In utero exposure to air pollution lowers erythrocyte antioxidant defense and decreases weight in adult mice

In this study, we tested the influence of ambient air pollution on different phases of development of adult mice. With respect to adult weight, the animals that had spent their in utero period exposed to pollution showed less weight gain over their lifetime, as well as lower activity levels of the antioxidant enzymes catalase, superoxide dismutase (SOD) and glutathione peroxidase (GPx). Our study suggests that contact with atmospheric pollutants during the foetal period produces important changes on enzymatic erythrocyte antioxidant defense and weight in adult mice. (C) 2011 Elsevier B.V. All rights reserved.

Immunization of neonatal mice with LAMP/p55 HIV gag DNA elicits robust immune responses that last to adulthood

Successful T cell priming in early postnatal life that can generate effective long-lasting responses until adulthood is critical in HIV vaccination strategies because it prevents early sexual initiation and breastfeeding transmission of HIV. A chimeric DNA vaccine encoding p55 HIV gag associated with lysosome-associated membrane protein 1 (LAMP-1; which drives the antigen to the MIIC compartment), has been used to enhance cellular and humoral antigen-specific responses in adult mice and macaques. Herein, we investigated LAMP-1/gag vaccine immunogenicity in the neonatal period in mice and its ability to generate long-lasting effects. Neonatal vaccination with chimeric LAMP/gag generated stronger Gag-specific immune responses, as measured by the breadth of the Gag peptide-specific IFN-gamma, proliferative responsiveness, cytokine production and antibody production, all of which revealed activation of CD4+ T cells as well as the generation of a more robust CTL response compared to gag vaccine alone. To induce long-lived T and B cell memory responses, it was necessary to immunize neonates with the chimeric IAMP/gag DNA vaccine. The LAMP/gag DNA vaccine strategy could be particularly useful for generating an anti-HIV immune response in the early postnatal period capable of inducing long-term immunological memory. (C) 2010 Elsevier Inc. All rights reserved.

Biological Effects and Dose-Response Assessment of Diesel Exhaust Particles on In Vitro Early Embryo Development in Mice

An increased risk of early pregnancy loss in women briefly exposed to high levels of ambient particulate matter during the preconceptional period was recently observed. The effects of this exposure on early embryo development are unknown. This study was designed to assess the dose-response and biological effects of diesel exhaust particles (DEP) on in vitro embryo development using the in vitro fertilization (IVF) mouse model. Zygotes obtained from superovulated mice after IVF were randomly cultured in different DEP concentrations (0, 0.2, 2, and 20 mu g/cm(2)) for 5 days and observed for their capacity to attach and develop on a fibronectin matrix until day 8. Main outcome measures included blastocyst rates 96 and 120 h after insemination, hatching discriminatory score, total cell count, proportion of cell allocation to inner cell mass (ICM) and trophectoderm (TE), ICM morphology, attachment rate and outgrowth area, apoptosis and necrosis rates, and Oct-4 and Cdx-2 expression. Multivariate analysis showed a negative dose-dependent effect on early embryo development and hatching process, blastocyst cell allocation, and ICM morphology. Although blastocyst attachment and outgrowth were not affected by DEP, a significant impairment of ICM integrity was observed in day 8 blastocysts. Cell death through apoptosis was significantly higher after DEP exposure. Oct-4 expression and the Oct-4/Cdx-2 ratio were significantly decreased in day 5 blastocysts irrespective of DEP concentration. Results suggest that DEP appear to play an important role in disrupting cell lineage segregation and ICM morphological integrity even at lower concentrations, compromising future growth and viability of the blastocyst.

Pulmonary function and histological impairment in mice after acute exposure to aluminum dust

