Mechanisms of cell accumulation induced by Mycobacterium bovis BCG

Mycobacteria, specially Mycobacterium tuberculosis are among the micro-organisms that are increasing dramatically the number of infections with death, all over the world. A great number of animal experimental models have been proposed to investigate the mechanisms involved in the host response against these intracellular parasites. Studies of airway infection in guinea-pigs and rabbits, as well as, in mice intravenously infected with BCG have made an important contribution to our understanding of the virulence, pathogenesis and the immunology of mycobacterial infections. Although, there are few models to study the mechanisms of the initial inflammatory process induced by the first contact with the Mycobacteria, and the relevance of the acute generation of inflammatory mediators, cytokines and leukocyte infiltration to the development of the mycobacterial infection. In this work we reviewed our results obtained with a model of M. bovis BCG-induced pleurisy in mice, describing the mechanisms involved in the leukocyte influx induced by BCG at 24 hr. Different mechanisms appear to be related with the influx of neutrophils, eosinophils and mononuclear cells and distinct inflammatory mediators, cytokines and adhesion molecules are involved in the BCG-induced cell accumulation.

The problem of the competitiveness of nuclear energy: a biophysical explanation

In this study I try to explain the systemic problem of the low economic competitiveness of nuclear energy for the production of electricity by carrying out a biophysical analysis of its production process. Given the fact that neither econometric approaches nor onedimensional methods of energy analyses are effective, I introduce the concept of biophysical explanation as a quantitative analysis capable of handling the inherent ambiguity associated with the concept of energy. In particular, the quantities of energy, considered as relevant for the assessment, can only be measured and aggregated after having agreed on a pre-analytical definition of a grammar characterizing a given set of finite transformations. Using this grammar it becomes possible to provide a biophysical explanation for the low economic competitiveness of nuclear energy in the production of electricity. When comparing the various unit operations of the process of production of electricity with nuclear energy to the analogous unit operations of the process of production of fossil energy, we see that the various phases of the process are the same. The only difference is related to characteristics of the process associated with the generation of heat which are completely different in the two systems. Since the cost of production of fossil energy provides the base line of economic competitiveness of electricity, the (lack of) economic competitiveness of the production of electricity from nuclear energy can be studied, by comparing the biophysical costs associated with the different unit operations taking place in nuclear and fossil power plants when generating process heat or net electricity. In particular, the analysis focuses on fossil-fuel requirements and labor requirements for those phases that both nuclear plants and fossil energy plants have in common: (i) mining; (ii) refining/enriching; (iii) generating heat/electricity; (iv) handling the pollution/radioactive wastes. By adopting this approach, it becomes possible to explain the systemic low economic competitiveness of nuclear energy in the production of electricity, because of: (i) its dependence on oil, limiting its possible role as a carbon-free alternative; (ii) the choices made in relation to its fuel cycle, especially whether it includes reprocessing operations or not; (iii) the unavoidable uncertainty in the definition of the characteristics of its process; (iv) its large inertia (lack of flexibility) due to issues of time scale; and (v) its low power level.

Genetic variability and identification of the intermediate snail hosts of Schistosoma mansoni

Studies based on shell or reproductive organ morphology and genetic considerations suggest extensive intraspecific variation in Biomphalaria snails. The high variability at the morphological and genetic levels, as well as the small size of some specimens and similarities between species complicate the correct identification of these snails. Here we review our work using methods based on polymerase chain reaction (PCR) amplification for analysis of genetic variation and identification of Biomphalaria snails from Brazil, Argentina, Uruguay and Paraguay. Arbitrarily primed-PCR revealed that the genome of B. glabrata exihibits a remarkable degree of intraespecific polymorphism. Low stringency-PCR using primers for 18S rRNA permited the identification of B. glabrata, B. tenagophila and B. occidentalis. The study of individuals obtained from geographically distinct populations exhibits significant intraspecific DNA polymorphism, however specimens from the same species, exhibit some species specific LSPs. We also showed that PCR-restriction fragment of length polymorphism of the internal transcribed spacer region of Biomphalaria rDNA, using DdeI permits the differentiation of the three intermediate hosts of Schistosoma mansoni. The molecular biological techniques used in our studies are very useful for the generation of new knowledge concerning the systematics and population genetics of Biomphalaria snails.

