Delayed plasma kallikrein inhibition fosters post-stroke recovery by reducing thrombo-inflammation.

Activation of the kallikrein-kinin system promotes vascular leakage, inflammation, and neurodegeneration in ischemic stroke. Inhibition of plasma kallikrein (PK) - a key component of the KKS - in the acute phase of ischemic stroke has been reported to reduce thrombosis, inflammation, and damage to the blood-brain barrier. However, the role of PK during the recovery phase after cerebral ischemia is unknown. To this end, we evaluated the effect of subacute PK inhibition starting from day 3 on the recovery process after transient middle artery occlusion (tMCAO). Our study demonstrated a protective effect of PK inhibition by reducing infarct volume and improving functional outcome at day 7 after tMCAO. In addition, we observed reduced thrombus formation in cerebral microvessels, fewer infiltrated immune cells, and an improvement in blood-brain barrier integrity. This protective effect was facilitated by promoting tight junction reintegration, reducing detrimental matrix metalloproteinases, and upregulating regenerative angiogenic markers. Our findings suggest that PK inhibition in the subacute phase might be a promising approach to accelerate the post-stroke recovery process.

Thromboinflammation in Brain Ischemia: Recent Updates and Future Perspectives.

Despite decades of promising preclinical validation and clinical translation, ischemic stroke still remains as one of the leading causes of death and disability worldwide. Within its complex pathophysiological signatures, thrombosis and inflammation, that is, thromboinflammation, are highly interconnected processes leading to cerebral vessel occlusion, inflammatory responses, and severe neuronal damage following the ischemic event. Hence, we here review the most recent updates on thromboinflammatory-dependent mediators relevant after stroke focusing on recent discoveries on platelet modulation, a potential regulation of the innate and adaptive immune system in thromboinflammation, utterly providing a thorough up-to-date overview of all therapeutic approaches currently undergoing clinical trial.

Smad7 in intestinal CD4+ T cells determines autoimmunity in a spontaneous model of multiple sclerosis.

Environmental triggers acting at the intestinal barrier are thought to contribute to the initiation of autoimmune disorders. The transforming growth factor beta inhibitor Smad7 determines the phenotype of CD4+ T cells. We hypothesized that Smad7 in intestinal CD4+ T cells controls initiation of opticospinal encephalomyelitis (OSE), a murine model of multiple sclerosis (MS), depending on the presence of gut microbiota. Smad7 was overexpressed or deleted in OSE CD4+ T cells to determine the effect on clinical progression, T cell differentiation, and T cell migration from the intestine to the central nervous system (CNS). Smad7 overexpression worsened the clinical course of OSE and increased CNS inflammation and demyelination. It favored expansion of intestinal CD4+ T cells toward an inflammatory phenotype and migration of intestinal CD4+ T cells to the CNS. Intestinal biopsies from MS patients revealed decreased transforming growth factor beta signaling with a shift toward inflammatory T cell subtypes. Smad7 in intestinal T cells might represent a valuable therapeutic target for MS to achieve immunologic tolerance in the intestine and suppress CNS inflammation.

Unexpected additive effects of minocycline and hydroxychloroquine in models of multiple sclerosis: Prospective combination treatment for progressive disease?

BACKGROUND: Most multiple sclerosis (MS) patients succumb to a progressive phenotype. Continued lymphocyte activity in the brain, microglia-mediated injury, iron deposition, and oxidative stress are characteristics of progressive MS. OBJECTIVE: As minocycline and hydroxychloroquine have been shown to inhibit microglia, we evaluated their effects on other outcomes relevant for progression. METHODS: Medications were evaluated in culture and in mice with acute and chronic experimental autoimmune encephalomyelitis (EAE). RESULTS: Both medications individually reduced iron neurotoxicity and a combination effect was not observed. Hydroxyl radical scavenging activity was manifested by minocycline only. Minocycline reduced T-cell proliferation more prominently than hydroxychloroquine; an aggregate effect occurred at low but not high concentrations. B-cell proliferation was mitigated to a greater extent by hydroxychloroquine and an additive effect was not evident. In EAE, suboptimal doses of minocycline and hydroxychloroquine individually delayed onset of clinical signs, while their combination suppressed clinical manifestations until treatment was stopped. In Biozzi ABH mice, a model of progressive MS, the chronic phase was beneficially altered using the combination. CONCLUSION: While minocycline and hydroxychloroquine did not manifest additive effects in most culture assays, their combination at suboptimal doses in EAE unexpectedly exceeded their individual activity. Minocycline and hydroxychloroquine combined are candidate treatments for progressive MS.

