Mitochondrial alterations near amyloid plaques in an Alzheimer's disease mouse model.

While accumulation of amyloid-ß (Aß) deposited as senile plaques is a hallmark feature of Alzheimer's disease (AD), the neurotoxicity of these deposits remains controversial. Recent in vitro studies suggested a link between elevated Aß and mitochondrial dysfunction that might contribute to the pathogenesis of AD. However, the in vivo evidence for mitochondria dysfunction caused by Aß is still missing. Using intravital multiphoton imaging with a range of fluorescent markers, we systematically surveyed mitochondrial structural and functional changes in AD mouse models. We observed severe impairments to be limited to the vicinity of Aß plaques, which included reduction of both numbers and membrane potential of mitochondria and the emergence of dystrophic and fragmented mitochondria. Both neuronal soma and neurites with oxidative stress show severe alterations in mitochondrial membrane potential in amyloid precursor protein mice. These results provide in vivo evidence revealing Aß plaques as focal sources of toxicity that lead to severe structural and functional abnormalities in mitochondria. These alterations may contribute to neuronal network dysfunction and warrant further investigation as possible targets for therapeutic intervention in AD.

P2Y6 receptors require an intact cysteinyl leukotriene synthetic and signaling system to induce survival and activation of mast cells.

Cysteinyl leukotrienes (cys-LTs) induce inflammatory responses through type 1 (CysLT1R) and type 2 (CysLT2R) cys-LT receptors and activate mast cells in vitro. We previously demonstrated that cys-LTs cross-desensitized IL-4-primed primary human mast cells (hMCs) to stimulation with the nucleotide uridine diphosphate (UDP). We now report that hMCs, mouse bone marrow-derived mast cells (mBMMCs), and the human MC line LAD2 all express UDP-selective P2Y6 receptors that cooperate with CysLT1R to promote cell survival and chemokine generation by a pathway involving reciprocal ligand-mediated cross-talk. Leukotriene (LT) D4, the most potent CysLT1R ligand, and UDP both induced phosphorylation of ERK and prolonged the survival of cytokine-starved hMCs and mBMMCs. ERK activation and cytoprotection in response to either ligand were attenuated by treatment of the cells with a selective P2Y6 receptor antagonist (MRS2578), which did not interfere with signaling through recombinant CysLT1R. Surprisingly, both UDP and LTD4-mediated ERK activation and cytoprotection were absent in mBMMCs lacking CysLT1R and the biosynthetic enzyme LTC4 synthase, implying a requirement for a cys-LT-mediated autocrine loop. In IL-4-primed LAD2 cells, LTD4 induced the generation of MIP-1beta, a response blocked by short hairpin RNA-mediated knockdown of CysLT1R or P2Y6 receptors, but not of CysLT2R. Thus, CysLT1R and P2Y6 receptors, which are coexpressed on many cell types of innate immunity, reciprocally amplify one another's function in mast cells through endogenous ligands.

Fatal stagger poisoning by consumption of Festuca argentina (Speg.) Parodi in goats from Argentine Patagonia.

The present study describes the spontaneous and experimental poisoning of goats by Festuca argentina in Argentine Patagonia. In April 2017, eight seven-month-old Creole male goats were accidentally introduced into a paddock that contained F. argentina. After four days, two of the goats were found dead and four out of the six remaining goats were clinically affected. Two of the latter had to be later euthanized in extremis. The main clinical signs were progressive nervous signs, starting with moderate muscle tremors, wide-based stance and ataxia. Postmortem examination was performed on the two euthanized goats. Epidermal fragments of F. argentina were found in the rumen samples from the necropsied goats and the fecal samples from the four affected goats. For the experimental poisoning, fresh sheaths of F. argentina collected from the paddock were offered to two goats at 10 g/kg body weight for 3 days. After 24-36 h, both animals exhibited severe muscle tremors, reluctance to move, tetanic convulsions, and opisthotonus. In both the spontaneously and experimentally poisoned goats, gross lesions were similar and consisted of dehydration, petechial hemorrhages in the epicardium and congestion. The main microscopic findings consisted of degeneration and loss of Purkinje cells and torpedoes in the granular layer of the cerebellum. The F. argentina sheaths collected from the pasture were found to contain tremorgenic indole-diterpene alkaloids. Taken together, the results of the present study suggest that the tremorgenic syndrome observed in the spontaneously poisoned goats was due to poisoning by F. argentina.

Matrix metalloproteinase inhibition reduces oxidative stress associated with cerebral amyloid angiopathy in vivo in transgenic mice.