Photodynamic induced uptake of liposomal doxorubicin to rat lung tumors parallels tumor vascular density.

BACKGROUND: Visudyne®-mediated photodynamic therapy (PDT) at low drug/light conditions has shown to selectively enhance the uptake of liposomal doxorubicin in subpleural localized sarcoma tumors grown on rodent lungs without causing morphological alterations of the lung. The present experiments explore the impact of low-dose PDT on liposomal doxorubicin (Liporubicin™) uptake to different tumor types grown on rodent lungs. MATERIAL AND METHODS: Three groups of Fischer rats underwent subpleural generation of sarcoma, mesothelioma, or adenocarcinoma tumors on the left lung. At least five animals of each group (sarcoma, n = 5; mesothelioma, n = 7; adenocarcinoma, n = 5) underwent intraoperative low-dose (10 J/cm(2) at 35 mW/cm(2) ) PDT with 0.0625 mg/kg Visudyne® of the tumor and the lower lobe. This was followed by intravenous (IV) administration of 400 µg Liporubicin™. After a circulation time of 60 min, the tumor-bearing lung was processed for HPLC analyses. At least five animals per group underwent the same procedure but without PDT (sarcoma, n = 5; mesothelioma, n = 5; adenocarcinoma, n = 6). Five untreated animals per group underwent CD31 immunostaining of their tumors with histomorphometrical assessment of the tumor vascularization. RESULTS: Low-dose PDT significantly enhanced Liporubicin™ uptake to all tumor types (sarcoma, P = 0.0007; mesothelioma, P = 0.001; adenocarcinoma, P = 0.02) but not to normal lung tissue compared to IV drug administration alone. PDT led to a significantly increased ratio of tumor to lung tissue drug uptake for all three tumor types (P < 0.05). However, the tumor drug uptake varied between tumor types and paralleled tumor vascular density. The vascular density was significantly higher in sarcoma than in adenocarcinoma (P < 0.001) and mesothelioma (P < 0.001), whereas there was no significant difference between adenocarcinoma and mesothelioma. CONCLUSION: Low-dose Visudyne®-mediated PDT selectively enhances the uptake of systemically administered liposomal doxorubicin in tumors without affecting the drug uptake to normal lung. However, drug uptake varied significantly between tumor types and paralleled tumor vascular density.

Production of an affinity-purified antibody against an aldehyde-treated neurofilament protein for use in immunocytochemistry.

Fixation enhances cellular morphology and reduces loss of molecules during tissue processing. Antibodies against fixation-resistant epitopes are very useful, because they allow an immunocytochemical detection in tissue of better preserved morphology. However, fixatives can alter antigenicity and adversely affect the result of immunohistochemical procedures. To address this problem, this study examined the feasibility of generating antibodies to a paraformaldehyde-fixed antigen for use in immunohistochemical procedures. The large subunit of neurofilament proteins was selected for this study. Crude neurofilament proteins were isolated and separated by SDS-polyacrylamide gel electrophoresis. The large subunit of neurofilaments (NF-H) was electroeluted from the electrophoresis gel and exposed to paraformaldehyde, and used for immunization of a rabbit. The rabbit antiserum was affinity purified on CNBr-sepharose immobilized neurofilament proteins. On Western blots, the antibody reacted with the NF-H protein in a phosphorylation-dependent manner. In aldehyde-fixed cerebellum, the antibody strongly stained axons. In contrast, in alcohol-fixed cryostat sections the immunocytochemical detection was substantially reduced. The procedure presented in this study, involving a simple pretreatment of the immunogen, allows for the generation of an antibody that may be used in immunohistochemical studies where localization of the immunogen may be reduced or even lost by aldehyde fixation.