Activation of TGF-ß-induced non-Smad signaling pathways during Th17 differentiation.

Although transforming growth factor-ß (TGF-ß) has been shown to positively regulate the development of murine T helper type 17 (Th17) cells, which of the intracellular signaling pathways are involved is controversial. We examined Smad-dependent and -independent signaling molecules downstream of the TGF-ß receptor (TGFßR) involved in Th17 differentiation of naive murine CD4(+)CD62L(+) T cells. During Th17 differentiation of wild-type T cells, Smad2/3 was phosphorylated, indicating activation of the canonical Smad pathway. T cells lacking TGFßRII did not differentiate into Th17, whereas T cells treated with a TGFßRI kinase inhibitor (SB-431542) or overexpression of inhibitory Smad7 retained a low amount of Th17 polarization despite absent Smad2/3 phosphorylation. Using protein antibody arrays we found an increase of expression and phosphorylation of the following Smad-independent signaling molecules in Th17-polarized wild-type T cells: AKT1(Tyr474), AKT2 (Ser474), ERK1-p44/42 MAPK(Tyr204), mTOR(Thr2446), p38 MAPK(Thr180), Rac1/cdc42(Ser71), SAPK/JNK(Tyr185) and SP1(Thr739). Pharmacological inhibition of AKT/mammalian target of rapamycin (mTOR) signaling with rapamycin or LY294002 decreased Th17 differentiation of wild-type T cells, and completely abolished interleukin-17 production in T cells with overexpression of Smad7. Rapamycin and LY294002 also decreased induced regulatory T cell differentiation, but only had minor additive effects to Smad7 overexpression. Finally, inhibitors of mitogen-activated protein kinase (MAPK) blocked in vitro polarization of Th17 cells. Our data show that Smad-dependent and -independent intracellular pathways contribute to murine Th17 differentiation.

Prophylactic Glatiramer Acetate Treatment Positively Attenuates Spontaneous Opticospinal Encephalomyelitis.

Background: Glatiramer acetate (GA) is a well-established treatment option for patients with clinically isolated syndrome and relapsing-remitting multiple sclerosis (MS) with few side effects. The double transgenic mouse model spontaneous opticospinal encephalomyelitis (OSE), based on recombinant myelin oligodendrocyte glycoprotein35-55 reactive T and B cells, mimicks features of chronic inflammation and degeneration in MS and related disorders. Here, we investigated the effects of prophylactic GA treatment on the clinical course, histological alterations and peripheral immune cells in OSE. Objective: To investigate the effects of prophylactic glatiramer acetate (GA) treatment in a mouse model of spontaneous opticospinal encephalomyelitis (OSE). Methods: OSE mice with a postnatal age of 21 to 28 days without signs of encephalomyelitis were treated once daily either with 150 µg GA or vehicle intraperitoneally (i. p.). The animals were scored daily regarding clinical signs and weight. The animals were sacrificed after 30 days of treatment or after having reached a score of 7.0 due to animal care guidelines. We performed immunohistochemistry of spinal cord sections and flow cytometry analysis of immune cells. Results: Preventive treatment with 150 µg GA i. p. once daily significantly reduced clinical disease progression with a mean score of 3.9 ± 1.0 compared to 6.2 ± 0.7 in control animals (p < 0.01) after 30 d in accordance with positive effects on weight (p < 0.001). The immunohistochemistry showed that general inflammation, demyelination or CD11c+ dendritic cell infiltration did not differ. There was, however, a modest reduction of the Iba1+ area (p < 0.05) and F4/80+ area upon GA treatment (p < 0.05). The immune cell composition of secondary lymphoid organs showed a trend towards an upregulation of regulatory T cells, which lacked significance. Conclusions: Preventive treatment with GA reduces disease progression in OSE in line with modest effects on microglia/macrophages. Due to the lack of established prophylactic treatment options for chronic autoimmune diseases with a high risk of disability, our study could provide valuable indications for translational medicine.

