Search

Integrins α4β1/ α9β1 are important in the pathogenesis and progression of inflammatory and autoimmune diseases by their roles in leukocyte activation and trafficking. Natalizumab, a monoclonal antibody selectively targeting α4β1 integrin and blocking leukocyte trafficking to the central nervous system, is an immunotherapy for multiple sclerosis (MS). However, due to its adverse effects associated with chronic treatment, alternative strategies using small peptide mimetic inhibitors are being sought. In the present study, we synthesized and characterized visabron c (4–4), a backbone cyclic octapeptide based on the sequence TMLD, a non-RGD unique α4β1 integrin recognition sequence motif derived from visabres, a proteinous disintegrin from the viper venom. Visabron c (4–4) was selected from a minilibrary with conformational diversity based on its potency and selectivity in functional adhesion cellular assays. Visabron c (4–4)’s serum stability, pharmacokinetics, and therapeutic effects following ip injection were assessed in an experimental autoimmune encephalomyelitis (EAE) animal model. Furthermore, visabron c (4–4)’s lack of toxic effects in mice was verified by blood analysis, tissue pathology, immunogenicity, and “off-target” effects, indicating its significant tolerability and lack of immunogenicity. Visabron c (4–4) can be delivered systemically. The in vitro and in vivo data justify visabron c (4–4) as a safe alternative peptidomimetic lead compound/drug to monoclonal anti-α4 integrin antibodies, steroids, and other immunosuppressant drugs. Moreover, visabron c (4–4) design may pave the way for developing new therapies for a variety of other inflammatory and/or autoimmune diseases.

Nitric oxide (NO) is acknowledged as a vital intercellular messenger in multiple systems in the body. Medicine has focused on its functions and therapeutic applications for decades, especially in cardiovascular and nervous systems, and its role in immunological responses. This review was composed to demonstrate the prevalence of NO in components of the ocular system, including corneal cells and multiple cells in the retina. It discussed NO’s assistance during the immune, inflammation and wound-healing processes. NO is identified as a vascular endothelial relaxant that can alter the choroidal blood flow and prompt or suppress vascular changes in age-related macular degeneration and diabetes, as well as the blood supply to the optic nerve, possibly influencing the progression of glaucoma. It will provide a deeper understanding of the role of NO in ocular homeostasis, the delicate balance between overproduction or underproduction and the effect on the processes from aqueous outflow and subsequent intraocular pressure to axial elongation and the development of myopia. This review also recognized the research and investigation of therapies being developed to target the NO complex and treat various ocular diseases.

The median survival time of patients with glioblastoma is still poor (14.6 month), partly due to a lack of effective treatment. We have observed that androgen receptor (AR) is amplified in glioblastomas at the DNA, RNA and protein levels. The AR gene was amplified in 27% of glioblastoma specimens from men (n=22) and of 38.2% from women (n=21). AR-RNA was overexpressed (>2.5 fold) in 93% (n=30), and AR-protein was induced (>two fold) in 56% of the glioblastomas samples (n=16). Thirty percent of the glioblastomas (n=21) also expressed a constitutively active AR-splice-variant (AR-V7/AR3) lacking the Ligand-Binding-Domain. Following these findings, we examined the effect of pharmacological inhibition of androgen receptor in vitro and in vivo, as well as of genetic silencing of the receptor in glioblastoma cell lines. AR antagonists, induced concentration-dependent death in three glioblastoma cell lines, as well as in two glioma initiating cell lines. Silencing of AR expression by siRNA induced cell death in the three tested glioblastoma cell lines. Enzalutamide given orally to nude mice bearing subcutaneous human glioma xenografts resulted in a 72% reduction in tumor volume (p=0.0027). The presence of AR-V7/AR3 in glioblastoma, together with the present data showing that genetic silencing of the full length AR in cell lines and pharmacological inhibition of AR, induce GBM cell death in vivo and in vitro, point to the important role of AR in GBM survival and render a potential therapeutic target for this devastating disease.

Despite the existence of potent anti-inflammatory biological drugs e.g., anti-TNF and anti IL-6 receptor antibodies, for treating chronic inflammatory and autoimmune diseases, these are costly and not specific. Cheaper oral available drugs remain an unmet need. Expression of the acute phase protein Serum Amyloid A (SAA) is dependent on release of pro-inflammatory cytokines IL-1, IL-6 and TNF-α during inflammation. Conversely, SAA induces pro-inflammatory cytokine secretion, including Th17, leading to a pathogenic vicious cycle and chronic inflammation. 5- MER peptide (5-MP) MTADV (methionine-threonine-alanine-aspartic acid-valine), also called Amilo-5MER, was originally derived from a sequence of a pro-inflammatory CD44 variant isolated from synovial fluid of a Rheumatoid Arthritis (RA) patient. This human peptide displays an efficient anti-inflammatory effects to ameliorate pathology and clinical symptoms in mouse models of RA, Inflammatory Bowel Disease (IBD) and Multiple Sclerosis (MS). Bioinformatics and qRT-PCR revealed that 5-MP, administrated to encephalomyelytic mice, up-regulates genes contributing to chronic inflammation resistance. Mass spectrometry of proteins that were pulled down from an RA synovial cell extract with biotinylated 5-MP, showed that it binds SAA. 5-MP disrupted SAA assembly, which is correlated with its pro-inflammatory activity. The peptide MTADV (but not scrambled TMVAD) significantly inhibited the release of pro-inflammatory cytokines IL-6 and IL-1β from SAA-activated human fibroblasts, THP-1 monocytes and peripheral blood mononuclear cells. 5-MP suppresses the pro-inflammatory IL-6 release from SAA-activated cells, but not from non-activated cells. 5-MP could not display therapeutic activity in rats, which are SAA deficient, but does inhibit inflammations in animal models of IBD and MS, both are SAA-dependent, as shown by others in SAA knockout mice. In conclusion, 5-MP suppresses chronic inflammation in animal models of RA, IBD and MS, which are SAA-dependent, but not in animal models, which are SAA-independent.

