Delivery of glutamine synthetase gene by baculovirus vectors: a proof of concept for the treatment of acute hyperammonemia.

Hyperammonemia, a condition present in patients with urea cycle disorders (UCDs) or liver diseases, can cause neuropsychiatric complications, which in the worst cases result in brain damage, coma or death. Diverse treatments exist for the treatment of hyperammonemia, but they have limited efficacy, adverse effects and elevated cost. Gene therapy is a promising alternative that is explored here. A baculovirus, termed Bac-GS, containing the glutamine synthetase (GS) gene was constructed for the in vitro and in vivo treatment of hyperammonemia. Transduction of MA104 epithelial or L6 myoblast/myotubes cells with Bac-GS resulted in a high expression of the GS gene, an increase in GS concentration, and a reduction of almost half of exogenously added ammonia. When Bac-GS was tested in an acute hyperammonemia rat model by intramuscularly injecting the rear legs, the concentration of ammonia in blood decreased 351 µM, in comparison with controls. A high GS concentration was detected in gastrocnemius muscles from the rats transduced with Bac-GS. These results show that gene delivery for overexpressing GS in muscle tissue is a promising alternative for the treatment of hyperammonemia in patients with acute or chronic liver diseases and hepatic encephalopathy or UCD.

Elevated arsenic and manganese in groundwaters of Murshidabad, West Bengal, India.

High levels of geogenic arsenic (As) and manganese (Mn) in drinking water has led to widespread health problems for the population of West Bengal, India. Here we delineate the extent of occurrences of As and Mn in Murshidabad, where the contaminated aquifers occur at shallow depths between 35 and 40 m and where access to safe drinking water is a critical issue for the local population. A total of 78 well-water samples were taken in 4 blocks on either side of the river Bhagirathi: Nabagram and Kandi (west, Pleistocene sediments), Hariharpara and Beldanga (east, Holocene sediments). High As, total iron (FeT) and low Mn concentrations were found in waters from the Holocene gray sediment aquifers east of the river Bhagirathi, while the opposite was found in the Pleistocene reddish-brown aquifer west of the river Bhagirathi in Murshidabad. Speciation of As in water samples from Holocene sediments revealed the dominant species to be As(III), with ratios of As(III):AsT ranging from 0.55 to 0.98 (average 0.74). There were indications from saturation index estimations that Mn solubility is limited by the precipitation of MnCO3. Tubewells from high As areas in proximity to anthropogenic waste influx sources showing high molar Cl/Br ratios, low SO4(2-) and low NO3(-) demonstrate relatively lower As concentrations, thereby reducing As pollution in those wells. Analyses of core samples (2 in each of the blocks) drilled to a depth of 45 m indicate that there is no significant variation in bulk As (5-20mg/kg) between the Holocene and Pleistocene sediments, indicating that favorable subsurface redox conditions conducive to mobilization are responsible for the release of As. The same applies to Mn, but concentrations vary more widely (20-2000 mg/kg). Sequential extraction of Holocene sediments showed As to be associated with 'specifically sorbed-phosphate-extractable' phases (10-15%) and with 'amorphous and well crystalline Fe-oxyhydroxide' phases (around 37%) at As-contaminated well depths, suggesting that the main As release mechanisms could be either competitive ion exchange with PO4(3-), or the dissolution of Fe oxyhydroxides. In the Pleistocene sediments Mn is predominantly found in the easily exchangeable fraction.

Melatonin-induced methylation of the ABCG2/BCRP promoter as a novel mechanism to overcome multidrug resistance in brain tumour stem cells.

BACKGROUND: Current evidence indicates that a stem cell-like sub-population within malignant glioblastomas, that overexpress members of the adenosine triphosphate-binding cassette (ABC) family transporters, is responsible for multidrug resistance and tumour relapse. Eradication of the brain tumour stem cell (BTSC) compartment is therefore essential to achieve a stable and long-lasting remission. METHODS: Melatonin actions were analysed by viability cell assays, flow cytometry, quantitative PCR for mRNA expression, western blot for protein expression and quantitative and qualitative promoter methylation methods. RESULTS: Combinations of melatonin and chemotherapeutic drugs (including temozolomide, current treatment for malignant gliomas) have a synergistic toxic effect on BTSCs and A172 malignant glioma cells. This effect is correlated with a downregulation of the expression and function of the ABC transporter ABCG2/BCRP. Melatonin increased the methylation levels of the ABCG2/BCRP promoter and the effects on ABCG2/BCRP expression and function were prevented by preincubation with a DNA methyltransferase inhibitor. CONCLUSION: Our results point out a possible relationship between the downregulation of ABCG2/BCRP function and the synergistic toxic effect of melatonin and chemotherapeutic drugs. Melatonin could be a promising candidate to overcome multidrug resistance in the treatment of glioblastomas, and thus improve the efficiency of current therapies.

