DNA in fresh urine supernatant is not affected by additional centrifugation and is protected against deoxyribonuclease.

Urinary DNA is widely studied as a non-invasive marker for monitoring of kidneys after transplantation or the progression of urinary tract tumors. The quantity of urinary DNA especially of mitochondrial origin has been reported to mirror kidney damage in various renal diseases and their models. Processing of samples might affect urinary DNA concentrations but the details are not clear. Samples of urine were collected from fifteen healthy volunteers. DNA was extracted from the whole urine, but also from the supernatant after centrifugation at 1600 g and 16000 g. In addition, we have analyzed the DNA in the microparticles in the pellet after the last spin. DNA was measured using fluorometry and real time PCR targeting nuclear and mitochondrial sequences. Addition of deoxyribonuclease to aliquots of samples enabled the characterization of DNA protection. Centrifugation at 1600 g decreased the concentration of extracted DNA by 66% at least in samples with higher DNA in whole urine. Interestingly, the additional spin at 16000 g did not result in a significant decrease in DNA concentration in the supernatant despite detectable microparticle-associated DNA. Deoxyribonuclease decreases total and nuclear DNA by 26% and 31% in whole urine. The majority of urinary mitochondrial DNA seems to be protected against deoxyribonuclease. Our results indicate high variability in urinary DNA even in healthy probands. Extracellular urinary DNA is partially bound to cell debris or microparticles, but a considerable part is still in the supernatant and is protected against cleavage. Further research should identify the nature of the protection, especially for mitochondrial DNA. Better understanding of the biology of urinary DNA should help its clinical interpretation.

Deoxyribonuclease activity negative correlates with extracellular DNA in uncomplicated singleton pregnancies in the third trimester.

OBJECTIVES: It is not clear, which factors affect extracellular DNA (ecDNA) concentrations in healthy women with singleton uncomplicated pregnancies, although deoxyribonucleases (DNases) are hypothesized to be responsible for the cleavage of plasma ecDNA. The aim of this study was to analyze potential determinants of total ecDNA including plasma DNase activity. METHODS: Plasma samples were collected from 48 healthy women with singleton uncomplicated pregnancies in the third trimester (gestation week 37). DNA was isolated and quantified using fluorometry and real time PCR. DNase activity was assessed using the single radial enzyme-diffusion method. RESULTS: Neither ecDNA, nor DNase activity were affected by maternal age or BMI. DNase activity negatively correlated with total plasma ecDNA (r=-0.40, p=0.007). Similar associations were found for ecDNA of nuclear and mitochondrial origin, but not with fetal DNA quantified using Y-targeted PCR in male fetus-bearing pregnancies. CONCLUSIONS: The role of plasma ecDNA of fetal and maternal origin is studied in the pathogenesis of pregnancy-complications. The results indicate that plasma DNase activity could negatively regulate ecDNA concentrations and should, thus, be analyzed in preeclampsia, preterm birth and other ecDNA-related pregnancy complications.

Formyl Peptide Receptor-1 Blockade Prevents Receptor Regulation by Mitochondrial Danger-Associated Molecular Patterns and Preserves Neutrophil Function After Trauma.

