Liver proteome of mice with different genetic susceptibilities to the effects of fluoride.

OBJECTIVE: In this study, we investigated the differential pattern of protein expression in the liver of these mice to provide insights on why they have different responses to F. MATERIAL AND METHODS: Weanling male A/J and 129P3/J mice (n=10 from each strain) were pared and housed in metabolic cages with ad libitum access to low-F food and deionized water for 42 days. Liver proteome profiles were examined using nLC-MS/MS. Protein function was classified by GO biological process (Cluego v2.0.7 + Clupedia v1.0.8) and protein-protein interaction network was constructed (PSICQUIC, Cytoscape). RESULTS: Most proteins with fold change were increased in A/J mice. The functional category with the highest percentage of altered genes was oxidation-reduction process (20%). Subnetwork analysis revealed that proteins with fold change interacted with Disks large homolog 4 and Calcium-activated potassium channel subunit alpha-1. A/J mice had an increase in proteins related to energy flux and oxidative stress. CONCLUSION: This could be a possible explanation for the high susceptibility of these mice to the effects of F, since the exposure also induces oxidative stress.

Liver proteome of mice with different genetic susceptibilities to the effects of fluoride

ABSTRACT A/J and 129P3/J mice strains have been widely studied over the last few years because they respond quite differently to fluoride (F) exposure. 129P3/J mice are remarkably resistant to the development of dental fluorosis, despite excreting less F in urine and having higher circulating F levels. These two strains also present different characteristics regardless of F exposure. Objective In this study, we investigated the differential pattern of protein expression in the liver of these mice to provide insights on why they have different responses to F. Material and Methods Weanling male A/J and 129P3/J mice (n=10 from each strain) were pared and housed in metabolic cages with ad libitum access to low-F food and deionized water for 42 days. Liver proteome profiles were examined using nLC-MS/MS. Protein function was classified by GO biological process (Cluego v2.0.7 + Clupedia v1.0.8) and protein-protein interaction network was constructed (PSICQUIC, Cytoscape). Results Most proteins with fold change were increased in A/J mice. The functional category with the highest percentage of altered genes was oxidation-reduction process (20%). Subnetwork analysis revealed that proteins with fold change interacted with Disks large homolog 4 and Calcium-activated potassium channel subunit alpha-1. A/J mice had an increase in proteins related to energy flux and oxidative stress. Conclusion This could be a possible explanation for the high susceptibility of these mice to the effects of F, since the exposure also induces oxidative stress.

Intra-uterine experimental infection by Ureaplasma diversum induces TNF-α mediated womb inflammation in mice.

Ureaplasma diversum is an opportunistic pathogen associated with uterine inflammation, impaired embryo implantation, infertility, abortions, premature birth of calves and neonatal pneumonia in cattle. It has been suggested that the intra-uterine infection by Ureaplasma diversum can cause vascular changes that hinder the success of pregnancy. Thus, the aim of this study was to evaluate the changes of intrauterine site of A/J mice in estrus or proestrus phase inoculated with Ureaplasma diversum. The infection was monitored at 24, 48 and 72 hours by the PCR methodology to detect the Ureaplasma in the inoculation site and the profile of circulating blood cells. Morphological changes, intensity of inflammation and the production of cytokines were compared. The infected mice showed local inflammation through the production of IFN-γ and TNF-α. Ureaplasma diversum infections in the reproductive tract of studied mice seemed to be associated with the production of pro-inflammatory cytokines in uterine parenchyma. The levels of TNF-α of infected mice were dependent on the bacterial load of inoculated Ureaplasma. Uterine experimental infections by Ureaplasma diversum have not been mentioned yet and herein we presented the first report of an intrauterine infection model in mice.

Lipoxin Inhibits Fungal Uptake by Macrophages and Reduces the Severity of Acute Pulmonary Infection Caused by Paracoccidioides brasiliensis.

