Silvia Maria Gomes Massironi

Isolation and Characterization of a Population of Immature Dental Pulp Stem Cells Expressing OCT-4 and Other Embryonic Stem Cell Markers

We report the isolation of a population of immature dental pulp stem cells (IDPSC), which express embryonic stem cell markers Oct-4, Nanog, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81 as well as several other mesenchymal stem cell markers during at least 25 passages while maintaining the normal karyotype and the rate of expansion characteristic of stem cells. The expression of these markers was maintained in subclones obtained from these cells. Moreover, in vitrothese cells can be induced to undergo uniform differentiation into smooth and skeletal muscles, neurons, cartilage, and bone under chemically defined culture conditions. After in vivo transplantation of these cells into immunocompromised mice, they showed dense engraftment in various tissues. The relative ease of recovery and the expression profiles of various markers justify further exploration of IDPSC for clinical therapy.

Differential Shh, Bmp and Wnt gene expressions during craniofacial development in mice.

In this study, Bmp-4, Wnt-5a and Shh gene expressions were compared during early craniofacial development in mice by comparative non-isotopic in situ hybridization. Wild-type C57BL/6J mice were studied at various stages of embryonic development (from 8.5- to 13.5-day-old embryos--E8.5-13.5). During early odontogenesis, transcripts for Bmp-4, Shh and Wnt-5a were co-localised at the tooth initiation stage. At E8.5, Shh mRNA expression was restricted to diencephalon and pharyngeal endoderm. Before maxillae and mandible ossification, Bmp-4 and Wnt-5a signals were detected in the mesenchymal cells and around Meckels cartilage. During palatogenesis, Shh was expressed only in the epithelium and Wnt-5a only in the mesenchyme of the elevating palatal shelves. During tongue development, Shh expression was found in mesenchyme, probably contributing to tongue miogenesis, while Wnt-5a signal was in the epithelium, possibly during placode development and papillae formation. Taken together, these findings suggest that Bmp-4, Shh and Wnt-5a gene expressions may act together on the epithelial-mesenchymal interactions occurring in several aspects of the early mouse craniofacial development, such as odontogenesis, neuronal development, maxillae and mandible ossification, palatogenesis and tongue formation.

A New Mouse Model for Marfan Syndrome Presents Phenotypic Variability Associated with the Genetic Background and Overall Levels of Fbn1 Expression

Marfan syndrome is an autosomal dominant disease of connective tissue caused by mutations in the fibrillin-1 encoding gene FBN1. Patients present cardiovascular, ocular and skeletal manifestations, and although being fully penetrant, MFS is characterized by a wide clinical variability both within and between families. Here we describe a new mouse model of MFS that recapitulates the clinical heterogeneity of the syndrome in humans. Heterozygotes for the mutant Fbn1 allele mgΔloxPneo, carrying the same internal deletion of exons 19–24 as the mgΔ mouse model, present defective microfibrillar deposition, emphysema, deterioration of aortic wall and kyphosis. However, the onset of a clinical phenotypes is earlier in the 129/Sv than in C57BL/6 background, indicating the existence of genetic modifiers of MFS between these two mouse strains. In addition, we characterized a wide clinical variability within the 129/Sv congenic heterozygotes, suggesting involvement of epigenetic factors in disease severity. Finally, we show a strong negative correlation between overall levels of Fbn1 expression and the severity of the phenotypes, corroborating the suggested protective role of normal fibrillin-1 in MFS pathogenesis, and supporting the development of therapies based on increasing Fbn1 expression.

Human IgG but not IgM Antibodies can Protect Mice from the Challenge with Live O6 Escherichia coli

We evaluated the ability of human anti‐lipopolysaccharide (LPS) O6 immunoglobulin G (IgG) and IgM antibodies to protect mice challenged with Escherichia coli serotype O6:K2ac. Purified whole IgG, commercial gammaglobulin, whole IgM‐effluent, pool of normal human serum (NHS), agammaglobulinaemic serum (test groups) or phosphate‐buffered saline (control group) was injected into adult male 18 h before a challenge with viable O6 E. coli. The mortality rate was assessed over a period of 72 h. To determine the opsonic and phagocytic activity of the antibody isotypes, we incubated peritoneal macrophages from the control and test groups collected at different times after challenge with the live bacteria with acridine orange for fluorescent analysis. Tumour necrosis factor (TNF)‐α and interleukin (IL)‐6 were quantified in serum of both the test and control groups. All mice that received commercial gammaglobulin or NHS survived. Purified whole IgG (containing 1.1 mg/l of anti‐LPS O6 IgG antibodies) protected 87.5% of the animals tested in this experiment, while whole IgM‐enriched effluent with 1.5 mg/l of anti‐LPS O6 IgM antibodies protected only 12.5%. The agamma serum showed no protective capacity compared with PBS (serving as control). The minimal concentration of anti‐LPS O6 IgG antibodies able to protect 50% of animals was 0.137 mg/l of purified whole IgG. Whole IgM‐enriched effluent showed no protective capacity independently of the concentration tested (0.048–17.0 mg/l of anti‐LPS O6 IgM antibodies). Fluorescent analysis of peritoneal macrophages from animals pretreated with purified whole IgG showed no bacteria at 8 h after the challenge. By contrast, whole IgM effluent showed an increasing number of live bacteria at the same time. Mice that had received whole IgM effluent (1.5 mg/l of anti‐LPS O6 IgM antibodies) before the challenge with LPS O6 presented 20.5 µg/l of IL‐6 and 1.5 µg/l of TNF‐α. Serum from animals pretreated with purified IgG did not present any detectable pro‐inflammatory cytokine. Our findings suggest that IgG but not IgM antibodies protect animals from a challenge with E. coli O6 serotype.

