Clonally expanded CD8 T cells characterize amyotrophic lateral sclerosis-4.

Amyotrophic lateral sclerosis (ALS) is a heterogenous neurodegenerative disorder that affects motor neurons and voluntary muscle control1. ALS heterogeneity includes the age of manifestation, the rate of progression and the anatomical sites of symptom onset. Disease-causing mutations in specific genes have been identified and define different subtypes of ALS1. Although several ALS-associated genes have been shown to affect immune functions2, whether specific immune features account for ALS heterogeneity is poorly understood. Amyotrophic lateral sclerosis-4 (ALS4) is characterized by juvenile onset and slow progression3. Patients with ALS4 show motor difficulties by the time that they are in their thirties, and most of them require devices to assist with walking by their fifties. ALS4 is caused by mutations in the senataxin gene (SETX). Here, using Setx knock-in mice that carry the ALS4-causative L389S mutation, we describe an immunological signature that consists of clonally expanded, terminally differentiated effector memory (TEMRA) CD8 T cells in the central nervous system and the blood of knock-in mice. Increased frequencies of antigen-specific CD8 T cells in knock-in mice mirror the progression of motor neuron disease and correlate with anti-glioma immunity. Furthermore, bone marrow transplantation experiments indicate that the immune system has a key role in ALS4 neurodegeneration. In patients with ALS4, clonally expanded TEMRA CD8 T cells circulate in the peripheral blood. Our results provide evidence of an antigen-specific CD8 T cell response in ALS4, which could be used to unravel disease mechanisms and as a potential biomarker of disease state.

Dianhydrogalactitol Overcomes Multiple Temozolomide Resistance Mechanisms in Glioblastoma.

Glioblastoma (GBM) is the most frequent and aggressive primary tumor type in the central nervous system in adults. Resistance to chemotherapy remains one of the major obstacles in GBM treatment. Identifying and overcoming the mechanisms of therapy resistance is instrumental to develop novel therapeutic approaches for patients with GBM. To determine the major drivers of temozolomide (TMZ) sensitivity, we performed shRNA screenings in GBM lines with different O6-methylguanine-DNA methyl-transferase (MGMT) status. We then evaluated dianhydrogalactitol (Val-083), a small alkylating molecule that induces interstrand DNA crosslinking, as a potential treatment to bypass TMZ-resistance mechanisms. We found that loss of mismatch repair (MMR) components and MGMT expression are mutually exclusive mechanisms driving TMZ resistance in vitro Treatment of established GBM cells and tumorsphere lines with Val-083 induces DNA damage and cell-cycle arrest in G2-M phase, independently of MGMT or MMR status, thus circumventing conventional resistance mechanisms to TMZ. Combination of TMZ and Val-083 shows a synergic cytotoxic effect in tumor cells in vitro, ex vivo, and in vivo We propose this combinatorial treatment as a potential approach for patients with GBM.

Circulating extracellular DNA is an independent predictor of mortality in elderly patients with venous thromboembolism.

BACKGROUND: Venous thromboembolism (VTE) is a major cause of morbidity and mortality in elderly patients. Extracellular DNA is a pro-inflammatory and pro-thrombotic mediator in vitro and in animal models. Levels of circulating extracellular DNA (ceDNA) are increased in VTE patients, but the association of ceDNA with VTE extent and clinical outcome is poorly understood. OBJECTIVES: We analyzed the association of ceDNA with the extent of VTE, categorized as distal and proximal deep vein thrombosis and pulmonary embolism, and with the clinical outcomes VTE recurrence and mortality. METHODS: We quantified ceDNA by a fluorescent probe, as well as circulating nucleosomes and neutrophil extracellular traps (NETs) by ELISA in plasma from 611 patients aged ≥ 65 years with acute VTE of a prospective cohort study (SWITCO65+). RESULTS: Levels of ceDNA and nucleosomes, but not NETs, correlated with VTE extent. Infectious comorbidities independently increased ceDNA levels in VTE. CeDNA strongly correlated with C-reactive protein and leukocytosis, suggesting an association of ceDNA with inflammation in VTE patients. CeDNA furthermore predicted PE-related and all-cause mortality, but not VTE recurrence, during a 3-year follow-up. CONCLUSIONS: Our study suggests that ceDNA levels in VTE patients reflect the degree of inflammation and may serve as a biomarker to stratify VTE patients at risk for mortality.

