Identification and characterization of aptameric inhibitors of human neutrophil elastase.

Human neutrophil elastase (HNE) plays a pivotal role in innate immunity, inflammation, and tissue remodeling. Aberrant proteolytic activity of HNE contributes to organ destruction in various chronic inflammatory diseases including emphysema, asthma, and cystic fibrosis. Therefore, elastase inhibitors could alleviate the progression of these disorders. Here, we used the systematic evolution of ligands by exponential enrichment to develop ssDNA aptamers that specifically target HNE. We determined the specificity of the designed inhibitors and their inhibitory efficacy against HNE using biochemical and in vitro methods, including an assay of neutrophil activity. Our aptamers inhibit the elastinolytic activity of HNE with nanomolar potency and are highly specific for HNE and do not target other tested human proteases. As such, this study provides lead compounds suitable for the evaluation of their tissue-protective potential in animal models.

The impact of periodontitis and periodontal treatment on rheumatoid arthritis outcomes: an exploratory clinical trial.

OBJECTIVE: Studies suggest rheumatoid arthritis (RA) patients could benefit from periodontal treatment. However, published data are inconsistent, and there is a need for better-controlled research. Our study aims to address these limitations. METHODS: In this exploratory randomised delayed-start study, 22 RA patients with moderate/severe periodontitis were subjected to full-mouth debridement. Periodontal and rheumatological assessments, including measuring anti-cyclic citrullinated peptide 2 (CCP2) IgG levels, were performed at baseline (V1), 2 months (V2) and 6 months (V3) after step 1 and 2 of periodontal therapy. Primary outcome was changes in disease activity score for 28 joints (DAS28) between V2 and V1. Secondary outcomes were changes in other rheumatological or periodontal clinical parameters (V2 or V3-V1). RESULTS: RA disease activity was significantly higher in RA patients with severe periodontitis compared to moderate periodontitis at baseline, with significant positive correlations between several rheumatological and periodontal parameters. After periodontal treatment, RA patients with severe, but not moderate, periodontitis demonstrated significant improvements in DAS28 (ΔV2-V1, p = 0.042; ΔV3-V1, p = 0.001) and significant reduction in anti-CCP2 IgG levels at V3 (p = 0.032). CONCLUSION: Periodontal treatment is locally effective in patients with RA and impacts RA disease activity and anti-CCP2 antibody levels in patients with severe periodontitis. Hence, our data suggest that periodontal assessment and treatment should be integrated in the management of RA patients within a treat-to-target strategy. CLINICAL TRIAL REGISTRATION: www.isrctn.com, ISRCTN 17950307.

Carbamylation of Integrin α IIb ß 3: The Mechanistic Link to Platelet Dysfunction in ESKD.

BACKGROUND: Bleeding diatheses, common among patients with ESKD, can lead to serious complications, particularly during invasive procedures. Chronic urea overload significantly increases cyanate concentrations in patients with ESKD, leading to carbamylation, an irreversible modification of proteins and peptides. METHODS: To investigate carbamylation as a potential mechanistic link between uremia and platelet dysfunction in ESKD, we used liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) to quantify total homocitrulline, and biotin-conjugated phenylglyoxal labeling and Western blot to detect carbamylated integrin α IIb ß 3 (a receptor required for platelet aggregation). Flow cytometry was used to study activation of isolated platelets and platelet-rich plasma. In a transient transfection system, we tested activity and fibrinogen binding of different mutated forms of the receptor. We assessed platelet adhesion and aggregation in microplate assays. RESULTS: Carbamylation inhibited platelet activation, adhesion, and aggregation. Patients on hemodialysis exhibited significantly reduced activation of α IIb ß 3 compared with healthy controls. We found significant carbamylation of both subunits of α IIb ß 3 on platelets from patients receiving hemodialysis versus only minor modification in controls. In the transient transfection system, modification of lysine 185 in the ß 3 subunit was associated with loss of receptor activity and fibrinogen binding. Supplementation of free amino acids, which was shown to protect plasma proteins from carbamylation-induced damage in patients on hemodialysis, prevented loss of α IIb ß 3 activity in vitro. CONCLUSIONS: Carbamylation of α IIb ß 3-specifically modification of the K185 residue-might represent a mechanistic link between uremia and dysfunctional primary hemostasis in patients on hemodialysis. The observation that free amino acids prevented the carbamylation-induced loss of α IIb ß 3 activity suggests amino acid administration during dialysis may help to normalize platelet function.

