Coagulopathy triggered autoimmunity: experimental antiphospholipid syndrome in factor V Leiden mice.

BACKGROUND: We investigated interactions between genetically and autoimmune-mediated coagulopathies by inducing experimental antiphospholipid syndrome (eAPS) in mice carrying the factor V Leiden (FVL) mutation. METHODS: eAPS was induced in heterozygous and homozygous FVL transgenic mice (C57BL/6 background) by immunization with ß(2)-glycoprotein I (ß(2)-GPI). Autoantibody levels were measured at 1 and 5 months post-immunization. Mice were tested at 4 months post-immunization for behavior and cognitive function in the staircase, elevated plus-maze, and swim T-maze tests. Brains were removed and analyzed by immunohistochemistry for inflammatory markers and neurodegenerative processes. RESULTS: A single immunization with ß(2)-GPI induced significantly higher and longer-lasting immune responses, and this was dependent on the number of FVL alleles. At 1 and 5 months post-immunization, levels of antibodies rose from 1.17 ± 0.07 to 1.62 ± 0.17 (optical density units; ODU) in homozygous FVL mice, compared with stable levels of 0.59 ± 0.17 and 0.48 ± 0.16 ODU in heterozygous FVL mice and a drop from 1.62 ± 0.21 to 0.61 ± 0.13 ODU in wild-type mice. Behavioral and cognitive clinical features of eAPS were also correlated with FVL allele load, as assessed by the elevated plus-maze (altered anxiety), staircase (hyperactivity and higher exploration), and swim T-maze (impaired learning) tests. Histological studies identified significant neurodegenerative changes in both grey and white matter in the eAPS-FVL brains. In spite of the potential interaction of two prothrombotic disease states, there were no ischemic lesions seen in this group. CONCLUSIONS: The results indicate that genetically mediated coagulopathies increase the risk of developing coagulation-targeted autoimmune responses, and suggest the importance of antibody-mediated neurodegenerative processes in the brain in APS.

Davunetide (NAP) as a preventative treatment for central nervous system complications in a diabetes rat model.

AIMS: Central nervous system complications including cognitive impairment are an early manifestation of diabetes mellitus, also evident in animal models. NAP (generic name, davunetide), a neuroprotective peptide was tested here for its ability to prevent diabetes-related brain pathologies in the streptozotocin injected diabetes rat model. METHODS: Diabetes was induced by an intraperitoneal streptozotocin injection (55 mg/kg). Intranasal NAP or vehicle was administered daily starting on the day following streptozotocin injection. Cognitive assessment was performed 12 weeks after diabetes induction, using the Morris water maze paradigm. Brain structural integrity was assessed on the 15th week of diabetes by magnetic resonance T2 scan. Characterization of cellular populations, apoptosis and synaptic density was performed 16 weeks after diabetes induction, using immunohistochemical markers and quantified in the prefrontal cortex, the cerebral cortex and the hippocampus of both hemispheres. RESULTS: Impaired spatial memory of the diabetic rats was observed in the water maze by attenuated learning curve and worsened performance in the probe memory test. NAP treatment significantly improved both measurements. T2 magnetic resonance imaging revealed atrophy in the prefrontal cortex of the diabetes rat group, which was prevented by NAP treatment. Immunohistochemical analysis showed that NAP treatment protected against major loss of the synaptic marker synaptophysin and astrocytic apoptosis, resulting from streptozotocin treatment. CONCLUSIONS: Our results show for the first time protective effects for NAP (davuentide) in a diabetes rat model at the behavioral and structural levels against one of the most severe complications of diabetes.

Deciphering anti-MOG IgG antibodies: Clinical and radiological spectrum, and comparison of antibody detection assays.

IgG antibodies to myelin oligodendrocyte glycoprotein (MOG) detected by cell based assays (CBA) have been identified in a constantly expanding spectrum of CNS demyelinating disorders. However, a universally accepted CBA has not been adopted yet. We aimed to analyze the clinical and radiological features of patients with anti-MOG IgG1-antibodies detected with a live-cell CBA and to compare the three most popular MOG-CBAs. We screened sera from 1300 Greek patients (including 426 patients referred by our 8 clinics) suspected for anti-MOG syndrome, and 120 controls with the live-cell MOG-CBA for IgG1-antibodies. 41 patients, versus 0 controls were seropositive. Clinical, serological and radiological data were available and analyzed for the 21 seropositive patients out of the 426 patients of our clinics. Their phenotypes were: 8 optic neuritis, 3 myelitis, 3 neuromyelitis optica, 2 encephalomyelitis, 2 autoimmune encephalitis and 3 atypical MS. We then retested all sera of our 426 patients with the other two most popular MOG-CBAs for total IgG (a live-cell and a commercial fixed-cell CBAs). Seven IgG1-seropositive patients were seronegative for one or both IgG-CBAs. Yet, all 21 patients had clinical and radiological findings previously described in MOG-antibody associated demyelination disease supporting the high specificity of the IgG1-CBA. In addition, all IgG1-CBA-negative sera were also negative by the IgG-CBAs. Also, all controls were negative by all three assays, except one serum found positive by the live IgG-CBA. Overall, our findings support the wide spectrum of anti-MOG associated demyelinating disorders and the superiority of the MOG-IgG1 CBA over other MOG-CBAs.

