An imbalance in the T-helper phenotypes displayed by senescent CD4+CD28null T cells is associated with erosive arthritis (rhupus syndrome) in systemic lupus erythematosus.

OBJECTIVE: The objective was to assess the proportion of Th1, Th2 and Th17 phenotypes in senescent CD4+CD28null cells from patients with systemic lupus erythematosus (SLE) and its association with the pattern of joint involvement. METHODS: This cross-sectional study was performed in SLE patients with erosive arthritis (rhupus) or nondeforming, nonerosive arthritis. Total CD4+CD28null cells as well as the proportion of these cells expressing T-bet, GATA3 or RORγt were analyzed by color-flow cytometry. Serum osteopontin levels were measured by ELISA. RESULTS: Eighteen SLE patients (nine with rhupus and nine with nonerosive arthritis) were studied. The percentage of CD4+CD28null/CD4+ cells (17.7%, 10.3-25.0% versus 9.4%, 8.1-22.4%; P = 0.386) as well as the osteopontin levels (5800, 5,134-5995 pg/ml versus 5578, 5171-5717 pg/ml; P > 0.05) were similar in both groups. A higher percentage of CD4+CD28nullT-bet+ cells (42.8%, 33.5-53.4% versus 30.0%, 23.3-34.2%) but a lower percentage of CD4+CD28nullGATA3+ cells (3.1%, 1.7-5.6% versus 6.2%, 2.6-18.4%) was observed in patients with rhupus than in their counterparts ( P = 0.016). The frequency of CD4+CD28nullRORγt+ cells was similar between groups. CONCLUSIONS: In patients with rhupus, senescent CD4+CD28null cells are preferentially polarized to a Th1 phenotype, whereas this is partial towards Th2 in lupus patients with a nonerosive arthritis pattern.

Enhanced survival from CLP-induced sepsis following late administration of low doses of anti-IFNγ F(ab')2 antibody fragments.

OBJECTIVE: To assess the impact of different doses of anti-interferon gamma (anti-IFNγ) F(ab')2 fragments, administered prophylactically, on survival and on serum concentration of cytokines in a murine model of sepsis induced by cecal ligation and puncture (CLP). We further explore the impact of therapeutic administration of the most protective dose on survival. SUBJECTS AND TREATMENT: Balb/c mice were prophylactically treated by the intraperitoneal route with anti-IFNγ initiated 2 h before CLP and every 24 h for a total of five times in each of the following doses: 0.01, 0.1, or 1 mg/kg. Sham and control groups received sterile saline solution in a similar scheme. METHODS: Serum tumor necrosis factor (TNF), interleukin (IL)-1ß, IL-6, IL-10 and IFNγ were measured at 3, 24 and 48 h after CLP by ELISA. Survival curves were compared using a Mantel-Haenzel method. RESULTS: Significant prophylactic protection was found only with 0.01 mg/kg, in association with regulation of IL-1ß and IL-10 concentrations. As therapy, anti-IFNγ fragments were protective only when initiated 24 h after CLP. CONCLUSIONS: Delicate modulation of IFNγ at the correct timing, even when the septic process has begun, is an exciting alternative to explore in the treatment of sepsis.

Norton scale score on admission and mortality of patients hospitalised in Internal Medicine departments. / Puntuación de la escala de Norton al ingreso y mortalidad en pacientes hospitalizados en Medicina Interna.

