Piotr Mydel

We assessed cross-sectional relationships of rheumatoid arthritis (RA) disease activity and cardio-metabolic comorbidities with serum peptidylarginine deiminase 4 (PAD4)-potentiating activity, domain-resolved angiotensin I converting enzyme (ACE1) enzymatic activity, and Porphyromonas gingivalis -specific antibodies. 177 RA patients and 147 healthy controls (HC) were included in the study. Clinical profiling included DAS28, CRP/ESR, treatments, and comorbidities. PAD4-potentiating activity, ACE1 activities, and anti-gingipain antibodies were measured using standardized in-house assays. Associations with RA status, treatments, and outcomes were analysed using covariate-adjusted models; discrimination for selected comorbidities was explored with ROC analyses. PAD4-potentiating activity, anti-Kgp/anti-RgpB and ACE1 activities did not differ between RA and controls. In RA, anti-Kgp showed a weak correlation with DAS28 (Rs=0.17, p=0.033) and was lower with biologic disease-modifying antirheumatic drugs (DMARDs); ACE1 activity was lower among synthetic-DMARDs users across all three substrates (all p≈0.01–0.04). None of the biomarkers showed independent associations with DAS28 in adjusted models. Overall discrimination for cardio-metabolic outcomes was limited; small, predefined strata showed exploratory signals, but low event counts and multiple comparisons constrain inference. In this cohort, between-group differences were limited overall. However, two exploratory within-RA signals emerged: anti-Kgp levels were weakly associated with disease activity and therapy, and lower ACE1 activity was associated with synthetic DMARDs use. Together, anti-Kgp and ACE1 (N-domain) may reflect mucosal-immune and vascular-remodeling axes that could relate to comorbidity patterns and warrant prospective, pre-specified validation.

Enzymes like peptidylarginine deiminase 4 (PAD4) generate citrullinated epitopes that are crucial in triggering and driving the anti‐citrullinated protein antibody (ACPA) response in rheumatoid arthritis. Here, we assessed whether serum PAD4‐potentiating activity is associated with subsequent arthritis development among ACPA‐positive individuals during the pre‐clinical phase. The study included 267 ACPA‐positive individuals from the Karolinska Risk‐RA cohort, among whom 101 (37.8%) developed arthritis within the observational period (range 22–60 months). We measured the ability of serum to potentiate PAD4 activity by quantifying the generation of citrullinated products using high‐performance HPLC. Study participants were then classified as PAD4 _act ‐positive or ‐negative, and PAD4 status analysed in relation to onset of arthritis, clinical characteristics (ESR, CRP and DAS28) and established risk factors (HLA‐DRB genotype, smoking). PAD4 _act ‐positivity was identified in 35 individuals, with a significant proportion (21, p = 0.0049) developing arthritis. Kaplan–Meier analysis demonstrated that individuals with PAD4 _act ‐positivity had increased rate of progressing to arthritis ( p = 0.0022). Additionally, PAD4 _act ‐positive individuals were more likely to possess at least one copy of the HLA‐DRB shared epitope allele ( p = 0.045). Notably, PAD4 _act ‐positivity did not associate with pre‐clinical CRP, ESR, DAS28, or smoking status. These findings suggest that serum PAD4‐potentiating activity may help stratify arthritis risk and time to onset among ACPA‐positive at‐risk individuals.

Background/Objectives: The aim of this study was to assess the relationship between the decline in rheumatoid arthritis (RA) disease activity—induced by periodontal treatment—and changes in the microbiology of subgingival plaque and serum antibody levels against the periodontal bacterium Porphyromonas gingivalis. Methods: Twenty-two RA patients with periodontitis underwent non-surgical periodontal treatment and assessment for the disease activity score of 28 joints (DAS28); antibody response to P. gingivalis virulence factors arginine (Rgp) and lysin (Kgp) gingipain; peptidyl arginine deiminase (PAD)2/4-activity; and the presence of P. gingivalis, Tannerella forsythia, and Prevotella intermedia in subgingival plaque through the evaluation of colony-forming units (CFUs) at baseline, two months, and six months post-treatment. Results: Periodontal treatment significantly reduced P. gingivalis CFUs at two and six months, and T. forsythia and P. intermedia CFUs at two months. Anti-RgpB IgG levels decreased at two months (p = 0.020). Higher baseline anti-RgpB IgG levels (r = −0.44, p = 0.039) and P. gingivalis CFU (r = −0.47, p = 0.028) correlated with greater reductions in DAS28. Greater reductions in P. gingivalis CFU were also associated with greater declines in DAS28 (r = 0.426, p = 0.048 and r = 0.467, p = 0.028, at two and six months, respectively). Anti-Kgp IgG and PAD2/PAD4 activity were not significantly affected by periodontal treatment. Conclusions: The impact of periodontal treatment on RA disease activity is more pronounced in patients with higher baseline P. gingivalis load and antibody response to RgpB. Better microbiological responses to periodontal treatment are associated with greater improvements in rheumatological symptoms. Further research is needed to confirm these findings and fully elucidate the underlying mechanisms.