Background Neuromyelitis-optica (NMO) and multiple-sclerosis (MS) are inflammatory- demyelinating-diseases of the central-nervous-system (CNS). In a previous study, we identified 17 miRNAs that were significantly upregulated in the peripheral blood of patients with NMO, relative to healthy controls (HCs). Target gene analysis have demonstrated that QKI is targeted by 70% of the upregulated miRNAs. QKI gene encodes for a RNA-binding-protein that plays a central role in myelination. QKI variants 5, 6, 7 (QKI-V5, QKI-V6, QKI-V7) are generated via alternative splicing. Given the role played by QKI in myelination we aimed to study the expression levels of QKI variants in the circulation of patients with NMO and MS and in the circulation and brain tissue of mice-model to CNS-inflammatory-demyelinating-disease. Methods RNA and protein expression levels of QKI variants QKI-V5, QKI-V6 and QKI-V7 were determined in the blood of patients with NMO (n = 23) or MS (n = 13). The effect of sera from patients on the expression of QKI in normal peripheral-blood-mononuclear-cells (PBMCs) or glial cells was explored. The mog-experimental-autoimmune-encephalomyelitis (EAE) mouse model was used to study the correlation between the changes in the expression levels of QKI in the blood to those in the brain. Results RNA and protein expression of QKI-V5 was decreased in the peripheral blood of patients with NMO and multiple-sclerosis. Incubation of normal peripheral-blood-mononuclear-cells or glial cells with sera of patients significantly reduced the expression of QKI-V5. The blood and brain of EAE mice exhibited a corresponding decrease in QKI-V5 expression. Conclusion The downregulation in the expression of QKI-V5 in the blood of patients with CNS-inflammatory-demyelinating-diseases and in the brain and blood of EAE mice is likely caused by a circulating factor and might promote re-myelination by regulation of myelin-associated genes. Key words: QKI variants, Multiple sclerosis (MS), Neuromyelitis optica (NMO), Astrocytes, Demyelination.

Androgen receptor (AR) gene is mapped to Xq11-12. It functions as a steroid-hormone-activated-transcription-factor. AR splice variants lacking the Ligand-Binding-Domain (LBD), such as AR-V7/AR3, which arise primarily through exon skipping and cryptic exon inclusion, are activated in a ligand-independent mechanism. The association between sex steroid receptors and brain tumors was described since 1983, but in contrast to the well-established oncogenic role played by androgen receptor (AR) in prostate cancer and the growing evidences on its role in breast cancer, the expression and significance of AR in GBM is controversial and poorly studied. Our previous presented data (Lavon, I. et al. CSIG-13. Neuro-Oncology 18, vi43-vi43 (2016)) and our current results have demonstrated for the first time amplification of AR at the DNA, RNA and protein levels in the majority of GBM samples in patients of both sexes. Our AR-RNA expression results were validated in 703 glioblastomas by analysis of several datasets including the TCGA. We also showed that 30% of glioblastomas express the constitutively active AR-splice-variant AR-V7/AR3. Based on the previously reported activation of AR-V7/AR3 by RTKs, including EGFR, we identified that combination therapy with the AR-antagonist Enzalutamide and EGFR inhibitor is more effective than AR-antagonists alone in cell line expressing AR-V7/AR3. A cell cycle analysis of glioma cells treated with Enzalutamide demonstrated a dose-dependent number of cells in sub-G1 phase suggesting apoptosis as the mechanism for cell death. Enzalutamide given orally (20 mg/kg three time per week) to nude mice bearing human gliomas (U78MG) resulted in reduction of 72% in tumor volume (p=0.0027) as compared to mice treated with vehicle. We hope that the results of this study together with continued laboratory efforts will lead to a new approach for the treatment of human glioblastoma.

Background: Screening of putative autoimmune targets in multiple sclerosis (MS) revealed a proportion of patients carrying antibodies (Abs) against KIR4.1, a potassium channel that shares functional properties with AQP4. Both are localized at the perivascular astrocytic processes. Aims: To measure anti-KIR4.1 Abs in the serum of MS and neuromyelitis optica (NMO) patients, and to identify the clinical and laboratory characteristics of patients harboring anti-KIR4.1 Abs. Methods: We measured anti-KIR4.1 Abs in serum, using the peptide KIR4.1 (83–120) enzyme-linked immunosorbent assay (ELISA). Results: Serum levels of anti-KIR4.1 Abs were significantly higher in MS and NMO patients than in healthy controls (HCs); with Abs detected in 21 of 80, 10 of 45, and 2 of 32 individuals, respectively (MS versus HC, p < 0.05). The level of anti-KIR4.1 Abs was significantly higher during MS relapse, versus remission ( p = 0.04). The clinical characteristics of our study patients did not vary based on KIR4.1 positivity. Conclusions: Anti-KIR4.1 Abs were found in similar proportions of patients with MS and NMO, at a significantly higher level than observed in HCs; consequently, the presence of Abs does not discriminate between these demyelinating diseases. However, anti-KIR4.1 Ab levels differed in MS patients during relapse and remission; as such, they may represent a marker of disease exacerbation.

Objectives/Hypothesis The objectives of this study were to examine the presence of β-2 transferrin (β2TRNSF) in cerebrospinal fluid (CSF) contaminated in vitro by various bacteria and explore the mechanism (passive or active) responsible for β2TRNSF elimination. Early diagnosis of CSF leakage may change treatment decisions and minimize the risk of meningitis and encephalitis. β2TRNSF is a protein present exclusively in CSF. Its detection is highly useful in cases of CSF leakage, although it has never been examined in the presence of central nervous system infection. Study Design Prospective patient analysis. Methods Sterile CSF drawn from patients was contaminated in vitro with several microorganisms chosen for their ability to cause neurosurgical-related infections: Streptococcus pneumoniae, methicillin-sensitive Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa. β2TRNSF was examined at two time points: following immediate inoculation (t0) and following an overnight incubation (t18) over various bacterial concentrations. Samples of CSF inoculated with S pneumoniae were also examined in the presence of ciprofloxacin. For β2TRNSF analysis we used immunoblotting electrophoresis and enzyme-linked immunosorbent assay (ELISA). Results CSF samples collected from nine patients were analyzed. β2TRNSF was not detected following S pneumoniae inoculation at both time points when immunoblotting electrophoresis was used. Quantitative analysis using ELISA demonstrated significant β2TRNSF concentration decrease. The addition of ciprofloxacin led to the same results. Conclusions CSF leak detection using β2TRNSF may be deceiving in the presence of a S pneumoniae cerebral nervous system infection. A passive process is suggested, as β2TRNSF disappeared either immediately or following incubation with inactive bacteria. Level of Evidence NA Laryngoscope, 125:556–560, 2015

Neurodegenerative diseases generate the accumulation of specific misfolded proteins, such as PrP Sc prions or A-beta in Alzheimer's diseases, and share common pathological features, like neuronal death and oxidative damage. To test whether reduced oxidation alters disease manifestation, we treated TgMHu2ME199K mice, modeling for genetic prion disease, with Nano-PSO, a nanodroplet formulation of pomegranate seed oil (PSO). PSO comprises large concentrations of a unique polyunsaturated fatty acid, Punicic acid, among the strongest natural antioxidants. Nano-PSO significantly delayed disease presentation when administered to asymptomatic TgMHu2ME199K mice and postponed disease aggravation in already sick mice. Analysis of brain samples revealed that Nano-PSO treatment did not decrease PrP Sc accumulation, but rather reduced lipid oxidation and neuronal loss, indicating a strong neuroprotective effect. We propose that Nano-PSO and alike formulations may be both beneficial and safe enough to be administered for long years to subjects at risk or to those already affected by neurodegenerative conditions. From the Clinical Editor This team of authors report that a nanoformulation of pomegranade seed oil, containing high levels of a strong antioxidant, can delay disease onset in a mouse model of genetic prion diseases, and the formulation also indicates a direct neuroprotective effect.