Acinar adipose tissue infiltration in salivary gland biopsy is associated with kynurenines-Interferon-γ pathway inflammation biomarkers.

OBJECTIVES: Assess if kynurenines metabolites are biomarkers of damage at labial salivary gland biopsy (LSGB). METHODS: This is a cross-sectional study including 99 patients with primary Sjögren's syndrome (AECG 2002 or ACR/EULAR 2017). Kynurenines were measured in plasma using liquid chromatography-tandem mass spectrometry. RESULTS: 95.9% were females, 51±12 years. Most had focal lymphocytic sialadenitis with focus score ≥1 (73.7%, n=73/99). The majority had mild to severe acinar atrophy (70.4%, n=57/81) and adipose infiltration (51.2%, n=39/80). Individuals with adipose infiltration were older (53.49±12.33 vs. 47.51±11.29 years, p=0.016), showed higher frequency of glandular dysfunction and higher kynurenines levels. Schirmer's test ≤ 5 mm/5min was found in 69.2% of individuals with adipose infiltration compared to 41% without (p=0.012) and unstimulated whole salivary flow (UWSF) was found in 87.2% compared to 70% without adipose infiltration (p=0.063). Additionally, individuals with adipose infiltration showed higher kynurenines metabolites compared with those without: quinolinic acid (503.35±193.30 vs. 427.35±285.76 nmol/L, p=0.029), kynurenine (1.99±0.6, 54 vs. 1.61±0.46 µmol/L, p=0.006), kynurenine/tryptophan ratio (KTR) (0.030±0.09 vs. 0.025±0.01, p=0.031) and anthranilic acid (03±4.96 vs. 16.46±5.24 nmol/L, p=0.022). CONCLUSIONS: Kynurenines are biomarkers of greater adipose infiltration in LSGB and glandular dysfunction suggesting that activation of interferon-γ pathway is involved in the salivary and lacrimal glands damage.

A Novel Biological Role for Peptidyl-Arginine Deiminases: Citrullination of Cathelicidin LL-37 Controls the Immunostimulatory Potential of Cell-Free DNA.

LL-37, the only human cathelicidin that is released during inflammation, is a potent regulator of immune responses by facilitating delivery of oligonucleotides to intracellular TLR-9, thereby enhancing the response of human plasmacytoid dendritic cells (pDCs) to extracellular DNA. Although important for pathogen recognition, this mechanism may facilitate development of autoimmune diseases. In this article, we show that citrullination of LL-37 by peptidyl-arginine deiminases (PADs) hindered peptide-dependent DNA uptake and sensing by pDCs. In contrast, carbamylation of the peptide (homocitrullination of Lys residues) had no effect. The efficiency of LL-37 binding to oligonucleotides and activation of pDCs was found to be inversely proportional to the number of citrullinated residues in the peptide. Similarly, preincubation of carbamylated LL-37 with PAD2 abrogated the peptide's ability to bind DNA. Conversely, LL-37 with Arg residues substituted by homoarginine, which cannot be deiminated, elicited full activity of native LL-37 regardless of PAD2 treatment. Taken together, the data showed that citrullination abolished LL-37 ability to bind DNA and altered the immunomodulatory function of the peptide. Both activities were dependent on the proper distribution of guanidinium side chains in the native peptide sequence. Moreover, our data suggest that cathelicidin/LL-37 is citrullinated by PADs during NET formation, thus affecting the inflammatory potential of NETs. Together this may represent a novel mechanism for preventing the breakdown of immunotolerance, which is dependent on the response of APCs to self-molecules (including cell-free DNA); overactivation may facilitate development of autoimmunity.

Development of a novel, high-affinity ssDNA trypsin inhibitor.

Inhibitors of serine proteases are not only extremely useful in the basic research but are also applied extensively in clinical settings. Using Systematic Evolution of Ligands by Exponential Enrichment (SELEX) approach we developed a family of novel, single-stranded DNA aptamers capable of specific trypsin inhibition. Our most potent candidate (T24) and its short version (T59) were thoroughly characterised in terms of efficacy. T24 and T59 efficiently inhibited bovine trypsin with Ki of 176 nM and 475 nM, respectively. Interestingly, in contrast to the majority of known trypsin inhibitors, the selected aptamers have superior specificity and did not interact with porcine trypsin or any human proteases tested. These included plasmin and thrombin characterised by trypsin-like substrate specificity. Our results demonstrate that SELEX may be successfully employed in the development of potent and specific DNA based protease inhibitors.