OBJECTIVE: To determine the association between the Norton scale score (which assesses the risk of pressure ulcers) and mortality in the short, medium and long term in patients hospitalised in Internal Medicine departments. PATIENTS AND METHODS: A prospective, single-centre cohort study was conducted on patients hospitalised in the months of October 2010 and January, May and October 2011. Data was collected on age, sex, Barthel index, Norton scale, presence of pressure ulcers, major diagnostic category, hospital stay and weight of the diagnosis-related group. The patients were divided according to the risk categories of the Norton scale. The follow-up was 3 years. RESULTS: The study included 624 patients with a median age (interquartile range) of 79 (17) years and a median Norton scale score of 16 (7). During hospitalisation, 74 (11.9%) patients died, 176 (28.2%) died at 6 months, 212 (34.0%) died at 1 year, and 296 (47.4%) died at 3 years. Mortality was greater in the higher risk categories of the Norton scale. The Norton score was independently associated with mortality at 6 months (p<.001), at 1 year (p=.005), and at 3 years (p=.002). The areas under the curve of the Norton scale were 0.746 (95% CI 0.686-0.806), 0.735 (95% CI 0.691-0.780) and 0.751 (95% CI 0.713-0.789), respectively (p<.001). CONCLUSIONS: The Norton scale is useful for predicting the prognosis in the short, medium and long term in patients hospitalized in internal medicine departments.

Mannose 6-phosphate-independent endocytosis of beta-glucuronidase by human fibroblasts. I. Evidence for the existence of a membrane-binding activity.

Prior work has shown that endocytosis of bovine beta-glucuronidase by human fibroblasts can be mediated by the existence of a Man6P-independent receptor for the recapture and targeting to lysosomes. In this study, we have isolated a peptide (IIIb2) from pronase digested bovine beta-glucuronidase that behaved as competitive inhibitor of the endocytosis of bovine beta-glucuronidase by human fibroblasts. This peptide contained a Ser-X-Ser sequence, where X is probably a posttranslational modified Trp. Antibodies raised against this peptide impaired the endocytosis of the bovine but not the human beta-glucuronidase, implying that the new recognition marker for the endocytosis of acid hydrolases might reside in a single discrete stretch of amino acid sequence. On the other hand, bovine beta-glucuronidase has been shown to bind specifically to receptors of human fibroblast membranes. The binding was saturable, divalent cation-dependent and was competitively inhibited by the IIIb2 peptide, but not by mannose 6-phosphate. Results presented suggested an interplay between manganese concentrations, temperature and pH on the dissociation of the beta-glucuronidase-receptor complexes. All together, these data reinforce the presence of two endocytic systems for the recapture and targeting of beta-glucuronidase in human fibroblasts.

Mechanical deformation inhibits IL-10 and stimulates IL-12 production by mouse calvarial osteoblasts in vitro.

The skeleton is continuously remodelled throughout life, a process that is orchestrated by cells of the osteoblast lineage. Remodelling involves a complex network of cell-cell signalling involving systemic hormones, locally produced cytokines, growth factors and the mechanical environment of the cells. Here, we report on the effect of mechanically-induced strain on the synthesis by mouse calvarial osteoblasts in monolayer culture of IL-10 and IL-12, two cytokines that inhibit osteoclast formation in bone marrow cultures; IL-10 also suppresses osteoblast differentiation suggesting a role for both cytokines in bone physiology. A tensile strain was applied to the cells intermittently for 6s, every 90s, for 2-96h. After 2-h culture, supernatants from deformed cells contained significantly less IL-10 than control cultures. In contrast, mechanical deformation had a stimulatory effect on IL-12 synthesis; however, by 48h both had returned to control levels. These data suggest that IL-10 and IL-12 can be added to the growing list of mechanical stress-responsive genes. The down-regulation of IL-10 and stimulation of IL-12 further suggests that the initial response of the cells to mechanical deformation was an osteogenic one.

Endothelin mediation of insulin and glucose-induced changes in vascular contractility.

