Generation of plant-derived recombinant DTP subunit vaccine.

The current diphtheria-tetanus-pertussis (DTP) pediatric vaccine is produced from the corresponding pathogenic bacteria Corynebacterium diphtheriae, Clostridium tetani and Bordetella pertussis; five injected doses of DTaP (acellular) vaccine are required for every child in the standard US vaccination schedule. Because the vaccine is derived from native live sources, adverse effects are possible and production is complex and costly. To address issues of safety, ease of renewability and expense, we used recombinant technology in an effort to develop a subunit DPT vaccine derived in non-pathogenic plant expression systems. Expression of diphtheria toxin (DT), tetanus fragment-C (TetC) and the non-toxic S1 subunit of pertussis toxin (PTX S1) antigenic proteins in soluble form in low-alkaloid tobacco plants and carrot cell cultures allowed efficient downstream purification to levels suitable for intramuscular injection in BALB/c mice. At working concentrations of 5mug per dose, these preparations induced high levels of antigen-specific IgGs in mouse sera. Our results clearly support the feasibility of producing recombinant pediatric vaccine components in plants.

Immunological assessment of plant-derived avian flu H5/HA1 variants.

Polypeptide variants of the HA1 antigenic domain of the H5N1 avian influenza virus hemagglutinin (HA) molecule were produced in plants using transient and stable expression systems and fused with His/c-myc tags or with mouse or human Fc antibody fragments. The resulting peptides were purified and used for intramuscular immunization of mice. While the recombinant HA1 variants induced a significant serum humoral immune response in the mice, none of the HA1 preparations induced virus-neutralizing antibodies. Fusion with the Fc fragment improved overall yield of the constructs and allowed purification requiring only a single step, but led to no detectable fusion-related enhancement of immunogenicity or quality of immune response.

Prevalencia y factores de riesgo de sobrepeso, obesidad y características bioquímicas en adolescentes de los colegios urbanos del cantón Cuenca en el año lectivo 2006-2007: Cuenca, 2006 / Prevalence and risk factors of overweight, obesity and biochemical characteristics in adolescents in urban schools in the canton Cuenca in the academic year 2006-2007: Cuenca, 2006

Estudio de corte transversal para calcular prevalencia de sobrepeso y obesidad e identificar los factores asociados en una muestra aleatoria de 696 estudiantes. la prevalencia de sobrepeso fue del 17,7% y de obesidad del 8,3%. El sobrepeso fue mayor en las mujeres con una razón de prevalencia (RP) de 1,08 (IC95%: 0,7 – 1,4) y la obesidad en los varones, RP de 1,9 (IC95%: 0,1 – 3,5). Hábitos nutricionales no saludables fue el factor asociado más relevante con una RP de 18,4 (IC95%: 12,8 – 26,4) en el grupo con sobrepeso y una RP de 36,1 (IC95%: 21,2 – 61,2) en el grupo con obesidad. La asociación fue altamente significativa. de las pruebas bioquímicas la insulina y la hipertrigliceridemia tuvieron cifras más elevadas en la obesidad que en el sobrepeso (P < 0,05) y la alteración de los niveles de HDL fue mayor del 65% en ambos subgrupos. la prevalencia de sobrepeso y obesidad en nuestra ciudad alcanza porcentajes similares a los reportados en la literatura médica y está asociada a los hábitos nutricionales no saludables con niveles elevados de insulina, triglicéridos y valores alterados de HDL.

Cross-sectional study to calculate prevalence of overweight and obesity and to identify associate factors in a random sample of 696 students. prevalence of overweight was 17,7% and obesity 8,3%. Overweight was bigger in the women with a prevalence rate (RP) 1,08 (IC95%: 0,7 - 1,4) and obesity in the males, RP 1,9 (IC95%: 0,1 - 3,5). Nutritional habits not healthy it was the most outstanding associate factor with a RP 18,4 (IC95%: 12,8 - 26,4) in overweight group and a RP 36,1 (IC95%: 21,2 - 61,2) in obesity group. The association was highly significant. of biochemical tests the insulin and hipertrigliceridemy had higher values in obesity that in overweight (P <0,05) and abnormalities levels of HDL was bigger than 65% in both subgroups. the prevalence of overweight and obesity in our city reach similar percentages to those reported in medical literature and it is associated to the nutritional habits no healthy with high levels of insulin, triglycerides and altered values of HDL.

Isomer-specific antidiabetic properties of conjugated linoleic acid. Improved glucose tolerance, skeletal muscle insulin action, and UCP-2 gene expression.

