[Hypomethylation and multiple sclerosis, the susceptibility factor?].

BACKGROUND: Azathioprine, off-label used long time ago to treat multiple sclerosis (MS) patients, has recently received approval from the Spanish Medicine Agency (Agencia Española del Medicamento) in relapsing-remitting (RR) forms of this condition. Clinical efficacy of azathioprine is due to the enzymatic conversion to 6-thioguanine (the active metabolite). The key enzyme in this process is thio purine methyl transferase (TPMT), converting 6-MP to 6-methylMP. A specific genetic polymorphism has been described affecting this enzyme. With the aim of optimizing purine therapy in a variety of autoimmune diseases, monitoring of TPMT phenotype has been performed in a vast number of patients. The TPMT activity frequency distribution histogram from a Spanish population sample has been compared with the corresponding ones to Crohn's disease, ulcerative colitis and MS patients. METHODS AND RESULTS: TPMT activity has been studied in red blood cells obtained from 3,640 clinical laboratory samples in Spain of which 1,249 corresponded to patients affected by Crohn's disease, 589 to ulcerative colitis, 348 to MS, 487 to several autoimmune diseases apart from the previously mentioned and 967 to a group of blood donors. The mean TPMT activity in the MS group (17.1 6.1 U/ml) was significantly lower (p < 0.001) than in Crohn's disease (20.0 5.8 U/ml), ulcerative colitis (19.7 6.1 U/ml) and donors group (19.9 6.3 U/ml). CONCLUSION: Defective methylation profile and subsequent hyperhomocysteinemia leading to a widespread impairment of the methyl-transferase activity (in this case affecting MBP methylation) is a vicious circle we propose as a MS susceptibility factor.

Hipometilación en la esclerosis múltiple: ¿factor de susceptibilidad? / Hypomethylation and multiple sclerosis, the susceptibility factor?

FUNDAMENTO: La azatioprina, ampliamente utilizada con carácter compasivo en el tratamiento de pacientes con esclerosis múltiple (EM), recibió recientemente aprobación por parte de la Agencia Española del Medicamento para su utilización en las formas recurrentes-remitentes de este proceso. La eficacia clínica de azatioprina se basa en la conversión enzimática en 6-tioguanina, su metabolito activo. La enzima clave en este proceso se denomina tio purina metiltransferasa (TPMT), que transforma la 6-mercaptopurina en 6-metilmercaptopurina. Se ha descrito un polimorfismo genético que afecta de manera específica a esta enzima. Con el objetivo de optimizar la terapia con purinas en varias enfermedades de carácter autoinmune, hemos llevado a cabo una monitorización fenotípica de la TPMT en un importante grupo de pacientes.Asimismo, hemos comparado el histograma de distribución de frecuencias de actividad TPMT procedente de una muestra de población española, con los correspondientes de pacientes afectados de enfermedad de Crohn, colitis ulcerosa y EM.MÉTODOS Y RESULTADOS: Hemos analizado la actividad TPMT en eritrocitos procedentes de 3.640 muestras clínicas, de las cuales 1.240 correspondían a pacientes con enfermedad de Crohn, 589 a pacientes con colitis ulcerosa, 348 a afectados de EM, 487 a enfermos con varios trastornos autoinmunes diferentes de los mencionados y 967 a un grupo de donantes. La actividad TPMT media en el grupo de pacientes con EM (17,1 ñ 6,1 U/ml) fue significativamente más baja (p < 0,001) que en los grupos con enfermedad de Crohn (20,0 ñ 5,8 U/ml), colitis ulcerosa (19,7 ñ 6,1 U/ml) y donante (19,9 ñ 6,3 U/ml).CONCLUSIÓN: Un perfil de metilación deficiente y la hiperhomocisteinemia secundaria conducen a una alteración generalizada de la actividad metiltransferasa (afectando, entre otras, a la proteína básica de mielina). Este círculo vicioso supondría un factor de susceptibilidad para padecer EM. (AU)

High ammonia diet: its effect on the glial fibrillary acidic protein (GFAP).

The effect of a recent hyperammonemic model, consisting of a high ammonia diet for 3, 7, 15, 45, and 90 days, on glial fibrillary acidic protein (GFAP) in the rat spinal cord and on blood ammonia levels has been studied. The high ammonia diet was prepared by mixing a standard diet with ammonium acetate (20% wt/wt); in addition, 5 mM of ammonium acetate was added to the water supply. GFAP contents were determined by means of immunoblotting analysis. The results demonstrated that this high ammonia diet model neither induces significant changes in GFAP immunoreactivity, nor modifies total protein concentration, and only induces significant blood hyperammonemic levels in the first days of treatment. An adaptive response to the diet is suggested and discussed to explain these results. A relation between ammonia and GFAP expression is suggested because transient hyperammonemia induces transient, although no significant, changes on GFAP expression.

Cysteamine normalizes cerebral somatostatin level and binding in pentylenetetrazol-kindled rats.

