Relaxed acceptor site specificity of bacterial oligosaccharyltransferase in vivo

A number of proteobacteria carry the genetic information to perform N-linked glycosylation, but only the protein glycosylation (pgl) pathway of Campylobacter jejuni has been studied to date. Here, we report that the pgl gene cluster of Campylobacter lari encodes for a functional glycosylation machinery that can be reconstituted in Escherichia coli. We determined that the N-glycan produced in this system consisted of a linear hexasaccharide. We found that the oligosaccharyltransferase (OST) of C. lari conserved a predominant specificity for the primary sequence D/E-X−1-N-X+1-S/T (where X−1 and X+1 can be any amino acid but proline). At the same time, we observed that this enzyme exhibited a relaxed specificity toward the acceptor site and modified asparagine residues of a protein at sequences DANSG and NNNST. Moreover, C. lari pgl glycosylated a native E. coli protein. Bacterial N-glycosylation appears as a useful tool to establish a molecular description of how single-subunit OSTs perform selection of glycosyl acceptor site

Relaxed acceptor site specificity of bacterial oligosaccharyltransferase in vivo

A number of proteobacteria carry the genetic information to perform N-linked glycosylation, but only the protein glycosylation (pgl) pathway of Campylobacter jejuni has been studied to date. Here, we report that the pgl gene cluster of Campylobacter lari encodes for a functional glycosylation machinery that can be reconstituted in Escherichia coli. We determined that the N-glycan produced in this system consisted of a linear hexasaccharide. We found that the oligosaccharyltransferase (OST) of C. lari conserved a predominant specificity for the primary sequence D/E-X−1-N-X+1-S/T (where X−1 and X+1 can be any amino acid but proline). At the same time, we observed that this enzyme exhibited a relaxed specificity toward the acceptor site and modified asparagine residues of a protein at sequences DANSG and NNNST. Moreover, C. lari pgl glycosylated a native E. coli protein. Bacterial N-glycosylation appears as a useful tool to establish a molecular description of how single-subunit OSTs perform selection of glycosyl acceptor site

Cytokine and immune cell profiling in the cerebrospinal fluid of patients with neuro-inflammatory diseases

Background Cytokines play multiple roles during neuro-inflammatory processes and several cytokines have been studied in the context of specific diseases. This study provides a comprehensive picture of cerebrospinal fluid (CSF) changes during neuro-inflammation by analyzing multiple cytokines in combination with immune cell subsets and standard CSF parameters. Methods Using multiplex assays, we simultaneously measured 36 cytokines (CCL1-3, CCL7, CCL8, CCL11, CCL13, CCL19, CCL20, CCL22-27, CXCL1, CXCL2, CXCL5, CXCL6, CXCL8, CXCL9, CXCL11-13, CXCL16, CX3CL1, IL2, IL4, IL6, IL10, IL16, GM-CSF, IFN gamma, MIF, TNF alpha, and MIB1 beta) in the CSF and serum of 75 subjects. Diagnoses included clinically isolated syndrome and relapsing-remitting multiple sclerosis (MS, n = 18), secondary progressive MS (n = 8), neuro-syphilis (n = 6), Lyme neuro-borreliosis (n = 13), bacterial and viral meningitis (n = 20), and patients with non-inflammatory neurological diseases (NIND, n = 10). Cytokine concentrations were correlated with CSF standard parameters and CSF immune cell subsets (CD4 and CD8 T cells, B cells, plasmablasts, monocytes, and NK cells) quantified by flow cytometry. Results We observed increased levels of multiple cytokines (26/36) in patients with neuro-inflammatory diseases when compared to NIND that consistently correlated with CSF cell count and Q(Albumin). Most CSF cytokine concentrations correlated with each other, but correlations between CSF and serum values were scarce (3/36). Within the CSF compartment, CXCL13 showed a strong association with B cells when analyzing all patients, as well as patients with an intact blood-brain barrier (BBB). NK cells positively correlated with CSF concentrations of multiple cytokines (22/36) when analyzing all patients. These correlations were maintained when looking at patients with a disrupted BBB but not detectable in patients with an intact BBB. Conclusions Under conditions of neuro-inflammation, multiple CSF cytokines are regulated in parallel and most likely produced locally. A combined increase of CSF CXCL13 levels and B cells occurs under conditions of an intact BBB. Under conditions of a disrupted BBB, CSF NK cells show significantly increased values and seem to have a major contribution to overall inflammatory processes, reflected by a strong correlation with multiple cytokines. Future studies are necessary to address the exact kinetics of these cytokines during neuro-inflammation and their relation to specific diseases phenotypes

Specific Induction of Double Negative B Cells During Protective and Pathogenic Immune Responses

Double negative (DN) (CD19(+)CD20(low)CD27(-)IgD(-)) B cells are expanded in patients with autoimmune and infectious diseases;however their role in the humoral immune response remains unclear. Using systematic flow cytometric analyses of peripheral blood B cell subsets, we observed an inflated DN B cell population in patients with variety of active inflammatory conditions: myasthenia gravis, Guillain-Barre syndrome, neuromyelitis optica spectrum disorder, meningitis/encephalitis, and rheumatic disorders. Furthermore, we were able to induce DN B cells in healthy subjects following vaccination against influenza and tick borne encephalitis virus. Transcriptome analysis revealed a gene expression profile in DN B cells that clustered with naive B cells, memory B cells, and plasmablasts. Immunoglobulin VH transcriptome sequencing and analysis of recombinant antibodies revealed clonal expansion of DN B cells that were targeted against the vaccine antigen. Our study suggests that DN B cells are expanded in multiple inflammatory neurologic diseases and represent an inducible B cell population that responds to antigenic stimulation, possibly through an extra-follicular maturation pathway

