Teriflunomide normalizes anti-anxiety effect in anti-ANXA2 APS mice model teriflunomide in anti-ANXA2 mice model.

Antiphospholipid syndrome (APS) affects the brain by both hypercoagulation and immunological mechanisms. APS is characterized by several autoantibodies binding to a thrombolytic complex including beta-2-glycoprotein I (ß2-GPI) and annexin A2 (ANXA2). Teriflunomide, an oral drug for the treatment of multiple sclerosis (MS), has a cytostatic effect on B cells and is therefore a potential antibody-targeting treatment for APS. In this study, we assessed the effect of teriflunomide in two APS mouse models by inducing autoantibody formation against ß2-GPI and ANXA2 in female BALB/c mice. The ANXA2 model displayed a behavioral change suggesting an anti-anxiety effect in open field and forced swim tests, early in the course of the disease. This effect was normalized following teriflunomide treatment. Conversely, behavioral tests done later during the study demonstrated depression-like behavior in the ANXA2 model. No behavioral changes were seen in the ß2-GPI model. Total brain IgG levels were significantly elevated in the ANXA2 model but not in the teriflunomide treated group. No such change was noted in the brains of the ß2-GPI model. High levels of serum autoantibodies were induced in both models, and their levels were not lowered by teriflunomide treatment. Teriflunomide ameliorated behavioral changes in mice immunized with ANXA2 without a concomitant change in serum antibody levels. These findings are compatible with the effect of teriflunomide on neuroinflammation.Teriflunomide ameliorated behavioral and brain IgG levels in mice immunized with ANXA2 without a concomitant change in serum antibody levels. These findings are compatible with an effect of teriflunomide on the IgG permeability to the brain and neuroinflammation.

Increased brain plasmin levels following experimental ischemic stroke in male mice.

Many coagulation factor proteases are increased in the brain during ischemic stroke. One of these proteases is plasmin. In this study we established a novel method for direct quantitative measurement of plasmin activity in male mouse brain slices using a sensitive fluorescent substrate in the presence of specific protease inhibitors. In both the ischemic and contralateral hemispheres, plasmin activity increased 3, 6, and 24 hr following stroke in comparison to healthy mice (F(3, 72) = 39.5, p < 0.0001, repeated measures ANOVA) after the induction of permanent middle cerebral artery occlusion (PMCAo). Plasmin activity was higher in the ischemic hemisphere (F(1,36) = 9.1, p = 0.005) and there was a significant interaction between time and ischemic hemisphere (F(3,36) = 4.4, p = 0.009). Plasmin activity was correlated with infarct volume (R2  = 0.5289, p = 0.0009 by Spearman). The specificity of the assay was verified utilizing tissue-type plasminogen activator (tPA)-deficient mice which, as expected, had significantly lower levels of plasmin 24 hr following ischemia compared to wild-type mice (ischemic (0.6 ± 0.23 and 1.94 ± 0.5, respectively), p = 0.049 and contralateral hemispheres (0.13 ± 0.14 and 0.75 ± 0.10, respectively), p = 0.018 by t test). There is a time-dependent increase in plasmin levels and an association of higher levels of plasmin with larger infarct volumes in an experimental stroke model. This suggests caution in the use of recombinant tPA (rtPA) and that plasmin inhibition in the brain may be a therapeutic target in acute ischemic stroke.

Autoantibodies to Annexin A2 and cerebral thrombosis: Insights from a mouse model.

INTRODUCTION: Antiphospholipid syndrome (APS) is an autoimmune disorder manifested by thromboembolic events, recurrent spontaneous abortions and elevated titers of circulating antiphospholipid antibodies. In addition, the presence of antiphospholipid antibodies seems to confer a fivefold higher risk for stroke or transient ischemic attack. Although the major antigen of APS is ß2 glycoprotein I, it is now well established that antiphospholipid antibodies are heterogeneous and bind to various targets. Recently, antibodies to Annexin A2 (ANXA2) have been reported in APS. This is of special interest since data indicated ANXA2 as a key player in fibrinolysis. Therefore, in the present study we assessed whether anti-ANXA2 antibodies play a pathological role in thrombosis associated disease. MATERIALS AND METHODS: Mice were induced to produce anti-ANXA2 antibodies by immunization with ANXA2 (iANXA2) and control mice were immunized with adjuvant only. A middle cerebral artery occlusion stroke model was applied to the mice. The outcome of stroke severity was assessed and compared between the two groups. RESULTS: Our results indicate that antibodies to ANXA2 lead to a more severe stroke as demonstrated by a significant larger stroke infarct volume (iANXA2 133.9 ± 3.3 mm3 and control 113.7 ± 7.4 mm3; p = 0.017) and a more severe neurological outcome (iANXA2 2.2 ± 0.2, and control 1.5 ± 0.18; p = 0.03). CONCLUSIONS: This study supports the hypothesis that auto-antibodies to ANXA2 are an independent risk factor for cerebral thrombosis. Consequently, we propose screening for anti-ANXA2 antibodies should be more widely used and patients that exhibit the manifestations of APS should be closely monitored by physicians.