Although the prevalence of hypertension in diabetic patients is high and many factors participate, hyperinsulinemia cannot be discarded as a contributing factor. Insulin could act directly on smooth muscle altering intracellular calcium levels that mediate contraction and glucose transport or could induce the secretion of endothelin by the endothelial cells lining the vessels. The aim of the present report was to study the effect of different glucose and insulin concentrations on rat vascular smooth-muscle contractile characteristics and to determine whether insulin effects are mediated by endothelin. Femoral arteries obtained from Wistar rats were placed in an in vitro chamber and superfused with different glucose and/or insulin solutions. The contractile response to KCl 80 mmol/L, measured by the force generated, showed a significant decrease with high extracellular glucose concentrations (11 mmol/L). Insulin caused a dose-dependent increase in arterial contraction induced by KCl. This increase was significant when arteries were stimulated with 80 mmol/L KCl in the presence of 5.5 mmol/L glucose, but when 40 mmol/L KCl was used, an increase was observed with both 5.5 and 11 mmol/L glucose. The insulin-induced contraction was significantly reduced in the presence of hyperimmune anti-endothelin serum and in the presence of endothelin receptor ET(A) and ET(B) antagonists PD 151,242 and BQ-788, respectively. These results suggest that hyperinsulinemia and hyperglycemia may contribute to hypertension in diabetes and that responses to insulin are mediated partially by endothelin, thus explaining why non-insulin-dependent diabetes mellitus patients show an increase in arterial pressure before the onset of nephropathy.

Effects of mebendazole on protein biosynthesis and secretion in human-derived fibroblast cultures.

Previous results of our group revealed that mebendazole, a broad spectrum anthelmintic drug with antimicrotubular properties, used for the treatment of liver cirrhosis, decreased total collagen content and biosynthesis in liver upon treatment. In the present study, we have evaluated the effects of mebendazole (5-50 micrograms/mL) on protein synthesis, secretion, and deposition in human-derived fibroblast cultures. The results showed a decrease in cell viability (18.5 +/- 0.9%) at 50 micrograms/mL. [3H]Thymidine incorporation diminished gradually with increasing mebendazole concentrations, reaching a plateau (53.67%) between 30 and 50 micrograms/mL. In late logarithmic phase cultures, the drug caused a decrease of [3H]proline incorporation (43.10%) and collagen biosynthesis (58.61%) in the extracellular matrix. This correlated with an increase in radioactivity in total proteins (51.28%) of the intracellular fraction. Similar results were obtained when mebendazole was assayed in post-confluent fibroblast cultures. The electrophoretic patterns of the extracellular matrix showed a decrease of radioactive collagenous components (alpha chains and beta dimers). By contrast, in the intracellular fraction an increase of radioactive collagen precursors (pro alpha chains) was observed. Immunofluorescence studies and immunotransfer analysis, using polyclonal anti-type I collagen antibodies, revealed an accumulation of intracellular collagen which included: collagen pro alpha chains, alpha chains, and low molecular weight peptides. The results obtained suggest that mebendazole interferes with the transcellular mobilization of proteins, resulting in a decrease of secretion and deposition of extracellular matrix proteins, and an accumulation of intracellular collagenous components. The intracellular accumulation of newly synthesized proteins could cause a feedback regulation in fibroblast cultures.

Antibody response of Mexican infants to Haemophilus influenzae type b capsular polyribosylribitol phosphate. Differences between natural and vaccine induced (oligosaccharide-CRM197 conjugated vaccine) immunization.

In this study we compared natural vs. induced Haemophilus influenzae type b (Hib) anti-capsular polyribosylribitol phosphate (PRP) antibody response in a low socioeconomic population. One hundred twenty five 2-month-old children received the complete HbOC vaccine immunization scheme and a booster dose at 15 months of age. One hundred twenty five non-immunized children served as the control group. Serum Hib anti-PRP antibody titers were determined by ELISA in all children. We found at the end of the primary immunization scheme an antibody concentration of 27.28 micrograms/ml in the immunized group vs. 7.48 micrograms/ml in the control group. The antibody response was mainly of the IgG1 class in both groups. After the booster dose the antibody concentration was 30.14 g/ml in the vaccinated group vs. 6.06 micrograms/ml in the control group (p < 0.01). Ninety nine percent of immunized and non-immunized infants had titers greater than 1 microgram/ml. These results confirm that immunization with the HbOC vaccine induces an important increase in anti-PRP specific antibody titer, but they also demonstrate that natural exposure induces responses higher than those referred as protective (1 microgram/ml).

Secretion of IFN-gamma by bovine peripheral blood mononuclear cells stimulated with Mycobacterium bovis protein fractions obtained by isoelectric-focusing.