Cysteinyl leukotrienes (CysLTs) and lipoxins (LXs) are lipid mediators that control inflammation, with the former inducing and the latter inhibiting this process. Because the role played by these mediators in paracoccidioidomycosis was not investigated, we aimed to characterize the role of CysLT in the pulmonary infection developed by resistant (A/J) and susceptible (B10.A) mice. 48 h after infection, elevated levels of pulmonary LTC4 and LXA4 were produced by both mouse strains, but higher levels were found in the lungs of susceptible mice. Blocking the CysLTs receptor by MTL reduced fungal loads in B10.A, but not in A/J mice. In susceptible mice, MLT treatment led to reduced influx of PMN leukocytes, increased recruitment of monocytes, predominant synthesis of anti-inflammatory cytokines, and augmented expression of 5- and 15-lipoxygenase mRNA, suggesting a prevalent LXA4 activity. In agreement, MTL-treated macrophages showed reduced fungal burdens associated with decreased ingestion of fungal cells. Furthermore, the addition of exogenous LX reduced, and the specific blockade of the LX receptor increased the fungal loads of B10.A macrophages. This study showed for the first time that inhibition of CysLTs signaling results in less severe pulmonary paracoccidioidomycosis that occurs in parallel with elevated LX activity and reduced infection of macrophages.

Modulation of Th1/Th2 immune responses by killed Propionibacterium acnes and its soluble polysaccharide fraction in a type I hypersensitivity murine model: induction of different activation status of antigen-presenting cells.

Propionibacterium acnes (P. acnes) is a gram-positive anaerobic bacillus present in normal human skin microbiota, which exerts important immunomodulatory effects, when used as heat- or phenol-killed suspensions. We previously demonstrated that heat-killed P. acnes or its soluble polysaccharide (PS), extracted from the bacterium cell wall, suppressed or potentiated the Th2 response to ovalbumin (OVA) in an immediate hypersensitivity model, depending on the treatment protocol. Herein, we investigated the mechanisms responsible for these effects, using the same model and focusing on the activation status of antigen-presenting cells (APCs). We verified that higher numbers of APCs expressing costimulatory molecules and higher expression levels of these molecules are probably related to potentiation of the Th2 response to OVA induced by P. acnes or PS, while higher expression of toll-like receptors (TLRs) seems to be related to Th2 suppression. In vitro cytokines production in cocultures of dendritic cells and T lymphocytes indicated that P. acnes and PS seem to perform their effects by acting directly on APCs. Our data suggest that P. acnes and PS directly act on APCs, modulating the expression of costimulatory molecules and TLRs, and these differently activated APCs drive distinct T helper patterns to OVA in our model.

Enamel crystals of mice susceptible or resistant to dental fluorosis: an AFM study.

OBJECTIVE: This study aimed to assess the overall apatite crystals profile in the enamel matrix of mice susceptible (A/J strain) or resistant (129P3/J strain) to dental fluorosis through analyses by atomic force microscopy (AFM). MATERIAL AND METHODS: Samples from the enamel matrix in the early stages of secretion and maturation were obtained from the incisors of mice from both strains. All detectable traces of matrix protein were removed from the samples by a sequential extraction procedure. The purified crystals (n=13 per strain) were analyzed qualitatively in the AFM. Surface roughness profile (Ra) was measured. RESULTS: The mean (±SD) Ra of the crystals of A/J strain (0.58±0.15 nm) was lower than the one found for the 129P3/J strain (0.66±0.21 nm) but the difference did not reach statistical significance (t=1.187, p=0.247). Crystals of the 129P3/J strain (70.42±6.79 nm) were found to be significantly narrower (t=4.013, p=0.0013) than the same parameter measured for the A/J strain (90.42±15.86 nm). CONCLUSION: enamel crystals of the 129P3/J strain are narrower, which is indicative of slower crystal growth and could interfere in the occurrence of dental fluorosis.

