HEV-Associated Neuralgic Amyotrophy: A Multicentric Case Series.

BACKGROUND: Neuralgic amyotrophy (NA) has been described as a possible extrahepatic manifestation of hepatitis E virus (HEV) infection. Usually, HEV-associated NA occurs bilaterally. The clinical characteristics determining the course of HEV-associated NA have still not been defined. METHODS: In this retrospective multicentric case series, 16 patients with HEV-associated NA were studied and compared to 176 HEV patients without NA in terms of their age, sex, and ALT levels. RESULTS: Neither gender distribution (75% vs. 67% male) nor age (47 vs. 48 years median) differed significantly between the NA patients and controls. Eight NA patients (50%) presented with bilateral involvement-seven of these had right-side dominance and one had left-side dominance. Thirteen cases (81%) were hospitalized. Eight of these patients stayed in hospital for five to seven days, and five patients stayed for up to two weeks. The time from the onset of NA to the HEV diagnosis, as well as the diagnostic and therapeutic proceedings, showed a large variability. In total, 13 (81%) patients received treatment: 1/13 (8%) received intravenous immunoglobulins, 8/13 (62%) received glucocorticoids, 3/13 (23%) received ribavirin, and 6/13 (46%) received pregabalin/gabapentin. Patients with ages above the median (47 years) were more likely to be treated (p = 0.001). CONCLUSION: HEV-associated NA causes a relevant morbidity. In our case series neither the type of treatment nor the time of initiation of therapy had a significant effect on the duration of hospitalization or the course of the disease. The clinical presentation, the common diagnostic and therapeutic procedures, and the patients' characteristics showed large variability, demonstrating the necessity of standardized protocols for this rare but relevant disease.

Defective cholesterol clearance limits remyelination in the aged central nervous system.

Age-associated decline in regeneration capacity limits the restoration of nervous system functionality after injury. In a model for demyelination, we found that old mice fail to resolve the inflammatory response initiated after myelin damage. Aged phagocytes accumulated excessive amounts of myelin debris, which triggered cholesterol crystal formation and phagolysosomal membrane rupture and stimulated inflammasomes. Myelin debris clearance required cholesterol transporters, including apolipoprotein E. Stimulation of reverse cholesterol transport was sufficient to restore the capacity of old mice to remyelinate lesioned tissue. Thus, cholesterol-rich myelin debris can overwhelm the efflux capacity of phagocytes, resulting in a phase transition of cholesterol into crystals and thereby inducing a maladaptive immune response that impedes tissue regeneration.

Evaluation of the Therapeutic Potential of Anti-TLR4-Antibody MTS510 in Experimental Stroke and Significance of Different Routes of Application.

Toll-like receptors (TLRs) are central sensors for the inflammatory response in ischemia-reperfusion injury. We therefore investigated whether TLR4 inhibition could be used to treat stroke in a standard model of focal cerebral ischemia. Anti-TLR4/MD2-antibody (mAb clone MTS510) blocked TLR4-induced cell activation in vitro, as reported previously. Here, different routes of MTS510 application in vivo were used to study the effects on stroke outcome up to 2d after occlusion of the middle cerebral artery (MCAO) for 45 min in adult male C57Bl/6 wild-type mice. Improved neurological performance, reduced infarct volumes, and reduced brain swelling showed that intravascular application of MTS510 had a protective effect in the model of 45 min MCAO. Evaluation of potential long-term adverse effects of anti-TLR4-mAb-treament revealed no significant deleterious effect on infarct volumes nor neurological deficit after 14d of reperfusion in a mild model of stroke (15 min MCAO). Interestingly, inhibition of TLR4 resulted in an altered adaptive immune response at 48 hours after reperfusion. We conclude that blocking TLR4 by the use of specific mAb is a promising strategy for stroke therapy. However, long-term studies with increased functional sensitivity, larger sampling sizes and use of other species are required before a clinical use could be envisaged.

Assessment of the Therapeutic Potential of Metallothionein-II Application in Focal Cerebral Ischemia In Vitro and In Vivo.

