Glucagon-like peptide-1 receptor signaling modifies the extent of diabetic kidney disease through dampening the receptor for advanced glycation end products-induced inflammation.

Glucagon like peptide-1 (GLP-1) is a hormone produced and released by cells of the gastrointestinal tract following meal ingestion. GLP-1 receptor agonists (GLP-1RA) exhibit kidney-protective actions through poorly understood mechanisms. Here we interrogated whether the receptor for advanced glycation end products (RAGE) plays a role in mediating the actions of GLP-1 on inflammation and diabetic kidney disease. Mice with deletion of the GLP-1 receptor displayed an abnormal kidney phenotype that was accelerated by diabetes and improved with co-deletion of RAGE in vivo. Activation of the GLP-1 receptor pathway with liraglutide, an anti-diabetic treatment, downregulated kidney RAGE, reduced the expansion of bone marrow myeloid progenitors, promoted M2-like macrophage polarization and lessened markers of kidney damage in diabetic mice. Single cell transcriptomics revealed that liraglutide induced distinct transcriptional changes in kidney endothelial, proximal tubular, podocyte and macrophage cells, which were dominated by pathways involved in nutrient transport and utilization, redox sensing and the resolution of inflammation. The kidney-protective action of liraglutide was corroborated in a non-diabetic model of chronic kidney disease, the subtotal nephrectomised rat. Thus, our findings identify a novel glucose-independent kidney-protective action of GLP-1-based therapies in diabetic kidney disease and provide a valuable resource for exploring the cell-specific kidney transcriptional response ensuing from pharmacological GLP-1R agonism.

Cutting Edge: Processing of Oxidized Peptides in Macrophages Regulates T Cell Activation and Development of Autoimmune Arthritis.

APCs are known to produce NADPH oxidase (NOX) 2-derived reactive oxygen species; however, whether and how NOX2-mediated oxidation affects redox-sensitive immunogenic peptides remains elusive. In this study, we investigated a major immunogenic peptide in glucose-6-phosphate isomerase (G6PI), a potential autoantigen in rheumatoid arthritis, which can form internal disulfide bonds. Ag presentation assays showed that presentation of this G6PI peptide was more efficient in NOX2-deficient (Ncf1m1J/m1J mutant) mice, compared with wild-type controls. IFN-γ-inducible lysosomal thiol reductase (GILT), which facilitates disulfide bond-containing Ag processing, was found to be upregulated in macrophages from Ncf1 mutant mice. Ncf1 mutant mice exhibited more severe G6PI peptide-induced arthritis, which was accompanied by the increased GILT expression in macrophages and enhanced Ag-specific T cell responses. Our results show that NOX2-dependent processing of the redox-sensitive autoantigens by APCs modify T cell activity and development of autoimmune arthritis.

Granulocyte colony-stimulating factor (G-CSF) plays an important role in immune complex-mediated arthritis.

Neutrophils are an abundant cell type in many chronic inflammatory diseases such as rheumatoid arthritis (RA); however, their contribution to the pathology of RA has not been widely studied. A key cytokine involved in neutrophil development and function is granulocyte-colony stimulating factor (G-CSF). In this study we used the K/BxN serum-transfer arthritis (STA) model, mimicking the effector phase of RA, to investigate the importance of G-CSF in arthritis development and its relation to neutrophils. Here, we show for the first time in this model that G-CSF levels are increased both in the serum and in inflamed paws of arthritic mice and importantly that G-CSF blockade leads to a profound reduction in arthritis severity, as well as reduced numbers of neutrophils in blood. Moreover, CXCL1 and CXCL2 levels in the arthritic joints were also lowered. Our data demonstrate that G-CSF is a pivotal driver of the disease progression in the K/BxN STA model and possibly acts in part by regulating neutrophil numbers in the circulation. Therefore, our findings suggest that G-CSF might be a suitable target in RA, and perhaps in other immune complex-driven pathologies.

Anti-IL-21 monoclonal antibody combined with liraglutide effectively reverses established hyperglycemia in mouse models of type 1 diabetes.

