Abstract Background This study aimed to evaluate the association of antiphospholipid antibodies (aPL) and conventional markers of coagulation with ischemic and bleeding risk in patients with atrial fibrillation (AF) undergoing percutaneous coronary intervention (PCI). Methods In this prospective two-center observational cohort study, patients with AF and an indication for oral anticoagulation (OAC) were enrolled after PCI. Blood was drawn on day 1–3 after PCI. Dilute Russell’s viper venom time was used to determine lupus anticoagulant (LA) in OAC-free plasma. Anti-cardiolipin (aCL) IgG, IgM, and anti-β2-Glycoprotein 1 (aβ2GP1) IgG were analyzed by enzyme-linked immunosorbent assay (ELISA). Fibrinogen (FIB), d-dimer, and prothrombin fragment 1 and 2 (PF 1 + 2) were measured in citrated plasma. The primary ischemic outcome was time to major adverse cardiovascular events (MACE; death, myocardial infarction, or stroke) assessed at 6 months. Bleeding was defined according to International Society on Thrombosis and Haemostasis. Results 158 patients were enrolled between May 2020 and May 2021 on day 1–3 after PCI. The median age was 78 years (interquartile range [IQR] 72–82), 111 (70%) were male, and 39 (25%) presented with acute coronary syndrome. D-dimer was elevated in 74 (47%) patients, FIB was increased in 40 (25%) and PF1 + 2 in 68 (43%) patients. 32 (20%) patients had ≥ 1 antiphospholipid antibody elevated (aPL; LA: 19 [12%], aCL: 14 [9%], aβ2GP1: 2 [1%]). The presence of aPL was neither significantly associated with MACE (HR 1.46, 95% CI [0.39–5.49], p = 0.579), nor bleeding (HR 1.07 [0.30–3.84], p = 0.917). Elevated d-dimer was significantly associated with higher risk for MACE (HR 5.06 [1.09–23.41], p = 0.038) and major bleeding (HR 7.04 [1.58–31.47], p = 0.011). Elevated D-dimer increased the predictive capacity of HAS-BLED for major bleedings (HAS-BLED: AUC 0.71 [0.60–0.83] vs. HAS-BLED + d-dimer: AUC 0.79 [0.70–0.88]; p = 0.025). Increased levels of FIB were associated with higher risk for MACE (HR 3.65 [1.11–11.96], p = 0.033). Conclusion Biomarkers of coagulation might be suitable to assess ischemic and bleeding risk in patients with AF following PCI.

ObjectivesAxial spondyloarthritis (axSpA) and psoriatic arthritis (PsA) may have a profound impact on sleep and health-related quality of life. The aim of this study was to assess sleep quality and quality of life and determine associated factors in patients treated with spondyloarthritides (SpA).MethodsCross-sectional questionnaire-based assessment of sleep behaviour, quality of life, functional impairment and depression (Regensburg Insomnia Scale, WHO Quality of Life questionnaire, Funktionsfragebogen Hannover questionnaire, Beck Depression Inventory II, Patient health questionnaire 9) and retrospective medical chart analysis of a monocentric cohort of 330 patients with SpA (n=168 PsA and n=162 axSpA).Results46.6% of patients with SpA demonstrated abnormal sleep behaviour. Linear regression models showed HLA-B27 positivity, Bath Ankylosing Spondylitis Disease Activity Index, depressive symptoms, functional capacity and disease duration to be predictive of insomnia symptoms in axSpA, respectively, depressive symptoms, female sex and Disease Activity Score 28 in patients with PsA. Patients with unrestful sleep had a significantly reduced health-related quality of life (p<0.001) as well as significantly more depressive symptoms (p<0.001). Satisfaction with health was rated significantly lower (p<0.001), indicating poor sleep as a burden on general well-being.In particular, female patients had a significantly worse sleep quality with a prolonged sleep latency (p=0.009), increased sleep disturbances (p=0.014) and unrestful sleep (p<0.001) as well as a reduced physical and mental health-related quality of life (p=0.015, p<0.001) and more depressive symptoms (p=0.015).ConclusionDespite treatment, many patients with SpA demonstrate abnormal sleep behaviour with symptoms of insomnia and a reduced quality of life with significant differences between male and female patients. An interdisciplinary and holistic approach may be needed to address unmet needs.