Measuring Patient Experience and Patient Satisfaction-How Are We Doing It and Why Does It Matter? A Comparison of European and U.S. American Approaches.

(1) Background: Patients' experiences and satisfaction with their treatment are becoming increasingly important in the context of quality assurance, but the measurement of these parameters is accompanied by several disadvantages such as poor cross-country comparability and methodological problems. The aim of this review is to describe and summarize the process of measuring, publishing, and utilizing patient experience and satisfaction data in countries with highly developed healthcare systems in Europe (Germany, Sweden, Finland, Norway, the United Kingdom) and the USA to identify possible approaches for improvement. (2) Methods: Articles published between 2000 and 2021 that address the topics described were identified. Furthermore, patient feedback in social media and the influence of sociodemographic and hospital characteristics on patient satisfaction and experience were evaluated. (3) Results: The literature reveals that all countries perform well in collecting patient satisfaction and experience data and making them publicly available. However, due to the use of various different questionnaires, comparability of the results is difficult, and consequences drawn from these data remain largely unclear. (4) Conclusions: Surveying patient experience and satisfaction with more unified as well as regularly updated questionnaires would be helpful to eliminate some of the described problems. Additionally, social media platforms must be considered as an increasingly important source to expand the range of patient feedback.

Clozapine Regulates Microglia and Is Effective in Chronic Experimental Autoimmune Encephalomyelitis.

Objective: Progressive multiple sclerosis is characterized by chronic inflammation with microglial activation, oxidative stress, accumulation of iron and continuous neurodegeneration with inadequate effectiveness of medications used so far. We now investigated effects of iron on microglia and used the previously identified neuroprotective antipsychotic clozapine in vitro and in chronic experimental autoimmune encephalomyelitis (EAE). Methods: Microglia were treated with iron and clozapine followed by analysis of cell death and response to oxidative stress, cytokine release and neuronal phagocytosis. Clozapine was investigated in chronic EAE regarding optimal dosing and therapeutic effectiveness in different treatment paradigms. Animals were scored clinically by blinded raters. Spinal cords were analyzed histologically for inflammation, demyelination, microglial activation and iron accumulation and for transcription changes of regulators of iron metabolism and inflammation. Effects on immune cells were analyzed using flow cytometry. Results: Iron impaired microglial function in vitro regarding phagocytosis and markers of inflammation; this was regulated by clozapine, reflected in reduced release of IL-6 and normalization of neuronal phagocytosis. In chronic EAE, clozapine dose-dependently attenuated clinical signs and still had an effect if applied in a therapeutic setting. Early mild sedative effects habituated over time. Histologically, demyelination was reduced by clozapine and positive effects on inflammation strongly correlated with reduced iron deposition. This was accompanied by reduced expression of DMT-1, an iron transport protein. Conclusions: Clozapine regulates microglial function and attenuates chronic EAE, even in a therapeutic treatment paradigm. This well-defined generic medication might therefore be considered as promising add-on therapeutic for further development in progressive MS.

Progressive Retinal and Optic Nerve Damage in a Mouse Model of Spontaneous Opticospinal Encephalomyelitis.

Neuromyelitis optica spectrum disorder (NMOSD) and myelin oligodendrocyte glycoprotein-antibody-associated disease (MOGAD) are antibody mediated CNS disorders mostly affecting the optic nerve and spinal cord with potential severe impact on the visual pathway. Here, we investigated inflammation and degeneration of the visual system in a spontaneous encephalomyelitis animal model. We used double-transgenic (2D2/Th) mice which develop a spontaneous opticospinal encephalomyelitis (OSE). Retinal morphology and its function were evaluated via spectral domain optical coherence tomography (SD-OCT) and electroretinography (ERG) in 6- and 8-week-old mice. Immunohistochemistry of retina and optic nerve and examination of the retina via RT-qPCR were performed using markers for inflammation, immune cells and the complement pathway. OSE mice showed clinical signs of encephalomyelitis with an incidence of 75% at day 38. A progressive retinal thinning was detected in OSE mice via SD-OCT. An impairment in photoreceptor signal transmission occurred. This was accompanied by cellular infiltration and demyelination of optic nerves. The number of microglia/macrophages was increased in OSE optic nerves and retinas. Analysis of the retina revealed a reduced retinal ganglion cell number and downregulated Pou4f1 mRNA expression in OSE retinas. RT-qPCR revealed an elevation of microglia markers and the cytokines Tnfa and Tgfb. We also documented an upregulation of the complement system via the classical pathway. In summary, we describe characteristics of inflammation and degeneration of the visual system in a spontaneous encephalomyelitis model, characterized by coinciding inflammatory and degenerative mechanisms in both retina and optic nerve with involvement of the complement system.