Rats were kindled by intraperitoneal injection of pentylenetetrazol (PTZ) (30 mg/Kg) every 48 h. Once kindled, some of the animals received a single injection of cysteamine (200 mg/Kg). Somatostatin-like immunoreactivity (SLI) and 125 I-Tyr11-somatostatin binding were measured in the frontoparietal cortex and hippocampus of the two experimental groups and the control rats. After PTZ kindling the following was observed: 1) SLI content was increased in the two areas; 2) Somatostatin receptor affinity decreased in the frontoparietal cortex and was unaltered in the hippocampus; 3) The number of somatostatin receptors decreased in the hippocampus and was unaltered in the frontoparietal cortex. Cysteamine, an agent which depletes brain somatostatin and suppresses kindled seizures in PTZ-treated rats, reversed the altered SLI levels and binding in these rats.

Effect of cysteamine on cytosolic somatostatin binding sites in rabbit duodenal mucosa.

Administration of cysteamine in rabbits elicited a rapid depletion of both duodenal mucosa and plasma somatostatin. A significant reduction was observed within 5 min, returning toward control values by 150 min. The depletion of somatostatin was associated with an increase in the binding capacity and a decrease in the affinity of both high- and low-affinity binding sites present in cytosol of duodenal mucosa. Incubation of cytosolic fraction from control rabbits with 1 mM cysteamine did not modify somatostatin binding. Furthermore, addition of cysteamine at the time of binding assay did not affect the integrity of 125I-Tyr11-somatostatin. It is concluded that in vivo administration of cysteamine to rabbits depletes both duodenal mucosa and plasma somatostatin and leads to up-regulation of duodenal somatostatin binding sites.

Modulation of somatostatin binding sites in cytosol of rabbit gastric fundic mucosa by cysteamine administration.

Cysteamine, when given in vivo to rabbits, depleted immunoreactive somatostatin in rabbit gastric fundic mucosa. Depletion of immunoreactive somatostatin was associated with both an increase in the number and a decrease in the affinity of the low-affinity somatostatin binding sites. The in vitro incubation of cysteamine (0.1 mM) with the cytosolic fraction did not result in any modification of somatostatin binding. These results suggest that a decrease in the endogenous immunoreactive somatostatin might lead to up-regulation of somatostatin binding sites in the gastric fundic mucosa.

Somatostatin binding sites in cytosolic fractions of parietal and non-parietal cells from rabbit fundic mucosa.

Specific binding sites for somatostatin have been found in the cytosolic fractions of both parietal and non-parietal cells from rabbit gastric fundic mucosa. The stoichiometric data suggested the presence of two classes of binding sites in both types of cells. The number of low-affinity binding sites was significantly higher in parietal cells than in non-parietal cells. The reverse was true for the high-affinity binding sites. However, the affinity of each class of binding sites was similar in the cytosolic fractions of both parietal and non-parietal cells. It thus appears that low-affinity somatostatin binding sites are mainly located in the parietal cells whereas the high-affinity sites occur principally in the non-parietal cells.

Somatostatin binding sites in cytosolic fraction of rabbit intestinal mucosa: distribution throughout the intestinal tract.

Specific binding sites for somatostatin have been identified in cytosolic fraction of both small and large intestinal mucosa. The stoichiometric data suggested the presence of two classes of binding sites in each part of the intestine. The binding capacity varied depending on the segment considered (rectum greater than duodenum = jejunum greater than ileum, caecum and colon). However, the affinities of the binding sites were similar throughout the whole intestinal mucosa, with the exception of rectum which showed higher Kd values. The binding sites were shown to be highly specific for somatostatin since neuropeptides such as vasoactive intestinal peptide, neurotensin, substance P and Leu-enkephalin did not show any effect upon somatostatin binding.

Characterization of somatostatin binding sites in cytosolic fraction of rat intestinal mucosa.

Specific binding sites for somatostatin have been characterized in cytosolic fraction of rat intestinal mucosa by using 125I-labelled Tyr11-somatostatin and a variety of physicochemical conditions. The binding depended on time, temperature and pH, and was reversible, saturable and specific. At apparent equilibrium, the specific binding of 125I-Tyr11-somatostatin was competitively inhibited by native somatostatin in the 1 nM-4 microM concentration range. Binding studies suggested the presence of two classes of binding sites: a class with high affinity (Kd = 0.07 microM) and low capacity (4.6 pmol/mg protein) and a class with low affinity (Kd = 1.05 microM) and high capacity (277 pmol/mg protein) at 25 degrees C. Somatostatin exhibited competitive inhibition of tracer binding, while neuropeptides such as neurotensin, substance P, Leu-enkephalin, and vasoactive intestinal peptide were ineffective. The presence of somatostatin binding sites in cytosolic fraction of intestinal mucosa, together with the known occurrence of somatostatin in D-cells and nerve endings in the small intestine, strongly suggest that this peptide may be involved in the physiology and physiopathology of intestinal epithelium.