Síndrome de Asia: revisión sistemática de literatura

Ha habido muchos casos de aparición de autoanticuerpos y síntomas de enfermedad después de la exposición a adyuvantes, no solo después del aumento de senos con implantes de silicona, sino también como un efecto secundario muy raro de la vacunación, como el síndrome de la guerra del Golfo o el síndrome de miofascitis macrofágica. Las enfermedades cuyos síntomas se desarrollaron después de dicha exposición adyuvante se denominan síndrome autoinmune/inflamatorio inducido por adyuvantes (ASIA). El grupo de adyuvantes incluye no solo implantes de silicona, sílice, escualeno y aluminio, sino también componentes de tinta utilizados para hacer tatuajes. Analizando los informes disponibles sobre la influencia de los adyuvantes en el desarrollo de enfermedades autoinmunes, se concluye que, además de la exposición prolongada a la silicona, también es necesaria la coexistencia de otros factores, como genéticos o ambientales. Los análisis claramente no confirman un mayor riesgo de desarrollar una enfermedad autoinmune después del aumento de senos con implantes de silicona o tatuajes, pero parece que entre estas pacientes hay un grupo que está más predestinado a desarrollar síntomas de la enfermedad. En la población general, los beneficios de la vacunación son obvios, y el riesgo de eventos adversos graves después de la inmunización es incomparablemente menor que el riesgo de desarrollar una enfermedad específica y sus complicaciones, también para pacientes con enfermedades autoinmunes diagnosticadas. Debido a la heterogeneidad de los datos en estudios previos y las dificultades para diagnosticar ASIA, parece necesario realizar más análisis de la influencia de los adyuvantes en el desarrollo de enfermedades autoinmunes y refinar los criterios de diagnóstico de ASIA, que ahora permiten un diagnóstico demasiado fácil de esta enfermedad. (provisto por Infomedic International)

There have been many cases of occurrence of autoantibodies and disease symptoms after adjuvant exposure, not only after breast augmentation with silicone implants, but also as a very rare side effect of vaccination, such as Gulf War syndrome or macrophagic myofasciitis syndrome. Diseases whose symptoms developed after such adjuvant exposure are called adjuvant-induced autoimmune/inflammatory syndrome (ASIA). The adjuvant group includes not only silicone, silica, squalene and aluminum implants, but also ink components used to make tattoos. Analyzing the available reports on the influence of adjuvants on the development of autoimmune diseases, it is concluded that, in addition to prolonged exposure to silicone, the coexistence of other factors, such as genetic or environmental, is also necessary. The analyses clearly do not confirm an increased risk of developing an autoimmune disease after breast augmentation with silicone implants or tattoos, but it seems that among these patients there is a group that is more predestined to develop symptoms of the disease. In the general population, the benefits of vaccination are obvious, and the risk of serious adverse events after immunization is incomparably lower than the risk of developing a specific disease and its complications, also for patients with diagnosed autoimmune diseases. Due to the heterogeneity of data in previous studies and the difficulties in diagnosing ASIA, further analysis of the influence of adjuvants on the development of autoimmune diseases and refinement of the diagnostic criteria for ASIA, which now allow too easy diagnosis of this disease, seems necessary. (provided by Infomedic International)

Infiltrating Lobular Carcinoma of the Breast Metastatic to the Duodenum, about a Case