Nuclear neutron-proton contact and the photoabsorption cross section.

The nuclear neutron-proton contact is introduced, generalizing Tan's work, and evaluated from medium energy nuclear photodisintegration experiments. To this end we reformulate the quasideuteron model of nuclear photodisintegration and establish the bridge between the Levinger constant and the contact. Using experimental evaluations of Levinger's constant, we extract the value of the neutron-proton contact in finite nuclei and in symmetric nuclear matter. Assuming isospin symmetry we propose to evaluate the neutron-neutron contact through the measurement of photonuclear spin correlated neutron-proton pairs.

Insulin-degrading enzyme deficiency accelerates cerebrovascular amyloidosis in an animal model.

Cerebrovascular amyloidosis (CA) may result in intraparenchymal bleeding and cognitive impairment. It was previously shown that transforming growth factor-ß1 (TGF-ß1) expression under an astrocyte promoter resulted in congophilic vascular deposits and vascular pathology. A reduction in insulin-degrading enzyme (IDE) activity was previously suggested to play a role in the accumulation of congophilic vascular deposits in the microvasculature of Alzheimer's disease (AD) cases. Here, we aim to investigate the link between TGF-ß1 and IDE activity in the development of CA. We found that TGF-ß1 can reduce IDE expression in a mouse brain endothelial cell line (ECs). Furthermore, we discovered that IDE activity in the brains of TGF-ß1 transgenic (Tg) mice was significantly reduced compared with that of the control mice in an age-dependent manner. In addition, TGF-ß1/IDE(-/-) mice showed significantly greater levels of cerebrovascular pathology compared with TGF-ß1 mice. We have previously shown that 16-month-old TGF-ß1 mice have a significant reduction in synaptophysin protein levels, which may lead to cognitive impairment. Here we discovered a significant reduction in synaptophysin protein already at the age of seven in the hippocampus of TGF-ß1/IDE(-/-) mice compared with TGF-ß1 mice. Further investigation of TGF-ß1-mediated IDE activity in ECs may provide useful therapeutic intervention targets for cerebrovascular diseases such as CA.

TGF-ß1 affects endothelial cell interaction with macrophages and T cells leading to the development of cerebrovascular amyloidosis.

Astrocyte-endothelial cell (EC) interactions play a major role in the function of the neurovascular unit. Dysfunction in these interactions may lead to amyloid accumulation in blood vessels and may cause microhemorrhage and cognitive impairment. Transforming growth factor-ß1 (TGF-ß1) expression levels positively correlate with the degree of cerebrovascular amyloid in Alzheimer's disease (AD) cases. Furthermore, expression of TGF-ß1 driven by the GFAP promoter in mice leads to an age-related deposition of amyloid, such as ß-amyloid (Aß), around cerebral blood vessels. Here, we demonstrate that TGF-ß1 affects the cross talk between EC and inflammation, leading to a reduction in macrophage activity as measured by protein levels and migration ability. Changes in EC secreted factors following TGF-ß1 stimulation also affect CD4(+) T cell activation, as shown by a reduction in the levels of IFN-γ. Moreover, while medium from EC can stimulate macrophages to clear insoluble cerebrovascular amyloid from an AD mouse brain, pre-incubation of EC with TGF-ß1 reduces the ability of EC to affect macrophage activity. Our findings support the importance of cross talk between EC, macrophages and CD4(+) T cells in preventing cerebrovascular amyloid deposition. Understanding EC-immune system interactions may pave the way to new therapeutic approaches for cerebrovascular amyloidosis diseases.

Human antibodies targeting a Mycobacterium transporter protein mediate protection against tuberculosis.

