Exploring immunogenicity of tick salivary AV422 protein in persons exposed to ticks: prospects for utilization.

In order to determine whether conserved tick salivary protein AV422 is immunogenic, the goal of our study was to detect specific IgG response within at-risk populations. Study groups included 76 individuals, differing in occurrence of recently recorded tick bites and health status. Western blotting with recombinant (r) protein derived from Ixodes ricinus (Ir) was performed. IgG response to Borrelia/Rickettsia, as indicators of previous tick infestations, was also assessed. Additionally, a detailed in silico AV422 protein sequence analysis was performed, followed by modelling of the interactions between peptides and corresponding MHC II molecules by molecular docking. Anti-rIrAV422 seroprevalences among individuals exposed to ticks were high (62.5, 57.9 and 66.7%) and anti-Borrelia/Rickettsia seroprevalences were 54.2, 15.8 and 44.4% among individuals with/without recent tick bite and patients suspected of tick-borne disease, respectively. In silico analysis of AV422 protein sequence showed a high level of conservation across tick genera, including also the predicted antigenic determinants specific for T and B cells. Docking to the restricted MHC II molecules was performed for all predicted AV422 T cell epitopes, and the most potent (highly immunogenic) epitope determinants were suggested. The epitope prediction reveals that tick salivary protein AV422 may elicit humoral immune response in humans, which is consistent with the high anti-rIrAV422 seroprevalence in tested at-risk subjects. Tick-borne diseases are a growing public health concern worldwide, and AV422 is potentially useful in clinical practice and epidemiological studies.

Effect of Vitamin B Complex Treatment on Macrophages to Schwann Cells Association during Neuroinflammation after Peripheral Nerve Injury.

Peripheral nerve injury (PNI) triggers a complex multi-cellular response involving the injured neurons, Schwann cells (SCs), and immune cells, often resulting in poor functional recovery. The aim of this study was to investigate the effects of the treatment with vitamin B (B1, B2, B3, B5, B6, and B12) complex on the interaction between macrophages and SCs during the recovery period after PNI. Transection of the motor branch of the femoral nerve followed by reconstruction by termino-terminal anastomosis was used as an experimental model. Isolated nerves from the sham (S), operated (O), and operated groups treated with the B vitamins (OT group) were used for immunofluorescence analysis. The obtained data indicated that PNI modulates interactions between macrophages and SCs in a time-dependent manner. The treatment with B vitamins complex promoted the M1-to M2-macrophage polarization and accelerated the transition from the non-myelin to myelin-forming SCs, an indicative of SCs maturation. The effect of B vitamins complex on both cell types was accompanied with an increase in macrophage/SC interactions, all of which correlated with the regeneration of the injured nerve. Clearly, the capacity of B vitamins to modulate macrophages-SCs interaction may be promising for the treatment of PNI.

Endogenous Gene Regulation as a Predicted Main Function of Type I-E CRISPR/Cas System in E. coli.

CRISPR/Cas is an adaptive bacterial immune system, whose CRISPR array can actively change in response to viral infections. However, Type I-E CRISPR/Cas in E. coli (an established model system), appears not to exhibit such active adaptation, which suggests that it might have functions other than immune response. Through computational analysis, we address the involvement of the system in non-canonical functions. To assess targets of CRISPR spacers, we align them against both E. coli genome and an exhaustive (~230) set of E. coli viruses. We systematically investigate the obtained alignments, such as hit distribution with respect to genome annotation, propensity to target mRNA, the target functional enrichment, conservation of CRISPR spacers and putative targets in related bacterial genomes. We find that CRISPR spacers have a statistically highly significant tendency to target i) host compared to phage genomes, ii) one of the two DNA strands, iii) genomic dsDNA rather than mRNA, iv) transcriptionally active regions, and v) sequences (cis-regulatory elements) with slower turn-over rate compared to CRISPR spacers (trans-factors). The results suggest that the Type I-E CRISPR/Cas system has a major role in transcription regulation of endogenous genes, with a potential to rapidly rewire these regulatory interactions, with targets being selected through naïve adaptation.

Vitamin B Complex Treatment Attenuates Local Inflammation after Peripheral Nerve Injury.

Peripheral nerve injury (PNI) leads to a series of cellular and molecular events necessary for axon regeneration and reinnervation of target tissues, among which inflammation is crucial for the orchestration of all these processes. Macrophage activation underlies the pathogenesis of PNI and is characterized by morphological/phenotype transformation from proinflammatory (M1) to an anti-inflammatory (M2) type with different functions in the inflammatory and reparative process. The aim of this study was to evaluate influence of the vitamin B (B1, B2, B3, B5, B6, and B12) complex on the process of neuroinflammation that is in part regulated by l-type CaV1.2 calcium channels. A controlled transection of the motor branch of the femoral peripheral nerve was used as an experimental model. Animals were sacrificed after 1, 3, 7, and 14 injections of vitamin B complex. Isolated nerves were used for immunofluorescence analysis. Treatment with vitamin B complex decreased expression of proinflammatory and increased expression of anti-inflammatory cytokines, thus contributing to the resolution of neuroinflammation. In parallel, B vitamins decreased the number of M1 macrophages that expressed the CaV1.2 channel, and increased the number of M2 macrophages that expressed this channel, suggesting their role in M1/M2 transition after PNI. In conclusion, B vitamins had the potential for treatment of neuroinflammation and neuroregeneration and thereby might be an effective therapy for PNI in humans.