Although the incidence of breast cancer metastasis (BCM) in brain has increased significantly in triple-negative breast cancer (TNBC), the mechanisms remain elusive. Using in vivo mouse models for BCM in brain, we observed that TNBC cells crossed the blood-brain barrier (BBB), lodged in the brain microvasculature and remained adjacent to brain microvascular endothelial cells (BMECs). Breaching of the BBB in vivo by TNBCs resulted in increased BBB permeability and changes in ZO-1 and claudin-5 tight junction (TJ) protein structures. Angiopoietin-2 expression was elevated in BMECs and was correlated with BBB disruption. Secreted Ang-2 impaired TJ structures and increased BBB permeability. Treatment of mice with the neutralizing Ang-2 peptibody trebananib prevented changes in the BBB integrity and BMEC destabilization, resulting in inhibition of TNBC colonization in brain. Thus, Ang-2 is involved in initial steps of brain metastasis cascade, and inhibitors for Ang-2 may serve as potential therapeutics for brain metastasis.

Background: Brain irradiation (BI) in humans may cause behavioral changes, cognitive impairment and neuroendocrine dysfunction. The effect of BI on the hypothalamic-pituitary-adrenal (HPA) axis is not fully understood. Objectives: To evaluate the effect of BI on HPA axis responses under basal and stressful conditions as well as following pretreatment with dexamethasone (Dex). Methods: Adult male rats were exposed to whole BI. HPA axis responses were examined at 2, 4, 9 and 20 weeks after BI. Histological evaluations of the irradiated rats and matched controls were conducted at 4 and 20 weeks after BI. Results: In contrast to the control group, the basal and stress-induced corticosterone levels were enhanced at 9 and 20 weeks after BI and the inhibitory effect of Dex was reduced. BI also caused hyposuppression of the adrenocortical response to stress. Histological assessment of the irradiated brains revealed hippocampal atrophy at 20 weeks after BI. The neuronal counts were lower only in the CA1 region of the irradiated brains. BI caused a decrease in the binding capacity of Dex to the hippocampal cytosolic fraction. Conclusions: Enhanced stress-induced HPA axis responses and the reduced effect of Dex suggest that BI has delayed effects on HPA axis responses as manifested by impairment of the negative feedback exerted by glucocorticoids (GCs). The mechanisms underlying these effects of BI are unknown. It is possible that the marked BI-induced damage in the hippocampus, which plays an important role in the regulation of the feedback effect of GCs, may cause abnormal HPA axis responses following BI.

We characterized the effect of acute ischemic stroke on the activation of the hypothalamic-pituitary-adrenal (HPA) axis and evaluated the role of glucocorticoids (GC) in the clinical outcome following ischemic stroke. Male spontaneous hypertensive rats underwent permanent middle cerebral artery occlusion (PMCAO) and developed a cortical infarct. At 4h post-PMCAO or sham operation, serum levels of ACTH and corticosterone (CS) were elevated 5 and 4 fold respectively as compared to controls and then returned to basal levels at 24h post surgery. In these experimental groups we found also a significant depletion of median eminence (ME)-CRH(41). In adrenalectomized (Adx) rats that underwent PMCAO the degree of motor disability and infarct volume was similar to that of intact rats. Administration of dexamethasone (Dex) to Adx-PMCAO rats significantly improved the motor disability and decreased the infarct volume. However, in sham-Adx with PMCAO, Dex had no effect on these two parameters. In rats with PMCAO or sham-PMCAO, brain production of PGE(2) was significantly increased. This effect was further enhanced in Adx-PMCAO rats and significantly inhibited by Dex. In conclusion, activation of the HPA axis following PMCAO is due to stress induced by surgery. This activation is mediated by hypothalamic CRH(41). Absence of endogenous GC or administration of Dex in naïve rats does not alter motor and pathological parameters in the acute stage following PMCAO. In contrast, administration of Dex significantly improved the outcome following cerebral ischemia in Adx rats which may be due to increased glucocorticoid receptors. Brain production of PGE(2) does not play an important role in the pathophysiology of the acute phase of cerebral ischemia.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system and is associated with demyelination, neurodegeneration, and sensitivity to oxidative stress. In this work, we administered a nanodroplet formulation of pomegranate seed oil (PSO), denominated Nano-PSO, to mice induced for experimental autoimmune encephalomyelitis (EAE), an established model of MS. PSO comprises high levels of punicic acid, a unique polyunsaturated fatty acid considered as one of the strongest natural antioxidants. We show here that while EAE-induced mice treated with natural PSO presented some reduction in disease burden, this beneficial effect increased significantly when EAE mice were treated with Nano-PSO of specific size nanodroplets at much lower concentrations of the oil. Pathological examinations revealed that Nano-PSO administration dramatically reduced demyelination and oxidation of lipids in the brains of the affected animals, which are hallmarks of this severe neurological disease. We propose that novel formulations of natural antioxidants such as Nano-PSO may be considered for the treatment of patients suffering from demyelinating diseases. On the mechanistic side, our results demonstrate that lipid oxidation may be a seminal feature in both demyelination and neurodegeneration.

Objective: Reciprocal pathways of interaction between the nervous and immune systems during stress may be regulated by stress-induced circulating glucocorticoids that act via type II glucocorticoid receptors (GRs). The aim of the present study was to investigate the effect of restraint stress on GRs in lymphocytes and the role of the sympathetic system in this effect. Methods: We used male Balb/c mice which were adrenalectomized 3 days before exposure to restraint stress (4 h). Specific binding of 3H-dexamethasone (Dex) and the expression of GR protein were measured in the cytosol of spleen cells. Results: Restraint stress caused a significant increase in the maximal binding of 3H-Dex to GRs in the cytosol of spleen cells but not in the binding affinity. In correlation with this increase in binding, restraint stress caused an increase in the amount of GR protein. To establish the relation of the nervous system in this stress response, we blocked the autonomic innervations to the spleen with the ganglionic blocker chlorisondamine. This blocker abrogated the stress-induced increase in the binding of 3H-Dex to GRs and in the GR protein levels. Abrogation of the stress response was also achieved by blocking β-adrenergic receptors. Conclusion: These results suggest that stress-induced increase in the level of GRs is mediated by the sympathetic nervous system via β-adrenergic receptors. It is possible that stress modulation of lymphocyte GR levels may be implicated in the bidirectional communication between the nervous and the immune systems.