Enamel crystals of mice susceptible or resistant to dental fluorosis: an AFM study

Objective: This study aimed to assess the overall apatite crystals profile in the enamel matrix of mice susceptible (A/J strain) or resistant (129P3/J strain) to dental fluorosis through analyses by atomic force microscopy (AFM). Material and Methods: Samples from the enamel matrix in the early stages of secretion and maturation were obtained from the incisors of mice from both strains. All detectable traces of matrix protein were removed from the samples by a sequential extraction procedure. The purified crystals (n=13 per strain) were analyzed qualitatively in the AFM. Surface roughness profile (Ra) was measured. Results: The mean (±SD) Ra of the crystals of A/J strain (0.58±0.15 nm) was lower than the one found for the 129P3/J strain (0.66±0.21 nm) but the difference did not reach statistical significance (t=1.187, p=0.247). Crystals of the 129P3/J strain (70.42±6.79 nm) were found to be significantly narrower (t=4.013, p=0.0013) than the same parameter measured for the A/J strain (90.42±15.86 nm). Conclusion: enamel crystals of the 129P3/J strain are narrower, which is indicative of slower crystal growth and could interfere in the occurrence of dental fluorosis. .

Biological and immunological characterization of recombinant Yellow Fever 17D viruses expressing a Trypanosoma cruzi Amastigote Surface Protein-2 CD8+ T cell epitope at two distinct regions of the genome.

BACKGROUND: The attenuated Yellow fever (YF) 17D vaccine virus is one of the safest and most effective viral vaccines administered to humans, in which it elicits a polyvalent immune response. Herein, we used the YF 17D backbone to express a Trypanosoma cruzi CD8+ T cell epitope from the Amastigote Surface Protein 2 (ASP-2) to provide further evidence for the potential of this virus to express foreign epitopes. The TEWETGQI CD8+ T cell epitope was cloned and expressed based on two different genomic insertion sites: in the fg loop of the viral Envelope protein and the protease cleavage site between the NS2B and NS3. We investigated whether the site of expression had any influence on immunogenicity of this model epitope. RESULTS: Recombinant viruses replicated similarly to vaccine virus YF 17D in cell culture and remained genetically stable after several serial passages in Vero cells. Immunogenicity studies revealed that both recombinant viruses elicited neutralizing antibodies to the YF virus as well as generated an antigen-specific gamma interferon mediated T-cell response in immunized mice. The recombinant viruses displayed a more attenuated phenotype than the YF 17DD vaccine counterpart in mice. Vaccination of a mouse lineage highly susceptible to infection by T. cruzi with a homologous prime-boost regimen of recombinant YF viruses elicited TEWETGQI specific CD8+ T cells which might be correlated with a delay in mouse mortality after a challenge with a lethal dose of T. cruzi. CONCLUSIONS: We conclude that the YF 17D platform is useful to express T. cruzi (Protozoan) antigens at different functional regions of its genome with minimal reduction of vector fitness. In addition, the model T. cruzi epitope expressed at different regions of the YF 17D genome elicited a similar T cell-based immune response, suggesting that both expression sites are useful. However, the epitope as such is not protective and it remains to be seen whether expression of larger domains of ASP-2, which include the TEWETGQI epitope, will elicit better T-CD8+ responses to the latter. It is likely that additional antigens and recombinant virus formulations will be necessary to generate a protective response.

Long-term maternal separation differentially alters serum corticosterone levels and blood neutrophil activity in A/J and C57BL/6 mouse offspring.