BackgroundJanus kinase (JAK) inhibitors have been approved for the treatment of several immune-mediated diseases (IMIDs) including rheumatoid arthritis (RA) and psoriatic arthritis and are in clinical trials for numerous other IMIDs. However, detailed studies investigating the effects of different JAK inhibitors on B cells are missing. Within this study, we therefore aimed to characterize the effect of JAK inhibition on the B cell compartment.MethodsTo this end, we investigated the B cell compartment under JAK inhibition and compared the specific effects of the different JAK inhibitors tofacitinib (pan-JAK), baricitinib (JAK1/2), ruxolitinib (JAK1/2), upadacitinib (JAK1/2) as well as filgotinib (selective JAK1) on in-vitro B cell activation, proliferation, and class switch recombination and involved pathways.ResultsWhile B cell phenotyping of RA patients showed an increase in marginal zone (MZ) B cells under JAK inhibition, comparison with healthy donors revealed that the relative frequency of MZ B cells was still lower compared to healthy controls. In an in-vitro model of T-cell-independent B cell activation we observed that JAK1/2 and selective JAK1 inhibitor treatment led to a dose-dependent decrease of total B cell numbers. We detected an altered B cell differentiation with a significant increase in MZ-like B cells and an increase in plasmablast differentiation in the first days of culture, most pronounced with the pan-JAK inhibitor tofacitinib, although there was no increase in immunoglobulin secretion in-vitro. Notably, we further observed a profound reduction of switched memory B cell formation, especially with JAK1/2 inhibition. JAK inhibitor treatment led to a dose-dependent reduction of STAT3 expression and phosphorylation as well as STAT3 target gene expression and modulated the secretion of pro- and anti-inflammatory cytokines by B cells.ConclusionJAK inhibition has a major effect on B cell activation and differentiation, with differential outcomes between JAK inhibitors hinting towards distinct and unique effects on B cell homeostasis.

Respiratory tract infections (RTIs) are the most common infections in patients with rheumatic diseases under immunosuppressive treatment and may contribute to morbidity and mortality as well as increased healthcare costs. However, to date only limited data on infection risk in spondyloarthritis (SpA) patients are available. In this study we assessed the occurrence of respiratory tract infections in a monocentric real-world cohort consisting of 330 patients (168 psoriatic arthritis and 162 axial spondyloarthritis patients) and determined factors associated with increased infection risk. Out of 330 SpA patients, 89.3% had suffered from ≥ 1 upper respiratory tract infection (URTI) and 31.1% from ≥ 1 lower respiratory tract infection (LRTI) within the last two years. The most common URTIs were rhinitis and laryngitis/pharyngitis with 87.3% and 36.1%, respectively. Bronchitis constituted the most common LRTI, reported in 29.7% of patients. In a multivariate binomial logistic regression model occurrence of LRTI was associated with chronic lung disease (OR 17.44, p=0.006), glucocorticoid therapy (OR 9.24, p=0.012), previous history of severe airway infections (OR 6.82, p=0.013), and number of previous biological therapies (OR 1.72, p=0.017), whereas HLA B27 positivity was negatively associated (OR 0.29, p=0.025). Female patients reported significantly more LRTIs than male patients (p=0.006) and had a higher rate of antibiotic therapy (p=0.009). There were no significant differences between axSpA and PsA patients regarding infection frequency or antibiotic use. 45.4% of patients had required antibiotics for respiratory tract infections. Antibiotic therapy was associated with smoking (OR 3.40, p=0.008), biological therapy (OR 3.38, p=0.004), sleep quality (OR 1.13, p<0.001) and age (OR 0.96, p=0.030). Hypogammaglobulinemia (IgG<7g/l) was rare (3.4%) in this SpA cohort despite continuous immunomodulatory treatment.Awareness of these risk factors will assist physicians to identify patients with an increased infection risk, who will benefit from additional preventive measures, such as vaccination and smoking cessation or adjustment of DMARD therapy.

Abstract Background Cytotoxic Natural Killer (NK) cells are increasingly recognized as a powerful tool to induce targeted cell death in cancer and autoimmune diseases. Still, basic blood NK cell parameters are poorly defined. The aims of this study were 1) to establish reference values of NK cell counts and percentages in healthy adults; 2) to describe these parameters in the prototype autoimmune disease group ANCA-associated vasculitis (AAV); and 3) to investigate whether NK cell counts and percentages may be used as activity biomarkers in the care of AAV patients, as suggested by a preceding study. Methods CD3-(CD56 or 16)+ NK cell counts and percentages were determined in 120 healthy adults. Lymphocyte subset and clinical data from two German vasculitis centers were analyzed retrospectively (in total 407 measurements, including 201/49/157 measurements from 64/16/39 patients with granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA) and eosinophilic granulomatosis with polyangiitis (EGPA), respectively). Results CD3-(CD56 or 16)+ NK cell counts and percentages in healthy adults were highly variable, not Gaussian distributed and independent of age and sex. NK cell percentages ranged from 1.9 to 37.9% of lymphocytes, and were significantly more dispersed in AAV (0.3 to 57.6%), while the median percentage was not different between AAV and healthy donors. In contrast, median NK cell counts were significantly lower in AAV compared to healthy donors. Sub-group analyses revealed that NK cell counts were low independent of AAV entity and disease activity. Azathioprine therapy was associated with significantly lower NK cell counts and percentages compared to non-azathioprine therapies. In 13.6% of azathioprine-treated patients, percentages were = 1% which may be interpreted as temporary NK cell deficiency. NK cell counts and percentages could not separate active from inactive AAV. Conclusions NK cell counts and percentages in blood are heterogeneous and can presently not be recommended as biomarker in clinical care of AAV patients. Azathioprine treatment was associated with significantly low NK cells. These findings may be relevant for the development of drugs that aim at exploiting NK cell cytotoxicity and may help to identify patients at risk to develop malignant or infectious co-morbidities.

