BackgroundObesity is a global health challenge linked to chronic non-communicable diseases. Low-grade inflammation and altered immune responses, including neutrophil extracellular trap (NET) formation, contribute to metabolic complications. Peptidyl arginine deiminase 4 (PAD4) is critical for NET formation, and its inhibition shows therapeutic potential. However, the sex-specific effects of PAD4 deficiency in diet-induced obesity remain unexplored.MethodsThis study investigated the impact of PAD4 deficiency on obesity-related metabolic pathologies in male and female C57BL/6 wild-type (WT) and Pad4(-/-) mice (n=5-6/group) fed a 22-week obesogenic cafeteria (CAF) diet. We hypothesized that PAD4 deficiency would ameliorate obesity-related metabolic and behavioral complications for both sexes. Body weight and composition, glucose and lipid metabolism, liver damage markers, and behavior were assessed. NETs were quantified via flow cytometry.ResultsPad4(-/-) males on CAF diet exhibited delayed obesity onset, lower body weight gain, and improved dyslipidemia than WT CAF males. This was associated with enhanced metabolic adaptation, indicated by higher brown adipose tissue temperature in Pad4(-/-) males. Conversely, Pad4(-/-) females on the CAF diet showed comparable weight gain to WT CAF females, similar or worsened dyslipidemia, impaired glucose metabolism, and higher liver lipid accumulation. While WT CAF females showed increased brown adipose tissue temperature, Pad4(-/-) CAF females did not.ConclusionPAD4 deficiency exerts sex-specific effects on obesity-related metabolic complications in mice. Inhibition of NET formation appears protective in males but not females on an obesogenic diet. These findings underscore the importance of considering sex as a biological variable in obesity research and developing sex-specific therapies.

Abstract Background Mitochondrial damage-associated molecular patterns (mtDAMPs) released after trauma can trigger neutrophil extracellular trap (NET) formation, potentially worsening tissue injury and increasing susceptibility to secondary infection. However, the mechanisms driving mitochondria-induced NETosis and whether this response can be selectively modulated without impairing antibacterial defense remain unclear. Methods We investigated the mechanisms of mitochondria-induced NET formation using in vitro, in vivo models and ex vivo, using trauma patient plasma. Neutrophils were exposed to intact mitochondria or mitochondrial components, and the roles of Toll-like receptor 9 (TLR9), formyl peptide receptor 1 (FPR1), PAD4, NOX2, p38α, and STAT6 were assessed. Effect of the synthetic FPR1 agonist FPRA14 on NET formation and antibacterial neutrophil functions was tested in double hit in vivo model of peritoneal mitochondria injection followed by S. aureus pneumonia. Results Intact mitochondria induced NET formation through coordinated activation of both TLR9 and FPR1, with dependence on PAD4, NOX2, and p38α signaling. Mitochondrial protein integrity was important for this response, likely by protecting mtDNA from degradation and enabling TLR9 activation. While insufficient to drive NETosis alone, FPR1 activation showed a concentration- and time-dependent modulatory effect: low-level activation promoted NETosis, whereas stronger or pre-exposure–induced signaling suppressed it through receptor desensitization. FPR1 agonist FPRA14 reduced mitochondria-driven NET formation in vitro and in a double hit murine model while preserving neutrophil antibacterial functions, including NET formation, phagocytosis, ROS generation, bacterial killing, and infection control. p38α acted as a positive regulator, whereas STAT6 functioned as a negative regulator of NET formation. Discussion These findings identify FPR1 as a key regulator of pathological mitochondria-driven NETosis and suggest that selective FPR1 receptor desensitization may suppress excessive sterile inflammation without compromising host defense. This work extends current understanding of mtDAMP signaling in neutrophils and supports prophylactic FPR1 agonism as a potential strategy for limiting inflammatory tissue damage after trauma or surgery. Conclusion Mitochondria-induced NETosis depends on TLR9/FPR1 signaling and can be reduced by FPR1 agonism without compromising antibacterial immunity. FPRA14 may represent a strategy to limit harmful sterile inflammation after trauma or surgery.

