Intracranial Assessment of Androgen Receptor Antagonists in Mice Bearing Human Glioblastoma Implants.

The median survival time of patients with an aggressive brain tumor, glioblastoma, is still poor due to ineffective treatment. The discovery of androgen receptor (AR) expression in 56% of cases offers a potential breakthrough. AR antagonists, including bicalutamide and enzalutamide, induce dose-dependent cell death in glioblastoma and glioblastoma-initiating cell lines (GIC). Oral enzalutamide at 20 mg/kg reduces subcutaneous human glioblastoma xenografts by 72% (p = 0.0027). We aimed to further investigate the efficacy of AR antagonists in intracranial models of human glioblastoma. In U87MG intracranial models, nude mice administered Xtandi (enzalutamide) at 20 mg/kg and 50 mg/kg demonstrated a significant improvement in survival compared to the control group (p = 0.24 and p < 0.001, respectively), confirming a dose-response relationship. Additionally, we developed a newly reformulated version of bicalutamide, named "soluble bicalutamide (Bic-sol)", with a remarkable 1000-fold increase in solubility. This reformulation significantly enhanced bicalutamide levels within brain tissue, reaching 176% of the control formulation's area under the curve. In the U87MG intracranial model, both 2 mg/kg and 4 mg/kg of Bic-sol exhibited significant efficacy compared to the vehicle-treated group (p = 0.0177 and p = 0.00364, respectively). Furthermore, combination therapy with 8 mg/kg Bic-sol and Temozolomide (TMZ) demonstrated superior efficacy compared to either Bic-sol or TMZ as monotherapies (p = 0.00706 and p = 0.0184, respectively). In the ZH-161 GIC mouse model, the group treated with 8 mg/kg Bic-sol as monotherapy had a significantly longer lifespan than the groups treated with TMZ or the vehicle (p < 0.001). Our study demonstrated the efficacy of androgen receptor antagonists in extending the lifespan of mice with intracranial human glioblastoma, suggesting a promising approach to enhance patient outcomes in the fight against this challenging disease.

MTADV 5-MER peptide suppresses chronic inflammations as well as autoimmune pathologies and unveils a new potential target-Serum Amyloid A.

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-1ß 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.

Nitric Oxide Interaction with the Eye.

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.

Synthesis and Pharmacological Characterization of Visabron, a Backbone Cyclic Peptide Dual Antagonist of α4ß1 (VLA-4)/α9ß1 Integrin for Therapy of Multiple Sclerosis.

Integrins α4ß1/ α9ß1 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 α4ß1 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 α4ß1 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.

QKI-V5 is downregulated in CNS inflammatory demyelinating diseases.

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: a potential therapeutic target for glioblastoma.

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.

T-cell responses to distinct AQP4 peptides in patients with neuromyelitis optica (NMO).

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.

Modulation of Hyperosmotic and Immune-Induced Disruption of the Blood-Brain Barrier by the Nitric Oxide System.

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.

Treatment of a multiple sclerosis animal model by a novel nanodrop formulation of a natural antioxidant.

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.

Nitric oxide secretion in human conjunctival fibroblasts is inhibited by alpha linolenic acid.

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-1ß 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-1ß induced the highest nitrite secretion (2.91 fold, P < 0.01) as compared to cells incubated in medium alone. nitrite secretion was reduced by 38.9 % (P < 0.05) after treatment with ALA alone. Co-culturing PBMC with HCF with and without ALA treatment demonstrated similar results in nitrite level as,compared to PBMC alone. In addition, ALA significantly decreased NOS-2 expression in HCF by 48.9 % (P < 0. 001) after 72 h. CONCLUSIONS: The decrease in nitrite release and inhibition of NOS-2 expression indicate that ALA may have an anti-inflammatory effect both on HCF and on peripheral immune cells. This indicates that ALA may serve as a potent anti-inflammatory agent in ocular surface inflammation.

Electrical stimulation of the amygdala modifies the negative feedback effect of glucocorticoids on the adrenocortical responses to stress.

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.

Nano-Drugs Based on Nano Sterically Stabilized Liposomes for the Treatment of Inflammatory Neurodegenerative Diseases.

The present study shows the advantages of liposome-based nano-drugs as a novel strategy of delivering active pharmaceutical ingredients for treatment of neurodegenerative diseases that involve neuroinflammation. We used the most common animal model for multiple sclerosis (MS), mice experimental autoimmune encephalomyelitis (EAE). The main challenges to overcome are the drugs' unfavorable pharmacokinetics and biodistribution, which result in inadequate therapeutic efficacy and in drug toxicity (due to high and repeated dosage). We designed two different liposomal nano-drugs, i.e., nano sterically stabilized liposomes (NSSL), remote loaded with: (a) a "water-soluble" amphipathic weak acid glucocorticosteroid prodrug, methylprednisolone hemisuccinate (MPS) or (b) the amphipathic weak base nitroxide, Tempamine (TMN). For the NSSL-MPS we also compared the effect of passive targeting alone and of active targeting based on short peptide fragments of ApoE or of ß-amyloid. Our results clearly show that for NSSL-MPS, active targeting is not superior to passive targeting. For the NSSL-MPS and the NSSL-TMN it was demonstrated that these nano-drugs ameliorate the clinical signs and the pathology of EAE. We have further investigated the MPS nano-drug's therapeutic efficacy and its mechanism of action in both the acute and the adoptive transfer EAE models, as well as optimizing the perfomance of the TMN nano-drug. The highly efficacious anti-inflammatory therapeutic feature of these two nano-drugs meets the criteria of disease-modifying drugs and supports further development and evaluation of these nano-drugs as potential therapeutic agents for diseases with an inflammatory component.