Along the aluminum refining process, alumina (Al(2)O(3)) constitutes the main source of dust. Although aluminum refinery workers present respiratory symptoms with lung functional changes, no conclusive data about lung function impairment after alumina exposure has been so far reported. We examined the pulmonary alterations of exposure to material collected in an aluminum refinery in Brazil. BALB/c mice were exposed in a whole-body chamber for 1 h to either saline (CTRL, n = 11) or to a suspension (in saline) of 8 mg/m(3) of the dust (ALUM, n = 11) both delivered by an ultrasonic nebulizer. Twenty-four hours after exposure lung mechanics were measured by the end-inflation method. Lungs were prepared for histology. ALUM showed significantly higher static elastance (34.61 +/- 5.76 cmH(2)O/mL), elastic component of viscoelasticity (8.16 +/- 1.20 cmH(2)O/mL), pressure used to overcome the resistive component of viscoelasticity (1.62 +/- 0.24 cmH(2)O), and total resistive pressure (2.21 +/- 0.49 cmH(2)O) than CTRL (27.95 +/- 3.63 cmH(2)O/mL, 6.12 +/- 0.99 cmH(2)O/mL, 1.23 +/- 0.19 cmH(2)O, and 1.68 +/- 0.23 cmH(2)O, respectively). ALUM also presented significantly higher fraction area of alveolar collapse (69.7 +/- 1.2%) and influx of polymorphonuclear cells (27.5 +/- 1.1%) in lung parenchyma than CTRL (27.2 +/- 1.1% and 14.6 +/- 0.7%, respectively). The composition analysis of the particulate matter showed high concentrations of aluminum. For the first time it was demonstrated in an experimental model that an acute exposure to dust collected in an aluminum producing facility impaired lung mechanics that could be associated with inflammation.

Humoral responses and immune protection in mice immunized with irradiated T. gondii tachyzoites and challenged with three genetically distinct strains of T. gondii

Toxoplasma gondii is an obligate intracellular parasite that infects a variety of mammals and birds. T. gondii also causes human toxoplasmosis; although toxoplasmosis is generally a benign disease, ocular, congenital or reactivated disease is associated with high numbers of disabled people. Infection occurs orally through the ingestion of meat containing cysts or by the intake of food or water contaminated with oocysts. Although the immune system responds to acute infection and mediates the clearance of tachyzoites, parasite cysts persist for the lifetime of the host in tissues such as the eye, muscle, and CNS. However, T. gondii RH strain tachyzoites irradiated with 255 Gy do not cause residual infection and induce the same immunity as a natural infection. To assess the humoral response in BALB/c and C57BL/6J mice immunized with irradiated tachyzoites either by oral gavage (p.o.) or intraperitoneal (i.p.) injection, we analyzed total and high-affinity IgG and IgA antibodies in the serum. High levels of antigen-specific IgG were detected in the serum of parenterally immunized mice, with lower levels in mice immunized via the oral route. However, most serum antibodies exhibited low affinity for antigen in both mice strain. We also found antigen specific IgA antibodies in the stools of the mice, especially in orally immunized BALB/c mice. Examination of bone marrow and spleen cells demonstrated that both groups of immunized mice clearly produced specific lgG, at levels comparable to chronic infection, suggesting the generation of IgG specific memory. Next, we challenged i.p. or p.o. immunized mice with cysts from ME49. VEG or P strains of T. gondii. Oral immunization resulted in partial protection as compared to challenged naive mice: these findings were more evident in highly pathogenic ME49 strain challenge. Additionally, we found that while mucosal IgA was important for protection against infection, antigen-specific IgG antibodies were involved with protection against disease and disease pathogenesis. Most antigen responsive cells in culture produced specific high-affinity IgG after immunization, diverse of the findings in serum IgG or from cells after infection, which produced low proportion of high-avidity IgG. (C) 2011 Elsevier B.V. All rights reserved.

CpG-Induced Th1-Type Response in the Downmodulation of Early Development of Allergy and Inhibition of B7 Expression on T Cells of Newborn Mice

Several differences have been described between neonatal and adult immune responses. The predisposition in early life to Th2-type response or tolerance makes it a susceptible period for infections and allergic sensitization. The aim of this work was to evaluate the effects of CpG-containing oligodeoxynucleotides on neonatal and adult immunization with ovalbumin and Blomia tropicalis extract and compare the CpG effects on B and T cells of neonatal and adult mice. Mice that received CpG showed reduced immunoglobulin E (IgE) antibody production in both neonatal and adult periods, in parallel to increased IgG2a antibody levels. We observed that spleen cells of mice that received CpG in early life produced increased amounts of interferon-gamma upon anti-CD3 stimulation. Negative regulation of IgE response was more pronounced in adult than neonate mice; further, CpG decreased anaphylactic antiovalbumin IgG1 only in adults. Also, an upregulation of toll-like receptor 9 expression was detected in adult B cells, but not in neonatal, upon CpG stimuli. Neonatal B cells showed enhanced interleukin (IL)-10 expression and decreased IL-6 levels than adult B cells in response to CpG. When we analyzed in vitro activation of CD4+ T cells, an increased expression of B7 molecules on T cells in neonates was suppressed by CpG. Altogether, we verified qualitative and quantitative evidences regarding CpG effect on neonatal and adult allergens immunizations, which points to the importance of understanding neonatal immune system to establish immunomodulatory strategies for prevention of allergic diseases.

