Deficient selenium status of a healthy adult Spanish population.

INTRODUCTION Selenium is an essential micronutrient for human health, being a cofactor for enzymes with antioxidant activity that protect the organism from oxidative damage. An inadequate intake of this mineral has been associated with the onset and progression of chronic diseases such as hypertension, diabetes, coronary diseases, asthma, and cancer. For this reason, knowledge of the plasma and erythrocyte selenium levels of a population makes a relevant contribution to assessment of its nutritional status. OBJECTIVE The objective of the present study was to determine the nutritional status of selenium and risk of selenium deficiency in a healthy adult population in Spain by examining food and nutrient intake and analyzing biochemical parameters related to selenium metabolism, including plasma and erythrocyte levels and selenium-dependent glutathione peroxidase (GPx) enzymatic activity. MATERIAL AND METHODS We studied 84 healthy adults (31 males and 53 females) from the province of Granada, determining their plasma and erythrocyte selenium concentrations and the association of these levels with the enzymatic activity of glutathione peroxidase (GPx) and with life style factors. We also gathered data on their food and nutrient intake and the results of biochemical analyses. Correlations were studied among all of these variables. RESULTS The mean plasma selenium concentration was 76.6 ± 17.3 μg/L (87.3 ± 17.4 μg/L in males, 67.3 ± 10.7 μg/L in females), whereas the mean erythrocyte selenium concentration was 104.6 μg/L (107.9 ± 26.1 μg/L in males and 101.7 ± 21.7 μg/L in females). The nutritional status of selenium was defined by the plasma concentration required to reach maximum GPx activity, establishing 90 μg/L as reference value. According to this criterion, 50% of the men and 53% of the women were selenium deficient. CONCLUSIONS Selenium is subjected to multiple regulation mechanisms. Erythrocyte selenium is a good marker of longer term selenium status, while plasma selenium appears to be a marker of short-term nutritional status. The present findings indicate a positive correlation between plasma selenium concentration and the practice of physical activity. Bioavailability studies are required to establish appropriate reference levels of this mineral for the Spanish population.

Discovery of catalases in members of the Chlamydiales order.

Catalase is an important virulence factor for survival in macrophages and other phagocytic cells. In Chlamydiaceae, no catalase had been described so far. With the sequencing and annotation of the full genomes of Chlamydia-related bacteria, the presence of different catalase-encoding genes has been documented. However, their distribution in the Chlamydiales order and the functionality of these catalases remain unknown. Phylogeny of chlamydial catalases was inferred using MrBayes, maximum likelihood, and maximum parsimony algorithms, allowing the description of three clade 3 and two clade 2 catalases. Only monofunctional catalases were found (no catalase-peroxidase or Mn-catalase). All presented a conserved catalytic domain and tertiary structure. Enzymatic activity of cloned chlamydial catalases was assessed by measuring hydrogen peroxide degradation. The catalases are enzymatically active with different efficiencies. The catalase of Parachlamydia acanthamoebae is the least efficient of all (its catalytic activity was 2 logs lower than that of Pseudomonas aeruginosa). Based on the phylogenetic analysis, we hypothesize that an ancestral class 2 catalase probably was present in the common ancestor of all current Chlamydiales but was retained only in Criblamydia sequanensis and Neochlamydia hartmannellae. The catalases of class 3, present in Estrella lausannensis and Parachlamydia acanthamoebae, probably were acquired by lateral gene transfer from Rhizobiales, whereas for Waddlia chondrophila they likely originated from Legionellales or Actinomycetales. The acquisition of catalases on several occasions in the Chlamydiales suggests the importance of this enzyme for the bacteria in their host environment.

Peroxisome proliferator-activated receptor beta/delta exerts a strong protection from ischemic acute renal failure.

Ischemic acute renal failure is characterized by damages to the proximal straight tubule in the outer medulla. Lesions include loss of polarity, shedding into the tubule lumen, and eventually necrotic or apoptotic death of epithelial cells. It was recently shown that peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) increases keratinocyte survival after an inflammatory reaction. Therefore, whether PPARbeta/delta could contribute also to the control of tubular epithelium death after renal ischemia/reperfusion was tested. It was found that PPARbeta/delta+/- and PPARbeta/delta-/- mutant mice exhibited much greater kidney dysfunction and injury than wild-type counterparts after a 30-min renal ischemia followed by a 36-h reperfusion. Conversely, wild-type mice that were given the specific PPARbeta/delta ligand L-165041 before renal ischemia were completely protected against renal dysfunction, as indicated by the lack of rise in serum creatinine and fractional excretion of Na+. This protective effect was accompanied by a significant reduction in medullary necrosis, apoptosis, and inflammation. On the basis of in vitro studies, PPARbeta/delta ligands seem to exert their role by activating the antiapoptotic Akt signaling pathway and, unexpectedly, by increasing the spreading of tubular epithelial cells, thus limiting potentially their shedding and anoikis. These results point to PPARbeta/delta as a remarkable new target for preconditioning strategies.

