Depletion of astrocytic transglutaminase 2 improves injury outcomes.

Astrocytes play an indispensable role in maintaining a healthy, functional neural network in the central nervous system (CNS). A primary function of CNS astrocytes is to support the survival and function of neurons. In response to injury, astrocytes take on a reactive phenotype, which alters their molecular functions. Reactive astrocytes have been reported to be both beneficial and harmful to the CNS recovery process subsequent to injury. Understanding the molecular processes and regulatory proteins that determine the extent to which an astrocyte hinders or supports neuronal survival is important within the context of CNS repair. One protein that plays a role in modulating cellular survival is transglutaminase 2 (TG2). Global deletion of TG2 results in beneficial outcomes subsequent to in vivo ischemic brain injury. Ex vivo studies have also implicated TG2 as a negative regulator of astrocyte viability subsequent to injury. In this study we show that knocking down TG2 in astrocytes significantly increases their ability to protect neurons from oxygen glucose deprivation (OGD)/reperfusion injury. To begin to understand how deletion of TG2 in astrocytes improves their ability to protect neurons from injury, we performed transcriptome analysis of wild type and TG2-/- astrocytes. TG2 deletion resulted in alterations in genes involved in extracellular matrix remodeling, cell adhesion and axon growth/guidance. In addition, the majority of genes that showed increases in the TG2-/- astrocytes had predicted cJun/AP-1 binding motifs in their promoters. Furthermore, phospho-cJun levels were robustly elevated in TG2-/- astrocytes, a finding which was consistent with the increase in expression of AP-1 responsive genes. These in vitro data were subsequently extended into an in vivo model to determine whether the absence of astrocytic TG2 improves outcomes after CNS injury. Our results show that, following a spinal cord injury, scar formation is significantly attenuated in mice with astrocyte-specific TG2 deletion compared to mice expressing normal TG2 levels. Taken together, these data indicate that TG2 plays a pivotal role in mediating reactive astrocyte properties following CNS injury. Further, the data suggest that limiting the AP-1 mediated pro-survival injury response may be a contributing factor to that the detrimental effects of astrocytic TG2.

Inhibition or ablation of transglutaminase 2 impairs astrocyte migration.

Astrocytes play numerous complex roles that support and facilitate the function of neurons. Further, when there is an injury to the central nervous system (CNS) they can both facilitate or ameliorate functional recovery depending on the location and severity of the injury. When a CNS injury is relatively severe a glial scar is formed, which is primarily composed of astrocytes. The glial scar can be both beneficial, by limiting inflammation, and detrimental, by preventing neuronal projections, to functional recovery. Thus, understanding the processes and proteins that regulate astrocyte migration in response to injury is still of fundamental importance. One protein that is likely involved in astrocyte migration is transglutaminase 2 (TG2); a multifunctional protein expressed ubiquitously throughout the brain. Its functions include transamidation and GTPase activity, among others, and previous studies have implicated TG2 as a regulator of migration. Therefore, we examined the role of TG2 in primary astrocyte migration subsequent to injury. Using wild type or TG2-/- astrocytes, we manipulated the different functions and conformation of TG2 with novel irreversible inhibitors or mutant versions of the protein. Results showed that both inhibition and ablation of TG2 in primary astrocytes significantly inhibit migration. Additionally, we show that the deficiency in migration caused by deletion of TG2 can only be rescued with the native protein and not with mutants. Finally, the addition of TGFß rescued the migration deficiency independent of TG2. Taken together, our study shows that transamidation and GTP/GDP-binding are necessary for inhibiting astrocyte migration and it is TGFß independent.

Functional inositol 1,4,5-trisphosphate receptors assembled from concatenated homo- and heteromeric subunits.

Vertebrate genomes code for three subtypes of inositol 1,4,5-trisphosphate (IP3) receptors (IP3R1, -2, and -3). Individual IP3R monomers are assembled to form homo- and heterotetrameric channels that mediate Ca(2+) release from intracellular stores. IP3R subtypes are regulated differentially by IP3, Ca(2+), ATP, and various other cellular factors and events. IP3R subtypes are seldom expressed in isolation in individual cell types, and cells often express different complements of IP3R subtypes. When multiple subtypes of IP3R are co-expressed, the subunit composition of channels cannot be specifically defined. Thus, how the subunit composition of heterotetrameric IP3R channels contributes to shaping the spatio-temporal properties of IP3-mediated Ca(2+) signals has been difficult to evaluate. To address this question, we created concatenated IP3R linked by short flexible linkers. Dimeric constructs were expressed in DT40-3KO cells, an IP3R null cell line. The dimeric proteins were localized to membranes, ran as intact dimeric proteins on SDS-PAGE, and migrated as an ∼1100-kDa band on blue native gels exactly as wild type IP3R. Importantly, IP3R channels formed from concatenated dimers were fully functional as indicated by agonist-induced Ca(2+) release. Using single channel "on-nucleus" patch clamp, the channels assembled from homodimers were essentially indistinguishable from those formed by the wild type receptor. However, the activity of channels formed from concatenated IP3R1 and IP3R2 heterodimers was dominated by IP3R2 in terms of the characteristics of regulation by ATP. These studies provide the first insight into the regulation of heterotetrameric IP3R of defined composition. Importantly, the results indicate that the properties of these channels are not simply a blend of those of the constituent IP3R monomers.

