Novel Adjuvant Based on the Pore-Forming Protein Sticholysin II Encapsulated into Liposomes Effectively Enhances the Antigen-Specific CTL-Mediated Immune Response.

Vaccine strategies to enhance CD8+ CTL responses remain a current challenge because they should overcome the plasmatic and endosomal membranes for favoring exogenous Ag access to the cytosol of APCs. As a way to avoid this hurdle, sticholysin (St) II, a pore-forming protein from the Caribbean Sea anemone Stichodactyla helianthus, was encapsulated with OVA into liposomes (Lp/OVA/StII) to assess their efficacy to induce a CTL response. OVA-specific CD8+ T cells transferred to mice immunized with Lp/OVA/StII experienced a greater expansion than when the recipients were injected with the vesicles without St, mostly exhibiting a memory phenotype. Consequently, Lp/OVA/StII induced a more potent effector function, as shown by CTLs, in vivo assays. Furthermore, treatment of E.G7-OVA tumor-bearing mice with Lp/OVA/StII significantly reduced tumor growth being more noticeable in the preventive assay. The contribution of CD4+ and CD8+ T cells to CTL and antitumor activity, respectively, was elucidated. Interestingly, the irreversibly inactive variant of the StI mutant StI W111C, encapsulated with OVA into Lp, elicited a similar OVA-specific CTL response to that observed with Lp/OVA/StII or vesicles encapsulating recombinant StI or the reversibly inactive StI W111C dimer. These findings suggest the relative independence between StII pore-forming activity and its immunomodulatory properties. In addition, StII-induced in vitro maturation of dendritic cells might be supporting these properties. These results are the first evidence, to our knowledge, that StII, a pore-forming protein from a marine eukaryotic organism, encapsulated into Lp functions as an adjuvant to induce a robust specific CTL response.

Role of B-1 cells in the immune response against an antigen encapsulated into phosphatidylcholine-containing liposomes.

B-1 lymphocytes comprise a unique subset of B cells that differ phenotypically, ontogenetically and functionally from conventional B-2 cells. A frequent specificity of the antibody repertoire of peritoneal B-1 cells is phosphatidylcholine. Liposomes containing phosphatidylcholine have been studied as adjuvants and their interaction with dendritic cells and macrophages has been demonstrated. However, the role of B-1 cells in the adjuvanticity of liposomes composed of phosphatidylcholine has not been explored. In the present work, we studied the contribution of B-1 cells to the humoral response against ovalbumin (OVA) encapsulated into dipalmitoylphosphatidylcholine (DPPC) and cholesterol-containing liposomes. BALB/X-linked immunodeficient (xid) mice, which are deficient in B-1 cells, showed quantitative and qualitative differences in the anti-OVA antibody response compared with wild-type animals after immunization with these liposomes. The OVA-specific immune response was significantly increased in the BALB/xid mice when reconstituted with B-1 cells from naive BALB/c mice. Our results indicate the internalization of DPPC-containing liposomes by these cells and their migration from the peritoneal cavity to the spleen. Phosphatidylcholine significantly contributed to the immunogenicity of liposomes, as DPPC-containing liposomes more effectively stimulated the anti-OVA response compared with vesicles composed of dipalmitoylphosphatidylglycerol. In conclusion, we present evidence for a cognate interaction between B-1 cells and phosphatidylcholine liposomes, modulating the immune response to encapsulated antigens. This provides a novel targeting approach to assess the role of B-1 cells in humoral immunity.

Oral administration of an enzymatic protein hydrolysate from the green microalga Chlorella vulgaris enhances the nutritional recovery of malnourished mice.

This study examined the effects of oral administration of an enzymatic protein hydrolysate from green microalga Chlorella vulgaris (Cv-PH) on the nutritional recovery of malnourished Balb/c mice after a 3-day fasting period. Mice were refed with commercial diet supplemented or not supplemented with Cv-PH (500 mg/kg) for 8 days. Regardless of the diet used during refeeding, animal body weights and serum protein concentrations did not differ between groups. Mice given Cv-PH had a significant increase in hemoglobin concentrations. Most serum amino acid levels were similar in the control and Cv-PH animals. Starved mice refed with Cv-PH showed normal liver functions, as judged by liver weight, protein concentration, and the enzymatic activities of cholinesterase and arginase. Cv-PH increased DNA, protein content, and gut-mucosal weight. In addition, brush-border oligosaccharidase activities were also higher in the Cv-PH group. These findings suggest that Chlorella protein hydrolysate can be used to develop specific formulations suitable for pharmacologic nutrition.

Effect of starvation and refeeding on biochemical and immunological status of Balb/c mice: an experimental model of malnutrition.