Fractura bilateral de calcáneo en edad pediátrica. A propósito de un caso

OBJECTIVE: Propose and debate the treatment management for unusual traumatic pathology, both due to the age of the patient and the bilateral location. CLINICAL CASE: Bilateral calcaneal fracture in a ten year old male, after falling three meters to land on his heels. Examination revealed pain and swelling at that level, with no distal neurovascular alterations. After the clinical study, x-rays were taken, as well as a CAT scan which revealed a complex fracture of both calcanei. After 72 hours and verifying that the soft tissue was in optimal condition, the left calcaneus was immobilized with a closed cast while the right was trans!xed with a Steinmann pin following the Essex-Lopresti technique, achieved under scope to attain an acceptable reduction of the fracture and the congruency of all a"ected joints.

Effect of a specific supplement enriched with n-3 polyunsaturated fatty acids on markers of inflammation, oxidative stress and metabolic status of ear, nose and throat cancer patients.

Malnutrition affects 40-50% of patients with ear, nose and throat (ENT) cancer. The aim of this study was to assess changes induced by a specific nutritional supplement enriched with n-3 polyunsaturated fatty acids, fiber and greater amounts of proteins and electrolytes, as compared with a standard nutritional supplement, on markers of inflammation, oxidative stress and metabolic status of ENT cancer patients undergoing radiotherapy (RT). Fourteen days after starting RT, 26 patients were randomly allocated to one of two groups, 13 supplemented with Prosure, an oncologic formula enriched with n-3 polyunsaturated fatty acids, fiber and greater amounts of proteins and electrolytes (specific supplement), and 13 supplemented with Standard-Isosource (standard supplement). Patients were evaluated before RT, and 14, 28 and 90 days after starting RT. The results showed that there were no significant differences between the groups, but greater changes were observed in the standard supplement group, such as a decline in body mass index (BMI), reductions in hematocrit, erythrocyte, eosinophil and albumin levels, and a rise in creatinine and urea levels. We concluded that metabolic, inflammatory and oxidative stress parameters were altered during RT, and began to normalize at the end of the study. Patients supplemented with Prosure showed an earlier normalization of these parameters, with more favorable changes in oxidative stress markers and a more balanced evolution, although the difference was not significant.

Immunotherapy reduces allergen-mediated CD66b expression and myeloperoxidase levels on human neutrophils from allergic patients.

CD66b is a member of the carcinoembryonic antigen family, which mediates the adhesion between neutrophils and to endothelial cells. Allergen-specific immunotherapy is widely used to treat allergic diseases, and the molecular mechanisms underlying this therapy are poorly understood. The present work was undertaken to analyze A) the in vitro effect of allergens and immunotherapy on cell-surface CD66b expression of neutrophils from patients with allergic asthma and rhinitis and B) the in vivo effect of immunotherapy on cell-surface CD66b expression of neutrophils from nasal lavage fluid during the spring season. Myeloperoxidase expression and activity was also analyzed in nasal lavage fluid as a general marker of neutrophil activation. RESULTS CD66b cell-surface expression is upregulated in vitro in response to allergens, and significantly reduced by immunotherapy (p<0.001). Myeloperoxidase activity in nasal lavage fluid was also significantly reduced by immunotherapy, as were the neutrophil cell-surface expression of CD66b and myeloperoxidase (p<0.001). Interestingly, CD66b expression was higher in neutrophils from nasal lavage fluid than those from peripheral blood, and immunotherapy reduced the number of CD66+MPO+ cells in nasal lavage fluid. Thus, immunotherapy positive effects might, at least in part, be mediated by the negative regulation of the CD66b and myeloperoxidase activity in human neutrophils.

Long-term effects of varying consumption of ω3 fatty acids in ear, nose and throat cancer patients: assessment 1 year after radiotherapy.