False negative ß-2 transferrin in the diagnosis of cerebrospinal fluid leak in the presence of Streptococcus pneumoniae.

OBJECTIVES/HYPOTHESIS: The objectives of this study were to examine the presence of ß-2 transferrin (ß2TRNSF) in cerebrospinal fluid (CSF) contaminated in vitro by various bacteria and explore the mechanism (passive or active) responsible for ß2TRNSF elimination. Early diagnosis of CSF leakage may change treatment decisions and minimize the risk of meningitis and encephalitis. ß2TRNSF 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. ß2TRNSF 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 ß2TRNSF analysis we used immunoblotting electrophoresis and enzyme-linked immunosorbent assay (ELISA). RESULTS: CSF samples collected from nine patients were analyzed. ß2TRNSF was not detected following S pneumoniae inoculation at both time points when immunoblotting electrophoresis was used. Quantitative analysis using ELISA demonstrated significant ß2TRNSF concentration decrease. The addition of ciprofloxacin led to the same results. CONCLUSIONS: CSF leak detection using ß2TRNSF may be deceiving in the presence of a S pneumoniae cerebral nervous system infection. A passive process is suggested, as ß2TRNSF disappeared either immediately or following incubation with inactive bacteria.

Increased anti-KIR4.1 antibodies in multiple sclerosis: could it be a marker of disease relapse?

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.

Antineoplastic effect of decoy oligonucleotide derived from MGMT enhancer.

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.

Pomegranate seed oil nanoemulsions for the prevention and treatment of neurodegenerative diseases: the case of genetic CJD.

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.

Anti-inflammatory effects of resolvin-D1 on human corneal epithelial cells: in vitro study.

BACKGROUND: This study evaluated the anti-inflammatory effects of Resolvin-D1 (RV-D1) and its mechanism of action in human corneal epithelial (HCE) cells. METHODS: HCE cells were incubated with different concentrations of RV-D1 for different time periods. Oleic acid (OA) and Dexamethasone (DM) served as negative and positive controls, respectively. Cells were stimulated with polyriboinosinic:polyribocytidylic acids (poly I:C). The protein contents and mRNA expression levels of Tumor necrosis factor-α (TNF-α), Interleukin (IL)-6, IL-1ß and IL-8 were evaluated with multiplex fluorescent bead immunoassay (FBI) and real time-PCR, respectively. In addition, the expression of inhibitory factor-κBα (I-κBα) was evaluated with real time-PCR. RESULTS: The protein level of pro-inflammatory cytokines TNF-α, IL-6, IL-1ß and IL-8 significantly increased after stimulation with Poly I:C. RV-D1 treatment at concentration of 1 µM decreased the protein level of TNF-α to 20.76 ± 9.3% (P < 0.05), IL-6 to 43.54 ± 14.16% (P < 0.001), IL-1ß to 46.73 ± 15.93% (P > 0.05) and IL-8 to 51.15 ± 13.01% (P < 0.05) compared with cells stimulated with poly I:C alone. Similarly, the mRNA levels of TNF-α, IL-6, IL-1ß and IL-8 were significantly reduced after treatment with RV-D1. A highly significant dose response curve was demonstrated for RV-D1 treated HCE cells for TNF-α and IL-1ß.DM treatment decreased the protein content for all of the pro-inflammatory cytokines, similar results were demonstrated at the mRNA level. The anti-inflammatory effects of RV-D1 were similar to those of DM for TNF-α, IL-6 and IL-8. CONCLUSIONS: RV-D1 may serve as a potent anti-inflammatory agent in ocular surface inflammation, as evaluated in cultured HCE cells. The anti-inflammatory effects of RV-D1 were comparable to those of DM, and were mediated through nuclear factor kappa B (NF-κB) signal transduction.

Genetic prion disease: no role for the immune system in disease pathogenesis?

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.

Expression and activation of toll-like receptor 3 and toll-like receptor 4 on human corneal epithelial and conjunctival fibroblasts.

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-1ß 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 < 0.001). Incubation with poly I:C lowered the percentage of HCE cells positive for TLR3 from 10.44% to 2.84% (P < 0.001). The mRNA expression of TLRs2, 4, 7 and 9 was detected in HCE only. Activation of HCE with LPS complex elicited protein secretion up to 4.51 ± 0.85-fold higher levels of IL-6 (P < 0.05), 2.5 ± 0.36-fold IL-8 (P > 0.05), 4.35 ± 1.12-fold IL-1ß (P > 0.05) and 29.35 ± 2.3-fold TNFα (P < 0.05) compared to cells incubated in medium. 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.