RESUMO
AIM: To identify dominant microorganisms in root filled teeth with apical periodontitis by Pan-PCRs in comparison with a culture-dependent approach, focusing on fungal species profiling. METHODOLOGY: The root filling material (gutta-percha) removed from 42 teeth with periapical radiolucencies undergoing root canal retreatments was analysed by molecular genetics techniques. Real-Time Pan-PCRs were conducted for the diagnosis of predominant bacteria (targeting 16S rDNA) and fungi (targeting ITS1-2 region). Identification of microorganisms was performed by Sanger sequencing of the PCR products and BLAST analysis. Additionally, subgingival plaque samples were collected and cultured to review the composition of the microbial flora. The McNemar test and the repeated measures anova were used for statistical analyses (significance level was set at P < 0.05). RESULTS: Overall, 42/42 plaque samples had bacterial growth, whereas 32/42 gutta-percha samples had bacterial growth with a dominance of Streptococcus spp. (12/42) and Enterococcus faecalis (9/42). The mean number of bacterial taxa per gutta-percha sample was 1.6 cultivatable taxa, significantly lower than in the plaque sample that had six taxa/sample (P < 0.001). Fungus-specific cultures were negative for gutta-percha samples, and only one plaque sample had growth of a fungus. In total, 36/42 plaque samples were positive in bacterial Pan-PCRs. In bacterial Pan-PCRs of 31/42 gutta-percha samples, dominant microorganisms were identified including Streptococcus spp. (5/42) and E. faecalis (4/42). Moreover, in 7/42 gutta-percha samples, DNA of bacteria which are difficult-to-cultivate in microbiology routine culture (Acinetobacter,Pyramidobacter,Bacteroidetes,Synergistes,Atopobium and Pseudoramibacter) was found. DNA of Candida spp. was detected in 5/42 root canals by fungal Pan-PCR (1/5) and genus-specific Candida-PCR (5/5). CONCLUSIONS: Pan-PCR assays remain appropriate as a broad-range approach for the detection of a dominant pathogen in gutta-percha samples which have less diverse microbial composition. The molecular genetic Pan-PCR approach has the advantage of detecting microorganisms that are as-yet-uncultivable or difficult-to-cultivate and should be therefore complement conventional microbiological diagnostics.
Assuntos
Cavidade Pulpar , Materiais Restauradores do Canal Radicular , Bactérias , Fungos , Guta-Percha , Projetos Piloto , Obturação do Canal Radicular , Preparo de Canal RadicularRESUMO
The role of Ureaplasma parvum in abnormal outcomes of human pregnancy has been discussed controversially in the past. Of the 14 known ureaplasma serovars, the Ureaplasma parvum serovars 1, 3, 6 and 14, have been found to derive from smaller genomes. Serovars 3 and 6 have been described more often to cause complications in pregnancy. To elucidate the serovar distribution in U. parvum positive specimens of 200 Mongolian mothers and their offspring, a new set of mba-targeting PCRs was developed enabling a fast and reliable serovar differentiation by melting peak analysis in a Real time PCR approach or by conventional agarose gel electrophoresis. 92% maternal and 55% neonatal samples were retrospectively genotyped and a dominance of serovars 3 and 6 was detected while serovar 14 was almost absent. Transmission from mothers to newborns was detected in 83% of U. parvum positive neonates exhibiting serovar patterns identical to their mothers. No statistically significant correlation between a distinct serovar and pregnancy outcome could be detected. However, neonatal colonization with serovar 1 declined with progressing pregnancy suggesting that a higher ureaplasma load shortened pregnancy and thereby had a potential negative effect on offspring health. Our novel mba-based Real time PCR approach, which can also be used in conventional PCR and gel electrophoretic analysis, provides the proof of principle that the four U. parvum serovars 1, 3, 6 and 14 can be differentially detected and quantified. A larger scale study outside the scope of this work should be conducted to clarify the impact of serovar 1 on pregnancy outcome.
Assuntos
Transmissão Vertical de Doenças Infecciosas , Complicações Infecciosas na Gravidez/microbiologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Infecções por Ureaplasma/diagnóstico , Infecções por Ureaplasma/transmissão , Ureaplasma/genética , DNA Bacteriano/genética , Feminino , Humanos , Recém-Nascido , Mongólia , Gravidez , Estudos Prospectivos , Inquéritos e Questionários , Ureaplasma/classificação , Ureaplasma/isolamento & purificação , Infecções por Ureaplasma/microbiologiaRESUMO
Mesenchymal stromal cells (MSCs) have a multilineage differentiation potential and provide immunosuppressive and antimicrobial functions. Murine as well as human MSCs restrict the proliferation of T cells. However, species-specific differences in the underlying molecular mechanisms have been described. Here, we analyzed the antiparasitic effector mechanisms active in murine MSCs. Murine MSCs, in contrast to human MSCs, could not restrict the growth of a highly virulent strain of Toxoplasma gondii (BK) after stimulation with IFN-γ. However, the growth of a type II strain of T. gondii (ME49) was strongly inhibited by IFN-γ-activated murine MSCs. Immunity-related GTPases (IRGs) as well as guanylate-binding proteins (GBPs) contributed to this antiparasitic effect. Further analysis showed that IFN-γ-activated mMSCs also inhibit the growth of Neospora caninum, a parasite belonging to the apicomplexan group as well. Detailed studies with murine IFN-γ-activated MSC indicated an involvement in IRGs like Irga6, Irgb6 and Irgd in the inhibition of N. caninum. Additional data showed that, furthermore, GBPs like mGBP1 and mGBP2 could have played a role in the anti-N. caninum effect of murine MSCs. These data underline that MSCs, in addition to their regenerative and immunosuppressive activity, function as antiparasitic effector cells as well. However, IRGs are not present in the human genome, indicating a species-specific difference in anti-T. gondii and anti-N. caninum effect between human and murine MSCs.