Fatigue in multiple sclerosis: Associations with clinical, MRI and CSF parameters

Background: Damage of different brain structures has been related to fatigue. Alternatively, functional alterations of central nervous system (CNS) cells by the inflammatory milieu within the CNS may be responsible for the development of fatigue. Aim: To investigate the effect of structural brain damage and inflammatory cerebrospinal fluid (CSF) changes on fatigue in multiple sclerosis (MS). Methods: We determined the association of different clinical, CSF and magnetic resonance imaging (MRI) parameters with prevalence and severity of fatigue, as measured by the Fatigue Scale for Motor and Cognitive Functions in 68 early MS patients (discovery cohort). We validated our findings in two MS cohorts: the MRI validation cohort (N=233) for the clinical and MRI parameters, and the CSF validation cohort (N=81) for the clinical and CSF parameters. Results: Fatigue was associated with clinical disability. Fatigue did not correlate with any CSF parameter but correlated negatively with total and cortical grey matter volume. However, when controlling for Expanded Disability Status Scale (EDSS) in a multivariate model, these associations lost significance. Conclusion: Disability and disease duration best explain fatigue severity but none of the tested MRI or CSF parameter was reliably associated with fatigue

Retrospective cohort study to devise a treatment decision score predicting adverse 24-month radiological activity in early multiple sclerosis

Background: Multiple sclerosis (MS) is a chronic neuroinflammatory disease affecting about 2.8 million people worldwide. Disease course after the most common diagnoses of relapsing-remitting multiple sclerosis (RRMS) and clinically isolated syndrome (CIS) is highly variable and cannot be reliably predicted. This impairs early personalized treatment decisions. Objectives: The main objective of this study was to algorithmically support clinical decision-making regarding the options of early platform medication or no immediate treatment of patients with early RRMS and CIS. Design: Retrospective monocentric cohort study within the Data Integration for Future Medicine (DIFUTURE) Consortium. Methods: Multiple data sources of routine clinical, imaging and laboratory data derived from a large and deeply characterized cohort of patients with MS were integrated to conduct a retrospective study to create and internally validate a treatment decision score [Multiple Sclerosis Treatment Decision Score (MS-TDS)] through model-based random forests (RFs). The MS-TDS predicts the probability of no new or enlarging lesions in cerebral magnetic resonance images (cMRIs) between 6 and 24 months after the first cMRI. Results: Data from 65 predictors collected for 475 patients between 2008 and 2017 were included. No medication and platform medication were administered to 277 (58.3%) and 198 (41.7%) patients. The MS-TDS predicted individual outcomes with a cross-validated area under the receiver operating characteristics curve (AUROC) of 0.624. The respective RF prediction model provides patient-specific MS-TDS and probabilities of treatment success. The latter may increase by 5–20% for half of the patients if the treatment considered superior by the MS-TDS is used. Conclusion: Routine clinical data from multiple sources can be successfully integrated to build prediction models to support treatment decision-making. In this study, the resulting MS-TDS estimates individualized treatment success probabilities that can identify patients who benefit from early platform medication. External validation of the MS-TDS is required, and a prospective study is currently being conducted. In addition, the clinical relevance of the MS-TDS needs to be established

Autologous haematopoietic stem cell transplantation for multiple sclerosis: a position paper and registry outline

Background: While substantial progress has been made in the development of disease-modifying medications for multiple sclerosis (MS), a high percentage of treated patients still show progression and persistent inflammatory activity. Autologous haematopoietic stem cell transplantation (AHSCT) aims at eliminating a pathogenic immune repertoire through intense short-term immunosuppression that enables subsequent regeneration of a new and healthy immune system to re-establish immune tolerance for a long period of time. A number of mostly open-label, uncontrolled studies conducted over the past 20 years collected about 4000 cases. They uniformly reported high efficacy of AHSCT in controlling MS inflammatory disease activity, more markedly beneficial in relapsing-remitting MS. Immunological studies provided evidence for qualitative immune resetting following AHSCT. These data and improved safety profiles of transplantation procedures spurred interest in using AHSCT as a treatment option for MS. Objective: To develop expert consensus recommendations on AHSCT in Germany and outline a registry study project. Methods: An open call among MS neurologists as well as among experts in stem cell transplantation in Germany started in December 2021 to join a series of virtual meetings. Results: We provide a consensus-based opinion paper authored by 25 experts on the up-to-date optimal use of AHSCT in managing MS based on the Swiss criteria. Current data indicate that patients who are most likely to benefit from AHSCT have relapsing-remitting MS and are young, ambulatory and have high disease activity. Treatment data with AHSCT will be collected within the German REgistry Cohort of autologous haematopoietic stem CeLl trAnsplantation In MS (RECLAIM). Conclusion: Further clinical trials, including registry-based analyses, are urgently needed to better define the patient characteristics, efficacy and safety profile of AHSCT compared with other high-efficacy therapies and to optimally position it as a treatment option in different MS disease stages. Keywords: Autologous haematopoietic stem cell transplantation (AHSCT), multiple sclerosis, registry study, treatment recommendation