Biomedical subjects

B Alvarez

Publications and source records attributed to B Alvarez.

At least 37 records · Page 2Linked to original sources

PubMed2001

Workshop studies on monoclonal antibodies in the myeloid panel with CD11 specificity.

Several putative anti-human and swine CD11-specific monoclonal antibodies (mAbs) were included in the myeloid section of the Third International Swine CD Workshop. Failure of clustering analysis to group these mAbs together prompted additional analyses to define the specificities of these mAb. Combination of one and two-color flow cytometry (FCM) and immunoprecipitation (IP) allowed the definition of the mAb into three CD11 groups. Cellular distribution of the molecules recognized by anti-human CD11b and c mAbs on swine cells proved to be significantly different from that found in humans.

Animals↗

PubMed2001

Overview of the Third International Workshop on Swine Leukocyte Differentiation Antigens.

The aim of the Third International Workshop on Swine Leukocyte Differentiation Antigens (CD workshop), supported by the Veterinary Immunology Committee (VIC) of the International Union of Immunological Societies (IUIS), was to standardize the assignment of monoclonal antibodies (mAb) reactive with porcine leukocyte differentiation antigens and to define new antibody clusters, using nomenclature in accordance with human and ruminant CD nomenclature, as agreed at the summary meeting of the Second International Swine CD Workshop in Davis, 1995: only mAb with proven reactivity for the orthologous porcine gene product or cross-reactivity for the human gene products, were given the full CD nomenclature, all other allocations were prefixed with "w". As in previous workshops, the overall organization was entrusted to the chair and first author, with support by the chair of the previous workshop and second author. In addition to the existing 26 pig leukocyte CD/SWC determinants established in previous workshops, this workshop established/confirmed another 11 CDs for pig leukocytes, identified by a total of 21 mAb: CD11R1 (2 mAb), CD11R2 (1 mAb), CD11R3 (4 mAb), wCD40 (1 mAb), wCD46 (4 mAb), wCD47 (3 mAb), wCD49d (1 mAb), CD61 (1 mAb), wCD92 (1 mAb), wCD93 (1 mAb) and CD163 (2 mAb).

Animals↗

PubMed2001

Interleukin-15 mediates reciprocal regulation of adipose and muscle mass: a potential role in body weight control.

Interleukin (IL)-15 is a cytokine which is highly expressed in skeletal muscle. Cell culture studies have indicated that IL-15 may have an important role in muscle fiber growth and anabolism. However, data concerning the metabolic effects of this cytokine in vivo are lacking. In the present study, IL-15 was administered to adult rats for 7 days. While IL-15 did not cause changes in either muscle mass or muscle protein content, it induced significant changes in the fractional rates of both muscle protein synthesis and degradation, with no net changes in protein accumulation. Additionally, IL-15 administration resulted in a 33% decrease in white adipose tissue mass and a 20% decrease in circulating triacylglycerols; this was associated with a 47% lower hepatic lipogenic rate and a 36% lower plasma VLDL triacylglycerol content. The decrease in white fat induced by IL-15 was in adipose tissue. No changes were observed in the rate of lipolysis as a result of cytokine administration. These findings indicate that IL-15 has significant effects on both protein and lipid metabolism, and suggest that this cytokine may participate in reciprocal regulation of muscle and adipose tissue mass.

Adipose Tissue↗

PubMed2001

Increased uncoupling protein-2 gene expression in brain of lipopolysaccharide-injected mice: role of tumour necrosis factor-alpha?

In order to understand the role of brain localized uncoupling proteins, we have examined the UCP2 and BMCP-1 gene expression in mice brain in two different catabolic states: administration of lipopolysaccharide (LPS) (2.5 mg/kg, i.p.) and tumour burden. Administration of LPS resulted in an increased UCP2 gene expression both in brain (208%) and cerebellum (77%). An increase in UCP2 gene expression was also observed after LPS treatment in double knockout mice for tumour necrosis factor-alpha (TNF) receptors 1 and 2 (75% in brain and 33% in cerebellum). Tumour growth also resulted in increased brain UCP2 gene expression (80%) in mice bearing the Lewis lung carcinoma as compared with the non-tumour-bearing controls. No changes were observed in BMCP-1 mRNA levels of either LPS-injected or tumour-bearing mice. From the results presented it may be suggested that: (a) the brain may contribute significantly to the increase in energy expenditure associated with hypermetabolic states such as fever and tumour burden, and (b) the regulation of UCP2 gene expression in brain does not seem to be influenced by TNF; therefore the action of other cytokines cannot be discarded.