Metallothionein-II (MT-II) is an ubiquitously expressed small-molecular-weight protein and highly induced in various species and tissues upon stress, inflammation, and ischemia. MT-deficiency exacerbates ischemic injury in rodent stroke models in vitro and in vivo. However, there is conflicting data on the potential neuroprotective effect of exogenously applied metallothionein. Thus, we applied MT-II in an in vitro stroke model and intraperitoneally (i.p.) in two in vivo standard models of transient middle cerebral artery occlusion (MCAO) (a 'stringent' one [60 min MCAO/48 h reperfusion] and a 'mild' one [30 min MCAO/72 h reperfusion]), as well as i.v. together with recombinant tissue plasminogen activator (rtPA) to evaluate if exogenous MT-II-application protects against ischemic stroke. Whereas MT-II did not protect against 60 min MCAO, there was a significant reduction of direct and indirect infarct volumes and neurological deficit in the MT-II (i.p.) treated animals in the 'mild' model at 3d after MCAO. Furthermore, MT-II also improved survival of the mice after MCAO, suppressed TNF-α mRNA induction in ischemic brain tissue, and protected primary neuronal cells against oxygen-glucose-deprivation in vitro. Thus, exogenous application of MT-II protects against ischemic injury in vitro and in vivo. However, long-term studies with different species and larger sampling sizes are required before a clinical use can be envisaged.

Elevated expression of the C-type lectin CD93 in the glioblastoma vasculature regulates cytoskeletal rearrangements that enhance vessel function and reduce host survival.

Glioblastoma is an aggressive brain tumor characterized by an abnormal blood vasculature that is hyperpermeable. Here, we report a novel role for CD93 in regulating angiogenesis in this setting by modulating cell-cell and cell-matrix adhesion of endothelial cells. Tissue microarray analysis demonstrated that vascular expression of CD93 was correlated with poor survival in a clinical cohort of patients with high-grade astrocytic glioma. Similarly, intracranial growth in the GL261 mouse model of glioma was delayed significantly in CD93(-/-) hosts, resulting in improved survival compared with wild-type mice. This effect was associated with increased vascular permeability and decreased vascular perfusion of tumors, indicating reduced vessel functionality in the absence of CD93. RNAi-mediated attenuation of CD93 in endothelial cells diminished VEGF-induced tube formation in a three-dimensional collagen gel. CD93 was required for efficient endothelial cell migration and proper cell polarization in vitro. Further, in endothelial cells where CD93 was attenuated, decreased cell spreading led to a severe reduction in cell adhesion, a lack of proper cell contacts, a loss of VE-cadherin, and aberrant actin stress fiber formation. Our results identify CD93 as a key regulator of glioma angiogenesis and vascular function, acting via cytoskeletal rearrangements required for cell-cell and cell-matrix adhesion.

Knockout of silent information regulator 2 (SIRT2) preserves neurological function after experimental stroke in mice.

Sirtuin-2 (Sirt2) is a member of the NAD(+)-dependent protein deacetylase family. Various members of the sirtuin class have been found to be involved in processes related to longevity, regulation of inflammation, and neuroprotection. Induction of Sirt2 mRNA was found in the whole hemisphere after experimental stroke in a recent screening approach. Moreover, Sirt2 protein is highly expressed in myelin-rich brain regions after stroke. To examine the effects of Sirt2 on ischemic stroke, we induced transient focal cerebral ischemia in adult male Sirt2-knockout and wild-type mice. Two stroke models with different occlusion times were applied: a severe ischemia (45 minutes of middle cerebral artery occlusion (MCAO)) and a mild one (15 minutes of MCAO), which was used to focus on subcortical infarcts. Neurological deficit was determined at 48 hours after 45 minutes of MCAO, and up to 7 days after induction of 15 minutes of cerebral ischemia. In contrast to recent data on Sirt1, Sirt2(-/-) mice showed less neurological deficits in both models of experimental stroke, with the strongest manifestation after 48 hours of reperfusion. However, we did not observe a significant difference of stroke volumes or inflammatory cell count between Sirt2-deficient and wild-type mice. Thus we postulate that Sirt2 mediates myelin-dependent neuronal dysfunction during the early phase after ischemic stroke.

