Search Results (38)

The effective suppression of inflammation using disease-modifying therapies is essential in the treatment of multiple sclerosis (MS). Anti-CD20 monoclonal antibodies are commonly used long-term as maintenance therapies, largely due to the lack of reliable biomarkers to guide dosing and evaluate treatment response. However, prolonged use increases the risk of infections and other immune-mediated side effects. The unique ability of brain-derived blood extracellular vesicles (EVs) to cross the blood–brain barrier and reflect the central nervous system (CNS) immune status has sparked interest in their potential as biomarkers. This study aimed to assess whether blood-derived L1CAM⁺ EVs could serve as biomarkers of treatment response to rituximab (RTX) in patients with relapsing-remitting MS (RRMS). Serum samples (n = 25) from the baseline (month 0) and after 6 months were analyzed from the RTX arm of the ongoing randomized clinical trial OVERLORD-MS (comparing anti-CD20 therapies in RRMS patients) and were compared with serum samples from healthy controls (n = 15). Baseline cerebrospinal fluid (CSF) samples from the same study cohort were also included. EVs from both serum and CSF samples were characterized, considering morphology, size, and concentration, using transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). The immunophenotyping of EV surface receptors was performed using flow cytometry with the MACSPlex exosome kit, while label-free quantitative proteomics of EV protein cargo was conducted using a proximity extension assay (PEA). TEM confirmed the presence of EVs with the expected round morphology with a diameter of 50–150 nm. NTA showed significantly higher concentrations of L1CAM⁺ EVs (p < 0.0001) in serum total EVs and EBNA1⁺ EVs (p < 0.01) in serum L1CAM⁺ EVs at baseline (untreated) compared to in healthy controls. After six months of RTX therapy, there was a significant reduction in L1CAM⁺ EV concentration (p < 0.0001) and the downregulation of TNFRSF13B (p = 0.0004; FC = −0.49) in serum total EVs. Additionally, non-significant changes were observed in CD79B and CCL2 levels in serum L1CAM⁺ EVs at baseline compared to in controls and after six months of RTX therapy. In conclusion, L1CAM⁺ EVs in serum showed distinct immunological profiles before and after rituximab treatment, underscoring their potential as dynamic biomarkers for individualized anti-CD20 therapy in MS. Full article

Background/Objectives: Anti-MAG polyneuropathy is a demyelinating peripheral neuropathy associated with IgM monoclonal gammopathies, particularly MGUS (monoclonal gammopathy of undetermined significance) and Waldenström macroglobulinemia. It is characterized by a subacute onset of distal sensory symptoms, with distal motor dysfunction typically appearing only in the later stages of the disease. The condition is caused by the presence of autoantibodies directed against myelin-associated glycoprotein, a structural protein of myelin. This leads to abnormalities in electrophysiological studies, such as markedly delayed distal latencies without conduction blocks or temporal dispersion of potentials. While rituximab (RTX) is the primary treatment, its efficacy is limited, with improvement seen in only 30–50% of patients. Recently, acute worsening of symptoms after RTX treatment has been increasingly reported. Methods: This systematic review compiles case reports and series from inception to June 2024 published on Scopus, PubMed or Cochrane, documenting acute exacerbations after RTX treatment in patients with anti-MAG polyneuropathy. Additionally, we present a case report from our institution that highlights this phenomenon. Results: We identified 13 clinical cases of acute deterioration in patients with anti-MAG polyneuropathy. Among these, eight patients (62%) achieved full recovery following additional treatment, while five patients (38%) did not return to their previous level of function. Plasmapheresis led to complete recovery in all four patients who received this intervention. Interestingly, many patients also experienced recovery after discontinuation of rituximab (RTX) treatment without the need for further therapeutic intervention. Conclusions: Acute clinical deterioration following RTX treatment in anti-MAG polyneuropathy is a possible occurrence. However, to date, no studies have assessed the true prevalence of this phenomenon. Further research is warranted to identify potential predictors of worsening following RTX treatment in this patient population. Full article