Introduction: Gastric outlet obstruction or pyloric syndrome can occur secondary to neoplastic involvement, and metastasis as an etiology is unusual. Breast neoplasms generally cause bone, liver, and lung metastases, rarely involving the gastrointestinal tract. Case presentation: A 69-year-old female patient with infiltrating lobular carcinoma of the right breast consulted for abdominal pain and postprandial emetic episodes with oral intolerance and dyspnea. Bilateral neoplastic breast involvement and dilation of the gastric chamber with thickening of the pylorus were recorded. She required antiemetic management and placement of a nasogastric tube. She was taken to an upper digestive tract endoscopy, which found an ulcerated lesion with an infiltrative appearance at the postpyloric level that circumferentially compromised the duodenal lumen. Then, a biopsy was taken, which was compatible with a breast carcinoma of a lobular type. This entity, called pyloric syndrome due to neoplasia, can be managed with gastrojejunostomy or an enteral prosthesis that improves the quality of life of patients with an ominous short-term prognosis. The patient in our case expressed advance directives not to receive invasive procedures, for which an uncovered metal prosthesis was placed for palliative purposes, achieving the re-establishment of the feeding route and resolution of dyspnea due to restriction. Conclusions: The metastatic involvement of neoplasms of the breast to the gastrointestinal tract is rare; however, it should be suspected in elderly patients with previously documented neoplasms.

Introducción: La obstrucción al tracto de salida gástrico o síndrome pilórico puede presentarse de manera secundaria a compromiso neoplásico, y es inusual el compromiso metastásico como etiología. Las neoplasias de la mama generalmente originan metástasis óseas, hepáticas y pulmonares, y es infrecuente el compromiso del tracto digestivo. Presentación del caso: Se trata de una paciente de 69 años con carcinoma lobulillar infiltrante de mama derecha que consultó por dolor abdominal y episodios eméticos posprandiales con intolerancia a la vía oral y disnea. Se registró un compromiso neoplásico mamario bilateral y dilatación de la cámara gástrica con engrosamiento del píloro. Se dio manejo antiemético y colocación de sonda nasogástrica. Fue llevada a una endoscopia de vías digestivas altas en la que se encontró una lesión ulcerada de aspecto infiltrativo a nivel pospilórico que comprometía de forma circunferencial la luz duodenal, y luego se tomó una biopsia a este nivel, que fue compatible con un carcinoma mamario de tipo lobulillar. Esta entidad, denominada síndrome pilórico por neoplasias, puede manejarse con gastroyeyunostomía o con prótesis enteral que mejore la calidad de vida en pacientes con pronóstico ominoso a corto plazo. La paciente de nuestro caso manifestó voluntades anticipadas para no recibir procedimientos invasivos, por lo cual se ofreció la colocación de una prótesis metálica descubierta con fines paliativos y se logró el restablecimiento de la vía de alimentación y resolución de la disnea por restricción. Conclusiones: El compromiso metastásico de neoplasias de la mama al tracto digestivo es poco frecuente; sin embargo, debe sospecharse en pacientes de edad avanzada y con neoplasias previamente documentadas.

Iron chelation alleviates multiple pathophysiological pathways in a rat model of cardiac pressure overload.

Iron dysmetabolism affects a great proportion of heart failure patients, while chronic hypertension is one of the most common risk factors for heart failure and death in industrialized countries. Serum data from reduced ejection fraction heart failure patients show a relative or absolute iron deficiency, whereas cellular myocardial analyses field equivocal data. An observed increase in organellar iron deposits was incriminated to cause reactive oxygen species formation, lipid peroxidation, and cell death. Therefore, we studied the effects of iron chelation on a rat model of cardiac hypertrophy. Suprarenal abdominal aortic constriction was achieved surgically, with a period of nine weeks to accommodate the development of chronic pressure overload. Next, deferiprone (100 mg/kg/day), a lipid-permeable iron chelator, was administered for two weeks. Pressure overload resulted in increased inflammation, fibrotic remodeling, lipid peroxidation, left ventricular hypertrophy and mitochondrial iron derangements. Deferiprone reduced cardiac inflammation, lipid peroxidation, mitochondrial iron levels, and hypertrophy, without affecting circulating iron levels or ejection fraction. In conclusion, metallic molecules may pose ambivalent effects within the cardiovascular system, with beneficial effects of iron redistribution, chiefly in the mitochondria.