Microglial dysregulation is increasingly recognized as a driver of Alzheimer’s disease (AD), yet how pathogen-specific cues sculpt microglial diversity remains unclear. Here we integrate high-dimensional single-cell cytometry in vitro with spatial proteomics in vivo to dissect the impact of two major periodontal pathogens on microglia. Using a 36-marker CyTOF panel, we exposed SIM-A9 microglia to wild-type Porphyromonas gingivalis (Pg) or Tannerella forsythia (Tf) and to gingipain-deficient or S-layer–deficient mutants, resolving 38 clusters. Virulence-factor “switches” redirected cells from homeostatic states toward i) oxidative, antigen-presenting programmes driven by Pg gingipains and ii) an immunosuppressive, exhausted-like state driven by the Tf S-layer. Complementary 37-marker imaging mass cytometry of 5xFAD × hTau knock-in mice chronically infected with Pg identified 21 microglial subclusters. The cortex—but not hippocampus—lost two Arg1⁺/IL-10⁺ immunoregulatory subsets (>2-fold decrease) while NADPH-oxidase-high microglia accumulated around amyloid-β and tau aggregates. These data demonstrate pathogen-specific reprogramming of microglia across model systems and brain regions, linking virulence-factor activity to AD-relevant neuroinflammation. By pinpointing gingipains and the bacterial S-layer as molecular “switches,” our study highlights tractable therapeutic targets for limiting infection-driven microglial dysfunction in Alzheimer’s disease.

Porphyromonas gingivalis (Pg) is a keystone pathogen in periodontitis, encoding a unique peptidyl arginine deiminase (PPAD) linked to protein citrullination, a process associated with rheumatoid arthritis (RA). Recently, we identified a super-active PPAD variant (T2) in Pg isolates. Here, we evaluated if the presence of the super-active T2 variant of PPAD affects the salivary microbiome, the severity of chronic periodontitis (CP), and subsequently CP's causative association with RA onset/progression. We examined 56 CP patients and 36 healthy volunteers. Pg and Tannerella forsythia counts were measured via RT-PCR, and PPAD variant was typed via PCR. 16S rRNA from salivary DNA sequencing characterized microbiota composition, while CP severity was assessed through bleeding on probing (BoP), clinical attachment loss (CAL), and pocket depth (PD) parameters. CP patients exhibited higher Pg and T. forsythia counts, with 30.7% harbouring the PPAD-T2 variant, compared to only one healthy volunteer. Clinical CP parameters were unaffected by the PPAD variant. However, PPAD-T2 influenced oral microbiota composition, enriching certain genera. While the PPAD variant did not affect CP severity, it influenced oral microbiota composition. Further research is needed to understand citrullination's role in oral microbiota and chronic inflammatory disease development.

Programmed death receptor 1, PD1, modulates the function of immune cells by providing inhibitory signals and constitutes the marker of immune exhaustion. Monitoring the level of PD1 promises a useful diagnostic approach in autoimmune diseases and cancer. Here we describe the development of an ssDNA aptamer-based molecular probe capable of specific recognition of human PD1 receptor. The aptamer was selected using SELEX, its sequence was further optimized, and the affinity and specificity were determined in biochemical assays. The aptamer was converted into a fluorescent probe and its potential in molecular imaging was demonstrated in a culture of human cells overexpressing PD1 and murine pancreatic organoids / immune cells mixed co-culture model. We conclude that the provided aptamers are suitable probes for imaging of PD1 expressing immune cells even in complex cellular models and may find future utility as diagnostic tools.