Due to the complexity and variety of biological effects found in Mycobacterium bovis (M. bovis) proteins analyzed solely on a molecular weight (MW) basis, we approached the purification of M. bovis proteins through their isoelectric point (pI). Twenty M. bovis culture filtrate protein extract (CFPE) isoelectric focused (IEF) protein fractions, confined between pI3 and 10, were isolated. The MW of the major proteins isolated in the various fractions correlated with protein already reported 14-, 18-, 20-, 25-, 31-, 38-, 45-, 64-, 67- and 70 kDa by SDS-PAGE. Since several different pI fractions showed proteins of the same MW we tested the ability of all IEF fractions to stimulate interferon-gamma (IFN-gamma) production by peripheral blood mononuclear cells (PBMC) isolated from cattle with well defined M. bovis tuberculosis (TB) infection. In animals with few lesions IFN-gamma inductive IEF fractions were in the acid range. As the number of lesions increased, neutral fractions were also inductive. Some fractions with relatively few proteins induced as much IFN-gamma production as others with abundant proteins. None of the 20 IEF fractions enhanced IFN-gamma production by anergic cells. We conclude that IFN-gamma production in diseased animals is induced mainly by acidic mycobacterial proteins and that the response towards these proteins is enhanced as the disease progresses, what coincides with higher PPD reactivity. However, the IFN-gamma production in anergic status was severely affected. We found that this cytokine production is spontaneous and antigen-independent.

Purification and characterization of an extracellular lipase from Penicillium candidum.

Penicillium candidum produces and secretes a single extracellular lipase with a monomer molecular weight of 29 kDa. However, this enzyme forms dimers and higher molecular weight aggregates under nondenaturing conditions. The lipase from P. candidum was purified 37-fold using Octyl-Sepharose CL-4B and DEAE-Sephadex columns. The optimal assay conditions for lipase activity were 35 degrees C and pH 9. The lipase was stable in the pH range of 5-6 with a pl of 5.5, but rapid loss of the enzyme activity was observed above 25 degrees C. Tributyrin was found to be the best substrate for the P. candidum lipase, among those tested. Metal ions such as Fe2+ and Cu2+ inhibited enzymatic activity and only Ca2+ was able to slightly enhance lipase activity. Ionic detergents inhibited the activity of the enzyme, whereas nonionic detergents stimulated lipase activity.

Differential effect of estradiol on antibody secretion of murine hybridomas.

The need for increased antibody production by hybridomas has been approached by the addition to cell cultures of different growth factors; in vitro addition of estradiol-17beta (E2) to human blood lymphocytes increases the accumulation of plasma-blasts and Ig-secreting cells. Four different murine-murine hybridomas secreting different monoclonal antibodies (MAbs) were treated with E2. Specific antibody concentration was measured by enzyme-linked immunoadsorbent assay (ELISA) in culture supernatants whereas expression of E2-receptor in the hybridoma cells was determined by polymerase chain reaction (PCR). When E2 was added as a growth supplement to alpha-estrogen receptor positive murine-murine hybridomas it enhanced MAb secretion by as much as 255%, in a dose-dependant manner. This effect lasted for as long as the alpha-estrogen receptor was detected in the hybridoma cells, was inhibited by tamoxifen and was not observed in alpha-estrogen receptor negative hybridomas. The synthetic estrogen analogue diethylstilbestrol had no effect. Estradiol-17beta should be added to the list of hybridoma-inducing growth factors.

[Anthropometric study of the school population of the autonomic community of Murcia]. / Estudio antropométrico de la poblacíon escolar de la Comunidad Autónoma de Murcia.

BACKGROUND: The aim of this study was to know the anthropometric study of school children in Murcia in order to obtain parameters of present reference for our country and region. METHODS: A transversal study of the school population of the community of Murcia at Preschool, EGB, BUP and Technical School levels was carried out including 1,930 children between the ages of 4 and 17. These children were selected from a total of 242,596 proportionally chosen with respect to demography of each of the twelve regions in the area with weight, height and bicipital, tricipital, subscapular and abdominal skin folds being measured. RESULTS: The values obtained and classified by age, sex and regions of the autonomic community of Murcia exposed in tables, demonstrate the differences existing in all the parameters measured with those routinely used at an international and national level. CONCLUSIONS: The differences found clearly justify the need to compare the regional antropometric alterations with the "reference parameters" obtained in Spain and Murcia for which other studies concerning the same would be advisable.