Cerebral amyloid angiopathy (CAA), characterized by extracellular beta-amyloid peptide (Abeta) deposits in vessel walls, is present in the majority of cases of Alzheimer's disease and is a major cause of hemorrhagic stroke. Although the molecular pathways activated by vascular Abeta are poorly understood, extracellular matrix metalloproteinases (MMP) and Abeta-induced oxidative stress appear to play important roles. We adapted fluorogenic assays for MMP activity and reactive oxygen species generation for use in vivo. Using multiphoton microscopy in APPswe/PS1dE9 and Tg-2576 transgenic mice, we observed strong associations between MMP activation, oxidative stress, and CAA deposition in leptomeningeal vessels. Antioxidant treatment with alpha-phenyl-N-tert-butyl-nitrone reduced oxidative stress associated with CAA (approximately 50% reduction) without affecting MMP activation. Conversely, a selection of agents that inhibit MMP by different mechanisms of action, including minocycline, simvastatin, and GM6001, reduced not only CAA-associated MMP activation (approximately 30-40% reduction) but also oxidative stress (approximately 40% reduction). The inhibitors of MMP did not have direct antioxidant effects. Treatment of animals with alpha-phenyl-N-tert-butyl-nitrone or minocycline did not have a significant effect on CAA progression rates. These data suggest a close association between Abeta-related MMP activation and oxidative stress in vivo and raise the possibility that treatment with MMP inhibitors may have beneficial effects by indirectly reducing the oxidative stress associated with CAA.

Spontaneous outbreak of Astragalus pehuenches (Fabaceae) poisoning in cattle in Argentina.

This is the first report of a spontaneous outbreak of Astragalus pehuenches poisoning on a farm in Argentine Patagonia, where 63 out of 70 cattle died. The main clinical signs of affected animals were ataxia, balance loss and progressive emaciation. Purkinje cells presented vacuolation and marginalization of the nucleus. Astragalus pehuenches was detected in the paddock as well as in the ruminal content and fecal matter samples of the affected animals. Swainsonine concentrations in Astragalus specimens were found to be as high as 0.096%.

Age-dependent cerebrovascular dysfunction in a transgenic mouse model of cerebral amyloid angiopathy.

The Tg2576 transgenic mouse model of human cerebral amyloid angiopathy is characterized by age-dependent cerebrovascular deposition of amyloid-beta (Abeta) starting from 9 months of age and progressively worsening to involve most pial arterioles by 18 months; soluble Abeta levels are elevated long before vascular deposition takes place in this model. It has been suggested that elevated soluble Abeta levels alone are sufficient to impair cerebral blood flow (CBF) regulation thereby contributing to the early progression of Alzheimer's disease. Using laser speckle flowmetry through an intact skull, we studied the impact of elevated soluble Abeta levels and vascular Abeta deposition on a wide range of CBF responses to evaluate vasodilation and vasoconstriction in young or aged Tg2576 mice. Nineteen-month-old Tg2576 with severe vascular Abeta deposits showed an attenuated hyperaemic response during hypercapnia and whisker stimulation compared to wild-type littermates. The anticipated increase in CBF due to isoflurane anaesthesia was also suppressed, as were the typical hypoperfusion responses during cortical spreading depression and alpha-chloralose anaesthesia. The responses of 8-month-old Tg2576 with elevated soluble Abeta levels, but without vascular Abeta deposition, did not differ from age-matched controls. In conclusion, our data suggest that vascular Abeta deposition is associated with impaired vasodilator as well as vasoconstrictor responses to a wide range of stimuli. These responses do not differ from controls when studied non-invasively prior to vascular Abeta deposition, thus challenging the view that elevated soluble Abeta levels are sufficient to cause cerebrovascular dysfunction.

Public epitope specificity of HLA class I antibodies induced by a failed kidney transplant: alloantibody characterization by flow cytometric techniques.