OBJECTIVES: Trauma predisposes to systemic sterile inflammation (systemic inflammatory response syndrome) as well as infection, but the mechanisms linking injury to infection are poorly understood. Mitochondrial debris contains formyl peptides. These bind formyl peptide receptor-1, trafficking neutrophils to wounds, initiating systemic inflammatory response syndrome, and wound healing. Bacterial formyl peptides, however, also attract neutrophils via formyl peptide receptor-1. Thus, mitochondrial formyl peptides might suppress neutrophils antimicrobial function. Also, formyl peptide receptor-1 blockade used to mitigate systemic inflammatory response syndrome might predispose to sepsis. We examined how mitochondrial formyl peptides impact neutrophils functions contributing to antimicrobial responses and how formyl peptide receptor-1 antagonists affect those functions. DESIGN: Prospective study of human and murine neutrophils and clinical cohort analysis. SETTING: University research laboratory and level 1 trauma center. PATIENTS: Trauma patients, volunteer controls. ANIMAL SUBJECTS: C57Bl/6, formyl peptide receptor-1, and formyl peptide receptor-2 knockout mice. INTERVENTIONS: Human and murine neutrophils functions were activated with autologous mitochondrial debris, mitochondrial formyl peptides, or bacterial formyl peptides followed by chemokines or leukotrienes. The experiments were repeated using formyl peptide receptor-1 antagonist cyclosporin H, "designer" human formyl peptide receptor-1 antagonists (POL7178 and POL7200), or anti-formyl peptide receptor-1 antibodies. Mouse injury/lung infection model was used to evaluate effect of formyl peptide receptor-1 inhibition. MEASUREMENTS AND MAIN RESULTS: Human neutrophils cytosolic calcium, chemotaxis, reactive oxygen species production, and phagocytosis were studied before and after exposure to mitochondrial debris, mitochondrial formyl peptides, and bacterial formyl peptides. Mitochondrial formyl peptide and bacterial formyl peptides had similar effects on neutrophils. Responses to chemokines and leukotrienes were suppressed by prior exposure to formyl peptides. POL7200 and POL7178 were specific antagonists of human formyl peptide receptor-1 and more effective than cyclosporin H or anti-formyl peptide receptor-1 antibodies. Formyl peptides inhibited mouse neutrophils responses to chemokines only if formyl peptide receptor-1 was present. Formyl peptide receptor-1 blockade did not inhibit neutrophils bacterial phagocytosis or reactive oxygen species production. Cyclosporin H increased bacterial clearance in lungs after injury. CONCLUSIONS: Formyl peptides both activate and desensitize neutrophils. Formyl peptide receptor-1 blockade prevents desensitization, potentially both diminishing systemic inflammatory response syndrome and protecting the host against secondary infection after tissue trauma or primary infection.

Sex, Age, and Bodyweight as Determinants of Extracellular DNA in the Plasma of Mice: A Cross-Sectional Study.

Extracellular DNA (ecDNA) is studied as a possible biomarker, but also as a trigger of the immune responses important for the pathogenesis of several diseases. Extracellular deoxyribonuclease (DNase) activity cleaves ecDNA. The aim of our study was to describe the interindividual variability of ecDNA and DNase activity in the plasma of healthy mice, and to analyze the potential determinants of the variability, including sex, age, and bodyweight. In this experiment, 58 adult CD1 mice (41 females and 31 males) of a variable age (3 to 16 months old) and bodyweight (females 25.7 to 52.1 g, males 24.6 to 49.6 g) were used. The plasma ecDNA was measured using a fluorometric method. The nuclear ecDNA and mitochondrial ecDNA were quantified using real-time PCR. The deoxyribonuclease activity was assessed using the single radial enzyme diffusion method. The coefficient of variance for plasma ecDNA was 139%, and for DNase 48%. Sex differences were not found in the plasma ecDNA (52.7 ± 73.0 ηg/mL), but in the DNase activity (74.5 ± 33.5 K.u./mL for males, and 47.0 ± 15.4 K.u./mL for females). There were no associations between plasma ecDNA and bodyweight or the age of mice. Our study shows that the variability of plasma ecDNA and DNase in adult healthy mice is very high. Sex, age, and bodyweight seem not to be major determinants of ecDNA variability in healthy mice. As ecDNA gains importance in the research of several diseases, it is of importance to understand its production and cleavage. Further studies should, thus, test other potential determinants, taking into account cleavage mechanisms other than DNase.

Cell-free DNA: the role in pathophysiology and as a biomarker in kidney diseases.