Abstract A prospective 1-year follow-up study in ear, nose, and throat (ENT) cancer patients was carried out one year after radiotherapy to assess the effect of varying consumption of ω3 fatty acid according to whether they consumed more or less than the 50th percentile of ω3 fatty acids. Clinical, analytical, inflammatory (CRP and IL-6), and oxidative variables (TAC, GPx, GST, and SOD) were evaluated. The study comprised 31 patients (87.1% men), with a mean age of 61.3 ± 9.1 years. Hematological variables showed significant differences in the patients with a lower consumption of ω3 fatty acids. A lower mortality and longer survival were found in the group with ω3 fatty acid consumption ≥50th percentile but the differences were not significant. No significant difference was reached in toxicity, inflammation, and oxidative stress markers. The group with ω3 fatty acid consumption <50th percentile significantly experienced more hematological and immune changes.

Peritalar dislocations: a retrospective study of 18 cases.

The purpose of this study is to retrospectively evaluate 18 consecutive cases of peritalar dislocations referred to our department during a period of 25 years and to delineate the factors influencing long-term prognosis. There were 13 (73%) medial and 5 (27%) lateral dislocations. Six patients (33%) suffered an open injury, including 2 of 13 (15%) medial and 4 of 5 (80%) lateral dislocations. Associated fractures involving the hindfoot or forefoot were noted in 7 feet, including 3 of 5 lateral dislocation cases. Reduction was accomplished under general anesthesia; in no case was open reduction necessary. In 4 of 6 open injuries with associated fractures, temporary fixation with Kirschner wires was performed. Patients were immobilized in a plaster cast for 4 weeks, or for 6 weeks in the presence of fracture, followed by weightbearing as tolerated. At a mean follow-up of 10.2 years (range, 4 to 26 years), 10 patients (56%) showed excellent results; all had sustained a closed medial low-energy dislocation. There were 3 cases (17%) with fair results and 5 cases (28%) with poor results. Forty-five percent of patients showed a restriction of activity, a reduction of subtalar range of motion, and moderate or severe radiographic signs of hindfoot degenerative arthritis. There were no cases of talar avascular necrosis, and in no case was secondary surgery necessary. Lateral dislocation and open medial dislocations with concomitant fractures showed a greater potential for poor prognosis. The results were independent from period of cast immobilization, suggesting that 4 to 6 weeks of immobilization provides acceptable long-term results.

The JNK binding domain of islet-brain 1 inhibits IL-1 induced JNK activity and apoptosis but not the transcription of key proapoptotic or protective genes in insulin-secreting cell lines.

The stress-activated protein kinase c-Jun NH2-terminal kinase (JNK) is a central signal for interleukin-1beta (IL-1beta)-induced apoptosis in insulin-producing beta-cells. The cell-permeable peptide inhibitor of JNK (JNKI1), that introduces the JNK binding domain (JBD) of the scaffold protein islet-brain 1 (IB1) inside cells, effectively prevents beta-cell death caused by this cytokine. To define the molecular targets of JNK involved in cytokine-induced beta-cell apoptosis we investigated whether JNKI1 or stable expression of JBD affected the expression of selected pro- and anti-apoptotic genes induced in rat (RIN-5AH-T2B) and mouse (betaTC3) insulinoma cells exposed to IL-1beta. Inhibition of JNK significantly reduced phosphorylation of the specific JNK substrate c-Jun (p&lt;0.05), IL-1beta-induced apoptosis (p&lt;0.001), and IL-1beta-mediated c-fos gene expression. However, neither JNKI1 nor JBD did influence IL-1beta-induced NO synthesis or iNOS expression or the transcription of the genes encoding mitochondrial manganese superoxide dismutase (MnSOD), catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase rho (GSTrho), heat shock protein (HSP) 70, IL-1beta-converting enzyme (ICE), caspase-3, apoptosis-inducing factor (AIF), Bcl-2 or Bcl-xL. We suggest that the anti-apoptotic effect of JNK inhibition by JBD is independent of the transcription of major pro- and anti-apoptotic genes, but may be exerted at the translational or posttranslational level.

Electrophoretic characteristics of monoclonal immunoglobulin G of different subclasses.

Monoclonal IgG are commonly observed in various B cell disorders, of which multiple myeloma is the most clinically relevant. In a series of serum samples, we identified by immunofixation 73 monoclonal IgG, including 63 IgG(1), 4 IgG(2), 5 IgG(3), and 1 IgG(4). The light chains were of kappa type in 45 cases, and of lambda type in 28 cases. These monoclonal IgG were further characterized by high resolution two-dimensional polyacrylamide gel electrophoresis (2-DE) in various isoelectric focusing conditions, as well as by 3-DE (2-DE of the proteins extracted from agarose after serum protein agarose electrophoresis). After 2-DE, 38 out of 73 monoclonal gamma chains (52%) were visualized using immobilized pH 3-10 gradients for isoelectric focusing. In 6 cases (8%), gamma chains were only detected using alkaline immobilized pH 6-11 gradients. In 3 cases (4%), 3-DE revealed monoclonal gamma chains hidden by polyclonal gamma chains. Finally, in 26 cases (36%), no monoclonal gamma chains were clearly visualized. Sixty-one monoclonal light chains (84%) were detected using immobilized pH 3-10 gradients, whereas 12 (16%) were not. Monoclonal gamma chains and light chains were highly heterogeneous in terms of pI and M(r). However, a statistically significant correlation (P<0.05) was observed between the position of the monoclonal IgG in agarose gel and the pI of their heavy and light chains (R=0.733, multiple linear regression). Because of the extreme diversity of their heavy and light chains, it appears that a classification of monoclonal IgG based only on their electrophoretic properties is not possible.