Conjugated linoleic acid (CLA) isomers have a number of beneficial health effects, as shown in biomedical studies with animal models. Previously, we reported that a mixture of CLA isomers improved glucose tolerance in ZDF rats and activated peroxisome proliferator-activated receptor (PPAR)-gamma response elements in vitro. Here, our aim was to elucidate the effect(s) of specific CLA isomers on whole-body glucose tolerance, insulin action in skeletal muscle, and expression of genes important in glucose and lipid metabolism. ZDF rats were fed either a control diet (CON), one of two CLA supplemented diets (1.5% CLA) containing differing isoforms of CLA (47% c9,t11; 47.9% c10,t12, 50:50; or 91% c9,t11, c9,t11 isomers), or were pair-fed CON diet to match the intake of 50:50. The 50:50 diet reduced adiposity and improved glucose tolerance compared with all other ZDF treatments. Insulin-stimulated glucose transport and glycogen synthase activity in skeletal muscle were improved with 50:50 compared with all other treatments. Neither phosphatidlyinositol 3-kinase activity nor Akt activity in muscle was affected by treatment. Uncoupling protein 2 in muscle and adipose tissue was upregulated by c9,t11 and 50:50 compared with ZDF controls. PPAR-gamma mRNA was downregulated in liver of c9,t11 and pair-fed ZDF rats. Thus, the improved glucose tolerance in 50:50 rats is attributable to, at least in part, improved insulin action in muscle, and CLA effects cannot be explained simply by reduced food intake.

Regulation of PPARgamma but not obese gene expression by dietary fat supplementation.

Leptin, the product of the obese gene, and peroxisome proliferator activated receptor gamma (PPARgamma) are important regulators of energy metabolism, adipogenesis, and immune function. In rodent models, both genes seem to respond at the mRNA and/or protein levels to dietary fat consumption. To determine the effect(s) of dietary saturated and polyunsaturated fatty acids on the expression (mRNA abundance) of these genes, adipose tissue was obtained from pigs fed three different dietary fat sources. Corn-soybean meal diets containing no added fat (NO, control) or 10% beef tallow (BT), safflower oil (SO), or fish oil (FO) were fed ad libitum (n = 12) for 12 weeks. The abundance of obese, PPARgamma1, and PPARgamma2 mRNA was quantified relative to 18S rRNA using ribonuclease protection assays. The gain:feed ratio was improved (P < 0.05) 21% by all fats with a corresponding reduction (P < 0.05) in feed intake. Relative to pigs fed NO, serum total cholesterol was increased (P < 0.01) in pigs fed BT and triglyceride and nonesterified fatty acid concentrations were increased (P < 0.01) by all supplemental fats. Serum insulin was increased (P < 0.10) only by SO. Neither obese nor PPARgamma1 mRNA abundance were responsive to added fat (P > 0.15). However, the abundance of PPARgamma2 mRNA was increased fourfold by SO compared with the NO diet. These data indicate that the abundance of obese mRNA is independent of dietary fat consumption per se, whether saturated or unsaturated, when feed consumption is reduced due to greater dietary caloric density. Furthermore, we provide evidence that expression of the PPARgamma2 gene in porcine adipose tissue is selectively responsive to SO (presumably linoleic acid, 18:2n-6).

Physiological response to acute endotoxemia in swine: effect of genotype on energy metabolites and leptin.

Certain high lean gain swine genotypes have greater sensitivity to pathogen and nonpathogen stressors evident by reduced productivity and increased mortality during disease stress or in suboptimal production environments. Saline (control) and an immunologic challenge (LPS; 25 microg lipopolysaccharide/kg BW) were administered to three genetic populations (each pig used as its own control): high lean (H), moderate lean terminal cross (MT), and moderate lean maternal cross (MM). LPS induced anorexia, and significantly increased body temperature and circulating TNF-alpha, cortisol, and NEFA in all genotypes (P < 0.0004). LPS reduced circulating glucose, insulin, and IGF-1 in all genotypes (P < 0.05). The LPS-induced hypoglycemia was significantly greater in MM versus MT and H pigs (P < 0.03). The hypoinsulinemia was significantly greater in MM versus H pigs (P < 0.02). MM pigs recovered from hypoinsulinemia slower than MT pigs (P < 0.03). Control insulin was higher in H versus MT pigs (P < 0.08), but relative to basal, the insulin response to LPS was similar. Plasma haptoglobin response to LPS was lower for MM versus MT and H pigs (P < 0.02), and tended to be lower in MT versus H pigs (P < 0.09). LPS treatment caused similar decreases in plasma IGF-1 concentrations among genotypes. Ten hours after LPS treatment, leptin mRNA abundance in adipose tissue was significantly reduced (relative to control) in MM and H pigs (P < 0.02) but not in MT pigs (P > 0.05). Physiological differences in leptin, a potent regulator of food intake and energy metabolism, may be important factors in the genetic variation in sensitivity to environmental stress.