Objective: Rheumatic diseases with involvement of the central nervous system (RDwCNS) may mimic multiple sclerosis (MS). Inversely, up to 60% of MS-patients have antinuclear autoantibodies (ANAs) and may be misdiagnosed as RDwCNS. The detection of antibodies against extractable nuclear antigens (ENA) and oligoclonal bands (OCB) are established valuable diagnostic tools in the differential diagnosis of RDwCNS and MS. The MRZ-reaction (MRZR) is defined by three antibody indices (AIs) against neurotropic viruses and is frequently positive in MS. To investigate the added value of MRZR combined with testing for antibodies against ENAs and OCB detection to distinguish RDwCNS from ANA positive MS. Methods: MRZR was evaluated in RDwCNS (n = 40) and 68 ANA positive MS-patients. Two stringency levels, MRZR-1 and MRZR-2 (at least one respectively two of three AIs positive) were applied. Autoantibody testing included ANA plus ENA and anti-dsDNA antibodies, antiphospholipid antibodies, and anti-neutrophil cytoplasmic antibodies. Results: Most of the RDwCNS patients (n = 32; 80%) suffered from systemic lupus erythematosus. Within the RDwCNS group 20% had a positive MRZR-1 and 8.5% a positive MRZR-2 compared to 80.9 and 60%, respectively within the MS-group (p < 0.0001 for both comparisons). Oligoclonal bands were found in 28.6% of the RDwCNS patients and 94.3% of the MS-patients (p < 0.0001). Conversely, autoantibodies to specific nuclear antigens or phospholipids were found more frequently in RDwCNS. A positive MRZR in conjunction with the absence of ENA autoantibodies distinguished MS from RDwCNS with high specificity (97.5%). Conclusions: We suggest combining MRZR, OCBs, and specific autoantibody diagnostics to differentiate RDwCNS from MS.

Background Some rheumatologic disorders (RD) may initially manifest with central nervous system (CNS) affection, mimicking the clinical, magnetic resonance imaging, and cerebrospinal fluid (CSF) findings of multiple sclerosis (MS). Vice versa MS might be difficult to separate from some RD because of the presence of autoantibodies (e.g. ANA) in up to 50%. The MRZ reaction (MRZR), composed of the three respective antibody indices (AI) against measles, rubella, and varicella zoster virus, has been found positive frequently in MS patients. However, it is unclear whether the MRZR is helpful to distinguish rheumatologic disorders with CNS involvement (RDwCNS) from MS. Objectives To investigate the MRZ reaction as a diagnostic tool to distinguish patients with RDwCNS from patients with MS. Methods The MRZR was evaluated in 35 patients with RDwCNS and compared to 70 sex- and age- matched MS patients. An AI result ≥1.5 was indicative for intrathecal IgG production against the respective pathogen. Two previously established stringency levels, MRZR-1 (≥1 of 3 AIs positive) and MRZR-2 (≥2 of 3 AIs positive), were applied. CNS involvement of RDwCNS was defined as clinical manifestation with neurological symptoms and signs of inflammation in CSF analysis and/or cerebral/spinal magnetic resonance imaging (MRI). MRZR results were compared using the Fisher’s exact test with p<0.05 for statistical significance. Results Within the RDwCNS group, 31 patients suffered from systemic lupus erythematosus, four had a small vessel vasculitis. In both groups 77.1% were female, mean age (±SD) was 43.2 years (±18.7) in RDwCNS and 47.5 years (±7.8) in MS (p=n .s.). All RDwCNS patients showed clinical symptoms indicative for CNS involvement and signs of inflammation in CSF analyses and/or MRI of the brain. In 52 MS patients autoantibody screening was performed. 42% were positive for ANA (n=20) or ANCA (n=2) in indirect immunofluorescence. Only 14.3% of RDwCNS patients had a positive MRZR-1 compared to 85.7% within the MS group (p<0.0001). The more specific MRZR-2 was positive in 60% of the MS patients compared to only 8.5% of the RDwCNS patients (p<0.0001). By using a higher threshold of >2.0 for a positive AI, the prevalence of positive MRZR-2 dropped to 5.7% (n=2) in the RDwCNS group compared to 54.3% (n=38) in the MS group (p<0.0001). Oligoclonal bands were found in 94.3% of the MS and 28.6% of the RDwCNS patients (p<0.0001). Conclusions Considering the high specificity of the MRZR-2 for MS confirmed in this study, this laboratory test may be a helpful diagnostic tool to distinguish RDwCNS from MS. Disclosure of Interest None declared