Reactive oxygen species are involved in the pathogenesis of multiple sclerosis (MS), Parkinson's disease and neurodegenerative diseases. Here we report that Tempamine (TMN), a stable radical with antioxidant and proapoptotic activities, when encapsulated in the intraliposome aqueous phase of pegylated (

Several microbial species, including probiotic lactic acid bacteria, have the ability to irreversibly bind a large variety of polyphenols (flavonoids) and anthocyanidins found in many colored fruits and vegetables and to enhance their total oxidant-scavenging capacities (TOSC). The binding of flavonoids to microbial surfaces was further increased by the cationic polyelectrolytes ligands poly-L-histidine, chlorhexidine and Copaxone®. This phenomenon was confirmed visually, by the FRAP, DPPH, cyclic voltammetry, Folin-Ciocalteu as well as by luminol-dependent chemiluminescence techniques employed to assay TOSC. The possibility is considered that clinically, microbial cells in the oral cavity and in the gastro intestinal tract, complexed with antioxidant polyphenols from nutrients and with cationic ligands, might increase the protection of mammalian cells against damage induced by excessive generation of reactive oxygen species during infections and inflammation.

The piperidine nitroxide tempamine (TMN) is a cell-permeable, stable radical having antioxidant, anticancer, and proapoptotic and/or pronecrotic activities, as was demonstrated by us in cell cultures. We also demonstrated synergism between TMN and doxorubicin in doxorubicin-sensitive and doxorubicin-resistant cell lines. Treatment of the C26 mouse colon carcinoma model in vivo also demonstrated synergism between TMN and doxorubicin in sterically stabilized liposomes (SSLs) containing TMN (SSL-TMN) and those containing doxorubicin. The above effects of TMN and SSL-TMN motivated us to develop and optimize the SSL-TMN formulation so that it will be able to reach the disease site with a sufficiently high TMN level and a release rate needed to achieve a therapeutic effect. Because TMN is an amphipathic weak base, it was remote loaded by an intraliposome high/extraliposome low transmembrane ammonium sulfate gradient. The kinetics and level of TMN loading were monitored by cyclic voltammetry (CV) and electron paramagnetic resonance (EPR); the latter also indicates TMN precipitation in the intraliposomal aqueous phase. The regeneration of the original CV and EPR signals by the ionophore nigericin indicates that TMN remained fully intact during loading and release. The cardinal role of the transmembrane ammonium ion gradient in the loading process was proven by the use of the selective ionophores nonactin (for NH4+) and nigericin (for H+). The anion of the ammonium salts affects loading stability and the rate of TMN release, both mediated through the TMN state of aggregation in the intraliposomal aqueous phase. The greater the TMN salt precipitation, the slower the TMN release rate. This was supported by measurement of osmolality, which is inversely related to TMN salt precipitate. Precipitation is in the order SO4(-2)>Cl-1>glucuronate-1. Liposome lipid composition, magnitude of the transmembrane ammonium ion gradient, and type of anion of the ammonium salt determine the amount of TMN loaded and its release rate.

Background Although antibodies to aquaporin-4(AQP4) are strongly associated with Neuromyelitis optica (NMO), the sole transfer of these antibodies is not sufficient to induce an NMO-like disease in experimental animals and T-cells and complement are also needed. Initial data indicating the presence of T-cell responses to AQP4 in patients with NMO, have beeen recently reported. Objective To evaluate the T-cell responses to specific AQP4 peptides/epitopes in patients with NMO and multiple sclerosis (MS). Methods Peripheral blood mononuclear cells (PBMCs) were obtained from 14 patients fulfilling the criteria for definite NMO and the proliferation responses to one of 15 distinct pentadecapeptides of AQP4, spanning the whole protein (except of its transmembrane parts) were tested by a standard [H3]-thymidine uptake assay and compared with those of 9 healthy controls and 7 MS patients. A cytometric bead array assay (CBA) and flow cytometry were used to evaluate cytokine (IFNγ, IL17, IL2, IL4, IL5, IL10 and TNFα) and chemokine (CXCL8, CCL5, CXCL10, CXCL9, CCL2) secretion by PHA-stimulated PBMCs and AQP4-specific T-cell lines. Results Four main immunodominant epitopes of the AQP4 protein (p137–151, p222–236, p217–231 and the p269–283) were identified in the NMO group. The first two epitopes (assigned as peptides 3 and 9) showed the highest sensitivity (~60% positivity), whereas the latter two (assigned as peptides 8 and 11), the higher specificity. Longitudinal follow up of 5 patients revealed changes in the epitope-specificities during the course of NMO. T-cell lines specific for the AQP4 peptides, produced from NMO patients (but not healthy donors) secreted mainly IL-17 and IL-10 and less IFNγ. Conclusions Our findings indicate that T-cells bearing characteristics of both Th1 and Th17 T-cells and targeting specific immunodominant epitopes of the AQP4 protein might be involved in the pathogenesis of NMO.

Purpose It is known that both human conjunctival fibroblasts (HCF) and corneal epithelial (HCE) cells contribute to the inflammatory process in the ocular surface by releasing inflammatory cytokines. In addition, nitric oxide (NO) has an important role in inflammatory responses in the ocular surface. In the present study, we aimed to characterize the capacity of these cells to release nitric oxide in response to cytokines and Lipopolysaccharide (LPS), and show that Alpha-linoleic acid (ALA) inhibits these responses. Methods HCF, HCE cells, peripheral blood mononuclear cells (PBMCs) and co-culture of HCF and PBMC were treated with different combinations of inflammatory inducers, including interleukin)IL- (6, tumor necrosis factors (TNF)-α, interferon (IFN)- γ and IL-1β and LPS. Nitrite levels were measured in cell supernatants with and without ALA by the Griess reaction test at 24, 48 and 72 h respectively. Expression of nitric oxide synthase 2 (NOS-2) was evaluated by real-time PCR. Results All cytokine combinations had an inducible effect on nitrite secretion in HCF, PBMC and co-cultured PBMC and HCF, but not in HCE cells. Treatment with a combination of IL-6, LPS, TNF-α, IFN- γ and IL-1β induced the highest nitrite secretion (2.91 fold, P

