Pyrimidine de novo synthesis inhibition selectively blocks effector but not memory T cell development.

Blocking pyrimidine de novo synthesis by inhibiting dihydroorotate dehydrogenase is used to treat autoimmunity and prevent expansion of rapidly dividing cell populations including activated T cells. Here we show memory T cell precursors are resistant to pyrimidine starvation. Although the treatment effectively blocked effector T cells, the number, function and transcriptional profile of memory T cells and their precursors were unaffected. This effect occurred in a narrow time window in the early T cell expansion phase when developing effector, but not memory precursor, T cells are vulnerable to pyrimidine starvation. This vulnerability stems from a higher proliferative rate of early effector T cells as well as lower pyrimidine synthesis capacity when compared with memory precursors. This differential sensitivity is a drug-targetable checkpoint that efficiently diminishes effector T cells without affecting the memory compartment. This cell fate checkpoint might therefore lead to new methods to safely manipulate effector T cell responses.

Fingolimod targeting protein phosphatase 2A differently affects IL-33 induced IL-2 and IFN-γ production in CD8(+) lymphocytes.

Multiple sclerosis patients are treated with fingolimod (FTY720), a prodrug that acts as an immune modulator. FTY720 is first phosphorylated to FTY720-P and then internalizes sphingosine-1-phosphate receptors, preventing lymphocyte sequestration. IL-33 is released from necrotic endothelial cells and contributes to MS severity by coactivating T cells. Herein we analyzed the influence of FTY720, FTY720-P, and S1P on IL-33 induced formation of IL-2 and IFN-γ, by using IL-33 receptor overexpressing EL4 cells, primary CD8(+) T cells, and splenocytes. EL4-ST2 cells released IL-2 after IL-33 stimulation that was inhibited dose-dependently by FTY720-P but not FTY720. In this system, S1P increased IL-2, and accordingly, inhibition of S1P producing sphingosine kinases diminished IL-2 release. In primary CD8(+) T cells and splenocytes IL-33/IL-12 stimulation induced IFN-γ, which was prevented by FTY720 but not FTY720-P, independently from intracellular phosphorylation. The inhibition of IFN-γ by nonphosphorylated FTY720 was mediated via the SET/protein phosphatase 2A (PP2A) pathway, since a SET peptide antagonist also prevented IFN-γ formation and the inhibition of IFN-γ by FTY720 was reversible by a PP2A inhibitor. While our findings directly improve the understanding of FTY720 therapy in MS, they could also contribute to side effects of FTY720 treatment, like progressive multifocal leukoencephalopathy, caused by an insufficient immune response to a viral infection.

Alpha-methylacyl-CoA racemase deletion has mutually counteracting effects on T-cell responses, associated with unchanged course of EAE.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system. Altering the metabolism of immune cells is an attractive strategy to modify their activity during autoimmunity in MS. We investigated the effect of modulating fatty acid metabolism in an animal model of MS, EAE. Alpha-methylacyl-CoA racemase (AMACR) converts R-configuration branched fatty acids into the S-configuration, thereby preparing them for ß-oxidation. We observed a significant, disease-dependent elevation of AMACR expression in monocytes and T cells from blood, draining lymph nodes and spleen of EAE mice during the preclinical phase. In vitro analysis revealed that the proliferation of T cells was inhibited in AMACR KO mice, but T-cell polarization was switched toward a pathogenic state involving the production of more IFN-γ and IL-17, but less IL-4. These opposing effects appeared to cancel out each other in vivo, because AMACR KO EAE mice showed a marginal increase in the severity of early clinical symptoms. AMACR was not regulated in the white blood cells of MS patients. Our data show that AMACR is regulated in immune cells during EAE, but it is not a suitable target for the treatment of MS due to its opposing effects.

Exacerbation of experimental autoimmune encephalomyelitis in ceramide synthase 6 knockout mice is associated with enhanced activation/migration of neutrophils.