In vitro fertilization, embryo development, and cell lineage segregation after pre- and/or postnatal exposure of female mice to ambient fine particulate matter

Objective: To evaluate effects of pre- and/or postnatal exposure to ambient fine particulate matter on fertilization, embryo development, and cell lineage segregation in preimplantation blastocysts using the IVF mouse model. Design: Animal model. Setting: Academic institution. Animal(S): Six-week-old, superovulated mice. Intervention(s): Pre- and postnatal exposure to filtered air (FA-FA), filtered-ambient air (FA-AA), or ambient air (AA-AA) in exposure chambers 24 hours a day for 9 weeks. Main Outcome Measure(S): Gestation length, litter size, sex ratio, ovarian response to superovulation, fertilization rate, embryo development, blastocyst and hatching rates, total cell count, and proportion of cell allocation to inner-cell mass (ICM) and trophectoderm (TE). Result(S): Gestation length, litter size and birth weight, live-birth index, and sex ratio were similar among exposure groups. Ovarian response was not affected by the exposure protocol. A multivariate effect for pre- and/or postnatal exposure to ambient fine particulate matter on IVF, embryo development, and blastocyst differential staining was found. Cell counts in ICM and ICM/TE ratios in blastocysts produced in the FA-FA protocol were significantly higher than in blastocysts produced in the FA-AA and AA-AA protocols. No difference in total cell count was observed among groups. Conclusion(S): Our study suggests that exposure to ambient fine particulate matter may negatively affect female reproductive health by disrupting the lineage specification at the blastocyst stage without interfering in early development of the mouse embryo. (Fertil Steril (R) 2009;92:1725-35. (C) 2009 by American Society for Reproductive Medicine.)

Effect of pre- and postnatal exposure to urban air pollution on myocardial lipid peroxidation levels in adult mice

Exposure to air pollution can elicit cardiovascular health effects. Children and unborn fetuses appear to be particularly vulnerable. However, the mechanisms involved in cardiovascular damage are poorly understood. It has been suggested that the oxidative stress generated by air pollution exposure triggers tissue injury. To investigate whether prenatal exposure can enhance oxidative stress in myocardium of adult animals, mice were placed in a clean chamber (CC, filtered urban air) and in a polluted chamber (PC, Sao Paulo city) during the gestational period and/or for 3 mo after birth, according to 4 protocols: control group-prenatal and postnatal life in CC; prenatal group-prenatal in PC and postnatal life in CC; postnatal group-prenatal in CC and postnatal life in PC; and pre-post group-prenatal and postnatal life in PC. As an indicator of oxidative stress, levels of lipid peroxidation in hearts were measured by malondialdehyde (MDA) quantification and by quantification of the myocardial immunoreactivity for 15-F2t-isoprostane. Ultrastructural studies were performed to detect cellular alterations related to oxidative stress. Concentration of MDA was significantly increased in postnatal (2.45 +/- 0.84 nmol/mg) and pre-post groups (3.84 +/- 1.39 nmol/mg) compared to the control group (0.31 +/- 0.10 nmol/mg) (p < .01). MDA values in the pre-post group were significantly increased compared to the prenatal group (0.71 +/- 0.15 nmol/mg) (p = .017). Myocardial isoprostane area fraction in the pre-post group was increased compared to other groups (p <= .01). Results show that ambient levels of air pollution elicit cardiac oxidative stress in adult mice, and that gestational exposure may enhance this effect.

Pre- and postnatal exposure to ambient levels of urban particulate matter (PM(2.5)) affects mice spermatogenesis

This work characterizes the effects of ambient levels of urban particulate matter (PM(2.5)) from the city of Sao Paulo on spermatogenesis using mice exposed during the embryo-fetal and/or postnatal phases of development. Parental generations (BALB/c mice) were exposed to air pollution in chambers with or without filtering PM(2.5) for 4 months. Animals were mated, and half of the 1-day-old offspring were moved between chambers, which yielded prenatal and postnatal groups. Remaining offspring comprised the non-exposed and pre+postnatal exposed groups. After 90 days, the animals were sacrificed for testis collection and weighing. Optical microscopy was used for the morphometric analyses of the cell counts, spermatogenic cycle, proliferation, and apoptosis. Prenatally exposed animals presented reduced body and testicular weight with an increased gonadosomatic index (GSI). Testicular volume also decreased, as well as the tubular diameter in testes of the same animals. Proliferation, apoptosis, and spermatogenic cycle analyses showed no significant differences among groups. However, the tubules at stage VII of pre- and postnatal animals presented a reduced number of elongated spermatids. Pre+postnatal group presented higher spermatid head retention at stages VIII-XII. These results show that ambient levels of PM(2.5) from Sao Paulo city affect spermatogenesis by damaging sperm production.