[Pathogenetic bases of epileptogenesis in cerebral cavernomas]. / Bases patogenicas de la epileptogenesis en los cavernomas cerebrales.

INTRODUCTION. Brain cavernoma are a type of arteriovenous malformation that clinically presenting seizures, neurological deficit or bleeding. Hypoxia, neoangiogenesis and metalloproteasas seems to be involved in seizures physiopathology. Our study aims to assess this potential relation by immunohistochemical methods, analyzing hypoxia inducible factor (HIF-1alpha) and metalloproteasa (MMP-9) in tissue surrounding cavernoma. PATIENTS AND METHODS. We selected 17 consecutive cases anatomopathologically diagnosed as cavernoma during 9 years. Immunohistochemical staining was performed for HIF-1alpha and MMP-9. We evaluated the relation between seizures and the scale of uptake of different tissues surrounding cavernoma. RESULTS. Cases with seizures had HIF-1alpha positive uptake in vascular endothelium in 31%, 17% in fibrous tissue and 34% in inflammatory tissue. Besides, it also shows MMP-9 positive uptake in vascular endothelium in 86%, 100% in fibrous tissue and 43% of brain tissue. Statistical analysis by chi-square and odds ratio shows a positive trend towards seizures and the presence of HIF-1alpha and MMP-9 in vascular tissue, fibrous tissue and brain tissue, but no for inflammatory tissue. CONCLUSION. HIF-1alpha and MMP-9, valued by immunohistochemical methods, are related to complications as seizures.

[Review of the target for the deep brain stimulation of chronic cluster headache]. / Revision de la diana para la estimulacion cerebral profunda de la cefalea en racimos cronica.

Cluster headache is included in the group of trigeminal autonomic cephalalgias. Although the pathophysiology of cluster headache has not yet been sufficiently established, the theory of a central origin tells us that this headache is produced by hypothalamic dysfunction. More than 50 patients have been treated with deep brain stimulation of the posterior nucleus of the hypothalamus from 2001. The results show clinical improvement in more than 60% of the cases, opening a promising issue for the treatment of the cluster headache persistent after medical treatment. The surgical target that have been used until now is based on the origin of the cluster headache in the hypothalamic dysfunction. Nevertheless, It has still some open questions as the lack of proving the posterior nucleus of the hypothalamus is the real origin of the cluster headache, the lack of consensus about the anatomy of the surgical target and the variability of the structures stimulated with the surgery. The aim of this article is a review of the target used and propose another surgical target based on physiopathological concepts to explain the improvement with the deep brain stimulation in these patients.

Tratamiento quirúrgico del ictus de la arteria cerebral media / Surgical treatment of the stroke in the middle cerebral artery

Introducción. La craniectomía descompresiva aumenta la supervivencia en los infartos malignos de la arteria cerebral media (ACM). Se analizan los signos radiológicos y clínicos que predicen la evolución maligna del infarto de la ACM,y factores asociados a un peor pronóstico. Pacientes y métodos. Se estudian 30 pacientes divididos en tres grupos: pacientes operados, y pacientes no operados con ingreso en cuidados intensivos o en planta de neurología. La técnica quirúrgica consistióen la creación de una ventana ósea de al menos 10 cm de diámetro y apertura dural. Para la valoración inicial del paciente se utilizó la escala de Glasgow y la escala de ictus del National Institute of Health, y para el seguimiento, la escala modificadade Rankin, el índice de Barthel y la Glasgow Outcome Scale a los seis meses. Resultados. Los pacientes más jóvenes tienen un mejor pronóstico funcional que los mayores de 60 años. La desviación de la línea media mayor de 10 mm se asocia con un peor pronóstico, al igual que volúmenes de tejido infartado mayores de 350 cm3. Menor puntuación en la escala de Glasgow al ingreso se asocia a peor pronóstico vital y a mayor número de secuelas, así como su disminución durante el ingreso. Conclusiones. La edad condiciona la presencia de secuelas en estos pacientes. La presencia de signos clínicos de herniación (anisocoria, menor puntuación inicial o descenso importante en la escala de Glasgow) y radiológicos (desplazamiento de la línea media, volumen infartado) implica un peor pronóstico. La cirugía precoz en aquellos pacientes en que estuviera indicada reduce el número de secuelas y aumenta la supervivencia (AU)