Animals↗

PubMed2001

Peroxynitrite decay in the presence of hydrogen peroxide, mannitol and ethanol: a reappraisal.

We have reported previously that the apparent rate of peroxynitrite (ONOO(-) ) decay, as followed from its absorbance at 302 nm, decreases in the presence of hydrogen peroxide, mannitol and ethanol (Alvarez et al., 1995, Chem. Res. Toxicol. 8:859-864; Alvarez et al., 1998, Free Radic. Biol. Med. 24:1331-1337). Recently, two papers confirmed the observation and proposed that this slowing effect was due to the formation of absorbing peroxynitrate (O(2) NOO(-) ) as intermediate (Goldstein and Czapski, 1998, J. Am. Chem. Soc. 120:3458-3463; Hodges and Ingold, 1999, J. Am. Chem. Soc. 121:10695-10701). Peroxynitrate would be formed from the reaction of peroxynitrite-derived nitrogen dioxide with superoxide. Superoxide, in turn, would arise from the one-electron oxidation of hydrogen peroxide, or from the reaction of reductive radicals derived from mannitol and ethanol with dioxygen. In agreement with this concept, we show herein that under the conditions of our previous work, the slowing effect is prevented by superoxide dismutase and, in the case of mannitol and ethanol, by reducing the dioxygen concentration of the reaction solutions. Thus, superoxide formation is necessary for the decrease in the rate of absorbance decay. In addition, by simulations using known rate constants and absorption coefficients, we show that the slowing effect can be quantitatively accounted for by the formation of peroxynitrate.

Biophysics↗

PubMed2001

Purification and characterization of a psychrophilic, calcium-induced, growth-phase-dependent metalloprotease from the fish pathogen Flavobacterium psychrophilum.

Flavobacterium psychrophilum is a fish pathogen that commonly affects salmonids. This bacterium produced an extracellular protease with an estimated molecular mass of 55 kDa. This enzyme, designated Fpp1 (F. psychrophilum protease 1), was purified to electrophoretic homogeneity from the culture supernatant by using ammonium sulfate precipitation, ion-exchange chromatography, hydrophobic chromatography, and size exclusion chromatography. On the basis of its biochemical characteristics, Fpp1 can be included in the group of metalloproteases that have an optimum pH for activity of 6.5 and are inhibited by 1,10-phenanthroline, EDTA, or EGTA but not by phenylmethylsulfonyl fluoride. Fpp1 activity was dependent on calcium ions not only for its activity but also for its thermal stability. In addition to calcium, strontium and barium can activate the protein. The enzyme showed typical psychrophilic behavior; it had an activation energy of 5.58 kcal/mol and was more active at temperatures between 25 and 40 degrees C, and its activity decreased rapidly at 45 degrees C. Fpp1 cleaved gelatin, laminin, fibronectin, fibrinogen, collagen type IV, and, to a lesser extent, collagen types I and II. Fpp1 also degraded actin and myosin, basic elements of the fish muscular system. The presence of this enzyme in culture media was specifically dependent on the calcium concentration. Fpp1 production started early in the exponential growth phase and reached a maximum during this period. Addition of calcium during the stationary phase did not induce Fpp1 production at all. Besides calcium and the growth phase, temperature also seems to play a role in production of Fpp1. In this study we found that production of Fpp1 depends on factors such as calcium concentration, growth phase of the culture, and temperature. The combination of these parameters corresponds to the combination in the natural host during outbreaks of disease caused by F. psychrophilum. Consequently, we suggest that environmental host factors govern Fpp1 production.

Animals↗

PubMed2000

Lack of effect of the cytokine suppressive agent FR167653 on tumour growth and cachexia in rats bearing the Yoshida AH-130 ascites hepatoma.

Daily s.c. administration of 6 mg/kg of FR167653 (an inhibitor of the synthesis of interleukin-1 and tumour necrosis factor-alpha) to rats bearing the ascites hepatoma Yoshida AH-130 (a highly cachectic tumour) did not prevent either the anorexia or the massive weight loss - affecting both adipose tissue and skeletal muscle - present in the cachectic animals. The compound did not affect the circulating levels of triacylglycerols or other metabolites such as glucose or lactate. Nor did the administration of FR167653 influence tumour growth. It is concluded that the drug is unable to reverse the cachectic state in this particular experimental tumour model.