Cell-free DNA (cfDNA) is present in various body fluids and originates mostly from blood cells. In specific conditions, circulating cfDNA might be derived from tumours, donor organs after transplantation or from the foetus during pregnancy. The analysis of cfDNA is mainly used for genetic analyses of the source tissue -tumour, foetus or for the early detection of graft rejection. It might serve also as a nonspecific biomarker of tissue damage in critical care medicine. In kidney diseases, cfDNA increases during haemodialysis and indicates cell damage. In patients with renal cell carcinoma, cfDNA in plasma and its integrity is studied for monitoring of tumour growth, the effects of chemotherapy and for prognosis. Urinary cfDNA is highly fragmented, but the technical hurdles can now be overcome and urinary cfDNA is being evaluated as a potential biomarker of renal injury and urinary tract tumours. Beyond its diagnostic application, cfDNA might also be involved in the pathogenesis of diseases affecting the kidneys as shown for systemic lupus, sepsis and some pregnancy-related pathologies. Recent data suggest that increased cfDNA is associated with acute kidney injury. In this review, we discuss the biological characteristics, sources of cfDNA, its potential use as a biomarker as well as its role in the pathogenesis of renal and urinary diseases.

Amniotic fluid cell-free DNA in preterm prelabor rupture of membranes.

INTRODUCTION: We evaluated the levels of cell-free nuclear DNA (nDNA) and cell-free mitochondrial DNA (mtDNA) in the amniotic fluid supernatant from pregnancies complicated by preterm prelabor rupture of membranes (PPROM) based on evidence of microbial invasion of the amniotic cavity (MIAC) and/or intra-amniotic inflammation (IAI). MATERIAL AND METHODS: A total of 155 women with PPROM were included in this study. Amniotic fluid samples were obtained by transabdominal amniocentesis. The levels of cell-free nDNA and mtDNA in the amniotic fluid supernatant were assessed and quantified by real-time polymerase chain reaction. RESULTS: The levels of cell-free nDNA and mtDNA were higher in women with MIAC and IAI than in women without these conditions (nDNA: with MIAC: median 3.9 × 104 genome equivalent [GE]/mL vs without MIAC: median 1.2 × 104  GE/mL, with IAI: median: 5.3 × 104  GE/mL vs without IAI: median 1.2 × 104  GE/mL; mtDNA: with MIAC: median 9.2 × 105  GE/mL vs without MIAC: median 2.5 × 105  GE/mL, with IAI: median 1.1 × 106  GE/mL vs without IAI: median 2.5 × 105 ; all P values ≤ 0.01). Women with the microbial-associated IAI showed the highest levels of cell-free nDNA and mtDNA. CONCLUSIONS: Cell-free nDNA and mtDNA are constituents of the amniotic fluid supernatant from PPROM pregnancies. Both cell-free nDNA and mtDNA are involved in the intra-amniotic inflammatory response in women with PPROM.

Anti-cytokine therapy and plasma DNA in patients with rheumatoid arthritis.

BACKGROUND: Extracellular DNA (ecDNA) is increased in inflammation and it also induces inflammation. In patients with rheumatoid arthritis (RA), plasma ecDNA is higher than in healthy controls. Due to low specificity, it cannot be used for screening, but it might be useful for monitoring and prognosis of therapy success. The effect of treatment with biological disease-modifying antirheumatic drugs (bDMARDs) on plasma ecDNA in RA patients with regards to its subcellular origin has not been analyzed yet. The aim of this study was to describe the effects of bDMARDs on plasma ecDNA and its nuclear (nDNA) and mitochondrial (mtDNA) fractions in patients with RA. METHODS: Plasma samples of 32 patients with RA were collected before, as well as 3 and 6 months after starting the treatment with bDMARDs. Total plasma ecDNA was quantified fluorometrically. The subcellular origin of ecDNA was assessed using real time PCR. Treatment success was monitored using DAS28 and C-reactive protein (CRP). RESULTS: The clinical status of patients improved. Both DAS28 and CRP decreased by 52 and 73% after 3 months of treatment. Plasma ecDNA decreased significantly only after 6 months (by 26%). Real-time PCR showed that both, nDNA and mtDNA decreased by 63 and by 45% after 6 months. CONCLUSION: Treatment with bDMARDs decreases plasma ecDNA of both nuclear and mitochondrial origin. Dynamics of ecDNA is slower than dynamics of standard clinical markers. Therefore, it is likely to be not useful for monitoring of the disease progress, at least for RA.

Deoxyribonuclease partially ameliorates thioacetamide-induced hepatorenal injury.