BACKGROUND: Patients whose kidney grafts fail develop alloantibodies that react with many HLA molecules. We analyzed the epitope specificity of HLA class I alloantibodies in the sera of 55 patients who had been sensitized by kidney grafts, and investigated the immunogenicity of various polymorphic epitopes. METHODS: HLA class I alloantibodies were detected and characterized by flow cytometry (FlowPRA beads). Potential "immunizing epitopes" were identified by comparing the amino acid sequences of HLA class I antigens/alleles of the donor, recipient and the antibody-reactivity pattern. RESULTS: In the 55 anti-HLA class I-positive patients, 82 different antibody reactivity patterns were identified; all but 5 (94%) were determined by a "public epitope" of donor HLA-A and/or -B molecules. Forty-five of 50 patients who showed HLA-A Res-MMs with their donors produced HLA-A antibodies, but only 31 of 51 subjects with HLA-B Res-MMs produced HLA-B antibodies (P=0.001; O.R.=5.81). The antibody patterns were specific for a "single" epitope of the mismatched donor molecules in 91% of patients. Forty-three of the 120 (36%) mismatched HLA-A and/or -B epitopes were positively correlated with antibody production. The polymorphic determinants of higher immunogenic capacity were b80N (Bw6-associated) and ab82-83LR (Bw4-associated) public epitopes. CONCLUSIONS: The humoral immune response against a kidney graft mainly produces HLA class I antibodies specific for "public epitopes" of mismatched donor molecules. A "single" donor-epitope may determine the production of a spread antibody pattern. In renal transplantation, epitope matching is better than HLA antigen matching for avoiding or minimizing development of HLA antibodies.

Four-dimensional microglia response to anti-Aß treatment in APP/PS1xCX3CR1/GFP mice.

Senile plaques, mainly composed of amyloid-ß (Aß), are a major hallmark of Alzheimer disease (AD), and immunotherapy is a leading therapeutic approach for Aß clearance. Although the ultimate mechanisms for Aß clearance are not well known, characteristic microglia clusters are observed in the surround of senile plaques, and are implicated both in the elimination of Aß as well as the deleterious inflammatory effects observed in AD patients after active immunization. Therefore, analyzing the direct effect of immunotherapy on microglia, using longitudinal in vivo multiphoton microscopy can provide important information regarding the role of microglia in immunotherapy. While microglia were observed to surround senile plaques, topical anti-Aß antibody administration, which led to a reduction in plaque size, directed microglia toward senile plaques, and the overall size of microglia and number of processes were increased. In some cases, we observed clusters of microglia in areas of the brain that did not have detectable amyloid aggregates, but this did not predict the deposition of new plaques in the area within a week of imaging, indicating that microglia react to but do not precipitate amyloid aggregation. The long-term presence of large microglial clusters in the surrounding area of senile plaques suggests that microglia cannot effectively remove Aß unless anti-Aß antibody is administered. All together, these data suggest that although there is a role for microglia in Aß clearance, it requires an intervention like immunotherapy to be effective.

Antioxidants have a rapid and long-lasting effect on neuritic abnormalities in APP:PS1 mice.

Senile plaques are a major pathological hallmark of Alzheimer's disease (AD). Compelling evidence suggests that senile plaques lead to structural alterations of neuronal processes and that local toxicity may be mediated by increased oxidative stress. Anti-oxidant therapy can alleviate the neuronal abnormalities in APP mice, but the time-course of this beneficial effect is unknown. We used multiphoton microscopy to assess in vivo the characteristics of antioxidant treatment on senile plaques and neurites in AD model mice (APPswe/PS1dE9). We observed that α-phenyl-N-tert-butyl nitrone (PBN), Ginkgo biloba extract (EGb 761) and Trolox had no effect on the size of existing senile plaques. However, all anti-oxidants had a straightening effect on curved neurites. This effect was detected as soon as 4 days after commencing the treatment, and was maintained after 1 month of daily treatment, with no further increase in the effect. The straightening of neurites persisted 15 days after stopping the treatment. These data indicate that neuronal plasticity is fast and still active in adult animals, and suggest that amelioration of the neuritic distortions associated with senile plaques with antioxidants is both rapid and long lasting.

Existing plaques and neuritic abnormalities in APP:PS1 mice are not affected by administration of the gamma-secretase inhibitor LY-411575.

The gamma-secretase complex is a major therapeutic target for the prevention and treatment of Alzheimer's disease. Previous studies have shown that treatment of young APP mice with specific inhibitors of gamma-secretase prevented formation of new plaques. It has not yet been shown directly whether existing plaques would be affected by gamma-secretase inhibitor treatment. Similarly, alterations in neuronal morphology in the immediate vicinity of plaques represent a plaque-specific neurotoxic effect. Reversal of these alterations is an important endpoint of successful therapy whether or not a treatment affects plaque size. In the present study we used longitudinal imaging in vivo with multiphoton microscopy to study the effects of the orally active gamma-secretase inhibitor LY-411575 in 10-11 month old APP:PS1 mice with established amyloid pathology and neuritic abnormalities. Neurons expressed YFP allowing fluorescent detection of morphology whereas plaques were labelled with methoxy-XO4. The same identified neurites and plaques were followed in weekly imaging sessions in living mice treated daily (5 mg/kg) for 3 weeks with the compound. Although LY-411575 reduced Abeta levels in plasma and brain, it did not have an effect on the size of existing plaques. There was also no effect on the abnormal neuritic curvature near plaques, or the dystrophies in very close proximity to senile plaques. Our results suggest that therapeutics aimed at inhibition of Abeta generation are less effective for reversal of existing plaques than for prevention of new plaque formation and have no effect on the plaque-mediated neuritic abnormalities, at least under these conditions where Abeta production is suppressed but not completely blocked. Therefore, a combination therapy of Abeta suppression with agents that increase clearance of amyloid and/or prevent neurotoxicity might be needed for a more effective treatment in patients with pre-existing pathology.

