Early Effects on T lymphocyte Response to Iron Deficiency in Mice. Short Communication

Iron deficiency is a common nutritional disorder, affecting about 30% of the world population. Deficits in iron functional compartments have suppressive effects on the immune system. Environmental problems, age, and other nutrient deficiencies are some of the situations which make human studies difficult and warrant the use of animal models. This study aimed to investigate alterations in the immune system by inducing iron deficiency and promoting recuperation in a mouse model. Hemoglobin concentration, hematocrit, liver iron store, and flow cytometry analyses of cell-surface transferrin receptor (CD71) on peripheral blood and spleen CD4+ and CD8+ T lymphocyte were performed in the control (C) and the iron-deficient (ID) groups of animals at the beginning and end of the experiment. Hematological indices of C and ID mice were not different but the iron stores of ID mice were significantly reduced. Although T cell subsets were not altered, the percentage of T cells expressing CD71 was significantly increased by ID. The results suggest that iron deficiency induced by our experimental model would mimic the early events in the onset of anemia, where thymus atrophy is not enough to influence subset composition of T cells, which can still respond to iron deficiency by upregulating the expression of transferrin receptor.

Overshadowing in conditioned taste aversion or in conditioned emotional response after neonatal ventral hippocampal lesions in rats

The neonatal hippocampus lesion thought to model schizophrenia should show the same modifications in behavioural tests as other models, especially pharmacological models. namely decreased latent inhibition, blocking and overshadowing. The present study is set out to evaluate overshadowing in order to complement our previous studies, which had tested latent inhibition. ""Overshadowing"" refers to the decreased conditioning that occurs when the to-be-conditioned stimulus is combined with another stimulus at the conditioning stage. We used the same two Pavlovian conditioning paradigms as in our previous works, namely conditioned taste aversion (CTA) and conditioned emotional response (CER). A sweet taste overshadowed a salty conditioned stimulus, and a tone overshadowed a flashing light. Totally different stimuli were used to counter possible sensory biases. The protocols were validated with two groups of Sprague Dawley rats. The same two protocols were then applied to a cohort of rats whose ventral hippocampus had been destroyed when they were 7 days old. Only rats with extended ventral hippocampus lesions were included. The overall effect of Pavlovian conditioning was attenuated, significantly so in the conditioned emotional response paradigm, but overshadowing appeared not to be modified in either the conditioned emotional response or the conditioned taste aversion paradigm. (C) 2008 Elsevier B.V. All rights reserved.

Immune response to vaccination with DNA-hsp65 in a phase I clinical trial with head and neck cancer patients

DNA-hsp65, a DNA vaccine encoding the 65-kDa heat-shock protein of Mycobacterium leprae (Hsp65) is capable of inducing the reduction of established tumors in mouse models. We conducted a phase I clinical trial of DNA-hsp65 in patients with advanced head and neck carcinoma. In this article, we report on the vaccine`s potential to induce immune responses to Hsp65 and to its human homologue, Hsp60, in these patients. Twenty-one patients with unresectable squamous cell carcinoma of the head and neck received three doses of 150, 400 or 600 mu g naked DNA-hsp65 plasmid by ultrasound-guided intratumoral injection. Vaccination did not increase levels of circulating anti-hsp65 IgG or IgM antibody, or lead to detectable Hsp65-specific cell proliferation or interferon-gamma (IFN-gamma) production by blood mononuclear cells. Frequency of antigen-induced IL-10-producing cells increased after vaccination in 4 of 13 patients analyzed. Five patients showed disease stability or regression following immunization; however, we were unable to detect significant differences between these patients and those with disease progression using these parameters. There was also no increase in antibody or IFN-gamma responses to human Hsp60 in these patients. Our results suggest that although DNA-hsp65 was able to induce some degree of immunostimulation with no evidence of pathological autoimmunity, we were unable to differentiate between patients with different clinical outcomes based on the parameters measured. Future studies should focus on characterizing more reliable correlations between immune response parameters and clinical outcome that may be used as predictors of vaccine success in immunosuppressed individuals. Cancer Gene Therapy (2009) 16, 598-608; doi:10.1038/cgt.2009.9; published online 6 February 2009

Development of an animal model for allergic conjunctivitis: influence of genetic factors and allergen concentration on immune response