Binding patterns and functional properties of human antibodies to AQP4 and MOG on murine optic nerve and retina.

Neuromyelitis optica spectrum disorder (NMOSD) is an autoimmune-inflammatory CNS disease affecting spinal cord and optic nerves, mediated by autoantibodies against aquaporin-4 (AQP4) and myelin-oligodendrocyte-glycoprotein (MOG). Effects of those immunoglobulins (Ig) on retina and optic nerve are incompletely understood. We investigated AQP4-IgG and MOG-IgG sera on retina and optic nerve ex vivo and in 2D2 mice, which harbor a transgenic MOG-specific T-cell receptor. Some sera reacted with murine retina and optic nerve showing distinct binding patterns, suggesting different epitopes being targeted in both subgroups. Transfer of total IgG from a MOG-IgG positive patient to 2D2 mice did neither enhance disability nor induce functional or histological alterations in the retina.

Induction of Regulatory Properties in the Intestinal Immune System by Dimethyl Fumarate in Lewis Rat Experimental Autoimmune Neuritis.

Objective: Dimethyl fumarate (DMF) exerts immunomodulatory and neuroprotective effects in the animal model of experimental autoimmune neuritis (EAN) in the Lewis rat. DMF has been shown to modulate gut microbiota in veterinary medicine, however the effects of oral DMF on the gut-associated lymphoid tissue (GALT) remain unknown. Methods: Lewis rats were treated orally twice daily with DMF up to day 10 after immunization with immunogenic P2 peptide. Histological, flow cytometric and RT-PCR analyses of the GALT (intraepithelial layer, lamina propria, and Peyer patches) in duodenum, jejunum, and ileum were performed ex vivo. Moreover, cell transfer experiments were used to examine the protective effects of GALT regulatory T cells of the Peyer patches. Results: In the upper layers of duodenum, DMF induced a reduction of the toll-like receptor 4 (TLR4) mRNA expression. This was combined by a decrease of the pro-inflammatory lamina propria IFN-γ mRNA expression. In the ileum, we detected an immunoregulatory phenotype characterized by an increase of FoxP3 mRNA expression and of the nuclear factor (erythroid-derived-2)- like 2 (Nrf2) downstream molecule heme oxygenase-1 (HO-1) mRNA. Finally, CD4+ CD25+ regulatory T cells were increased in the Peyer patches. In vivo, the protective effect of these regulatory cells was verified by cell transfer into recipient EAN rats. Conclusions: Our results identified a novel immunomodulatory effect of DMF through the different regions and layers of the small intestine, which led to an increase of regulatory T cells, exerting a protective role in experimental neuritis.

Fingolimod for Irradiation-Induced Neurodegeneration.

