different biochemical problems and enables other research groups to use HiTSEE

IgG Antibodies against Measles, Rubella, and Varicella Zoster Virus Predict Conversion to Multiple Sclerosis in Clinically Isolated Syndrome

BACKGROUND:Multiple sclerosis (MS) is characterized by a polyspecific B-cell response to neurotropic viruses such as measles, rubella and varicella zoster, with the corresponding antibodies measurable in CSF as the so-called "MRZ reaction" (MRZR). We aimed to evaluate the relevance of MRZR to predict conversion of patients with clinically isolated syndrome (CIS) to MS, and to compare it to oligoclonal bands (OCB) and MRI. METHODOLOGY/PRINCIPAL FINDINGS:MRZR was determined in a prospective study over 2 years including 40 patients that remained CIS over follow-up (CIS-CIS) and 49 patients that developed MS (CIS-RRMS) using ELISA. Using logistic regression, a score (MRZS) balancing the predictive value of the antibody indices included in MRZR was defined (9 points measles, 8 points rubella, 1 point varicella zoster, cutpoint: sum of scores greater 10). MRZR and MRZS were significantly more frequent in CIS-RRMS as compared to CIS-CIS (p=0.04 and p=0.02). MRZS showed the best positive predictive value (PPV) of all parameters investigated (79%, 95%-CI: 54-94%), which could be further increased by combination with MRI (91%, 95%-CI: 59-99%). CONCLUSIONS/SIGNIFICANCE:Our data indicate the relevance of MRZR to predict conversion to MS. It furthermore shows the importance of weighting the different antibody indices included in MRZR and suggest that patients with positive MRZR are candidates for an early begin of immunomodulatory therapy

Modulation of Cytochrome P450 Metabolism and Transport across Intestinal Epithelial Barrier by Ginger Biophenolics

Natural and complementary therapies in conjunction with mainstream cancer care are steadily gaining popularity. Ginger extract (GE) confers significant health-promoting benefits owing to complex additive and/or synergistic interactions between its bioactive constituents. Recently, we showed that preservation of natural ‘‘milieu’’ confers superior anticancer activity on GE over its constituent phytochemicals, 6-gingerol (6G), 8-gingerol (8G), 10-gingerol (10G) and 6-shogaol (6S), through enterohepatic recirculation. Here we further evaluate and compare the effects of GE and its major bioactive constituents on cytochrome P450 (CYP) enzyme activity in human liver microsomes by monitoring metabolites of CYPspecific substrates using LC/MS/MS detection methods. Our data demonstrate that individual gingerols are potent inhibitors of CYP isozymes, whereas GE exhibits a much higher half-maximal inhibition value, indicating no possible herb-drug interactions. However, GE’s inhibition of CYP1A2 and CYP2C8 reflects additive interactions among the constituents. In addition, studies performed to evaluate transporter-mediated intestinal efflux using Caco-2 cells revealed that GE and its phenolics are not substrates of P-glycoprotein (Pgp). Intriguingly, however, 10G and 6S were not detected in the receiver compartment, indicating possible biotransformation across the Caco-2 monolayer. These data strengthen the notion that an interplay of complex interactions among ginger phytochemicals when fed as whole extract dictates its bioactivity highlighting the importance of consuming whole foods over single agents. Our study substantiates the need for an indepth analysis of hepatic biotransformation events and distribution profiles of GE and its active phenolics for the design of safe regimens

B Cell Antigen Presentation Promotes Th2 Responses and Immunopathology during Chronic Allergic Lung Disease

Background: The role of B cells in allergic asthma remains undefined. One mechanism by which B cells clearly contribute to allergic disease is via the production of specific immunoglobulin, and especially IgE. Cognate interactions with specific T cells result in T cell help for B cells, resulting in differentiation and immunoglobulin secretion. Proximal to (and required for) T cell-dependent immunoglobulin production, however, is antigen presentation by B cells. While interaction with T cells clearly has implications for B cell function and differentiation, this study investigated the role that B cells have in shaping the T cell response during chronic allergic lung disease. Methodology/Principal Findings: In these studies, we used a clinically relevant mouse model of chronic allergic lung disease to study the role of B cells and B cell antigen presentation in this disease. In these studies we present several novel findings: 1) Lung B cells from chronically allergen challenged mice up-regulated MHC II and costimulatory molecules CD40, CD80 and CD86. 2) Using in vitro studies, B cells from the lungs of allergen challenged mice could present antigen to T cells, as assessed by T cell proliferation and the preferential production of Th2 cytokines. 3) Following chronic allergen challenge, the levels of Th2 cytokines IL-4 and IL-5 in the lungs and airways were significantly attenuated in B cell 2/2 mice, relative to controls. 4) B cell driven Th2 responses and mucus hyper secretion in the lungs were dependent upon MHC II expression by B cells. Conclusions/Significance: Collectively, these results provide evidence for antigen presentation as a novel mechanism b