Background The detection of antinuclear autoantibodies by immunofluorescence (ANA-IFT) supports the diagnosis of many different autoimmune and rheumatological diseases (ARE). In combination with the detection of specific autoantibodies against known extracted nuclear antigens (ENA) ANAs have high diagnostic sensitivity and specificity. However, positive ANA-IFT may also occur in disease states not related to ARE and even in healthy individuals. Especially in the latter group this may lead to unnecessary and repeated hospital visits, as well as avoidable diagnostic and therapeutic interventions. Anti-DFS70 autoantibodies were introduced as a biomarker for the exclusion of ARE in ANA-IFT +patients without additional ENAs and non-specific clinical history. Objectives To evaluate if the diagnostic pathway for suspected or established ARE patients in our centre benefits from the addition of anti-DFS70 autoantibodies to an existing ENA test profile. Methods Serum from patients was tested for anti-DFS70 autoantibodies by the ANA-Profil 3 plus DFS70 line blot (Euroimmun, Lübeck Germany). Results In 2017 the Freiburg university rheumatology centre tested 671 patients, referred to for diagnosis or follow up of ARE, for anti-DFS70 and 126 patients were found positive. Descriptive statistics of the anti-DFS70 positive vs negative cohorts are summarised in table 1. Of the 126 anti-DFS70 positive patients, 53 (42%) had one or more additional ENAs positively tested (4 snRNP/Sm,1 Sm,15 SS-A/Ro,15 Ro-52,2 SS-B/La,4 Scl-70,8 PM-Scl,2 PM-Scl75,1 SRP,2 Ku,1 Jo-1,4 Centromer,2 PCNA,2 Nukleosomes,9 Histones,1 ribos-P-Prot,2 AMA-M2,6 ds-DNA) and 3 (2%) patients had anti-DFS70 in conjunction with anti CCP autoantibodies. 70 (56%) patients were tested single positive for anti-DFS70. 81 (64%) of anti-DFS70 positive patients either had an established diagnosis of ARE or were newly diagnosed with ARE based on clinical criteria and laboratory results. The majority of these patients had additional ENAs or other autoantibodies tested positive, as described above. A diagnosis of ARE was excluded or revised with help of the anti-DFS70 result in 45 (36%) patients, all of these were anti-DFS70 positive only. In 10 of these patients (20%) preexisting therapies were discontinued (9 Hydroxychloroquin, 1 MTX/sulfasalazine). Anti-DFS70 positive(n=126) Anti-DFS70 negative(n=544) p-value Male, n (%) 19 (15.1) 111 (20.4) n.s. Mean age, years (min-max) 47 (18–77) 52 (14–88) n.s. DFS70 +++, n (%) 82 (65.1) NA NA DFS70 ++, n (%) 13 (10.3) NA NA DFS70 +, n (%) 10 (7.9) NA NA DFS70 (+), n (%) 21 (16.7) NA NAAbstract THU0380 – Table 1 Descriptive statistics of the patient cohorts tested for anti-DFS70-antibodies Conclusions We found a high added diagnostical value of anti-DFS70 autoantibodies testing, since they were helpful to rule out or revise a previously suspected diagnosis of ARE in more than a third all patients tested anti-DFS70 positive at our centre. However, patients referred to a university rheumatology centre are a highly selected cohort and have a higher probability being finally diagnosed with ARE, and thus consideration of clinical criteria and thorough testing for additional autoantibodies is recommended in such a setting since also many patients with confirmed ARE will test positive for anti-DFS70. Disclosure of Interest None declared