Delivery of chemically glycosylated cytochrome c immobilized in mesoporous silica nanoparticles induces apoptosis in HeLa cancer cells.

Cytochrome c (Cyt c) is a small mitochondrial heme protein involved in the intrinsic apoptotic pathway. Once Cyt c is released into the cytosol, the caspase mediated apoptosis cascade is activated resulting in programmed cell death. Herein, we explore the covalent immobilization of Cyt c into mesoporous silica nanoparticles (MSN) to generate a smart delivery system for intracellular drug delivery to cancer cells aiming at affording subsequent cell death. Cyt c was modified with sulfosuccinimidyl-6-[3'-(2-pyridyldithio)-propionamido] hexanoate (SPDP) and incorporated into SH-functionalized MSN by thiol-disulfide interchange. Unfortunately, the delivery of Cyt c from the MSN was not efficient in inducing apoptosis in human cervical cancer HeLa cells. We tested whether chemical Cyt c glycosylation could be useful in overcoming the efficacy problems by potentially improving Cyt c thermodynamic stability and reducing proteolytic degradation. Cyt c lysine residues were modified with lactose at a lactose-to-protein molar ratio of 3.7 ± 0.9 using mono(lactosylamido)-mono(succinimidyl) suberate linker chemistry. Circular dichroism (CD) spectra demonstrated that part of the activity loss of Cyt c was due to conformational changes upon its modification with the SPDP linker. These conformational changes were prevented in the glycoconjugate. In agreement with the unfolding of Cyt c by the linker, a proteolytic assay demonstrated that the Cyt c-SPDP conjugate was more susceptible to proteolysis than Cyt c. Attachment of the four lactose molecules reversed this increased susceptibility and protected Cyt c from proteolytic degradation. Furthermore, a cell-free caspase-3 assay revealed 47% and 87% of relative caspase activation by Cyt c-SPDP and the Cyt c-lactose bioconjugate, respectively, when compared to Cyt c. This again demonstrates the efficiency of the glycosylation to improve maintaining Cyt c structure and thus function. To test for cytotoxicity, HeLa cells were incubated with Cyt c loaded MSN at different Cyt c concentrations (12.5, 25.0, and 37.5 µg/mL) for 24-72 h and cellular metabolic activity determined by a cell proliferation assay. While MSN-SPDP-Cyt c did not induced cell death, the Cyt c-lactose bioconjugate induced significant cell death after 72 h, reducing HeLa cell viability to 67% and 45% at the 25 µg/mL and 37.5 µg/mL concentrations, respectively. Confocal microscopy confirmed that the MSN immobilized Cyt c-lactose bioconjugate was internalized by HeLa cells and that the bioconjugate was capable of endosomal escape. The results clearly demonstrate that chemical glycosylation stabilized Cyt c upon formulation of a smart drug delivery system and upon delivery into cancer cells and highlight the general potential of chemical protein glycosylation to improve the stability of protein drugs.

Stimulus-responsive controlled release system by covalent immobilization of an enzyme into mesoporous silica nanoparticles.

Mesoporous silica nanoparticles (MSN) have emerged as an attractive class of drug delivery carriers for therapeutic agents. Herein, we explored the covalent immobilization of proteins into MSN to generate a stimulus-responsive controlled release system. First, MSN were functionalized with thiol groups using (mercaptopropyl)-trimethoxysilane (MPTMS). Functionalization was verified by X-ray photoelectron spectroscopy (XPS), Fourier-transform infrared (FTIR) spectroscopy, and dynamic light scattering. The model enzyme carbonic anhydrase (CA) was coupled to sulfosuccinimidyl 6-[3'(2-pyridyldithio)-propionamido]hexanoate (Sulfo-LC-SPDP) at a low ratio of 1:1 to prevent enzyme inactivation and subsequently covalently immobilized into MSN via thiol-disulfide interchange. The enzyme could be released from MSN with 10 mM glutathione, which represents intracellular redox conditions, while it remained bound to the MSN at extracellular redox conditions represented by 1 µM glutathione. The activity of the released enzyme was >80% demonstrating that the enzyme was still largely functional and active after immobilization and release. Human cervical cancer (HeLa) cells were incubated with the MSN-CA bioconjugates at various concentrations for 24 h and the data show good biocompatibility. In summary, we demonstrate the potential of MSN as drug delivery systems for proteins.