Purpose: Animal models of diseases are extremely important in the study of the physiopathogenesis of human diseases and for testing novel therapeutic interventions. The present study aimed to develop an animal model that simulates human allergic conjunctivitis and to study how allergic response may be influenced by the allergen dose used for immunization and by genetic factors. Methods: Sixty C57Bl/6 mice and 60 BALB/c mice were immunized with placebo, or 5 mu g or 500 mu g of allergen derived from Dermatophagoides pteronyssinus. After ocular challenge, the mice were examined in order to clinically verify the occurrence or not of conjunctivitis. Material obtained from animals was used for total and specific IgE and IgG1 dosage, for assays of Der p-specific lymphocyte proliferation and supernatant cytokine dosage, and for histopathological evaluation of conjunctiva. Results: We developed a murine model of allergic conjunctivitis induced by D. pteronyssinus. The model is similar to human disease both clinically and according to laboratory findings. In mouse, conjunctivitis was associated with a Th2 cytokine profile. However, IL-10 appeared to be involved with disease blockade. Mice of different strains have distinct immune responses, depending on the sensitization dose. Conclusions: The murine model developed is suitable for the study of immunopathogenesis and as a template for future therapies. Using BALB/c and C57BL/6 mice, we demonstrated that genetic factors play a role in determining susceptibility and resistance, as well as in establishing the allergen concentration needed to induce or to block disease development.

Pharmacodynamics of PEG-IFN-alpha-2a and HCV Response as a Function of IL28B Polymorphism in HIV/HCV-Coinfected Patients

We examined the association between IL28B single-nucleotide polymorphism rs12979860, hepatitis C virus (HCV) kinetic, and pegylated interferon alpha-2a pharmacodynamic parameters in HIV/HCV-coinfected patients from South America. Twenty-six subjects received pegylated interferon alpha-2a + ribavirin. Serum HCV-RNA and interferon concentrations were measured frequently during the first 12 weeks of therapy and analyzed using mathematical models. African Americans and whites had a similar distribution of IL28B genotypes (P = 0.5). The IL28B CC genotype was overrepresented (P = 0.015) in patients infected with HCV genotype-3 compared with genotype-1. In both genotype-1 and genotype-3, the first-phase viral decline and the average pegylated interferon-alpha-2a effectiveness during the first week of therapy were larger (trend P <= 0.12) in genotype-CC compared with genotypes-TC/TT. In genotype-1 patients, the second slower phase of viral decline (days 2-29) and infected cells loss rate, delta, were larger (P = 0.02 and 0.11, respectively) in genotype-CC than in genotypes-TC/TT. These associations were not observed in genotype-3 patients.

Methylprednisolone improves lung mechanics and reduces the inflammatory response in pulmonary but not in extrapulmonary mild acute lung injury in mice

Objective: Gorticosteroids have been proposed to be effective in modulating the inflammatory response and pulmonary tissue remodeling in acute lung injury (ALI). We hypothesized that steroid treatment might act differently in models of pulmonary (p) or extrapulmonary (exp) ALI with similar mechanical compromise. Design: Prospective, randomized, controlled experimental study. Setting: University research laboratory. Subjects: One hundred twenty-eight BALB/c mice (20-25 g). Interventions: Mice were divided into six groups. In control animals sterile saline solution was intratracheally (0.05 mL, Cp) or intraperitoneally (0.5 mL, Gexp) injected, whereas ALI animals received Escherichia coli lipopolysaccharide intratracheally (10 mu g, ALIp) or intraperitoneally (125 mu g, ALIexp). Six hours after lipopolysaccharide administration, ALIp and ALlexp animals were further randomized into subgroups receiving saline (0.1 mL intravenously) or methylprednisolone (2 mg/kg intravenously, Mp and Mexp, respectively). Measurements and Main Results: At 24 hrs, lung state elastance, resistive and viscoelastic pressures, lung morphometry, and collagen fiber content were similar in both ALI groups. KC, interieukin-6, and transforming growth factor (TGF)-beta levels in bronchoatveolar lavage fluid, as well as tumor necrosis factor (TNF)-alpha, migration inhibitory factor (MIF), interferon (IFN)-gamma, TGF-beta 1 and TGF-beta 2 messenger RNA expression in lung tissue were higher in ALIp than in ALIexp animals. Methylprednisolone attenuated mechanical and morphometric changes, cytokine levels, and TNF-alpha, MIF, IFN gamma, and TGF-beta 2 messenger RNA expression only in ALIp animals, but prevented any changes in collagen fiber content in both ALI groups. Conclusions. Methylprednisolone is effective to inhibit fibrogenesis independent of the etiology of ALI, but its ability to attenuate inflammatory responses and lung mechanical changes varies according to the cause of ALI.