Immunotherapy for type 1 diabetes (T1D) has previously focused on suppressing the autoimmune response against pancreatic beta cells to preserve endogenous insulin production and regulate glucose levels. With increased attention toward combination therapy strategies, studies indicate the multifunctional cytokine interleukin-21 (IL-21) may be a suitable target as an immuno-modulatory arm, while glucagon-like peptide-1 receptor (GLP-1R) agonists may be appropriate as a beta cell protective arm in combination therapy for T1D. We report here that treatment with anti-IL-21 monoclonal antibody delays diabetes onset in the spontaneous non-obese diabetic (NOD) and NOD.scid adoptive transfer models, while its effect in reversing recent-onset hyperglycemia is limited. However, the combination of anti-IL-21 plus the GLP-1R agonist liraglutide is effective in reversing established disease compared to either monotherapy in both the NOD and rat insulin promotor-lymphocytic choriomeningitis virus glycoprotein (RIP-LCMV-GP) models of autoimmune diabetes. Enhanced efficacy is particularly evident in severely hyperglycemic mice, with return to normoglycemia remaining stable for the majority of mice even after therapy is withdrawn. Importantly, increased beta cell proliferation does not appear to be the predominant mechanism. In conclusion, combination therapy with anti-IL-21 and liraglutide is able to consistently reverse disease in mouse models of T1D. The observed effects rival the most effective experimental disease-modifying treatments tested in preclinical studies.

Dexamethasone and levetiracetam reduce hetero-cellular gap-junctional coupling between F98 glioma cells and glial cells in vitro.

Gap junctions (GJs) in astrocytes and glioma cells are important channels for cell-to-cell communication that contribute to homo- and heterocellular coupling. According to recent studies, heterocellular gap-junctional communication (H-GJC) between glioma cells and their surrounding environment enhances glioma progression. Therefore, we developed a new in vitro model to examine H-GJC between glioma cells, astrocytes and microglia. Consequently, F98 rat glioma cells were double-labeled with GJ-impermeable (CM-DiI) and GJ-permeable dye (calcein AM) and were seeded on unlabeled astrocyte-microglia co-cultures. Dual whole cell voltage clamp recordings were carried out on selected cell pairs to characterize the functional properties of H-GJC in vitro. The expression of four types of connexins (Cxs), including Cx32, Cx36, Cx43 and Cx45, and microglial phenotypes were analyzed by immunocytochemistry. The H-GJC between glioma cells and astrocytes/microglia increased after a longer incubation period with a higher number of glioma cells. We provided evidence for the direct GJ coupling of microglia and glioma cells under native in vitro conditions. In addition, we exploited this model to evaluate H-GJC after incubation with levetiracetam (LEV) and/or dexamethasone (DEX). Previous in vitro studies suggest that LEV and DEX are frequently used to control seizure and edema in glioma. Our findings showed that LEV and/or DEX decrease the number of heterocellular coupled cells significantly. In conclusion, our newly developed model demonstrated H-GJC between glioma cells and both astrocytes and microglia. The reduced H-GJC by LEV and DEX suggests a potential effect of both drugs on glioma progression.

Glia and epilepsy: experimental investigation of antiepileptic drugs in an astroglia/microglia co-culture model of inflammation.