Hemolysis is associated with the release of damage-associated molecular patterns, including free heme and extracellular DNA (ecDNA). Using several mouse models of bleeding anemia and hemolysis, we demonstrate a significant increase in plasma ecDNA, independent of neutrophil extracellular trap formation. This ecDNA forms G-quadruplex (G4) structures, which we detected in both mice and patients with systemic lupus erythematosus. Catalytic complexes (DNAzymes) formed by G4 ecDNA and heme drive oxidative stress, tissue injury, and inflammation. In anemic mice lacking deoxyribonuclease 1L3 (DNase1l3-/-), we found elevated polynucleosomal ecDNA in the plasma, reduced expression of the heme-degrading enzyme heme oxygenase-1 in macrophages, but also increased plasma creatinine, renal iron accumulation, and complement C3 deposition along elevated apoptosis and DNA damage. ecDNA isolated from these mice also triggered toll-like receptor 9-dependent inflammatory responses in vitro and in vivo. In summary, these findings suggest that concurrent release of heme and ecDNA during hemolysis promotes inflammation and tissue damage, contributing to lupus pathogenesis.

Autism Spectrum Disorder (ASD) is a neurodevelopmental condition with a strong genetic basis, yet its etiology is not fully understood. Neuroinflammation is emerging as a critical secondary factor. Since peripheral organ injury, such as acute liver failure, can induce neuroinflammation, we aimed to investigate whether liver injury could exacerbate ASD-like phenotypes in a genetically susceptible model. We used adult (12-month-old) Shank3−/− mice, a genetic model of ASD, and their wild-type (WT) counterparts. A single high dose of acetaminophen (APAP; 350 mg/kg) was administered intraperitoneally to induce acute liver injury. We assessed acute (24-h) and long-term (1-month) consequences on liver health, neuroinflammation, blood–brain barrier integrity, and brain edema using plasma biomarkers, immunohistochemistry, and in-vivo MRI, as well as behavioral phenotypes, including sociability, repetitive behaviors and exploration. Acutely, APAP induced severe liver damage, cerebral edema, and neuroinflammation in Shank3−/− mice. One month later, despite liver recovery, both APAP-treated WT and Shank3−/− mice exhibited brain edema, elevated apparent transverse relaxation (R2*) rates, and lasting neuroinflammation, evidenced by microgliosis and reactive astrocytosis. These differences were more extensive in the Shank3−/− group. Behaviorally, this was accompanied by increased social deficits in both genotypes. Critically, only the APAP-treated Shank3−/− mice displayed significantly increased repetitive behaviors, and reduced exploratory activity. Here, we show that transient liver injury is associated with long-term neuroinflammation, markers of blood–brain barrier disruption, and behavioral deficits. These findings demonstrate that while peripheral organ damage can induce lasting behavioral changes in healthy animals, the severity and spectrum of these deficits are exacerbated by ASD-associated genetic predisposition. These findings support a preclinical gene-environment interaction framework relevant to ASD-associated vulnerability.