Tofacitinib Suppresses Natural Killer Cells In Vitro and In Vivo: Implications for Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal and incurable neurodegenerative disease with few therapeutic options. However, the immune system, including natural killer (NK) cells, is linked to ALS progression and may constitute a viable therapeutic ALS target. Tofacitinib is an FDA-approved immunomodulating small molecule which suppresses immune cell function by blocking proinflammatory cytokine signaling. This includes the cytokine IL-15 which is the primary cytokine associated with NK cell function and proliferation. However, the impact of tofacitinib on NK activation and cytotoxicity has not been thoroughly investigated, particularly in ALS. We therefore tested the ability of tofacitinib to suppress cytotoxicity and cytokine production in an NK cell line and in primary NK cells derived from control and ALS participants. We also investigated whether tofacitinib protected ALS neurons from NK cell cytotoxicity. Finally, we conducted a comprehensive pharmacokinetic study of tofacitinib in mice and tested the feasibility of administration formulated in chow. Success was assessed through the impact of tofacitinib on peripheral NK cell levels in mice. We found tofacitinib suppressed IL-15-induced activation as measured by STAT1 phosphorylation, cytotoxicity, pro-inflammatory gene expression, and pro-inflammatory cytokine secretion in both an NK cell line and primary NK cells. Furthermore, tofacitinib protected ALS neurons from NK cell-mediated cytotoxicity. In mice, we found tofacitinib bioavailability was 37% in both male and female mice; using these data we formulated mouse containing low and high doses of tofacitinib and found that the drug suppressed peripheral NK cell levels in a dose-dependent manner. These results demonstrate that tofacitinib can suppress NK cell function and may be a viable therapeutic strategy for ALS.

Cromolyn inhibits the secretion of inflammatory cytokines by human microglia (HMC3).

Cromolyn is a known mast cell stabilizer and is approved for treatment of asthma and for other allergic indications. Cromolyn, in a new redesigned dry powder formulation, is being tested in a pivotal clinical trial in combination with low dose ibuprofen to treat early Alzheimer's Disease (AD) subjects. To better understand the mechanistic effect cromolyn has in slowing down or halting the neuroinflammatory response associated with AD progression, we tested the effect of cromolyn to dampen the inflammatory response in the human HMC3 microglia cell line. The direct effect of cromolyn on HMC3 microglia is on cytokines and chemokines production following their activation by the inflammatory cytokine TNF-α. Cromolyn and a new fluorinated analog dramatically reduced the secretion of a wide spectrum of inflammatory mediators, which included cytokines such as IL-1ß, IL-6, IL-8 and IFN-γ, and chemokines such as CXCL10, CCL2, CCL3 and CCL4. These results bolster our understanding of how our cromolyn platform modulates toxic microglia behavior as a dynamic future treatment option for neurodegenerative disorders.

Cerebral Small Vessel Disease Associated with Subclinical Vascular Damage Indicators in Asymptomatic Hypertensive Patients.

Background: Cerebral small vessel disease (CSVD) is frequent in patients with cardiovascular risk factors including arterial hypertension, and it is associated with vascular damage in other organs and the risk of stroke, cognitive impairment, and dementia. Early diagnosis of CSVD could prevent deleterious consequences. Objective: To characterize CSVD associated with indicators of subclinical vascular damage in asymptomatic hypertensive patients. Materials and Methods: Participants were hypertensive (HT) and non-hypertensive (non-HT) individuals; without signs of cerebrovascular disease, dementia, and chronic renal failure. For CSVD, white matter hyperintensities (WMH), enlarged Virchow-Robin perivascular spaces (EVRPS), lacunar infarcts, and microbleeds were investigated. Subclinical vascular damage was evaluated (hypertensive retinopathy, microalbuminuria, and extracranial carotid morphology: intima media thickness (IMT) and atheroma plaque). Results: CSVD MRI findings were more frequent in HT; as well as greater intimal thickening. The IMT + plaque was significantly associated with all MRI variables; but retinopathy was correlated with EVRPS and lacunar infarcts. Only microalbuminuria was related to the greater severity of WMH in HT. Multivariate analysis evidenced that CSVD was independently associated with the combination of indicators of vascular damage and systolic blood pressure. Conclusions: Combining indicators of subclinical vascular damage, such as carotid morphological variables, microalbuminuria, and hypertensive retinopathy for early detection of CSVD in asymptomatic hypertensive patients could prove to be useful to take actions for the prevention of irreversible brain damage, which could lead to cognitive impairment, dementia and stroke.

Proteína-C reactiva de alta sensibilidad y homocisteína en pacientes con enfermedad cerebrovascular isquémica

Fundamento: la proteína C reactiva de alta sensibilidad (PCR-as) y la homocisteína (Hci) parecen relacionarse con la enfermedad cerebrovascular isquémica, pero sus hallazgos sobre el riesgo y pronóstico de esta enfermedad resultan controversiales y no concluyentes. Objetivo caracterizar la proteína C reactiva de alta sensibilidad y homocisteína en pacientes con enfermedad cerebrovascular isquémica. Métodos: se realizó un estudio descriptivo y retrospectivo de corte transversal en pacientes con enfermedad cerebrovascular isquémica, ingresados en el Servicio de Ictus del Instituto de Neurología y Neurocirugía entre 2016 y 2019. Se recogieron variables demográficas, manifestaciones clínicas, tiempo de evolución, etiología y localización del infarto y factores riesgo. Se cuantificaron la PCR-as (riesgo cardiovascular) y la Hci. Resultados las medias de PCR-as (7,0±8,3 mg/L) y Hci (17,1±7,3 µM) fueron elevadas. El riesgo cardiovascular moderado y alto se presentaron en igual proporción (46,8 %). Hubo diferencias estadísticas en la relación entre el riesgo cardiovascular y la edad (p=0,00); pero ni el tiempo de evolución ni los factores de riesgo de la enfermedad mostraron este comportamiento. Los pacientes con riesgo cardiovascular alto (PCR-as >3 mg/L) y elevada Hci (>15 (M) exhibieron mayores frecuencias de etiologías aterotrombótica o cardioembólica. Conclusiones el riesgo cardiovascular aumenta en la medida que se incrementa la edad de pacientes con enfermedad cerebrovascular isquémica. Las características demográficas, clínicas y neurológicas no mostraron relación con el alto riesgo cardiovascular y los valores elevados de Hci, aunque se encontró una tendencia asociativa de la etiología aterotrombótica con el incremento de PCR-as y Hci.