Afectación esofágica y pulmonar por paracoccidioidomicosis en paciente inmunocompetente: reporte de caso / Esophageal and Pulmonary Involvement Caused by Paracoccidioidomycosis in Immunocompromised Patient: Case Report

Resumen La paracoccidioidomicosis es una infección fúngica endémica de América del Sur, que afecta predominantemente a los hombres y, según su campo laboral, granjeros y agricultores. Es ocasionada por la aspiración del hongo en su forma micelar y debuta en tres formas de presentación: aguda, subaguda y crónica; esta última es más frecuente en adultos, cuyo tratamiento dependerá de los azoles, anfotericina B y sulfonamidas. El presente caso trata de un hombre de 57 años, colombiano, agricultor, sin antecedentes patológicos, quien presentaba dos meses de disfagia para sólidos que progresó a líquidos, sialorrea y pérdida de peso, a quien se le realizó endoscopia de vías digestivas altas y se observaron lesiones blanquecinas, por lo cual se realizó una biopsia que evidenció levaduras en múltiple gemación compatibles con paracoccidioidomicosis; a su vez, se observó en una tomografía de tórax compromiso parenquimatoso intersticial generalizado; posteriormente, recibió tratamiento con itraconazol, con el que mostró mejoría y resolución del cuadro clínico. En vista de que América del Sur es endémica de la patología descrita y puede presentarse de forma diseminada en inmunocompetentes, se debe tener en cuenta en aquellos pacientes que poseen factores de riesgo, sintomatología y hallazgos en estudios de extensión sugestivos de dicha enfermedad, dado el gran espectro de presentación de la infección, para así dar tratamiento oportuno y dirigido.

Abstract Paracoccidioidomycosis (PCM) is a fungal infection endemic to South America. It predominantly affects men, depending on their work field: farmers and agriculturists. Paracoccidioidomycosis is caused by the aspiration of the fungus in its micellar form and manifests in three conditions: acute, subacute, and chronic; the latter is more frequent in adults, whose treatment will depend on azoles, amphotericin B, and sulfonamides. This case concerns a 57-year-old Colombian man, a farmer with no pathological history who showed dysphagia for solids that progressed to liquids, sialorrhea, and weight loss for two months. He underwent upper GI endoscopy, and whitish lesions were observed; thus, he was biopsied, displaying yeasts in multiple gemmations compatible with paracoccidioidomycosis. In turn, a chest CT scan showed generalized interstitial parenchymal involvement. Subsequently, he was treated with itraconazole, showing improvement and resolution in his clinical picture. Since the pathology described is endemic in South America and can be disseminated in immunocompromised patients. Given the broad infection spectrum, consideration should be given to patients with risk factors, symptomatology, and findings in extension studies suggesting this disease to provide timely and specific treatment.

Low Doses of Celecoxib Might Promote Phenotype Switching in Cutaneous Melanoma Treated with Dabrafenib-Preliminary Study.

Background: Cutaneous melanoma is a heterogeneous tumor with a rapidly switching molecular and cellular phenotype. The invasive phenotype switching characterized by MITFlow/AXLhigh predicts early resistance to multiple targeted drugs in melanoma. Celecoxib proved to be a valuable adjuvant in cutaneous melanoma in preclinical studies. Our in vitro study evaluated for the first time whether celecoxib could prevent phenotype switching in two human melanoma cell lines treated with dabrafenib. Methods: All in vitro experiments were carried out on BRAF-V600E-positive A375 and SK-MEL-28 human melanoma cell lines, and subjected to a celecoxib and dabrafenib drug combination for 72 h. Melanoma cells were already in the MITFlow/AXLhigh end of the spectrum. Of main interest was the evaluation of the key proteins expressed in phenotype switching (TGF-ß, MITF, AXL, YAP, TAZ), as well as cell death mechanisms correlated with oxidative stress production. Results: Celecoxib significantly enhanced the apoptotic effect of dabrafenib in each melanoma cell line compared to the dabrafenib group (p < 0.0001). Even though celecoxib promoted low MITF expression, this was correlated with high receptor tyrosine kinase AXL levels in A375 and SK-MEL-28 cell lines (p < 0.0001), a positive marker for the phenotype switch to an invasive state. Conclusion: This preliminary study highlighted that celecoxib might promote MITFlow/AXLhigh expression in cutaneous melanoma treated with dabrafenib, facilitating phenotype switching in vitro. Our results need further confirmation, as this finding could represent an important limitation of celecoxib as an antineoplastic drug.