Vibrio ordalii is the causative agent of atypical vibriosis in salmonids cultured in Chile. While extensive research provides insights into V. ordalii through phenotypic, antigenic, and genetic typing, as well as various virulence mechanisms, proteomic characterization remains largely unexplored. This study aimed to advance the proteomic knowledge of Chilean V. ordalii Vo-LM-18 and its OMVs, which have known virulence. Using Nano-UHPLC-LC-MS/MS, we identified 2242 proteins and 1755 proteins in its OMVs. Of these, 644 unique proteins were detected in V. ordalii Vo-LM-18, namely 156 unique proteins in its OMVs and 1596 shared proteins. The major categories for the OMVs were like those in the bacteria (i.e., cytoplasmic and cytoplasmic membrane proteins). Functional annotation identified 37 biological pathways in V. ordalii Vo-LM-18 and 28 in its OMVs. Proteins associated with transport, transcription, and virulence were predominant in both. Evident differences in protein expression were found. OMVs expressed a higher number of virulence-associated proteins, including those related to iron- and heme-uptake mechanisms. Notable pathways in the bacteria included flagellum assembly, heme group-associated proteins, and protein biosynthesis. This proteomic analysis is the first to detect the RTX toxin in a V. ordalii strain (Vo-LM-18) and its vesicles. Our results highlight the crucial role of OMVs in the pathogenesis and adaptation of V. ordalii, suggesting use as potential diagnostic biomarkers and therapeutic targets for bacterial infections. Full article

Background: Many Gram-negative enterobacteria translocate virulence proteins (effectors) into intestinal epithelial cells using a type III secretion system (T3SS) to subvert the activity of various cell functions possess. Many T3SS effectors have been extensively characterized, but there are still some effector proteins whose functional information is completely unknown. Methods: In this study, two predicted effectors of unknown function, EspN and EspS (Escherichia coli secreted protein N and S), were selected for analysis of translocation, distribution and structure prediction. Results: The TEM1 (β-lactamase) translocation assay was performed, which showed that EspN and EspS are translocated into host cells in a T3SS-dependent manner during bacterial infection. A phylogenetic tree analysis revealed that homologs of EspN and EspS are widely distributed in pathogenic bacteria. Multiple sequence alignment revealed that EspN and its homologs share a conserved C-terminal region (673–1133 a.a.). Furthermore, the structure of EspN (673–1133 a.a.) was also predicted and well-defined, which showed that it has three subdomains connected by a loop region. EspS and its homologs share a sequence-conserved C-terminal (146–291 a.a.). The predicted structure of EspS (146–291 a.a.) is composed of a β-sheet consisting of four β-strands and several short helices, which has a TM score of 0.5014 with the structure of the Vibrio cholerae RTX cysteine protease domain (PDBID: 3eeb). Conclusions: These results suggest that EspN and EspS may represent two important classes of T3SS effectors associated with pathogen virulence, and our findings provide important clues to understanding the potential functions of EspN and EspS. Full article

Bone cancer and its related chronic pain are huge clinical problems since the available drugs are often ineffective or cannot be used long term due to a broad range of side effects. The mechanisms, mediators and targets need to be identified to determine potential novel therapies. Here, we characterize a mouse bone cancer model induced by intratibial injection of K7M2 osteosarcoma cells using an integrative approach and investigate the role of capsaicin-sensitive peptidergic sensory nerves. The mechanical pain threshold was assessed by dynamic plantar aesthesiometry, limb loading by dynamic weight bearing, spontaneous pain-related behaviors via observation, knee diameter with a digital caliper, and structural changes by micro-CT and glia cell activation by immunohistochemistry in BALB/c mice of both sexes. Capsaicin-sensitive peptidergic sensory neurons were defunctionalized by systemic pretreatment with a high dose of the transient receptor potential vanilloid 1 (TRPV1) agonist resiniferatoxin (RTX). During the 14- and 28-day experiments, weight bearing on the affected limb and the paw mechanonociceptive thresholds significantly decreased, demonstrating secondary mechanical hyperalgesia. Signs of spontaneous pain and osteoplastic bone remodeling were detected both in male and female mice without any sex differences. Microglia activation was shown by the increased ionized calcium-binding adapter molecule 1 (Iba1) immunopositivity on day 14 and astrocyte activation by the enhanced glial fibrillary acidic protein (GFAP)-positive cell density on day 28 in the ipsilateral spinal dorsal horn. Interestingly, defunctionalization of the capsaicin-sensitive afferents representing approximately 2/3 of the nociceptive fibers did not alter any functional parameters. Here, we provide the first complex functional and morphological characterization of the K7M2 mouse osteosarcoma model. Bone-cancer-related chronic pain and hyperalgesia are likely to be mediated by central sensitization involving neuroinflammation via glial cell activation in the spinal dorsal horn, but not the capsaicin-sensitive sensory neuronal system. Full article