Host DNases prevent vascular occlusion by neutrophil extracellular traps.

Platelet and fibrin clots occlude blood vessels in hemostasis and thrombosis. Here we report a noncanonical mechanism for vascular occlusion based on neutrophil extracellular traps (NETs), DNA fibers released by neutrophils during inflammation. We investigated which host factors control NETs in vivo and found that two deoxyribonucleases (DNases), DNase1 and DNase1-like 3, degraded NETs in circulation during sterile neutrophilia and septicemia. In the absence of both DNases, intravascular NETs formed clots that obstructed blood vessels and caused organ damage. Vascular occlusions in patients with severe bacterial infections were associated with a defect to degrade NETs ex vivo and the formation of intravascular NET clots. DNase1 and DNase1-like 3 are independently expressed and thus provide dual host protection against deleterious effects of intravascular NETs.

Therapeutic targeting of extracellular DNA improves the outcome of intestinal ischemic reperfusion injury in neonatal rats.

Thrombosis and inflammation cooperate in the development of intestinal infarction. Recent studies suggest that extracellular DNA released by damaged cells or neutrophils in form of extracellular traps (NETs) contributes to organ damage in experimental models of ischemia-reperfusion injury. Here we compared the therapeutic effects of targeting fibrin or extracellular DNA in intestinal infarction after midgut volvulus in rats. Following iatrogenic midgut volvulus induction for 3 hours, we treated animals with a combination of tissue plasminogen activator (tPA) and low molecular weight heparin (LMWH) to target fibrin or with DNase1 to degrade extracellular DNA. The therapeutic effects of tPA/LMWH and DNase1 were analyzed after 7 days. We observed that both therapeutic interventions ameliorated tissue injury, apoptosis, and oxidative stress in the intestine. DNase1, but not tPA/LMWH, reduced intestinal neutrophil infiltration and histone-myeloperoxidase-complexes, a surrogate marker of NETs, in circulation. Importantly, tPA/LMWH, but not DNase1, interfered with hemostasis as evidenced by a prolonged tail bleeding time. In conclusion, our data suggest that the therapeutic targeting of fibrin and extracellular DNA improves the outcome of midgut volvulus in rats. DNase1 therapy reduces the inflammatory response including NETs without increasing the risk of bleeding. Thus, targeting of extracellular DNA may provide a safe therapy for patients with intestinal infarction in future.

Degradation of Extracellular DNA by DNase1 Significantly Reduces Testicular Damage After Testicular Torsion in Rats.

OBJECTIVE: To examine the effects of DNase1 treatment on testicular damage after testicular torsion (TT). It has been demonstrated that TT induces thrombus formation and that anticoagulation significantly reduces testicular damage after TT. It was hypothesized that these thrombi are dependent on neutrophil extracellular traps (NETs) and thus NETs disintegration would reduce testicular cell damage. METHODS: A sham operation was performed in 10 rats. Thirty-four rats underwent induction of iatrogenic TT for 3 hours. After de-torsion and randomization, 24 rats received DNase1 or inactivated DNase1. The following parameters were assessed: testicular damage via Cosentino grading; spermatogenesis via Johnsen score; stem cell factor and c-Kit, apoptosis via Bax, Bcl2, Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling assay, and cleaved caspase3 staining; oxidative stress via superoxide dismutase, catalase, glutathione peroxidase, and malondialdehyde; neutrophil recruitment via myeloperoxidase and neutrophil elastase staining; and NET formation via cell-free DNA. RESULTS: Forty-three rats were included in the study. Subjects treated with DNase1 showed significantly less cellular damage, oxidative stress, and apoptosis. Further, DNase1-treated rats demonstrated a significant improvement of spermatogenesis, compared with the controls. CONCLUSION: The results of the study indicate that thrombus formation during TT is quite likely NET associated, and that dissolution of cell-free DNA (including NETs) significantly improves testicular damage in rats. As treatment with DNase1 reduced apoptosis, oxidative stress, and inflammation, without adversely affecting coagulation, it might be a suitable treatment for (neonatal) TT and ought to be evaluated in humans.