OBJECTIVES: In this work, we searched for maternal separation effects on serum corticosterone levels and blood neutrophil activity in adult male A/J and C57BL/6 mouse offspring. METHODS: 40 male A/J mice and 40 male C57BL/6 mice were divided within each strain into two groups. Mice in the maternal separation group were separated from their mothers (1 h/day) on postnatal days 0-13. Mice in the control group were left undisturbed. On postnatal day 45, blood was drawn from all mice and used to assess neutrophil activity by flow cytometry and serum corticosterone levels by radioimmunoassay. RESULTS: The results showed that each mouse strain responded differently to maternal separation, but in both cases, serum corticosterone levels were affected. In both strains, adult mice that experienced maternal separation showed lower serum corticosterone levels than control mice. In relation to control mice kept together with their mothers, the levels of serum corticosterone were 72.7 and 36.36% lower in A/J and C57BL/6 mice submitted to maternal separation, respectively. The current findings showed that maternal separation increased neutrophil activity in mice after reaching adulthood. The observed effects, although in the same direction, differed between A/J and C57BL/6 mice. Maternal separation increased both the percentage and intensity of phagocytosis in C57BL/6 mice, but had no effects on A/J mice. Furthermore, maternal separation increased basal and propidium iodide-labeled Staphylococcus aureus-induced oxidative burst in A/J mice but did not affect oxidative burst in C57BL/6 mice. Finally, phorbol myristate acetate-induced oxidative burst increased in both strains. CONCLUSION: These results indicate that early maternal separation increases innate immunity, most likely by modifying hypothalamus-pituitary-adrenal axis activity. This suggests that maternal separation is a good model for stress which produces long-term neuroimmune changes whatever the animal species and strain used.

Effects of Cissampelos sympodialis Eichl. and its alkaloid, warifteine, in an experimental model of respiratory allergy to Blomia tropicalis.

Asthma is one of the most prevalent chronic diseases worldwide. Medicinal plants are historically used in its treatment. The plant Cissampelos sympodialis, known in Northeastern Brazil as "Jarrinha" or "Milona", is used to treat some inflammatory conditions, including asthma. The objective of this study was to evaluate the potential of Cissampelos sympodialis EICHL. extract (CsE) and its isolated alkaloids, especially warifteine (Wa) on a Blomia tropicalis extract (BtE)-induced experimental model of allergy. The respiratory allergy was induced in AJ mice by the administration of BtE. Mice were orally treated with the 400 mg/kg of CsE or 8 mg/kg of total alkaloids fraction (TAF) or 4 mg/kg of Wa and the following parameters were analyzed: (a) total cell numbers in bronchoalveolar fluid (BAF); (b) differential cell numbers in BAF; (c) eosinophil peroxidase (EPO) activity in BAF; (d) IgE serum levels by passive cutaneous anaphylaxis; (e) IL-5, IL-13, IL-10, and IFN-γ levels in BAF; (f) histopathological alterations in the lung. The treatment of the animals with CsE, Wa or TAF led to a reduction in the numbers of total cells and eosinophils in BAF. The same reduction was observed in EPO levels in the BAF. The levels of IL-5 and IL-13 were also reduced in animals treated with Cissampelos sympodialis, while IL-10 levels were significantly increased in the BAF of CsE-treated animals. The treatment also decreased the density of inflammatory cells in the lung by histopathological examination demonstrating the potential of this medicinal plant as new agent for asthma treatment.

Betamethasone inhibits tumor development, microvessel density and prolongs survival in mice with a multiresistant adenocarcinoma TA3.

Tumor resistance to traditional cancer treatments poses an important challenge to modern science. Thus, angiogenesis inhibition is an important emerging cancer treatment. Many drugs are tested and corticosteroids have shown interesting results. Herein we investigate the effect on microvessel density, survival time and tumoral volume of mice with TA3-MTX-R tumors. Twenty six mice were inoculated with l x l06 tumor cells; 4-5 days after injection, six mice were injected with PBS (group A) and twenty mice were treated with ß-met (group B). All animals from Group A died on day 22. Group B was divided into Bl (treated discontinued) and B2 (treated daily) and observed until day 88. All mice were processed for histo-immunohistochemical analysis and the blood vessels were counted. A decrease in microvessel density and tumoral volume and longer survival times were observed in the treated group. We propose that the antiangiogenic ß-met effect explains, at least partially, its tumor inhibitory properties. As an important perspective, we will experimentally combine these strategies with those recently described by us with regard to the important antiangiogenic-antitumor effects of Trypanosoma cruzi calreticulin. Since the molecular targets of these strategies are most likely different, additive or synergic effects are envisaged.