Biophysical requirements of power-supply systems: nuclear energy and fossil energy

The report presents a grammar capable of analyzing the process of production of electricity in modular elements for different power-supply systems, defined using semantic and formal categories. In this way it becomes possible to individuate similarities and differences in the process of production of electricity, and then measure and compare “apples” with “apples” and “oranges” with “oranges”. For instance, when comparing the various unit operations of the process of production of electricity with nuclear energy to the analogous unit operations of the process of production of fossil energy, we see that the various phases of the process are the same. The only difference is related to characteristics of the process associated with the generation of heat which are completely different in the two systems. As a matter of facts, the performance of the production of electricity from nuclear energy can be studied, by comparing the biophysical costs associated with the different unit operations taking place in nuclear and fossil power plants when generating process heat or net electricity. By adopting this approach, it becomes possible to compare the performance of the two power-supply systems by comparing their relative biophysical requirements for the phases that both nuclear energy power plants and fossil energy power plants have in common: (i) mining; (ii) refining/enriching; (iii) generating heat/electricity; (iv) handling the pollution/radioactive wastes. This report presents the evaluation of the biophysical requirements for the two powersupply systems: nuclear energy and fossil energy. In particular, the report focuses on the following requirements: (i) electricity; (ii) fossil-fuels, (iii) labor; and (iv) materials.

Life cycle and reproductive parameters of Clerada apicicornis signoret (Hemiptera: Lygaeidae) under laboratory conditions

The life cycle of Clerada apicicornis was determined under laboratory conditions. Mean development times in days were: egg 27.2, nymph I 12.5, nymph II 12, nymph III 13.4, nymph IV 16.4, nymph V 26. The life expectancy of adults ranged from 117 to 317 days (mean 196 days). Based on a cohort of 29 females of C. apicicornis, a horizontal life table was constructed. The following predictive parameters were obtained: net rate of reproduction (Ro = 48.31), intrinsic rate of population increase (r m = 0.153), generation time (Tc = 28.20 weeks), and finite rate of population increment (lambda = 1.16). The reproductive value (Vx) for each age class of the cohort females was calculated. The following observed parameters were calculated after mortality in each stage: net rate of reproduction (R'o=13.4), intrinsic rate of population increase (r c' =0.09 ), and finite rate of population increment (lambda' =1.1). The generation time (Tc' =27.4) was estimated using the methods of Laughlin and Bengstron. A vertical life table was elaborated and mortality was described for one generation of the cohort.

Circadian regulation of renal function.

Urinary excretion of water and all major electrolytes exhibit robust circadian oscillations. The 24-h periodicity has been well documented for several important determinants of urine formation, including renal blood flow, glomerular filtration, tubular reabsorption, and tubular secretion. Disturbance of the renal circadian rhythms is increasingly recognized as a risk factor for hypertension, polyuria, and other diseases and may contribute to renal fibrosis. The origin of these rhythms has been attributed to the reactive response of the kidney to circadian changes in volume and/or in the composition of extracellular fluids that are entrained by rest/activity and feeding/fasting cycles. However, numerous studies have shown that most of the renal excretory rhythms persist for long periods of time, even in the absence of periodic environmental cues. These observations led to the hypothesis of the existence of a self-sustained mechanism, enabling the kidney to anticipate various predictable circadian challenges to homeostasis. The molecular basis of this mechanism remained unknown until the recent discovery of the mammalian circadian clock made of a system of autoregulatory transcriptional/translational feedback loops, which have been found in all tissues studied, including the kidney. Here, we present a review of the growing evidence showing the involvement of the molecular clock in the generation of renal excretory rhythms.

Highly diverse TCRα chain repertoire of pre-immune CD8(+) T cells reveals new insights in gene recombination.