Cutibacterium acnes as an overseen autoimmunity trigger: Unearthing heat-shock driven molecular mimicry.

Cutibacterium acnes, common resident of the human skin, can establish both commensal and pathogenic relations with the human host; however, long-term consequences of C. acnes-induced inflammation remained un(der)explored. To infer the capacity of triggering autoimmunity in humans via molecular mimicry, a comprehensive immunoinformatics analysis of the experimentally characterized C. acnes proteome was performed. The protocol included homology screening between the C. acnes and the human proteome, and validation of shared specificity regions against the collection of experimentally characterized T-cell epitopes, related to autoimmunity. To obtain highly reliable predictions, the results were subjected to additional cross-validation by a dedicated MHC-restriction analysis, including a docking study of C. acnes mimotopes and human counterparts with the highest degree of sequence similarity to MHCII molecules representing the highest risk for detected autoimmune pathologies. Due to mimicking of highly immunogenic, but also evolutionary conserved autoantigens from the Heat Shock protein family, association between C. acnes and the pathogenesis of highly incident autoimmune diseases: Type 1 Diabetes, Rheumatoid Arthritis, and Juvenile Idiopathic Arthritis, was found. To the best of our knowledge, this study is the first one to provide preliminary information and a mechanistic link on the putative involvement of C. acnes in the pathogenesis of autoimmunity in humans.

Mining the capacity of human-associated microorganisms to trigger rheumatoid arthritis-A systematic immunoinformatics analysis of T cell epitopes.

Autoimmune diseases, often triggered by infection, affect ~5% of the worldwide population. Rheumatoid Arthritis (RA)-a painful condition characterized by the chronic inflammation of joints-comprises up to 20% of known autoimmune pathologies, with the tendency of increasing prevalence. Molecular mimicry is recognized as the leading mechanism underlying infection-mediated autoimmunity, which assumes sequence similarity between microbial and self-peptides driving the activation of autoreactive lymphocytes. T lymphocytes are leading immune cells in the RA-development. Therefore, deeper understanding of the capacity of microorganisms (both pathogens and commensals) to trigger autoreactive T cells is needed, calling for more systematic approaches. In the present study, we address this problem through a comprehensive immunoinformatics analysis of experimentally determined RA-related T cell epitopes against the proteomes of Bacteria, Fungi, and Viruses, to identify the scope of organisms providing homologous antigenic peptide determinants. By this, initial homology screening was complemented with de novo T cell epitope prediction and another round of homology search, to enable: i) the confirmation of homologous microbial peptides as T cell epitopes based on the predicted binding affinity to RA-related HLA polymorphisms; ii) sequence similarity inference for top de novo T cell epitope predictions to the RA-related autoantigens to reveal the robustness of RA-triggering capacity for identified (micro/myco)organisms. Our study reveals a much larger repertoire of candidate RA-triggering organisms, than previously recognized, providing insights into the underestimated role of Fungi in autoimmunity and the possibility of a more direct involvement of bacterial commensals in RA-pathology. Finally, our study pinpoints Endoplasmic reticulum chaperone BiP as the most potent (most likely mimicked) RA-related autoantigen, opening an avenue for identifying the most potent autoantigens in a variety of different autoimmune pathologies, with possible implications in the design of next-generation therapeutics aiming to induce self-tolerance by affecting highly reactive autoantigens.

A Simple Criterion for Inferring CRISPR Array Direction.

Inferring transcriptional direction (orientation) of the CRISPR array is essential for many applications, including systematically investigating non-canonical CRISPR/Cas functions. The standard method, CRISPRDirection (embedded within CRISPRCasFinder), fails to predict the orientation (ND predictions) for ∼37% of the classified CRISPR arrays (>2200 loci); this goes up to >70% for the II-B subtype where non-canonical functions were first experimentally discovered. Alternatively, Potential Orientation (also embedded within CRISPRCasFinder), has a much smaller frequency of ND predictions but might have significantly lower accuracy. We propose a novel simple criterion, where the CRISPR array direction is assigned according to the direction of its associated cas genes (Cas Orientation). We systematically assess the performance of the three methods (Cas Orientation, CRISPRDirection, and Potential Orientation) across all CRISPR/Cas subtypes, by a mutual crosscheck of their predictions, and by comparing them to the experimental dataset. Interestingly, CRISPRDirection agrees much better with Cas Orientation than with Potential Orientation, despite CRISPRDirection and Potential Orientation being mutually related - Potential Orientation corresponding to one of six (heterogeneous) predictors employed by CRISPRDirection - and being unrelated to Cas Orientation. We find that Cas Orientation has much higher accuracy compared to Potential Orientation and comparable accuracy to CRISPRDirection - while accurately assigning an orientation to ∼95% of the CRISPR arrays that are non-determined by CRISPRDirection. Cas Orientation is, at the same time, simple to employ, requiring only (routine for prokaryotes) the prediction of the associated protein coding gene direction.