BackgroundCisplatin-induced peripheral neuropathy (CIPN) is a frequent and often persistent complication in survivors of testicular germ cell tumors (GCT) treated with curative therapy. Although several mechanisms have been proposed, the biological drivers of long-term neurotoxicity remain incompletely understood. Disruption of intestinal barrier integrity during chemotherapy or radiotherapy may promote gut microbial translocation (GMT), leading to systemic immune activation and chronic inflammation that could contribute to neuropathy development. This study investigated the relationship between circulating biomarkers of GMT and symptoms of CIPN in long-term GCT survivors.MethodsA total of 170 GCT survivors (median age 41 years) from the National Cancer Institute of Slovakia were included, with a median follow-up of 10 years after treatment. Participants completed the EORTC QLQ-CIPN20 questionnaire assessing sensory, motor, and autonomic neuropathy. Peripheral blood samples were analyzed for plasma biomarkers associated with gut microbial translocation and innate immune activation, including soluble CD14 (sCD14), high-mobility group box-1 (HMGB1), lipopolysaccharide (LPS), and D-lactate. Associations between biomarker concentrations and CIPN scores were evaluated across treatment groups: orchiectomy only (active surveillance, n=28), cisplatin-based chemotherapy (n=119), radiotherapy (n=14), and combined chemoradiotherapy (n=9).ResultsPatients with higher plasma sCD14 levels had significantly higher overall CIPN scores (7.6% increase, p=0.019) and worse sensory function (9.5% increase, p=0.019) compared with those with lower levels. Patients treated with chemotherapy exhibited significantly higher plasma sCD14 levels than those under active surveillance (6613 vs. 3768 μg/L, p = 0.009). Among chemotherapy-treated patients, elevated sCD14 was associated with a higher risk of motor neuropathy (RR = 3.5, 95% CI 1.21–10.14, p=0.020). In survivors receiving combined chemotherapy and radiotherapy, increased sCD14 levels were associated with a higher risk of autonomic neuropathy (RR = 2.85, 95% CI 1.11–7.30, p=0.029). Elevated HMGB1 was also associated with an increased probability of autonomic dysfunction (RR = 2.15, 95% CI 1.07–4.33, p=0.015). No significant associations were observed between cumulative cisplatin dose and GMT biomarkers.ConclusionElevated biomarkers of gut microbial translocation, particularly sCD14, are associated with increased severity of CIPN in long-term GCT survivors. These findings support the hypothesis that treatment-related intestinal barrier disruption and subsequent immune activation may contribute to persistent neurotoxicity in cancer survivorship.

Neutrophil extracellular traps (NETs) are suggested to play a role in chronic kidney disease (CKD). Whether they are indeed involved in the pathogenesis of animal models of CKD has not been proved. This study tested the hypothesis that a genetic deficiency of peptidylarginine deiminase 4 (PAD4) - a regulator of NETs production will protect mice from developing adenine-induced nephropathy as a model of CKD. Adult male Padi4-/- mice and their wild-type counterparts received 100 mg/kg adenine or saline i.p. daily for 14 days. Markers of renal function and NETs-related biomarkers were assessed during the CKD induction in plasma and urine. In wild-type mice, adenine injections decreased body weight by 20% and increased plasma creatinine (twofold), neutrophil gelatinase-associated lipocalin (21-fold), and neutrophil elastase (4-fold). No significant differences were found between the Padi4-/- and wild-type mice except the earlier increase in plasma creatinine in Padi4-/- mice (day 3 vs. day 7). Analyses of neutrophil elastase and myeloperoxidase in plasma and urine suggest that neutrophils are activated in adenine-induced nephropathy, but the production of NETs seems not to be directly involved in the pathogenesis of this CKD model. Further studies should clear the role of NETs in other kidney disease models with clinical relevance.

Bleomycin, etoposide, and cisplatin (BEP) remains the cornerstone of curative first-line treatment for metastatic germ cell tumors (GCTs) across all prognostic risk groups. Although etoposide-cisplatin (EP) and etoposide-ifosfamide-cisplatin (VIP) are validated alternatives in selected clinical scenarios, oncologic efficacy among standard first-line cisplatin-based regimens is largely comparable within their respective indications. Consequently, treatment selection is increasingly driven by differences in toxicity profiles rather than antitumor effectiveness. Bleomycin exposure distinguishes BEP from other cisplatin-based regimens and underlies its distinct spectrum of adverse effects. Among these, pulmonary toxicity is the most clinically consequential and potentially irreversible complication. Bleomycin-induced pulmonary toxicity is characterized by marked interindividual variability, clinically significant toxicity occurring even at relatively low cumulative exposure despite cumulative dose remaining an important risk factor, and clinically meaningful morbidity and mortality in a predominantly young and otherwise curable population. Accumulating evidence indicates that bleomycin lung injury represents an immune-mediated process driven by dysregulated alveolar-immune crosstalk, innate immune activation, and profibrotic signaling. This review provides a toxicity-focused comparison of standard first-line cisplatin-based regimens used in the curative treatment of metastatic GCTs, particularly BEP, EP, and VIP regimens within the framework of the International Germ Cell Cancer Collaborative Group (IGCCCG) risk stratification and repositions bleomycin pulmonary toxicity as a model of immune-mediated chemotherapy toxicity. We integrate clinical and experimental data on immunopathogenesis and discuss emerging biomarkers of alveolar injury and immune activation, as promising investigational tools that require prospective validation before routine implementation in first-line treatment selection or pulmonary toxicity monitoring.

