Genomic testing for copy number and single nucleotide variants in spermatogenic failure.

PURPOSE: To identify clinically significant genomic copy number (CNV) and single nucleotide variants (SNV) in males with unexplained spermatogenic failure (SPGF). MATERIALS AND METHODS: Peripheral blood DNA from 97/102 study participants diagnosed with oligozoospermia, severe oligozoospermia, or non-obstructive azoospermia (NOA) was analyzed for CNVs via array comparative genomic hybridization (aCGH) and SNVs using whole-exome sequencing (WES). RESULTS: Of the 2544 CNVs identified in individuals with SPGF, > 90% were small, ranging from 0.6 to 75 kb. Thirty, clinically relevant genomic aberrations, were detected in 28 patients (~ 29%). These included likely diagnostic CNVs in 3/41 NOA patients (~ 7%): 1 hemizygous, intragenic TEX11 deletion, 1 hemizygous DDX53 full gene deletion, and 1 homozygous, intragenic STK11 deletion. High-level mosaicism for X chromosome disomy (~ 10% 46,XY and ~ 90% 47,XXY) was also identified in 3 of 41 NOA patients who previously tested normal with conventional karyotyping. The remaining 24 CNVs detected were heterozygous, autosomal recessive carrier variants. Follow-up WES analysis confirmed 8 of 27 (30%) CNVs (X chromosome disomy excluded). WES analysis additionally identified 13 significant SNVs and/or indels in 9 patients (~ 9%) including X-linked AR, KAL1, and NR0B1 variants. CONCLUSION: Using a combined genome-wide aCGH/WES approach, we identified pathogenic and likely pathogenic SNVs and CNVs in 15 patients (15%) with unexplained SPGF. This value equals the detection rate of conventional testing for aneuploidies and is considerably higher than the prevalence of Y chromosome microdeletions. Our results underscore the importance of comprehensive genomic analysis in emerging diagnostic testing of complex conditions like male infertility.

Proteomic identification of sperm antigens using serum samples from individuals with and without antisperm antibodies.

The aim of the study was to identify human sperm antigens reacting with polyclonal antisperm antibodies. Protein sperm extracts were subjected to electrofocusing, and next immune reactions (immunoblotting) were carried out with positive for antisperm antibodies and control (not containing antisperm antibodies) serum samples. Proteomic analysis of human sperm proteins resulted in identification of 80 sperm antigens that could be divided into three groups: antigens specific for patients with antisperm antibodies (32), antigens recognised by both infertile patients and control sera (35) and antigens detected by control serum samples only (13). Among antigens specific for infertile patients, there were 12 sperm entities known to be involved in fertilisation process. We have also characterised three protein entities identified only by sera of infertile women. Altogether, the proteomic analysis resulted in identification of 27 sperm entities not reported previously in human sperm proteome. Identified proteins are sperm antigens that could be potentially responsible for immunological infertility. The study also sheds new light on the sperm antigens in aspect of gender specificity. The investigation of human sperm proteome by the use of antisperm antibodies-containing sera of infertile individuals not only may indicate new proteins but also can draft their immunological nature.

Implantation of autologous muscle-derived stem cells in treatment of fecal incontinence: results of an experimental pilot study.

BACKGROUND: The aim of this study is to present results of the implantation of autologous myoblasts into the external anal sphincter (EAS) in ten patients with fecal incontinence. METHODS: After anatomical and functional assessment of the patients' EAS, a vastus lateralis muscle open biopsy was performed. Stem cells were extracted from the biopsy specimens and cultured in vitro. Cell suspensions were then administered to the EAS. Patients were scheduled for follow-up visits in 6-week intervals. Total follow-up was 12 months. RESULTS: All biopsy and cell implantation procedures were performed without complications. Nine of the patients completed a full 12-month follow-up. There was subjective improvement in six patients (66.7 %). In manometric examinations 18 weeks after implantation, squeeze anal pressures and high-pressure zone length increased in all patients, with particularly significant sphincter function recovery in five patients (55.6 %). Electromyographic (EMG) examination showed an increase in signal amplitude in all patients, detecting elevated numbers of propagating action potentials. Twelve months after implantation two patients experienced deterioration of continence, which was also reflected in the deterioration of manometric and EMG parameters. The remaining four patients (44.4 %) still described their continence as better than before implantation and retained satisfactory functional examination parameters. CONCLUSIONS: Implantation of autologous myoblasts gives good short-term results not only in a subjective assessment, but also in objective functional tests. It seems that this promising technology can improve the quality of life of patients with fecal incontinence, but further study is required to achieve better and more persistent results.