Leptin expression is reduced with acute endotoxemia in the pig: correlation with glucose, insulin, and insulin-like growth factor-1 (IGF-1).

Leptin has been implicated in the regulation of anorexia associated with cachexia in rodents and humans. Regulation of leptin expression is under complex endocrine and metabolic control. To determine if leptin expression is regulated by acute inflammation and to define the endocrine and metabolic factor(s) that regulates leptin expression during acute inflammation, castrate male pigs (ad libitum fed, used as their own controls) were treated with saline (control period) and endotoxin (lipopolysaccharide [LPS] period). Frequent blood samples were collected to identify dynamic changes in hormones and metabolites that are known to regulate leptin expression. LPS caused fever and elevated plasma cortisol (p < 0.0004), tumor necrosis factor-alpha (TNF-alpha) (p < 0.0001), and plasma nonesterified fatty acids (NEFA) (p < 0.001) compared with control. Circulating insulin (p < 0.01), glucose (p < 0.003), and insulin-like growth factor-1 (IGF-1) (p < 0.0001), as well as adipose leptin mRNA abundance (p < 0.01), were profoundly reduced following LPS treatment compared with control. Our data indicate that during acute endotoxemia (1-10 h after injection), leptin gene expression is decreased compared with ad libitum fed animals and is more closely related to energy homeostasis than cytokine profiles in plasma.

Growth hormone regulates leptin gene expression in bovine adipose tissue: correlation with adipose IGF-1 expression.

Leptin, the product of the ob gene, is secreted from white adipocytes and regulates food intake and whole-body energy metabolism. In rodents and humans, leptin gene expression is under complex endocrine and metabolic control, and is strongly influenced by energy balance. Growth hormone (GH) has myriad effects on adipose tissue metabolism. The primary aim of this study was to determine the ability of GH to regulate leptin mRNA expression in bovine adipose tissue in vitro and in vivo. Incubation of subcutaneous adipose tissue explants for 24 h with GH alone had no effect on bovine leptin gene expression, whereas high concentrations of insulin or dexamethasone (DEX) potently stimulated bovine leptin mRNA abundance. GH, in combination with high concentrations of insulin, DEX, or both, attenuated the ability of insulin or DEX to stimulate leptin expression in vitro. These data indicate that GH can indirectly regulate leptin expression in vitro by altering the adipose tissue response to insulin or DEX. We extended these studies to examine the ability of GH to regulate leptin expression in vivo, using young castrate male cattle treated with no hormone (control) or GH (200 micrograms/kg body weight per day) for 3 days. GH increased plasma GH and insulin concentrations, but not those of cortisol or non-esterified fatty acid (NEFA) concentrations. GH treatment increased adipose tissue leptin and IGF-1 mRNA concentrations (n=9, P>0.001). In addition, leptin abundance was highly correlated with adipose tissue IGF-1 mRNA in GH-treated animals (P>0.001). The timing of GH-induced changes in leptin gene expression preceded measurable GH effects on adiposity.

Peroxisome proliferator-activated receptor gamma1 expression in porcine white blood cells: dynamic regulation with acute endotoxemia.

Peroxisome proliferator-activated receptor gamma (PPARgamma), a primary regulator of adipocyte differentiation, has been implicated in the regulation of monocyte and macrophage function in vitro. We report that PPARgamma protein is expressed in porcine peripheral white blood cells (WBC), and that PPARgamma1 but not gamma2 mRNA predominates. Additionally, we provide the first evidence that in vivo lipopolysaccharide challenge (LPS, 25 microg/kg BW) causes a dynamic increase in PPARgamma protein expression in peripheral WBC (P < 0.05). PPARgamma expression was increased 2-fold over basal (1 hr post-LPS), was maximal by 4 hr (3-fold), and was normalized to control by 8 hr post-LPS. Changes in PPARgamma expression coincided with or closely followed LPS-induced changes in plasma cortisol, TNF-alpha, insulin, IGF-1, glucose, and free fatty acids. These data suggest that induction of PPARgamma expression in WBC may play a role in host response to acute inflammatory challenge and may prove to be an important target of anti-inflammatory therapies.