EFFECTS OF A POTENT PEROXYNITRITE DECOMPOSITION CATALYST IN MURINE MODELS OF ENDOTOXEMIA AND SEPSIS

Excessive free-radical production due to various bacterial components released during bacterial infection has been linked to cell death and tissue injury. Peroxynitrite is a highly reactive oxidant produced by the combination of nitric oxide (NO) and superoxide anion, which has been implicated in cell death and tissue injury in various forms of critical illness. Pharmacological decomposition of peroxynitrite may represent a potential therapeutic approach in diseases associated with the overproduction of NO and superoxide. In the present study, we tested the effect of a potent peroxynitrite decomposition catalyst in murine models of endotoxemia and sepsis. Mice were injected i.p. with LPS 40 mg/kg with or without FP15 [Fe(III) tetrakis-2-(N-triethylene glycol monomethyl ether) pyridyl porphyrin] (0.1, 0.3, 1, 3, or 10 mg/kg per hour). Mice were killed 12 h later, followed by the harvesting of samples from the lung, liver, and gut for malondialdehyde and myeloperoxidase measurements. In other subsets of animals, blood samples were obtained by cardiac puncture at 1.5, 4, and 8 h after LPS administration for cytokine (TNF-alpha, IL-1 beta, and IL-10), nitrite/nitrate, alanine aminotransferase, and blood urea nitrogen measurements. Endotoxemic animals showed an increase in survival from 25% to 80% at the FP15 doses of 0.3 and 1 mg/kg per hour. The same dose of FP15 had no effect on plasma levels of nitrite/nitrate. There was a reduction in liver and lung malondialdehyde in the endotoxemic animals pretreated with FP15, as well as in hepatic myeloperoxidase and biochemical markers of liver and kidney damage (alanine aminotransferase and blood urea nitrogen). In a bacterial model of sepsis induced by cecal ligation and puncture, FP15 treatment (0.3 mg/kg per day) significantly protected against mortality. The current data support the view that peroxynitrite is a critical factor mediating liver, gut, and lung injury in endotoxemia and septic shock: its pharmacological neutralization may be of therapeutic benefit.

Intra-dorsal periaqueductal gray administration of cannabidiol blocks panic-like response by activating 5-HT1A receptors

Activation of 5-HT1A receptors in the dorsal periaqueductal gray (dPAG) impairs escape behavior, suggesting a panicolytic-like effect. Cannabidiol (CBD), a major non-psychotomimetic compound present in Cannabis sativa, causes anxiolytic-like effects after intra-dPAG microinjections by activating 5-HT1A receptors. In the present work we tested the hypothesis that CBD could also impair escape responses evoked by two proposed animal models of panic: the elevated T-maze (ETM) and electric stimulation of dPAG. In experiment 1 male Wistar rats with a single cannula implanted in the dPAG received a microinjection of CBD or vehicle and, 10 min later, were submitted to the ETM and open field tests. In experiment 2 escape electrical threshold was measured in rats with chemitrodes implanted in the dPAG before and 10 min after CBD microinjection. In experiment 3 similar to experiment 2 except that the animals received a previous intra-dPAG administration of WAY-100635, a 5-HT1A receptor antagonist, before CBD treatment. In the ETM microinjection of CBD into the dPAG impaired inhibitory avoidance acquisition, an anxiolytic-like effect, and inhibited escape response, a panicolytic-like effect. The drug also increased escape electrical threshold, an effect that was prevented by WAY-100635. Together, the results suggest that CBD causes panicolytic effects in the dPAG by activating 5-HT1A receptors. (C) 2010 Elsevier B.V. All rights reserved.

The suitability of different FEA models for studying root fractures caused by wedge effect