BACKGROUND: Cranial irradiation is a common therapy for the treatment of brain tumors, but unfortunately patients suffer from side effects, particularly cognitive impairment, caused by neurodegenerative and neuroinflammatory mechanisms. Finding a therapeutic agent protecting hippocampal neurons would be beneficial. Fingolimod (FTY720), a sphingosine-1-phosphate receptor modulator approved for multiple sclerosis, is an immunosuppressant and known to enhance proliferation and differentiation of neuronal precursor cells (NPCs). OBJECTIVES: To investigate whether pre-treatment with FTY720 protects NPCs in vitro and in vivo from irradiation-induced damage. METHODS: Neuronal precursor cells were isolated from E13 C57BL/6 wildtype mice, treated at day 0 of differentiation with FTY720 and irradiated on day 6 with 1 Gy. NPCs were analyzed for markers of cell death (PI, caspase-3), proliferation (Ki67), and differentiation (DCX, ßIII-tubulin). Adult C57BL/6 wildtype mice were treated with FTY720 (1 mg/kg) and received a single dose of 6 Gy cranial irradiation at day 7. Using immunohistochemistry, we analyzed DCX and BrdU as markers of neurogenesis and Iba1, GFAP, and CD3 to visualize inflammation in the dentate gyrus (DG) and the subventricular zone (SVZ). B6(Cg)-Tyrc-2J/J DCX-luc reporter mice were used for bioluminescence imaging to evaluate the effect of FTY720 on neurogenesis in the DG and the spinal cord of naïve mice. RESULTS: FTY720 protected NPCs against irradiation induced cell death in vitro. Treatment with FTY720 dose-dependently reduced the number of PI+ cells 24 and 96 h after irradiation without effecting proliferation or neuronal differentiation. In vivo treatment resulted in a significant survival of DCX+ neurons in the DG and the SVZ 4 weeks after irradiation as well as a slight increase of proliferating cells. FTY720 inhibited microglia activation 24 h after X-ray exposure in the DG, while astrocyte activation was unaffected and no lymphocyte infiltrations were found. In naïve mice, FTY720 treatment for 4 weeks had no effect on neurogenesis. CONCLUSION: FTY720 treatment of NPCs prior to X-ray exposure and of mice prior to cranial irradiation is neuroprotective. No effects on neurogenesis were found.

1,25-OH2 vitamin D3 and AKT-inhibition increase glucocorticoid induced apoptosis in a model of T-cell acute lymphoblastic leukemia (ALL).

In acute lymphoblastic leukemia (ALL), steroid resistance and hypovitaminosis D are both associated with a poor prognosis. We show that methylprednisolone, calcitriol and the AKT-inhibitor MK-2206 have a synergistic effect on the apoptosis of steroid resistant T-ALL cells. Compared to methylprednisolone monotherapy, calcitriol increases methylprednisolone induced apoptosis dose-dependently (1.37-1.92-fold; p < 0.05). Pre-incubation with calcitriol increases the apoptotic effect of MK-2206 even further (3.6-fold; p < 0.05). It also potentiates synergism between MK-2206 and methylprednisolone (vehicle control 38% vs. calcitriol 58%, p < 0.01). The combination of calcitriol and AKT inhibition should be investigated further as treatment options for steroid resistance in T-ALL.

Aquaporin-4 antibodies in patients treated with natalizumab for suspected MS.

OBJECTIVE: To evaluate (1) the frequency of aquaporin-4 antibody (AQP4-ab)-seropositive cases among patients treated with natalizumab (NAT) and previously diagnosed with MS (MSNAT) in a nationwide cohort, (2) the clinical course of NAT-treated AQP4-ab-seropositive neuromyelitis optica spectrum disorder (NMOSD) patients (NMONAT), (3) AQP4-ab titers in NMONAT and AQP4-ab-seropositive NMOSD treated with other immunotherapies (NMOIT), and (4) immune mechanisms influencing disease activity in NMONAT. METHODS: MSNAT serum samples were retrospectively screened with a cell-based assay for AQP4-IgG and titers determined by ELISA. The annualized relapse rate (ARR) and disability progression were assessed. Serum levels of proinflammatory cytokines (interleukin [IL]-1ß, IL-4, IL-6, IL-8, IL-10, IL-17, IL-21, and interferon [IFN]-γ) and the chemokine CXCL-10 of NMONAT patients identified in this (n = 4) and a previous study (n = 5) were measured by cytometric bead array and ELISA. RESULTS: Of the 1,183 MSNAT patients (851 female, median 9 NAT infusions), only 4 (0.33%; 3 female, 1 male) had AQP4-IgG. Of these, 2 fulfilled the 2006 NMO criteria and all met the 2015 NMOSD criteria. The ARR was higher in NMONAT vs MSNAT (p = 0.0182). All 4 NMONAT patients had relapses and 2 had an increase of disability. AQP4-ab titers were higher in NMONAT (n = 9) vs NMOIT (n = 13; p = 0.0059). IL-8, IL-1ß, and IFN-γ serum levels were significantly higher, and CXCL-10 was significantly lower in NMONAT vs NMOIT. CONCLUSIONS: Misdiagnosis of NMOSD with MS is rare. NAT was not able to control disease activity in NMONAT patients, who had higher serum levels of AQP4-IgG and proinflammatory cytokines than patients with NMOSD treated with other immunotherapies.