Leptin and its receptors: regulators of whole-body energy homeostasis.

Leptin is the adipocyte-specific product of the ob gene. Expression of leptin in fully fed animals reflects adipocyte size and body-fat mass. Leptin signals the status of body energy stores to the brain, where signals emanate to regulate food intake and whole-body energy expenditure. The leptin gene was identified in the leptin-deficient, obese ob/ob mouse by positional cloning techniques. Recently, leptin has been cloned in domestic species including pigs, cattle, and chickens. The leptin receptor has at least five splice variants; the long form of the receptor is primarily expressed in the hypothalamus and is thought to be the predominant signaling isoform. Leptin receptors are members of the cytokine family of receptors and signal via janus-activated kinases (JAK)/signal transducers and activators of transcription (STAT) and mitogen-activated protein kinase (MAPK) pathways. Mutations in the leptin or leptin receptor genes results in morbid obesity, infertility, and insulin resistance in rodents and humans. Leptin regulates food intake and energy expenditure via central and peripheral mechanisms. Leptin receptors are expressed in most tissues, and in vitro evidence suggests that leptin may have direct effects on some tissues such as adipose tissue, the adrenal cortex, and the pancreatic beta-cell. Leptin is thought to influence whole-body glucose homeostasis and insulin action. Studies are underway to determine the role that leptin plays in the biology of domestic animals.

Dietary conjugated linoleic acid normalizes impaired glucose tolerance in the Zucker diabetic fatty fa/fa rat.

Conjugated linoleic acid (CLA) is a naturally occurring fatty acid which has anti-carcinogenic and anti-atherogenic properties. CLA activates PPAR alpha in liver, and shares functional similarities to ligands of PPAR gamma, the thiazolidinediones, which are potent insulin sensitizers. We provide the first evidence that CLA is able to normalize impaired glucose tolerance and improve hyperinsulinemia in the pre-diabetic ZDF rat. Additionally, dietary CLA increased steady state levels of aP2 mRNA in adipose tissue of fatty ZDF rats compared to controls, consistent with activation of PPAR gamma. The insulin sensitizing effects of CLA are due, at least in part, to activation of PPAR gamma since increasing levels of CLA induced a dose-dependent transactivation of PPAR gamma in CV-1 cells cotransfected with PPAR gamma and PPRE X 3-luciferase reporter construct. CLA effects on glucose tolerance and glucose homeostasis indicate that dietary CLA may prove to be an important therapy for the prevention and treatment of NIDDM.

Expression and cDNA cloning of porcine peroxisome proliferator-activated receptor gamma (PPARgamma).

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a member of the PPAR subfamily of nuclear hormone receptors. In rodents and humans, expression of PPARgamma is predominantly found in adipose tissue where it regulates adipocyte differentiation and the expression of multiple adipocyte genes. The primary aim of this work was to clone the porcine PPARgamma cDNA and examine the regulation of gene expression in porcine subcutaneous adipose tissue. The porcine PPARgamma gene encodes a 1.8-kb mRNA transcript and shares 99, 96 and 97% amino acid sequence identity to the human, mouse and cow PPARgamma molecules, respectively. Both isoforms of PPARgamma (gamma1 and gamma2) are highly expressed in porcine adipose tissue. The gamma2 isoform is expressed in low abundance in porcine spleen, whereas the gamma1 isoform is highly expressed in spleen and lung and at a low abundance in several other tissues. Western blot analysis confirmed a high level of PPARgamma protein expression in porcine adipose tissue compared to other tissues. Both caloric restriction and fasting significantly reduced PPARgamma2 but not gamma1 mRNA and PPARgamma protein abundance in subcutaneous adipose tissue compared to ad-libitum fed controls. We provide the first evidence that PPARgamma is abundantly expressed in porcine subcutaneous adipose tissue, and that expression is regulated by caloric intake. Thus, PPARgamma may play an important role in adipogenesis and hormone action in porcine adipocytes.

Leptin is present in human milk and is related to maternal plasma leptin concentration and adiposity.

Leptin is elevated during pregnancy and may be involved in the regulation of milk production in women. Immunoreactive leptin was quantified in human milk by modified radioimmunoassay. Leptin concentration was higher in whole vs. skim milk fractions; however, leptin concentration was not correlated with percentage milk fat. Leptin concentrations in whole and skim milk were correlated with maternal plasma leptin concentrations, maternal body weight, body mass index, and tricep skinfold thickness, but not with plasma insulin concentration. These data provide the first evidence for the presence of leptin in human milk in the range of concentrations found in human plasma and indicate that the concentration of leptin in milk reflects maternal adiposity. Determining the biological role(s) of milk-borne leptin could add to our understanding of neonatal metabolism and the mechanisms underlying the development of body fat and obesity in humans.