Adipose Tissue↗

PubMed2000

Oxidation of ubiquinol by peroxynitrite: implications for protection of mitochondria against nitrosative damage.

A major pathway of nitric oxide utilization in mitochondria is its conversion to peroxynitrite, a species involved in biomolecule damage via oxidation, hydroxylation and nitration reactions. In the present study the potential role of mitochondrial ubiquinol in protecting against peroxynitrite-mediated damage is examined and the requirements of the mitochondrial redox status that support this function of ubiquinol are established. (1) Absorption and EPR spectroscopy studies revealed that the reactions involved in the ubiquinol/peroxynitrite interaction were first-order in peroxynitrite and zero-order in ubiquinol, in agreement with the rate-limiting formation of a reactive intermediate formed during the isomerization of peroxynitrite to nitrate. Ubiquinol oxidation occurred in one-electron transfer steps as indicated by the formation of ubisemiquinone. (2) Peroxynitrite promoted, in a concentration-dependent manner, the formation of superoxide anion by mitochondrial membranes. (3) Ubiquinol protected against peroxynitrite-mediated nitration of tyrosine residues in albumin and mitochondrial membranes, as suggested by experimental models, entailing either addition of ubiquinol or expansion of the mitochondrial ubiquinol pool caused by selective inhibitors of complexes III and IV. (4) Increase in membrane-bound ubiquinol partially prevented the loss of mitochondrial respiratory function induced by peroxynitrite. These findings are analysed in terms of the redox transitions of ubiquinone linked to both nitrogen-centred radical scavenging and oxygen-centred radical production. It may be concluded that the reaction of mitochondrial ubiquinol with peroxynitrite is part of a complex regulatory mechanism with implications for mitochondrial function and integrity.

Albumins↗

PubMed2000

Calpain-3 gene expression is decreased during experimental cancer cachexia.

The Yoshida AH-130 rat ascites hepatoma is a model system for studying the mechanisms involved in the protein hypercatabolism associated with cancer cachexia. The present study was aimed at investigating if the calpain-3 gene expression in skeletal muscle was affected by tumor growth. The results presented clearly show that calpain-3 gene expression is considerably reduced in experimental cancer cachexia, while there is a reciprocal change in the expression of the ubiquitin-dependent proteolytic system and in the ubiquitous m-calpain. The results, observed during cancer cachexia, suggest a potential counterregulatory role of calpain-3 in muscle proteolysis.

Animals↗

PubMed2000

DNA fragmentation occurs in skeletal muscle during tumor growth: A link with cancer cachexia?

In two different experimental models of cancer cachexia, the rat Yoshida AH-130 ascites hepatoma and the mouse Lewis lung carcinoma, the implantation of the tumor caused a loss of body weight which was associated with a reduction in the weight of different skeletal muscles, as well as with their protein content. The decrease in protein content was accompanied by a reduction in DNA content. Interestingly, the protein/DNA ratio was unchanged in the skeletal muscle of the tumor-bearing animals as compared with the non-tumor-bearing controls. Analysis of DNA fragmentation in skeletal muscle clearly showed enhanced laddering in the skeletal muscle of tumor-bearing animals, suggesting an apoptotic phenomenon. Interestingly, the degree of laddering (total DNA fragmented) increased with tumor burden. These results suggest that DNA fragmentation may be a primary event in cancer-associated cachexia.

Animals↗

PubMed2000

Branched-chain amino acids inhibit proteolysis in rat skeletal muscle: mechanisms involved.

Incubation of isolated rat soleus and EDL muscles in the presence of 10 mM leucine resulted in a decreased proteolytic rate as measured by the release of tyrosine into the incubation medium. The effect of this branched-chain amino acid (BCAA) is associated with a decreased activity of the lysosomal proteases and a decreased expression of the genes of the ATP-ubiquitin-dependent proteolysis (ubiquitin and C8). Incubation of muscles in the presence of actinomycin D revealed that the effects of the amino acid can be accounted for by an inhibition of the transcription rate. The presence of leucine did not influence the gene expression of other nonlysosomal (m-calpain) and lysosomal (cathepsin B) proteolytic systems. It is concluded that the well-known effect of BCAA on muscle proteolysis is mediated, in the short term, by the inhibition of lysosomal proteolysis. In a longer period, based on the inhibition of gene transcription observed, an involvement of the ATP-dependent proteolytic system is also likely to occur.