RTX toxins are important virulence factors produced by a wide range of Gram-negative bacteria. They are secreted as water-soluble proteins that are able to bind to the host cell membrane and insert hydrophobic segments into the lipid bilayer that ultimately contribute to the formation of transmembrane pores. Ion diffusion through these pores leads then to cytotoxic and cytolytic effects on the hosts. Several reports have evidenced that the binding of several RTX toxins to the target cell membrane may take place through a high-affinity interaction with integrins of the β₂ family that is highly expressed in immune cells of the myeloid lineage. However, at higher toxin doses, cytotoxicity by most RTX toxins has been observed also on β₂-deficient cells in which toxin binding to the cell membrane has been proposed to occur through interaction with glycans of glycosylated lipids or proteins present in the membrane. More recently, cumulative pieces of evidence show that membrane cholesterol is essential for the mechanism of action of several RTX toxins. Here, we summarize the most important aspects of the RTX toxin interaction with the target cell membrane, including the cholesterol dependence, the recent identification in the sequences of several RTX toxins of linear motifs coined as the Cholesterol Recognition/interaction Amino acid Consensus (CRAC), and the reverse or mirror CARC motif, which is involved in the toxin–cholesterol interaction. Full article

The aims of the present study were to document the presence of Aggregatibacter actinomyctemcomitans and the emerging oral pathogen Filifactor alocis, as well as to identify genotypes of these bacterial species with enhanced virulence. In addition, these data were analyzed in relation to periodontal pocket depth (PPD) and the progression of PPD from the sampled periodontal sites during a two-year period. Subgingival plaque samples were collected from 172 periodontal pockets of 68 Ghanaian adolescents. PPD at sampling varied from 3–14 mm and the progression from baseline, i.e., two years earlier up to 8 mm. The levels of A. actinomycetemcomitans and F. alocis were determined with quantitative PCR. The highly leukotoxic JP2-genotype of A. actinomycetemcomitans and the ftxA a gene of F. alocis, encoding a putative Repeats-in-Toxin (RTX) protein, were detected with conventional PCR. The prevalence of A. actinomycetemcomitans was 57%, and 14% of the samples contained the JP2 genotype. F. alocis was detected in 92% of the samples and the ftxA gene in 52%. The levels of these bacterial species were significantly associated with enhanced PPD and progression, with a more pronounced impact in sites positive for the JP2 genotype or the ftxA gene. Taken together, the results indicate that the presence of both A. actinomycetemcomitans and F. alocis with their RTX proteins are linked to increased PPD and progression of disease. Full article

Fifth lumbar (L5) nerve injury in rodent produces neuropathic manifestations in the corresponding hind paw. The aim of this study was to investigate the effect of cutaneous injection of resiniferatoxin (RTX), a TRPV1 receptor agonist, in the rat’s hind paw on the neuropathic pain induced by L5 nerve injury. The results showed that intraplantar injection of RTX (0.002%, 100 µL) (1) completely reversed the development of chronic thermal and mechanical hypersensitivity; (2) completely prevented the development of nerve-injury-induced thermal and mechanical hypersensitivity when applied one week earlier; (3) caused downregulation of nociceptive pain markers, including TRPV1, IB4 and CGRP, and upregulation of VIP in the ipsilateral dorsal horn of spinal cord and dorsal root ganglion (DRG) immunohistochemically and a significant reduction in the expression of TRPV1 mRNA and protein in the ipsilateral DRG using Western blot and qRT-PCR techniques; (4) caused downregulation of PGP 9.5- and CGRP-immunoreactivity in the injected skin; (5) produced significant suppression of c-fos expression, as a neuronal activity marker, in the spinal neurons in response to a second intraplantar RTX injection two weeks later. This work identifies the ability of cutaneous injection of RTX to completely alleviate and prevent the development of different types of neuropathic pain in animals and humans. Full article