PURPOSE: The contribution of glial cells, mainly astrocytes and microglia, to the pathophysiology of epilepsy is increasingly appreciated. Glia play a pivotal role in the initiation and maintenance of the central nervous system (CNS) immune response and neuronal metabolic and trophic supply. Recent clinical and experimental evidence suggests a direct relationship between epileptic activity and CNS inflammation, which is characterized by accumulation, activation, and proliferation of microglia and astrocytes. Concomitant glia-mediated mechanisms of action of several antiepileptic drugs (AEDs) have been proposed. However, their direct effects on glial cells have been rarely investigated. We aimed to investigate the effect of commonly used AEDs on glial viability, the gap junctional network, the microglial activation, and cytokine expression in an in vitro astroglia/microglia co-culture model. METHODS: Primary astrocytic cultures were prepared from brains of postnatal (P0-P2) Wistar rats and co-cultured with a physiologic amount of 5%, as well as 30% microglia in order to mimic inflammatory conditions. Co-cultures were treated with valproic acid (VPA), carbamazepine (CBZ), phenytoin (PHE), and gabapentin (GBT). Viability and proliferation were measured using the tetrazolium (MTT) assay. The microglial activation state was determined by immunocytochemical labeling. The astroglial connexin 43 (Cx43) expression was measured by Western blot analysis. The transforming growth factor-ß1 (TGF-ß1) and tumor necrosis factor-α (TNF-α) cytokine levels were measured by the quantitative sandwich enzyme immunosorbent assay (ELISA). KEY FINDINGS: Astrocytes, co-cultured with 5% microglia (M5 co-cultures), showed a dose-dependent, significant reduction in glial viability after incubation with PHE and CBZ. Furthermore, VPA led to highly significant microglial activation at all doses examined. The antiinflammatory cytokine TGF-ß1 release was induced by high doses of GBT and PHE. Astrocytes co-cultured with 30% microglia (M30 co-cultures) revealed a dose-dependent significant reduction in glial viability after incubation with PHE, accompanied by increased TGF-ß1 and TNF-α levels. However, CBZ significantly reduced the amount of activated microglial cells and increased the total number of inactivated microglia. Finally, CBZ resulted in reduced viability at all doses examined. SIGNIFICANCE: CNS inflammation is characterized by a disturbance of glial cell functions. Strong microglial activation, a typical hallmark of inflammation, was induced by VPA in M5 and continued in M30 co-cultures. With regard to the direct relation between CNS inflammation and seizures, VPA seems to be unsuitable for reducing inflammatory conditions. The reverse effect was achieved after CBZ. We noticed significant microglial inactivation, after incubation of the M30 co-cultures. In conclusion, we suggest that AEDs with antiinflammatory glial features are beneficial for seizures caused by persistent brain inflammation.

Autoantibodies to glutamate receptor antigens in multiple sclerosis and Rasmussen's encephalitis.

BACKGROUND: Glutamate and its specific ionotropic receptors, including N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors, are supposed to play an important role in neurodegeneration as well as neuronal regeneration. Although autoantibodies (aab) to glutamate receptors (GluR) have been identified in several neurologic diseases, including paraneoplastic encephalitis and Rasmussen's encephalitis (RE) with an increasing prevalence, the presence and role of anti-GluR aab in multiple sclerosis (MS) have not been studied yet. OBJECTIVES AND METHODS: In this study, we tested the serum samples of 56 subjects, including patients with relapsing-remitting MS (n = 25), patients with RE (n = 8), and healthy donors (HD; n = 23), for anti-GluR aab by immunoblot analysis of a panel of recombinantly expressed GluR proteins, including GluN1, GluN2C, GluA3, GluK2, and GluD2. RESULTS: aab were mainly found directed against GluN1 and, except for one aab positive to GluK2 in 1 MS patient and 2 HD controls positive for GluA3, no other anti-GluR aab were detected. In the sera of RE patients, no anti-GluR aab were found. In patients with MS, 8 of the 25 sera (32%) tested positive for GluN1. Compared to the HD (6/23; 26%), this difference was not statistically significant (p = 0.28). CONCLUSIONS: Our study showed that if anti-GluR aab were detectable in HD and MS patients, they were mainly directed against GluN1 (in particular to oligomeric protein complexes) and were not found in RE. Those antibodies may have low titers and low affinities and might be considered an immune epiphenomenon. Hence, further studies will have to clarify their potential role as a surrogate marker for the extent of neuronal destruction or regeneration, respectively.

Do post-translational beta cell protein modifications trigger type 1 diabetes?

Type 1 diabetes is considered an autoimmune disease characterised by specific T cell-mediated destruction of the insulin-producing beta cells. Yet, except for insulin, no beta cell-specific antigens have been discovered. This may imply that the autoantigens in type 1 diabetes exist in modified forms capable of specifically triggering beta cell destruction. In other immune-mediated diseases, autoantigens targeted by the immune system have undergone post-translational modification (PTM), thereby creating tissue-specific neo-epitopes. In a similar manner, PTM of beta cell proteins might create beta cell-specific neo-epitopes. We suggest that the current paradigm of type 1 diabetes as a classical autoimmune disease should be reconsidered since the immune response may not be directed against native beta cell proteins. A modified model for the pathogenetic events taking place in islets leading to the T cell attack against beta cells is presented. In this model, PTM plays a prominent role in triggering beta cell destruction. We discuss literature of relevance and perform genetic and human islet gene expression analyses. Both direct and circumstantial support for the involvement of PTM in type 1 diabetes exists in the published literature. Furthermore, we report that cytokines change the expression levels of several genes encoding proteins involved in PTM processes in human islets, and that there are type 1 diabetes-associated polymorphisms in a number of these. In conclusion, data from the literature and presented experimental data support the notion that PTM of beta cell proteins may be involved in triggering beta cell destruction in type 1 diabetes. If the beta cell antigens recognised by the immune system foremost come from modified proteins rather than native ones, the concept of type 1 diabetes as a classical autoimmune disease is open for debate.