Discovery of Novel Potential Reversible Peptidyl Arginine Deiminase Inhibitor.

Citrullination, a posttranslational modification, is catalyzed by peptidylarginine deiminases (PADs), a unique family of enzymes that converts peptidyl-arginine to peptidyl-citrulline. Overexpression and/or increased PAD activity is observed in rheumatoid arthritis (RA), Alzheimer's disease, multiple sclerosis, and cancer. Moreover, bacterial PADs, such as Porphyromonas gingivalis PAD (PPAD), may have a role in the pathogenesis of RA, indicating PADs as promising therapeutic targets. Herein, six novel compounds were examined as potential inhibitors of human PAD4 and PPAD, and compared to an irreversible PAD inhibitor, Cl-amidine. Four of the tested compounds (compounds 2, 3, 4, and 6) exhibited a micromolar-range inhibition potency against PAD4 and no effect against PPAD in the in vitro assays. Compound 4 was able to inhibit the PAD4-induced citrullination of H3 histone with higher efficiency than Cl-amidine. In conclusion, compound 4 was highly effective and presents a promising direction in the search for novel RA treatment strategies.

Transcriptional, post-transcriptional and chromatin-associated regulation of pri-miRNAs, pre-miRNAs and moRNAs.

MicroRNAs (miRNAs) play a major role in the post-transcriptional regulation of target genes, especially in development and differentiation. Our understanding about the transcriptional regulation of miRNA genes is limited by inadequate annotation of primary miRNA (pri-miRNA) transcripts. Here, we used CAGE-seq and RNA-seq to provide genome-wide identification of the pri-miRNA core promoter repertoire and its dynamic usage during zebrafish embryogenesis. We assigned pri-miRNA promoters to 152 precursor-miRNAs (pre-miRNAs), the majority of which were supported by promoter associated post-translational histone modifications (H3K4me3, H2A.Z) and RNA polymerase II (RNAPII) occupancy. We validated seven miR-9 pri-miRNAs by in situ hybridization and showed similar expression patterns as mature miR-9. In addition, processing of an alternative intronic promoter of miR-9-5 was validated by 5' RACE PCR. Developmental profiling revealed a subset of pri-miRNAs that are maternally inherited. Moreover, we show that promoter-associated H3K4me3, H2A.Z and RNAPII marks are not only present at pri-miRNA promoters but are also specifically enriched at pre-miRNAs, suggesting chromatin level regulation of pre-miRNAs. Furthermore, we demonstrated that CAGE-seq also detects 3'-end processing of pre-miRNAs on Drosha cleavage site that correlates with miRNA-offset RNAs (moRNAs) production and provides a new tool for detecting Drosha processing events and predicting pre-miRNA processing by a genome-wide assay.

Antibodies against carbamylated proteins are present in primary Sjögren's syndrome and are associated with disease severity.

OBJECTIVES: Herein, we investigate the presence and prognostic value of autoantibodies against carbamylated proteins (anti-CarP) in the serum of patients with primary Sjögren's syndrome (pSS). PATIENTS AND METHODS: Serum levels of anti-CarP antibodies were measured in Norwegian patients with pSS (n=78) and corresponding controls (n=74) using ELISA and analysed in relation with exocrine gland function, degree of salivary gland inflammation, signs of ectopic germinal centre (GC) formation and immunological markers. For univariate comparisons, the Mann-Whitney U test and χ(2) or Fisher's exact tests were used. Correlations were assessed with Spearman's rank testing. Multivariate regression analyses were used to assess the effect of anti-CarP positivity on clinical manifestations. RESULTS: Of the patients with pSS, 27% were positive for anti-CarP IgG antibodies. Levels of anti-CarP correlated positively with total IgG, IgM, rheumatoid factor and ß2-microglobulin. Importantly, after adjusting for confounding factors, patients positive for anti-CarP had significantly higher focus score. Furthermore, positive anti-CarP status coincided with 9.2-fold higher odds of having developed GC-like structures in the minor salivary glands. As a patient group considered having worse disease outcome, individuals with ectopic GC-like structures also presented with significantly higher levels of anti-CarP antibodies. CONCLUSIONS: Presence of anti-CarP in patients with pSS is strongly associated with increased focal lymphocytic infiltration, formation of ectopic GC-like structures in minor salivary glands, and diminished salivary gland function. Even taking into consideration our relatively small cohort we believe that anti-CarP antibodies offer new possibilities for identifying patients with more active disease and at risk of developing additional comorbidity.