Ceramides are mediators of inflammatory processes. In experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), we observed that CerS6 mRNA expression was upregulated 15-fold in peripheral blood leukocytes before the onset of EAE symptoms. In peripheral blood leukocytes from MS patients, a 3.9-fold upregulation was found. Total genetic deletion of CerS6 and the selective deletion of CerS6 in peripheral blood leucocytes exacerbated the progression of clinical symptoms in EAE mice. This was associated with enhanced leukocyte, predominantly neutrophil infiltration and enhanced demyelination in the lumbar spinal cord of EAE mice. Interferon-gamma/tumor necrosis factor alpha (IFN-γ/TNF-α) and granulocyte colony-stimulating factor (G-CSF) both drive EAE development and induce expression of the integrin CD11b and the chemokine receptor C-X-C motif chemokine receptor 2 (CXCR2), and we found they also induce CerS6 expression. In vivo, the genetic deletion of CerS6 enhanced the activation/migration of neutrophils, as reflected by an enhanced upregulation of CD11b and CXCR2. In vitro, the genetic deletion of CerS6 enhanced the activation status of IFN-γ/TNF-α-stimulated neutrophils, as shown by increased expression of nitric oxide and CD11b and an increased adhesion capacity. In G-CSF-stimulated neutrophils, the migration status was enhanced, as reflected by an elevated level of CXCR2 and an increased migration capacity. These data suggest that CerS6/C16-Cer mediates feedback regulation by inhibiting the formation of CD11b and CXCR2, which are induced either by IFN-γ/TNF-α or by G-CSF, respectively. We conclude that CerS6/C16-Cer mediates anti-inflammatory effects during the development of EAE and MS possibly by suppressing the migration and deactivation of neutrophils.

Lack of ceramide synthase 2 suppresses the development of experimental autoimmune encephalomyelitis by impairing the migratory capacity of neutrophils.

Ceramide synthases (CerS) synthesise ceramides of defined acyl chain lengths, which are thought to mediate cellular processes in a chain length-dependent manner. In experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), we observed a significant elevation of CerS2 and its products, C24-ceramides, in CD11b(+) cells (monocytes and neutrophils) isolated from blood. This result correlates with the clinical finding that CerS2 mRNA expression and C24-ceramide levels were significantly increased by 2.2- and 1.5-fold, respectively, in white blood cells of MS patients. The increased CerS2 mRNA/C24-ceramide expression in neutrophils/monocytes seems to mediate pro-inflammatory effects, since a specific genetic deletion of CerS2 in blood cells or a total genetic deletion of CerS2 significantly delayed the onset of clinical symptoms, due to a reduced infiltration of immune cells, in particular neutrophils, into the central nervous system. CXCR2 chemokine receptors, expressed on neutrophils, promote the migration of neutrophils into the central nervous system, which is a prerequisite for the recruitment of further immune cells and the inflammatory process that leads to the development of MS. Interestingly, neutrophils isolated from CerS2 null EAE mice, as opposed to WT EAE mice, were characterised by significantly lower CXCR2 receptor mRNA expression resulting in their reduced migratory capacity towards CXCL2. Most importantly, G-CSF-induced CXCR2 expression was significantly reduced in CerS2 null neutrophils and their migratory capacity was significantly impaired. In conclusion, our data strongly indicate that G-CSF-induced CXCR2 expression is regulated in a CerS2-dependent manner and that CerS2 thereby promotes the migration of neutrophils, thus, contributing to inflammation and the development of EAE and MS.

Contrasting disease patterns in seropositive and seronegative neuromyelitis optica: A multicentre study of 175 patients.

BACKGROUND: The diagnostic and pathophysiological relevance of antibodies to aquaporin-4 (AQP4-Ab) in patients with neuromyelitis optica spectrum disorders (NMOSD) has been intensively studied. However, little is known so far about the clinical impact of AQP4-Ab seropositivity. OBJECTIVE: To analyse systematically the clinical and paraclinical features associated with NMO spectrum disorders in Caucasians in a stratified fashion according to the patients' AQP4-Ab serostatus. METHODS: Retrospective study of 175 Caucasian patients (AQP4-Ab positive in 78.3%). RESULTS: Seropositive patients were found to be predominantly female (p < 0.0003), to more often have signs of co-existing autoimmunity (p < 0.00001), and to experience more severe clinical attacks. A visual acuity of ≤ 0.1 during acute optic neuritis (ON) attacks was more frequent among seropositives (p < 0.002). Similarly, motor symptoms were more common in seropositive patients, the median Medical Research Council scale (MRC) grade worse, and MRC grades ≤ 2 more frequent, in particular if patients met the 2006 revised criteria (p < 0.005, p < 0.006 and p < 0.01, respectively), the total spinal cord lesion load was higher (p < 0.006), and lesions ≥ 6 vertebral segments as well as entire spinal cord involvement more frequent (p < 0.003 and p < 0.043). By contrast, bilateral ON at onset was more common in seronegatives (p < 0.007), as was simultaneous ON and myelitis (p < 0.001); accordingly, the time to diagnosis of NMO was shorter in the seronegative group (p < 0.029). The course of disease was more often monophasic in seronegatives (p < 0.008). Seropositives and seronegatives did not differ significantly with regard to age at onset, time to relapse, annualized relapse rates, outcome from relapse (complete, partial, no recovery), annualized EDSS increase, mortality rate, supratentorial brain lesions, brainstem lesions, history of carcinoma, frequency of preceding infections, oligoclonal bands, or CSF pleocytosis. Both the time to relapse and the time to diagnosis was longer if the disease started with ON (p < 0.002 and p < 0.013). Motor symptoms or tetraparesis at first myelitis and > 1 myelitis attacks in the first year were identified as possible predictors of a worse outcome. CONCLUSION: This study provides an overview of the clinical and paraclinical features of NMOSD in Caucasians and demonstrates a number of distinct disease characteristics in seropositive and seronegative patients.

