Production of functional active human growth factors in insects used as living biofactories.

Growth factors (GFs) are naturally signalling proteins, which bind to specific receptors on the cell surface. Numerous families of GFs have already been identified and remarkable progresses have been made in understanding the pathways that these proteins use to activate/regulate the complex signalling network involved in cell proliferation or wound healing processes. The bottleneck for a wider clinical and commercial application of these factors relay on their scalable cost-efficient production as bioactive molecules. The present work describes the capacity of Trichoplusia ni insect larvae used as living bioreactors in combination with the baculovirus vector expression system to produce three fully functional human GFs, the human epidermal growth factor (huEGF), the human fibroblast growth factor 2 (huFGF2) and the human keratinocyte growth factor 1 (huKGF1). The expression levels obtained per g of insect biomass were of 9.1, 2.6 and 3mg for huEGF, huFGF2 and huKGF1, respectively. Attempts to increase the productivity of the insect/baculovirus system we have used different modifications to optimize their production. Additionally, recombinant proteins were expressed fused to different tags to facilitate their purification. Interestingly, the expression of huKGF1 was significantly improved when expressed fused to the fragment crystallizable region (Fc) of the human antibody IgG. The insect-derived recombinant GFs were finally characterized in terms of biological activity in keratinocytes and fibroblasts. The present work opens the possibility of a cost-efficient and scalable production of these highly valuable molecules in a system that favours its wide use in therapeutic or cosmetic applications.

Cross-sectional survey of the prevalence of reduced estimated glomerular filtration rate, albuminuria and cardiovascular risk in a native Spanish population.

OBJECTIVES: HERMEX is a population-based study which tries to evaluate the relative weight of cardiovascular risk factors in inhabitants of Extremadura, Spain. This report presents the data about chronic kidney disease (CKD) in a Spanish population sample. METHODS: For an observational cross-sectional population-based study, 3,402 subjects were randomly selected from health care system records. The final sample included 2,813 participants (mean age 51.2 years, 53.5% women). Renal function was estimated from serum creatinine using the 4-variable Modification of Diet in Renal Disease (MDRD-4) Study and Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equations. Individual renal risk was calculated using the Kidney Disease Improving Global Outcomes (KDIGO) table. RESULTS: Using the CKD-EPI formula, 3.6% of participants had a glomerular filtration rate (GFR) <60 ml/min. MDRD-4 gave a result of 4.0%. Prevalence of albuminuria was 5.5%. Taken together, in patients with albuminuria and/or reduced GFR, the prevalence of renal disease was 8.1%. The KDIGO renal risk table suggested that 0.05% of patients were at high or very high risk of CKD progression and 1.6% at medium risk. CKD was more common in those who were obese, hypertensive, dyslipidemic or had diabetes. Multivariate analysis showed an independent negative association of CKD as dependent variable with systolic blood pressure and body mass index, but a positive correlation with diastolic blood pressure and male sex. CONCLUSIONS: A low frequency of abnormal GFR was detected in a randomly selected sample of the Spanish general population. This finding agreed with the low rates of cardiovascular mortality and morbidity observed in Spain in spite of a high prevalence of classic cardiovascular risk factors.

Prevalence of abnormal urinary albumin excretion in a population-based study in Spain: results from the HERMEX Study.

BACKGROUND: HERMEX is a population-based study which try to evaluate the relative weight of cardiovascular risk factors in the population of Extremadura. This report provides the data obtained about microalbuminuria in a large Spanish population. DESIGN AND METHODS: Observational, cross-sectional, population-based study. A total of 3402 subjects were randomly selected from the Health Care System of Extremadura. The final sample included 2813 subjects (mean age 51·2 years, 53·5% female). Urinary albumin excretion rate (UAER) in first morning urine sample was analysed. Microalbuminuria was diagnosed when UAER was ≥ 22 in men or ≥ 31 mg/g in women. RESULTS: Prevalence of abnormal UAER in general population was 5·5% (microalbuminuria: 4·7%; proteinuria 0·8%). Microalbuminuria grew slightly in patients between 65 and 74 years and showed a dramatic increase in subject older than 75 years (P < 0·001). Men showed a high prevalence of microalbuminuria (5·8% vs. women 3·6%; P = 0·006 chi-squared test). Increased UAER was more common in obese subjects (6·7% vs. 2·3%, P < 0·001), hypertensive patients (8·3% vs. 2·3%, P < 0·001) and diabetic ones (10·9% vs. 3·7%, P < 0·001). The multivariate analysis showed a positive correlation of abnormal UAER with body mass index (BMI), systolic blood pressure, plasma creatinine and triglyceride levels. CONCLUSIONS: A low frequency of abnormal UAER was detected in a randomly selected sample of Spanish general population. This finding agreed with the low rates of cardiovascular mortality and morbidity observed in Spain in spite of a high prevalence of classic cardiovascular risk factors.