Circulating Extracellular DNA: Cause or Consequence of Thrombosis?

Thrombosis leads to ischemic organ damage in cardiovascular and thromboembolic diseases. Neutrophils promote thrombosis in vitro and in vivo by releasing neutrophil extracellular traps (NETs). NETs are composed of DNA filaments coated with histones and neutrophil enzymes such as myeloperoxidase (MPO). Circulating extracellular DNA (ceDNA) is widely used as a surrogate marker to monitor NET formation in thrombosis. This narrative review summarizes the association of ceDNA with human thrombosis. Levels of ceDNA indicate the extent and outcome of several cardiovascular and thromboembolic diseases, including myocardial infarction, stroke, and venous thromboembolism. ceDNA correlates with markers of coagulation and platelet consumption, thus supporting the hypothesis that ceDNA may be a surrogate marker of thrombus formation. In addition, ceDNA levels correlate with markers of cell injury and size of ischemic lesions, suggesting that ceDNA does not derive from NETs but is probably released from damaged organs. Few studies identified NET-specific biomarkers such as DNA-MPO complexes in the blood of patients with thrombosis. In conclusion, it remains to be established whether ceDNA in patients derives from NETs and is a cause or consequence of thrombosis.

The Chromatin Remodeling Complex Chd4/NuRD Controls Striated Muscle Identity and Metabolic Homeostasis.

Heart muscle maintains blood circulation, while skeletal muscle powers skeletal movement. Despite having similar myofibrilar sarcomeric structures, these striated muscles differentially express specific sarcomere components to meet their distinct contractile requirements. The mechanism responsible is still unclear. We show here that preservation of the identity of the two striated muscle types depends on epigenetic repression of the alternate lineage gene program by the chromatin remodeling complex Chd4/NuRD. Loss of Chd4 in the heart triggers aberrant expression of the skeletal muscle program, causing severe cardiomyopathy and sudden death. Conversely, genetic depletion of Chd4 in skeletal muscle causes inappropriate expression of cardiac genes and myopathy. In both striated tissues, mitochondrial function was also dependent on the Chd4/NuRD complex. We conclude that an epigenetic mechanism controls cardiac and skeletal muscle structural and metabolic identities and that loss of this regulation leads to hybrid striated muscle tissues incompatible with life.

Modulation of Thrombosis Significantly Reduces Testicular Damage after Testicular Torsion in Rats: Anti-Thrombotic Treatment and Testicular Torsion.

OBJECTIVE: To evaluate the effects of thrombolysis and/or anticoagulation on testicular viability after testicular tortion (TT) was the aim of this study. It has been suggested that alterations of circulation during TT result in thrombus formation that might prevent sufficient perfusion after detorsion. Due to the narrow safety margin of testicular perfusion, even moderate disturbances in blood supply can cause major testicular damage. METHODS: In 112 rats, the right testicle was torsed for 3 or 6 hours. After detorsion and randomization, they received either enoxaparin, alteplase, both, or placebo, according to their subgroup. Thrombus formation was accessed via D-dimers, pDNA, oxidative testicular damage was evaluated via glutathione peroxidase and malondialdehyde, and cellular damage via inhibin B, testosterone, histological analysis (Johnsen score, Cosetino grading), and TUNEL assay. RESULTS: One hundred and twelve rats were included in the study. The treatment with alteplase or enoxaparin showed significantly less testicular damage and significantly improved Sertoli cell function. Enoxaparin significantly reduced oxidative impairment. CONCLUSION: The results of the study indicate that TT induces thrombus formation and demonstrate that modulation of thrombosis significantly ameliorates testicular damage in rats. Hence, this treatment option after TT ought to be evaluated in humans.