NKG2D blockade facilitates diabetes prevention by antigen-specific Tregs in a virus-induced model of diabetes.

It is thought that viral infections might jeopardize regulatory T cell therapy in type 1 diabetes. Viral infections can lead to surface expression of ligands for the activating NKG2D receptor, such as retinoic acid early transcript 1 (Rae-1), whose expression on beta-cells recruits NKG2D(+) autoreactive CD8(+) T cells. Both in men and mice, autoreactive cytotoxic T cells express NKG2D. We showed that NKG2D expression increased on CD4(+) and CD8(+) T cells during virus-induced diabetes development in the rat insulin promotor (RIP) Lymphocytic Choriomeningitis Virus (LCMV) model. Combination treatment with anti-NKG2D and antigen-specific regulatory T cells (Treg), at doses inefficacious in mono-treatment, synergized to prevent diabetes in 75% of the virus-infected RIP-LCMV mice. Nevertheless, NKG2D blockade alone failed to reverse recent-onset diabetes in non-obese diabetic (NOD) mice, despite downregulation of NKG2D on NK cells in the blood and CD8(+) T cells in the spleen and pancreatic lymph nodes. Our data suggest that blocking the interaction of NKG2D with it ligands is insufficient to protect against diabetes when a strong inflammatory process actively drives NKG2D upregulation, but should be considered to help maintaining Treg functionality during ongoing pancreatic inflammation.

Blockade of NKG2D ameliorates disease in mice with collagen-induced arthritis: a potential pathogenic role in chronic inflammatory arthritis.

OBJECTIVE: To assess the role of the activating receptor NKG2D in arthritis. METHODS: Levels of NKG2D and its ligands were determined by fluorescence-activated cell sorting, real-time polymerase chain reaction, and immunohistochemistry in rheumatoid arthritis (RA) synovial membrane tissue and in paw tissue from arthritic mice. Arthritis was induced in DBA/1 mice by immunization with type II collagen, and mice were treated intraperitoneally with a blocking anti-NKG2D antibody (CX5) on days 1, 5, and 8 after clinical onset and were monitored for 10 days. RESULTS: We demonstrated expression of NKG2D and its ligands on human RA synovial cells and extended this finding to the paws of arthritic mice. Expression of messenger RNA for the NKG2D ligand Rae-1 was up-regulated, and NKG2D was present predominantly on natural killer (NK) and CD4+ T cells, in arthritic paw cell isolates. NKG2D was down-modulated during the progression of collagen-induced arthritis (CIA). NKG2D expression in arthritic paws was demonstrated by immunohistochemistry. Blockade of NKG2D ameliorated established CIA, with significant reductions in clinical scores and paw swelling. Histologic analysis of arthritic joints from anti-NKG2D-treated mice demonstrated significant joint protection, compared with control mice. Moreover, anti-NKG2D treatment significantly reduced both interleukin-17 production from CD4+ T cells in arthritic paws and splenic NK cell cytotoxic effector functions in vivo and in vitro. CONCLUSION: Our findings indicate that blockade of NKG2D in a murine model and in human explants has beneficial therapeutic potential that merits further investigation in RA.

Pharyngeal electrical stimulation for neurogenic dysphagia following stroke, traumatic brain injury or other causes: Main results from the PHADER cohort study.