Long-term cryopreservation of dental stem cells.

Cryopreservation is a procedure of a long-term storage of cells and/or tissues at a temperature that prevents cell divisions and metabolic processes. Due to ability to self-renewal and differentiation into more specialised cells, stem cells may be helpful in repairing of other damaged organs or tissues. Cryopreservation allows the frozen genetic material to maintain its biological properties for a long time. Therefore, there is a real chance for some samples to be used in the future therapy of the pathological conditions that at present remain incurable because of the current state of knowledge. The purpose of this review is to describe the modern methods of extraction, preservation, and storage of dental stem cells at low temperatures in particular procedure of collecting and transporting tissues intended for freezing, precise characteristics of stem cells of dentary origin and methods of their isolation using Enzymatic Digestion and Spontaneous Outgrowth. In the paper are also presented technical details of the protocols of rapid rate freezing, controlled rate milling and freezing in a magnetic field (magnetic freezing) which provides precise information about procedures of thawing cells and unfavourable effect of negative temperature on the biological properties of stem cells. Dental tissues may constitute a rich source of stem cells. The inexpensive, simple and quick procedure of their extraction is minimally invasive and does not pose a threat to the donor's organism. Transferring autologous cells within the same organism does not present a potential risk of transplant rejection and thereof does not raise ethical controversies. Laboratory procedures including cell preparation, its characteristics and genetic features, basics on the process of freezing, thawing, as well as quality control essentials have been also outlined.

Membrane stability and mitochondrial activity of human-ejaculated spermatozoa during in vitro experimental infection with Escherichia coli, Staphylococcus haemolyticus and Bacteroides ureolyticus.

The aim of the study was to examine an in vitro effect of the three bacterial strains (Escherichia coli, Staphylococcus haemolyticus and Bacteroides ureolyticus) on ejaculated spermatozoa with reference to sperm membrane integrity and mitochondrial activity. The study was carried out on swim-up-separated spermatozoa from 12 normozoospermic volunteers. Sperm plasma membrane stability was evaluated by the LIVE/DEAD Sperm Viability Kit and by the merocyanine 540 test. Mitochondrial activity was evaluated using the JC-1 test as well as the NADH-dependent NBT assay. The percentage of dead cells was significantly higher in spermatozoa treated with B. ureolyticus as compared to that of control spermatozoa (P < 0.01). All the bacterial strains applied affected sperm plasma membrane architecture measured by M540 test (P < 0.01). Moreover, the presence of E. coli or B. ureolyticus was connected with significant decrease in both the number of cells with high mitochondrial transmembrane potential (ΔΨm) and the cells with normal oxidoreductive function of mitochondria (P < 0.05 as compared to untreated cells). To conclude, the contact of bacteria with ejaculated spermatozoa can be a reason for severe injury of sperm membrane stability and mitochondrial activity with potential consequences for male fertility.

Varicocelectomy improves sperm parameters, sperm DNA integrity as well as the other critical semen features.

Varicocele is a major entity defined within male infertility. In this report we have studied the influence of laparoscopic varicocelectomy on semen quality, biochemical parameters of seminal plasma and sperm DNA fragmentation. In this study, the semen samples from patients with left-side varicocele of grade II-III before and after laparoscopic varicocelectomy were compared to healthy individuals and separated into three groups. The volume of semen, sperm concentration (106/ml), motility (%), viability (%) and normal morphology (%) were assessed. Total antioxidant capacity (TAC), catalase (CAT), superoxide dismutase (SOD) and malondialdehyde (MDA) together with other biochemical substances in seminal plasma as alpha-glucosidase (α-Glu), fructose (Fr) and citric acid (CA) were determined by ELISA method. The spermatozoa activity including ion-transports through sodium, potassium ATPase (Na+, K+-ATPase) and calcium, magnesium ATPase (Ca2+, Mg2+-ATPase) were determined by using spectrophotometry. In addition, flow cytometry method for detection of sperm DNA fragmentation was used. The results showed, that three months after varicocelectomy such intervention led to significant postoperative improvement in volume of semen (p<0.001), total sperm count (p<0.001), sperm motility (p<0.001) and spermatozoa with normal morphology (p<0.001). We found decreased α-Glu levels due to varicocelectomy (p<0.05). There has been shown a high positive correlation between Na+, K+-ATPase and Ca2+, Mg2+-ATPase activity with total number of spermatozoa (p<0.05). The TAC levels and DNA fragmentation values after varicocelectomy can be considered as significant indicators of good prognosis after surgical intervention. It has to be emphasized that α-Glu levels and total sperm count expressed statistically significant both positive and negative predictive values for semen assessment. Varicocelectomy may lead to significant improvement of semen quality although the observations must be correlated with clinical pregnancies observed thereafter.