Effects by periodontitis on pristane-induced arthritis in rats.

BACKGROUND: An infection-immune association of periodontal disease with rheumatoid arthritis has been suggested. This study aimed to investigate the effect of pre-existing periodontitis on the development and the immune/inflammatory response of pristane-induced arthritis. METHODS: We investigated the effect of periodontitis induced by ligature placement and Porphyromonas gingivalis (P. gingivalis) infection, in combination with Fusobacterium nucleatum to promote its colonization, on the development of pristane-induced arthritis (PIA) in rats (Dark Agouti). Disease progression and severity of periodontitis and arthritis was monitored using clinical assessment, micro-computed tomography (micro-CT)/intraoral radiographs, antibody response, the inflammatory markers such as α-1-acid glycoprotein (α-1-AGP) and c-reactive protein (CRP) as well as cytokine multiplex profiling at different time intervals after induction. RESULTS: Experimentally induced periodontitis manifested clinically (P < 0.05) prior to pristane injection and progressed steadily until the end of experiments (15 weeks), as compared to the non-ligated arthritis group. Injection of pristane 8 weeks after periodontitis-induction led to severe arthritis in all rats demonstrating that the severity of arthritis was not affected by the pre-existence of periodontitis. Endpoint analysis showed that 89% of the periodontitis-affected animals were positive for antibodies against arginine gingipain B and furthermore, the plasma antibody levels to a citrullinated P. gingivalis peptidylarginine deiminase (PPAD) peptide (denoted CPP3) were significantly (P < 0.05) higher in periodontitis rats with PIA. Additionally, there was a trend towards increased pro-inflammatory and anti-inflammatory cytokine levels, and increased α-1-AGP levels in plasma from periodontitis-challenged PIA rats. CONCLUSIONS: Pre-existence of periodontitis induced antibodies against citrullinated peptide derived from PPAD in rats with PIA. However, there were no differences in the development or severity of PIA between periodontitis challenged and periodontitis free rats.

Sjögren's syndrome patients with ectopic germinal centers present with a distinct salivary proteome.

OBJECTIVE: Clinical expression of SS shows considerable interpatient heterogeneity. Thus, the aim of this study was to assess whether individual salivary proteomic profiles provide a framework for identification of disease-phenotype-driven biomarker signatures. METHODS: Using a 187-plex capture antibody-based assay, proteomic biomarker profiles from unstimulated whole saliva were generated from a SS-cohort representing six clinically distinct disease phenotypes. Discriminant function analyses identified the most powerful biomarker signatures for correct recapitulation of each patient's status with respect to hyposalivation and histopathological features of salivary gland inflammation. In addition, gene ontology-based network analyses allowed systematic interpretation of the molecular patterns underlying these specific disease features. RESULTS: Presentation of hyposalivation was associated with significant alteration in 22 out of 119 reliably detectable biomarkers. Thereof, a 4-plex signature allowed accurate prediction of salivary gland function for >80% of the cases. With respect to histopathological features, the most distinct profiles were identified in conjunction with ectopic germinal centres. Selected from the 13 analytes relevant here, pregnancy-associated plasma protein A, thrombospondin 1 and peptide YY would recapitulate the presence or absence of tertiary lymphoid organization for 93.8% of the patients. Whereas functional annotation of alterations associated with hyposalivation identified the IL1 system as a dominant pro-inflammatory component, changes observed in context with ectopic lymphoid organization revealed specific shifts in chemotactic profiles and altered regulation of apoptotic processes. CONCLUSION: Multivariate analyses of a patient's salivary proteome could reliably recapitulate specific aspects of SS disease. Accessible and repetitively collectable, such biomarker signatures harbour great potential for patient subclassification and subsequent follow-up.

Atherosclerosis in Sjögren's syndrome: evidence, possible mechanisms and knowledge gaps.