Molecular regulation of cell fate in cerebral ischemia: role of the inflammasome and connected pathways.

Analogous to Toll-like receptors, NOD-like receptors represent a class of pattern recognition receptors, which are cytosolic and constitute part of different inflammasomes. These large protein complexes are activated not only by different pathogens, but also by sterile inflammation or by specific metabolic conditions. Mutations can cause hereditary autoinflammatory systemic diseases, and inflammasome activation has been linked to many multifactorial diseases, such as diabetes or cardiovascular diseases. Increasing data also support an important role in different central nervous diseases such as stroke. Thus, the current knowledge of the functional role of this intracellular 'master switch' of inflammation is discussed with a focus on its role in ischemic stroke, neurodegeneration, and also with regard to the recent data which argues for a relevant role in other organs or biologic systems which influence stroke incidence or prognosis.

Invasive pressure monitoring saves from tuberculous meningitis with fulminant generalized brain edema.

We report a 57-year old female patient with a rapid and dramatic dynamic of whole brain edema caused by tuberculous meningitis. After initiation of tuberculostatic medication, general condition of the patient worsened and finally she was intubated due to a progredient loss of consciousness and respiratory insufficiency. Repeated cerebral computer tomography (CCT) revealed a global brain edema with slit ventricles and a dramatic progress of generalized brain swelling. Highly interesting, a rapid expanded regime of brain pressure monitoring and treatment according to a neurosurgical intensive standard ICP/CPP management protocol, which was complemented by the tuberculostatic therapy and high dose steroid application, dramatically improved the general conditions, so that the patient is now in a general condition which corresponds that before the occurrence of tuberculous meningitis. Thus, it is mandatory in situations with a rapid progressive brain swelling caused by bacterial meningitis to consider an intensified cerebral monitoring and stratified treatment protocol in order to avoid the devasting effects of a long lasting increase in intracranical pressure.

The NLRP3 inflammasome is differentially activated by pneumolysin variants and contributes to host defense in pneumococcal pneumonia.

Streptococcus pneumoniae is a leading cause of pneumonia, meningitis, and sepsis. Pneumococci can be divided into >90 serotypes that show differences in the pathogenicity and invasiveness. We tested the hypotheses that the innate immune inflammasome pathway is involved in fighting pneumococcal pneumonia and that some invasive pneumococcal types are not recognized by this pathway. We show that human and murine mononuclear cells responded to S. pneumoniae expressing hemolytic pneumolysin by producing IL-1ß. This IL-1ß production depended on the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome. Some serotype 1, serotype 8, and serotype 7F bacteria, which have previously been associated with increased invasiveness and with production of toxins with reduced hemolytic activity, or bacterial mutants lacking pneumolysin did not stimulate notable IL-1ß production. We further found that NLRP3 was beneficial for mice during pneumonia caused by pneumococci expressing hemolytic pneumolysin and was involved in cytokine production and maintenance of the pulmonary microvascular barrier. Overall, the inflammasome pathway is protective in pneumonia caused by pneumococci expressing hemolytic toxin but is not activated by clinically important pneumococcal sequence types causing invasive disease. The study indicates that a virulence factor polymorphism may substantially affect the recognition of bacteria by the innate immune system.

Blocking TLR2 in vivo protects against accumulation of inflammatory cells and neuronal injury in experimental stroke.