Vigilancia de la resistencia a los medicamentos antituberculosos en el Perú, 1995-96 / Surveillance about the antituberculosis drugs resistance in Peru, 1995-96

La tuberculosis es un importante y serio problema de salud en el Perú. La resistencia antibiótica constituye un problema emergente de magnitud no totalmente definida. A fin de determinar la prevalencia de la resistencia del M. tuberculosis a los medicamentos antituberculosos e iniciar un estudio de vigilancia, se efectuó un proyecto multicéntrico en 31 subregiones de salud del país, que incluyó muestras de esputo de pacientes diagnosticados de TB, con baciloscopia positiva precedentes de 814 hospitales y centros de salud. Se completó una muestra de 1958 pacientes nuevos y antes trabajos de quienes se obtuvo muestras de esputo, las cuales se cultivaron en los medios de Lowenstein-Jensen y Ogawa y los aislamientos fueron sometidos a pruebas de sensibilidad a los medicamentos anti TB por el método de las proporciones. Los resultados fueron: resistencia a uno o más medicamentos en el 15,4 de 1500 casos de TB no tratados previamente (NT), y en el 36,0 de 458 casos ya tratados (AT). la multirresistencia (MR) afectó a 2,4 de los pacientes NT y un 15,7 de los AT. En 9 casis (0,4) se reportó positividad para HIV, de los cuales seis no tratados (NT) fueron sensibles a los medicamentos anti TB y los otros 3 ya tratados (AT) un (1) caso fue sensible y 2 resistentes de éstos uno de ellos MR. El estudio permitió el fortalecimiento de la Red Nacional de Laboratorios en TB del país, contribuyendo a la seguridad y a la oportunidad en el diagnóstico y control de la enfermedad

Onchocerciasis and immunity in humans: enhanced T cell responsiveness to parasite antigen in putatively immune individuals.

To identify possible immune mechanisms in human onchocerciasis, we compared a group of 12 individuals who had no clinical or parasitological evidence of infection, despite ongoing exposure to the parasite, with a group of 16 individuals from the same area who had active Onchocerca volvulus infection. Despite having less parasite-specific serum antibody, the infection-free ("putatively immune") individuals showed greater lymphocyte responsiveness, especially interleukin-2 (IL-2) production, to O. volvulus antigen (OVA) than did the infected subjects; lymphocyte responses (including IL-2 production) to mitogens and nonparasite antigen in both study groups were equivalent and normal. Our findings define differences in parasite-specific T cell subpopulations between infected and putatively immune subjects that could be a central element in developing or maintaining protective immunity to O. volvulus infection.

The metabolic and respiratory alterations of heat stroke.

The metabolic and respiratory changes of 21 patients with heat stroke were studied. Admission arterial blood gas levels were measured, and serum bicarbonate, lactate, calcium, phosphorus, and anion gap determinations were performed. Seven patients had a metabolic acidosis (pH 7.20 +/- 0.04, PCO2 32 +/- 2 mm Hg, and bicarbonate 12 +/- 1 mEq/L), seven a combined metabolic acidosis and respiratory alkalosis (pH 7.39 +/- 0.01, PCO2 25 +/- 1 mm Hg, and bicarbonate 15 +/- 1 mEq/L), four a respiratory alkalosis (pH 7.45 +/- 0.01, PCO2 30 +/- 1 mm Hg, and bicarbonate 20 +/- 1 mEq/L), one a metabolic and respiratory acidosis (pH 7.13, PCO2 52 mm Hg, and bicarbonate 17 mEq/L), and one a respiratory acidosis (pH 7.30, PCO2 56 MM Hg, and bicarbonate 27 mEq/L). The 15 patients with a metabolic acidosis had a pH of 7.28 +/- 0.03, PCO2 of 30 +/- 2 mm Hg, bicarbonate level of 14 +/- 1 mEq/L, lactate concentration of 6.5 +/- 1.0 mEq/L, and an anion gap of 26 +/- 4 mEq/L. Nine patients were hypocalcemic (7.8 +/- 0.3 mg/dL), and five patients were hypophosphatemic (2.0 +/- 0.2 mg/dL). The predominant metabolic change in heat stroke is a metabolic acidosis secondary to increased lactate content and/or a respiratory alkalosis. Hypocalcemia is common and hypophosphatemia is not infrequent.