Although the T-cell receptor αδ (TCRαδ) locus harbours large libraries of variable (TRAV) and junctional (TRAJ) gene segments, according to previous studies the TCRα chain repertoire is of limited diversity due to restrictions imposed by sequential coordinate TRAV-TRAJ recombinations. By sequencing tens of millions of TCRα chain transcripts from naive mouse CD8(+) T cells, we observed a hugely diverse repertoire, comprising nearly all possible TRAV-TRAJ combinations. Our findings are not compatible with sequential coordinate gene recombination, but rather with a model in which contraction and DNA looping in the TCRαδ locus provide equal access to TRAV and TRAJ gene segments, similarly to that demonstrated for IgH gene recombination. Generation of the observed highly diverse TCRα chain repertoire necessitates deletion of failed attempts by thymic-positive selection and is essential for the formation of highly diverse TCRαβ repertoires, capable of providing good protective immunity.

Evaluation of the early changes occurring in the draining lymph node upon subcutaneous stimulation

Adjuvants have been shown since many years to have an important role in enhancing the immune responses against the co-administered antigens used as vaccines. The continuous study of the mechanism of action of adjuvants is necessary to develop further safe and efficacious vaccines. Complete Freund's adjuvant (CFA) is currently in use as adjuvant to induce some autoimmune diseases in murine models, therefore the study of the mechanisms involved in the generation of the related immune responses could be instrumental for the understanding of the induction of inflammatory Thl7 responses. In the present work, we showed in C57B1/6 mice that CFA peripheral administration induces very early, at 6 h, a potent influx of CDllb+ cells, mainly neutrophils (CD11b+Ly6GhighLy6Cint) and monocytes (CD11b+Ly6GlowLy6Chigh), in the draining lymph node. By investigating the route by which neutrophils reach the lymph node we observed that, around 20% of them arrive from the afferent lymph and the majority stains positive for Mycobacterium tuberculosis. We also observed a correlation between the influx of neutrophils and an increase in IL-23 and IL-Ιβ, together with several inflammatory chemokines, in the draining lymph node. Concomitantly, we detected the expression of the IL-23 receptor on CDllc+ DCs. Moreover, we confirmed the ability of murine neutrophils to express IL-23 both, in vitro by stimulating bone-marrow extracted PMNs with Mycobacterium tuberculosis, and on total cells from draining lymph node by immunohistochemistry. We also observed by in vivo priming a reduction in the percentage of IFN-γ and CXCR3 expressing Τ cells upon depletion of neutrophils. Altogether, we show that upon stimulation from the periphery, the draining lymph node undergo changes in cytokine/chemokine production leading to the recruitment of different leukocytes subpopulations. Here we show that CFA induces a rapid influx of neutrophils which are responsible for the production of IL-23 that in turn influences the generation of Τ helper cells.

Enriched basaltic andesites from mid-crustal fractional crystallization, recharge, and assimilation (Pilavo Volcano, Western Cordillera of Ecuador)