Betamethasone inhibits tumor development, microvessel density and prolongs survival in mice with a multiresistant adenocarcinoma TA3

Tumor resistance to traditional cancer treatments poses an important challenge to modern science. Thus, angiogenesis inhibition is an important emerging cancer treatment. Many drugs are tested and corticosteroids have shown interesting results. Herein we investigate the effect on microvessel density, survival time and tumoral volume of mice with TA3-MTX-R tumors. Twenty six mice were inoculated with lxlO6 tumor cells, 4-5 days after injection, six mice were injected with PBS (group A) and twenty mice were treated with p-met (group B). All animals from Group A died on day 22. Group B was divided into Bl (treated discontinued) and B2 (treated daily) and observed until day 88. All mice were processed for histo-immunohistochemical analysis and the blood vessels were counted. A decrease in microvessel density and tumoral volume and longer survival times were observed in the treated group. We propose that the antiangiogenic p-met effect explains, at least partially, its tumor inhibitory properties. As an important perspective, we will experimentally combine these strategies with those recently described by us with regard to the important antiangiogenic-antitumor effects of Trypanosoma cruzi calreticulin. Since the molecular targets of these strategies are most likely different, additive or synergic effects are envisaged.

Influence of genetic background on fluoride metabolism in mice.

A/J and 129P3/J mouse strains have different susceptibilities to dental fluorosis, due to their genetic backgrounds. This study tested whether these differences are due to variations in water intake and/or F metabolism. A/J (susceptible to dental fluorosis) and 129P3/J mice (resistant) received drinking water containing 0, 10, or 50 ppm F. Weekly F intake, excretion and retention, and terminal plasma and femur F levels were determined. Dental fluorosis was evaluated clinically and by quantitative fluorescence (QF). Data were tested by two-way ANOVA. Although F intakes by the strains were similar, excretion by A/J mice was significantly higher due to greater urinary F excretion, which resulted in lower plasma and femur F levels. Compared with 129P3/J mice given 50 ppm F, significantly higher QF scores were recorded for A/J mice. In conclusion, these strains differ with respect to several features of F metabolism, and amelogenesis in the 129P3/J strain seems to be unaffected by high F exposure.

Different strains of mice present distinct lung tissue mechanics and extracellular matrix composition in a model of chronic allergic asthma.

The impact of genetic factors on asthma is well recognized but poorly understood. We tested the hypothesis that different mouse strains present different lung tissue strip mechanics in a model of chronic allergic asthma and that these mechanical differences may be potentially related to changes of extracellular matrix composition and/or contractile elements in lung parenchyma. Oscillatory mechanics were analysed before and after acetylcholine (ACh) in C57BL/10, BALB/c, and A/J mice, subjected or not to ovalbumin sensitization and challenge. In controls, tissue elastance (E) and resistance (R), collagen and elastic fibres' content, and alpha-actin were higher in A/J compared to BALB/c mice, which, in turn, were more elevated than in C57BL/10. A similar response pattern was observed in ovalbumin-challenged animals irrespective of mouse strain. E and R augmented more in ovalbumin-challenged A/J [E: 22%, R: 18%] than C57BL/10 mice [E: 9.4%, R: 11%] after ACh In conclusion, lung parenchyma remodelled differently yielding distinct in vitro mechanics according to mouse strain.

Pathogenic effector T cell enrichment overcomes regulatory T cell control and generates autoimmune gastritis.

Regulatory T cells (Treg) deficiency leads to a severe, systemic, and lethal disease, as showed in immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome patients, and scurfy mouse. Postneonatal thymectomy autoimmune gastritis has also been attributed to the absence of Tregs. In this case however, disease is mild, organ-specific, and, more important, it is not an obligatory outcome. We addressed this paradox comparing T cell compartments in gastritis-susceptible and resistant animals. We found that neonatal thymectomy-induced gastritis is not caused by the absence of Tregs. Instead of this, it is the presence of gastritogenic T cell clones that determines susceptibility to disease. The expansion of such clones under lymphopenic conditions results in a reduced Treg:effector T cell ratio that is not enough to control gastritis development. Finally, the presence of gastritogenic clones is determined by the amount of gastric Ag expressed in the neonatal thymus, emphasizing the importance of effector repertoire variability, present even in genetically identical subjects, to organ-specific autoimmune disease susceptibility.