Basal physiological parameters of two congenic mice strains: C5 deficient C57BL/6 and C5 sufficient A/J

To investigate the in vivo role of complement component C5 it is common to compare the inflammatory response between C5-normal and C5-deficient inbred mice strains. Nevertheless, it should be expected that differences in the genetic backgrounds between those strains may affect several physiological parameters, complicating the correct interpretation of results. The use of congenic mice, developed by backcrossing, is therefore preferred. Still, several physiological parameters may be distinctive in the normal and deficient strains and therefore require careful analysis before animals are selected for investigation. We generated two congenic mouse strains: C57BL/6 (B6) C5(-), derived from the parental B6 C5(+) strain and A/J C5(+) mice derived from the parental A/J C5(-) strain. After confirmation by nucleotide sequencing, immunodiffusion and hemolytic activity analysis, several basal physiological parameters were analyzed in the congenic and parental strains before antigen exposition. Serum levels of liver alanine aminotransferase, alkaline phosphatase, albumin, triglycerides, cholesterol and uric acid were found to be different in C5-sufficient and C5-deficient mice from one or both genetic backgrounds (B6 and/or A/J). On the other hand, serum levels of liver aspartate aminotransferase, glucose and urea were not affected by the presence of C5 in either strain. Furthermore, in some cases, C5-dependent variations in these parameters were more evident in mice of the same gender. We conclude here that C5-deficient mice strains may present distinct systemic behaviors which should be taken in consideration before differences in the immune responses are attributed solely to the lack of circulating C5.

The circling mutant Pcdh15roda is a new mouse model for hearing loss

Mouse mutagenesis is a key tool for studying gene function and several mutant alleles have been described and constitute mouse models for human hereditary diseases. Genetic hearing loss represents over 50% of all hearing loss cases in children and, due to the heterogeneity of the disorder, there is still a demand for the isolation and characterization of new genes and alleles. Here we report phenotypic and molecular characterization of a new mouse model for hereditary hearing loss. The mutant rodador, isolated by Massironi and colleagues in 2006, presents an autosomal recessive disorder characterized by deafness and balance dysfunction associated with abnormal stereocilia in the inner ear. The mutation was mapped to mouse chromosome 10, and characterization of the gene Pcdh15 revealed an AT-to-GC transition in intron 23 of mutant animals. The alteration led to the switch of a dinucleotide ApA for ApG, creating a novel intronic acceptor splice site, which leads to incorporation of eight intronic bases into the processed mRNA and alteration of the downstream reading frame. In silico analysis indicated that the mutated protein is truncated and lacks two cadherin domains, and the transmembrane and cytoplasmic domains. Real Time PCR analyses revealed a significantly reduced Pcdh15 mRNA level in the brain of mutant mice, which might be due to the mechanism of non-sense mediated decay. In man, mutations in the orthologue PCDH15 cause non-syndromic deafness and Usher Syndrome Type 1F, a genetic disorder characterized by hearing loss and retinitis pigmentosa. Rodador mouse constitutes a new model for studying deafness in these conditions and may help in the comprehension of the pathogeneses of the disease, as well as of the mechanisms involved in the morphogenesis and function of inner ear stereocilia. This is a new ENU-induced allele and the first isolated in a BALB/c background.

Localization of Bmp-4, Shh and Wnt-5a transcripts during early mice tooth development by in situ hybridization

A comparative nonisotopic in situ hybridization (ISH) analysis was carried out for the detection of Bmp-4, Shh and Wnt-5a transcripts during mice odontogenesis from initiation to cap stage. Bmp-4 was expressed early in the epithelium and then in the underlying mesenchyme. Shh expression was seen in the odontogenic epithelial lining thickening, being stronger in the enamel knot area, during the cap stage. Wnt-5a transcripts were expressed only in the mesenchyme during the initiation, bud and cap stages, with strong expression in the dental mesenchyme during the bud stage. The present results showed that Bmp-4, Shh and Wnt-5a are expressed since the very early stages of tooth development, and they suggest that the Wnt-5a gene is expressed in different cell populations than Bmp-4 and Shh.