BackgroundBleomycin-induced pulmonary toxicity remains a significant complication of curative BEP chemotherapy for germ cell tumours, and no validated circulating biomarker enabling individual risk prediction or early detection of lung damage is currently available.MethodsThis exploratory two-centre cohort study enrolled 60 patients with germ cell tumours receiving therapeutic three or four cycles of bleomycin, etoposide, and cisplatin (BEP) chemotherapy. A separate cohort of 18 patients with germ cell tumours receiving adjuvant single-cycle BEP was analysed independently. Plasma and urinary concentrations of surfactant protein D (SP-D) and soluble receptor for advanced glycation end-products (sRAGE) were evaluated before, during, and after BEP chemotherapy in relation to pulmonary toxicity, diffusing capacity of the lung for carbon monoxide (DLCO), pulmonary metastases, renal function, inflammatory indices, grade ≥3 neutropenia, febrile neutropenia, and other non-pulmonary toxicities.ResultsDocumented pulmonary toxicity developed in thirteen patients during or after BEP chemotherapy. Baseline plasma SP-D and sRAGE were not significantly associated with pulmonary toxicity (OR per doubling 0.60 [95% CI 0.27–1.37]; p=0.230 and OR 1.20 [95% CI 0.55–2.62]; p=0.640, respectively). The longitudinal increase of plasma SP-D during BEP chemotherapy was statistically significant (p=0.00018; n=30 with complete measurements), with a more pronounced increase associated with HRCT-confirmed pulmonary toxicity. Higher baseline urinary SP-D was significantly associated with lower post-treatment DLCO (Spearman coefficient rho=−0.609; p=0.001; FDR q=0.014). Plasma SP-D trajectories differed significantly according to the presence of pulmonary metastases (time × metastases interaction: p=0.012; adjusted p=0.027). Maximum plasma sRAGE was statistically significantly associated with neutropenia and/or febrile neutropenia during BEP chemotherapy (OR per doubling 12.13 [95% CI 2.59–56.93]; p=0.0016; FDR q=0.013), with this association remaining significant after correction for multiple testing.ConclusionsIn patients with germ cell tumours receiving BEP chemotherapy, SP-D showed potential as a candidate monitoring biomarker of pulmonary injury during BEP chemotherapy. Greater SP-D increases were associated with HRCT-documented pulmonary toxicity in an exploratory analysis, whereas higher baseline urinary SP-D was significantly associated with impaired diffusing capacity of the lungs after treatment completion. Higher maximum plasma sRAGE was associated with neutropenia and/or febrile neutropenia. These results support prospective validation of compartment-specific monitoring of SP-D and sRAGE during BEP chemotherapy.

Chronic kidney disease (CKD) is associated with chronic low-grade inflammation, but the primary factors triggering this inflammation remain unclear. Extracellular or cell-free DNA (exDNA) originates from virtually all tissues, being released during cell death, and stimulates the innate immune system. Our study was designed as an observational, cross-sectional cohort study of children with CKD (both before and after kidney transplantation) and controls to analyze associations between exDNA, markers of inflammation, and cardiovascular health. Extracellular DNA (total, nuclear, and mitochondrial) was analyzed in plasma using fluorometry and real-time PCR. We found that children with CKD after kidney transplantation had higher concentrations of total and nuclear extracellular DNA (total exDNA and nc_exDNA) in plasma compared to controls. In univariate analysis, levels of interleukin-6 (IL-6), antimicrobial peptide cathelicidin (LL-37), soluble vascular cell adhesion molecule-1 (VCAM-1) and left ventricular mass index (LVMI) were positively correlated with total exDNA and nc_exDNA concentrations. Multivariate analysis revealed LVMI as the only independent variable associated with high levels of both total exDNA and nc_exDNA. We believe that our results contribute new knowledge to the pathogenesis of CKD and its complications and may help identify new treatment targets.