Exacerbation of Leishmania (Viannia) shawi infection in BALB/c mice after immunization with soluble antigen from amastigote forms

The present study aimed to evaluate the effects of immunization with soluble amastigote (AmaAg) and promastigote (ProAg) antigens from Leishmania (Viannia) shawi on the course of infection in BALB/c mice. After immunization with AmaAg, the challenged group showed greater lesion size and parasite load in the skin and lymph nodes, associated with diminished interleukin (IL)-2, IL-4, IL-10, interferon (IFN)-gamma and nitrate levels in the supernatant of lymph node cell cultures, together with increases in transforming growth factor (TGF)-beta concentrations and humoral immune response. In contrast, immunization with ProAg led to smaller lesion size with reduced numbers of viable parasites in the skin. Protection was associated with increases in IL-12, IFN-gamma, TGF-beta and nitrates and decreases in IL-4 and IL-10 levels. Concerning humoral immune response, a significant reduction in anti-leishmania immunoglobulin G was verified in the ProAg-challenged group. Analysis of these results suggests that AmaAg induced a suppressive cellular immune response in mice, favouring the spread of infection, whereas ProAg induced partial protection associated with increased cellular immune response.

Oral tolerance reduces Th17 cells as well as the overall inflammation in the central nervous system of EAE mice

Multiple sclerosis (MS) is an autoimmune disease characterized by inflammatory immune response directed against myelin antigens of the central nervous system. In its murine model, EAE, Th17 cells play an important role in disease pathogenesis. These cells can induce blood-brain barrier disruption and CNS immune cells activation, due to the capacity to secrete high levels of IL-17 and IL-22 in an IL-6 + TGF-beta dependent manner. Thus, using the oral tolerance model, by which 200 mu g of MOG 35-55 is given orally to C57BL/6 mice prior to immunization, we showed that the percentage of Th17 cells as well as IL-17 secretion is reduced both in the periphery and also in the CNS of orally tolerated animals. Altogether, our data corroborates with the pathogenic role of IL-17 and IFN-gamma in EAE, as its reduction after oral tolerance, leads to an overall reduction of pro-inflammatory cytokines, such as IL-1 alpha, IL-6, IL-9, IL-12p70 and the chemokines MIP-1 beta, RANTES, Eotaxin and KC in the CNS. It is noteworthy that this was associated to an increase in IL-10 levels. Thus, our data clearly show that disease suppression after oral tolerance induction, correlates with reduction in target organ inflammation, that may be caused by a reduced Th1/Th17 response. Crown Copyright (c) 2010 Published by Elsevier B.V. All rights reserved.

Subchronic effects of nasally instilled diesel exhaust particulates on the nasal and airway epithelia in mice

Diesel exhaust is the major source of ultrafine particles released during traffic-related pollution. Subjects with chronic respiratory diseases are at greater risk for exacerbations during exposure to air pollution. This study evaluated the effects of subchronic exposure to a low-dose of diesel exhaust particles (DEP). Sixty male BALB/c mice were divided into two groups: (a) Saline: nasal instillation of saline (n = 30); and (b) DEP: nasal instillation of 30 mu g of DEP/10 mu l of saline (n = 30). Nasal instillations were performed 5 days a week, over 30 and 60 days. Animals were anesthetized with pentobarbital sodium (50 mg/kg intraperitoneal [i.p.]) and sacrificed by exsanguination. Bronchoalveolar lavage (BAL) fluid was performed to evaluate the inflammatory cell count and the concentrations of the interleukin (IL)-4, IL-10, and IL-13 by enzyme-linked immunosorbent assay (ELISA). The gene expression of oligomeric mucus/gel-forming (Muc5ac) was evaluated by real-time polymerase chain reaction (PCR). Histological analysis in the nasal septum and bronchioles was used to evaluate the bronchial and nasal epithelium thickness as well as the acidic and neutral nasal mucus content. The saline group (30 and 60 days) did not show any changes in any of the parameters. However, the instillation of DEP over 60 days increased the expression of Muc5ac in the lungs and the acid mucus content in the nose compared with the 30-day treatment, and it increased the total leukocytes in the BAL and the nasal epithelium thickness compared with saline for 60 days. Cytokines concentrations in the BAL were detectable, with no differences among the groups. Our data suggest that a low-dose of DEP over 60 days induces respiratory tract inflammation.