Introduction. Decompressive craniectomy increases the survival rate in cases of malignant middle cerebral artery (MCA) stroke. The imaging and clinical signs that predict a malignant progression of stroke of the MCA are analysed, together with factors associated with a poorer prognosis. Patients and methods. The study involved 30 patients, who were divided into three groups: patients who had undergone surgery, and patients who had not undergone surgery but were admitted to intensive care or to neurology wards. The surgical procedure consisted in creating a bone window with a diameter of at least 10 cm and a dural opening. The initial evaluation of the patient was performed using the Glasgow scale and the National Institute of Health stroke scale; follow-up was carried out using the modified Rankin scale, the Barthel index and the Glasgow Outcome Scale at six months. Results. Younger patients have a better functional prognosis than those over 60 years of age. A deviation of more than 10 mm from the mean line is associated with a poorer prognosis, as are volumes of infarcted tissue above 350 cm3. Lower scores on the Glasgow scale on admission are associated with a poorer prognosis for survival and a higher number of sequelae, as well as their reduction during hospitalisation. Conclusions. Age conditions the presence of sequelae in these patients. The presence of clinical signs of herniation (anisocoria, lower initial score or important drop on the Glasgow scale) and imaging signs (displacement of the mean line, volume of infarcted tissue) imply a poorer prognosis. Early surgery in those patients in whom it is indicated reduces the number of sequelae and increases the rate of survival (AU)

[Surgical treatment of the stroke in the middle cerebral artery]. / Tratamiento quirúrgico del ictus de la arteria cerebral media.

INTRODUCTION: Decompressive craniectomy increases the survival rate in cases of malignant middle cerebral artery (MCA) stroke. The imaging and clinical signs that predict a malignant progression of stroke of the MCA are analysed, together with factors associated with a poorer prognosis. PATIENTS AND METHODS: The study involved 30 patients, who were divided into three groups: patients who had undergone surgery, and patients who had not undergone surgery but were admitted to intensive care or to neurology wards. The surgical procedure consisted in creating a bone window with a diameter of at least 10 cm and a dural opening. The initial evaluation of the patient was performed using the Glasgow scale and the National Institute of Health stroke scale; follow-up was carried out using the modified Rankin scale, the Barthel index and the Glasgow Outcome Scale at six months. RESULTS: Younger patients have a better functional prognosis than those over 60 years of age. A deviation of more than 10 mm from the mean line is associated with a poorer prognosis, as are volumes of infarcted tissue above 350 cm3. Lower scores on the Glasgow scale on admission are associated with a poorer prognosis for survival and a higher number of sequelae, as well as their reduction during hospitalisation. CONCLUSIONS: Age conditions the presence of sequelae in these patients. The presence of clinical signs of herniation (anisocoria, lower initial score or important drop on the Glasgow scale) and imaging signs (displacement of the mean line, volume of infarcted tissue) imply a poorer prognosis. Early surgery in those patients in whom it is indicated reduces the number of sequelae and increases the rate of survival.

Gabapentina en el tratamiento de la neuromiotonía ocular / Gabapentin in the treatment of ocular neuromyotonia

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[A proposed new target for deep brain stimulation in obsessive-compulsive disorder]. / Propuesta de una nueva diana para la estimulación cerebral profunda en el trastorno obsesivo-compulsivo.