Finite element analysis (FEA) utilizing models with different levels of complexity are found in the literature to study the tendency to vertical root fracture caused by post intrusion (""wedge effect""). The objective of this investigation was to verify if some simplifications used in bi-dimensional FEA models are acceptable regarding the analysis of stresses caused by wedge effect. Three plane strain (PS) and two axisymmtric (Axi) models were studied. One PS model represented the apical third of the root entirely, in dentin (PS-nG). The other models included gutta-percha in the apical third, and differed regarding dentin-post relationship: bonded (PS-B and Axi-B) or nonbonded (PS-nB and Axi-nB). Mesh discretization and material properties were similar for all cases. Maximum principal stress (sigma(max)) was analyzed as a response to a 165 N longitudinal load. Stress magnitude and orientation varied widely (PS-nG: 10.3 MPa; PS-B: 0.8 MPa; PS-nB: 10.4 MPa; Axi-13: 0.2 MPa, Axi-nB: 10.8 MPa). Axi-nB was the only model where all (sigma(max) vectors at the apical third were perpendicular to the model plane. Therefore, it is adequate to demonstrate the tendency to vertical root fractures caused by wedge effect. Axi-13 showed only part of the (sigma(max) perpendicular to the model plane while PS models showed sigma(max) on the model plane. In these models, sigma(max) orientation did not represent a situation where vertical root fracture would occur due to wedge effect. Adhesion between post and dentin significantly reduced (c) 2007 Wiley Periodicals, Inc.

Effect of extrusion on the emulsifying properties of soybean proteins and pectin mixtures modelled by response surface methodology

The objective of this study was to apply response surface methodology to estimate the emulsifying capacity and stability of mixtures containing isolated and textured soybean proteins combined with pectin and to evaluate if the extrusion process affects these interfacial properties. A simplex-centroid design was applied to the model emulsifying activity index (EAI), average droplet size (D-[4.3]) and creaming inhibition (Cl%) of the mixtures. All models were significant and able to explain more than 86% of the variation. The high predictive capacity of the models was also confirmed. The mean values for EAI, D-[4.3] and Cl% observed in all assays were 0.173 +/- 0.015 mn, 19.2 +/- 1.0 mu m and 53.3 +/- 2.6%, respectively. No synergism was observed between the three compounds. This result can be attributed to the low soybean protein solubility at pH 6.2 (<35%). Pectin was the most important variable for improving all responses. The emulsifying capacity of the mixture increased 41% after extrusion. Our results showed that pectin could substitute or improve the emulsifying properties of the soybean proteins and that the extrusion brings additional advantage to interfacial properties of this combination. (C) 2008 Elsevier Ltd. All rights reserved.

Seasonal drought stress in the Amazon: Reconciling models and observations

The Amazon Basin is crucial to global circulatory and carbon patterns due to the large areal extent and large flux magnitude. Biogeophysical models have had difficulty reproducing the annual cycle of net ecosystem exchange (NEE) of carbon in some regions of the Amazon, generally simulating uptake during the wet season and efflux during seasonal drought. In reality, the opposite occurs. Observational and modeling studies have identified several mechanisms that explain the observed annual cycle, including: (1) deep soil columns that can store large water amount, (2) the ability of deep roots to access moisture at depth when near-surface soil dries during annual drought, (3) movement of water in the soil via hydraulic redistribution, allowing for more efficient uptake of water during the wet season, and moistening of near-surface soil during the annual drought, and (4) photosynthetic response to elevated light levels as cloudiness decreases during the dry season. We incorporate these mechanisms into the third version of the Simple Biosphere model (SiB3) both singly and collectively, and confront the results with observations. For the forest to maintain function through seasonal drought, there must be sufficient water storage in the soil to sustain transpiration through the dry season in addition to the ability of the roots to access the stored water. We find that individually, none of these mechanisms by themselves produces a simulation of the annual cycle of NEE that matches the observed. When these mechanisms are combined into the model, NEE follows the general trend of the observations, showing efflux during the wet season and uptake during seasonal drought.

THE ROLE OF DIFFUSIVITY QUENCHING IN FLUX-TRANSPORT DYNAMO MODELS

In the nonlinear phase of a dynamo process, the back-reaction of the magnetic field upon the turbulent motion results in a decrease of the turbulence level and therefore in a suppression of both the magnetic field amplification (the alpha-quenching effect) and the turbulent magnetic diffusivity (the eta-quenching effect). While the former has been widely explored, the effects of eta-quenching in the magnetic field evolution have rarely been considered. In this work, we investigate the role of the suppression of diffusivity in a flux-transport solar dynamo model that also includes a nonlinear alpha-quenching term. Our results indicate that, although for alpha-quenching the dependence of the magnetic field amplification with the quenching factor is nearly linear, the magnetic field response to eta-quenching is nonlinear and spatially nonuniform. We have found that the magnetic field can be locally amplified in this case, forming long-lived structures whose maximum amplitude can be up to similar to 2.5 times larger at the tachocline and up to similar to 2 times larger at the center of the convection zone than in models without quenching. However, this amplification leads to unobservable effects and to a worse distribution of the magnetic field in the butterfly diagram. Since the dynamo cycle period increases when the efficiency of the quenching increases, we have also explored whether the eta-quenching can cause a diffusion-dominated model to drift into an advection-dominated regime. We have found that models undergoing a large suppression in eta produce a strong segregation of magnetic fields that may lead to unsteady dynamo-oscillations. On the other hand, an initially diffusion-dominated model undergoing a small suppression in eta remains in the diffusion-dominated regime.