BACKGROUND: Neurogenic dysphagia is common and has no definitive treatment. We assessed whether pharyngeal electrical stimulation (PES) is associated with reduced dysphagia. METHODS: The PHAryngeal electrical stimulation for treatment of neurogenic Dysphagia European Registry (PHADER) was a prospective single-arm observational cohort study. Participants were recruited with neurogenic dysphagia (comprising five groups - stroke not needing ventilation; stroke needing ventilation; ventilation acquired; traumatic brain injury; other neurological causes). PES was administered once daily for three days. The primary outcome was the validated dysphagia severity rating scale (DSRS, score best-worst 0-12) at 3 months. FINDINGS: Of 255 enrolled patients from 14 centres in Austria, Germany and UK, 10 failed screening. At baseline, mean (standard deviation) or median [interquartile range]: age 68 (14) years, male 71%, DSRS 11·4 (1·7), time from onset to treatment 32 [44] days; age, time and DSRS differed between diagnostic groups. Insertion of PES catheters was successfully inserted in 239/245 (98%) participants, and was typically easy taking 11·8 min. 9 participants withdrew before the end of treatment. DSRS improved significantly in all dysphagia groups, difference in means (95% confidence intervals, CI) from 0 to 3 months: stroke (n = 79) -6·7 (-7·8, -5·5), ventilated stroke (n = 98) -6·5 (-7·6, -5·5); ventilation acquired (n = 35) -6·6 (-8·4, -4·8); traumatic brain injury (n = 24) -4·5 (-6·6, -2·4). The results for DSRS were mirrored for instrumentally assessed penetration aspiration scale scores. DSRS improved in both supratentorial and infratentorial stroke, with no difference between them (p = 0·32). In previously ventilated participants with tracheotomy, DSRS improved more in participants who could be decannulated (n = 66) -7·5 (-8·6, -6·5) versus not decannulated (n = 33) -2·1 (-3·2, -1·0) (p<0·001). 74 serious adverse events (SAE) occurred in 60 participants with pneumonia (9·2%) the most frequent SAE. INTERPRETATION: In patients with neurogenic dysphagia, PES was safe and associated with reduced measures of dysphagia and penetration/aspiration. FUNDING: Phagenesis Ltd.

Antagonizing dabigatran by idarucizumab in cases of ischemic stroke or intracranial hemorrhage in Germany-Updated series of 120 cases.

BACKGROUND: Idarucizumab is a monoclonal antibody fragment with high affinity for dabigatran reversing its anticoagulant effects within minutes. Thereby, patients with acute ischemic stroke who are on dabigatran treatment may become eligible for thrombolysis with recombinant tissue-type plasminogen activator (rt-PA). In patients on dabigatran with intracerebral hemorrhage idarucizumab could prevent lesion growth. AIMS: To provide insights into the clinical use of idarucizumab in patients under effective dabigatran anticoagulation presenting with signs of acute ischemic stroke or intracranial hemorrhage. METHODS: Retrospective data collected from German neurological/neurosurgical departments administering idarucizumab following product launch from January 2016 to August 2018 were used. RESULTS: One-hundred and twenty stroke patients received idarucizumab in 61 stroke centers. Eighty patients treated with dabigatran presented with ischemic stroke and 40 patients suffered intracranial bleeding (intracerebral hemorrhage (ICH) in n = 27). In patients receiving intravenous thrombolysis with rt-PA following idarucizumab, 78% showed a median improvement of 7 points in National Institutes of Health Stroke Scale. No bleeding complications were reported. Hematoma growth was observed in 3 out of 27 patients with ICH. Outcome was favorable with a median National Institutes of Health Stroke Scale improvement of 4 points and modified Rankin score 0-3 in 61%. Six out of 40 individuals (15%) with intracranial bleeding died during hospital stay. CONCLUSION: Administration of rt-PA after reversal of dabigatran activity with idarucizumab in case of acute ischemic stroke seems feasible, effective, and safe. In dabigatran-associated intracranial hemorrhage, idarucizumab appears to prevent hematoma growth and to improve outcome.

Executive deficits in generalized and extrafrontal partial epilepsy: long versus short seizure-free periods.