INTRODUCTION: The obsessive-compulsive disorder (OCD) has an incidence in general population of 1.5-3%. If we consider as a positive respond a diminution of the 25-35% in the symptoms of OCD according to the Y-BOCS, and we add the cognitive-behavioral therapy to the pharmacological treatment, only a 40-60% of treated patients would have significant improvement and a 10% of patients with OCD, would be refractory to all type of medical treatment. DEVELOPMENT: Current neurosurgical techniques for resistant cases of OCD interrupt the connections between the frontal lobes and subcortical structures (cingulotomy, capsulotomy). These techniques are ablative and irreversible. It shows the importance of finding a less aggressive technique with better clinical results. Deep brain stimulation (DBS) is an alternative to traditional neurosurgery based in neuromodulation methods. It's considered that the physiopathology of the OCD consists of a dysfunction of the direct and indirect vias that control the extrapiramidal limbic circuit. On the other hand, it had been obtained positive results after DBS of the subthalamic nucleus of three patients with Parkinson's disease and OCD. CONCLUSION: This article has as target the demonstration that bilateral DBS of the limbic part of the subthalamic nucleus is an alternative for the treatment of refractory OCD.

Propuesta de una nueva diana para la estimulación cerebral profunda en el trastorno obsesivo-compulsivo / A proposed new target for deep brain stimulation in obsessive-compulsive disorder

La incidencia del trastorno obsesivo-compulsivo (TOC) en la población general es del 1,5-3%, y secalcula que sólo un 40-60% de los pacientes tratados farmacológicamente tiene mejoría significativa, y un 10% de ellos es refractario a dicho tratamiento. Actualmente, el TOC tiene dos tipos de tratamiento neuroquirúrgico: uno ablativo (cingulotomía,capsulotomía) y otro neuromodulador –estimulación cerebral profunda (ECP)–. Desarrollo. Partiendo del hecho de que la fisiopatología del TOC consiste en una disfunción de las vías directa e indirecta que regulan el circuito límbico extrapiramidal,y de los resultados clínicos de tres pacientes con enfermedad de Parkinson y TOC que, tras recibir ECP en el núcleo subtalámico, han mejorado de las dos patologías, proponemos el área límbica del núcleo subtalámico como diana quirúrgicapara la ECP en el TOC. Las coordenadas estereotáxicas sugeridas serían: x, 8-9 mm lateral a la línea media comisura anterior-comisura posterior; y, 1 mm por delante del punto intermedio comisural; z, 3 mm por debajo de la línea media comisuraanterior-comisura posterior. Conclusión. Este artículo tiene como objetivo demostrar que la ECP bilateral de la parte límbica de los núcleos subtalámicos puede ser una alternativa para el tratamiento del TOC refractario a tratamiento farmacológico

The obsessive-compulsive disorder (OCD) has an incidence in general population of 1.5-3%. If we consider as a positive respond a diminution of the 25-35% in the symptoms of OCD according to the Y-BOCS, and we add thecognitive-behavioral therapy to the pharmacological treatment, only a 40-60% of treated patients would have significant improvement and a 10% of patients with OCD, would be refractory to all type of medical treatment. Development. Current neurosurgical techniques for resistant cases of OCD interrupt the connections between the frontal lobes and subcortical structures (cingulotomy, capsulotomy). These techniques are ablative and irreversible. It shows the importance of finding a less aggressive technique with better clinical results. Deep brain stimulation (DBS) is an alternative to traditional neurosurgery based in neuromodulation methods. It’s considered that the physiopathology of the OCD consists of a dysfunction of the direct and indirect vias that control the extrapiramidal limbic circuit. On the other hand, it had been obtained positiveresults after DBS of the subthalamic nucleus of three patients with Parkinson’s disease and OCD. Conclusion. This article has as target the demonstration that bilateral DBS of the limbic part of the subthalamic nucleus is an alternative for the treatmentof refractory OCD

Complications in subthalamic nucleus stimulation surgery for treatment of Parkinson's disease. Review of 272 procedures.