Background The cytokine Flt3 ligand is an important cofactor for early hematopoiesis by mainly driving the development of lymphoid and early B-cell precursors. In the periphery functions of Flt3 are more pleiotropic involving the differentiation of regulatory T-cells, dendritic cells as well as peripheral B-cells. Besides its well-known roles in hematological disorders and as an indicator of bone marrow (BM) output capacity, the possible involvement of FLT3 ligand in autoimmune disorders was only discovered recently. Objectives Our primary aim was to analyze if FLT3 ligand serum concentrations are affected in AAV patients. Secondary aims were to correlate the FLT3 ligand serum concentrations with clinical and laboratory parameters. And, since FLT3 ligand concentrations are elevated in different states of bone marrow failure, we also wanted to evaluate FLT 3ligand as a marker for treatment related BM toxicity in AAV patients. Methods We performed a cross sectional study using a sandwich ELISA to determine FLT3 ligand concentrations in the serum of 98 well characterized AAV patients (69 GPA, 20 MPA and 9 EGPA) and 144 healthy controls (HC). Statistical evaluation was done using Mann-Whitney or unpaired, two-tailed Student9s t-test. Results In patients with AAV, FLT3 ligand concentrations were significantly elevated (207 pg/ml +/- 116.2 in AAV versus 142.5 pg/ml +/- 65.98 in HC; p<0.0001). Disease specific analysis revealed significantly elevated FLT3 ligand concentrations in GPA (217.6 pg/ml +/- 123; p<0.0001), but no significant differences for MPA and EGPA when compared to HC. FLT3 ligand concentrations did not correlate to serological or clinical markers of disease activity, however, overall disease activity was low in the studied cohort. To assess the influences of treatment regimen on FLT3 ligand concentrations, we focused our analysis on treatment histories of cyclophosphamide (CYC) and azathioprine (AZA) and grouped the patient cohort according to the cumulative CYC dose (< or >5g) and/or duration of AZA therapy (< or >6 months). AZA and CYC naïve patients (n=10) showed FLT3 concentrations comparable to HC (121 pg/ml +/- 42.3), but in patients with low dose CYC and short term AZA therapy FLT3 concentrations were significantly higher (176.6 pg/ml +/- 51.6), both compared to treatment naïve patients and HC (p=0.0095 vs. AZA/CYC naïve, p=0.0105 vs HC). Intensified treatment was associated with even further increased concentrations of FLT 3 ligand with highest concentrations found in patients treated with >5g CYC cumulative dose or AZA treatment for >6 months (263.4 pg/ml +/- 172.6; p=0.0024 vs. AZA/CYC naïve, p<0.0001 vs HC). Conclusions Flt3 ligand concentrations are elevated in patients with AAV, especially in patients with GPA. The elevation more likely reflects the therapeutic regimen and history than disease activity as we could show that patients with more intensive treatment including both CYC and AZA show higher FLT3 ligand serum levels when compared to patients with less intense therapy. References Ramos MI et al. FMS-related tyrosine kinase 3 ligand (Flt3L)/CD135 axis in rheumatoid arthritis. Arthritis Res Ther. 2013; 15:R209. Giri N et al. Immune status of patients with inherited bone marrow failure syndromes.Am J Hematol. 2015; 90:702–8. Disclosure of Interest None declared

BACKGROUND: Rituximab (RTX) is approved for induction therapy of granulomatosis with polyangiitis and microscopic polyangiitis. In eosinophilic granulomatosis with polyangiitis (EGPA), organ-threatening manifestations are mainly treated with cyclophosphamide (CYC). RTX as treatment in EGPA has been described in small case series; however long-term data and the efficacy of RTX in EGPA refractory to CYC have not been reported yet. OBJECTIVES: To investigate the efficacy and safety of RTX and conventional immunosuppressive therapy with CYC in EGPA as induction therapy and during long-term follow-up. METHODS: Retrospective analysis of 28 patients with EGPA was done. Treatment response and disease activity were determined by Birmingham Vasculitis Activity Score, C-reactive protein, eosinophils, antineutrophil cytoplasmic antibody, and peripheral CD19(+) B cells. RESULTS: Fourteen patients with EGPA treated with RTX were compared with 14 age-and sex-matched patients with EGPA treated with CYC for remission induction; 64% of the RTX-treated patients with EGPA had previously failed CYC treatment. Disease duration was longer and the number of previous immunosuppressants higher in RTX-treated patients. Five RTX-treated patients (36%) and 4 CYC-treated patients (29%) achieved complete remission. All other patients were in partial remission. There was no difference between both groups in respect to treatment response and partial and complete remission. In both treatment groups, eosinophils, C-reactive protein, and IgE levels dropped. Relapse-free survival within an observation period of 36 months was comparable between RTX- and CYC-treated patients. RTX was well tolerated, but resulted in a decline in serum immunoglobulin levels. CONCLUSIONS: RTX was effective in inducing remission and during long-term follow-up in patients with EGPA, even when previously refractory to standard immunosuppressive therapy including CYC. RTX-treated patients should be monitored for hypogammaglobulinemia. (C) 2017 American Academy of Allergy, Asthma & Immunology