The origin of andesite is an important issue in petrology because andesite is the main eruptive product at convergent margins, corresponds to the average crustal composition and is often associated with major Cu-Au mineralization. In this study we present petrographic, mineralogical, geochemical and isotopic data for basaltic andesites of the latest Pleistocene Pilavo volcano, one of the most frontal volcanoes of the Ecuadorian Quaternary arc, situated upon thick (30-50 km) mafic crust composed of accreted Cretaceous oceanic plateau rocks and overlying mafic to intermediate Late Cretaceous-Late Tertiary magmatic arcs. The Pilavo rocks are basaltic andesites (54-57 center dot 5 wt % SiO(2)) with a tholeiitic affinity as opposed to the typical calc-alkaline high-silica andesites and dacites (SiO(2) 59-66 wt %) of other frontal arc volcanoes of Ecuador (e.g. Pichincha, Pululahua). They have much higher incompatible element contents (e.g. Sr 650-1350 ppm, Ba 650-1800 ppm, Zr 100-225 ppm, Th 5-25 ppm, La 15-65 ppm) and Th/La ratios (0 center dot 28-0 center dot 36) than Pichincha and Pululahua, and more primitive Sr ((87)Sr/(86)Sr similar to 0 center dot 7038-0 center dot 7039) and Nd (epsilon(Nd) similar to +5 center dot 5 to +6 center dot 1) isotopic signatures. Pilavo andesites have geochemical affinities with modern and recent high-MgO andesites (e.g. low-silica adakites, Setouchi sanukites) and, especially, with Archean sanukitoids, for both of which incompatible element enrichments are believed to result from interactions of slab melts with peridotitic mantle. Petrographic, mineral chemistry, bulk-rock geochemical and isotopic data indicate that the Pilavo magmatic rocks have evolved through three main stages: (1) generation of a basaltic magma in the mantle wedge region by flux melting induced by slab-derived fluids (aqueous, supercritical or melts); (2) high-pressure differentiation of the basaltic melt (at the mantle-crust boundary or at lower crustal levels) through sustained fractionation of olivine and clinopyroxene, leading to hydrous, high-alumina basaltic andesite melts with a tholeiitic affinity, enriched in incompatible elements and strongly impoverished in Ni and Cr; (3) establishment of one or more mid-crustal magma storage reservoirs in which the magmas evolved through dominant amphibole and clinopyroxene (but no plagioclase) fractionation accompanied by assimilation of the modified plutonic roots of the arc and recharge by incoming batches of more primitive magma from depth. The latter process has resulted in strongly increasing incompatible element concentrations in the Pilavo basaltic andesites, coupled with slightly increasing crustal isotopic signatures and a shift towards a more calc-alkaline affinity. Our data show that, although ultimately originating from the slab, incompatible element abundances in arc andesites with primitive isotopic signatures can be significantly enhanced by intra-crustal processes within a thick juvenile mafic crust, thus providing an additional process for the generation of enriched andesites.

Regional-scale integration of multi-scale hydrological and geophysical data using a two-step Bayesian sequential simulation approach

Significant progress has been made with regard to the quantitative integration of geophysical and hydrological data at the local scale for the purpose of improving predictions of groundwater flow and solute transport. However, extending corresponding approaches to the regional scale still represents one of the major challenges in the domain of hydrogeophysics. To address this problem, we have developed a regional-scale data integration methodology based on a two-step Bayesian sequential simulation approach. Our objective is to generate high-resolution stochastic realizations of the regional-scale hydraulic conductivity field in the common case where there exist spatially exhaustive but poorly resolved measurements of a related geophysical parameter, as well as highly resolved but spatially sparse collocated measurements of this geophysical parameter and the hydraulic conductivity. To integrate this multi-scale, multi-parameter database, we first link the low- and high-resolution geophysical data via a stochastic downscaling procedure. This is followed by relating the downscaled geophysical data to the high-resolution hydraulic conductivity distribution. After outlining the general methodology of the approach, we demonstrate its application to a realistic synthetic example where we consider as data high-resolution measurements of the hydraulic and electrical conductivities at a small number of borehole locations, as well as spatially exhaustive, low-resolution estimates of the electrical conductivity obtained from surface-based electrical resistivity tomography. The different stochastic realizations of the hydraulic conductivity field obtained using our procedure are validated by comparing their solute transport behaviour with that of the underlying ?true? hydraulic conductivity field. We find that, even in the presence of strong subsurface heterogeneity, our proposed procedure allows for the generation of faithful representations of the regional-scale hydraulic conductivity structure and reliable predictions of solute transport over long, regional-scale distances.