Background: Immune cells from rheumatoid arthritis (RA) patients display a reduced in vitro response to Porphyromonas gingivalis (P. gingivalis), which may have functional immune consequences. The aim of this study was to characterize, by flow cytometry, the frequency/activity of monocytes and naturally occurring myeloid dendritic cells (mDCs) in peripheral blood samples from patients with periodontitis and patients with periodontitis and RA. Methods: The relative frequency of monocytes and mDCs in the whole blood, the frequency of these cells producing TNFα or IL-6 and the protein expression levels for each cytokine, before and after stimulation with lipopolysaccharide (LPS) from Escherichia coli plus interferon-γ (IFN-γ), were assessed by flow cytometry, in peripheral blood samples from 10 healthy individuals (HEALTHY), 10 patients with periodontitis (PERIO) and 17 patients with periodontitis and RA (PERIO+RA). Results: The frequency of monocytes and mDCs producing IL-6 or TNF-α and the expression of IL-6 and TNF-α in the PERIO group were generally higher. Within the PERIO+RA group, P. gingivalis and related antibodies were negatively correlated with the monocyte and mDC expression of IL-6. A subgroup of the PERIO+RA patients that displayed statistically significantly lower frequencies of monocytes producing IL-6 after activation presented statistically significantly higher peptidylarginine deiminase (PAD)2/4 activity, anti-arg-gingipain (RgpB) IgG levels, mean probing depth (PD), periodontal inflamed surface area (PISA) and bleeding on probing (BoP). Conclusions: In the patients with PERIO+RA, innate immune cells seemed to produce lower amounts of pro-inflammatory cytokines, which are correlated with worse periodontitis-related clinical and microbiological parameters.

Peptidylarginine deiminase 4 (PAD4) is a citrullinating enzyme that is gathering increasing attention due to its possible involvement in physiological processes as well as in the pathogenesis of diseases like rheumatoid arthritis or thrombosis. PAD4 is activated by calcium ions, but the details of this mechanism are elusive, because in the human body, Ca ²⁺ concentrations are too low for full activity. Given that glycosaminoglycans (GAGs) are also implicated in the development and progression of rheumatoid arthritis, we investigated the activation of PAD4 by GAGs using heparin as a model. We employed activity assays, chromatography techniques, molecular interaction measurements (MST and SPR), FACS, and immunocytochemistry to demonstrate the activation of PAD4 by GAGs. Our data show that PAD4 binds heparin with high affinity and forms high molecular weight complexes with heparin, consistent with heparin-bound tetramer formation. Heparin activates PAD4 by increasing the enzyme’s Ca ²⁺ affinity threefold. We also show that the effectiveness of activation with heparin depends on the length of GAG used and its negative charge. Direct measurement of heparin binding to PAD4 confirmed tight interaction with nanomolar affinity. Mutagenesis of regions likely responsible for heparin binding showed that dimerization of PAD4 is necessary for efficient activation, but the distinct binding site was not determined as interaction with heparin likely occurs over larger surface of PAD4. Furthermore, we show that other GAG family members, including heparan and chondroitin sulphates, are also able to activate PAD4. We also found that disturbed production of GAGs by CHO cells results in reduced PAD4 binding efficiency. Finally, heparin induces NETosis in hPMNs in concentration-dependent manner, as measured by the release of DNA and citrullination of histone H3. In summary, we identify the first natural coactivator of PAD4, which is present in all individuals, potentially explaining the regulation of PAD4 activity in physiological conditions, and providing new insight into the development of rheumatoid arthritis and other PAD4-related diseases.

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 β 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 β ₃ compared with healthy controls. We found significant carbamylation of both subunits of α _IIb β ₃ on platelets from patients receiving hemodialysis versus only minor modification in controls. In the transient transfection system, modification of lysine 185 in the β 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 β ₃ activity in vitro. Conclusions: Carbamylation of α _IIb β 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 β ₃ activity suggests amino acid administration during dialysis may help to normalize platelet function.

Immune checkpoint targeting immunotherapy has revolutionized the treatment of certain cancers in the recent years. Determination of the status of immune checkpoint expression in particular cancers may assist decision making. Here, we describe the development of a single-stranded aptamer-based molecular probe specifically recognizing human PD-L1. Target engaging aptamers are selected by iterative enrichment from a random ssDNA pool and the binding is characterized biochemically. Specificity and dose dependence is demonstrated in vitro in the cell culture using human kidney tumor cells (786-0), human melanoma cells (WM115 and WM266.4) and human glioblastoma LN18 cancer cells. The utility of the probe in vivo is demonstrated using two mouse tumor models, where we show that the probe exhibits excellent potential in imaging. We postulate that further development of the probe may allow universal imaging of different types of tumors depending on their PD-L1 status, which may find utility in cancer diagnosis.