Activation of CD4+ and CD8+ parasite -specific T-cells by macrophages infected with live T. cruzi amastigotes.

T. cruzi-infected macrophages are potential candidates for the presentation of parasite antigens to T. cruzi-specific T lymphocytes. To assess this question, we examine the ability of peritoneal exudate macrophages to process exogenous live or dead parasites and to activate defined populations of T. cruzi-specific immune T-cells. Macrophages infected with live amastigotes activated both lymph node CD4+ and spleen CD8 + T-primed cells that proliferated and secreted cytokines. Lymph node CD4+ T-cells produced IFN-gamma and IL-10 while CD8 + T-cells produced IFN-gamma. In contrast, macrophages pulsed with dead parasites activated only lymph node CD4+ T-cells, which proliferated and secreted IFN-gamma. Interestingly, the immunization with heat-killed parasites primed mice for CD8+ T-cells which were expanded in vitro by recognition of infected macrophages. Taken together, these results demonstrated that amastigote infected macrophages present parasite peptides associated with MHC I and II molecules, activating both CD4 + and CD8+ T-cells. Furthermore, the development of T. cruzi-specific CD8+ T-cells in vivo using the immunization protocol with non-living parasites as described in this report could be explored for further studies on the role of CTL in the outcome of infection.

Consequences of pilocarpine-induced status epilepticus in immunodeficient mice

Systemic injection of pilocarpine in rodents induces status epilepticus (SE) and reproduces the main characteristics of temporal lobe epilepsy (TLE). Different mechanisms are activated by SE contributing to cell death and immune system activation. We used BALB/c nude mice, a mutant that is severely immunocompromised, to characterize seizure pattern, neurochemical changes, cell death and c-Fos activation secondarily to pilocarpine-induced SE. The behavioral seizures were less severe in BALB/c nude than in BALB/c wild type mice. However, nude mice presented more tonic-clonic episodes and higher mortality rate during SE. The c-Fos expression was most prominent in the caudate-putamen, CA3 (p<0.05), dentate gyrus, entorhinal cortex (p<0.001), basolateral nucleus of amygdala (p<0.01) and piriform cortex (p<0.05) of BALB/c nude mice than of BALB/c. Besides, nude mice subjected to SE presented high number of Fluorojade-B (FJB) stained cells in the piriform cortex, amygdala (p<0.05) and hilus (p<0.001) in comparison with BALB/c mice. A significant increase in the level of glutamate and GABA was found in the hippocampus and cortex of BALB/c mice presenting SE in comparison to controls. However, the level of glutamate was higher in the brains of BALB nude mice than in the brains of BALB/c wild type mice, while the levels of GABA were unchanged. These results indicate that the brains of immunodeficient nude mice are more vulnerable to the deleterious effects of pilocarpine-induced SE as they present intense activation, increased glutamate levels and more cell death.

Minimal concentration of human IgM and IgG antibodies necessary to protect mice from challenges with live O6 Escherichia coli

This work evaluated the ability of human anti-lipopolysaccharide O6 IgM and IgG antibodies to protect mice challenged with Escherichia coli serotype O6 : K2ac. Purified IgM-effluent, purified IgG, pools of normal human serum (NHS), or control group were injected into mice 18 h before challenges with O6 E. coli. Interleukin 6 and tumor necrosis factor alpha were quantified in the sera of test and control groups. All mice receiving purified IgM-effluent (66.6 mg L(-1) of anti-lipopolysaccharide O6 IgM antibodies) and NHS survived. Purified IgG (1.1 mg L(-1) of anti-lipopolysaccharide O6 IgG antibodies) protected 87.5% of the animals. The control group showed no protective ability. The minimal concentration of anti-lipopolysaccharide O6 IgM antibodies, able to protect 50% of the animals was 33.3 mg L(-1) of purified IgM-effluent, whereas purified IgG was able to protect 50% of the animals with only 1.1 mg L(-1) of anti-lipopolysaccharide O6 IgG antibodies. Serum from animals pretreated with purified IgM-effluent and purified IgG before challenges with lipopolysaccharide O6 did not have detectable pro-inflammatory cytokines. Hepatocytes of the control group were completely invaded by bacteria, whereas none was found in animals pretreated with purified IgM-effluent and purified IgG. Higher concentrations of anti-lipopolysaccharide O6 IgM antibodies as compared to anti-lipopolysaccharide O6 IgG antibodies were needed to protect mice from challenges with E. coli O6 serotype.