The perfect crime? CCSVI not leaving a trace in MS.

BACKGROUND: Multiple sclerosis (MS) is a chronic, inflammatory demyelinating disease of the central nervous system, believed to be triggered by an autoimmune reaction to myelin. Recently, a fundamentally different pathomechanism termed 'chronic cerebrospinal venous insufficiency' (CCSVI) was proposed, provoking significant attention in the media and scientific community. METHODS: Twenty MS patients (mean age 42.2 ± 13.3 years; median Extended Disability Status Scale 3.0, range 0-6.5) were compared with 20 healthy controls. Extra- and intracranial venous flow direction was assessed by colour-coded duplex sonography, and extracranial venous cross-sectional area (VCSA) of the internal jugular and vertebral veins (IJV/VV) was measured in B-mode to assess the five previously proposed CCSVI criteria. IJV-VCSA ≤ 0.3 cm(2) indicated 'stenosis,' and IJV-VCSA decrease from supine to upright position 'reverted postural control.' The sonographer, data analyser and statistician were blinded to the patient/control status of the participants. RESULTS: No participant showed retrograde flow of cervical or intracranial veins. IJV-VCSA ≤ 0.3 cm(2) was found in 13 MS patients versus 16 controls (p=0.48). A decrease in IJV-VCSA from supine to upright position was observed in all participants, but this denotes a physiological finding. No MS patient and one control had undetectable IJV flow despite deep inspiration (p=0.49). Only one healthy control and no MS patients fulfilled at least two criteria for CCSVI. CONCLUSIONS: This triple-blinded extra- and transcranial duplex sonographic assessment of cervical and cerebral veins does not provide supportive evidence for the presence of CCSVI in MS patients. The findings cast serious doubt on the concept of CCSVI in MS.

Use of complementary and alternative medicine in patients with multiple sclerosis in Germany.

BACKGROUND: The use of complementary and alternative medicine (CAM) for chronic diseases such as multiple sclerosis (MS) is becoming an increasingly important issue for those affected. Especially in Germany there are only a few studies dealing with CAM, as yet. The aim of this study was to assess the prevalence, the methods used, the subjective benefits as well as physician/patient communication. METHODS: A structured questionnaire including demographic and disease-specific data, CAM use, perceived benefits as well as physician/patient communication was sent to real and web-based self-help groups for MS in Germany. RESULTS: 343 answers could be evaluated. 77.3% of the participants were females. The mean age was 45.0 ±â€¯11.9 years and the duration of the disease was 12.0 ±â€¯9.6 years. 81.9% said they were using CAM, nearly half (44.8%) used it alternatively to conventional medicine. The average number of CAM- methods used were 3.6. The most popular methods were vitamin supplements, Yoga/Thai chi/Qi Gong, relaxation techniques and meditation. Approximately half (139/49.5%) of CAM users disclosed this to their treating neurologist. Yet, 37,6% have doubts on the competence of the respective physician. CONCLUSION: Patients with MS have a strong interest in CAM. Usage as alternative therapy is widespread and puts patients at risk of progress of the disease. As patient/physician communication on the topic is increasing, neurologists should be attentive to guiding their patients through safe complementary methods.

