Differential effects of formaldehyde exposure on the cell influx and vascular permeability in a rat model of allergic lung inflammation

Exposure to air pollutants such as formaldehyde (FA) leads to inflammation, oxidative stress and immune-modulation in the airways and is associated with airway inflammatory disorders such as asthma. The purpose of our study was to investigate the effects of exposure to FA on the allergic lung inflammation. The hypothesized link between reactive oxygen species and the effects of FA was also studied. To do so, male Wistar rats were exposed to FA inhalation (1%, 90 min daily) for 3 days. and subsequently sensitized with ovalbumin (OVA)-alum by subcutaneous route One week later the rats received another OVA-alum injection by the same route (booster). Two weeks later the rats were challenged with aerosolized OVA. The OVA challenge of rats upon FA exposure induced an elevated release of LTB(4). TXB(2), IL-1 beta, IL-6 and VEGF in lung cells, increased phagocytosis and lung vascular permeability, whereas the cell recruitment into lung was reduced. FA inhalation induced the oxidative burst and the nitration of proteins in the lung Vitamins C, E and apocynin reduced the levels of LTB(4) in BAL-cultured cells of the FA and FA/OVA groups, but Increased the cell influx into the lung of the FA/OVA rats. In OVA-challenged rats, the exposure to FA was associated to a reduced lung endothelial cells expression of intercellular cell adhesion molecule 1 (ICAM-1) In conclusion, our findings suggest that FA down regulate the cellular migration into the lungs after an allergic challenge and increase the ability of resident lung cells likely macrophages to generate inflammatory mediators, explaining the increased lung vascular permeability Our data are indicative that the actions of FA involve mechanisms related to endothelium-leukocyte interactions and oxidative stress, as far as the deleterious effects of this air pollutant on airways are concerned. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

CD28 Exerts Protective and Detrimental Effects in a Pulmonary Model of Paracoccidioidomycosis

T-cell immunity has been claimed as the main immunoprotective mechanism against Paracoccidioides brasiliensis infection, the most important fungal infection in Latin America. As the initial events that control T-cell activation in paracoccidioidomycosis (PCM) are not well established, we decided to investigate the role of CD28, an important costimulatory molecule for the activation of effector and regulatory T cells, in the immunity against this pulmonary pathogen. Using CD28-deficient (CD28(-/-)) and normal wild-type (WT) C57BL/6 mice, we were able to demonstrate that CD28 costimulation determines in pulmonary paracoccidioidomycosis an early immunoprotection but a late deleterious effect associated with impaired immunity and uncontrolled fungal growth. Up to week 10 postinfection, CD28(-/-) mice presented increased pulmonary and hepatic fungal loads allied with diminished production of antibodies and pro-and anti-inflammatory cytokines besides impaired activation and migration of effector and regulatory T (Treg) cells to the lungs. Unexpectedly, CD28-sufficient mice progressively lost the control of fungal growth, resulting in an increased mortality associated with persistent presence of Treg cells, deactivation of inflammatory macrophages and T cells, prevalent presence of anti-inflammatory cytokines, elevated fungal burdens, and extensive hepatic lesions. As a whole, our findings suggest that CD28 is required for the early protective T-cell responses to P. brasiliensis infection, but it also induces the expansion of regulatory circuits that lately impair adaptive immunity, allowing uncontrolled fungal growth and overwhelming infection, which leads to precocious mortality of mice.

Bradykinin B(1) receptor antagonist R954 inhibits eosinophil activation/proliferation/migration and increases TGF-beta and VEGF in a murine model of asthma

In the present study the effects of bradykinin receptor antagonists were investigated in a murine model of asthma using BALB/c mice immunized with ovalbumin/alum and challenged twice with aerosolized ovalbumin. Twenty four hours later eosinophil proliferation in the bone marrow, activation (lipid bodies formation), migration to lung parenchyma and airways and the contents of the pro-angiogenic and pro-fibrotic cytokines TGF-beta and VEGF were determined. The antagonists of the constitutive B(2) (HOE 140) and inducible B(1) (R954) receptors were administered intraperitoneally 30 min before each challenge. In sensitized mice, the antigen challenge induced eosinophil proliferation in the bone marrow, their migration into the lungs and increased the number of lipid bodies in these cells. These events were reduced by treatment of the mice with the B(1) receptor antagonist. The B(2) antagonist increased the number of eosinophils and lipid bodies in the airways without affecting eosinophil counts in the other compartments. After challenge the airway levels of VEGF and TGF-beta significantly increased and the B(1) receptor antagonist caused a further increase. By immunohistochemistry techniques TGF-beta was found to be expressed in the muscular layer of small blood vessels and VEGF in bronchial epithelial cells. The B(1) receptors were expressed in the endothelial cells. These results showed that in a murine model of asthma the B(1) receptor antagonist has an inhibitory effect on eosinophils in selected compartments and increases the production of cytokines involved in tissue repair. It remains to be determined whether this effects of the B(1) antagonist would modify the progression of the allergic inflammation towards resolution or rather towards fibrosis. (C) 2009 Elsevier Ltd. All rights reserved.