Celecoxib as a Valuable Adjuvant in Cutaneous Melanoma Treated with Trametinib.

BACKGROUND: Melanoma patients stop responding to targeted therapies mainly due to mitogen activated protein kinase (MAPK) pathway re-activation, phosphoinositide 3 kinase/the mechanistic target of rapamycin (PI3K/mTOR) pathway activation or stromal cell influence. The future of melanoma treatment lies in combinational approaches. To address this, our in vitro study evaluated if lower concentrations of Celecoxib (IC50 in nM range) could still preserve the chemopreventive effect on melanoma cells treated with trametinib. MATERIALS AND METHODS: All experiments were conducted on SK-MEL-28 human melanoma cells and BJ human fibroblasts, used as co-culture. Co-culture cells were subjected to a celecoxib and trametinib drug combination for 72 h. We focused on the evaluation of cell death mechanisms, melanogenesis, angiogenesis, inflammation and resistance pathways. RESULTS: Low-dose celecoxib significantly enhanced the melanoma response to trametinib. The therapeutic combination reduced nuclear transcription factor (NF)-kB (p < 0.0001) and caspase-8/caspase-3 activation (p < 0.0001), inhibited microphthalmia transcription factor (MITF) and tyrosinase (p < 0.05) expression and strongly down-regulated the phosphatidylinositol-3-kinase/protein kinase B (PI3K/AKT) signaling pathway more significantly than the control or trametinib group (p < 0.0001). CONCLUSION: Low concentrations of celecoxib (IC50 in nM range) sufficed to exert antineoplastic capabilities and enhanced the therapeutic response of metastatic melanoma treated with trametinib.

Características microbiológicas de la periimplantitis y la periodontitis / Microbiologic characteristics of periimplantitis and periodontitis

Se presenta una revisión sistemática de la bibliografía para esclarecer diferencias entre el conjunto de características microbiológicas de la periimplantitis y el de la periodontitis. De acuerdo a la declaración PRISMA, se realizaron búsquedas en bases de datos (PubMed, EBSCOhost, LILACS, Web Of Science y Clinical Key) y se seleccionaron artículos originales y revisiones sistemáticas donde se compararan o analizaran datos microbiológicos obtenidos de muestras de biopelículas subgingivales de pacientes con periodontitis y periimplantitis. El peso de la evidencia se evaluó por medio de las escalas de Newcastle-Ottawa y PRISMA. De los 335 artículos identificados, se incluyeron 12, de los cuales 9 fueron estudios observacionales y 3 revisiones sistemáticas. Se obtuvo que las características microbiológicas asociadas a la periimplantitis son similares a las de la periodontitis debido a que comparten un porcentaje de su microbiota, como es el caso de los agentes periodontopatógenos; sin embargo, se encontraron bacterias relacionadas únicamente al surco periimplantar. Finalmente, se destacó que en la periimplantitis figuran bacterias que en su mayoría son gramnegativas anaerobias, periodontopatógenas, oportunistas y no cultivables; es decir, que sus características microbiológicas resultan complejas y difieren de las específicas de la periodontitis.