BACKGROUND: Deep brain stimulation (DBS) is a surgical technique used to alleviate symptoms in patients with advanced Parkinson's disease (PD). It is a reversible procedure and its effect is based on electrical modulation of the nervous system and has considerable advantages in morbidity-mortality when compared to lesion techniques such as thalamotomy and/or pallidotomy. The objective was to evaluate the adverse events during the surgical placement of leads in the subthalamic nucleus for the treatment of Parkinson's disease. METHODS: A retrospective data collection was made in a total of 130 patients in whom we performed 272 procedures for the implant of leads in the subthalamic nucleus between May 1998 and December 2005. All the patients were operated by the same surgeon, in the same institution and with the same surgical methodology. The complications under evaluation were: aborted procedure, misplaced leads, intracranial haemorrhage, seizures, hardware complications and other complications. RESULTS: 130 patients were treated (62 women, 68 men; average age 62 (36-74) years). The average duration of disease from the time of diagnosis to operation was 15.3 years (4-28 years) and the mean follow-up was of 37 months (3-93 months). One hundred and twenty four patients were implanted bilaterally and 6 unilaterally. 62% did not present any complications, 30% had one complication, and 8% more than one complication. Aborted procedures amounted to 5.14% of all procedures, misplaced leads 2.2%, intracranial haemorrhage 3.3%, seizures 4.7%, hardware complications 1.8% and other complications 5.1%. CONCLUSION: Deep brain stimulation surgery is an effective and safe method to treat Parkinson's disease with a low incidence of permanent adverse events.

Localization of the lipid receptors CD36 and CLA-1/SR-BI in the human gastrointestinal tract: towards the identification of receptors mediating the intestinal absorption of dietary lipids.

The scavenger receptors CLA-1/SR-BI and CD36 interact with native and modified lipoproteins and with some anionic phospholipids. In addition, CD36 binds/transports long-chain free fatty acids. Recent biochemical evidences indicates that the rabbit CLA-1/SR-BI receptor can be detected in enterocytes, and previous studies showed the presence of mRNA for both CLA-1/SR-BI and CD36 in some segments of the intestinal tract. These findings prompted us to study their respective localization and distribution from the human stomach to the colorectal segments, using immunohistochemical methods. Their expression in the colorectal carcinoma-derived cell line Caco-2 was analyzed by Northern blotting. In the human intestinal tract, CLA-1/SR-BI was found in the brush-border membrane of enterocytes from the duodenum to the rectum. However, CD36 was found only in the duodenal and jejunal epithelium, whereas enterocytes from other intestinal segments were not stained. In the duodenum and jejunum, CD36 co-localized with CLA-1/SR-BI in the apical membrane of enterocytes. The gastric epithelium was immunonegative for both glycoproteins. We also found that CLA-1/SR-BI mRNA was expressed in Caco-2 cells and that its expression levels increased concomitantly with their differentiation. In contrast, the CD36 transcript was not found in this colon cell line, in agreement with the absence of this protein in colon epithelium. The specific localization of CLA-1/SR-BI and CD36 along the human gastrointestinal tract and their ability to interact with a large variety of lipids strongly support a physiological role for them in absorption of dietary lipids.

[Rheumatoid arthritis among mapuche aborigines. A 16 years experience in the IX Region of the Chile]. / Artritis reumatoidea en población mapuche. Una experiencia de 16 años en la IX región de Chile.

BACKGROUND: Mapuche, Chilean natives, represent approximately 9.8% of Chilean population and in the IX region of the country, they account for 18.4% of population over 15 years old. They preserve some socio-cultural characteristics that make them different to the rest of the population. AIM: To describe the epidemiological characteristics rheumatoid arthritis among Mapuche natives. SUBJECTS AND METHODS: Retrospective review of patients of Mapuche origin with rheumatoid arthritis, seen at Temuco Hospital between 1980 and 1996. RESULTS: Among 308 cases gathered, only 106 (93 women, aged 55 +/- 10 years old) complied with 1987 American College of Rheumatology (ACR) criteria for rheumatoid arthritis. The disease began between 29 and 52 years old in 73% of patients and the mean delay in diagnosis was 4.4 years. At diagnosis, 99% had symmetric poliarthritis, 28.3% had either fatigue, fever or weight loss, and 46.9% were in class III or in class IV of ACR-1991. Fifty three percent of patients developed Sicca syndrome, 36% developed nodules, 23% developed Raynaud phenomenon, 11% developed pulmonary involvement, 7% developed vasculitis, 5% developed neurological manifestations and 19% developed ophthalmologic involvement. Rheumatoid factor was positive in 78% and 73% had erosions. HLA DR4 was (+) in 60% of 30 patients. Thirty percent required 3 or more disease modifying drugs and prednisone over 10 mg/day. There was no correlation between functional capacity and several other features of the disease. CONCLUSIONS: Mapuche rheumatoid arthritis patients are detected late and have a poor functional capacity at the time of diagnosis. They also have a higher proportion of extraarticular manifestations, more erosions and require more aggressive treatments.