Tissue factor-dependent and -independent pathways of systemic coagulation activation in acute myeloid leukemia: a single-center cohort study.

BACKGROUND: In acute myeloid leukemia (AML), disseminated intravascular coagulation (DIC) contributes to morbidity and mortality, but the underlying pathomechanisms remain incompletely understood. METHODS: We conducted a prospective study on 69 patients with newly diagnosed AML to further define the correlates of systemic coagulation activation in this hematological malignancy. Tissue factor procoagulant activity (TF PCA) of isolated peripheral blood mononuclear cells (PBMCs) and TF expression by circulating microparticles (MPs) were assessed by single-stage clotting and thrombin generation assay, respectively. Soluble plasma TF antigen and secretion of vascular endothelial growth factor (VEGF) by cultured PBMCs were measured by ELISA. Cell-free plasma DNA was quantified by staining with a fluorescent dye. RESULT: TF PCA of PBMCs was significantly increased in AML patients as compared to healthy controls. Furthermore, TF PCA was significantly associated with decompensated DIC at presentation, as defined by a plasma fibrinogen level of ≤1 g/L (n = 11). In addition to TF PCA and circulating blasts, serum lactate dehydrogenase, a surrogate marker for leukemic cell turnover, correlated with plasma D-Dimer in the total patient cohort and was significantly increased in DIC patients, suggesting a role for myeloblast apoptosis/necrosis in activation of the TF-dependent coagulation pathway. Consistently, TF-bearing plasma MPs were more frequently detected and levels of soluble TF antigen were significantly higher in DIC vs. non-DIC patients. No association was found between TF PCA expression and VEGF secretion by isolated PBMCs, but significantly increased levels of cell-free plasma DNA pointed to a contribution of the intrinsic contact pathway to systemic coagulation activation in the total patient cohort and in patients with lower TF PCA expression. While PBMC-associated TF PCA had no effect on long-term survival, DIC occurrence at presentation increased the risk of early mortality. CONCLUSION: In newly diagnosed AML, TF expression by PBMCs and shedding of TF-bearing plasma MPs are central to the pathogenesis of DIC, but additional pathways, such as DNA liberation, may contribute to systemic coagulation activation.

Complement factor H, FHR-3 and FHR-1 variants associate in an extended haplotype conferring increased risk of atypical hemolytic uremic syndrome.

Atypical hemolytic uremic syndrome (aHUS) is a severe thrombotic microangiopathy affecting the renal microvasculature and is associated with complement dysregulation caused by mutations or autoantibodies. Disease penetrance and severity is modulated by inheritance of "risk" polymorphisms in the complement genes MCP, CFH and CFHR1. We describe the prevalence of mutations, the frequency of risk polymorphisms and the occurrence of anti-FH autoantibodies in a Spanish aHUS cohort (n=367). We also report the identification of a polymorphism in CFHR3 (c.721C>T; rs379370) that is associated with increased risk of aHUS (OR=1.78; CI 1.22-2.59; p=0.002), and is most frequently included in an extended risk haplotype spanning the CFH-CFHR3-CFHR1 genes. This extended haplotype integrates polymorphisms in the promoter region of CFH and CFHR3, and is associated with poorer evolution of renal function and decreased FH levels. The CFH-CFHR3-CFHR1 aHUS-risk haplotype seems to be the same as was previously associated with protection against meningococcal infections, suggesting that the genetic variability in this region is limited to a few extended haplotypes, each with opposite effects in various human diseases. These results suggest that the combination of quantitative and qualitative variations in the complement proteins encoded by CFH, CFHR3 and CFHR1 genes is key for the association of these haplotypes with disease.