Objective: The amygdala (AMG) plays a facilitatory role in the hypothalamic-pituitary-adrenal (HPA) axis. The effect of the AMG on the negative feedback exerted by glucocorticoids (GC) is not clear. We investigated the effect of repeated electrical stimulation of the AMG on the feedback action of GC upon the adrenocortical (AC) response to stressful stimuli. Methods: Rats received electrical stimulation into the central amygdalar nucleus once daily for 4 days. At days 5 and 12 after the onset of stimulation, rats were treated with dexamethasone (Dex) or vehicle and were exposed to either photic or acoustic stress stimuli, and serum corticosterone (CS) was measured. In another group of rats, we measured the binding of Dex to the hippocampal cytosol at 5 and 12 days after the AMG stimulation. Results: At 5 and 12 days after the onset of stimulation or a sham control, stress increased the serum CS level. In the sham group, Dex completely inhibited the CS response, but at 5 days after stimulation, it was significantly less effective in doing this. At day 12, Dex was as effective as in the control group. AMG stimulation delayed the return of CS response to basal levels and caused a significant decrease in the binding capacity of Dex to hippocampal cytosol. Conclusion: Electrical stimulation of the AMG caused a transient impairment of the feedback action of GC upon the stress response. This effect may be due to the decrease in hippocampal corticosteroid receptors. This suggests that the impaired GC feedback caused by AMG stimulation may be involved in the facilitatory effect of the AMG on the function of the AC axis.

Objectives: The role of nitric oxide (NO) in modulating the blood-brain barrier (BBB) is not entirely clear. We examined the effect of different NO synthase (NOS) inhibitors and NO donors on the permeability of the BBB in animals with normally functioning brain blood vessels, following disruption by hyperosmotic mannitol and during immune inflammation. Methods: We administered L-NAME, aminoguanidine, S-methyl-thiocitrulline (SMT) and 7-indazole (NOS inhibitors) and NOR-4 (an NO donor) into the cerebral ventricle of rats. Disruption of the BBB was induced by intracarotid injection of mannitol (25%). Experimental autoimmune encephalomyelitis (EAE) was induced by brain homogenate. The extent of disruption was evaluated by Evans blue (2%) dye extravasation. Results: L-NAME (a nonspecific NOS inhibitor) and SMT (a neuronal and endothelial NOS inhibitor) increased mannitol-induced disruption of BBB. This effect was inhibited by NO donors. In animals with a normally functioning BBB, none of these inhibitors or NO donors caused a change in the permeability. 7-indazole (a specific neuronal NOS inhibitor) and aminoguanidine (an inducible NOS inhibitor) had no facilitatory effect on BBB permeability, either alone or in combination with hyperosmotic mannitol. Administration of L-NAME and SMT to rats with EAE significantly aggravated the clinical outcome. In contrast, the administration of NOR-4 diminished clinical signs of EAE. Conclusion: The NOS system does not play a role in BBB permeability in naïve animals. Only endothelial NOS takes part in the facilitation of BBB compromised by mannitol and EAE. Extrinsic NO decreases this facilitatory effect.

Background: Several studies have suggested that an early increase in renal nitric oxide (NO) production or activity mediates pathophysiologic and morphologic changes in diabetic nephropathy. To evaluate the role of NO in developing diabetic kidney disease, we studied the NO system in streptozotocin (STZ)-induced diabetic rats for a period of 8 weeks. Methods: Control rats, STZ-induced diabetic rats, and STZ-induced diabetic rats treated with insulin were monitored and sacrificed at 1, 2, and 8 weeks. Urinary cGMP was measured, and the levels and activity of NO synthase (NOS) isoforms in the kidney cortex were determined at specific times by immunoblotting and diaphorase staining. Results: Diabetic rats had increased kidney weight, urinary volume, glucose, sodium and potassium excretion, which was precluded by insulin treatment. Creatinine clearance was increased in the diabetic group and reversed by insulin treatment. Urinary cGMP decreased by 71, 93, and 92% at 1, 2, and 8 weeks of diabetes, respectively, compared with the control animals. Insulin treatment curtailed the urinary cGMP reduction in diabetic animals. Total NOS activity in the renal cortex was reduced by 65, 52, and 44% after 1, 2, and 8 weeks of diabetes, respectively, and returned to normal levels upon insulin treatment. NADPH diaphorase staining of renal cortical slices showed a 77, 63, and 70% decrease in neuronal NOS isoform activity in the macula densa after 1, 2, and 8 weeks of diabetes, respectively, compared with control non-diabetic animals. This reduction was normalized by insulin treatment. Endothelial NOS protein expression in the kidney cortex tended to increase after 1 week of diabetes and its level was elevated significantly after 2 and 8 weeks of diabetes. However, neuronal NOS protein expression in the kidney cortex was reduced by 52% in 2-week diabetic animals, but this reduction was normalized by insulin treatment. Conclusions: The decreased renal NOS activity during the late phase of diabetes is partially associated with a decrease in neuronal NOS activity and protein expression in kidney macula densa.

We present a method of mapping the water molecule distribution in mouse brain tissues using injected bis-N-carbazolyl-distyrylbenzene and the FLIM technique. The fluorescence lifetime of this two-photon absorbing chromophore diminishes when the amount of water in the surrounding area increases. The fluorescence lifetime of the injected in vivo chromophore strongly depends on the content of water in different areas. Thus, lifetimes of 900 ± 50 ps in the hippocampus (extracellular fluid), 520 ± 50 ps in the lateral ventricle (choroid plexus, cerebrospinal fluid), and 400–150 ps in blood vessels were observed. Moreover, the fluorescence lifetime distribution undergoes drastic changes when mice are deprived of water. Statistical analysis of the investigated samples showed that upon water deprivation water content decreased at the border of the hippocampus/lateral ventricle areas and increased in blood vessels.

"Negative selection" and "death by neglect" are governed by apoptotic processes occurring in the thymus that shape the repertoire of maturing T cells. We have previously developed an in vitro model that recapitulates "death by neglect": Co-cultivation of double positive (DP) thymocytes or thymic lymphoma cells (PD1.6) with thymic epithelial cells (TEC) caused TcR-independent apoptosis of the former. We further demonstrated that this apoptosis could be attenuated by aminoglutethimide, an inhibitor of steroid synthesis, suggesting a role of TEC-derived glucocorticoids (GC) in this death process. We have now substantiated the role of the GC-glucocorticoid receptor (GR) axis by using a GC-resistant subline (PD1.6Dex(-)) obtained from the GC-sensitive PD1.6 cells by repeated exposures to increasing doses of dexamethasone (Dex). The PD1.6Dex(-) cells barely express GR and are much less sensitive to TEC-induced apoptosis. Re-expression of GR in PD1.6Dex(-) cells restored their sensitivity to both Dex and TEC, highlighting the central role of GR in these apoptotic processes. Likewise, repeated exposures of PD1.6 cells to TEC led to the selection of TEC-resistant cells (PD1.6TEC(-)) that are insensitive to corticosterone and less sensitive to Dex, though their GR level was only moderately reduced. This is in line with the low levels of corticosterone secreted by TEC. Altogether, our data show that TEC eliminates DP thymic lymphoma cells in a GR-dependent manner and modulates the GC sensitivity of the surviving cells.