Endovascular Treatment of a Severe Stroke Occurring in a Commercial Airplane Over the Sahara.

BACKGROUND: The occurrence of an acute stroke syndrome during a long-distance flight demands critical decisions from the crew and attendant physicians. One in particular is whether the flight should be continued or be detoured to a nearby airport. CASE REPORT: We describe a 42-yr-old woman who suffered from a severe stroke in a commercial airplane over the Sahara. The captain, together with a physician aboard and the next of kin, decided not to detour to north African or south European airports, but to continue flying to Frankfurt, Germany (final destination; remaining flight time 4 h), where an efficient transfer infrastructure and an advanced medical standard were presumed. At the hospital, the patient was successfully treated by means of catheter-based mechanical thrombus extraction. The patient was free of neurological deficits at discharge. DISCUSSION: The geographically unbalanced availability of complex but highly effective therapies such as mechanical recanalization in acute stroke challenges decision making in aviation medicine. In selected cases it might be beneficial to continue flying to cities with advanced medical standards instead of deviating to nearby airports. Foerch C, Mayer CA, Berkefeld J, You S-J. Endovascular treatment of a severe stroke occurring in a commercial airplane over the Sahara. Aerosp Med Hum Perform. 2016; 87(9):825-829.

Interferon-Beta Increases Plasma Ceramides of Specific Chain Length in Multiple Sclerosis Patients, Unlike Fingolimod or Natalizumab.

Fingolimod is used for the treatment of multiple sclerosis (MS) and targets receptors for the bioactive sphingolipid sphingosine-1-phosphate (S1P). Whether fingolimod or other MS therapies conversely affect plasma concentrations of sphingolipids has, however, not yet been analyzed. Herein, we quantified 15 representative sphingolipid species by mass spectrometry in plasma from relapsing-remitting MS patients currently under fingolimod (n = 24), natalizumab (n = 16), or IFN-ß (n = 18) treatment. Healthy controls (n = 21) and untreated MS patients (n = 11) served as control groups. IFN-ß treatment strongly increased plasma level of C16:0, C18:0, C20:0, and C24:1 ceramides compared to healthy controls, untreated patients, or patients receiving fingolimod or natalizumab medication. Natalizumab treatment increased plasma concentrations of both S1P and sphinganine-1-phosphate, whereas fingolimod treatment did not affect any of these lipids. Correlations of sphingolipids with the Expanded Disability Status Scale and other disease specific parameters revealed no systemic change of sphingolipids in MS, independent of the respective treatment regime. These results indicate type I interferon treatment to cause a strong and specific increase in ceramide level. If confirmed in larger cohorts, these data have implications for the efficacy and adverse effects of IFN-ß. Moreover, quantification of ceramides soon after therapy initiation may help to identify therapy-responsive patients.

Blood levels of glial fibrillary acidic protein (GFAP) in patients with neurological diseases.

BACKGROUND AND PURPOSE: The brain-specific astroglial protein GFAP is a blood biomarker candidate indicative of intracerebral hemorrhage in patients with symptoms suspicious of acute stroke. Comparably little, however, is known about GFAP release in other neurological disorders. In order to identify potential "specificity gaps" of a future GFAP test used to diagnose intracerebral hemorrhage, we measured GFAP in the blood of a large and rather unselected collective of patients with neurological diseases. METHODS: Within a one-year period, we randomly selected in-patients of our university hospital for study inclusion. Patients with ischemic stroke, transient ischemic attack and intracerebral hemorrhage were excluded. Primary endpoint was the ICD-10 coded diagnosis reached at discharge. During hospital stay, blood was collected, and GFAP plasma levels were determined using an advanced prototype immunoassay at Roche Diagnostics. RESULTS: A total of 331 patients were included, covering a broad spectrum of neurological diseases. GFAP levels were low in the vast majority of patients, with 98.5% of cases lying below the cut-off that was previously defined for the differentiation of intracerebral hemorrhage and ischemic stroke. No diagnosis or group of diagnoses was identified that showed consistently increased GFAP values. No association with age and sex was found. CONCLUSION: Most acute and chronic neurological diseases, including typical stroke mimics, are not associated with detectable GFAP levels in the bloodstream. Our findings underline the hypothesis that rapid astroglial destruction as in acute intracerebral hemorrhage is mandatory for GFAP increase. A future GFAP blood test applied to identify patients with intracerebral hemorrhage is likely to have a high specificity.