BACKGROUND:The use of zidovudine is associated with a loss of subcutaneous adipose tissue (SAT). We assessed if zidovudine treatment also affects visceral adipose tissue (VAT) and if uridine supplementation abrogates the adverse effects of zidovudine on VAT.METHODS:Rats were fed zidovudine for 21 weeks with or without simultaneous uridine supplementation. Control animals did not receive zidovudine, or were treated with uridine alone. Changes in SAT and VAT were monitored by magnetic resonance imaging. Adipose tissue was examined for structural and molecular signs of mitochondrial toxicity.RESULTS:Zidovudine induced lipoatrophy in SAT and fat hypertrophy in VAT. Compared with controls zidovudine-exposed VAT adipocytes had increased diameters, microvesicular steatosis and enlarged mitochondria with disrupted crystal architecture on electron microscopy. VAT adipocyte mitochondrial DNA (mtDNA) copy numbers were diminished, as were mtDNA-encoded respiratory chain proteins. The 'common' mtDNA deletion was detected in high frequencies in zidovudine treated animals, but not in the controls. Although mtDNA depletion was more profound in SAT compared with VAT, the 'common' deletion tended to be more frequent in the VAT than in the SAT. Uridine coadministration abrogated all effects of zidovudine on VAT and SAT pathology.CONCLUSIONS:Zidovudine induces a gain of intra-abdominal fat in association with quantitative and qualitative alterations of the mitochondrial genome and impaired expression of mtDNA-encoded respiratory chain components, indicating that zidovudine may contribute to abdominal fat hypertrophy in HIV-infected patients. In this rodent model, uridine supplementation abrogates both SAT and VAT pathology induced by zidovudine.

INTRODUCTION:Skeletal muscle fiber composition and muscle energetics are not static and change in muscle disease. This study was performed to determine whether a mitochondrial myopathy is associated with adjustments in skeletal muscle fiber-type composition.METHODS:Ten rats were treated with zidovudine, an antiretroviral nucleoside reverse transcriptase inhibitor that induces a myopathy by interfering with mitochondrial functions. Soleus muscles were examined after 21 weeks of treatment. Ten untreated rats served as controls.RESULTS:Zidovudine induced a myopathy with mitochondrial DNA depletion, abnormalities in mitochondrial ultrastructure, and reduced cytochrome c oxidase activity. Mitochondrial DNA was disproportionally more diminished in type I compared with type II fibers, whereas atrophy predominated in type II fibers. Compared with those of controls, zidovudine-exposed soleus muscles contained an increased proportion (256%) of type II fibers, whereas neonatal myosin heavy chains remained repressed, indicating fiber-type transformation in the absence of regeneration. Microarray gene-expression analysis confirmed enhanced fast-fiber isoforms, repressed slow-fiber transcripts, and reduced neonatal fiber transcripts in the mitochondrial myopathy. Respiratory chain transcripts were diminished, whereas the enzymes of glycolysis and glycogenolysis were enhanced, indicating a metabolic adjustment from oxidative to glycolytic capacities. A coordinated regulation was found of transcription factors known to orchestrate type II fiber formation (upregulation of MyoD, Six1, Six2, Eya1, and Sox6, and downregulation of myogenin and ERRγ).CONCLUSIONS:The type I to type II fiber transformation in mitochondrial myopathy implicates mitochondrial function as a new regulator of skeletal muscle fiber type.

OBJECTIVE:Long-term antiretroviral treatment with nucleoside analogue reverse transcriptase inhibitors (NRTI) may result in a cardiomyopathy due to mitochondrial DNA (mtDNA) depletion. An intact mitochondrial function is required for the synthesis of intramyocardial pyrimidine nucleotides, which in turn are building blocks of mtDNA. We investigated if NRTI-related cardiomyopathy can be prevented with pyrimidine precursors.METHODS:Mice were fed with zidovudine or zalcitabine with or without simultaneous Mitocnol, a dietary supplement with high uridine bioavailability. Myocardia were examined after 9 weeks.RESULTS:Both NRTI induced a cardiomyopathy with mitochondrial enlargement, a disrupted cristal architecture on electron microscopy and diminished myocardial mtDNA copy numbers. The myocardial mtDNA-encoded cytochrome c-oxidase I subunit was impaired more profoundly than the nucleus-encoded cytochrome c-oxidase IV subunit. The myocardial formation of reactive oxygen species and mtDNA mutations was enhanced in zidovudine and zalcitabine treated animals. Mitocnol attenuated or normalized all myocardial pathology when given with both NRTI, but by itself had no intrinsic effects and no apparent adverse effects.CONCLUSIONS:Zidovudine and zalcitabine induce a mitochondrial cardiomyopathy, which is antagonized with uridine supplementation, implicating pyrimidine pool depletion in its pathogenesis. Pyrimidine pool replenishment may be exploited clinically because uridine is well tolerated.

Objective:Peripheral neuropathy and central nervous system neurodegeneration may result from the mitochondrial toxicity of some antiretroviral nucleoside analogues. We investigated whether this neuropathology may be antagonized by uridine supplementation in vivo. Design:Because of the obvious difficulties in obtaining human neural tissues, the mitochondrial neurotoxicity of the nucleoside analogues was studied in mice. Methods:BALB/C mice (7 weeks of age) were fed for 9 weeks with zalcitabine (13 mg/kg per day) or zidovudine (100 mg/kg per day) with or without mitocnol (340 mg/kg per day), a dietary supplement with high uridine bioavailability. Hippocampal and sciatic nerve mitochondria were analyzed. Results:Zalcitabine and to a lesser extent zidovudine induced a significant peripheral neuropathy and encephalopathy with disrupted mitochondrial ultrastructure, depleted mitochondrial DNA, reduced levels of cytochrome c oxidase activity and diminished expression of mitochondrial DNA-encoded cytochrome c oxidase subunit I. Mitocnol had no intrinsic effects but attenuated or fully normalized all measured disorder of the peripheral and central nervous system. Conclusion:Zidovudine and zalcitabine induce a mitochondrial disorder in the peripheral and central nervous system, both of which are antagonized by uridine supplementation.