Several recent studies have shown that liver injury is associated with the release of DNA from hepatocytes. This DNA stimulates innate immunity and induces sterile inflammation, exacerbating liver damage. Similar mechanisms have been described for acute renal injury. Deoxyribonuclease degrades cell-free DNA and can potentially prevent some of the induced tissue damage. This study analyzed the effects of thioacetamide-induced hepatorenal injury on plasma DNA in rats. Plasma DNA of both nuclear and mitochondrial origin was higher in thioacetamide-treated animals. Administration of deoxyribonuclease resulted in a mild, nonsignificant decrease in total plasma DNA and plasma DNA of mitochondrial origin but not of nuclear origin. This was accompanied by a decrease in bilirubin, creatinine, and blood urea nitrogen as markers of renal function. In conclusion, the study confirmed the hepatotoxic and nephrotoxic effect of thioacetamide. The associated increase in cell-free DNA seems to be involved in hepatorenal pathogenesis because treatment with deoxyribonuclease resulted in a partial prevention of hepatorenal injury. Further experiments will focus on the effects of long-term treatment with deoxyribonuclease in other clinically more relevant models. Clinical studies should test endogenous deoxyribonuclease activity as a potential risk determinant for kidney or liver failure.NEW & NOTEWORTHY Thioacetamide-induced hepatorenal injury resulted in higher plasma cell-free DNA. Deoxyribonuclease decreased average cell-free DNA of mitochondrial origin but not nuclear origin. Deoxyribonuclease partially prevented hepatorenal injury in rats.

Paediatric Home Parenteral Nutrition in the Czech Republic and Its Development: Multicentre Retrospective Study 1995-2011.

BACKGROUND: Treatment quality and outcomes of paediatric home parenteral nutrition (HPN) program during its development in the Czech Republic. METHODS: A retrospective study of patients receiving HPN from May 1995 till June 2011. RESULTS: Sixty-six patients were treated in 8 centres. In 48 patients, long-term PN began in the first year of life and in 35 of them in the first month. Sixty children had gastrointestinal and 6 had non-gastrointestinal disease. In a majority of the patients, the Broviac catheter was used. Thirty-two (48.5%) patients were weaned from PN after 1-117 months, 21 (32.8%) continued on HPN after 7-183 months, and 13 (19.7%) patients died, all on PN. The mortality in patients with primary gastrointestinal disease was significantly lower than in patients with non-gastrointestinal disease. Thirty-one paediatric patients were receiving HPN for 14,480 catheter days in 2009-2010. Fourteen patients had 23 Catheter Related Blood Stream Infections (CRBSI) episodes. The incidence of CRBSI in 2009-2010 was 1.58/1,000 catheter days. CONCLUSION: Submitted data showed that even in the absence of expert centres, patient care may achieve results comparable to countries with well-developed HPN program. A majority of Czech HPN patients are at present treated in specialized centres, following the most desirable pattern of care.

Relationship between circulating tumor cells, blood coagulation, and urokinase-plasminogen-activator system in early breast cancer patients.

Cancer is a risk factor for venous thromboembolism (VTE) and plasma d-dimer (DD) and tissue factor (TF) are established VTE associated markers. Circulating tumor cells (CTCs) are associated with the risk of VTE in metastatic breast cancer. This study aimed to correlate CTCs, blood coagulation and the urokinase plasminogen activator (uPA) system in primary breast cancer (PBC) patients. This prospective study included 116 PBC patients treated by primary surgery. CTCs were detected by quantitative RT-PCR assay for expression of epithelial (CK19) or epithelial-mesenchymal transition (EMT) genes (TWIST1, SNAIL1, SLUG, ZEB1, FOXC2). Plasma DD, TF, uPA system proteins were detected by enzyme-linked immunosorbent assays, while expressions of uPA system in surgical specimens were evaluated by immunohistochemistry. CTCs were detected in 27.6% patients. Patients with CTCs had a significantly higher mean plasma DD (ng/mL) than those of patients without CTCs (632.4 versus 365.4, p = 0.000004). There was no association between plasma TF and CTCs. Epithelial CTCs exhibit higher expression of uPA system genes compared to EMT_CTCs. Patients with CTCs had higher plasma uPA proteins than those of patients without CTCs; there was no correlation between tissue expression of uPA system, CTCs, DD or TF levels. In multivariate analysis CTCs and patients age were independent factors associated with plasma DD. We found association between plasma DD and CTCs indicating a potential role for activation of the coagulation cascade in the early metastatic process. CTCs could be directly involved in coagulation activation or increased CTCs could be marker of aggressive disease and increased VTE risk.