Reduced infarct volume in TLR2-knockout mice compared with C57Bl/6 wild-type mice has recently been shown in experimental stroke and confirmed in this study. We now also show a significant decrease of CD11b-positive cell counts and decreased neuronal death in the ischemic hemispheres of TLR2-deficient mice compared with C57Bl/6wt mice 2 days after transient focal cerebral ischemia. To examine the potential benefit of intravascular TLR2 inhibition, C57Bl/6wt mice were treated intraarterially with TLR2-blocking anti-TLR2 antibody (clone T2.5) after 45 minutes of cerebral ischemia and compared with control antibody (isotype) treated wild-type mice. Whereas T2.5-treated mice had no reduction in infarct volumes at 48 hours after reperfusion, they did have decreased numbers of CD11b-positive inflammatory cells and decreased neuronal death compared with isotype-treated control mice. Comparison of the isotype antibody treatment to control (saline) treatment showed no effects on infarct volumes or neuronal survival. However, mice treated with the control isotype antibody had increased numbers of CD11b-positive inflammatory cells compared with saline-treated animals. Thus, antibody treatment itself (i.e., control isotype antibody, but potentially of any antibody) may have adverse effects and limit therapeutic benefit of anti-TLR2-antibody therapy. We conclude that TLR2 mediates leukocyte and microglial infiltration and neuronal death, which can be attenuated by TLR2 inhibition. The TLR2 inhibition in vivo improves neuronal survival and may represent a future stroke therapy.

Spinal cord atrophy in triple A syndrome associated with a novel compound heterozygous mutation.

A 38-year-old male patient was admitted with slowly progressive spastic gait disturbance. Imaging revealed general spinal cord atrophy. Because of adrenal insufficiency, alacrima and achalasia, triple A syndrome was suspected. This is a case report of a triple A syndrome patient with a predominance of neurological features and a new heterozygous compound mutation in triple A syndrome gene.

CD93/AA4.1: a novel regulator of inflammation in murine focal cerebral ischemia.

The stem-cell marker CD93 (AA4.1/C1qRp) has been described as a potential complement C1q-receptor. Its exact molecular function, however, remains unknown. By using global expression profiling we showed that CD93-mRNA is highly induced after transient focal cerebral ischemia. CD93 protein is upregulated in endothelial cells, but also in selected macrophages and microglia. To elucidate the potential functional role of CD93 in postischemic brain damage, we used mice with a targeted deletion of the CD93 gene. After 30 min of occlusion of the middle cerebral artery and 3 d of reperfusion these mice displayed increased leukocyte infiltration into the brain, increased edema, and significantly larger infarct volumes (60.8 +/- 52.2 versus 23.9 +/- 16.6 mm(3)) when compared with wild-type (WT) mice. When the MCA was occluded for 60 min, after 2 d of reperfusion the CD93 knockout mice still showed more leukocytes in the brain, but the infarct volumes were not different from those seen in WT animals. To further explore CD93-dependent signaling pathways, we determined global transcription profiles and compared CD93-deficient and WT mice at various time points after induction of focal cerebral ischemia. We found a highly significant upregulation of the chemokine CCL21/Exodus-2 in untreated and treated CD93-deficient mice at all time points. Induction of CCL21 mRNA and protein was confirmed by PCR and immunohistochemistry. CCL21, which was formerly shown to be released by damaged neurons and to activate microglia, contributes to neurodegeneration. Thus, we speculate that CD93-neuroprotection is mediated via suppression of the neuroinflammatory response through downregulation of CCL21.

Membrane attack complex inhibitor CD59a protects against focal cerebral ischemia in mice.

BACKGROUND: The complement system is a crucial mediator of inflammation and cell lysis after cerebral ischemia. However, there is little information about the exact contribution of the membrane attack complex (MAC) and its inhibitor-protein CD59. METHODS: Transient focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) in young male and female CD59a knockout and wild-type mice. Two models of MCAO were applied: 60 min MCAO and 48 h reperfusion, as well as 30 min MCAO and 72 h reperfusion. CD59a knockout animals were compared to wild-type animals in terms of infarct size, edema, neurological deficit, and cell death. RESULTS AND DISCUSSION: CD59a-deficiency in male mice caused significantly increased infarct volumes and brain swelling when compared to wild-type mice at 72 h after 30 min-occlusion time, whereas no significant difference was observed after 1 h-MCAO. Moreover, CD59a-deficient mice had impaired neurological function when compared to wild-type mice after 30 min MCAO. CONCLUSION: We conclude that CD59a protects against ischemic brain damage, but depending on the gender and the stroke model used.