Foundation: High-sensitivity C-reactive protein and homocysteine seem to be related to ischemic cerebrovascular disease, but their findings on the risk and prognosis of this disease are controversial and inconclusive. Objective: to characterize high sensitivity C-reactive protein and homocysteine in patients with ischemic cerebrovascular disease. Methods: a descriptive and retrospective cross-sectional study was carried out in patients with ischemic cerebrovascular disease, admitted to the Stroke Service of the Neurology and Neurosurgery Institute between 2016 and 2019. Demographic variables, clinical manifestations, time of evolution, etiology and infarction location, risk factors. High-sensitivity C-reactive protein (cardiovascular risk) and homocysteine were quantified. Results: the means of C-reactive protein (7.0±8.3 mg/L) and homocysteine (17.1±7.3 µM) were high. Moderate and high cardiovascular risk occurred in equal proportions (46.8%). There were statistical differences in the relationship between cardiovascular risk and age (p=0.00); but neither the time of evolution nor the risk factors of the disease showed this behavior. Patients with high cardiovascular risk (hs-CRP >3 mg/L) and high homocysteine (>15 (M), exhibited higher frequencies of atherothrombotic or cardioembolic etiologies. Conclusions: cardiovascular risk increases as the age of patients with ischemic cerebrovascular disease increases. Demographic, clinical and neurological characteristics did not show a relationship with high cardiovascular risk and high homocysteine values, although an associative trend of atherothrombotic etiology was found with increased high-sensitivity C-reactive protein and homocysteine.

Biomarcadores de estrés oxidativo en la enfermedad de Huntington y otros desórdenes neurológicos: estudio comparativo

Fundamento: los biomarcadores de estrés oxidativo en la enfermedad de Huntington pudieran predecir el curso de la enfermedad y evaluar nuevos tratamientos, pero su naturaleza inespecífica parece impedir la identificación de algún marcador útil. Esclarecer similitudes y diferencias de este fenómeno y su comportamiento con características clínicas puede ser esencial. Objetivo comparar biomarcadores de estrés oxidativo entre pacientes con enfermedad de Huntington y con otros desórdenes neurológicos. Métodos se realizó un estudio analítico, retrospectivo y caso-control (enfermedad de Huntington esclerosis lateral amiotrófica, ataxia espinocerebelosa tipo 2 e ictus isquémico: en etapa aguda y crónica). Se recogieron variables demográficas, clínicas y marcadores de daño oxidativo (malonildialdehído, productos avanzados de oxidación a proteínas) y antioxidantes (superóxido dismutasa: catalasa: glutatión peroxidasa, capacidad antioxidante del plasma). Resultados hubo diferencias significativas del malonildialdehído en la enfermedad de Huntington respecto al control (p=0,02), pero no con el resto de los grupos. La enzima superóxido dismutasa en enfermedad de Huntington fue menor estadísticamente en comparación con esclerosis lateral amiotrófica, aunque para la catalasa fue superior en relación con el resto de los pacientes. El FRAP en enfermedad de Huntington resultó menor significativamente versus esclerosis lateral amiotrófica e ictus isquémico agudo. Los productos avanzados de la oxidación de proteína se correlacionaron directamente con las edades biológicas y de inicio de la enfermedad de Huntington. La actividad motora en la esclerosis lateral amiotrófica y el déficit neurológico en el ictus isquémico agudo se correlacionaron con el malonildialdehído y glutatión peroxidasa respectivamente. Conclusiones la enfermedad de Huntington parece mostrar características específicas en su sistema antioxidante. La oxidación de proteínas pudiera estar relacionada con la acumulación de huntingtina mutada en el tiempo.

Foundation: biomarkers of oxidative stress in Huntington's disease could predict the course of the disease and evaluate new treatments, but their nonspecific nature seems to prevent the identification of any useful marker. Clarifying similarities and differences of this phenomenon and its behavior with clinical characteristics may be essential. Objective: compare biomarkers of oxidative stress between patients with Huntington's disease and other neurological disorders. Methods: an analytical, retrospective and case-control study was carried out (Huntington's disease, amyotrophic lateral sclerosis, spinocerebellar ataxia type 2 and ischemic stroke: acute and chronic stage). Demographic and clinical variables and markers of oxidative damage (malonildialdehyde, advanced protein oxidation products) and antioxidants (superoxide dismutase: catalase: glutathione peroxidase, plasma antioxidant capacity) were collected. Results: there were significant differences in malonyldialdehyde in Huntington's disease compared to the control (p=0.02), but not with the rest of the groups. The enzyme superoxide dismutase in Huntington's disease was statistically lower compared to amyotrophic lateral sclerosis, although for catalase it was higher in relation to the rest of the patients. FRAP in Huntington's disease was significantly lower versus amyotrophic lateral sclerosis and acute ischemic stroke. Advanced products of protein oxidation were directly correlated with the biological and onset ages of Huntington's disease. Motor activity in amyotrophic lateral sclerosis and neurological deficit in acute ischemic stroke were correlated with malonyldialdehyde and glutathione peroxidase, respectively. Conclusions: huntington's disease seems to show specific characteristics in its antioxidant system. Protein oxidation could be related to the accumulation of mutated huntingtin over time.