NAP protects memory, increases soluble tau and reduces tau hyperphosphorylation in a tauopathy model.

NAP (NAPVSIPQ) provides broad neuroprotection through microtubule interaction. Here, NAP was investigated for neuroprotection in an in vivo tauopathy model. Transgenic mice (2-month-old) that express the human double mutant tau protein [P301S;K257T] fused to the tau promoter, were subjected to daily intranasal drug treatment for approximately 5 months. Results showed increased performance in the NAP-treated mice compared to controls, as demonstrated in the Morris water maze, (p<0.05). Treatment continued for 5 additional months and mouse cortices were biochemically analyzed. Protein extraction identified increased tau protein content in the heat-stable soluble fraction, which contains microtubule-associated tau, in the 1-year-old NAP-treated mice as compared to vehicle-controls. Tau phosphorylation (Ser 202) increased in the tau-transgenic mice compared to control mice, and was significantly reduced in NAP-treated mice. The current studies show for the first time activity for NAP in a "pure" tauopathy model, positioning it as a promising drug candidate in multiple neurodegenerative tauopathies.

Interaction of inflammation, thrombosis, aspirin and enoxaparin in CNS experimental antiphospholipid syndrome.

Experimental antiphospholipid syndrome (eAPS) induced by immunization with beta(2)-glycoprotein I (beta(2)-GPI) causes behavioral hyperactivity. We assessed the role of thrombotic and inflammatory perivascular factors and standard APS therapies for CNS manifestations. Groups of mice (n=10 per group) were immunized once with beta(2)-GPI (eAPS) or adjuvant (controls) and treated daily from 1 month after immunization with either sham injections, aspirin (1.2 mg/kg) or enoxaparin (1 mg/kg) for 3 months. Serum antiphospholipid antibodies (aPL) and brain levels of tissue necrosis factor-alpha (TNF-alpha) and prostaglandin E (PGE) were then measured by ELISA and thrombin inhibitors by immunoblot. Behavioral hyperactivity was assessed by the staircase test. The eAPS mice had higher levels of aPL than adjuvant immunized controls. Inflammatory markers were found to be twofold higher and intrinsic brain thrombin inhibitors 50% lower in eAPS brains compared to controls. aPL titers were unaffected by treatment. Both aspirin and enoxaparin normalized brain concentrations of PGE and TNF-alpha and elevated thrombin inhibitors, the latter effect being more pronounced for enoxaparin. The increased activity and rearing exploratory behavior in eAPS (138.6+/-13.6 and 141.9+/-13.9% of controls, respectively) were attenuated significantly more by treatment with enoxaparin (91.5+/-12.3 and 95.0+/-9.8%) than by aspirin (167.0+/-18.4 and 114.7+/-13.1%, p<0.01, ANOVA). Together, these results demonstrate that eAPS is associated with significant brain inflammation and thrombosis that are well treated with both standard therapies for human APS. Enoxaparin attenuates behavior better than aspirin possibly by reducing thrombosis or stabilization of the blood brain barrier, suggesting an advantage for similar drugs in CNS APS.

Cannabinoids and capsaicin improve liver function following thioacetamide-induced acute injury in mice.