To elucidate the mechanisms underlying the EAE-associated behavioral syndrome (EBS), we examined the temporal correlation between the behavioral alterations and inflammatory processes. Onset of the behavioral syndrome was associated with the onset of brain infiltration, as well as mRNA expression of interleukin 1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF-alpha) and elevated production of interleukin 1 beta protein and prostaglandin E(2) (PGE(2)). Sickness behavior symptoms coincided with peak cytokine expression. Behavioral recovery was associated with a reduction of cytokine expression, but not infiltration, PGE(2) production or motor disturbances. These results suggest that inflammatory processes in general, and the production of pro-inflammatory cytokines in particular, are involved in mediating EAE-associated sickness behavior.

EAE is associated with sickness behavior symptoms that are temporally correlated with inflammatory processes. To further elucidate the role of inflammatory mediators in the behavioral syndrome, EAE mice were injected daily with anti-inflammatory drugs, beginning at disease onset. Dexamethasone or interleukin-1 (IL-1) receptor antagonist or the prostaglandins synthesis inhibitor indomethacin attenuated the behavioral symptoms. Administration of the tumor necrosis-factor alpha (TNF-alpha) synthesis inhibitor pentoxifylline or targeted deletion of the type I TNF receptor had no behavioral effects whereas administration of pentoxifylline in IL-1ra-treated mice further reversed the behavioral depression. These findings demonstrate the critical involvement of pro-inflammatory cytokines and prostaglandins in the EAE-associated behavioral syndrome, and may have implications for understanding and treating the neuropsychiatric disturbances in multiple sclerosis (MS) patients.

Many medical conditions, including inflammatory diseases such as multiple sclerosis (MS), are often accompanied by a high prevalence of depressive episodes. Inflammatory mediators, such as cytokines, were implicated in illness-associated depressive conditions, both in humans and in animals. For example, MS-associated depression (MSD) was attributed to pathophysiological processes such as immune dysregulation and cerebral inflammation. We have recently documented a depressive-like behavioral syndrome in mice with experimental autoimmune encephalomyelitis (EAE), an established model of MS. In the present paper, we discuss the similarities between the EAE-associated behavioral syndrome (EBS) and MSD, in terms of phenomenology, putative mechanisms and responsiveness to anti-depressive therapy. In particular, we show that: (1) EAE and depression are associated not only with similar behavioral symptomatology, but also with common physiological alterations, including impaired serotonergic neurotransmission, and activation of neuroendocrine (e.g., adrenocortical) and inflammatory cytokine systems; (2) the EBS precedes any neurological deficit during the initial EAE attack, as well as further exacerbations, and remits during recovery and between relapses. Similarly, in many MS patients depression precedes and accompanies the attacks and wanes during remissions; (3) females show increased susceptibility to EBS. Similarly, depression is much more prevalent in women than in men; (4) chronic treatment with the tricyclic anti-depressant imipramine reduced EAE-induced mortality, body-weight loss and behavioral suppression. Similarly, anti-depressant drugs have been used effectively in treating MSD. These findings suggest that the EBS may serve as an animal model for MSD.

Background and Purpose — We sought to determine the cerebroprotective potential of NAP, a synthetic octapeptide related to vasoactive intestinal peptide. Activity-dependent neuroprotective protein mediates some of the protective effects of vasoactive intestinal peptide. The neuroprotective NAP sequence is derived from activity-dependent neuroprotective protein. Methods — Spontaneously hypertensive rats underwent permanent middle cerebral artery occlusion by craniotomy and electrocoagulation. After dose-response and time-course experiments, the animals were injected with NAP (3 μg/kg) or vehicle intravenously 1 hour after stroke onset. Another group of rats was injected with the d -amino acid isomer of NAP (D-NAP) and served as a negative control. Rats were examined for motor and behavioral deficits 24 hours to 30 days later, and infarct volumes were determined. The effect of NAP administration on apoptotic death was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and caspase-3 stainings. Results — NAP significantly reduced motor disability and infarct volumes compared with vehicle or D-NAP when tested at 24 hours after stroke onset (9.67±1.4% versus 17.04±1.18% and 19.19±1.9% of hemispheric volume, respectively; P P Conclusions — Our results indicate that the durable cerebroprotection by NAP involves antiapoptotic mechanisms.

INTRODUCTION: Silencing of O(6)-methylguanine-DNA-methyltransferase (MGMT) in tumors, correlates with a better therapeutic response and with increased survival. Our previous results demonstrated the pivotal role of NF-kappaB in MGMT expression, mediated mainly through binding of p65/NF-kappaB homodimers to the non-canonical NF-KappaB motif (MGMT-kappaB1) within MGMT enhancer. METHODS AND RESULTS: In an attempt to attenuate the transcription activity of MGMT in tumors we designed locked nucleic acids (LNA) modified decoy oligonucleotides corresponding to the specific sequence of MGMT-kappaB1 (MGMT-kB1-LODN). Following confirmation of the ability of MGMT-kB1-LODN to interfere with the binding of p65/NF-kappaB to MGMT enhancer, the potential of the MGMT-kB1-LODN to enhance cell killing was studied in vitro in two glioma cell lines (T98G and U87) and a melanoma cell line (A375P). All three cell lines manifested a significant enhanced cell killing effect following exposure to temozolomide (TMZ) when first transfected with MGMT-kb1-LODN, and also induced a significant cell killing when administered as monotherapy. These results were confirmed also in-vivo on A375P Melanoma xenografts. Intratumoral (Intralesional - IL) injection of MGMT-kB1-LODN with or without IP injection of TMZ induced significant tumor growth inhibition either as a monotherapy or in combination with TMZ. The long-term effect of MGMT-kB1-LODN monotherapy was evaluated using a repetitive IL injection every 4 to 5 days for 55 days with either MGMT-κB1 LODN or control ODN or vehicle. A significant difference (p < 0.01) in tumor volume was obtained by MGMT-κB1-LODN compared to both control groups. Moreover, two out of the seven mice treated with MGMT-κB1-LODN demonstrated tumor regression by day 55 and no tumor recurrence was observed five months later. CONCLUSION: The results of these experiments show that the MGMT-kB1-LODN has a substantial antineoplastic effect when used either in combination with temozolomide or as monotherapy. Our results suggest that MGMT-kB1-LODN may provide a novel strategy for cancer therapy.