Mrp-8 and -14 mediate CNS injury in focal cerebral ischemia.

Several reports have recently demonstrated a detrimental role of Toll-like receptors (TLR) in cerebral ischemia, while there is little information about the endogenous ligands which activate TLR-signaling. The myeloid related proteins-8 and-14 (Mrp8/S100A8; Mrp14/S100A9) have recently been characterized as endogenous TLR4-agonists, and thus may mediate TLR-activation in cerebral ischemia. Interestingly, not only TLR-mRNAs, but also Mrp8 and Mrp14 mRNA were found to be induced in mouse brain between 3 and 48 h after transient 1 h focal cerebral ischemia/reperfusion. Mrp-protein was expressed in the ischemic hemisphere, and co-labeled with CD11b-positive cells. To test the hypothesis that Mrp-signaling contributes to the postischemic brain damage, we subjected Mrp14-deficient mice, which also lack Mrp8 protein expression, to focal cerebral ischemia. Mrp14-deficient mice had significantly smaller lesion volumes when compared to wild-type littermates (130+/-16 mm(3) vs. 105+/-28 mm(3)) at 2 days after transient focal cerebral ischemia (1 h), less brain swelling, and a reduced macrophage/microglia cell count in the ischemic hemisphere. We conclude that upregulation and signaling of Mrp-8 and-14 contribute to neuroinflammation and the progression of ischemic damage.

Acute neurodegeneration and the inflammasome: central processor for danger signals and the inflammatory response?

Activation of the inflammatory response is a crucial event in the adverse outcome of cerebral ischemia, which is promoted by proinflammatory cytokines such as interleukin (IL)-1beta. Although caspase-1 is necessary for IL-1beta processing, the 'upstream' signaling pathways were, until recently, essentially unknown. Fortunately, the inflammasome, a multiprotein complex responsible for activating caspase-1 and caspase-5, has recently been characterized. The activation of the inflammasome can result in one of several consequences such as cytokine secretion, cell death, or the development of a stress-resistant state. The significance of the inflammasome for the initiation of the inflammatory response during systemic diseases has already been shown and members of the inflammasome complex were recently found to be induced in acute brain injury. However, the specific pathophysiologic role of the inflammasome in neurodegenerative disorders still remains to be clarified. The underlying theories (e.g., danger signal theory) along with the signaling pathways that link the inflammasome to acute neurodegeneration will be discussed here. Furthermore, the stimuli that potentially activate the inflammasome in cerebral ischemia will be specified, as well as their relation to well-known pathways activating the innate immune response (e.g., Toll-like receptor signaling) and the consequences that result from their activation (beneficial versus deleterious).

TLR2 has a detrimental role in mouse transient focal cerebral ischemia.

A significant up-regulation of Toll-like-receptor (TLR) mRNAs between 3 and 48 h reperfusion time after induction of transient focal cerebral ischemia for 1h was revealed by applying global gene expression profiling in postischemic mouse brains. Compared to TLR4 and TLR9, TLR2 proved to be the most significantly up-regulated TLR in the ipsilateral brain hemisphere. TLR2-protein was found to be expressed mainly in microglia in the postischemic brain tissue, but also in selected endothelial cells, neurons, and astrocytes. Additionally, TLR2-related genes with pro-inflammatory and pro-apoptotic capabilities were induced. Therefore we hypothesized that TLR2-signaling could exacerbate the primary brain damage after ischemia. Two days after induction of transient focal cerebral ischemia (1h), we found a significant decrease of the infarct volume in TLR2 deficient mice compared to wild type mice (75+/-5 vs. 42+/-7 mm(3)). We conclude that TLR2 up-regulation and TLR2-signaling are important events in focal cerebral ischemia and contribute to the deterioration of ischemic damage.