Acrylamide, advanced glycation end products (AGEs), and alpha-dicarbonyls are formed during the thermal processing of foods. Their dietary intake raises potential health concerns. Using food frequency questionnaires on acrylamide-rich Slovak foods, we estimated dietary acrylamide intake in 107 students aged 19-to-30 years and correlated it with salivary, plasma, skin autofluorescence; plasma levels of soluble receptor for advanced glycated end-products, and oxidative status markers (thiobarbituric acid reacting substances, ferric-reducing ability of plasma). No significant relationship was revealed between estimated daily acrylamide intake and analyzed biomarkers. As the extent of exposure to alpha-dicarbonyls and AGEs when consuming acrylamide-rich food remains unknown, we aligned acrylamide intake with that of glyoxal, methylglyoxal, 3-deoxyglucosone, and Nε-carboxymethyllysine, Nε-carboxyethyllysine, or methylglyoxal-derived hydroimidazolone. Correlation coefficients between intakes of acrylamide and alpha-dicarbonyls or AGEs reached 0.7-to-0.8 (p < 0.001, all), but, at individual levels, high intake of acrylamide was not unequivocally associated with high intake of AGEs or alpha-dicarbonyls. Our data suggest that the restriction of dietary AGEs recommended to patients with chronic non-communicable diseases must not simultaneously mitigate acrylamide intake. Nutritional research should explore the potential cumulative or synergistic adverse health effects of concurrent dietary intakes of acrylamide, AGEs, and alpha-dicarbonyls.

The increasing incidence of autism spectrum disorder (ASD) increases the urgency of establishing the mechanism of its development for effective prevention and treatment. ASD's etiology includes genetic predisposition and environmental triggers, both of which can play a role in the changed microbiota. Recent research has proved the impact of maternal microbiota on the neurodevelopment of the child. To investigate the co-play of genetic and microbiota factors in ASD development, we performed fecal microbiota transplantation (FMT) from children with ASD to female Shank3b+/- mice and studied the autism-like symptoms in the male Shank3b-/- and wild-type (WT) offspring. WT animals with prenatal exposure to ASD microbiota had delayed neurodevelopment and impaired food intake behavior, but also elevated plasma leptin concentration and body weight. Shank3b-/- mice after FMT ASD exhibited impaired learning and exacerbated anxiety-like behavior in adulthood. Interestingly, FMT ASD improved learning in adolescent Shank3b-/- mice. Prenatal exposure to ASD microbiota decreased the activity of hypocretin neurons of the lateral hypothalamic area in both genotypes. The combination of genetic predisposition and FMT ASD led to an increased colon permeability, evaluated by zonula occludens (ZO1, ZO3) and claudin factors. These results suggest the effect of parental FMT exposure on shaping offspring behavior in Shank3b-/- mice and the potential of microbiota in the modulation of ASD.

BackgroundGerm cell tumor (GCT) patients with unfavourable response to first-line therapy still lack reliable diagnostic and effective treatment Detailed correlation of total extracellular DNA (ecDNA), other DNA species and endogenous DNase levels in GCT patients' plasma and translational utility remains under- investigated.Study aim and methodsWe determined DNase plasma levels, ecDNA of different subcellular origin and neutrophil extracellular trap (NETs)-associated markers. Next, we determined the associations of these parameters with a level of the DNA damage, immune inflammatory index, specific immune cell subpopulations in a cohort of the 117 GCT patients and 19 matched healthy donors (HDs). Moreover, we investigated how exogenous DNase affects antitumor effect of cisplatin in GCT model of cisplatin-resistant embryonal carcinoma NTERA-2 CisR.ResultsOur data demonstrate that high level of ecDNA and low level of DNase in GCT patients' plasma is associated with significantly worse progression-free survival and overall survival. The level of the plasma ecDNA was five times higher in the GCT patients compared to the HDs. The patients with higher total ecDNA and ncDNA, but not mtDNA, had inferior PFS and OS compared to the patients with lower ecDNA (all p < 0.05). There was an inverse correlation between plasma DNase and ecDNA levels, and between plasma DNase level and clinical outcome. Importantly, combined treatment with cisplatin and human recombinant DNase I delayed growth of the NTERA-2 CisR xenografts and prolonged animal survival. Importantly, Pulmozyme significantly reduced intratumoral microvascular density in our preclinical model.ConclusionOur data confirm the association between low plasma DNase activity and worse overall survival for the first time in GCT patients. This study further validated the prognostic value of total ecDNA in GCT patients. More importantly, our preclinical data substantiated beneficial effect of Pulmozyme combination with cisplatin treatment to improve the therapeutic outcome in refractory disease.