CONTEXT: Although new methods for the induction of malnutrition disorders in laboratory animals have been developed, the bulk of the models described in the literature are essentially based on dietary restriction/starvation principle. In this context, little data are available about the metabolic and the immune system parameters of Balb/c mice under starvation/refeeding. OBJECTIVE: This study examined the effects of starvation and refeeding on the biochemical and immunological status of undernourished Balb/c mice. METHODS: Female Balb/c mice, weighing 20 g, were starved for 3 days and then refed with commercial pelleted diet for 8 days. The variables considered were as follows: body weight; serum protein and amino acid concentrations; liver protein content, and cholinesterase and arginase activities; jejunal protein and DNA contents as well as oligosaccharidase levels; hematological parameters (bone marrow and peripheral blood cellularity); peritoneal macrophage activation; and humoral and cell-mediated immune functions. RESULTS: Profound alterations in both biochemical and immunological conditions appeared after the starvation period. Refeeding resulted in the normalization of serum albumin levels, the intestinal DNA content and the gut-mucosal associated enzymatic activities, the blood lymphocyte counts, and the number of peritoneal macrophages. The markers of liver metabolic function (cholinesterase and arginase activities), and those of bone marrow hemopoiesis and the adaptive immune response (T-dependent antibody titres and delayed-type hypersensitivity response) remained altered after refeeding compared with control mice. CONCLUSION: These findings suggest that fasted mice can be used as an animal model of acute starvation that might prove useful in evaluating the effectiveness of nutritional and immunopharmacological interventions.

Características de las encapsulación de EGP, P64K y el conjugado de ambas proteínas en liposomas obtenidos mediante congelación-descongelación / Encapsulation characteristics of EGF, P64K and the conjugate of both proteins into liposomes obtained by freeze and thaw procedure

Se determinó la influencia de la composición fosfolipídica sobre la eficiencia de encapsulación de las siguientes proteínas recombinantes: factor de crecimiento epidérmico(EGF), P64K y el conjugado de ambas en liposomas obtenidos mediante el procedimiento tecnológico de congelación-descongelación. Se determinó además, la estabilidad de estos preparados durante el almacenamimento a 4 grados céntigrados. Se prepararon liposomas de dipalmitoi fosfatidilcolina, diesteaoril fosfatidilcolina y fosfatidilcolina de yema de huevo en todos los casos, con colesterol en proporción molar 1:1. Estos liposomas contenían al EGP y la P64K marcadas con I 125 o el conjugado marcado en la molécula del factor de crecimento. La capacidad de las vesículas liposomales para retener su contenido dependió no sólo de la composición fosfolipídica, en términos de su temperatura de transición, sino también de la naturaleza del soluto encapsulado. El EGP fue la entidad molecular que experimentó una mayor liberación de los liposomas al medio acuoso. Las vesículas de dipalmitoil fosfatidilcolina: colesterol resultaron las más estables en cuanto a su capacidad de retención de las proteínas recombinantes mientras que el conjugado se comportó como una entidad molecular diferente, al menos en relación a la composición fosfolipídica requerida para alcanzar una mayor estabilidad de los liposomas que lo contienen. La adicción de sacarosa, trealosa, maltosa o glucosa a las vesículas de fosfatidilcolina de yema de huevo: colesterol redujo notablemente la salida de EGP, independiente del tipo de azúcar empleado, en comparación con aquellas vesículas preparadas en ausencia de azúcares(AU)

Juvenile myoclonic epilepsy: linkage to chromosome 6p12 in Mexico families.

Juvenile myoclonic epilepsy is a common subtype of idiopathic epilepsy accounting for 4-11% of all epilepsies. We reported previously significant evidence of linkage between chromosome 6p12-11 microsatellites and the clinical epilepsy and EEG traits of JME families from Belize and Los Angeles. To narrow the JME region, we ascertained and genotyped 31 new JME families from Mexico using a later generation of Généthon microsatellites. Two point linkage analyses obtained significant Z(max) values of 3.70 for D6S1573 and 2.65 for D6S1714 at theta(m = f) = 0.10, and 3.49 for D6S465, 2.11 for D6S1960 at theta(m = f) = 0.05 assuming autosomal dominant inheritance with 70% age-dependent penetrance. Multipoint LOD score curve peaked at 4.21 for D6S1573. Haplotype and recombination analysis reduced the JME region to 3.5 cM flanked by D6S272 and D6S1573. These results provide confirmatory evidence that a major susceptibility gene for JME exists in chromosome 6p12 in Spanish-Amerinds of Mexico.

Deteccion del bebedor problema / Detection of trouble drinker

Se realizó un estudio de la población adulta de dos consultorios del médico de la familia con la finalidad de detectar, mediante entrevistas personales y colectivas a familiares, así como a dirigentes de los organismos de masa, los individuos que ingerian reiteradamente bebidas alcohólica. A la muestra seleccionada se le aplicó encuesta para el pesquisaje y caracterización de los bebedores. Se encontró que el 4 porciento de la población estudiada es alcohólica, con un predominio del sexo masculino, entre las edades de 19 a 44 años y con bajo nivel de escolaridad. El 50 porciento de estos pacientes tenia problemas con su pareja y el 33,3 porciento ha estado arrestado por su conducta anormal durante la embriaguez(AU)