Mycobacterium tuberculosis (Mtb) exposure drives antibody responses, but whether patients with active tuberculosis elicit protective antibodies, and against which antigens, is still unclear. Here we generate monoclonal antibodies from memory B cells of one patient to investigate the B cell responses during active infection. The antibodies, members of four distinct B cell clones, are directed against the Mtb phosphate transporter subunit PstS1. Antibodies p4-36 and p4-163 reduce Mycobacterium bovis-BCG and Mtb levels in an ex vivo human whole blood growth inhibition assay in an FcR-dependent manner; meanwhile, germline versions of p4-36 and p4-163 do not bind Mtb. Crystal structures of p4-36 and p4-170, complexed to PstS1, are determined at 2.1 Å and 2.4 Å resolution, respectively, to reveal two distinctive PstS1 epitopes. Lastly, a prophylactic p4-36 and p4-163 treatment in Mtb-infected Balb/c mice reduces bacterial lung burden by 50%. Our study shows that inhibitory anti-PstS1 B cell responses arise during active tuberculosis.

Decreased hippocampal cell proliferation in mice with experimental antiphospholipid syndrome.

The antiphospholipid syndrome (APS) is an autoimmune disease characterized by the presence of antiphospholipid antibodies, which may trigger vascular thrombosis with consecutive infarcts. However, cognitive dysfunctions representing one of the most commonest neuropsychiatric symptoms are frequently present despite the absence of any ischemic brain lesions. Data on the structural and functional basis of the neuropsychiatric symptoms are sparse. To examine the effect of APS on hippocampal neurogenesis and on white matter, we induced experimental APS (eAPS) in adult female Balb/C mice by immunization with ß2-glycoprotein 1. To investigate cell proliferation in the dentate gyrus granular cell layer (DG GCL), eAPS and control mice (n = 5, each) were injected with 5-bromo-2'-deoxyuridine (BrdU) once a day for 10 subsequent days. Sixteen weeks after immunization, eAPS resulted in a significant reduction of BrdU-positive cells in the DG GCL compared to control animals. However, double staining with doublecortin and NeuN revealed a largely preserved neurogenesis. Ultrastructural analysis of corpus callosum (CC) axons in eAPS (n = 6) and control mice (n = 7) revealed no significant changes in CC axon diameter or g-ratio. In conclusion, decreased cellular proliferation in the hippocampus of eAPS mice indicates a limited regenerative potential and may represent one neuropathological substrate of cognitive changes in APS while evidence for alterations of white matter integrity is lacking.

Behavioral abnormalities in female mice following administration of aluminum adjuvants and the human papillomavirus (HPV) vaccine Gardasil.

Vaccine adjuvants and vaccines may induce autoimmune and inflammatory manifestations in susceptible individuals. To date most human vaccine trials utilize aluminum (Al) adjuvants as placebos despite much evidence showing that Al in vaccine-relevant exposures can be toxic to humans and animals. We sought to evaluate the effects of Al adjuvant and the HPV vaccine Gardasil versus the true placebo on behavioral and inflammatory parameters in female mice. Six-week-old C57BL/6 female mice were injected with either, Gardasil, Gardasil + pertussis toxin (Pt), Al hydroxide, or, vehicle control in amounts equivalent to human exposure. At 7.5 months of age, Gardasil and Al-injected mice spent significantly more time floating in the forced swimming test (FST) in comparison with vehicle-injected mice (Al, p = 0.009; Gardasil, p = 0.025; Gardasil + Pt, p = 0.005). The increase in floating time was already highly significant at 4.5 months of age for the Gardasil and Gardasil + Pt group (p ≤ 0.0001). No significant differences were observed in the number of stairs climbed in the staircase test which measures locomotor activity. These results indicate that differences observed in the FST were unlikely due to locomotor dysfunction, but rather due to depression. Moreover, anti-HPV antibodies from the sera of Gardasil and Gardasil + Pt-injected mice showed cross-reactivity with the mouse brain protein extract. Immunohistochemistry analysis revealed microglial activation in the CA1 area of the hippocampus of Gardasil-injected mice. It appears that Gardasil via its Al adjuvant and HPV antigens has the ability to trigger neuroinflammation and autoimmune reactions, further leading to behavioral changes.

Rheumatic diseases and autoimmune vascular dementia.

Vascular dementia (VD) comes second after Alzheimer's disease (AD) as a cause of impaired cognition. VD is not a specific nosological entity, but rather a syndrome encompassing a number of diseases caused by impaired supply of blood to the brain. Systemic autoimmune disorders such as systemic lupus erythematosus, rheumatoid arthritis, vasculitis and antiphospholipid syndrome (APS) can be associated with dementia. VD is often related to the presence of traditional cardiovascular risk factors, but it may also be associated with a host of disorders affecting the brain blood vessels, neuronal cells, or both. It is important to entertain in the differential diagnosis of VD, to recognize and to cure them accurately in order to preserve life's quality of our patients.

Thrombin and protein C pathway in peripheral nerve Schwann cells.