OBJECTIVE: The goal of this study was to examine the influence of seizure freedom on executive function in outpatients with generalized epilepsy and extrafrontal partial epilepsy. Recent investigations of cognitive function in epilepsy have revealed executive deficits in persons with focal temporal as well as generalized epilepsies. Additional studies have suggested an influence of seizure freedom on cognitive function. METHODS: Thirty-five consecutive outpatients were divided into seizure free 3 months (n=17). The neuropsychological tests administered included: verbal fluency tasks, the Cognitive Estimation Test, the Hayling and Brixton Test, and the Behavioural Assessment of the Dysexecutive Syndrome (BADS) battery. Both patient groups were compared with matched healthy controls (n=16). RESULTS: The extensive testing revealed significant differences between patients with shorter seizure-free periods and healthy controls with respect to overall errors and phonemic verbal fluency, response suppression, and BADS overall profile scores. Subjects seizure free >3 months exhibited a trend toward impairment in the phonemic fluency task only. CONCLUSIONS: The results suggest that deficits in executive function were present in patients with extrafrontal partial epilepsy and generalized epilepsy, indicating the potential influence of epileptic activity on the ability to focus on relevant information and switch attention to other relevant information, to plan tasks and subtasks, and to check on and encode working memory content. The results also suggest that those deficits may be more pronounced in patients with relatively short seizure-free periods.

Evaluating IL-21 as a Potential Therapeutic Target in Crohn's Disease.

BACKGROUND AND AIM: Interleukin-21 (IL-21) is primarily a T cell-derived cytokine; it is upregulated in patients with Crohn's Disease (CD) and could be a potential new therapeutic target in CD. METHODS: In human material, IL-21 and IL-21R expression was investigated by in situ hybridization (ISH) and immunohistochemistry (IHC) in noninflammatory bowel disease (non-IBD) controls and patients with CD. The pathologic role of IL-21 was examined in murine models of T cell-dependent and T cell-independent colitis, either with a neutralizing monoclonal antibody against IL-21 or with the transfer of CD4+CD45RBhighIL-21R-/- T cells. Colonic pathology was examined by endoscopy, histopathology, IHC, ELISA, and Luminex. RESULTS: In the human intestine, IL-21 and IL-21R mRNA and protein-expressing cells were observed in the mucosa, in lymphoid aggregates of submucosa in non-IBD controls, and in lymphoid aggregates of muscularis externa in patients with CD. IL-21 expression was most abundant in germinal centers (GCs) of the lymphoid aggregates, and IL-21R expression assessed semiquantitatively, was significantly higher in patients with CD compared to non-IBD controls. Following prophylactic and interventive anti-IL-21 mAb treatment in the adoptive transfer (AdTr) model, clinical and pathological parameters were significantly reduced. The most persistent finding was a reduction in colonic infiltrating neutrophils. As well, Rag2-/- mice receiving CD4+CD45RBhighIL-21R-/- T cells developed less severe colitis compared to Rag2-/- mice receiving CD4+CD45RBhighIL-21R+/+ T cells. No effect of reduced IL-21 signalling was observed in T cell-independent colitis. CONCLUSION: Our study shows that patients with CD have significant expression of IL-21 and IL-21R in the gut. As well, we show that neutralization of IL-21 in experimental T cell-driven colitis is associated with a reduction in clinical and pathological findings. This amelioration seems to be associated with a reduction in colon-infiltrating neutrophils.

CD40 is required for development of islet inflammation in the RIP-CD154 transgenic mouse model of type 1 diabetes.

Type 1 diabetes is believed to be an autoimmune disease where cells of the immune system destroy the insulin-producing beta cells in the islets of Langerhans. The trigger(s) of the inflammatory reaction is yet unknown, but both genetic and environmental factors, including viruses or other pathogens, are thought to play a role. We have recently described a transgenic mouse model--the RIP-CD154 mouse--in which beta-cell-specific expression of CD154 (CD40 ligand) mediates immune activation, insulitis, and diabetes on a non-diabetes-prone background. By the use of bone marrow chimeric mice, we now demonstrate that a functional Cd40 gene is necessary for islet inflammation and we show that CD40 expression on bone marrow-derived cells is sufficient to trigger activation of the immune system and development of insulitis.

Antagonizing dabigatran by idarucizumab in cases of ischemic stroke or intracranial hemorrhage in Germany - A national case collection.