A systematic literature review is presented to clarify differences between the group of microbiologic characteristics of peri-Implantitis and periodontitis. According to the PRISMA declaration, searches in databases were carried out (PubMed, EBSCOhost, LILACS, Web Of Science and Clinical Key) and original works and systematic reviews were selected where comparing or analyzing microbiologic data obtained from samples of subgingival biofilms of patients with periodontitis and peri-Implantitis. The weight of evidence was evaluated by means of the Newcastle-Ottawa and PRISMA scales. Of the 335 identified works, 12 were included, of which 9 were observational studies and 3 systematic reviews. It was obtained that the microbiologic characteristics associated with peri-Implantitis are similar to those of periodontitis because they share a percentage of their microbiota, as the case of the periodontopathogen agents; however, bacterias only related to the peri-implantar line were found. Finally, it is remarkable that in peri-Implantitis are bacterias that are mostly gramnegative anaerobias, periodontopathogens, opportunists and noncultivable; that is to say that their microbiologic characteristics are complex and differ from the specific characteristics of periodontitis.

COX-2 as a potential biomarker and therapeutic target in melanoma.

With a constantly increasing incidence, cutaneous melanoma has raised the need for a better understanding of its complex microenvironment that may further guide therapeutic options. Melanoma is a model tumor in immuno-oncology. Inflammation represents an important hallmark of cancer capable of inducing and sustaining tumor development. The inflammatory process also orchestrates the adaptative immunosuppression of tumor cells that helps them to evade immune destruction. Besides its role in proliferation, angiogenesis, and apoptosis, cyclooxygenase-2 (COX-2) is a well-known promoter of immune suppression in melanoma. COX-2 inhibitors are closely involved in this condition. This review attempts to answer two controversial questions: is COX-2 a valuable prognostic factor? Among all COX-2 inhibitors, is celecoxib a suitable adjuvant in melanoma therapy?

Influence of mitochondrial and systemic iron levels in heart failure pathology.

Iron deficiency or overload poses an increasingly complex issue in cardiovascular disease, especially heart failure. The potential benefits and side effects of iron supplementation are still a matter of concern, even though current guidelines suggest therapeutic management of iron deficiency. In this review, we sought to examine the iron metabolism and to identify the rationale behind iron supplementation and iron chelation. Cardiovascular disease is increasingly linked with iron dysmetabolism, with an increased proportion of heart failure patients being affected by decreased plasma iron levels and in turn, by the decreased quality of life. Multiple studies have concluded on a benefit of iron administration, even if just for symptomatic relief. However, new studies field evidence for negative effects of dysregulated non-bound iron and its reactive oxygen species production, with concern to heart diseases. The molecular targets of iron usage, such as the mitochondria, are prone to deleterious effects of the polyvalent metal, added by the scarcely described processes of iron elimination. Iron supplementation and iron chelation show promise of therapeutic benefit in heart failure, with the extent and mechanisms of both prospects not being entirely understood. It may be that a state of decreased systemic and increased mitochondrial iron levels proves to be a useful frame for future advancements in understanding the interconnection of heart failure and iron metabolism.

Dose dependent side effect of superparamagnetic iron oxide nanoparticle labeling on cell motility in two fetal stem cell populations.

Multipotent stem cells (SCs) could substitute damaged cells and also rescue degeneration through the secretion of trophic factors able to activate the endogenous SC compartment. Therefore, fetal SCs, characterized by high proliferation rate and devoid of ethical concern, appear promising candidate, particularly for the treatment of neurodegenerative diseases. Super Paramagnetic Iron Oxide nanoparticles (SPIOn), routinely used for pre-clinical cell imaging and already approved for clinical practice, allow tracking of transplanted SCs and characterization of their fate within the host tissue, when combined with Magnetic Resonance Imaging (MRI). In this work we investigated how SPIOn could influence cell migration after internalization in two fetal SC populations: human amniotic fluid and chorial villi SCs were labeled with SPIOn and their motility was evaluated. We found that SPIOn loading significantly reduced SC movements without increasing production of Reactive Oxygen Species (ROS). Moreover, motility impairment was directly proportional to the amount of loaded SPIOn while a chemoattractant-induced recovery was obtained by increasing serum levels. Interestingly, the migration rate of SPIOn labeled cells was also significantly influenced by a degenerative surrounding. In conclusion, this work highlights how SPIOn labeling affects SC motility in vitro in a dose-dependent manner, shedding the light on an important parameter for the creation of clinical protocols. Establishment of an optimal SPIOn dose that enables both a good visualization of grafted cells by MRI and the physiological migration rate is a main step in order to maximize the effects of SC therapy in both animal models of neurodegeneration and clinical studies.