Thrombin and activated protein C (aPC) bound to the endothelial protein C receptor (EPCR) both activate protease-activated receptor 1 (PAR1) generating either harmful or protective signaling respectively. In the present study we examined the localization of PAR-1 and EPCR and thrombin activity in Schwann glial cells of normal and crushed peripheral nerve and in Schwannoma cell lines. In the sciatic crush model nerves were excised 1h, 1, 4, and 7days after the injury. Schwannoma cell lines produced high levels of prothrombin which is converted to active thrombin and expressed both EPCR and PAR-1 which co-localized. In the injured sciatic nerve thrombin levels were elevated as early as 1h after injury, reached their peak 1day after injury which was significantly higher (24.4±4.1mU/ml) compared to contralateral uninjured nerves (2.6±7mU/ml, t-test p<0.001) and declined linearly reaching baseline levels by day 7. EPCR was found to be located at the microvilli of Schwann cells at the node of Ranvier and in cytoplasm surrounding the nucleus. Four days after sciatic injury, EPCR levels increased significantly (57,785±16602AU versus 4790±1294AU in the contralateral uninjured nerves, p<0.001 by t-test) mainly distal to the site of injury, where axon degeneration is followed by proliferation of Schwann cells which are diffusely stained for EPCR. EPCR seems to be located to cytoplasmic component of Schwann cells and not to compact myelin component, and is highly increased following injury.

WITHDRAWN: Behavioral abnormalities in young female mice following administration of aluminum adjuvants and the human papillomavirus (HPV) vaccine Gardasil.

This article has been withdrawn at the request of the Editor-in-Chief due to serious concerns regarding the scientific soundness of the article. Review by the Editor-in-Chief and evaluation by outside experts, confirmed that the methodology is seriously flawed, and the claims that the article makes are unjustified. As an international peer-reviewed journal we believe it is our duty to withdraw the article from further circulation, and to notify the community of this issue. The full Elsevier Policy on Article Withdrawal can be found at http://www.elsevier.com/locate/withdrawalpolicy.

Scavenger receptor A deficiency accelerates cerebrovascular amyloidosis in an animal model.

Cerebrovascular amyloidosis caused by amyloid accumulation in blood vessel walls may lead to hemorrhagic stroke and cognitive impairment. Expression of TGF-ß1 under glial fibrillary acidic protein promoter in mice leads to age-related deposition of amyloid, including ß-amyloid (Aß), around cerebral blood vessels, leading to vascular pathology starting at age of 7 months. We have recently shown the important role of macrophages in clearing cerebrovascular amyloid. Scavenger receptor A (SRA) is a multi-ligand and multifunctional receptor expressed on macrophages, and it has been suggested to play a role in meditating phagocytosis of different types of antigens. We investigated the role of SRA in mediating cerebrovascular amyloid clearance. We bred TGF-ß1 mice with SRA(-/-) mice and discovered that TGF-ß1/SRA(-/-) mice showed cerebrovascular pathology at an earlier age (3 months) compared with TGF-ß1 mice. Furthermore, SRA deficiency in macrophages led to impaired clearing of congophilic cerebrovascular amyloid from amyloid precursor protein mouse model and led to reduced phagocytosis of both soluble and insoluble Aß in vivo as compared with macrophages from wild-type mice. Our findings demonstrate the important role of SRA in cerebrovascular amyloid pathology and suggest targeting SRA for future diagnostic and therapeutic approaches for cerebral amyloid angiopathy.

Immunotherapy of cerebrovascular amyloidosis in a transgenic mouse model.

Cerebrovascular amyloidosis is caused by amyloid accumulation in walls of blood vessel walls leading to hemorrhagic stroke and cognitive impairment. Transforming growth factor-ß1 (TGF-ß1) expression levels correlate with the degree of cerebrovascular amyloid deposition in Alzheimer's disease (AD) and TGF-ß1 immunoreactivity in such cases is increased along the cerebral blood vessels. Here we show that a nasally administered proteosome-based adjuvant activates macrophages and decreases vascular amyloid in TGF-ß1 mice. Animals were nasally treated with a proteosome-based adjuvant on a weekly basis for 3 months beginning at age 13 months. Using magnetic resonance imaging (MRI) we found that while control animals showed a significant cerebrovascular pathology, proteosome-based adjuvant prevents further brain damage and prevents pathological changes in the blood-brain barrier. Using an object recognition test and Y-maze, we found significant improvement in cognition in the treated group. Our findings support the potential use of a macrophage immunomodulator as a novel approach to reduce cerebrovascular amyloid, prevent microhemorrhage, and improve cognition.