The role of mitochondria in Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is an X-linked lethal disorder caused by mutations in the dystrophin gene. Progression of this disease may lead to cardiomyopathy and respiratory failure, which are the main causes of death among DMD patients. Lack of dystrophin affects cellular myogenic function and related organelles. Dystrophin deficiency results in intracellular Ca2+ dysregulation, mitochondrial dysfunction and induces elevated production of reactive oxygen species (ROS). Due to current findings, mitochondria may be also a potential target for DMD therapy. In this review we attempted to provide an insight into the role of mitochondria in perpetuation of DMD disease.

HLA-DQA1*0505 sharing and killer immunoglobulin-like receptors in sub fertile couples: report from the 15th International Histocompatibility Workshop.

This aim of the study was to investigate whether human leukocyte antigen (HLA)-DQA1*0505 sharing or the maternal killer immunoglobulin-like receptor (KIR) repertoire is associated with recurrent spontaneous abortion (RSA) or repeated implantation failure (RIF). The study included 224 couples with RSA, 61 couples with RIF, 182 fertile couples, and 10 couples with successful in vitro fertilization and embryo transfer (IVF)/ET at first cycle. HLA-DQA1*0505 typing using polymerase chain reaction-sequence-specific oligonucleotide (PCR-SSO) was performed in 185 RSA (117 with alloimmune abnormalities and 68 of autoimmune etiology), 61 RIF and 182 control couples, and KIR genotyping using polymerase chain reaction-sequence-specific primer (PCR-SSP) in 167 RSA and 55 RIF cases as well as 46 RSA and 10 IVF controls. No differences in DQA1*0505 sharing were found between patients and controls. In RSA and RIF women, the ratio of inhibitory to activating KIRs was slightly lower (1.53 and 1.85 vs 2.03 in controls). The analysis of maternal inhKIR and fetal HLA-C molecule pairs showed that the 'less inhibiting' combination KIR2DL3-C1 was found in higher percentage in subfertile (mainly RIF) than in fertile couples. In contrast, the percentage of cases possessing the 'strong inhibiting' combination KIR2DL1-C2 was lower in the RSA and RIF groups in comparison with that in the control groups (17.36% vs 23.91 and 16.36% vs 40%, respectively). In women with >or= 6 implantation failures, the KIR2DL1-C2 combination was not found in any of them (P = 0.0014), and the KIR2DL3-C1 combination was not found in the control IVF group. The results oppose the suggestion that increased HLA-DQA1*0505 sharing predispose to RSA or RIF. The KIR2DL3-C1 combination (or lack of the KIR2DL1-C2 one) is associated with implantation failure.

SCF and c-kit expression profiles in male individuals with normal and impaired spermatogenesis.

The transcription levels of stem cell factor (SCF) and c-kit were examined using real-time RT PCR in interstitial and intratubular cell fractions, as well as in tissue homogenates from normal, azoospermic and neoplasmic patients. Peripheral blood mononuclear cells (PBMC) were used as a systemic control. The observed level of c-kit expression in all investigated groups was generally higher than the expression of SCF. The highest (statistically significant) level of c-kit was noted in testicular tumours (the greater part of which were represented by seminomas) in contrast to SCF mRNA, which may indicate an association between c-kit overexpression and seminoma development. In Sertoli cell only syndrome, almost equal levels of SCF and c-kit transcripts were noted. These results may indicate Leydig cells as the alternative source of c-kit gene transcription. SCF transcript values were low and comparable among the analysed subgroups except that in maturation arrest at spermatocyte stage, the SCF gene expression was statistically higher than in testicular tumours. It appears from the study that c-kit has been a dynamic gene, changing its activity in a variety of testicular pathologies while being expressed in all testicular compartments but clearly overexpressed in testicular tumours of seminomatous origin.