Background Idarucizumab is a monoclonal antibody fragment with high affinity for dabigatran that reverses its anticoagulant effects within minutes. It may exhibit the potential for patients under dabigatran therapy suffering ischemic stroke to regain eligibility for thrombolysis with rt-PA and may inhibit lesion growth in patients with intracerebral hemorrhage on dabigatran. Aims To provide insights into the clinical use of idarucizumab in patients under effective dabigatran anticoagulation presenting with signs of ischemic stroke or intracranial hemorrhage. Methods Retrospective data collected from German neurological/neurosurgical departments administering idarucizumab following product launch from January to August 2016 were used. Results Thirty-one patients presenting with signs of stroke received idarucizumab in 22 stroke centers. Nineteen patients treated with dabigatran presented with ischemic stroke and 12 patients suffered from intracranial bleeding. In patients receiving rt-PA thrombolysis following idarucizumab, 79% benefitted from i.v. thrombolysis with a median improvement of five points in NIHSS. No bleeding complications occurred. Hematoma growth was observed in 2 out of 12 patients with intracranial hemorrhage. The outcome was favorable with a median NIHSS improvement of 5.5 points and mRS 0-3 in 67%. Overall, mortality was low with 6.5% (one patient in each group). Conclusion Administration of rt-PA after reversing dabigatran activity with idarucizumab in case of ischemic stroke is feasible, easy to manage, effective, and appears to be safe. In dabigatran-associated intracranial hemorrhage, idarucizumab has the potential to prevent hematoma growth and improve outcome. Idarucizumab represents a new therapeutic option for patients under dabigatran treatment presenting with ischemic stroke or intracranial hemorrhage.

Phase I clinical and pharmacokinetic study of the Novel Raf kinase and vascular endothelial growth factor receptor inhibitor BAY 43-9006 in patients with advanced refractory solid tumors.

PURPOSE: BAY 43-9006 is a novel dual-action Raf kinase and vascular endothelial growth factor receptor inhibitor that inhibits tumor cell proliferation and angiogenesis. This study established the safety and pharmacokinetics of BAY 43-9006 in 69 patients with advanced refractory solid tumors. PATIENTS AND METHODS: BAY 43-9006 (50 to 800 mg) was administered once or twice daily on a varying weekly schedule. Pharmacokinetic sampling was performed in all patients; preliminary tumor response was also assessed. The effect of BAY 43-9006 on phorbol myristate acetate-stimulated ERK phosphorylation in peripheral blood lymphocytes was studied using flow cytometry. RESULTS: Mild to moderate diarrhea was the most common (55%) treatment-related adverse event. The maximum-tolerated dose was 400 mg bid continuous. Dose-limiting toxicities were grade 3 diarrhea and fatigue at 800 mg bid, and grade 3 skin toxicity at 600 mg bid. BAY 43-9006 pharmacokinetics were highly variable for single and multiple dosing, and toxicity did not appear to be dose dependent. Significant decreases of phorbol myristate acetate-stimulated ERK phosphorylation (P < .01) were identified at doses >/= 200 mg bid continuous. Forty-five patients were assessable for efficacy; one patient had a partial response (hepatocellular carcinoma at 400 mg bid continuous), 25 patients had stable disease, with eight lasting > 6 months and five for >12 months. Eighteen patients had progressive disease, and tumor response could not be evaluated in one patient. CONCLUSION: Oral BAY 43-9006 was well tolerated and appeared to provide some clinical benefits. Based on the results of this study, BAY 43-9006 at 400 mg bid continuous is recommended for ongoing and future studies.

K/BxN Serum-Transfer Arthritis as a Model for Human Inflammatory Arthritis.

The K/BxN serum-transfer arthritis (STA) model is a murine model in which the immunological mechanisms occurring in rheumatoid arthritis (RA) and other arthritides can be studied. To induce K/BxN STA, serum from arthritic transgenic K/BxN mice is transferred to naive mice and manifestations of arthritis occur a few days later. The inflammatory response in the model is driven by autoantibodies against the ubiquitously expressed self-antigen, glucose-6-phosphate isomerase (G6PI), leading to the formation of immune complexes that drive the activation of different innate immune cells such as neutrophils, macrophages, and possibly mast cells. The pathogenesis further involves a range of immune mediators including cytokines, chemokines, complement factors, Toll-like receptors, Fc receptors, and integrins, as well as factors involved in pain and bone erosion. Hence, even though the K/BxN STA model mimics only the effector phase of RA, it still involves a wide range of relevant disease mediators. Additionally, as a murine model for arthritis, the K/BxN STA model has some obvious advantages. First, it has a rapid and robust onset of arthritis with 100% incidence in genetically identical animals. Second, it can be induced in a wide range of strain backgrounds and can therefore also be induced in gene-deficient strains to study the specific importance of disease mediators. Even though G6PI might not be an essential autoantigen, for example, in RA, the K/BxN STA model is a useful tool to understand how autoantibodies, in general, drive the progression of arthritis by interacting with downstream components of the innate immune system. Finally, the model has also proven useful as a model wherein arthritic pain can be studied. Taken together, these features make the K/BxN STA model a relevant one for RA, and it is a potentially valuable tool, especially for the preclinical screening of new therapeutic targets for RA and perhaps other forms of inflammatory arthritis. Here, we describe the molecular and cellular pathways in the development of K/BxN STA focusing on the recent advances in the understanding of the important mechanisms. Additionally, this review provides a comparison of the K/BxN STA model to some other arthritis models.