Fosalvudine tidoxil is a prodrug derived from the nucleoside reverse transcriptase inhibitor 3-deoxy-3-fluorothymidine (FLT; alovudine). FLT effectively inhibits resistant human immunodeficiency virus type 1, but its clinical development was stopped due to bone marrow and liver toxicity. In this study, we examined the long-term in vivo effects of fosalvudine tidoxil on the mitochondrial DNA (mtDNA) contents in rats. Sprague-Dawley rats received fosalvudine tidoxil (15, 40, or 100 mg/kg of body weight/day) by oral gavage during a period of 8 weeks. Didanosine (100 mg/kg/day) was used as a positive control for mitochondrial toxicity. mtDNA levels in liver, gastrocnemius muscle, sciatic nerve, and inguinal fat pad tissues were quantified by real-time PCR. In hepatic mitochondria, fosalvudine tidoxil induced significant mtDNA depletion. At doses of 15, 40, and 100 mg/kg, the mean hepatic mtDNA values were 62, 64, and 47% of control values, respectively. Rats exposed to 100 mg/kg of fosalvudine tidoxil, unlike all other groups, had slightly elevated levels of glutamate pyruvate transaminase in sera. Didanosine induced a loss of mtDNA (to 48% of the control level) similar to that induced by fosalvudine tidoxil. mtDNA levels in skeletal, neural, and adipose tissues in the negative control and treatment groups were similar. Our results suggest that fosalvudine tidoxil induces mitochondrial hepatotoxicity and that this effect warrants scrutiny in clinical trials.

Objectives: Tenofovir disoproxil fumarate (tenofovir DF) use has been associated with renal dysfunction and Fanconi syndrome. Tenofovir is taken up into renal tubules by anion transporters where high intracellular drug concentration may induce a functionally relevant depletion of mitochondrial DNA (mtDNA). We investigated if tenofovir may induce renal mtDNA depletion and respiratory chain dysfunction.Methods: Rats (n = 8) were gavaged daily with 100 mg.kg(-1).d(-1) of tenofovir DF or didanosine. Kidneys and livers were examined after 8 weeks of treatment.Results: The tenofovir group had significantly lower body and kidney weights than rats exposed to water or didanosine. Proximal but not distal tubules were of increased diameter and contained small lipid droplets. Tubular mitochondria were enlarged, and their crystal architecture was disrupted. Tenofovir-exposed kidneys contained low mtDNA copy numbers and impaired expression of mtDNA-encoded cytochrome c oxidase (COX) I but not nucleus-encoded COX IV subunits. Histochemistry demonstrated low tubular COX and nicotinamide adenine dinucleotide dehydrogenase (NADH-DH) activities, whereas succinate dehydrogenase activity was preserved. COX activity was preserved in the glomeruli of tenofovir-exposed rats. Didanosine did not elicit renal effects but, unlike tenofovir.. depleted mtDNA in liver (by 52%).Conclusions: Tenofovir DF induces an organ-specific nephrotoxicity with mtDNA depletion and dysfunction of mtDNA-encoded respiratory chain Subunits. The data do not support nephrotoxicity of didanosine.