Silencing of O(6)-methylguanine-DNA-methyltransferase (MGMT) in tumors, mainly through promoter methylation, correlates with a better therapeutic response and with increased survival. Therefore, it is conceivable to consider MGMT as a potential therapeutic target for the treatment of cancers. Our previous results demonstrated the pivotal role of NF-kappaB in MGMT expression, mediated mainly through p65/NF-kappaB homodimers. Here we show that the non-canonical NF-KappaB motif (MGMT-kappaB1) within MGMT enhancer is probably the major inducer of MGMT expression following NF-kappaB activation. Thus, in an attempt to attenuate the transcription activity of MGMT in tumors we designed locked nucleic acids (LNA) modified decoy oligonucleotides corresponding to the specific sequence of MGMT-kappaB1 (MGMT-kB1-LODN). Following confirmation of the ability of MGMT-kB1-LODN to interfere with the binding of p65/NF-kappaB to the NF-KappaB motif within MGMT enhancer, the efficacy of the decoy was studied in-vitro and in-vivo. The results of these experiments show that the decoy MGMT-kB1-LODN have a substantial antineoplastic effect when used either in combination with temozolomide or as monotherapy. Our results suggest that MGMT-kB1-LODN may provide a novel strategy for cancer therapy.

The objectives of this study were to examine the presence of β-2 transferrin (β2TRNSF) in cerebrospinal fluid (CSF) contaminated in vitro by various bacteria and explore the mechanism (passive or active) responsible for β2TRNSF elimination. Early diagnosis of CSF leakage may change treatment decisions and minimize the risk of meningitis and encephalitis. β2TRNSF is a protein present exclusively in CSF. Its detection is highly useful in cases of CSF leakage, although it has never been examined in the presence of central nervous system infection.Prospective patient analysis.Sterile CSF drawn from patients was contaminated in vitro with several microorganisms chosen for their ability to cause neurosurgical-related infections: Streptococcus pneumoniae, methicillin-sensitive Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa. β2TRNSF was examined at two time points: following immediate inoculation (t0) and following an overnight incubation (t18) over various bacterial concentrations. Samples of CSF inoculated with S pneumoniae were also examined in the presence of ciprofloxacin. For β2TRNSF analysis we used immunoblotting electrophoresis and enzyme-linked immunosorbent assay (ELISA).CSF samples collected from nine patients were analyzed. β2TRNSF was not detected following S pneumoniae inoculation at both time points when immunoblotting electrophoresis was used. Quantitative analysis using ELISA demonstrated significant β2TRNSF concentration decrease. The addition of ciprofloxacin led to the same results.CSF leak detection using β2TRNSF may be deceiving in the presence of a S pneumoniae cerebral nervous system infection. A passive process is suggested, as β2TRNSF disappeared either immediately or following incubation with inactive bacteria.

Prion diseases, which can manifest by transmissible, sporadic or genetic etiologies, share several common features, such as a fatal neurodegenerative outcome and the aberrant accumulation of proteinase K (PK)-resistant PrP forms in the CNS. In infectious prion diseases, such as scrapie in mice, prions first replicate in immune organs, then invade the CNS via ascending peripheral tracts, finally causing death. Accelerated neuroinvasion and death occurs when activated prion-infected immune cells infiltrate into the CNS, as is the case for scrapie-infected mice induced for experimental autoimmune encephalomyelitis (EAE), a CNS inflammatory insult. To establish whether the immune system plays such a central role also in genetic prion diseases, we induced EAE in TgMHu2ME199K mice, a line mimicking for late onset genetic Creutzfeldt Jacob disease (gCJD), a human prion disease. We show here that EAE induction of TgMHu2ME199K mice neither accelerated nor aggravated prion disease manifestation. Concomitantly, we present evidence that PK-resistant PrP forms were absent from CNS immune infiltrates, and most surprisingly also from lymph nodes and spleens of TgMHu2ME199K mice at all ages and stages of disease. These results imply that the mechanism of genetic prion disease differs widely from that of the infectious presentation, and that the conversion of mutant PrPs into PK resistant forms occurs mostly/only in the CNS. If the absence of pathogenic PrP forms form immune organs is also true for gCJD patients, it may suggest their blood is devoid of prion infectivity.

In order to test the long-term cerebroprotective effects of dexanabinol, a synthetic non-competitive NMDA antagonist that also has anti-TNFalpha effects, spontaneously hypertensive rats underwent permanent middle cerebral artery occlusion (PMCAO). Rats were given vehicle or dexanabinol (4.5 mg/kg) 1, 3 or 6 h after PMCAO. The research consisted of 2 stages. In the short-term set of experiments animals (n=5/group), were tested with a motor disability scale 24 h post PMCAO, then sacrificed and the infarct volume was measured using 2,3,5-Triphenyltetrazolium chloride (TTC) staining. In the long-term set of experiments the rats (n=7/group) were examined daily with a motor disability scale up to 30 days after PMCAO and then sacrificed and infarct volumes were determined using TTC staining. Motor scores were significantly improved in the dexanabinol treated rats (P

The permanent middle cerebral artery occlusion (PMCAO) model was used to investigate the cerebroprotective effects of the synthetic cannabinoid, dexanabinol (HU-211). Dexanabinol is a noncompetitive N-methyl-D-aspartate antagonist, with antioxidant and anti-TNFalpha properties. Twenty hypertensive rats were subjected to PMCAO. Eight were given 4 mg/kg dexanabinol, i.v., 1 h after PMCAO, eight received vehicle and four were not injected Five rats underwent sham surgery. Infarct volumes were assessed, as well as TNFalpha concentrations and NOS activity in brain homogenates. Dexanabinol significantly decreased infarct volumes. It also significantly lowered TNFalpha levels in the ipsilateral hemisphere although not to the level of sham operated rats. No effect could be demonstrated on NOS activity. In conclusion, dexanabinol may be a pluripotent cerebroprotective agent.

It has been shown that the hypothalamic-pituitary-adrenal (HPA) axis responses to immune-derived stimuli in particular can be modulated by nitric oxide (NO). In the present study we examined the effect of endogenous and exogenous NO on the HPA axis responses to neural stimuli which are not related to immune functions. Intracerebroventricular injection of NOR-3, a donor of NO, had no effect on basal HPA axis activity but significantly attenuated the secretion of median eminence (ME) CRH-41 as well as the serum ACTH and corticosterone (CS) in response to acute photic stimulation in a dose-dependent manner. Intracerebroventricular administration of N-ω-nitro-L-arginine methyl ester (L-NAME), a general NOS inhibitor, significantly enhanced ACTH and CS responses to this stress but did not change the basal levels of these hormones. On the other hand, i.c.v. injection of aminoguanidine, an inhibitor of inducible NO synthase (NOS) but not of neuronal NOS, did not affect the HPA axis responses to photic stimulation. These results suggest that: (1) NO is involved in modulation of the HPA axis responses to neural stimuli which are not dependent on immune factors, (2) the effect of NO is mediated by inhibition of hypothalamic ME CRH-41 secretion, and (3) this effect is probably mediated by neuronal NOS and not by inducible NOS.