Crystal structure of the complete integrin alphaVbeta3 ectodomain plus an alpha/beta transmembrane fragment.

We determined the crystal structure of 1TM-alphaVbeta3, which represents the complete unconstrained ectodomain plus short C-terminal transmembrane stretches of the alphaV and beta3 subunits. 1TM-alphaVbeta3 is more compact and less active in solution when compared with DeltaTM-alphaVbeta3, which lacks the short C-terminal stretches. The structure reveals a bent conformation and defines the alpha-beta interface between IE2 (EGF-like 2) and the thigh domains. Modifying this interface by site-directed mutagenesis leads to robust integrin activation. Fluorescent lifetime imaging microscopy of inactive full-length alphaVbeta3 on live cells yields a donor-membrane acceptor distance, which is consistent with the bent conformation and does not change in the activated integrin. These data are the first direct demonstration of conformational coupling of the integrin leg and head domains, identify the IE2-thigh interface as a critical steric barrier in integrin activation, and suggest that inside-out activation in intact cells may involve conformational changes other than the postulated switch to a genu-linear state.

CysLT2 receptors interact with CysLT1 receptors and down-modulate cysteinyl leukotriene dependent mitogenic responses of mast cells.

Cysteinyl leukotrienes (cys-LTs) induce inflammation through 2 G protein-coupled receptors (GPCRs), CysLT(1) and CysLT(2), which are coexpressed by most myeloid cells. Cys-LTs induce proliferation of mast cells (MCs), transactivate c-Kit, and phosphorylate extracellular signal-regulated kinase (ERK). Although MCs express CysLT(2), their responses to cys-LTs are blocked by antagonists of CysLT(1). We demonstrate that CysLT(2) interacts with CysLT(1), and that knockdown of CysLT(2) increases CysLT(1) surface expression and CysLT(1)-dependent proliferation of cord blood-derived human MCs (hMCs). Cys-LT-mediated responses were absent in MCs from mice lacking CysLT(1) receptors, but enhanced by the absence of CysLT(2) receptors. CysLT(1) and CysLT(2) receptors colocalized to the plasma membranes and nuclei of a human MC line, LAD2. Antibody-based fluorescent lifetime imaging microscopy confirmed complexes containing both receptors based on fluorescence energy transfer. Negative regulation of CysLT(1)-induced mitogenic signaling responses of MCs by CysLT(2) demonstrates physiologically relevant functions for GPCR heterodimers on primary cells central to inflammation.

Post-transplant donor-specific antibody production and graft outcome in kidney transplantation: results of sixteen-year monitoring by flow cytometry.

Our data show that monitoring by sensitive flow cytometric techniques of the de novo production of anti-HLA antibodies in patients receiving kidney transplantation is a useful and noninvasive tool to identify the onset of an immune response towards the graft before any clinical manifestation of antibody-mediated graft injury. Consequently prospective posttransplant monitoring of anti-HLA donor-directed antibodies may offer the chance to realize an effective clinical intervention in order to prevent graft dysfunction and to prolong graft survival. The long follow-up period of the study allowed us to demonstrate a very low graft survival rate in patients who developed donor-specific HLA antibodies in comparison with patients who did not have antibodies, thus confirming the "humoral theory of transplantation". The posttransplant production of anti-HLA antibodies can predict not only graft failure but also chronic dysfunction of the graft. Moreover, our findings suggest that graft survival is influenced by the epitope- and locus-specificity of anti-HLA donor-directed antibodies. The interval between antibody appearance and loss of graft function was short in some patients but reached several years in others. Moreover, some patients showed consistent production of antibodies for many years and an uneventful clinical status. These findings suggest a mechanism of graft "accommodation" or the production of "harmless" antibodies. Immunosuppressive drug combinations able to inhibit T and B cell activation are useful tools to prevent the humoral immune response against graft and consequently to prolong graft survival.