Inflammation has been associated with higher cardiovascular risk in rheumatic autoimmune diseases like rheumatoid arthritis (RA) and systemic lupus erythematosus. More recently, primary Sjögren's syndrome (pSS) was also demonstrated as an independent risk factor for cardiovascular disease, emerging as a new interesting model to study atherosclerosis in autoimmune diseases. Patients with pSS have a higher prevalence of developing traditional cardiovascular risk factors like hypertension and dyslipidaemia predisposing for endothelial dysfunction and premature atherosclerosis. However, the disease-specific mechanisms for premature atherosclerosis in pSS are not fully understood. The aim of this review was to critically analyse the current literature on cardiovascular risks in pSS and to discuss the traditional and disease-associated risk factors. We also suggest possible new mechanisms that should be explored in future research to close the current knowledge gaps on the association of pSS, premature atherosclerosis, and clinical cardiovascular disease.

Serum calprotectin is a biomarker of carotid atherosclerosis in patients with primary Sjögren's syndrome.

OBJECTIVES: We aimed to identify the association of carotid atherosclerosis with the traditional risk factors, disease features, cytokine profile, and calprotectin in patients with primary Sjögren's syndrome (pSS). METHODS: 63 primary pSS patients and 63 age- and sex-matched healthy controls underwent carotid ultrasound, clinical and laboratory examination. The presence of carotid plaques was taken as carotid atherosclerosis. The covariates of carotid atherosclerosis were identified in univariate and multivariate regressions. RESULTS: Patients with pSS had higher prevalence of carotid atherosclerosis (13% vs. 2%, p<0.05) and higher serum levels of calprotectin, tumour necrosis factor receptor 2 (TNF-R2), hepatocyte growth factor (HGF), and monocyte chemoattractant protein-1 (MCP-1) than controls. Sex, menopause, and the prevalence of traditional cardiovascular did not differ between groups (all p>0.05). In univariate analyses, serum calprotectin, most traditional cardiovascular (age, male sex, metabolic syndrome, hypertension, hypertriglyceridaemia, and serum creatinine), and some disease-associated risk factors (glucocorticoid or saliva substitute use, constitutional domain of Eular-Sjögren's syndrome disease activity index - EULAR) were associated with a higher risk for plaque. In a multivariate analysis, having pSS and higher serum calprotectin were associated with carotid atherosclerosis independent of traditional risk factors. CONCLUSIONS: pSS have a higher prevalence of carotid atherosclerosis, which is associated with higher serum calprotectin level independent of traditional cardiovascular risk factors. Our findings suggest calprotectin as a biomarker of subclinical atherosclerosis in pSS.

Carbamylation of immunoglobulin abrogates activation of the classical complement pathway.

Post-translational modifications of proteins significantly affect their structure and function. The carbamylation of positively charged lysine residues to form neutral homoitrulline occurs primarily under inflammatory conditions through myeloperoxidase-dependent cyanate (CNO-) formation. We analyzed the pattern of human IgG1 carbamylation under inflammatory conditions and the effects that this modification has on the ability of antibodies to trigger complement activation via the classical pathway. We found that the lysine residues of IgG1 are rapidly modified after brief exposure to CNO- . Interestingly, modifications were not random, but instead limited to only few lysines within the hinge area and the N-terminal fragment of the CH2 domain. A complement activation assay combined with mass spectrometry analysis revealed a highly significant inverse correlation between carbamylation of several key lysine residues within the hinge region and N-terminus of the CH2 domain and the proper binding of C1q to human IgG1 followed by subsequent complement activation. This severely hindered complement-dependent cytotoxicity of therapeutic IgG1 . The reaction can apparently occur in vivo, as we found carbamylated antibodies in synovial fluid from rheumatoid arthritis patients. Taken together, our data suggest that carbamylation has a profound impact on the complement-activating ability of IgG1 and reveals a pivotal role for previously uncharacterized lysine residues in this process.

Porphyromonas gingivalis facilitates the development and progression of destructive arthritis through its unique bacterial peptidylarginine deiminase (PAD).