The C-terminal domain of rotavirus NSP5 is essential for its multimerization, hyperphosphorylation and interaction with NSP6.

Rotavirus NSP5 is a non-structural phosphoprotein with putative autocatalytic kinase activity, and is present in infected cells as various isoforms having molecular masses of 26, 28 and 30-34 kDa. We have previously shown that NSP5 forms oligomers and interacts with NSP6 in yeast cells. Here we have mapped the domains of NSP5 responsible for these associations. Deletion mutants of the rotavirus YM NSP5 were constructed and assayed for their ability to interact with full-length NSP5 and NSP6 using the yeast two-hybrid assay. The homomultimerization domain was mapped to the 20 C-terminal aa of the protein, which have a predicted alpha-helical structure. A deletion mutant lacking the 10 C-terminal aa (DeltaC10) failed to multimerize both in yeast cells and in an in vitro affinity assay. When transiently expressed in MA104 cells, NSP5 became hyperphosphorylated (30-34 kDa isoforms). In contrast, the DeltaC10 mutant produced forms equivalent to the 26 and 28 kDa species, but was poorly hyperphosphorylated, suggesting that multimerization is important for this proposed activity of the protein. The interaction domain with NSP6 was found to be present in the 35 C-terminal aa of NSP5, overlapping the multimerization domain of the protein, and suggesting that NSP6 might have a regulatory role in the self-association of NSP5. NSP6 was also found to interact with wild-type NSP5, but not with its mutant DeltaC10, in cells transiently transfected with plasmids encoding these proteins, confirming the relevance of the 10 C-terminal aa for the formation of the heterocomplex.

In vivo interactions among rotavirus nonstructural proteins.

The rotavirus genome encodes six nonstructural (NS) proteins, five of which (NSP1, NSP2, NSP3, NSP5, and NSP6) have been suggested to be involved in a variety of events, such as genome replication, regulation of gene expression, and gene assortment. These NS proteins have been found to be associated with replication complexes that are precursors of the viral core, however, little information is available about the intermolecular interactions that may exist among them. Using the yeast two-hybrid system, which allows the detection of protein-protein interactions in vivo, all possible combinations among the rotavirus NS proteins were tested, and several interactions were observed. NSP1 interacted with the other four proteins tested; NSP3 associated with itself; and NSP5 was found to form homodimers and to interact with NSP6. Co-immunoprecipitation of proteins from rotavirus-infected cells, using hyperimmune sera monospecific for the NS proteins, showed the same interactions for NSP1 as those observed in yeast. Immunofluorescence co-localization analysis of virus-infected epithelial cells revealed that the intracellular distribution of proteins that were seen to interact in yeast had patterns of distribution that would allow such intermolecular interactions to occur. These findings should contribute to the understanding of the role these proteins play in different aspects of the virus replication cycle.

Human CD36 is a high affinity receptor for the native lipoproteins HDL, LDL, and VLDL.

Mouse and hamster SR-BI glycoproteins and their putative human counterpart CLA-I are so far the only scavenger receptors known to bind both native and modified lipoproteins. CD36, a multigland glycoprotein structurally related to SR-BI and CLA-1, has been reported to bind oxidized low density lipoprotein (OxLDL) and acetylated LDL (AcLDL). In this report, we have studied the ability of CD36 to bind native lipoproteins. By transient expression of human CD36 in mammalian and insect cells, we demonstrate that CD36 is a high affinity receptor for the native lipoproteins HDL, LDL, VLDL, and, as previously reported, for OxLDL and AcLDL. The specificity of these interactions is supported by the dose-dependent inhibiton, effect of a monoclonal antibody against CD36. Furthermore, at least for HDL, binding to CD36 does not require the presence of apoE. These findings, together with preferential expression of CD36 in tissues performing very active fatty acid metabolism (skeletal muscle, heart, mammary epithelium, and adipose tissue) and its involvement in foam cell formation (macrophages), suggest that binding of lipoproteins to CD36 might contribute to the regulation of lipid metabolism, and to the pathogenesis of atherosclerosis.