Assuntos
GTP Fosfo-Hidrolases/metabolismo , Proteínas de Ligação ao GTP/imunologia , Proteínas de Ligação ao GTP/metabolismo , Células-Tronco Mesenquimais/enzimologia , Células-Tronco Mesenquimais/imunologia , Neospora/imunologia , Toxoplasma/imunologia , Animais , Interferon gama/metabolismo , Camundongos , Neospora/crescimento & desenvolvimento , Toxoplasma/crescimento & desenvolvimentoRESUMO
In the post-genome era, the mouse will have a major role as a model system for functional genome analysis. This requires a large number of mutants similar to the collections available from other model organisms such as Drosophila melanogaster and Caenorhabditis elegans. Here we report on a systematic, genome-wide, mutagenesis screen in mice. As part of the German Human Genome Project, we have undertaken a large-scale ENU-mutagenesis screen for dominant mutations and a limited screen for recessive mutations. In screening over 14,000 mice for a large number of clinically relevant parameters, we recovered 182 mouse mutants for a variety of phenotypes. In addition, 247 variant mouse mutants are currently in genetic confirmation testing and will result in additional new mutant lines. This mutagenesis screen, along with the screen described in the accompanying paper, leads to a significant increase in the number of mouse models available to the scientific community. Our mutant lines are freely accessible to non-commercial users (for information, see http://www.gsf.de/ieg/groups/enu-mouse.html).
Assuntos
Etilnitrosoureia/farmacologia , Genoma , Mutagênicos/farmacologia , Mutação/efeitos dos fármacos , Animais , Cruzamentos Genéticos , Criopreservação , Feminino , Membro Anterior/anormalidades , Imunidade/genética , Imunidade/imunologia , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Mutagênese , Mutação/genética , Mutação/imunologia , FenótipoRESUMO
Successful transplantation of allogeneic organs is an important objective in modern medicine. However, sophisticated immune defense mechanisms, primarily evolved to combat infections, often work against medical transplantation. To investigate the roles of natural and adaptive immune responses in transplant rejection, we functionally inactivated key effector systems of the innate (NK cells) and the adaptive immune system (CD28-mediated costimulation of T cells) in mice. Neither of these interventions alone led to acceptance of allogeneic vascularized cardiac grafts. In contrast, inhibition of NK-receptor-bearing cells combined with CD28-costimulation blockade established long-term graft acceptance. These results indicate a concerted interplay between innate and adaptive immune surveillance for graft rejection. Thus we suggest that inactivation of NK-receptor-bearing cells could be a new strategy for successful survival of solid-organ transplants.
Assuntos
Antígenos CD28/fisiologia , Sobrevivência de Enxerto/imunologia , Transplante de Coração , Células Matadoras Naturais/imunologia , Animais , Antígenos CD28/genética , Citocinas/genética , Depleção Linfocítica , Camundongos , Camundongos Knockout , RNA Mensageiro/genética , Transplante HomólogoRESUMO
Lymphotoxin alpha (LT-alpha) may form secreted homotrimers binding to p55 and p75 tumor necrosis factor (TNF) receptors or cell surface-bound heterotrimers with LT-beta that interact with the LT-beta receptor. Genetic ablation of LT-alpha revealed that mutant mice have no detectable lymph nodes or Peyer's patches and that the organization of the splenic white pulp in T and B cell areas is disturbed. In this report we describe a novel function for the p55 TNF receptor during ontogeny and demonstrate that mice deficient for p55 completely lack organized Peyer's patches. In contrast, lymph nodes and spleen are present in p55-deficient mice and lymphocytes segregate normally into B and T cell areas in these organs. Lamina propria and intraepithelial lymphocytes of the small intestine were detected in normal number and distribution in p55 mutant mice. Lymphocytes and endothelial cells from p55-deficient mice express normal levels of adhesion molecules considered important for lymphocyte migration to mucosal organs; this indicates that the lack of Peyer's patches does not result from a defect in lymphocyte homing. In summary, the p55 receptor for TNF selectively mediates organogenesis of Peyer's patches throughout ontogeny, suggesting that the effects of LT-alpha on the development of lymphoid organs may be mediated by distinct receptors, each functioning in an organ-specific context.