Rheumatoid arthritis and periodontitis are two prevalent chronic inflammatory diseases in humans and are associated with each other both clinically and epidemiologically. Recent findings suggest a causative link between periodontal infection and rheumatoid arthritis via bacteria-dependent induction of a pathogenic autoimmune response to citrullinated epitopes. Here we showed that infection with viable periodontal pathogen Porphyromonas gingivalis strain W83 exacerbated collagen-induced arthritis (CIA) in a mouse model, as manifested by earlier onset, accelerated progression and enhanced severity of the disease, including significantly increased bone and cartilage destruction. The ability of P. gingivalis to augment CIA was dependent on the expression of a unique P. gingivalis peptidylarginine deiminase (PPAD), which converts arginine residues in proteins to citrulline. Infection with wild type P. gingivalis was responsible for significantly increased levels of autoantibodies to collagen type II and citrullinated epitopes as a PPAD-null mutant did not elicit similar host response. High level of citrullinated proteins was also detected at the site of infection with wild-type P. gingivalis. Together, these results suggest bacterial PAD as the mechanistic link between P. gingivalis periodontal infection and rheumatoid arthritis.

The link between periodontal disease and rheumatoid arthritis: an updated review.

Porphyromonas gingivalis is a leading pathogen in chronic periodontitis, a disease process involving progressive destruction of the tissues that support the teeth. Recently, the organism has been reported to produce a unique bacterial enzyme, P. gingivalis peptidyl-arginine deiminase (PPAD), which has the ability to convert arginine residues in proteins to citrulline. Protein citrullination alters protein structure and function; hence, PPAD may be involved in deregulation of the host's signalling network and immune evasion. Further, accumulating evidence suggests a role for autoimmunity against citrullinated proteins in the development of rheumatoid arthritis (RA). As inflammatory conditions in the lungs of cigarette smokers contribute to the breakdown of immune tolerance to citrullinated epitopes, chronic exposure to citrullinated proteins at periodontitis sites may also predispose susceptible individuals to the development of autoantibodies and the initiation of RA. In this review, we discuss evidence that PPAD may represent a mechanistic link between periodontitis and RA, diseases that are known to be significantly associated at the epidemiological level.

Glutaminyl cyclases as novel targets for the treatment of septic arthritis.

BACKGROUND: Septic arthritis is a severe and rapidly debilitating disease mainly caused by Staphylococcus aureus. Here, we assess the antiarthritic efficiency of glutaminyl cyclase (QC) inhibitors. METHODS: Mice were inoculated with an arthritogenic amount of S. aureus intravenously or by local administration into the knee joint. Animals were treated with QC inhibitors (PBD155 and PQ529) via chow during the experiment. QC and isoQC knockout mice were also analyzed for arthritis symptoms after local administration of bacteria. RESULTS: Both QC inhibitors significantly delayed the onset of clinical signs of arthritis, and inhibitors significantly decreased weight loss in treated animals. Following intraarticular injection of S. aureus, PBD155-treated mice had lower levels of synovitis and bone erosion, as well as less myeloperoxidase in synovial tissue. Fluorescence-activated cell sorter analysis revealed that PBD155 treatment affected the expression pattern of adhesion molecules, preventing the upregulation of cells expressing CD11b/CD18. CONCLUSION: The compounds investigated here represent a novel class of small molecular antiarthritic inhibitors. In our studies, they exerted strong antiinflammatory actions, and therefore they might be suited for disease-modifying treatment of infectious arthritis.

Carbamylation Is Instrumental in End-Stage Kidney Disease Coagulopathies: The Impact on von Willebrand Factor and Platelet Functionality.

BACKGROUND: Chronic kidney disease (CKD) is a progressive, irreversible, and incurable condition characterized by high morbidity and mortality, affecting approximately one-tenth of the global population. Rise of urea-derived cyanate levels in CKD patients, severalfold higher in comparison to those found in healthy individuals, leads to an increased rate of carbamylation of lysine residues of proteins and peptides. This posttranslational modification plays an important role in the progression of kidney failure but also in the onset of CKD-related complications, including previously reported coagulopathies. In this study, we have explored the impact of carbamylation on the functionality of von Willebrand factor (vWF), a pivotal player in hemostasis, and its implications for platelet adhesion. MATERIALS AND METHODS: We have explored carbamylated vWF's interactions with its partner proteins via ELISA. Mass spectrometry was employed to identify modified lysine residues. Blood platelets isolated from healthy donors were carbamylated, and their activation, binding to endothelium and thromboxane release were evaluated using flow cytometry, adhesion assays and ELISA, respectively. RESULTS: Using mass spectrometry we detected the vWF's lysine residue smost susceptible to carbamylation. This modification has in turn affected vWF's interactions with its key binding partners: decreased binding to collagen types I/III but increased the affinity to factor FVIII, while its binding to fibrinogen remained unchanged. Carbamylation of vWF impeded vWF-blood platelet binding, but carbamylation of platelets led to their increased thrombin-dependent activation as observed by enhanced phosphatidylserine exposure, improved their binding to vascular endothelium, at the same time decreasing the production of the prothrombotic mediator, thromboxane A2. CONCLUSION: Our findings highlight the multifaceted impact of carbamylation on vWF and platelets, disturbing the delicate balance of coagulation cascade. These alterations could contribute to the complex hemostatic imbalance in ESKD, underscoring the need for further research to fully understand these mechanisms and their clinical implications.