Asociación de marcadores inflamatorios y de disfunción endotelial con la depresión en la enfermedad cerebrovascular isquémica

Fundamento: la depresión es una de las complicaciones no neurológicas más frecuentes en la enfermedad cerebrovascular isquémica. Objetivo: determinar la asociación de marcadores inflamatorios y de disfunción endotelial con la depresión en pacientes con enfermedad cerebrovascular isquémica. Métodos: se realizó un estudio analítico, prospectivo de corte transversal en pacientes con enfermedad cerebrovascular isquémica en fase aguda (N=22) y no aguda (N=37); atendidos en el Instituto de Neurología y Neurocirugía y el Hospital Manuel Fajardo, de La Habana, Cuba. Se recogieron variables demográficas, factores de riesgo, etiología y localización del infarto, deficiencia neurológica, discapacidad para las actividades de la vida diaria (índice de Barthel), neuropsicológicas (depresión por inventario de Beck y test de Hamilton). Se determinó proteína C-reactiva, alfa-1-antitripsina, complementos C3 y C4 y microalbuminuria. Resultados: las puntuaciones de las pruebas neuropsicológicas no tuvieron diferencias significativas entre la fase aguda y no aguda, pero hubo un aumento estadístico de la frecuencia de pacientes sin depresión y con ligera depresión en la fase no aguda. En la fase aguda, el complemento C4 y en la fase no aguda el complemento C3, la proteína C-reactiva y el alfa-1-antitripsina se correlacionaron directamente con la puntuación del inventario de Beck. La proteína C-reactiva y C3 se correlacionaron estadísticamente con la puntuación del test de Hamilton. En el análisis multivariado, la proteína C-reactiva mostró asociación independiente con el grado de depresión por el test de Hamilton. Conclusiones: la proteína C-reactiva pudiera estar relacionada con la severidad de la depresión, quizás por asociación con la discapacidad para las actividades de vida diaria.

Foundation: depression in ischemic cerebrovascular disease is one of the most frequent non-neurological complications. Objective: to determine the association of inflammatory markers and endothelial dysfunction with depression in patients with ischemic cerebrovascular disease. Methods: an analytical, prospective, cross-sectional study was carried out in patients with acute (N=22) and non-acute (N=37) ischemic cerebrovascular disease; treated at the Institute of Neurology and Neurosurgery; and the Manuel Fajardo Hospital, in Havana, Cuba. Demographic variables, risk factors, etiology and location of the infarction, neurological deficiency, disability for activities of daily living (Barthel index), neuropsychological (depression by Beck inventory and Hamilton test) were collected. C-reactive protein, alpha-1-antitrypsin, C3 and C4 complements, and microalbuminuria were determined. Results: the scores of the neuropsychological tests did not have significant differences between the acute and non-acute phase, but there was a statistical increase in the frequency of patients without depression and with slight depression in the non-acute phase. In the acute phase, C4, and in the non-acute phase, C3, C-reactive protein and alpha-1-antitrypsin were directly correlated with the Beck inventory score. C-reactive protein and C3 were statistically correlated with the Hamilton test score. In the multivariate analysis, C-reactive protein showed an independent association with the degree of depression by the Hamilton test. Conclusions: C-reactive protein could be related to the severity of depression, perhaps by association with the disability for activities of daily living.

The complex role of transglutaminase 2 in glioblastoma proliferation.

BACKGROUND: Glioblastomas (GBMs) are a heterogeneous group of primary brain tumors. These tumors are resistant to therapeutic interventions and invariably recur after surgical resection. The multifunctional protein transglutaminase 2 (TG2) has been shown to promote cell survival in a number of different tumors. There is also evidence that TG2 may be a pro-survival factor in GBMs. However, the roles that TG2 plays in facilitating GBM survival and proliferation have not yet been clearly delineated . METHODS: The functions of TG2 are often cell- and context-specific. Therefore, in this study we examined the ability of TG2 to facilitate GBM proliferation using colony formation assays and 5-ethynyl-2'-deoxyuridine (EdU) incorporation in several different GBM cell lines as well as neurospheres derived from patient tumors representing the 3 major subtypes of GBM tumors (mesenchymal, proneural, and classical) and maintained in the absence of serum. TG2 knockdown or selective TG2 inhibitors were used to modulate TG2 expression and activity. RESULTS: We show that TG2 plays differential roles in the proliferative process depending on the cell type. In most, but not all, GBM models TG2 plays a crucial role in the proliferative process, and some but not all TG2 inhibitors were highly effective at reducing proliferation in a large subset of the GBM models. CONCLUSION: Our results show that TG2 plays an important-but notoriously context-specific-role in GBM cell biology. Nonetheless, as future studies unravel the genetic "fingerprints" that make TG2 inhibitors effective, this information could be exploited to develop TG2 inhibitors into personalized GBM therapies.