Biocompatible fluorescent nanoparticles for in vivo stem cell tracking.

Efficient application of stem cells to the treatment of neurodegenerative diseases requires safe cell tracking to follow stem cell fate over time in the host environment after transplantation. In this work, for the first time, fluorescent and biocompatible methyl methacrylate (MMA)-based nanoparticles (fluoNPs) were synthesized through a free-radical co-polymerization process with a fluorescent macromonomer obtained by linking Rhodamine B and hydroxyethyl methacrylate. We demonstrate that the fluoNPs produced by polymerization of MMA-Rhodamine complexes (1) were efficient for the labeling and tracking of multipotent human amniotic fluid cells (hAFCs); (2) did not alter the main biological features of hAFCs (such as viability, cell growth and metabolic activity); (3) enabled us to determine the longitudinal bio-distribution of hAFCs in different brain areas after graft in the brain ventricles of healthy mice by a direct fluorescence-based technique. The reliability of our approach was furthermore confirmed by magnetic resonance imaging analyses, carried out by incubating hAFCs with both superparamagnetic iron oxide nanoparticles and fluoNPs. Our data suggest that these finely tunable and biocompatible fluoNPs can be exploited for the longitudinal tracking of stem cells.

A reliable indirect cell-labelling protocol for optical imaging allows ex vivo visualisation of mesenchymal stem cells after transplantation.

We set out to assess the feasibility of exploiting expression of the mCherry gene, after lentiviral infection, in order visualise bone marrow-derived human mesenchymal stem cells (hMSCs) by optical imaging, and to provide proof of principle of this approach as a method for cell tracking and quantification in pre-clinical models. Commercial hMSCs were infected with a lentiviral vector carrying the mCherry gene under the control of the phosphoglycerate kinase promoter. After extensive in vitro culture, infected hMSCs were analysed for viability, morphology, differentiation capability, and maintenance of fluorescence. Thereafter, mCherry-positive cells were transplanted into unilaterally 6-hydroxy-dopamine lesioned rats (an experimental model of Parkinson's disease). Our analysis showed that hMSCs can be efficiently transduced with the lentiviral vector, retaining their biological features even in the long term. Intrastriatally transplanted mCherry-positive hMSCs can be detected ex vivo by a sensitive cooled CCD camera, both in the whole brain and in serial slices, and relatively quantified. Our protocol was found to be a reliable means of studying the viability of implanted hMSCs. mCherry labelling appears to be readily applicable in the post-transplantation tracking of stem cells and could favour the rapid development of new therapeutic targets for clinical treatments.

Longitudinal tracking of human fetal cells labeled with super paramagnetic iron oxide nanoparticles in the brain of mice with motor neuron disease.

Stem Cell (SC) therapy is one of the most promising approaches for the treatment of Amyotrophic Lateral Sclerosis (ALS). Here we employed Super Paramagnetic Iron Oxide nanoparticles (SPIOn) and Hoechst 33258 to track human Amniotic Fluid Cells (hAFCs) after transplantation in the lateral ventricles of wobbler (a murine model of ALS) and healthy mice. By in vitro, in vivo and ex vivo approaches we found that: 1) the main physical parameters of SPIOn were maintained over time; 2) hAFCs efficiently internalized SPIOn into the cytoplasm while Hoechst 33258 labeled nuclei; 3) SPIOn internalization did not alter survival, cell cycle, proliferation, metabolism and phenotype of hAFCs; 4) after transplantation hAFCs rapidly spread to the whole ventricular system, but did not migrate into the brain parenchyma; 5) hAFCs survived for a long time in the ventricles of both wobbler and healthy mice; 6) the transplantation of double-labeled hAFCs did not influence mice survival.