Adenosine Triphosphate↗

PubMed2000

Phenotypic characterization of monocyte subpopulations in the pig.

We have recently described the existence of two subsets of porcine monocytes based on the expression of CD163. In this study we compare the expression of a number of cell surface antigens in CD163+ and CD163- monocyte subsets using three-color flow cytometry. These monocyte subsets show differences with respect to the expression of MHC class II antigens (SLA-DR and DQ) and a variety of adhesion molecules (CD11a, wCD11c, wCD29, CD49d) that are expressed at higher levels on CD163+ monocytes, and of CD14 that is higher expressed by CD 163- cells. These differences on phenotype could reflect differences in the ability of these two subsets to migrate to tissues and may account for the higher allostimulatory capacity of CD163+ cells. In some aspects, the phenotype of CD163+ monocytes resembles that of mature macrophages. In vitro serum-induced maturation of monocytes into macrophages lead to the expression of SWC9 together with an increase in the expression of CD163 and a reduction in that of CD14. These results delineate a maturation pathway where CD14hiCD163-SWC9- monocytes develop into CD14loCD163+SWC9- monocytes and these cells into CD14loCD163+SWC9+ macrophages.

Animals↗

PubMed2000

Short-term effects of leptin on skeletal muscle protein metabolism in the rat.

We have examined the short-term effects of leptin on protein metabolism in the rat. Indeed, an intravenous leptin administration (100 microg/kg body weight), which resulted in no changes in circulating insulin in the time interval studied, induced a decrease in the incorporation of (14)C-leucine to (14)C-skeletal muscle protein. No changes were observed in relation to muscle protein degradation (either measured in vivo following isotope preloading or in vitro as tyrosine released into the incubation medium) and gene expression associated with the different proteolytic systems (cathepsin B, m-calpain and ubiquitin-proteasome system). The effects of leptin on amino acid incorporation into muscle protein do not seem to be direct because incubation of isolated EDL muscles in the presence of 10 microg/ml of leptin did not modify either the protein incorporation or the oxidation of (14)C-leucine. It may, therefore, be suggested that leptin is able to influence protein synthesis in skeletal muscle through the action of an unknown mediator.

Journal Article↗

PubMed2000

A porcine cell surface receptor identified by monoclonal antibodies to SWC3 is a member of the signal regulatory protein family and associates with protein-tyrosine phosphatase SHP-1.

SWC3 was defined at the First International Swine CD Workshop as a specific myelomonocytic antigen of 230 kDa with mAbs 74-22-15, 6F3 and DH59B. In this report, we describe two new mAbs (BL1H7 and BA1C11) that react selectively with granulocytes, monocytes and macrophages. These monoclonal antibodies (mAbs) recognize a molecule in the range of 90-115 kDa in immunoprecipitation and/or Western blotting analyses. Two-colour FACS analyses showed that the distribution of BL1H7 and BA1C11 antigens was identical to that of SWC3. Moreover, in this assay, mAb 74-22-15 appeared to partially block the binding of mAbs BL1H7 and BA1C11, suggesting that all these mAbs reacted with the same or spatially close epitopes. Cross-blocking analyses indicated that it was the case with mAbs 74-22-15 and BL1H7. Immunoprecipitation experiments with mAbs 74-22-15, BL1H7 and BA1C11, followed by immunoblotting with mAb BL1H7 confirmed that all three mAbs recognize the same molecule. Analysis of the N-terminal sequence carried out on the affinity purified protein revealed homology with members of signal regulatory protein (SIRP) family. Like other members of this family, after treatment with sodium pervanadate, SWC3 became phosphorylated in tyrosines, and associated with the protein-tyrosine phosphatase SHP-1.

Animals↗

PubMed2000

Molecular and functional characterization of porcine LFA-1 using monoclonal antibodies to CD11a and CD18.