Increased C-reactive protein in acute ischemic stroke patients is age dependent / El incremento de la proteína C reactiva en pacientes con ictus isquémico agudo varía con la edad

Introduction: Several studies investigating blood biomarkers such as C-reactive protein (CRP) in the prognosis and mortality of stroke have not been conclusive. This may be related to the fact that age has not been taken into account for these analyses. Objective: In the present study, we evaluated the possible relationship of blood markers with the age and clinical characteristics of ischemic stroke patients. Methods: Two groups of acute ischemic stroke patients (≤ 55 years and > 55 years of age) who were paired with a control group were included. CRP, alpha 1 antitrypsin (AAT), complements C3 and C4, microalbuminura, ceruloplasmin, glucose, cholesterol, triglycerides, glutamic-piruvic transaminase (GPT), glutamic-oxalacetic transaminase (GOT), gamma glutamiltranspeptidase (GGT), creatinine, and uric acid were determined. Other clinical information, including NIH stroke scale was collected. Results: AAT, ceruloplasmin, microalbuminuria, GPT, GOT and GGT were significantly increased with respect to control subjects in both age groups. Nevertheless, CRP was increased only in patients older than 55 years. CRP and age were directly correlated in stroke patients, but not in the control group joint analysis of age and NIHSS revealed a tendency towards even higher CRP values in older patients with more severe neurological impairment. Levels of CRP increased significantly with age according to NIH score. Conclusions: Age should be considered when evaluating the usefulness of CRP and other blood biomarkers as clinical tools for predicting long or short-term neurological outcome or stroke recurrence events in ischemic stroke patients(AU)

Introducción: Los estudios sobre marcadores sanguíneos incluido la proteína C reactiva (PCR) en el pronóstico y mortalidad del ictus no han sido concluyentes, quizás porque en sus análisis no se ha tenido en cuenta la edad los pacientes. Objetivo: Evaluar la relación de los marcadores sanguíneos con la edad y características clínicas de pacientes con ictus isquémico. Métodos: Se incluyeron en el estudio 2 grupos de pacientes con ictus isquémico (( y > 55 años) quienes fueron pareados con grupos controles. Fueron determinados: PCR, alfa 1 antitripsina (AAT), complementos C3 y C4, microalbuminuria, ceruloplasmina, glucosa, colesterol, triglicéridos, transaminasa glutámico-pirúvico (TGP), transaminasa glutámico-oxalacético (TGO), gamma glutamiltranspeptidasa (GGT), creatinina, y ácido úrico. También, se recogió información clínica (escala neurológica, etiología y localización del ictus). Resultados: La AAT, ceruloplasmina, microalbuminuria, TGP, TGO y GGT aumentaron significativamente respecto al grupo control de ambos grupos de estudio. Sin embargo, la PCR se incrementó solamente en pacientes mayores de 55 años. La PCR se correlacionó directamente con la edad de los pacientes, pero no en el grupo control. A su vez, se observó una tendencia hacia el aumento de la PCR en pacientes mayores de 55 años con mayor la severidad neurológica. Los valores de PCR se incrementaron estadísticamente con la edad de acuerdo al déficit neurológico. Conclusiones: La edad debiera ser considerada en la evaluación de la utilidad de la PCR y de otros marcadores como herramientas clínicas para predecir un desenlace neurológico fatal o recurrencia de nuevos eventos en pacientes con ictus isquémico(AU)

Comportamiento de la GM-1 gangliosidosis en Cuba / Diagnosis of GM-1 gangliodosis in Cuba

Fundamento: La GM1 gangliosidosis se encuentra incluida dentro del grupo de las enfermedades lisosomales. Se caracteriza por la deficiencia de la enzima b-galactosidasa, lo cual tiene como consecuencia la acumulación del gangliosido GM1 en células nerviosas y galactosil oligosacáridos y productos de degradación de keratán sulfato en otros tejidos. Clínicamente esta enfermedad se expresa por un temprano deterioro psicomotor, mancha rojo-cereza en la mácula, dismorfia facial, deformidades óseas y hepatoesplenomegalia. Se transmite con un patrón de herencia autosómico-recesivo. Objetivo: Determinar la actividad enzimática de la â-galactosidasa ácida en pacientes con sospecha de GM-1 gangliosidosis. Métodos: Se atendieron 1851 pacientes procedentes de todo el país en el período comprendido entre 1986-2006, de ellos 851 con la impresión diagnóstica de padecer alguna forma de GM1 gangliosidosis. Para la determinación de la actividad enzimática en leucocitos de pacientes, controles y progenitores se empleó una técnica fluorimétrica (derivados metilumbelliferil) y las proteínas leucocitarias se determinaron por el método de Lowry. Resultados y conclusiones: Se pudo llegar al diagnóstico de GM-1 gangliosidosis en 11 de los pacientes, siendo la actividad enzimática tres veces menor respecto a sus padres y cinco veces menor respecto al grupo control. La actividad de b-galactosidasa no resultó dependiente del sexo ni de la edad(AU)