Background: Obesity is a risk factor for several non-communicable diseases and premature death. The Western-type diet, rich in calories and diverse in tastes, smells, and textures, promotes the onset and progression of obesity. We compared the effects of two Western-style palatable obesogenic diets—the cafeteria (CAF) diet, which allows for self-selection of calorie-dense food items consumed by humans, and the fast-food diet (FFD)—composed of a fixed combination of cheeseburgers and fries—on the manifestation of obesity-related complications. Methods: 3-month-old female rats consumed either the control (CTRL), FFD, or CAF diet for 12 months. Body weight was monitored weekly. At the end of the experiment, rats underwent metabolic and behavioral testing. Cardiometabolic markers and those characterizing glycoxidative and carbonyl stress, inflammatory status, and tryptophan metabolism were determined. Results: The CAF rats gain most weight (CTRL: +111 ± 40 g; FFD: +211 ± 77 g; CAF: 316 ± 87 g). CAF feeding produced a classical metabolic syndrome–like profile with severe obesity, insulin resistance, dyslipidemia, and liver steatosis, whereas the FFD model led to moderate obesity with preserved insulin sensitivity but elevated blood pressure and hepatic cholesterol accumulation. Thus, the CAF group developed a severe metabolic syndrome-like pathology assessed as continuous metabolic syndrome z-core (CTRL: −2.3 ± 1.0; FFD: −0.4 ± 1.9; CAF: 3.0 ± 2.4). Despite these differences, both diets promoted neuroinflammation and social deficits, likely mediated through gut microbiota–derived metabolites such as 5-HIAA and indoxyl sulfate. Conclusions: In female rats, self-selected CAF diet drives more severe and distinct pattern of metabolic syndrome-like pathology than a fixed FFD.

BACKGROUND:High heritability (80-90%) of the autism spectrum disorder (ASD) and sex-biased incidence (3-4 times more boys than girls) suggest the roles of genetic predisposition and sex in the etiopathogenesis of the disorder. As ASD is commonly diagnosed in early childhood, most of the research is focused on children, yet animal research predominantly uses adult-aged animals. The effect of aging on the core and secondary ASD symptomatology is understudied, both in patients and animal models of ASD. METHODS:To investigate the effect of aging on sociability, repetitive behavior, exploration, locomotor activity, anxiety-like behavior, and object-avoidance behavior, behavioral phenotyping was conducted in Shank3B-/- (n = 67) and C57BL/6J wild-type (WT, n = 68) mice of both sexes (female n = 70, male n = 65) in adolescence (1-2 months of age, n = 42), adulthood (3-6 months of age, n = 40), and old age (12-18 months of age, n = 53). RESULTS:Social deficits were observed only in old Shank3B-/- males. Anxiety-like behavior peaked in adulthood with Shank3B-/- mice roughly 20% more anxious than controls. Repetitive grooming and object-induced avoidance behavior were twice more prevalent in Shank3B-/- mice consistently across the lifespan. Hypoactivity (20% less distance moved) and reduced exploration (30% less rearing behavior) were recorded in Shank3B-/- mice and were more prevalent in female animals (30% less rearing behavior). Data were analyzed using the Three-way ANOVA (genotype, sex, age), followed by a posthoc Bonferroni correction to compare respective subgroups. CONCLUSIONS:Present study shows that aging affects ASD-like phenotype in the Shank3B-mutant mouse model, even though the effect size seems to be small. The mechanisms underlying these partially sex-specific effects should be the subject of further research with potential translational implications.