Vanishing twin as a potential source of bias in non-invasive fetal sex determination: a case report.

Detection of fetal sex based on fetal DNA present in maternal plasma is already in clinical use. Here we present a case of false positivity during the first trimester which may be attributable to a vanishing twin. The presence of Y-chromosome-specific sequences is used as a marker to indicate a male fetus and the absence of a female fetus. Fetal sex determination was conducted in a pregnant woman at gestational week 10. The sample was positive in all triplicates. Ultrasonography at gestational week 20 revealed female sex. Analysis of sample taken at gestational week 22 indicated a female fetus. According to recently published meta-analyses, non-invasive prenatal diagnosis of fetal sex has high sensitivity and specificity values. Nevertheless, false negative and false positive cases occur. Future studies focusing on the dynamics of fetal DNA are needed. Vanishing twin might be one of the possible causes of false positivity in fetal sex determination.

Pre-Analytical Factors Affecting Extracellular DNA in Saliva.

Salivary DNA is widely used for genetic analyses because of its easy collection. However, its extracellular fraction in particular, similar to the extracellular DNA (ecDNA) in plasma, could be a promising biomarker for oral or systemic diseases. In contrast to genetics, the quantity of salivary ecDNA is of importance and can be affected by the pre-analytical processing of samples, but the details are not known. The aim of our study was to analyze the effects of centrifugation and freezing of saliva on the concentration of ecDNA in saliva. Fifteen healthy volunteers, free of any known systemic or oral diseases, were asked to collect unstimulated saliva samples. Aliquots were centrifuged at 1600× g and frozen or directly processed. The fresh or thawed cell-free saliva samples underwent subsequent centrifugation at 16,000× g. The supernatants were used for DNA isolation and quantification using fluorometry and real-time PCR. While freezing had minimal effects on the salivary ecDNA concentration, another centrifugation step decreased ecDNA considerably in both fresh and frozen samples (by 97.8% and 98.4%, respectively). This was mirrored in the quantitative PCR targeting a nuclear (decrease by 93.5%) and mitochondrial (decrease by 97.7%) ecDNA sequence. In conclusion, in this first study focusing on the technical aspects of salivary ecDNA quantitation, we show that, regardless of its subcellular origin, the concentration of ecDNA in saliva is mainly affected by additional centrifugation and not by the freezing of centrifuged cell-free saliva samples. This suggests that most salivary ecDNA likely is associated with cell debris and apoptotic bodies. Which fraction is affected by a particular disease should be the focus of further targeted studies.

Extracellular DNA and Markers of Neutrophil Extracellular Traps in Saliva from Patients with Periodontitis-A Case-Control Study.

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.

Effect of SARS-CoV-2 Infection and COVID-19 Vaccination on Oxidative Status of Human Placenta: A Preliminary Study.

Infection with SARS-CoV-2 during pregnancy increases the risk of pregnancy complications associated with inflammation, which could lead to oxidative stress in the placenta. Whether vaccination against COVID-19 has any effect is unclear. This study aimed to analyze the effects of SARS-CoV-2 infection and vaccination against COVID-19 during pregnancy on oxidative stress in the placenta and on extracellular DNA (ecDNA) in umbilical cord plasma. Placenta samples from healthy uninfected and unvaccinated control patients who recovered from COVID-19 and women vaccinated against COVID-19 during pregnancy were collected. Biomarkers of oxidative damage and antioxidant capacity were assessed in the placenta homogenates. EcDNA and deoxyribonuclease activity were quantified in umbilical cord plasma using real-time PCR and the single radial enzyme diffusion method, respectively. Markers of oxidative damage to lipids and proteins as well as antioxidant capacity in the placenta did not differ between the study groups. No differences were observed in total, nuclear or mitochondrial ecDNA, or deoxyribonuclease activity in the umbilical cord plasma. Taking into account the limits of a small observational study, our results suggest that the infection with SARS-CoV-2 and vaccination against COVID-19 do not induce any major disturbances in the balance between the production of free radicals and antioxidant activity in the placenta. This is in line with the minor effects on fetal outcomes and ecDNA as a suggested marker of fetal well-being.