We describe in this report the production and characterization of monoclonal antibodies (mAb) to the swine homologues of CD11a and CD18 antigens, and their use for phenotypic and functional analysis of porcine leukocytes. Monoclonal antibodies BL1H8 and BL2F1 precipitated two bands of approximately 170 and 95 kDa, whereas mAb BA3H2 brought down three bands of 170, 155 and 95 kDa, from alveolar macrophage lysates. Clearance of macrophage lysates with mAbs BL1H8 and BL2F1 resulted in complete removal of the 170-kDa band. The cell distribution of the molecules recognized by these mAbs was similar to that of human LFA-1. It was found on all leukocytes, although its expression varied among the different leukocyte subpopulations, with monocytes, granulocytes and a subset of CD8+ cells expressing the highest levels. Cross-blocking studies showed that these antibodies recognize different epitopes on porcine LFA-1. Both anti-LFA-1 mAbs strongly inhibited the mitogenic response of PBMC to ConA, whereas the anti-CD18 mAb had no effect. These anti-LFA-1 mAbs also inhibited the mixed lymphocyte reaction (MLR) and the NK cell-mediated lysis of K-562 cells.

Animals↗

PubMed2000

Interleukin-15 antagonizes muscle protein waste in tumour-bearing rats.

Tissue protein hypercatabolism (TPH) is an important feature in cancer cachexia, particularly with regard to the skeletal muscle. The Yoshida AH-130 rat ascites hepatoma is a model system for studying the mechanisms involved in the processes that lead to tissue depletion, since it induces in the host a rapid and progressive muscle wasting, primarily due to TPH. The present study was aimed at investigating if IL-15, which is known to favour muscle fibre hypertrophy, could antagonize the enhanced muscle protein breakdown in this cancer cachexia model. Indeed, IL-15 treatment partly inhibited skeletal muscle wasting in AH-130-bearing rats by decreasing (8-fold) protein degradative rates (as measured by 14C-bicarbonate pre-loading of muscle proteins) to values even lower than those observed in non-tumour-bearing animals. These alterations in protein breakdown rates were associated with an inhibition of the ATP-ubiquitin-dependent proteolytic pathway (35% and 41% for 2.4 and 1.2 kb ubiquitin mRNA, and 57% for the C8 proteasome subunit, respectively). The cytokine did not modify the plasma levels of corticosterone and insulin in the tumour hosts. The present data give new insights into the mechanisms by which IL-15 exerts its preventive effect on muscle protein wasting and seem to warrant the implementation of experimental protocols involving the use of the cytokine in the treatment of pathological states characterized by TPH, particularly in skeletal muscle, such as in the present model of cancer cachexia.

Animals↗

PubMed2000

Exacerbation of endometriosis as a result of premenopausal tamoxifen exposure.

A 41-year-old woman with a history of endometriosis had breast carcinoma diagnosed and treated with tamoxifen. A pelvic mass was subsequently diagnosed and observed on serial ultrasonographic examinations to increase in size. At surgery extensive endometriosis was found. Exacerbation of endometriosis by tamoxifen may become more common as tamoxifen is used more frequently to reduce the risk of breast cancer among premenopausal women.

Adult↗

PubMed2000

Increased phosphoinositide 3-kinase activity induces a lymphoproliferative disorder and contributes to tumor generation in vivo.

Alterations in the cell division:cell death ratio induce multiple autoimmune and transformation processes. Phosphoinositide 3-kinase (PI3K) controls cell division and cell death in vitro, but its effect on the function of the cellular immune system and on tumor formation in mammals is poorly characterized. Here we show that transgenic mice expressing in T lymphocytes an active form of PI3K derived from a thymic lymphoma, p65(PI3K), developed an infiltrating lymphoproliferative disorder and autoimmune renal disease with an increased number of T lymphocytes exhibiting a memory phenotype and reduced apoptosis. This pathology was strikingly similar to that described in mice exhibiting heterozygous loss of the tumor suppressor PTEN, a lipid and protein phosphatase. We show that overexpression of PTEN selectively blocks p65(PI3K)-induced 3T3 fibroblast transformation. Moreover, the early development of T cell lymphomas in p65(PI3K) Tg p53(-/-) mice indicated that PI3K contributes to tumor development. These observations demonstrate that constitutive activation of PI3K extends T cell survival in vivo, affects T cell homeostasis, and contributes to tumor generation, supporting a model in which selective increases in one type of PTEN substrate, the PI3K-derived lipid products, induce tumorigenesis. PI3K thus emerges as a potential target in autoimmune disease and cancer therapy.

Animals↗