Lipidomic and metabolomic characterization of a genetically modified mouse model of the early stages of human type 1 diabetes pathogenesis.

The early mechanisms regulating progression towards beta cell failure in type 1 diabetes (T1D) are poorly understood, but it is generally acknowledged that genetic and environmental components are involved. The metabolomic phenotype is sensitive to minor variations in both, and accordingly reflects changes that may lead to the development of T1D. We used two different extraction methods in combination with both liquid- and gas chromatographic techniques coupled to mass spectrometry to profile the metabolites in a transgenic non-diabetes prone C57BL/6 mouse expressing CD154 under the control of the rat insulin promoter (RIP) crossed into the immuno-deficient recombination-activating gene (RAG) knockout (-/-) C57BL/6 mouse, resembling the early stages of human T1D. We hypothesized that alterations in the metabolomic phenotype would characterize the early pathogenesis of T1D, thus metabolomic profiling could provide new insight to the development of T1D. Comparison of the metabolome of the RIP CD154 × RAG-/- mice to RAG-/- mice and C57BL/6 mice revealed alterations of >100 different lipids and metabolites in serum. Low lysophosphatidylcholine levels, accumulation of ceramides as well as methionine deficits were detected in the pre-type 1 diabetic mice. Additionally higher lysophosphatidylinositol levels and low phosphatidylglycerol levels where novel findings in the pre-type 1 diabetic mice. These observations suggest that metabolomic disturbances precede the onset of T1D.

Anti-RANKL treatment inhibits erosive joint destruction and lowers inflammation but has no effect on bone formation in the delayed-type hypersensitivity arthritis (DTHA) model.

BACKGROUND: The aims of the present study were to determine the relationship between bone destruction and bone formation in the delayed-type hypersensitivity arthritis (DTHA) model and to evaluate the effect of receptor activator of nuclear factor κB ligand (RANKL) blockade on severity of arthritis, bone destruction, and bone formation. METHODS: DTHA was induced in C57BL/6 mice. Inflammation, erosive joint damage, and new bone formation were semiquantitatively scored by histology. Osteoclast activity was assessed in vivo, and messenger RNA (mRNA) expression of mediators of bone destruction and bone formation were analyzed by mRNA deep sequencing. Serum concentrations of tartrate-resistant acid phosphatase 5b, carboxy-terminal telopeptide I (CTX-I), matrix metalloproteinase 3 (MMP3), and serum amyloid P component (SAP) were determined by enzyme-linked immunosorbent assay. Anti-RANKL monoclonal antibody treatment was initiated at the time of immunization. RESULTS: Bone destruction (MMP3 serum levels, cathepsin B activity, and RANKL mRNA) peaked at day 3 after arthritis induction, followed by a peak in cartilage destruction and bone erosion on day 5 after arthritis induction. Periarticular bone formation was observed from day 10. Induction of new bone formation indicated by enhanced Runx2, collagen X, osteocalcin, MMP2, MMP9, and MMP13 mRNA expression was observed only between days 8 and 11. Anti-RANKL treatment resulted in a modest reduction in paw and ankle swelling and a reduction of serum levels of SAP, MMP3, and CTX-I. Destruction of the subchondral bone was significantly reduced, while no effect on bone formation was seen. CONCLUSIONS: Anti-RANKL treatment prevents joint destruction but does not prevent new bone formation in the DTHA model. Thus, although occurring sequentially during the course of DTHA, bone destruction and bone formation are apparently not linked in this model.