TLR2 Is a Negative Regulator of Th17 Cells and Tissue Pathology in a Pulmonary Model of Fungal Infection

To study the role of TLR2 in a experimental model of chronic pulmonary infection, TLR2-deficient and wild-type mice were intratracheally infected with Paracoccidioides brasiliensis, a primary fungal pathogen. Compared with control, TLR2(-/-) mice developed a less severe pulmonary infection and decreased NO synthesis. Equivalent results were detected with in vitro-infected macrophages. Unexpectedly, despite the differences in fungal loads both mouse strains showed equivalent survival times and severe pulmonary inflammatory reactions. Studies on lung-infiltrating leukocytes of TLR2(-/-) mice demonstrated an increased presence of polymorphonuclear neutrophils that control fungal loads but were associated with diminished numbers of activated CD4(+) and CD8(+) T lymphocytes. TLR2 deficiency leads to minor differences in the levels of pulmonary type 1 and type 2 cytokines, but results in increased production of KC, a CXC chemokine involved in neutrophils chemotaxis, as well as TGF-beta, IL-6, IL-23, and IL-17 skewing T cell immunity to a Th17 pattern. In addition, the preferential Th17 immunity of TLR2(-/-) mice was associated with impaired expansion of regulatory CD4(+)CD25(+)FoxP3(+) T cells. This is the first study to show that TLR2 activation controls innate and adaptive immunity to P. brasiliensis infection. TLR2 deficiency results in increased Th17 immunity associated with diminished expansion of regulatory T cells and increased lung pathology due to unrestrained inflammatory reactions. The Journal of Immunology, 2009, 183: 1279-1290.

A new model of outbred genetically selected mice which present a strong acute inflammatory response in the absence of complement component C5

Mice selected for a strong (AIRmax) or weak (AIRmin) acute inflammatory response present different susceptibilities to bacterial infections, autoimmune diseases and carcinogenesis. Variations in these phenotypes have been also detected in AIRmax and AIRmin mice rendered homozygous for Slc11a1 resistant (R) and susceptible (S) alleles. Our aim was to investigate if the phenotypic differences observed in these mice was related to the complement system. AIRmax and AIRmin mice and AIRmax and AIRmin groups homozygous for the resistance (R) or susceptibility (S) alleles of the solute carrier family 11a1 member (Slc11a1) gene, formerly designated Nramp-1. While no difference in complement activity was detected in sera from AIRmax and AIRmin strains, all sera from AIRmax Slc11a1 resistant mice (AIRmax(RR)) presented no complement-dependent hemolytic activity. Furthermore, C5 was not found in their sera by immunodiffusion and, polymerase chain reaction and DNA sequencing of its gene demonstrated that AIRmax(RR) mice are homozygous for the C5 deficient (D) mutation previously described in A/J. Therefore, the C5D allele was fixed in homozygosis in AIRmax(RR) line. The AIRmax(RR) line is a new experimental mouse model in which a strong inflammatory response can be triggered in vivo in the absence of C5.

Correlation of the in vitro antifungal drug susceptibility with the in vivo activity of fluconazole in a murine model of cerebral infection caused by Cryptococcus gattii

Forty Cryptococcus gattii strains were submitted to antifungal susceptibility testing with fluconazole, itraconazole, amphotericin B and terbinafine. The minimum inhibitory concentration (MIC) ranges were 0.5-64.0 for fluconazole, < 0.015-0.25 for itraconazole, 0.015-0.5 for amphotericin B and 0.062-2.0 for terbinafine. A bioassay for the quantitation of fluconazole in murine brain tissue was developed. Swiss mice received daily injections of the antifungal, and their brains were withdrawn at different times over the 14-day study period. The drug concentrations varied from 12.98 to 44.60 mu g/mL. This assay was used to evaluate the therapy with fluconazole in a model of infection caused by C. gattii. Swiss mice were infected intracranially and treated with fluconazole for 7, 10 or 14 days. The treatment reduced the fungal burden, but an increase in fungal growth was observed on day 14. The MIC for fluconazole against sequential isolates was 16 mu g/mL, except for the isolates obtained from animals treated for 14 days (MIC = 64 mu g/mL). The quantitation of cytokines revealed a predominance of IFN-gamma and IL-12 in the non-treated group and elevation of IL-4 and IL-10 in the treated group. Our data revealed the possibility of acquired resistance during the antifungal drug therapy.