Alveolar macrophages from susceptible mice are more competent than those of resistant mice to control initial Paracoccidioides brasiliensis infection.

Alveolar macrophages (AM) are the first host cells to interact with Paracoccidioides brasiliensis (Pb), a primary human pathogen that causes severe pulmonary infections in Latin America. To better understand innate immunity in pulmonary paracoccidioidomycosis, we decided to study the fungicidal and secretory abilities of AM from resistant (A/J) and susceptible (B10.A) mice to infection. Untreated, IFN-gamma and IL-12 primed AM from B10.A and A/J mice were challenged with P. brasiliensis yeasts and cocultured for 72 h. B10.A macrophages presented an efficient fungicidal ability, were easily activated by both cytokines, produced high levels of nitric oxide (NO), IL-12, and MCP-1 associated with low amounts of IL-10 and GM-CSF. In contrast, A/J AM showed impaired cytokine activation and fungal killing, secreted high levels of IL-10 and GM-CSF but low concentrations of NO, IL-12, and MCP-1. The fungicidal ability of B10.A but not of A/J macrophages was diminished by aminoguanidine treatment, although only the neutralization of TGF-beta restored the fungicidal activity of A/J cells. This pattern of macrophage activation resulted in high expression of MHC class II antigens by A/J cells, while B10.A macrophages expressed elevated levels of CD40. Unexpectedly, our results demonstrated that susceptibility to a fungal pathogen can be associated with an efficient innate immunity, while a deficient innate response can ultimately favor the development of a resistant pattern to infection. Moreover, our data suggest that different pathogen recognition receptors are used by resistant and susceptible hosts to interact with P. brasiliensis yeasts, resulting in divergent antigen presentation, acquired immunity, and disease outcomes.

Monoclonal antibody to fungal glucosylceramide protects mice against lethal Cryptococcus neoformans infection.

Glucosylceramides (GlcCer) are involved in the regulation of Cryptococcus neoformans virulence. In the present study, we demonstrate that passive immunization with a monoclonal antibody to GlcCer significantly reduces host inflammation and prolongs the survival of mice lethally infected with C. neoformans, revealing a potential therapeutic strategy to control cryptococcosis.

Increased apoptosis during the early phase of experimental paracoccidioidomycosis as a phenotypic marker of resistance.

Paracoccidiodomycosis (PCM) is a systemic mycosis that presents a wide spectrum of clinical manifestations caused by Paracoccidiodes brasiliensis. The experimental murine model has been used to approach the disease with susceptible and resistant mouse strains that reproduce most of the main human immunological features. We investigated whether the pattern of apoptosis of peritoneal cells from two polar strains of mice after infection with P. brasiliensis could be associated with the susceptibility or resistance to this pathogen. Apoptosis of A/J mouse cells (resistant), cultured in the presence or absence of LPS as stimuli, was observed as early as on the first day of infection. Cells from the infected susceptible strain BALB/c did not exhibit apoptosis in absence of LPS and persistently at a lesser degree than that observed in resistant mice. The apoptosis induced by the infection in resistant mice was not due to nitric oxide, since its blockage either in vitro or in vivo did not revert it. Analysis of additional strains of polar susceptibilities to PCM assured the dissociation of NO production and apoptosis. Interestingly, IL-6 and IL-10 were secreted in high amounts, by BALB/c cells and might be involved in shielding cells from apoptosis induced by P. brasiliensis. Furthermore, IFNgamma(-/-) mice did not show apoptosis of peritoneal cells while the Wt controls presented levels similar to those of A/J strain that secreted high amounts of IFNgamma and IL-1beta. The expression of Fas was increased in both strains and in Wt mice, whereas FasL was decreased in the susceptible strain and not significantly modulated in TNFRI and IFNgamma KO mice. These results suggest that apoptosis might be a mechanism of control of engagement of cells that could otherwise contribute to the susceptible phenotype observed in some strains of mice.