Complex cytogenetic and molecular-genetic analysis of males with spermatogenesis failure.

The chromosomal anomalies, microdeletions of AZF region of Y-chromosome and CFTR gene mutations have been studied among 80 infertile men with idiopathic spermatogenetic failure: 36 (45%) patients with aspermia, 19 (24%) patients with azoospermia and 25 (31%) patients with severe oligoasthenoteratozoospermia. In total 30% males with spermatogenetic failure genetic factor of infertility was observed. Karyotype anomalies were observed in 17.5% of infertile men, within 16.2% numerical and structural gonosomal anomalies and in 1.3%--Robertsonian translocation were revealed. In 11% males with spermatogenetic failure, Y-chromosome AZF region microdeletions were detected. The frequency of CFTR major mutation F508del among infertile men was 6.25%. 5T allele of polymorphic locus IVS8polyT was detected in 7.5% of examined men. The results obtained indicate the high complexity of cytogenetic and molecular-genetic studies of male infertility.

Frequencies of killer immunoglobulin-like receptor genotypes influence susceptibility to spontaneous abortion.

Natural killer (NK) cells are the most abundant lymphocyte population in the decidua. These cells express killer immunoglobulin-like receptors (KIRs), which upon recognition of HLA class I molecules on trophoblasts may either stimulate NK cells (activating KIRs) or inhibit them (inhibitory KIRs) to produce soluble factors necessary for the maintenance of pregnancy. KIR genes exhibit extensive haplotype polymorphism; individuals differ in both the number and kind (activating vs. inhibitory) of KIR genes. This polymorphism affects NK cell reactivity and susceptibility to diseases, including gynecological disorders. Therefore we KIR-genotyped 149 spontaneously aborting women and 117 control multiparae (at least 2 healthy-born children). Several genotypes (i.e. combinations of various KIR genes) were differently distributed among the patients and control subjects. Differences were observed in the numbers and the ratios of activating to inhibitory KIRs between patients and healthy women: (i) genotypes containing 6 activating KIR genes were less frequent and those containing 6 inhibitory KIR genes were more frequent in patients than in control subjects, and (ii) an excess of inhibitory KIRs (activating-to-inhibitory KIR gene ratios of 0.33 to 0.83) was associated with miscarriage, whereas ratios close to equilibrium (0.86-1.25) seemed to be protective. In addition, the results suggest for the first time that sporadic and recurrent spontaneous abortions as well as miscarriage in the presence or absence of autoantibodies may have different KIR genotypic backgrounds.

Human skeletal muscle-derived stem/progenitor cells modified with connexin-43 prevent arrhythmia in rat post-infarction hearts and influence gene expression in the myocardium.

Stem cell therapy in combination with genetic modification (e.g., transfection with the coding sequence for the connexion 43 gene, GJA1) may solve the problems associated with the occurrence of additional (secondary) stimulation in the post-infarcted heart (arrhythmia). Human skeletal muscle-derived stem/progenitor cells (SkMDS/PCs) were transfected with the pCiNeo-GJA1 plasmid at an efficiency of approximately 96%. Gene overexpression was assessed using qPCR, and subsequent analysis revealed that GJA1 expression increased more than 40-fold in SkMDS/PCs transfected with the appropriate coding sequence (SkMDS/PCsCX43) compared to that of the 'native' SkMDS/PCs control (SkMDS/PCsWT). Enhanced (4-fold) protein expression of connexin-43 was also confirmed by Western immunoblotting. Furthermore, using the arrhythmic score, we demonstrated the positive effects of SkMDS/PCsCX43 cell intervention in reducing additional secondary stimulations in rat post-infarcted hearts compared with that of wild-type cell delivery. Selected gene responses (Kcnq1, Cacna1c, Ncx1, Serca2a, and Tgfb1) showed significantly altered expression profiles in the rat myocardium upon intervention with SkMDS/PCsCX43. The genetic modification of human skeletal muscle-derived stem/progenitor cells with connexin-43 prevented the pro-arrhythmic effects of myogenic implanted stem cells on the host myocardium and positively influenced myocardial gene expression profiles in respect to myocardium conductivity.

Application of stem cells in dentistry for bone regeneration.