Filifactor alocis is a Gram-positive asaccharolytic, obligate anaerobic rod of the Firmicutes phylum, which has recently been implicated in oral infections. Extracellular vesicles (EVs) are crucial conveyors of microbial virulence in bacteria and archaea. Previously, in highly purified EVs from the F. alocis reference strain ATCC 35896 (CCUG 47790), 28 proteins were identified. The present study aimed to use label-free quantification proteomics in order to chart these EV proteins, in the reference strain, and in nine less-well-characterized clinical F. alocis isolates. In total, 25 of the EV proteins were identified and 24 were quantified. Sixteen of those were differentially expressed between the ten strains and the novel FtxA RTX toxin and one lipoprotein were among them. Consistent expression was observed among ribosomal proteins and proteins involved in L-arginine biosynthesis and type IV pilin, demonstrating a degree of EV protein expression preservation among strains. In terms of protein–protein interaction analysis, 21 functional associations were revealed between 19 EV proteins. Interestingly, FtxA did not display predicted interactions with any other EV protein. In conclusion, the present study charted 25 EV proteins in ten F. alocis strains. While most EV proteins were consistently identified among the strains, several of them were also differentially expressed, which justifies that there may be potential variations in the virulence potential among EVs of different F. alocis strains. Full article

Adenylate Cyclase Toxin (ACT or CyaA) is one of the important virulence factors secreted by Bordetella pertussis, the bacterium causative of whooping cough. ACT debilitates host defenses by production of unregulated levels of cAMP into the cell cytosol upon delivery of its N-terminal domain with adenylate cyclase activity (AC domain) and by forming pores in the plasma membrane of macrophages. Binding of soluble toxin monomers to the plasma membrane of target cells and conversion into membrane-integrated proteins are the first and last step for these toxin activities; however, the molecular determinants in the protein or the target membrane that govern this conversion to an active toxin form are fully unknown. It was previously reported that cytotoxic and cytolytic activities of ACT depend on membrane cholesterol. Here we show that ACT specifically interacts with membrane cholesterol, and find in two membrane-interacting ACT domains, four cholesterol-binding motifs that are essential for AC domain translocation and lytic activities. We hypothesize that direct ACT interaction with membrane cholesterol through those four cholesterol-binding motifs drives insertion and stabilizes the transmembrane topology of several helical elements that ultimately build the ACT structure for AC delivery and pore-formation, thereby explaining the cholesterol-dependence of the ACT activities. The requirement for lipid-mediated stabilization of transmembrane helices appears to be a unifying mechanism to modulate toxicity in pore-forming toxins. Full article

Filifactor alocis is a Gram-positive asaccharolytic, obligate anaerobic rod that has been isolated from a variety of oral infections including periodontitis, peri-implantitis, and odontogenic abscesses. As a newly emerging pathogen, its type strain has been investigated for pathogenic properties, yet little is known about its virulence variations among strains. We previously screened the whole genome of nine clinical oral isolates and a reference strain of F. alocis, and they expressed a novel RTX toxin, FtxA. In the present study, we aimed to use label-free quantification proteomics to characterize the full proteome of those ten F. alocis strains. A total of 872 proteins were quantified, and 97 among them were differentially expressed in FtxA-positive strains compared with the negative strains. In addition, 44 of these differentially expressed proteins formed 66 pairs of associations based on their predicted functions, which included clusters of proteins with DNA repair/mediated transformation and catalytic activity-related function, indicating different biosynthetic activities among strains. FtxA displayed specific interactions with another six intracellular proteins, forming a functional cluster that could discriminate between FtxA-producing and non-producing strains. Among them were FtxB and FtxD, predicted to be encoded by the same operon as FtxA. While revealing the broader qualitative and quantitative proteomic landscape of F. alocis, this study also sheds light on the deeper functional inter-relationships of FtxA, thus placing this RTX family member into context as a major virulence factor of this species. Full article

The receptor tyrosine kinase AXL (RTK-AXL) is implicated in therapy resistance and tumor progression in glioblastoma multiforme (GBM). Here, we investigated therapy-induced receptor modifications and how endogenous RTK-AXL expression and RTK-AXL inhibition contribute to therapy resistance in GBM. GBM cell lines U118MG and SF126 were exposed to temozolomide (TMZ) and radiation (RTX). Receptor modifications in response to therapy were investigated on protein and mRNA levels. TMZ-resistant and RTK-AXL overexpressing cell lines were exposed to increasing doses of TMZ and RTX, with and without RTK-AXL tyrosine kinase inhibitor (TKI). Colorimetric microtiter (MTT) assay and colony formation assay (CFA) were used to assess cell viability. Results showed that the RTK-AXL shedding product, C-terminal AXL (CT-AXL), rises in response to repeated TMZ doses and under hypoxia, acts as a surrogate marker for radio-resistance. Endogenous RTX-AXL overexpression leads to therapy resistance, whereas combination therapy of TZM and RTX with TKI R428 significantly increases therapeutic effects. This data proves the role of RTK-AXL in acquired and intrinsic therapy resistance. By demonstrating that therapy resistance may be overcome by combining AXL TKI with standard treatments, we have provided a rationale for future study designs investigating AXL TKIs in GBM. Full article