Background: GM-1 gangliosidosis is included in the group of lysosomal diseases and is characterized by a deficiency of the enzyme b-galactosidase, which as a consequence produces accumulation of GM1 ganglioside in nervous cells and galactosil oligosaccharides and products of keratan sulfate degradation in other tissues. Clinically this disease presents with an early psychomotor impairment, macular cherry red spots, facial dysmorphia, bone deformities and hepatosplenomegaly. It has a hereditary autosomic-recessive pattern. Objective: To determine of â-galactosidase acid activity in patients with suspicion of GM-1 gangliosidosis. Methods: From 1986 to 2006, 1851 patients have been received in our laboratory from all the country, of which 851 had the diagnostic impression of GM-1 gangliosidosis. In samples from patients, parents and controls, the activity of leukocyte b-galactosidase was determined by a fluorimetric technique (metilumbelliferil derivatives) and leukocyte protein level was determined according to Lowrys method. Results and conclusions: The diagnosis of GM-1 gangliosidosis was attained in 11 patients, and the enzymatic activity was three times lower in patients with respect to their parents and five times lower with respect to controls. The enzymatic activity was not related with gender or age(AU)

Metaloproteinasas de matríz en pacientes con esclerosis múltiple / Matrix metalloproteinases in patients with multiple sclerosis

Fundamento: La ruptura proteolítica de la matriz extracelular por las metaloproteinasas 2 y 9 es uno de los aspectos que puede influir en la alteración de la permeabilidad de la barrera hematoencefálica en la esclerosis múltiple. Objetivo: Determinar la actividad de las metaloproteinasas con actividad gelatinasa en pacientes con esclerosis múltiple. Métodos: Las muestras de líquido cefalorraquídeo se obtuvieron de 31 pacientes con esclerosis múltiple y un grupo control conformado por 21 pacientes sin enfermedad neurológica. Las actividades de las metaloproteinasas 2 y 9 en el líquido cefalorraquídeo se determinaron mediante técnica zimográfica por electroforesis en gel de poliacrilamida, después las bandas fueron identificadas por sus pesos moleculares y se calculó la actividad relativa de la metaloproteinasa 9. Se evaluaron las concentraciones de proteínas totales, albúmina e IgG, el índice IgG y el índice Q para evaluar síntesis intratecal de IgG y estado funcional de la barrera hematoencefálica. Resultados: Se detectó actividad de metaloproteinasa 2 en el líquido cefalorraquídeo de todos los pacientes y controles, mientras que la 9 solo se detectó en los pacientes (61, 3 por ciento). La presencia de actividad relativa de metaloproteinasa 9 no se asoció con las variables clínicas, ni con las variables de laboratorio. Se encontró asociación entre su presencia y las bandas oligoclonales en los pacientes con esclerosis múltiple, en aquellos sometidos a tratamiento inmunomodulador esta se presentó con menor frecuencia. Conclusiones: Existe una posible participación de la metaloproteinasa 9 en los mecanismos inmunopatológicos de la esclerosis múltiple(AU)

Fundament: The proteolitic rupture of the extracellular matrix due to metalloproteinase 2 and 9 is one of the aspects that can influence in the alteration of the permeability of the blood-brain barrier (BBB) in multiple sclerosis. Objective: To determine metalloproteinase activity with gelatinous activity in patients suffering from multiple sclerosis. Methods: the cerebrospinal fluid (CSF) samples taken from 31 patients suffering from multiple sclerosis and a control group formed by 21 patients without neurological disease. The metalloproteinase 2 and 9 activities in the cerebrospinal fluid were determined by zimográfica technique through polyacrylamide gel electrophoresis. The bands were later analysed by their molecular weight and the relative metalloproteinase 9 activity was calculated. Total protein concentrations, albumin and immunoglobulin G (IgG), the IgG rate and the Q rate were assessed to evaluate the IgG intrathecal and the functional state of the blood-brain barrier. Results: metalloproteinase 2 activity was detected in the cerebrospinal fluid of all patients and control group. Metalloproteinase 9 activity was only found in the 61.3 percent of the patients. The presence of relative metalloproteinase 9 activity was neither associated with the clinical variables nor the laboratory ones. An association was found between its presence and the oligoclonal bands in patients with multiple sclerosis. In those patients under immunomodular treatment it was presented with less frequency. Conclusions: There is a possible participation of Metalloproteinase 9 in the immunopathological mechanisms of the multiple sclerosis(AU)

Efectos adversos por el uso de la biomodulina T y la corticotropina en pacientes con esclerosis múltiple / Adverse effects due to the usage of T biomodulina and corticotropin in patients suffering from Multiple Sclerosis