Using gap-crossing capacity to evaluate functional connectivity of two Atlantic rainforest birds and their response to fragmentation

One of the main consequences of habitat loss and fragmentation is the increase in patch isolation and the consequent decrease in landscape connectivity. In this context, species persistence depends on their responses to this new landscape configuration, particularly on their capacity to move through the interhabitat matrix. Here, we aimed first to determine gap-crossing probabilities related to different gap widths for two forest birds (Thamnophilus caerulescens, Thamnophilidae, and Basileuterus culicivorus, Parulidae) from the Brazilian Atlantic rainforest. These values were defined with a playback technique and then used in analyses based on graph theory to determine functional connections among forest patches. Both species were capable of crossing forest gaps between patches, and these movements were related to gap width. The probability of crossing 40 m gaps was 50% for both species. This probability falls to 10% when the gaps are 60 m (for B. culicivorus) or 80 m (for T caerulescens). Actually, birds responded to stimulation about two times more distant inside forest trials (control) than in gap-crossing trials. Models that included gap-crossing capacity improved the explanatory power of species abundance variation in comparison to strictly structural models based merely on patch area and distance measurements. These results highlighted that even very simple functional connectivity measurements related to gap-crossing capacity can improve the understanding of the effect of habitat fragmentation on bird occurrence and abundance.

Relation of pretreatment sequence diversity in NS5A region of HCV genotype 1 with immune response between pegylated-INF/ribavirin therapy outcomes

Hepatitis C virus (HCV) infection frequently persists despite substantial virus-specific immune responses and the combination of pegylated interferon (INF)-alpha and ribavirin therapy. Major histocompatibility complex class I restricted CD8+ T cells are responsible for the control of viraemia in HCV infection, and several studies suggest protection against viral infection associated with specific HLAs. The reason for low rates of sustained viral response (SVR) in HCV patients remains unknown. Escape mutations in response to cytotoxic T lymphocyte are widely described; however, its influence in the treatment outcome is ill understood. Here, we investigate the differences in CD8 epitopes frequencies from the Los Alamos database between groups of patients that showed distinct response to pegylated alpha-INF with ribavirin therapy and test evidence of natural selection on the virus in those who failed treatment, using five maximum likelihood evolutionary models from PAML package. The group of sustained virological responders showed three epitopes with frequencies higher than Non-responders group, all had statistical support, and we observed evidence of selection pressure in the last group. No escape mutation was observed. Interestingly, the epitope VLSDFKTWL was 100% conserved in SVR group. These results suggest that the response to treatment can be explained by the increase in immune pressure, induced by interferon therapy, and the presence of those epitopes may represent an important factor in determining the outcome of therapy.

Saturated Fatty Acids Produce an Inflammatory Response Predominantly through the Activation of TLR4 Signaling in Hypothalamus: Implications for the Pathogenesis of Obesity

In animal models of diet-induced obesity, the activation of an inflammatory response in the hypothalamus produces molecular and functional resistance to the anorexigenic hormones insulin and leptin. The primary events triggered by dietary fats that ultimately lead to hypothalamic cytokine expression and inflammatory signaling are unknown. Here, we test the hypothesis that dietary fats act through the activation of toll-like receptors 2/4 and endoplasmic reticulum stress to induce cytokine expression in the hypothalamus of rodents. According to our results, long-chain saturated fatty acids activate predominantly toll-like receptor 4 signaling, which determines not only the induction of local cytokine expression but also promotes endoplasmic reticulum stress. Rats fed on a monounsaturated fat-rich diet do not develop hypothalamic leptin resistance, whereas toll-like receptor 4 loss-of-function mutation and immunopharmacological inhibition of toll-like receptor 4 protects mice from diet-induced obesity. Thus, toll-like receptor 4 acts as a predominant molecular target for saturated fatty acids in the hypothalamus, triggering the intracellular signaling network that induces an inflammatory response, and determines the resistance to anorexigenic signals.