Cytokine targeting in tumors using a bispecific antibody directed against carcinoembryonic antigen and tumor necrosis factor alpha.

The use of tumor necrosis factor alpha (TNFalpha) in cancer therapy is limited by its short circulatory half-life and its severe systemic side effects. To overcome these limitations, we evaluated the capability of a bispecific antibody (BAb) directed against carcinoembryonic antigen (CEA) and human TNFalpha to target this cytokine in tumors. A BAb was constructed by coupling the Fab' fragments from an anti-CEA monoclonal antibody (MAb) to the Fab' fragments from an anti-TNFalpha MAb via a stable thioether linkage. The double specificity of the BAb for CEA and TNFalpha was demonstrated using a BIAcoreTM two-step analysis. The affinity constants of the BAb for CEA immobilized on a sensor chip and for soluble TNFalpha added to the CEA-BAb complex were as high as those of the parental MAbs (1.7 x 10(9) M-1 and 6.6 x 10(8) M-1, respectively). The radiolabeled 125I-labeled BAb retained high immunoreactivity with both CEA and TNFalpha immobilized on a solid phase. In nude mice xenografted with the human colorectal carcinoma T380, the 125I-labeled BAb showed a tumor localization and biodistribution comparable to that of 131I-labeled anti-CEA parental F(ab')2 with 25-30% of the injected dose (ID)/g tumor at 24 h and 20% ID/g tumor at 48 h. To target TNFalpha to the tumor, a two-step i.v. injection protocol was used first, in which a variable dose of 125I-labeled BAb was injected, followed 24 or 48 h later by a constant dose of 131I-labeled TNFalpha (1 microg). Mice pretreated with 3 microg of BAb and sacrificed 2, 4, 6, or 8 h after the injection of TNFalpha showed a 1.5- to 2-fold increased concentration of 131I-labeled TNFalpha in the tumor as compared to control mice, which received TNFalpha alone. With a higher dose of BAb (25 microg), mice showed a better targeting of TNFalpha with a 3.2-fold increased concentration of 131I-labeled TNFalpha in the tumor: 9.3% versus 2.9% ID/g in control mice 6 h after TNFa injection. In a one-step injection protocol using a premixed BAb-TNFalpha preparation, similar results were obtained 6 h postinjection (3.5-fold increased TNFalpha tumor concentration). A longer retention time of TNFalpha was observed leading to an 8.1-fold increased concentration of TNFalpha in the tumor 14 h postinjection (4.4 versus 0.5% ID/g tumor for BAb-treated and control mice, respectively). These results show that our BAb is able, first, to localize in a human colon carcinoma and, there, to immunoabsorb the i.v.-injected TNFalpha, leading to its increased concentration at the tumor site.

Induction of transendothelial migration in normal and malignant human T lymphocytes.

Activated CD 3+ enriched human peripheral blood T cells exhibited potent capacity for transendothelial migration through HUVEC layers in the absence of T cell ***. In contrast, malignant human T cell lines *** no or negligible ability of transendothelial migration in the absence of chemoattractants. Time lapse studies of transendothelial migration of activated CD 3+ enriched peripheral blood T cells through a HUVEC layer showed that the first T cells were detected in the lower compartment of a tissue culture insert after 1 hour and that migration increased to reach a maximum of 25 x 10(4) T cells/hr after 24 hours. Adhesion assays of human T cell lines demonstrated that all T cell lines were capable of adhesion to HUVEC and that adhesion of T cells to HUVECs was primarily mediated by CD11a/CD18 and ICAM-1 interactions. Furthermore, transendothelial migration of CD 3+ enriched human peripheral blood T cells was inhibited by pretreating the T cells with anti-CD 18 monoclonal antibodies. The inability of malignant T cells to migrate through HUVEC layers in the absence of chemoattractants was not due to poor motility per se, since both normal and malignant T cells migrated well on extracellular matrix components as determined by using Boyden chambers. Crosslinking of alpha 1 beta 2 and alpha 4 beta 1 with immobilized monoclonal antibodies induced motile behaviour in activated CD 3 enriched human peripheral blood T cells but not in malignant T cell lines. In conclusion, the differences in the ability of transendothelial migration between normal and malignant human T cells in the absence of chemoattractants is primarily due to the differences in the capacity of alpha 1 beta 2 and alpha 4 beta 1 to trigger motile behaviour in the separate cell types.