Experimental autoimmune neuritis (EAN) is an animal model that shares clinical, pathological and electrophysiological features with the human disease Guillain–Barre syndrome. The synthetic immunomodulatory substance linomide has been successfully used to prevent the development of several experimental autoimmune models in laboratory animals and has been proved to be beneficial in modulating the course of multiple sclerosis in humans. In the present study we demonstrate that oral administration of linomide prevents the development of clinical and histopathological signs of EAN in Lewis rats, inoculated with the P2 (60–70) synthetic peptide. The immunomodulatory effect of linomide on this experimental model of disease was associated with marked apoptosis of lymphocytes in thymus and spleen early after starting the treatment. Furthermore, a downregulation of the endothelial expression of the adhesion molecules ICAM-1 at the target site and LFA-1 on lymphocytes could also contribute to the absence of inflammatory cells in the neuraxis.

Background Toll-like receptors (TLRs) are recognized as important contributors to the initiation and modulation of the inflammatory response in the eye. This study investigated the precise expression patterns and functionality of TLRs in human corneal epithelial cells (HCE) and in conjunctival fibroblasts (HCF). Methods The cell surface expression of TLRs 2-4, TLR7 and TLR9 in HCE and HCF was examined by flow cytometry with or without stimulation with lipopolysaccharide (LPS) or polyinosinic:polycytidylic acid (poly I:C). The mRNA expression of the TLRs was determined by real-time PCR. The protein content levels of interleukin (IL)-6, IL-8, IL-1β and tumor necrosis factor-α (TNF-α) were measured in HCE and HCF using multiplex fluorescent bead immunoassay (FBI). Results The surface expression of TLR3 and TLR4 was detected on both HCE and HCF. Following incubation with LPS, the percentage of HCE cells staining for TLR4 decreased from 10.18% to 0.62% (P 0.05), 4.35 ± 1.12-fold IL-1β (P > 0.05) and 29.35 ± 2.3-fold TNFα (P Conclusions HCF and HCE both express TLRs that respond to specific ligands by increasing cytokine expression. Following activation, the surface expression of TLR3 and TLR4 on HCE is decreased, thus creating a negative feedback loop, mitigating the effect of TLR activation.

Linomide (quinoline-3-carboxamide, LS-2616), a synthetic immunomodulator, protects animals against a variety of experimental autoimmune diseases. In experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis (MS), linomide blocks both the clinical and histological signs of the disease, without inducing generalized immunosuppression. In the first clinical trial in patients with MS, linomide was shown to inhibit the progression of the disease. In the present study we investigated several aspects of the mechanisms of action of this immunomodulator. We found that linomide can inhibit acute EAE even when given as pretreatment, prior to induction of disease (days −10 to 0). This inhibitory effect was reversed by adoptive transfer of naive spleen cells. A short course (7 days) of linomide treatment also inhibited EAE, especially when administered immediately after disease induction. Spleen cells from linomide-treated mice failed to present myelin antigens to T-cell lines in vitro. The defective antigen presentation was normalized by anti-oxidants such as 2-mercaptoethanol. The proportion of Mac1 + cells in the spleens of linomide-treated mice was significantly reduced and macrophage growth was inhibited in long term cultures of spleen cells derived from linomide-treated animals. Our findings suggest that the effect of linomide on EAE may be attributed, at least in part, to inactivation of antigen presenting cells, possibly following a short period of over-stimulation and increased oxidant production. This mechanism may play a universal role in the regulation of autoimmune reactivity and merits further investigation.

Although the incidence of breast cancer metastasis (BCM) in brain has increased significantly in triple-negative breast cancer (TNBC), the mechanisms remain elusive. Using in vivo mouse models for BCM in brain, we observed that TNBC cells crossed the blood-brain barrier (BBB), lodged in the brain microvasculature and remained adjacent to brain microvascular endothelial cells (BMECs). Breaching of the BBB in vivo by TNBCs resulted in increased BBB permeability and changes in ZO-1 and claudin-5 tight junction (TJ) protein structures. Angiopoietin-2 expression was elevated in BMECs and was correlated with BBB disruption. Secreted Ang-2 impaired TJ structures and increased BBB permeability. Treatment of mice with the neutralizing Ang-2 peptibody trebananib prevented changes in the BBB integrity and BMEC destabilization, resulting in inhibition of TNBC colonization in brain. Thus, Ang-2 is involved in initial steps of brain metastasis cascade, and inhibitors for Ang-2 may serve as potential therapeutics for brain metastasis.

Inflammatory demyelinative diseases of the central nervous system are mostly idiopathic and represent the major cause of neurological disability in young adults. These diseases differ in terms of clinical symptoms, severity, pathological characteristics and epidemiology. However, there are also significant similarities between these diseases, which sometimes bring to a misleading diagnosis. Neuromyelitis optica (NMO) is a demyelinative disease in which the optic nerve and the spinal cord are predominantly affected. The detection of specific antibodies to aquaporin-4 (NMO-IgG) led to a modification of the diagnostic criteria for NMO.We performed a retrospective study on NMO-IgG positive patients referred to the Department of Neurology MS Center (2006-2011) with suspected NMO. Based on the presenting symptomatology of the patients, we identified the cases with optic neuritis and various parameters that may differentiate between NMO and MS. NMO-IgG were evaluated by ELISA.A total of 50% of the 107 patients with NMO-IgG fulfilled the revised criteria of NMO; 38 patients had a single attack of optic neuritis or long lesion in the spinal cord and 15 patients presented with an opticospinal type of MS. The visual acuity following a single attack of optic neuritis remained significantly lower in NMO patients as compared to MS patients. Most of the NMO patients with NMO-IgG had additional attacks of optic neuritis within a short time from the initial event.The finding of NMO-IgG in patients with optic neuritis foreshadows a bad prognosis and relapses. These patients are at high risk of experiencing a second event in the central nervous system and fulfilling the clinical criteria for NMO. Due to the difference in the severity of inflammation of the optic nerve between NMO and MS, it is highly recommended to seek a laboratory check-up for NMO-IgG in serum, immediately after the first event, in order to determine the necessity and the kind of treatment for the patient.