Stem cells constitute a non-specialised cell pool able for proliferation, self-renewal and differentiation into progeny constantly replacing used up tissue and/or organ fragments. They have been observed to be present in many tissue reservoirs, including stomatognathic system. Oral cavity seems to be a particularly attractive stem cells' source as these cells are richly present and easily accessible in dental and periodontal tissues and they can be used for therapeutic purposes. Their application is also morally and ethically non-controversial. Bone tissue structure restoration together with restoring its weight-bearing and nutritive function depend on the proper function of stem cells supported with other techniques including tissue engineering. Traditionally, bone regeneration means bone restoration supported by newly formed bones supplied by stem cells of tissue reservoir. It may be also indispensable to create a scaffold which may support the bone formation facilitating the transportation of cells and cell stimulating molecules involving both the matrix and bone-forming cells. The regenerative potential of stem cells present in oral cavity can be used, for instance, to restore maxillary and mandibular bones. The bones support natural teeth or prostheses and regress as soon as at least the one tooth is lost or extracted. Bone mass loss makes it difficult to conduct effective dental treatment and reduces the chances of obtaining positive, long-lasting therapeutic effects. It seems that modern and innovative therapies based on stem cells application may bring spectacular effects especially in patients in whom routine medical activities did not lead to satisfactory results.

Influence of hypoxia prevailing in post-infarction heart on proangiogenic gene expression and biological features of human myoblast cells applied as a pro-regenerative therapeutic tool.

Cardiovascular diseases along with MI (myocardial infarction) lead to regional ischaemia and hypoxic conditions, which prevail after infarction. Diminished O2 saturation which is related to elevated level of hypoxia inducible factor 1 (HIF-1) transcription factor, may switch the expression of many genes. To maximize effect of therapies proposed by regenerative medicine, it is essential to verify (within different time points after MI) the expression of proangiogenic genes and their receptors that are regulated, along with the expression of HIF-1α. We demonstrated a connection between the expression of Hif-1α (in murine post infarcted heart model) and the proangiogenic genes Vegf-a; and Plgf and their receptors during myocardial hypoxia. The innovative part of the study required establishment of the most accurate in vitro O2 level corresponding to the hypoxia level prevailing in myocardium after MI. We determined the influence of hypoxia on the biology of human myoblasts in in vitro oxygen conditions (3%), corresponding to those prevailing in the heart after an infarction using a murine model. We also tested myoblasts that were genetically modified with VEGF-A/FGF-4 and PlGF under hypoxic conditions and compared their characteristics with cells cultured under normoxia and hyperoxia (standard in vitro conditions) with respect to myogenic gene expression, cell proliferation, fusion potential and proangiogenic function. The examination of genetically modified myoblasts under optimized in vitro hypoxia conditions led to the conclusion that hypoxia did not negatively influence the biological functions of the myoblasts, such as cell proliferation and/or proangiogenic characteristics. These results support the expected increased proregenerative effects of such genetically modified human myoblasts.

Sperm mitochondrial dysfunction and oxidative stress as possible reasons for isolated asthenozoospermia.

Reduced sperm motility, defined as asthenozoospermia, is a frequent cause of male infertility, and is mainly connected with the dysfunction of sperm mitochondria. The aim of this study was to identify the proteins, and thereby the metabolic pathways, responsible for asthenozoospermia, using 2-DE and MALDI-TOF MS, and correlate the results obtained with those of two mitochondrial tests: JC-1 and MitoSox Red. The JC-1 test was performed to test sperm mitochondrial activity, and the MitoSox Red test was performed to check whether the observed sperm poor motility is associated with mitochondrial reactive oxygen species (ROS) production. To identify proteins strictly connected with reduced sperm motility, men with isolated asthenozoospermia (n = 4 versus 10 normozoospermic controls) alone were included in the study. The proteomic analyses resulted in the identification of 25 sperm proteins that are differentially expressed in asthenozoospermic individuals. Most of the identified proteins were downregulated and were involved in energy production; however, we have also identified structural sperm proteins and proteins secreted by the epididymis. The latter, together with the results from MitoSox Red assay, may provide insights into the pathophysiological basis of asthenozoospermia.

Differential effect of IFN-tau on proliferation and distribution of lymphocyte subsets in one-way mixed lymphocyte reaction in cows and heifers.