Prevalencia de síndrome metabólico según las nuevas recomendaciones de la OMS. Estudio HERMEX / Prevalence of metabolic syndrome estimated with the new World Health Organization recommendations. The HERMEX study

Objetivos La unificación de criterios para el diagnóstico del síndrome metabólico, junto con la propuesta de la Organización Mundial de la Salud de eliminar de ellos a los pacientes con diabetes o con enfermedades cardiovasculares, cambiará la estimación de su prevalencia. Nuestro objetivo fue determinar la prevalencia del síndrome metabólico en un área de salud extremeña siguiendo ambas recomendaciones. Métodos Estudio transversal, poblacional, con selección aleatoria de individuos entre 25 y 79 años de edad, en un Área de Salud de Badajoz. Se recogieron los antecedentes de factores de riesgo cardiovascular, la presión arterial y el perímetro abdominal, y una muestra de sangre en ayunas. Se comparó la prevalencia del síndrome metabólico siguiendo los recientes criterios, por sexo y edad. Resultados Se reclutaron 2.833 personas, el 46,5% eran hombres, y la edad media 51,2 años. La prevalencia del síndrome metabólico fue del 33,6%, significativamente mayor en los hombres (36,7% frente a 30,9%; p<0,001) y con una disminución significativa al excluir la diabetes y la enfermedad cardiovascular (20,8%; p<0,001). La diferencia de prevalencia con los distintos criterios fue significativa para el total y por sexo (p<0,001), a partir del decenio de edad de 45-54 años en los hombres y de 55-64 años en las mujeres. Conclusiones La prevalencia de síndrome metabólico en el área estudiada es de las más altas halladas en España en estudios poblacionales. Aunque se reduce con las nuevas recomendaciones internacionales, indica una población considerable y joven en la cual aplicar medidas preventivas (AU)

Objectives The unification of criteria for the diagnosis of metabolic syndrome, together with the subsequent World Health Organization (WHO) proposal to eliminate diabetes and cardiovascular diseases from the diagnostic criteria, will change estimates of the known prevalence of this syndrome. The aim of this study was to determine the prevalence of metabolic syndrome in a health area of Badajoz (Spain) using the latest consensus criteria and eliminating diabetes and cardiovascular disease. Methods We performed a cross-sectional population-wide study of randomly selected individuals aged between 25 and 79 years old in a health area of Badajoz. In all patients, data on their history of cardiovascular risk factors were gathered, waist circumference and blood pressure were measured and a fasting blood sample was collected. The prevalence of metabolic syndrome, following recent criteria, was compared by age and gender. Results We recruited 2,833 individuals (46.5% men). The mean age was 51.2 years The prevalence of metabolic syndrome was 33.6% and was significantly higher in men (36.7% vs 30.9%; p<0.001). The prevalence of metabolic syndrome fell significantly after exclusion of patients with diabetes or cardiovascular disease (20.8%; p<0.001). The difference in prevalence between the distinct criteria was significant for the whole population and by sex (p<0.000). A significant difference in prevalence between genders was observed from the age of 45-54 years in men and 55-64 years in women Conclusions The prevalence of metabolic syndrome in a health area of Badajoz is among the highest reported in population-based studies in Spain. Although estimates of the prevalence are decreased by the new international recommendations, a considerable proportion of the young population requires preventive measures (AU)

[Prevalence of metabolic syndrome estimated with the new World Health Organization recommendations. The HERMEX study]. / Prevalencia de síndrome metabólico según las nuevas recomendaciones de la OMS. Estudio HERMEX.