To the Editor: The supplementation of uridine prevents and treats the mitochondrial toxicity of pyrimidine nucleoside reverse transcriptase inhibitors (NRTIs) by abrogating mitochondrial DNA (mtDNA) depletion in vitro1-3 and in vivo.4-6 Uridine completely abrogates the lipoatrophic phenotype of adipocytes exposed to zidovudine and stavudine by preventing apoptosis, loss of lipids, mtDNA depletion, and mitochondrial depolarization.1 Uridine also normalizes the proliferation of blood cell precursors exposed to zidovudine.3 A recent randomized, double-blind, and placebo-controlled trial of dietary uridine supplementation demonstrated a markedly rapid and quantitatively significant gain of subcutaneous fat in HIV-infected lipoatrophic subjects under continued treatment with thymidine analogues.4 NucleomaxX (Pharma Nord, Vojens, Denmark) is a food supplement that has a high bioavailability of uridine.7 Product analysis revealed that each 36-g sachet of NucleomaxX contains 1.61% of uridine and 15.0% of 2′,3′,5′-tri-O-acetyluridine, the latter of which can be converted to uridine by plasma esterases.8 Previous studies have shown that compared with pure uridine, 2′,3′,5′-tri-O-acetyluridine exhibits enhanced gastrointestinal tract absorption and is resistant to catabolism by uridine phosphorylase.9 The beneficial effects of uridine were dose dependent and observed at concentrations of 50 to 200 μM, depending on the system studied.1-3 Competition of uridine or its metabolites with NRTIs at polymerase-γ or at enzymes responsible for NRTI activation and transport is the most plausible mechanism of action.2 Such a competitive mechanism, however, also raises the question about the safety of uridine supplementation in HIV-infected patients. Whereas interference of uridine with the antiretroviral efficacy of NRTIs at the intracellular level is unlikely based on phenotypic HIV resistance assays,10 it has not yet been examined whether uridine interferes with competitive processes involved in the gastrointestinal absorption or plasma elimination of pyrimidine NRTIs. The purpose of this study was therefore to assess the effect of uridine supplementation on the pharmacokinetic profile of lamivudine and zidovudine. After ethics committee approval and informed written consent, healthy fasting adult human volunteers (4 male and 4 female, mean body weight of 69.9 ± 9.0 kg) took 1 tablet of zidovudine/lamivudine (300 mg/150 mg) and 2 NRTIs (in a fixed-dose combination [FDC]) that are both pyrimidine analogues. After an 8-day wash-out period, the probands swallowed another tablet of zidovudine/lamivudine (FDC) and simultaneously consumed 36 g of NucleomaxX (1 sachet) dissolved in 200 mL of orange juice. Proband sera were assayed for NRTI concentrations by high-performance liquid chromatography/ultraviolet detection using a Symmetry Octyl 15-cm column (Waters, Milford, MA) with a phosphate buffer/acetonitrile mobile phase. Without uridine supplementation, zidovudine and lamivudine serum levels rose sharply after ingestion of the tablet and peaked after 0.91 hour (SEM = 0.20) and 1.66 hours (SEM = 0.09), respectively (Fig. 1). The mean maximal serum concentration (Cmax) was 1.53 μg/mL (SEM = 0.27) for zidovudine and 1.21 μg/mL (SEM = 0.14) for lamivudine. The mean Cmax of zidovudine was similar in men (1.59 μg/mL) and women (1.48 μg/mL) (P = 0.91). The mean Cmax for lamivudine was 1.05 μg/mL in men and 1.37 μg/mL in women (P = 0.53). Zidovudine was eliminated from the serum with a half-life of 1.0 hour (SEM = 0.12), whereas the half-life of lamivudine was 4.1 hours (SEM = 0.81). There was no significant difference in the elimination time between men and women. The mean AUC0-∞ for zidovudine, calculated with the linear trapezoidal rule between the time points in the time data range, was 2.02 μg/mL × h (SEM = 0.29). The mean area under the curve from zero to infinity (AUC0-∞) for lamivudine was 4.95 μg/mL × h (SEM = 0.88). The pharmacokinetic findings for lamivudine and zidovudine in this study of healthy subjects are consistent with those observed in previous zidovudine/lamivudine FDC studies.11,12FIGURE 1: Lamivudine and zidovudine serum concentrations after consumption of 1 tablet of lamivudine/zidovudine combination with or without simultaneous oral uridine supplementation with NucleomaxX (1 36-g sachet). Data are mean ± SEM.Coadministration of NucleomaxX did not significantly affect any of the pharmacokinetic parameters of zidovudine. Lamivudine Cmax values were significantly higher (P = 0.01) and peaked faster (time to maximum concentration [tmax]; P = 0.02) after NucleomaxX coadministration, but there were no significant differences in the AUC0-∞ or half-life. Adverse events were not observed. We conclude that NucleomaxX does not negatively affect the bioavailability of lamivudine and zidovudine. These safety results, the so far sustained HIV suppression in clinical studies,4,5,13 and the efficacy data provide the rationale for a currently recruiting phase 3 trial of NucleomaxX in lipoatrophy. Nils Venhoff, MD* Ana C. Venhoff, PhD* Anura L. Jayewardene, PhD† Fran Aweeka, PharmD† Dirk Lebrecht, PhD* Ulrich A. Walker, MD* *Department of Rheumatology and Clinical Immunology Medizinische Universitätsklinik Freiburg, Germany †Drug Research Unit Department of Clinical Pharmacy School of Pharmacy University of California, San Francisco San Francisco, CA

Purpose of the study3-methylhistidine (3-MH) acts as an in vivo label of the rate of myofibrillar protein breakdown.The aim of this study was to evaluate 3-MH measurement in spot urine as a simple screening method for muscle protein degradation in HIV-infected persons compared to creatine kinase (CK) measurement in blood. MethodsWe prospectively measured serum creatinine (mg/dl) and serum CK (U/L) and 3-MH (μmol/L) in spot urine from 162 HIV-positive subjects at each visit.3-MH was determined by HPLC after derivatization with fluorescamine.