IFN-tau is a signaling protein secreted by the bovine conceptus during the peri-implantation period and responsible for pregnancy recognition. Its main role is the prevention of pulsatile release of luteolytic PGF2alpha, but it also exerts immunomodulatory activities characteristic for other type I interferons. The aim of the study was to examine the effect of IFN-tau on the proliferation and distribution of peripheral blood lymphocyte subsets during one-way mixed lymphocyte reaction (MLR) in cows and heifers. IFN-tau inhibited the proliferative response of lymphocytes in MLR both in cows and heifers in a dose-dependent manner, but cow lymphocytes were less susceptible than those ones from heifers. It was also showed that IFN-tau differentially changed lymphocyte subsets distribution in MLR in cows and heifers. In cows, the relative percentage of CD8(+) cells after MRL in the presence of IFN-tau was significantly lower than in heifers. Differential effect of rIFN-tau on proliferation and lymphocyte subsets distribution in a one-way MRL in cows and heifers indicated that the age of the mother is an important factor in immunomodulatory effect towards developing bovine embryo.

Human myoblast transplantation in mice infarcted heart alters the expression profile of cardiac genes associated with left ventricle remodeling.

BACKGROUND: Myocardial infarction (MI) and left ventricle remodeling (LVR) are two of the most challenging disease entities in developed societies. Since conventional treatment cannot fully restore heart function new approaches were attempted to develop new strategies and technologies that could be used for myocardial regeneration. One of these strategies pursued was a cell therapy--particularly applying skeletal muscle stem cells (SkMCs). METHODS AND RESULTS: Using NOD-SCID murine model of MI and human skeletal myoblast transplantation we were able to show that SkMC administration significantly affected gene expression profile (p<0.05) (NPPB, CTGF, GATA4, SERCA2a, PLB) of the heart ventricular tissue and this change was beneficial for the heart function. We have also shown, that the level of heart biomarker, NT-proBNP, decreased in animals receiving implanted cells and that the NT-proBNP level negatively correlated with left ventricle area fraction change (LVFAC) index which makes NT-proBNP an attractive tool in assessing the efficacy of cell therapy both in the animal model and prospectively in clinical trials. CONCLUSIONS: The results obtained suggest that transplanted SkMCs exerted beneficial effect on heart regeneration and were able to inhibit LVR which was confirmed on the molecular level, giving hope for new ways of monitoring novel cellular therapies for MI.

Bacterial infection and semen quality.

We have analyzed two infertile male cohorts with (n=39) and without genital tract infection (n=14) comparing their selected seminological parameters with healthy controls (n=30). Genital tract infection (GTI) has been defined by the presence of leukocytes and pathological bacterial strains identified with Bio-Merieux tests. We have found statistically significant deteriorated semen volume, sperm concentration, motility, morphology and vitality in ejaculated samples of patients with genital tract infection in comparison to healthy controls. Statistically significant negative influence towards sperm reproductive potential has been revealed in case of Escherichia coli, Ureaplasma urealyticum and Staphylococcus aureus.

Suppression of inflammatory arthritis by simultaneous inhibition of nitric oxide synthase and NADPH oxidase.

TH1-type proinflammatory cytokines induce the expression of phagocytic nitric oxide synthase (NOS) and prime the membrane-bound NADPH oxidase of neutrophils and monocytes of mice so as to attain an activated state, which upon a second stimulus releases up to 6-fold increased levels of reactive oxygen species (ROS) than do unprimed phagocytes. Enhanced levels of ROS and NO deregulate inflammatory signal transduction pathways, which play a crucial role in the pathogenesis of arthritis. The antiarthritic reactivity of diphenylene iodoniumchloride (DPI), an irreversible inhibitor of NADPH oxidase and NOS, was tested in male DBA/1xB10A(4R) hybrid mice suffering from potassium peroxochromate-induced arthritis. Daily doses of 2.8 mu mol/kg of DPI sufficed to inhibit the arthritis by 50%. A complete inhibition was obtained with 10 mu mol/kg of DPI. The reduction of overt arthritic symptoms correlated well with both the reduced levels of ROS and NO in plasma of DPI-treated mice. Our data support the hypothesis that oxidative stress and nitric oxides play a pivotal role in the pathology of arthritis, which can be therapeutically targetted by NADPH oxidase- and NO synthase-inhibitors.