Fundamento: La biomodulina T es unproducto natural tímico con acción antinflamatoria y acción inmunomoduladora. La corticotropina es un esteroide utilizado también en el tratamiento de la esclerosis múltiple. Objetivo: Comparar el comportamiento de efectos adversos de la biomodulina y la corticotropina en el tratamiento de la esclerosis múltiple. Métodos: Ensayo clínico fase II, abierto, aleatorizado y controlado, sobre 17 pacientes con esclerosis múltiple a los que se les aplicó el siguiente tratamiento: a un grupo se le suministró 100 miligramos por vía endovenosa de biomodulina por 10 días, 20 miligramos los siguientes 20 días; al otro grupo 1 miligramo de corticotropina por 10 días, seguido de 0, 5 mg por los restantes 20 días. Se evaluaron los eventos adversos a los 10 y 30 días, clasificando su intensidad como ausentes, ligeros, moderados, severos, muy severos, así como duración y tipo de evento. Resultados: Se evaluó la seguridad en 8 pacientes con biomodulina y 7 con corticotropina. Ocurrieron 40 eventos adversos: 24 con corticotropina y 16 con biomodulina (80 y 53,3 por ciento respectivamente). Fueron más frecuentes los eventos ligeros con biomodulina que con corticotropina (56 y 18 por ciento respectivamente), mientras fueron más frecuentes los moderados con corticotropina. Fue más corto el tiempo de duración de los eventos producidos por biomodulina. Conclusiones: La biomodulina fue segura en el tratamiento de la esclerosis múltiples pues sus efectos adversos fueron menos intensos y de menor duración(AU)

Background: T biomodulina is a thymic natural product with anti-inflammatory and immunomodulator action. Corticotropin is a steroid which is also used in the treatment of multiple sclerosis. Objetives: To compare the adverse effects of the biomodulina and corticotroprin in the treatment of multiple sclerosis. Methods: Phase II clinical trial, open, randomized and controlled on 17 patients suffering from multiple sclerosis to whom the following treatment was applied: group one, 100mg IV biomodulina during 10 days, 20 mg the following 20 days; group two: 1 mg of corticotroprin during 10 days followed by 0,5 mg the very next 20 days. The adverse events were evaluated from the 10th day up to the 30th day classifying its intensity as absent, mild, moderate, severe, very severe. The duration and the type of event were also classified. Results: Safeness on 8 patients treated with biomodulina and 7 patients treated with cortcotropin were assessed. 40 adverse events took place: 24 patients in whose corticotropin was used, 16 in the treatment with biomodulina (80 and 53, 3 percent respectively), while the moderate adverse reactions in the usage of corticotropin were more frequent. The shorter period of time of the events was produced by biomodulina. Conclusions: The usage of biomodulina was safer in the treatment of multiple sclerosis because the adverse events as well as the period of time were less intense(AU)

Errores innatos del metabolismo. Enfermedades lisosomales / Metabolism inborn errors. Lysosomal storage diseases

Dentro de los errores innatos del metabolismo se encuentran las enfermedades de almacenamiento lisosomal o enzimopatías lisosomales, las cuáles se caracterizan por un déficit enzimático específico, la excreción de metabolitos por la orina y la acumulación de los compuestos no degradados en diferentes órganos y tejidos que ocasionan la disfución de éstos. Tienen un patrón de herencia autosómico recesivo, excepto para la enfermedad de Fabry y la enfermedad de Hunter en las que el patrón de herencia está ligado al cromosoma X. Estas enfermedades tienen una baja incidencia en general, aunque hay poblaciones donde algunas de ellas tienen una alta incidencia. Su importancia radica en la magnitud que representan como problema de salud, por la pobre calidad de vida de esos pacientes, así como su fallecimiento prematuro, motivo por el cual hay que evitar los nacimientos de nuevos niños afectados(AU)

Errores innatos del metabolismo. Enfermedades lisosomales / Metabolism inborn errors. Lysosomal storage diseases

Dentro de los errores innatos del metabolismo se encuentran las enfermedades de almacenamiento lisosomal o enzimopatías lisosomales, las cuáles se caracterizan por un déficit enzimático específico, la excreción de metabolitos por la orina y la acumulación de los compuestos no degradados en diferentes órganos y tejidos que ocasionan la disfución de éstos. Tienen un patrón de herencia autosómico recesivo, excepto para la enfermedad de Fabry y la enfermedad de Hunter en las que el patrón de herencia está ligado al cromosoma X. Estas enfermedades tienen una baja incidencia en general, aunque hay poblaciones donde algunas de ellas tienen una alta incidencia. Su importancia radica en la magnitud que representan como problema de salud, por la pobre calidad de vida de esos pacientes, así como su fallecimiento prematuro, motivo por el cual hay que evitar los nacimientos de nuevos niños afectados

Las alteraciones lipídicas en el infarto cerebral / Lipid alterations in cerebral infarction

Se realizó un estudio prospectivo tipo caso-control en pacientes y controles, para definir los principales desórdenes lipídicos en pacientes con infarto cerebral y comparar la relación de estos con factores de riesgo vascular. Se observaron valores significativamente más elevados de triglicéridos, lipoproteínas de baja densidad e índice aterogénico, en los pacientes respecto de los controles. No hubo diferencias para el colesterol total ni las lipoproteínas de muy baja densidad entre los 2 grupos. La hipertensión arterial como factor de riesgo se asoció con valores más bajos de lipoproteínas de alta densidad, y más elevados de lipoproteínas de baja densidad, triglicéridos, lipoproteínas de muy baja densidad e índice aterogénico. No se encontró relación entre la hipertensión arterial y el colesterol total. Se concluyó que era necesario controlar los lípidos en el infarto cerebral