OBJECTIVES: The unification of criteria for the diagnosis of metabolic syndrome, together with the subsequent World Health Organization (WHO) proposal to eliminate diabetes and cardiovascular diseases from the diagnostic criteria, will change estimates of the known prevalence of this syndrome. The aim of this study was to determine the prevalence of metabolic syndrome in a health area of Badajoz (Spain) using the latest consensus criteria and eliminating diabetes and cardiovascular disease. METHODS: We performed a cross-sectional population-wide study of randomly selected individuals aged between 25 and 79 years old in a health area of Badajoz. In all patients, data on their history of cardiovascular risk factors were gathered, waist circumference and blood pressure were measured and a fasting blood sample was collected. The prevalence of metabolic syndrome, following recent criteria, was compared by age and gender. RESULTS: We recruited 2,833 individuals (46.5% men). The mean age was 51.2 years The prevalence of metabolic syndrome was 33.6% and was significantly higher in men (36.7% vs 30.9%; p < 0.001). The prevalence of metabolic syndrome fell significantly after exclusion of patients with diabetes or cardiovascular disease (20.8%; p < 0.001). The difference in prevalence between the distinct criteria was significant for the whole population and by sex (p < 0.000). A significant difference in prevalence between genders was observed from the age of 45-54 years in men and 55-64 years in women CONCLUSIONS: The prevalence of metabolic syndrome in a health area of Badajoz is among the highest reported in population-based studies in Spain. Although estimates of the prevalence are decreased by the new international recommendations, a considerable proportion of the young population requires preventive measures.

Calcium-dependent nitric oxide production is involved in the cytoprotective properties of n-acetylcysteine in glycochenodeoxycholic acid-induced cell death in hepatocytes.

The intracellular oxidative stress has been involved in bile acid-induced cell death in hepatocytes. Nitric oxide (NO) exerts cytoprotective properties in glycochenodeoxycholic acid (GCDCA)-treated hepatocytes. The study evaluated the involvement of Ca2+ on the regulation of NO synthase (NOS)-3 expression during N-acetylcysteine (NAC) cytoprotection against GCDCA-induced cell death in hepatocytes. The regulation of Ca2+ pools (EGTA or BAPTA-AM) and NO (L-NAME or NO donor) production was assessed during NAC cytoprotection in GCDCA-treated HepG2 cells. The stimulation of Ca2+ entrance was induced by A23187 in HepG2. Cell death, Ca2+ mobilization, NOS-1, -2 and -3 expression, AP-1 activation, and NO production were evaluated. GCDCA reduced intracellular Ca2+ concentration and NOS-3 expression, and enhanced cell death in HepG2. NO donor prevented, and l-NAME enhanced, GCDCA-induced cell death. The reduction of Ca2+ entry by EGTA, but not its release from intracellular stores by BAPTA-AM, enhanced cell death in GCDCA-treated cells. The stimulation of Ca2+ entrance by A23187 reduced cell death and enhanced NOS-3 expression in GCDCA-treated HepG2 cells. The cytoprotective properties of NAC were related to the recovery of intracellular Ca2+ concentration, NOS-3 expression and NO production induced by GCDCA-treated HepG2 cells. The increase of NO production by Ca2+-dependent NOS-3 expression during NAC administration reduces cell death in GCDCA-treated hepatocytes.

Mitochondrial-driven ubiquinone enhances extracellular calcium-dependent nitric oxide production and reduces glycochenodeoxycholic acid-induced cell death in hepatocytes.

Ca(2+) mobilization, nitric oxide (NO), and oxidative stress have been involved in cell death induced by hydrophobic bile acid in hepatocytes. The aim of the study was the elucidation of the effect of the antioxidant mitochondrial-driven ubiquinone (Mito Q) on the intracellular Ca(2+) concentration, NO production, and cell death in glycochenodeoxycholic acid (GCDCA)-treated HepG2 cells. The role of the regulation of the intracellular Ca(2+) concentration by Ca(2+) chelators (EGTA or BAPTA-AM), agonist of Ca(2+) entrance (A23187) or NO (L-NAME or NO donor), was assessed during Mito Q cytoprotection in GCDCA-treated HepG2 cells. Cell death, NO synthase (NOS)-1, -2, and -3 expression, Ca(2+) mobilization, and NO production were evaluated. GCDCA reduced the intracellular Ca(2+) concentration and NOS-3 expression and enhanced cell death in HepG2. NO donor prevented and L-NAME enhanced GCDCA-induced cell death. The reduction of Ca(2+) entry by EGTA, but not its release from intracellular stores by BAPTA-AM, reduced the expression of NOS-3 and enhanced cell death in control and GCDCA-treated cells. Mito Q prevented the reduction of intracellular Ca(2+) concentration, NOS-3 expression, NO production, and cell death in GCDCA-treated HepG2 cells. The conclusion is that the recovery of Ca(2+)-dependent NOS-3 expression by Mito Q may be considered an additional cytoprotective property of an antioxidant.