[Genetic analysis of RET mutations in families with multiple endocrine neoplasia type II in the community of Murcia]. / Análisis genético de las mutaciones de RET en las familias con neoplasia endocrina múltiple tipo 2 de la Comunidad de Murcia.

BACKGROUND: Multiple endocrine neoplasia type 2 (MEN 2) syndromes are inherited following an autosomal dominant pattern. RET protooncogen mutations have been associated with MEN 2. The identification of these mutations enables us to diagnose MEN 2. The objectives were to recognize RET mutations and gene carriers in the area of Murcia and to sep up the relationship between genotype and phenotype. PATIENTS AND METHODS: 284 subjects from 14 MEN 2A kindreds and one MEN 2B family from the Community of Murcia, Spain, were studied. 48 out of them had MEN 2 tumours and 236 subjects were at risk. The initial screening test was single-strand conformation polymorphism (SSCP) in 8 MEN 2A families and denaturing gradient gel electrophoresis (DGGE) in 6 MEN 2A families; the results in all the subjects were confirmed with restriction analysis. The MEN 2A family in which the Cfo-I enzyme detected but did not specify the type of mutation received DNA sequence assay. The MEN 2B kindred was studied with restriction analysis. RESULTS: TGC-->TAC and TGC-->CGC mutations of codon 634 were found in 13 and one MEN 2A kindreds, respectively. ATG-->ACG mutation of codon 918 was present in the MEN 2B family. Clinical diagnosis was confirmed in the 48 patients, 44 new gene carriers were detected and 192 carriers of normal alleles were ruled out. The incidence of hyperparathyroidism was highest if RET mutation was TGC-->CGC. CONCLUSIONS: Community of Murcia is one of the areas with the highest prevalence of MEN 2. The risk of hyperparathyroidism is increased if TGC-->CGC is present.

[Effect of obesity on the morphology of the left ventricle]. / Influencia de la obesidad sobre la morfología del ventrículo izquierdo.

BACKGROUND: To analyze the relationship between obesity in its different degrees and the left ventricle morphology. PATIENTS AND METHODS: M-mode echocardiography was used to estimate the mass, wall thickness and internal dimension of left ventricle in 48 obese women with different degrees of obesity, defined according to the body mass index. 25 women with normal weight were used as controls. RESULTS: The body mass index was correlated with left ventricular mass, as well as with both the wall thickness of the left ventricle and its diastolic internal dimension. The abnormalities in the heart morphology increased according to the obesity degree, ranging from a 59% in the lesser obesity group up to a 100% in the more obese women. The incidence of the left ventricular hypertrophy determined by echocardiography also increased along with the body mass index, ranging from a 29% in the lesser degree of obesity women up to an 82% in the patients with a body mass index > 35 kg/m2. CONCLUSIONS: Obesity, even in its lowest degrees, shows frequent alterations in the heart morphology. This is related with a left ventricular mass increase and a higher incidence of the left ventricular hypertrophy. The left ventricular mass increase is due to an increase in the left ventricular walls thickness and also to a dilatation of its cavity.

[Role of tumor necrosis factor alpha in atherosclerosis]. / Participación del factor de necrosis tumoral-alpha en la aterosclerosis.

We describe tumour necrosis factor alpha and its role in the development of the atherosclerotic lesion, and detail the effects of this cytokine upon vascular endothelial cells under normal and high risk conditions. We propose that TNF-alpha performs a central role in the progression of the lesion since, once the endothelial cell feedback regulatory mechanisms are altered, there is an increase in the microenvironment TNF-alpha concentration, which together with some of the already well known risk factors, generates an environment that favours and perpetuates the development of the atheromatous lesion.