OBJECTIVES: We have shown the beneficial effects of cannabinoids in a murine model of hepatic encephalopathy following thioacetamide and now report their effects on the liver injury. METHODS: Fulminant hepatic failure (FHF) was induced by administration of 200 mg/kg thioacetamide to wild-type (WT) and CB2 Knockout (KO) mice. Twenty-four hours later, mice were injected with 2-arachidonoylglycerol (CB1, CB2, and TRPV1 agonist), HU308 (CB2 agonist), SR141716 A (CB1 receptor blocker), SR141716 A+2-AG, and SR144528 (CB2 receptor blocker), capsaicin and capsazepine (TRPV1 agonist and antagonist receptors). Mice were sacrificed 2 days after thioacetamide administration (day 3) and liver biochemistry and histopathology as well as evaluation of 2-arachidonoylglycerol levels were performed on liver tissue. RESULTS: Liver histopathology undertaken 48 h after thioacetamide showed evidence of necrosis and inflammation. SR141716 A, HU308, and 2-arachidonoylglycerol reduced inflammation and promoted regeneration 1 day after their administration. Liver enzymes increased after thioacetamide administration and were reversed after SR141716 A and 2-arachidonoylglycerol administered alone or combined, HU-308, but not SR144528. Thus, the beneficial effects mediated through CB2 receptors. However, CB2 KO mice still modulated liver function via the TRPV1 receptors. Capsaicin improved both liver pathology and function in WT thioacetamide-treated mice, while capsazepine impaired it. CONCLUSIONS: The similar pattern found between the effect of cannabinoids and their antagonists on brain and liver indicated that the therapeutic effect might be directed by the improvement in both organs through CB2 receptors and/or TRPV1 receptors. Modulation of these systems may have therapeutic potential.

Immune Parameters That Distinguish Multiple Sclerosis Patients from Patients with Other Neurological Disorders at Presentation.

BACKGROUND/AIM: Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system. Effector T helper cells, mainly Th1 and Th17, cytotoxic T-cells, B-cells, macrophages, microglia, and the cytokines they secrete, are implicated in the initiation and maintenance of a deregulated immune response to myelin antigens and the ensuing immune-mediated demyelination. In this study, we investigated whether signature cytokines exist in MS patients at presentation to gain an insight into the underlying immunopathogenic processes at the early stage of the disease. METHODS: We collected serum and cerebrospinal fluid (CSF) samples from 123 patients at presentation, eventually diagnosed with MS or non-inflammatory (NIND) or inflammatory neurological diseases (IND) or symptomatic controls (SC). The levels of cytokines IFN-γ, TNF-α, TGF-ß1, IL-2, IL-4, IL-6, IL-10 and IL-17 were measured, and cytokine ratios, such as Th1/Th2, Th1/Th17, and Type-1/Type-2, were calculated. All parameters were tested for their correlations with the intrathecal IgG synthesis. RESULTS: Cytokine levels in CSF were lower than in serum in all the patients, with the exception of IL-6. Serum or CSF cytokine levels of MS patients did not differ significantly from NIND or SC, with the exception of serum IFN-γ and TNF-α that were significantly higher in NIND. IND patients presented with the highest levels of all cytokines in serum and CSF, with the exception of serum IL-10 and CSF IL-17. MS patients had a significantly lower serum Th1/Th2 ratio compared to the NIND and IND groups, and significantly lower serum Type-1/Type-2, IFN-γ/IL-10 and CSF Th1/Th17 ratios compared to IND patients. MS patients had a significantly higher CSF IL-17/IL-10 ratio compared to IND patients. The IgG index was higher in MS patients compared to the control groups; the differences reached statistical significance between the MS and the NIND and SC groups. Reiber-Felgenhauer analysis of the QIgG and QAlb indices revealed higher intrathecal IgG synthesis in MS patients, and higher blood-CSF barrier dysfunction in IND patients. The IgG index correlated with CSF IL-4 in MS patients only. CONCLUSIONS: We found no signature cytokines or profiles thereof in MS patients at presentation. Only IND patients presented with a clear Th1 cytokine polarization in serum and CSF. The parameters that distinguished MS patients from patients with other neurological disorders were IgG intrathecal synthesis, the IgG index and its correlation with CSF IL-4 levels.

New horizons in schizophrenia treatment: autophagy protection is coupled with behavioral improvements in a mouse model of schizophrenia.

Autophagy plays a key role in the pathophysiology of schizophrenia as manifested by a 40% decrease in BECN1/Beclin 1 mRNA in postmortem hippocampal tissues relative to controls. This decrease was coupled with the deregulation of the essential ADNP (activity-dependent neuroprotector homeobox), a binding partner of MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 ß) another major constituent of autophagy. The drug candidate NAP (davunetide), a peptide fragment from ADNP, enhanced the ADNP-LC3B interaction. Parallel genetic studies have linked allelic variation in the gene encoding MAP6/STOP (microtubule-associated protein 6) to schizophrenia, along with altered MAP6/STOP protein expression in the schizophrenic brain and schizophrenic-like behaviors in Map6-deficient mice. In this study, for the first time, we reveal significant decreases in hippocampal Becn1 mRNA and reversal by NAP but not by the antipsychotic clozapine (CLZ) in Map6-deficient (Map6(+/-)) mice. Normalization of Becn1 expression by NAP was coupled with behavioral protection against hyperlocomotion and cognitive deficits measured in the object recognition test. CLZ reduced hyperlocomotion below control levels and did not significantly affect object recognition. The combination of CLZ and NAP resulted in normalized outcome behaviors. Phase II clinical studies have shown NAP-dependent augmentation of functional activities of daily living coupled with brain protection. The current studies provide a new mechanistic pathway and a novel avenue for drug development.