The reduction of cell death and proliferation by p27(Kip1) minimizes DNA damage in an experimental model of genotoxicity.

Hepatocellular carcinoma (HCC) is the fifth most commonly occurring cancer worldwide. The expression of p27 has been related to reduced severity of tumor grade and recurrence of HCC. The study assessed the role of p27 on the cell proliferation and death, and DNA mutagenesis in experimental genotoxicity induced by aflatoxin B1 (AFB(1)) in cultured hepatocytes obtained from control and p27(Kip1) deficient mice. The overexpression of p27 was assessed with wild type p27(Kip1) expression vector in HepG2 cells. The expression of p27, p21 and p53 was assessed in well and poorly-differentiated liver tumors. DNA damage and cell death induced by AFB(1) were related to a reduction of p27 and p21 expression in cultured hepatocytes. AFB(1)-induced nuclear phosphorylated (Ser 10) p27 degradation was related to a rise of nuclear KIST, Rsk-1 and Rsk-2 expression and cytoplasm phosphorylated (Thr 198) p27 expression. The overexpression of p27 reduced cell proliferation, cell death and DNA damage in AFB(1)-treated hepatocytes. The enhanced survival of patients with well differentiated compared to poorly-differentiated tumors was related to high expression of p27, p21 and p53 in liver sections. The study showed that the p27 reduced cell proliferation and death, as well as the accumulation of DNA damage in hepatocarcinogenesis.

Interleukin-6 is associated with liver lipid homeostasis but not with cell death in experimental hepatic steatosis.

Hepatic steatosis is a risk factor for the progression of non-alcoholic fatty liver disease. The role of pro-inflammatory interleukin (IL)-6 in hepatic steatosis etiology is controversial. We investigated in vivo and in primary hepatocyte cultures whether IL-6 has a modulator role in liver and mitochondria lipid composition and cell death in a choline-deficient (CD) diet rat model of hepatic steatosis. Dietary choline deficiency increased triglycerides and cholesterol, and reduced phosphatidylcholine (PC), phosphatidylethanolamine (PE) and the membrane integrity marker PC:PE ratio in liver. Choline-deficient diet enhanced systemic IL-6, and IL-6 receptor expression and cell death vulnerability in hepatocytes. Derangement of the mitochondrial electron transport chain and of its phospholipid environment was found in CD rat liver mitochondria, which exhibited elevated concentrations of triglycerides, cardiolipin and PC and elevated PC:PE ratio. The cell treatment with IL-6, but not PC, eliminated much of the CD-promoted lipid imbalance in mitochondria but not tumor-necrosis factor (TNF)-alpha-induced cell death. However, PC supplementation prevented the TNF-alpha-induced DNA fragmentation, cytochrome-c release and caspase-3 activity in control and CD hepatocytes. In conclusion, IL-6 ameliorated the mitochondria lipid disturbance in hepatocytes isolated from steatotic animals. Furthermore, PC is identified as a new survival agent that reverses several TNFalpha-inducible responses that are likely to promote steatosis and necrosis.

N-acetylcysteine, coenzyme Q10 and superoxide dismutase mimetic prevent mitochondrial cell dysfunction and cell death induced by d-galactosamine in primary culture of human hepatocytes.