Metabolic Effects of Anti-TNF-α Treatment in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is associated with high cardiovascular mortality. It is not clear whether the metabolic consequences of chronic inflammation are involved. Biological disease-modifying anti-rheumatic drugs (bDMARDs) are highly efficient in the treatment of inflammation in RA. In this study, we aimed to describe the metabolic effects of anti-TNF-α treatment in RA patients. The clinical status of 16 patients was assessed using disease activity score-28 (DAS28) and C-reactive protein (CRP). Plasma samples were collected before treatment with anti-TNF-α treatment as well as after three and six months of treatment. Markers of lipid and glucose metabolism, as well as renal biomarkers, were assessed using standard biochemistry. ELISA was used for the quantification of insulin, leptin, and adiponectin. Although fasting insulin decreased by 14% at the end of the study, most of the analyzed parameters did not show any statistically or clinically significant dynamics. The exception was total bilirubin and cholesterol, which increased by 53% and 14%, respectively, after six months of treatment with anti-TNF-α treatment. Anti-TNF-α treatment did not induce major metabolic changes despite the strong anti-inflammatory and clinical symptoms of RA. Further studies will show whether longer observations are required for the detection of the metabolic effects of the anti-inflammatory treatment. Additional research is needed to understand the observed effect of bilirubin as an important endogenous antioxidant.

Mitochondria-induced formation of neutrophil extracellular traps is enhanced in the elderly via Toll-like receptor 9.

Neutrophil extracellular traps are potent antimicrobial weapons; however, their formation during sterile inflammation is detrimental, and the mechanism of induction is still unclear. Since advanced age is the primary clinical risk factor for poor outcomes in inflammatory diseases, we hypothesized that sterile stimuli, represented by mitochondria, would induce neutrophil extracellular trap formation in an age-dependent manner. Therefore, we analyzed induction of neutrophil extracellular traps in patients grouped according to age or immune status and observed that neutrophils from elderly patients responded to the presence of mitochondria with enhanced neutrophil extracellular trap formation. These neutrophil extracellular traps were also found to be more oxidized and exhibited higher resistance to DNase I degradation. Additionally, a higher concentration of residual neutrophil extracellular traps was detected in the plasma of the elderly. This plasma was capable of priming neutrophils through TLR9-mediated signaling, leading to further neutrophil extracellular trap formation, which was successfully inhibited with chloroquine. Finally, in a mouse model of mitochondria-induced acute lung injury, we observed that neutrophils from aged mice displayed impaired chemotactic activity but exhibited a trend of higher neutrophil extracellular trap formation. Thus, we propose that residual neutrophil extracellular traps circulating in the elderly preactivate neutrophils, making them more prone to enhanced neutrophil extracellular trap formation when exposed to mitochondria during sterile inflammation. Further investigation is needed to determine whether this vicious circle could be a suitable therapeutic target.

Sex differences in long-term effects of collagen-induced arthritis in middle-aged mice.