Evaluation of five screening tests licensed in Argentina for detection of hepatitis C virus antibodies

This study was conducted to compare among the most recent generation of five screening tests licensed in Argentina, in order to evaluate which of the tests has the best sensitivity for detection of antibodies against hepatitis C virus (HCV). The tests analyzed were: Detect-HCV™ (3.0) Biochem ImmunoSystems, Canada; Hepatitis C EIA Wiener Lab., Argentina; Equipar HCV Ab, Italy; Murex HCV 4.0, UK and Serodia-HCV particles agglutination test, Japan. The results obtained showed high discrepancy between the different kits used and show that some of the tests assessed have a low sensitivity for anti-HCV detection in both chronic infections and early seroconversion, and indicate that among the commercially available kits in Argentina, Murex HCV 4.0 (UK) and Serodia-HCV particles agglutination test (Japan) have the best sensitivity for HCV screening. Although the sensitivity of the assays is the first parameter to be considered for blood screening, more studies should be carried out to assess the specificity of such assays.

The fou2 gain-of-function allele and the wild-type allele of Two Pore Channel 1 contribute to different extents or by different mechanisms to defense gene expression in Arabidopsis.

The fatty acid oxygenation up-regulated 2 (fou2) mutant in Arabidopsis thaliana creates a gain-of-function allele in a non-selective cation channel encoded by the Two Pore Channel 1 (TPC1) gene. This mutant genetically implicates cation fluxes in the control of the positive feedback loop whereby jasmonic acid (JA) stimulates its own synthesis. In this study we observed extensive transcriptome reprogramming in healthy fou2 leaves closely resembling that induced by treatment with methyl jasmonate, biotic stresses and the potassium starvation response. Proteomic analysis of fou2 leaves identified increased levels of seven biotic stress- and JA-inducible proteins. In agreement with these analyses, epistasis studies performed by crossing fou2 with aos indicated that elevated levels of JA in fou2 are the major determinant of the mutant phenotype. In addition, generation of fou2 aba1-5, fou2 etr1-1 and fou2 npr1-1 double mutants showed that the fou2 phenotype was only weakly affected by ABA levels and unaffected by mutations in NPR1 and ETR1. The results now suggest possible mechanisms whereby fou2 could induce JA synthesis/signaling early in the wound response. In contrast to fou2, transcriptome analysis of a loss-of-function allele of TPC1, tpc1-2, revealed no differential expression of JA biosynthesis genes in resting leaves. However, the analysis disclosed reduced mRNA levels of the pathogenesis-related genes PDF1.2a and THI2.1 in healthy and diseased tpc1-2 leaves. The results suggest that wild-type TPC1 contributes to their expression by mechanisms somewhat different from those affecting their expression in fou2.

Selective PDE4 inhibitors as potent anti-inflammatory drugs for the treatment of airway diseases

Phosphodiesterases (PDEs) are responsible for the breakdown of intracellular cyclic nucleotides, from which PDE4 are the major cyclic AMP metabolizing isoenzymes found in inflammatory and immune cells. This generated greatest interest on PDE4 as a potential target to treat lung inflammatory diseases. For example, cigarette smoke-induced neutrophilia in BAL was dose and time dependently reduced by cilomilast. Beside the undesired side effects associated with the first generation of PDE4 inhibitors, the second generation of selective inhibitors such as cilomilast and roflumilast showed clinical efficacy in asthma and chronic obstrutive pulmonary diseases trials, thus re-enhancing the interest on these classes of compounds. However, the ability of PDE4 inhibitors to prevent or modulate the airway remodelling remains relatively unexplored. We demonstrated that selective PDE4 inhibitor RP 73-401 reduced matrix metalloproteinase (MMP)-9 activity and TGF-beta1 release during LPS-induced lung injury in mice and that CI-1044 inhibited the production of MMP-1 and MMP-2 from human lung fibroblasts stimulated by pro-inflammatory cytokines. Since inflammatory diseases of the bronchial airways are associated with destruction of normal tissue structure, our data suggest a therapeutic benefit for PDE4 inhibitors in tissue remodelling associated with chronic lung diseases.