Neutrophil role in pulmonary paracoccidioidomycosis depends on the resistance pattern of hosts.

The immunoprotective and immunomodulatory role of neutrophils during pulmonary infection of resistant (A/J) and susceptible (B10.A) mice to Paracoccidioides brasiliensis was investigated. First, comparative studies about early cellular influx to the lungs demonstrated higher numbers of neutrophils in susceptible rather than in resistant mice. Neutrophil depletion resulted in decreased survival times of susceptible but not resistant mice. In both mouse strains, depletion led to increased fungal burdens at Week 1 of infection; however, only susceptible mice remained with increased pulmonary fungal loads and presented a dramatic fungal dissemination to liver and spleen. At Week 1 of infection, treated and untreated B10.A and A/J mice were negative for delayed-type hypersensitivity (DTH) reactions, which remained negative for the susceptible strain. In contrast, from the second week onward, control and neutrophil-depleted, resistant mice became positive for DTH reactions. In B10.A mice, neutrophil depletion resulted in increased levels of interleukin (IL)-12 and IL-4 in the lungs, high levels of hepatic cytokines, and increased synthesis of T helper cell type 1 (Th1)- and Th2-regulated antibodies [immunoglobulin G1 (IgG1), IgA, and IgG3]. In neutrophil-depleted A/J mice, high levels of pulmonary IL-12 and granulocyte macrophage-colony stimulating factor were concomitant to diminished levels of hepatic cytokines and increased amounts of Th1-regulated isotypes (IgG2a, IgG2b, and IgG3). Differently from primary infection, neutrophil depletion did not alter immunoprotection in secondary paracoccidioidomycosis. As a whole, our data showed that the genetic patterns of hosts exert an important influence on the immunoprotective and immunoregulatory functions of neutrophils, which appear to be essential in situations devoid of cell-mediated immunity.

Contribution of NK, NK T, gamma delta T, and alpha beta T cells to the gamma interferon response required for liver protection against Trypanosoma cruzi.

In the present work, we show that intracellular Trypanosoma cruzi is rarely found in the livers of acutely infected mice, but inflammation is commonly observed. The presence of numerous intrahepatic amastigotes in infected gamma interferon (IFN-gamma)-deficient mice corroborates the notion that the liver is protected by an efficient local immunity. The contribution of different cell populations was suggested by data showing that CD4- and CD8-deficient mice were able to restrain liver parasite growth. Therefore, we have characterized the liver-infiltrating lymphocytes and determined the sources of IFN-gamma during acute T. cruzi infection. We observed that natural killer (NK) cells increased by day 7, while T and B cells increased by day 14. Among CD3+ cells, CD4+, CD8+, and CD4- CD8- cell populations were greatly expanded. A large fraction of CD3+ cells were positive for PanNK, a beta1 integrin expressed by NK and NK T cells. However, these lymphocytes were not classic NK T cells because they did not express NK1.1 and showed no preferential usage of Vbeta8. Otherwise, liver NK T (CD3+ NK1.1+) cells were not increased in acutely infected mice. The majority of PanNK+ CD4+ and PanNK+ CD8+ cells expressed T-cell receptor alphabeta (TCRalphabeta), whereas PanNK+ CD4- CD8- cells were positive for TCRgammadelta. In fact, gammadelta T cells showed the most remarkable increase (40- to 100-fold) among liver lymphocytes. Most importantly, intracellular analysis revealed high levels of IFN-gamma production at day 7 by NK cells and at day 14 by CD4+, CD8+, and CD4- CD8- TCRgammadelta+ cells. We concluded that NK cells are a precocious source of IFN-gamma in the livers of acutely infected mice, and, as the disease progresses, conventional CD4+ and CD8+ T cells and gammadelta T cells, but not classic NK-T cells, may provide the IFN-gamma required for liver protection against T. cruzi.