The role of Mcl-1 in S. aureus-induced cytoprotection of infected macrophages.

As a facultative intracellular pathogen, Staphylococcus aureus invades macrophages and then promotes the cytoprotection of infected cells thus stabilizing safe niche for silent persistence. This process occurs through the upregulation of crucial antiapoptotic genes, in particular, myeloid cell leukemia-1 (MCL-1). Here, we investigated the underlying mechanism and signal transduction pathways leading to increased MCL-1 expression in infected macrophages. Live S. aureus not only stimulated de novo synthesis of Mcl-1, but also prolonged the stability of this antiapoptotic protein. Consistent with this, we proved a crucial role of Mcl-1 in S. aureus-induced cytoprotection, since silencing of MCL1 by siRNA profoundly reversed the cytoprotection of infected cells leading to apoptosis. Increased MCL1 expression in infected cells was associated with enhanced NFκB activation and subsequent IL-6 secretion, since the inhibition of both NFκB and IL-6 signalling pathways abrogated Mcl-1 induction and cytoprotection. Finally, we confirmed our observation in vivo in murine model of septic arthritis showing the association between the severity of arthritis and Mcl-1 expression. Therefore, we propose that S. aureus is hijacking the Mcl-1-dependent inhibition of apoptosis to prevent the elimination of infected host cells, thus allowing the intracellular persistence of the pathogen, its dissemination by infected macrophages, and the progression of staphylococci diseases.

Transcriptomic analysis reveals partial epithelial-mesenchymal transition and inflammation as common pathogenic mechanisms in hypertensive nephrosclerosis and Type 2 diabetic nephropathy.

Hypertensive nephrosclerosis (HN) and Type 2 diabetic nephropathy (T2DN) are the leading causes of chronic kidney disease (CKD). To explore shared pathogenetic mechanisms, we analyzed transcriptomes of kidney biopsies from patients with HN or T2DN. Total RNA was extracted from 10 µm whole kidney sections from patients with HN, T2DN, and normal controls (Ctrl) (n = 6 for each group) and processed for RNA sequencing. Differentially expressed (log2 fold change >1, adjusted p < 0.05) genes (DEG) and molecular pathways were analyzed, and selected results were validated by immunohistochemistry (IHC). ELISA on serum samples was performed on a related cohort consisting of patients with biopsy-proven HN (n = 13) and DN (n = 9), and a normal control group (n = 14). Cluster analysis on RNA sequencing data separated diseased and normal tissues. RNA sequencing revealed that 88% (341 out of 384) of DEG in HN were also altered in T2DN, while gene set enrichment analysis (GSEA) showed that over 90% of affected molecular pathways, including those related to inflammation, immune response, and cell-cycle regulation, were similarly impacted in both HN and T2DN samples. The increased expression of genes tied to interleukin signaling and lymphocyte activation was more pronounced in HN, while genes associated with extracellular matrix organization were more evident in T2DN. Both HN and T2DN tissues exhibited significant upregulation of genes connected with inflammatory responses, T-cell activity, and partial epithelial to mesenchymal transition (p-EMT). Immunohistochemistry (IHC) further confirmed T-cell (CD4+ and CD8+ ) infiltration in the diseased tissues. Additionally, IHC revealed heightened AXL protein expression, a key regulator of inflammation and p-EMT, in both HN and T2DN, while serum analysis indicated elevated soluble AXL levels in patients with both conditions. These findings underline the shared molecular mechanisms between HN and T2DN, hinting at the potential for common therapeutic strategies targeting both diseases.