The research of novel markers in urinary samples, for the description of renal damage, is of high interest, and several works demonstrated the value of urinary mRNA quantification for the search of events related to renal disease or affecting the outcome of transplant kidneys. In the present pilot study, a comparison of the urine mRNA expression of specific podocyte markers among patients who had undergone clinical indication to renal transplanted (RTx, n = 20) and native (N, n = 18) renal biopsy was performed. The aim of this work was to identify genes involved in podocytes signaling and cytoskeletal regulation (NPHS1, NPHS2, SYNPO, WT1, TRPC6, GRM1, and NEUROD) in respect to glomerular pathology. We considered some genes relevant for podocytes signaling and for the function of the glomerular filter applying an alternative normalization approach. Our results demonstrate the WT1 urinary mRNA increases in both groups and it is helpful for podocyte normalization. Furthermore, an increase in the expression of TRPC6 after all kinds of normalizations was observed. According to our data, WT1 normalization might be considered an alternative approach to correct the expression of urinary mRNA. In addition, our study underlines the importance of slit diaphragm proteins involved in calcium disequilibrium, such as TRPC6. Full article

The prevalence and the factors related to cytomegalovirus (CMV) disease (CMVd) during the 1st year of renal transplantation (RTx) and the relationship between CMVd and early and long-term graft and RTx-patient (RTx-p) survival were evaluated. In 505 RTx-p, followed up for 8(5–11) years, data were recorded after 1-(T1) and 12-(T12) months of RTx. CMVd was defined either by CMV replication without clinical signs of disease (CMVr, 43%), or CMV replication with signs of disease (CMVs, 57%). During the 1st year of RTx, 45% of RTx-p had CMVd (CMVd+). CMVd+ patients were older than CMVd− patients. Female gender and Donor CMV-IgG+ (CMV IgG−D+)/recipient IgG- (CMV IgG−R-) status were more prevalent in CMVd+. At T1, CMVd+ had lower albumin, haemoglobin, and higher uric-acid and reactive C-protein than CMVd− and, at T1 and T12, received more steroids. Albumin-T1 was the unique factor in determining CMVd+, maintaining its significance also after the inclusion of IgG−D+/IgG−R− status to the model. CMVs had higher prevalence of CMV IgG-D+/IgG-R- than CMVr. CMVd, CMVr, and CMVs had no impact on graft loss (11% of RTx-p) and RTx-p death (8% of RTx-p). CMVd is highly prevalent during the 1st year of RTx. Albumin-T1 influences CMVd insurgence. CMVd did not impact on RTx and RTx-p loss. Full article

Drought is the largest stress affecting agricultural crops, resulting in substantial reductions in yield. Plant adaptation to water stress is a complex trait involving changes in hormone signaling, physiology, and morphology. Sorghum (Sorghum bicolor (L.) Moench) is a C4 cereal grass; it is an agricultural staple, and it is particularly drought-tolerant. To better understand drought adaptation strategies, we compared the cytosolic- and organelle-enriched protein profiles of leaves from two Sorghum bicolor genotypes, RTx430 and BTx642, with differing preflowering drought tolerances after 8 weeks of growth under water limitation in the field. In agreement with previous findings, we observed significant drought-induced changes in the abundance of multiple heat shock proteins and dehydrins in both genotypes. Interestingly, our data suggest a larger genotype-specific drought response in protein profiles of organelles, while cytosolic responses are largely similar between genotypes. Organelle-enriched proteins whose abundance significantly changed exclusively in the preflowering drought-tolerant genotype RTx430 upon drought stress suggest multiple mechanisms of drought tolerance. These include an RTx430-specific change in proteins associated with ABA metabolism and signal transduction, Rubisco activation, reactive oxygen species scavenging, flowering time regulation, and epicuticular wax production. We discuss the current understanding of these processes in relation to drought tolerance and their potential implications. Full article