Cytotoxic effects of FGF2-saporin on bovine epithelial lens cells in vitro.

PURPOSE: To test the ability of two preparations of FGF2-saporin, either FGF2 chemically conjugated to saporin (FGF2-SAP) or genetically engineered FGF2-saporin (rFGF2-SAP) to inhibit the growth of bovine epithelial lens (BEL) cells in vitro when in solution and when immobilized on heparin surface-modified (HSM) polymethylmethacrylate (PMMA) intraocular lenses (IOLs). METHOD: Bovine epithelial lens cells were incubated with various concentrations FGF2-saporin for as long as 4 days. The number of surviving cells was determined by counting the number of nuclei. Because FGF2 binds to heparin, FGF2-saporin was incubated with HSM PMMA IOLs; excess toxin was washed off, and the BEL cells were grown on the FGF2-saporin-treated IOLs (HSM and non-HSM) for 4 days. Cell density was determined by image analysis. RESULTS: Both FGF2-SAP and rFGF2-SAP were highly cytotoxic (nM range), with rFGF2-SAP 10 times less active than FGF2-SAP. FGF2-saporin bound to the surface of HSM IOLs and eluted by 2M NaCl retained its activity. Toxin bound to HSM IOLs killed more than 90% of the BEL cells placed on the IOL surface within 4 days. The ability of FGF2-saporin to prevent the growth of cells on the IOL surface was strictly dependent on the presence of heparin on the IOL. CONCLUSIONS: FGF2-saporin is bound to HSM PMMA IOLs and prevents the growth of epithelial cells on the surface of the lens.

Can GAP-43 interact with brain spectrin?

The growth-associated and presynaptic protein GAP-43 is important for axonal growth during brain development, for synaptic plasticity and in axonal regeneration [Benowitz, Routtenberg, TINS 12 (1987) 527]. It has been speculated that such growth may be mediated by cytoskeletal proteins. However, the interaction of GAP-43 with proteins of the presynaptic terminals is poorly characterized. Here, we analyze GAP-43 binding to cytoskeletal proteins by two different biochemical assays, by blot overlay and sedimentation. We find that immobilized brain spectrin (BS) is able to bind GAP-43. In contrast, little binding was observed to microtubule proteins and other elements of the cytoskeleton. Since GAP-43 is located presynaptically, it may bind to the presynaptic form of BS (SpIISigma1). It is attractive to think that such an interaction would participate in the structural plasticity observed in growth cones and adult synapses.

Synthetic RGD-containing alpha-helical coiled coil peptides promote integrin-dependent cell adhesion.

Integrin receptors are the main mediators of cell adhesion to the extracellular matrix. They bind to their ligands by interacting with short amino acid sequences, such as the RGD sequence. Soluble, small RGD-based peptides have been used to block integrin-binding to ligands, thereby interfering with cell adhesion, migration and survival, while substrate-immobilized RGD sequences have been used to enhance cell binding to artificial surfaces. This approach has several important medical applications, e.g. in suppression of tumor angiogenesis or stimulation of bone formation around implants. However, the relatively weak affinity of short RGD-containing peptides often results in incomplete integrin inhibition or ineffective ligation. In this work, we designed and synthesized several new multivalent RGD-containing molecules and tested their ability to inhibit or to promote integrin-dependent cell adhesion when used in solution or immobilized on substrates, respectively. These molecules consist of an oligomeric structure formed by alpha-helical coiled coil peptides fused at their amino-terminal ends with an RGD-containing fragment. When immobilized on a substrate, these peptides specifically promoted integrin alphaVbeta3-dependent cell adhesion, but when used in solution, they blocked alphaVbeta3-dependent cell adhesion to the natural substrates fibronectin and vitronectin. One of the peptides was nearly 10-fold more efficient than fibronectin or vitronectin in promoting cell adhesion, and almost 100-fold more efficient than a linear RGD tripeptide in blocking adhesion. These results indicate that alpha-helical coiled coil peptides carrying an amino-terminal RGD motif can be used as soluble antagonists or surface-immobilized agonists to efficiently inhibit or promote integrin alphaVbeta3-mediated cell adhesion, respectively.