Periodontitis is a chronic inflammatory disease. We have previously shown that salivary DNA is higher in patients with periodontitis. Neutrophil extracellular traps (NETs) are involved in the pathogenesis of chronic inflammatory diseases. The objective of this case-control study was to compare patients with periodontitis and healthy controls regarding the salivary concentrations of extracellular DNA and NET components. Unstimulated saliva samples were collected from 49 patients with periodontitis and 71 controls before an oral examination. Salivary extracellular DNA was isolated and quantified fluorometrically and using PCR. NET-associated markers were assessed using ELISA. We have found significantly higher concentrations of salivary extracellular DNA in samples from periodontitis patients (five-times higher for supernatant and three times for pellet). Our results show that patients also have three-times-higher salivary nucleosomes and NET-associated enzymes-myeloperoxidase and neutrophil elastase (both two-times higher). Neutrophil elastase and salivary DNA in the pellet correlated positively with the pocket depth/clinical attachment level in periodontitis patients (r = 0.31-weak correlation; p = 0.03 and r = 0.41-moderate correlation, p = 0.004). Correlations between salivary extracellular DNA and NET enzymes were positive and significant. Based on our results, the higher salivary extracellular DNA in periodontitis seems to be related to components of NETs, albeit with weak to moderate correlations indicating that NETs are produced in periodontitis and can play a role in its pathogenesis similarly to other inflammatory diseases. Further studies should prove this assumption with potential diagnostic and therapeutic consequences.

Background: The etiopathogenesis of rheumatoid arthritis (RA) is unclear [1]. Extracellular DNA (ecDNA) originating mainly from neutrophil extracellular traps (NETs) induces inflammation and is higher in patients with RA [2, 3]. However, whether it is directly involved in the pathogenesis of the disease is not known. Objectives: Our aim was to prove whether anti-inflammatory treatment in RA patients affects plasma ecDNA and whether its enzymatic cleavage modulates the course of collage-induced arthritis in mice. Methods: Clinical status was assessed, and plasma samples were collected from patients treated with biologic disease-modifying anti-rheumatic drugs (bDMARDs) before treatment and 3 and 6 months after treatment induction. Baseline plasma collection was performed in rheumatoid arthritis patients with high disease activity (DAS28 > 5,1) treated with conventional synthetic disease-modifying anti-rheumatic drugs (csDMARDs), and later 3 and 6 months after they start the treatment with biologic DMARDs (bDMARDs). Plasma concentrations of NETs components were quantified using real time PCR and ELISA. Adult male DBA/IJ mice with collagen antibody-induced arthritis (CAIA) were treated with DNase I (10 mg/kg) or Cl-amidine (2 mg/kg). Arthritis score, the temperature of paws, swelling of paws and the extent of inflammation quantified using bioluminescence were assessed. Results: In the clinical study, the anti-inflammatory treatment led to a significant decrease of C-reactive protein, DAS28 and sedimentation rate by 60%, 43% and 40% resp. already after three months. EcDNA decreased by 31% (p<0.05) after 6 months of treatment. In mice, treatment with DNase I had no effect on the progress of the CAIA model. Mice treated with the NETs production inhibitor Cl-amidine had a lower arthritis score in comparison to untreated CAIA controls (0 vs. 6 arthritis score points, p<0.05) (Figure 1). Conclusion: Treatment with bDMARDs reduces concentration of NETs components including ecDNA. No effect of DNase I and reduced arthritis severity with the peptidylarginine deiminase inhibitor Cl-amidine suggest that NETs rather than ecDNA are involved in the etiopathogenesis of RA. This is compatible with the early presence of antibodies against citrullinated peptides originating from NETs in RA. Taking into account the differences between the disease and its animal model, our results show that NETs production could be a potential novel treatment target. Further studies are needed to find the causes of NETs production. REFERENCES: [1] Smolen, J.S., et al., Rheumatoid arthritis. Nat Rev Dis Primers, 2018. 4: p. 18001.[2] Apel, F., A. Zychlinsky, and E.F. Kenny, The role of neutrophil extracellular traps in rheumatic diseases. Nature Reviews Rheumatology, 2018. 14(8): p. 467-475.[3] Boyapati, R.K., et al., Advances in the understanding of mitochondrial DNA as a pathogenic factor in inflammatory diseases. F1000Res, 2017. 6: p. 169. Acknowledgements: APVV-22-0554 Disclosure of Interests: None declared.Figure 1Dynamic of extracellular DNA concentration and arthritis score after the application of DNase I and NETs inhibitor.