Global attractor for a model of extensible beam with nonlinear damping and source terms

This paper is concerned with the existence of a global attractor for the nonlinear beam equation, with nonlinear damping and source terms, u(tt) + Delta(2)u -M (integral(Omega)vertical bar del u vertical bar(2)dx) Delta u + f(u) + g(u(t)) = h in Omega x R(+), where Omega is a bounded domain of R(N), M is a nonnegative real function and h is an element of L(2)(Omega). The nonlinearities f(u) and g(u(t)) are essentially vertical bar u vertical bar(rho) u - vertical bar u vertical bar(sigma) u and vertical bar u(t)vertical bar(r) u(t) respectively, with rho, sigma, r > 0 and sigma < rho. This kind of problem models vibrations of extensible beams and plates. (C) 2010 Elsevier Ltd. All rights reserved.

Fluorescence spectroscopy to diagnose hepatic steatosis in a rat model of fatty liver

Steatosis is diagnosed on the basis of the macroscopic aspect of the liver evaluated by the surgeon at the time of organ extraction or by means of a frozen biopsy. In the present study, the applicability of laser-induced fluorescence (LIF) spectroscopy was investigated as a method for the diagnosis of different degrees of steatosis experimentally induced in rats. Rats received a high-lipid diet for different periods of time. The animals were divided into groups according to the degree of induced steatosis diagnosis by histology. The concentration of fat in the liver was correlated with LIF by means of the steatosis fluorescence factor (SFF). The histology classification, according to liver fat concentration was, Severe Steatosis, Moderate Steatosis, Mild Steatosis and Control (no liver steatosis). Fluorescence intensity could be directly correlated with fat content. It was possible to estimate an average of fluorescence intensity variable by means of different confidence intervals (P=95%) for each steatosis group. SFF was significantly higher in the Severe Steatosis group (P < 0.001) compared with the Moderate Steatosis, Mild Steatosis and Control groups. The various degrees of steatosis could be directly correlated with SFF. LIF spectroscopy proved to be a method capable of identifying the degree of hepatic steatosis in this animal model, and has the potential of clinical application for non-invasive evaluation of the degree of steatosis.

Pharmacophore Model of Cruzain Inhibitors

Chagas disease, caused by the protozoan Trypanosoma cruzi, is one of the most serious amongst the so-called neglected diseases in Latin America, specially in Brazil. So far there has been no effective treatment for the chronic phase of this disease. Cruzain is a major cysteine protease of T cruzi and it is recognized as a valid target for Chagas disease chemotherapy. The mechanism of cruzain action is associated with the nucleophilic attack of an activated sulfur atom towards electrophilic groups. In this report, features of a putative pharmacophore model of the enzyme, developed as a virtual screening tool for the selection of potential cruzain inhibitors, are described. The final proposed model was applied to the ZINC v.7 database and afterwards experimentally validated by an enzymatic inhibition assay. One of the compounds selected by the model showed cruzain inhibition in the low micromolar range.

Increased SOD1 association with chromatin, DNA damage, p53 activation, and apoptosis in a cellular model of SOD1-linked ALS

Mutations in the gene encoding cytosolic Cu,Zn-superoxide dismutase (SOD1) have been linked to familial amyotrophic lateral sclerosis (FALS). However the molecular mechanisms of motor neuron death are multifactorial and remain unclear. Here we examined DNA damage;p53 activity and apoptosis in SH-SY5Y human neuroblastoma cells transfected to achieve low-level expression of either wild-type or mutant Gly(93) --> Ala (G93A) SOD1, typical of FALS. DNA damage was investigated by evaluating the levels of 8-oxo-7,8-dihydro-2`-deoxyguanosine (8-oxodGuo) and DNA strand breaks. Significantly higher levels of DNA damage, increased p53 activity, and a greater percentage of apoptotic cells were observed in SH-SY5Y cells transfected with G93A SOD1 when compared to cells overexpressing wild-type SOD1 and untransfected cells. Western blot, FACS, and confocal microscopy analysis demonstrated that G93A SOD1 is present in the nucleus in association with DNA. Nuclear G93A SOD1 has identical superoxide dismutase activity but displays increased peroxidase activity when compared to wild-type SOD1. These results indicate that the G93A mutant SOD1 association with DNA might induce DNA damage and trigger the apoptotic response by activating p53. This toxic activity of mutant SOD1 in the nucleus may play an important role in the complex mechanisms associated with motor neuron death observed in ALS pathogenesis. (C) 2010 Elsevier B.V. All rights reserved.

A Model of Normative Power

A power describes the ability of an agent to act in some way. While this notion of power is critical in the context of organisational dynamics, and has been studied by others in this light, it must be constrained so as to be useful in any practical application. In particular, we are concerned with how power may be used by agents to govern the imposition and management of norms, and how agents may dynamically assign norms to other agents within a multi-agent system. We approach the problem by defining a syntax and semantics for powers governing the creation, deletion, or modification of norms within a system, which we refer to as normative powers. We then extend this basic model to accommodate more general powers that can modify other powers within the system, and describe how agents playing certain roles are able to apply powers, changing the system’s norms, and also the powers themselves. We examine how the powers found within a system may change as the status of norms change, and show how standard norm modification operations — such as the derogation, annulment and modification of norms— may be represented within our system.