D-Galactosamine (D-GalN) induces reactive oxygen species (ROS) generation and cell death in cultured hepatocytes. The aim of the study was to evaluate the cytoprotective properties of N-acetylcysteine (NAC), coenzyme Q(10) (Q(10)) and the superoxide dismutase (SOD) mimetic against the mitochondrial dysfunction and cell death in D-GalN-treated hepatocytes. Hepatocytes were isolated from liver resections. NAC (0.5 mM), Q(10) (30 microM) or MnTBAP (Mn(III)tetrakis(4-benzoic acid) porphyrin chloride (1mg/mL) were co-administered with D-GalN (40 mM) in hepatocytes. Cell death, oxidative stress, mitochondrial transmembrane potential (MTP), ATP, mitochondrial oxidized/reduced glutathione (GSH) and Q(10) ratios, electronic transport chain (ETC) activity, and nuclear- and mitochondria-encoded expression of complex I subunits were determined in hepatocytes. d-GalN induced a transient increase of mitochondrial hyperpolarization and oxidative stress, followed by an increase of oxidized/reduced GSH and Q(10) ratios, mitochondrial dysfunction and cell death in hepatocytes. The cytoprotective properties of NAC supplementation were related to a reduction of ROS generation and oxidized/reduced GSH and Q(10) ratios, and a recovery of mitochondrial complexes I+III and II+III activities and cellular ATP content. The co-administration of Q(10) or MnTBAP recovered oxidized/reduced GSH ratio, and reduced ROS generation, ETC dysfunction and cell death induced by D-GalN. The cytoprotective properties of studied antioxidants were related to an increase of the protein expression of nuclear- and mitochondrial-encoded subunits of complex I. In conclusion, the co-administration of NAC, Q(10) and MnTBAP enhanced the expression of complex I subunits, and reduced ROS production, oxidized/reduced GSH ratio, mitochondrial dysfunction and cell death induced by D-GalN in cultured hepatocytes.

Alteration of S-nitrosothiol homeostasis and targets for protein S-nitrosation in human hepatocytes.

The liver is one organ clearly influenced by nitric oxide (NO), and acute and chronic exposure to this substance has been associated with distinct patterns of liver disease. Disruption or deregulation of S-nitrosothiol (SNO) signalling leads to impairment of cellular function and disease, and this study was aimed to identify potential targets for protein S-nitrosation during alteration of SNO homeostasis in human hepatocytes. Cells were treated with S-nitroso-L-cysteine (CSNO), an effective physiological nitrosothiol for delivering NO bioactivity to cells. Treatment with CSNO augmented the levels of S-nitrosoproteins detected both by chemiluminescence and the biotin switch method. CSNO treatment also increased S-nitrosoglutathione reductase (GSNOR) activity that returned SNO content to basal levels. This increased enzymatic activity was related to augmented levels of ADH-5 mRNA, the gene encoding for GSNOR in humans. In addition, the treatment with the SNO also increased cell death. Twenty S-nitrosoproteins were identified in CSNO-treated hepatocytes, including mitochondrial aldehyde dehydrogenase, protein disulphide isomerase, Hsp60, GRP75 and Raf kinase inhibitor protein. The identification in the S-nitrosatable proteome of proteins involved in metabolism, maintenance of cellular homeostasis and signalling points to the relevance of protein S-nitrosation to the physiology and pathophysiology of human hepatocytes.

Common sources and estimated intake of plant sterols in the Spanish diet.

Plant sterols (PS) are minor lipid components of plants, which may have potential health benefits, mainly based in their cholesterol-lowering effect. The aim of this study was to determine the composition and content of PS in plant-based foods commonly consumed in Spain and to estimate the PS intake in the Spanish diet. For this purpose, the determination of PS content, using a modern methodology to measure free, esterified, and glycosidic sterol forms, was done. Second, an estimation of the intake of PS, using the Spanish National Food Consumption data, was made. The daily intake per person of PS--campesterol, beta-sitosterol, stigmasterol, and stigmastanol--in the Spanish diet was estimated at 276 mg, the largest component being beta-sitosterol (79.7%). Other unknown compounds, tentatively identified as PS, may constitute a considerable potential intake (99 mg). When the daily PS intake among European diets was compared in terms of campesterol, beta-sitosterol, stigmasterol, and stigmastanol, the PS intake in the Spanish diet was in the same range of other countries such as Finland (15.7% higher) or The Netherlands (equal). However, some qualitative differences in the PS sources were detected, that is, the predominant brown bread and vegetable fat consumption in the northern diets versus the white bread and vegetable oil consumption in the Spanish diet. These differences may help to provide a link between the consumption of PS and healthy effects of the diet.