Neuroprotective effects of human mesenchymal stem cells on neural cultures exposed to 6-hydroxydopamine: implications for reparative therapy in Parkinson's disease.

Stem cell (SC) transplantation represents a promising tool to treat neurodegenerative disorders, such as Parkinson's disease (PD), but positive therapeutic outcomes require elucidation of the biological mechanisms involved. Therefore, we investigated human Mesenchymal SCs (hMSCs) ability to protect murine differentiated Neural SCs (mdNSCs) against the cytotoxic effects of 6-hydroxydopamine (6-OHDA) in a co-culture model mimicking the in vivo neurovascular niche. The internalization of 6-OHDA mainly relies on its uptake by the dopamine active transporter (DAT), but its toxicity could also involve other pathways. We demonstrated that mdNSCs consistently expressed DAT along the differentiative process. Exposure to 6-OHDA did not affect hMSCs, but induced DAT-independent apoptosis in mdNSCs with generation of reactive oxygen species and caspases 3/7 activation. The potential neuroprotective action of hMSCs on mdNSCs exposed to 6-OHDA was tested in different co-culture conditions, in which hMSCs were added to mdNSCs prior to, simultaneously, or after 6-OHDA treatment. In the presence of the neurotoxin, the majority of mdNSCs acquired an apoptotic phenotype, while co-cultures with hMSCs significantly increased their survival (up to 70%) in all conditions. Multiplex human angiogenic array analysis on the conditioned media demonstrated that cytokine release by hMSCs was finely modulated. Moreover, sole growth factor addition yielded a similar neuroprotective effect on mdNSCs. In conclusion, our findings demonstrate that hMSCs protect mdNSCs against 6-OHDA neurotoxicity, and rescue cells from ongoing neurodegeneration likely through the release of multiple cytokines. Our findings provide novel insights for the development of therapeutic strategies designed to counteract the neurodegenerative processes of PD.

The molecular assembly of amyloid aß controls its neurotoxicity and binding to cellular proteins.

Accumulation of ß-sheet-rich peptide (Aß) is strongly associated with Alzheimer's disease, characterized by reduction in synapse density, structural alterations of dendritic spines, modification of synaptic protein expression, loss of long-term potentiation and neuronal cell death. Aß species are potent neurotoxins, however the molecular mechanism responsible for Aß toxicity is still unknown. Numerous mechanisms of toxicity were proposed, although there is no agreement about their relative importance in disease pathogenesis. Here, the toxicity of Aß 1-40 and Aß 1-42 monomers, oligomers or fibrils, was evaluated using the N2a cell line. A structure-function relationship between peptide aggregation state and toxic properties was established. Moreover, we demonstrated that Aß toxic species cross the plasma membrane, accumulate in cells and bind to a variety of internal proteins, especially on the cytoskeleton and in the endoplasmatic reticulum (ER). Based on these data we suggest that numerous proteins act as Aß receptors in N2a cells, triggering a multi factorial toxicity.

Neural precursor-derived astrocytes of wobbler mice induce apoptotic death of motor neurons through reduced glutamate uptake.