Introduction: Rheumatoid arthritis (RA) is a chronic inflammatory disorder with high prevalence among middle-aged women. Collagen-induced arthritis (CIA) is the most widely used animal model of RA, however, sex differences and long-term effects of CIA in mice are poorly described in the literature. Aim: Therefore, the present study aimed to analyze the long-term effects of CIA on the joints of middle-aged mice of both sexes and to describe potential sex differences. Materials and methods: CIA was induced in middle-aged DBA/1J mice by immunization with bovine type II collagen and complete Freund's adjuvant. Saline was administered to control mice. Arthritis score assessment, plethysmometry, and thermal imaging of the joints were performed weekly for 15 weeks. Locomotor activity, micro-computed tomography, joint histology and biochemical analyses were performed at the end of the experiment. Results: Our results indicate a similar prevalence of arthritis in both sexes of mice-67% (8/12) of females and 89% (8/9) males with an earlier onset in males (day 14 vs. day 35). After the arthritis scores peaked on day 56 for males and day 63 for females, they steadily declined until the end of the experiment on day 105. A similar dynamics was observed in paw volume and temperature analyzing different aspects of joint inflammation. Long-term consequences including higher proteinuria (by 116%), loss of bone density (by 33.5%) and joint damage in terms of synovial hyperplasia as well as bone and cartilage erosions were more severe in CIA males compared to CIA females. There were no significant differences in locomotor activity between CIA mice and CTRL mice of any sex. Conclusion: This is the first study to describe the long-term effects of the CIA model in terms of sex differences in DBA/1J mice. Our results indicate sex differences in the dynamics, but not in the extent of arthritis. An earlier onset of arthritis and more severe consequences on joints, bones and kidneys were found in males. The underlying immune pathomechanisms responsible for the limited duration of the arthritis symptoms and the opposite sex difference in comparison to RA patients require further investigation.

Cytomegalovirus durably primes neutrophil oxidative burst.

Cytomegalovirus (CMV) is a ubiquitous herpes virus that infects most humans, thereafter persisting lifelong in tissues of the host. It is a known pathogen in immunosuppressed patients, but its impact on immunocompetent hosts remains less understood. Recent data have shown that CMV leaves a significant and long-lasting imprint in host immunity that may confer some protection against subsequent bacterial infection. Such innate immune activation may come at a cost, however, with potential to cause immunopathology. Neutrophils are central to many models of immunopathology, and while acute CMV infection is known to influence neutrophil biology, the impact of chronic CMV infection on neutrophil function remains unreported. Using our murine model of CMV infection and latency, we show that chronic CMV causes persistent enhancement of neutrophil oxidative burst well after resolution of acute infection. Moreover, this in vivo priming of marrow neutrophils is associated with enhanced formyl peptide receptor expression, and ultimately constitutive c-Jun N-terminal kinase phosphorylation and enhanced CD14 expression in/on circulating neutrophils. Finally, we show that neutrophil priming is dependent on viral load, suggesting that naturally infected human hosts will show variability in CMV-related neutrophil priming. Altogether, these findings represent a previously unrecognized and potentially important impact of chronic CMV infection on neutrophil responsiveness in immunocompetent hosts.

Prognostic role of plasma vitamin D and its association with disease characteristics in germ cell tumours.

Background: Testicular cancer is the most common malignancy among young men. Vitamin D has pluripotent effects on cancer pathogenesis and plays a role in the metastatic cascade. The aim of this study is to analyze plasma vitamin D in association with clinico-pathological findings and prognosis in patients with germ-cell tumors (GCTs). Methods: This study included 120 newly diagnosed and/or relapsed GCT patients treated from April 2013 to July 2020, for whom plasma was available in the biobank. Blood samples were drawn the 1st chemotherapy cycle as well as before the 2nd cycle. Plasma vitamin D was measured using ELISA and correlated with disease characteristics and the outcome. For survival analysis, the cohort was dichotomized into "low" and "high" based on median vitamin D. Results: There was no significant difference in vitamin D plasma levels between healthy donors and GCT patients (p = 0.71). Vitamin D level was not associated with disease characteristics except for brain metastases, where patients with brain metastases had a vitamin D level that was 32% lower compared to patients without brain metastases, p = 0.03. Vitamin D was also associated with response to chemotherapy, with an approximately 32% lower value in patients with an unfavorable response compared to a favorable response, p = 0.02. Moreover, low plasma levels of vitamin D were significantly associated with disease recurrence and inferior progression-free survival (PFS), but not with overall survival (OS) (HR = 3.02, 95% CI 1.36-6.71, p = 0.01 for PFS and HR = 2.06, 95% CI 0.84-5.06, p = 0.14 for OS, respectively). Conclusion: Our study suggests the prognostic value of pretreatment vitamin D concentrations in GCT patients. Low plasma vitamin D was associated with an unfavorable response to therapy and disease recurrence. However, it remains to be determined whether the biology of the disease confirms a causative role for low vitamin D and whether its supplementation affects the outcome.