Salbutamol improves markers of epithelial function in mice with chronic allergic pulmonary inflammation

We investigated the effects of salbutamol on the markers of epithelial function in a murine model of chronic allergic pulmonary inflammation by recording the ciliary beat frequency (CBF) and the transepithelial potential difference (PD) in vivo. Mice were sensitized and received four challenges of ovalbumin (OVA group) or 0.9% saline (control group). Forty-eight hours after the 4th inhalation, we observed eosinophilia in the bronchoalveolar lavage and epithelium remodeling with stored acid mucus in the OVA group (P < 0.001). No difference in the baseline CBF was noticed between the groups; however, the OVA group had a significantly lower baseline PD (P = 0.013). Salbutamol increased the CBF in all groups studied, and the dose response curve to salbutamol increased the PD in the OVA group from 10(-4) M to 10(-2) M. We suggest that salbutamol affects the CBF and the depth of the periciliary layer, which, in great part, determines the ability of the cilia to propel the mucus layer. This effect may have a positive impact on airway mucociliary transport in asthma and may have clinical implications. (C) 2011 Elsevier B.V. All rights reserved.

Individual vulnerability to escalated aggressive behavior by a low dose of alcohol: decreased serotonin receptor mRNA in the prefrontal cortex of male mice

Low to moderate doses of alcohol consumption induce heightened aggressive behavior in some, but not all individuals. Individual vulnerability for this nonadaptive behavior may be determined by an interaction of genetic and environmental factors with the sensitivity of alcohol`s effects on brain and behavior. We used a previously established protocol for alcohol oral self-administration and characterized alcohol-heightened aggressive (AHA) mice as compared with alcohol non-heightened (ANA) counterparts. A week later, we quantified mRNA steady state levels of several candidate genes in the serotonin [5-hydroxytryptamine (5-HT)] system in different brain areas. We report a regionally selective and significant reduction of all 5-HT receptor subtype transcripts, except for 5-HT(3), in the prefrontal cortex of AHA mice. Comparable gene expression profile was previously observed in aggressive mice induced by social isolation or by an anabolic androgenic steroid. Additional change in the 5-HT(1B) receptor transcripts was seen in the amygdala and hypothalamus of AHA mice. In both these areas, 5-HT(1B) mRNA was elevated when compared with ANA mice. In the hypothalamus, AHA mice also showed increased transcripts for 5-HT(2A) receptor. In the midbrain, 5-HT synthetic enzyme, 5-HT transporter and 5-HT receptors mRNA levels were similar between groups. Our results emphasize a role for postsynaptic over presynaptic 5-HT receptors in mice which showed escalated aggression after the consumption of a moderate dose of alcohol. This gene expression profile of 5-HT neurotransmission components in the brain of mice may suggest a vulnerability trait for alcohol-heightened aggression.

Erection induced by Tx2-6 toxin of Phoneutria nigriventer spider: Expression profile of genes in the nitric oxide pathway of penile tissue of mice

The peptides Tx2-5 and Tx2-6, isolated from the whole venom of ""armed-spider"" Phoneutria nigniventer venom, are directly linked with the induction of persistent and painful erection in the penis of mammals. The erection induced by Tx2-6 has been associated with the activation of nitric oxide synthases. There is a scarcity of studies focusing on the outcome of Tx2-6 at the molecular level, by this reason we evaluated the gene profile activity of this toxin at the nitric oxide (NO) pathway. After microarray analyses on cavernous tissue of mice inoculated with Tx2-6 we found that only 10.4% (10/96) of these genes were differentially expressed, showing a limited effect of the toxin on the NO pathway. We found the genes sparc, ednrb, junb, cdkn1a, bcl2, ccl5, abcc1 over-expressed and the genes sod1, s100a10 and fth1 under-expressed after inoculation of Tx2-6. The differential expressions of sparc and ednrb genes were further confirmed using real-time PCR. Interestingly, ednrb activates the L-arginine/NO/cGMP pathway that is involved in the relaxation of the cavernous body. Therefore the priapism induced by Tx2-6 is a consequence of a highly specific interference of this neurotoxin with the NO pathway. (C) 2009 Elsevier Ltd. All rights reserved.