Background/Objectives: Sepsis is characterized by a dysregulated immune response to infection and is associated with high lethality. Extracellular DNA (ecDNA) has drawn significant interest as a damage-associated molecular pattern because of its potential involvement in the pathophysiology of sepsis. Methods: In this study, we examined the ecDNA concentration in 27 adult patients admitted to the intensive care unit. Fluorometry and quantitative PCR were used for the assessment of ecDNA. In addition, deoxyribonuclease activity was measured as a potential modulator of ecDNA. Results: Our findings reveal nearly 5-fold higher concentrations of ecDNA in non-survivors, suggesting its potential as a prognostic indicator for sepsis outcomes on day 7. Interestingly, the subcellular origin of ecDNA was similar between patients diagnosed with systemic inflammatory response syndrome, sepsis, and septic shock. Deoxyribonuclease activity, implicated in the cleavage of ecDNA, was comparable across all patient groups. Conclusions: To establish the prognostic value of ecDNA as a biomarker, further investigations within a larger patient cohort are needed. Nevertheless, our results suggest that high ecDNA in sepsis patients represents a negative prognostic biomarker.

The collection and examination method of vaginal smears is the standard for the determination of ovulation or phases of the estrous cycle of rodents used in research. However, this method is time consuming and may not be amenable to continual monitoring of a large number of animals. Infrared thermography has recently emerged as a noninvasive technique that requires relatively little handling of animals. The body temperature of rodents has been shown to correlate with the ocular surface temperature. This study aimed to evaluate the use of thermographic monitoring of the ocular surface for the identification of estrus in rats. Vaginal smears were collected from female Wistar rats (n = 22) for 14 consecutive days. Core body temperature was estimated by measuring ocular surface temperature using a thermal camera; vaginal temperature was measured using a digital thermometer. Average temperatures were calculated for each rat for each phase of the estrous cycle. The highest core body and vaginal temperature were measured during the estrus phase (37.2 ± 0.6 °C and 37.7 ± 0.6 °C, respectively). The temperatures then fell as the rat entered the diestrus phase (36.8 ± 0.5 °C and 37 ± 0.5 °C). The core body temperature was positively correlated with vaginal temperature (r = 0.697, P < 0.001). In conclusion, thermography is a less invasive method of determining estrus in rats as compared with vaginal smear collection. However, thermography is less accurate and requires at least a 12-d period of measurement.

Increasing evidence of sexual dimorphism in the pathophysiology of metabolic complications caused by sex steroids is under investigation. The gut microbiota represents a complex microbial ecosystem involved in energy metabolism, immune response, nutrition acquisition, and the health of host organisms. Gender-specific differences in composition are present between females and males. The purpose of this study was to use cross-sex fecal microbiota transplantation (FMT) for the detection of sex-dependent metabolic, hormonal, and gut microbiota changes in female and male recipients. Healthy non-obese female and male Wistar rats were divided into donor, same-sex, and cross-sex recipient groups. After a 30-day period of FMT administration, biochemical markers (glucose and lipid metabolism) and sex hormones were measured, and the gut microbiota was analyzed. The cross-sex male recipients displayed a significantly lower testosterone concentration compared to the males that received same-sex FMT. Sex-dependent changes caused by cross-sex FMT were detected, while several bacterial taxa correlated with plasma testosterone levels. This study represents the first to study the effect of cross-sex changes in the gut microbiome concerning metabolic and hormonal changes/status in adult non-obese Wistar rats. Herein, we present cross-sex FMT as a potential tool to modify sex-specific pathologies.