Immunohistochemical analysis of sympathetic involvement in the SOD1-G93A transgenic mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal disorder caused by degeneration of motor neurons in the cerebral cortex, brainstem and spinal cord. Several clinical reports indicated sympathetic and parasympathetic dysfunction in ALS patients. In addition, we have recently reported elevated heart rate and blood pressure in transgenic (TG) mice carrying the SOD1 mutant form of the human SOD1 transgene (SOD1-G93A) even prior to the appearance of motor symptoms. In order to further elucidate the mechanisms underlying autonomic impairment in ALS we performed an immunohistochemical study of the intermediolateral nucleus (IML) column neurons (T2 - L2), superior cervical ganglia (SCG) and adrenal glands (AG) in TG and littermate wild-type (WT) mice at the age of 75 - 80 days. IML column neurons sections were stained with antibodies against choline acetyltransferase (ChAT), SOD1, ubiquitin and SMI31. SCG and AG sections were stained with antibodies against ChAT and tyrosine hydroxylase (TH). ChAT is predominantly located at preganglionic nerve terminals that innervate the AG, while TH is located in sympathetic neurons. Results showed that ChAT, SOD1 and ubiquitin expressions in IML column were significantly lower in the SOD1-G93A group compared to WT (p < 0.0001, p = 0.0042 and p < 0.0001, respectively). SMI31 measurements did not reveal any statistical differences between the two groups (p = 0.6187). TH expression in AG revealed a 24% decrease in the SOD1-G93A group compared to WT (p < 0.0001), while ChAT expression in the SCG was reduced by 28% (p < 0.0001). No significant differences were found for TH in SCG or for ChAT in AG. In conclusion, these results are consistent with preganglionic sympathetic denervation as a potential contributor to the abnormal sympathetic regulation in ALS.

Novel acylethanolamide derivatives that modulate body weight through enhancement of hypothalamic pro-opiomelanocortin (POMC) and/or decreased neuropeptide Y (NPY).

Newly synthesized acylethanolamide derivatives oleoyl-L-valinolamide (1), oleoyl-D-valinolamide (2), elaidoyl-L-valinolamide (3), elaidoyl-D-valinolamide (4) stearoyl-L-valinolamide (5), and palmitoyl-L-valinolamide (6) were investigated in mice as antiobesity compounds. Compounds 1, 2, 5, 6 significantly decreased body weight by 6.57% following eight injections of 1 mg/kg i.p. during 39 days, while 3 and 4 showed no such activity. Receptor binding indicated that no compound activated CB1, CB2, PPARα, or TRPV1 receptors. Hypothalamic RT-PCR showed that mRNA expression of the anorexigenic genes POMC and CART was up-regulated by 1, 2, 5 and 1, 2, respectively, while that of the orexigenic genes NPY and CaMKK2 was down-regulated by the respective compounds 1, 5, 6 and 1, 2, 5. Oleoyl-L-valinolamide enhances anorectic pathways and lead to decreased glucose levels, enhanced locomotor activity, and improved cognition. Effects of oleoyl-L-valinolamide on weight were dose-dependent, and it could be given orally. 1, 2, 4, 5 down-regulated FAAH mRNA expression.

Urine from scrapie-infected hamsters comprises low levels of prion infectivity.

The question of whether prion diseases can be transmitted by body fluids has important epidemiological, environmental and economical implications. In this work, we set to investigate whether urine collected from scrapie-infected hamsters can transmit fatal or subclinical infectivity to normal hamsters. After prolonged incubation times ranging from 300 to 700 days, a small number of animals inoculated with scrapie urine succumbed to scrapie disease, and several asymptomatic hamsters presented low levels of PrP(Sc) in their brains. In addition, most of the asymptomatic hamsters inoculated with scrapie urine, as opposed to those inoculated with normal urine, presented extensive gliosis as well as protease-resistant light chain IgG in their urine, a molecule shown by us and others to be a surrogate marker for prion infection. Our results suggest that urine from scrapie-infected hamsters can transmit a widespread subclinical disease that in some cases develops into fatal scrapie.