In the present study, we investigated whether cultured astrocytes derived from adult neural precursor cells (NPCs) obtained from the subventricular zone (SVZ) of wobbler mice display metabolic traits of the wobbler astrocytes in situ and in primary culture. We also utilized NPC-derived astrocytes as a tool to investigate the involvement of astrocytes in the molecular mechanism of MND focusing on the possible alteration of glutamate reuptake since excitotoxicity glutamate-mediated may be a contributory pathway. NPC-derived wobbler astrocytes are characterized by high immunoreactivity for GFAP, significant decrease of glutamate uptake and reduced immunoreactivity for glutamate transporters GLT1 and GLAST. Spinal cord motor neurons obtained from healthy mouse embryos, when co-cultured with wobbler NPC-derived astrocytes, show reduced viability and morphologic alterations. These suffering motor neurons are caspase-7 positive, and treatment with anti-apoptotic drug V5 increases cell survival. Physical contact with wobbler astrocytes is not essential because purified motor neurons display reduced survival also when treated with the medium conditioned by wobbler NPC-derived astrocytes. Toxic levels of glutamate were revealed by HPLC assay in the extracellular medium of wobbler NPC-derived astrocytes, whereas the level of intracellular glutamate is reduced if compared with controls. Moreover, glutamate receptor antagonists are able to enhance motor neuron survival. Therefore, our results demonstrate that astrocytes derived from wobbler neural precursor cells display impaired glutamate homeostasis that may play a crucial role in motor neuron degeneration. Finally, the cultured astrocytes derived from NPCs of adult mice may offer a useful alternative in vitro model to study the molecular mechanisms involved in neurodegeneration.

Morphological features and responses to AMPA receptor-mediated excitotoxicity of mouse motor neurons: comparison in purified, mixed anterior horn or motor neuron/glia cocultures.

Primary motor neuron cultures are widely used as in vitro model to study the early mechanisms involved in the aetiology of amyotrophic lateral sclerosis. In this study, we directly compared the morphological features and the responses to AMPA receptor (AMPAR) activation of mouse spinal cord motor neurons under different culture conditions (OptiPrep-purified, mixed anterior horn or motor neuron/glia cocultures). Motor neurons cocultured with a confluent glial layer had significant improvements in axonal length and in somata perimeter and area, compared both to mixed anterior horn cultures and to purified cultures, suggesting that the presence of more "mature" glial cells was determinant to obtain healthier motor neurons. By immuno-cytochemical assays we found that both in mixed anterior horn cultures and in cocultures, lower AMPA (0.3 microM) or kainate (5 microM) concentrations, but not the higher (1 or 15 microM, respectively), induced classical apoptotic events such as the nuclear fragmentation, the membrane externalization of phosphatidylserine residues and the activation of caspases-9 and -3. The morphological features and the different degenerative pathways induced by AMPAR agonist concentrations suggest that the experimental conditions used for in vitro studies are key factors that should be deeply considered to obtain more valid and reproducible results.

Structure-activity study of 2,3-benzodiazepin-4-ones noncompetitive AMPAR antagonists: identification of the 1-(4-amino-3-methylphenyl)-3,5-dihydro-7,8-ethylenedioxy-4H-2,3-benzodiazepin-4-one as neuroprotective agent.

In the search for AMPA receptor (AMPAR) antagonists, 2,3-benzodiazepines represent a family of specific noncompetitive antagonists with anticonvulsant and neuroprotective properties. We have previously shown that 2,3-benzodiazepin-4-ones possess marked anticonvulsant properties and high affinity for the noncompetitive binding site of the AMPAR complex. In this paper, we report the synthesis and pharmacological characterization of a full set of 2,3-benzodiazepin-4-ones in order to better define the structure-activity relationship (SAR) of this class of compounds. Binding assays and functional tests were performed to evaluate the antagonistic activity at the AMPARs. Through these results we have identified a potent AMPAR antagonist, 1-(4-amino-3-methylphenyl)-3,5-dihydro-7,8-ethylenedioxy-4H-2,3-benzodiazepin-4-one (5c). This compound noncompetitively inhibited AMPAR-mediated toxicity in primary mouse hippocampal cultures with an IC(50) of 1.6muM and blocked kainate-induced calcium influx in rat cerebellar granule cells with an IC(50) of 6.4muM. Thus, 5c has the in vitro potential as therapeutic drug in the treatment of various neurological disorders.