Assuntos
Cadeias beta de Integrinas , Nódulos Linfáticos Agregados/anormalidades , Receptores do Fator de Necrose Tumoral/deficiência , Animais , Antígenos CD/fisiologia , Integrina alfa4 , Integrina beta1/fisiologia , Integrinas/fisiologia , Selectina L/metabolismo , Antígeno-1 Associado à Função Linfocitária/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peso Molecular , Nódulos Linfáticos Agregados/crescimento & desenvolvimento , Receptores do Fator de Necrose Tumoral/química , Baço/patologiaRESUMO
T cell receptor recognition of antigen can lead either to T lymphocyte differentiation and proliferation or to a state of unresponsiveness, which is dependent on whether appropriate costimulatory signals are provided to the mature T cell. We have investigated a novel intracellular signaling pathway provided by the costimulatory molecule CD28. CD28 engagement triggers the activation of an acidic sphingomyelinase (A-SMase), which results in the generation of ceramide, an important lipid messenger intermediate. A-SMase activation by CD28 occurred in resting as well as in activated primary T cells or leukemic Jurkat cells. In contrast, ligation of either CD3 or CD2 did not result in A-SMase activation. Overexpression of recombinant A-SMase in Jurkat T cells substituted for CD28 with regard to nuclear factor-kB activation. These data suggest that CD28 provides an important costimulatory signal by activation of an acidic sphingomyelinase pathway.
Assuntos
Antígenos CD28/fisiologia , Receptores de Antígenos de Linfócitos T/fisiologia , Transdução de Sinais , Esfingomielina Fosfodiesterase/metabolismo , Linfócitos T/fisiologia , Animais , Diferenciação Celular , Linhagem Celular , Cricetinae , Ativação Enzimática , Feminino , Humanos , Cinética , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Fito-Hemaglutininas , Linfócitos T/citologia , Linfócitos T/enzimologiaRESUMO
The formation of germinal centers (GCs) represents a crucial step in the humoral immune response. Recent studies using gene-targeted mice have revealed that the cytokines tumor necrosis factor (TNF), lymphotoxin (LT) alpha, and LTbeta, as well as their receptors TNF receptor p55 (TNFRp55) and LTbetaR play essential roles in the development of GCs. To establish in which cell types expression of LTbetaR, LTbeta, and TNF is required for GC formation, LTbetaR-/-, LTbeta-/-, TNF-/-, B cell-deficient (BCR-/-), and wild-type mice were used to generate reciprocal or mixed bone marrow (BM) chimeric mice. GCs, herein defined as peanut agglutinin-binding (PNA+) clusters of centroblasts/centrocytes in association with follicular dendritic cell (FDC) networks, were not detectable in LTbetaR-/- hosts after transfer of wild-type BM. In contrast, the GC reaction was restored in LTbeta-/- hosts reconstituted with either wild-type or LTbetaR-/- BM. In BCR-/- recipients reconstituted with compound LTbeta-/-/BCR-/- or TNF-/-/BCR-/- BM grafts, PNA+ cell clusters formed in splenic follicles, but associated FDC networks were strongly reduced or absent. Thus, development of splenic FDC networks depends on expression of LTbeta and TNF by B lymphocytes and LTbetaR by radioresistant stromal cells.
Assuntos
Linfócitos B/metabolismo , Células Dendríticas/metabolismo , Linfotoxina-alfa/metabolismo , Proteínas de Membrana/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Baço/crescimento & desenvolvimento , Células Estromais/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Transplante de Medula Óssea , Regulação da Expressão Gênica no Desenvolvimento/genética , Centro Germinativo/metabolismo , Imuno-Histoquímica , Receptor beta de Linfotoxina , Linfotoxina-beta , Camundongos , Camundongos Knockout , Baço/metabolismo , Células Estromais/efeitos da radiação , Irradiação Corporal TotalRESUMO
T cell receptor stimulation without costimulation is insufficient for the induction of an optimal immune response. It is thought that engagement of the CD28 molecule with its ligand B7 provides an essential costimulatory signal without which full activation of T cells cannot occur. A mouse strain with a defective CD28 gene was established. Development of T and B cells in the CD28-deficient mice appeared normal. However, T lymphocytes derived from CD28-/- mutant mice had impaired responses to lectins. Lectin stimulation did not trigger interleukin-2 (IL-2) production, IL-2 receptor alpha expression was significantly decreased, and exogenous IL-2 only partially rescued the CD28 defect. Basal immunoglobulin (Ig) concentrations in CD28-deficient mice were about one-fifth of those found in wild-type controls, with low titers of IgG1 and IgG2b but an increase in IgG2a. In addition, activity of T helper cells in CD28-/- mice was reduced and immunoglobulin class switching was diminished after infection with vesicular stomatitis virus. However, cytotoxic T cells could still be induced and the mice showed delayed-type hypersensitivity after infection with lymphocytic choriomeningitis virus. Thus, CD28 is not required for all T cell responses in vivo, suggesting that alternative costimulatory pathways may exist.
Assuntos
Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos T/imunologia , Ativação Linfocitária , Linfócitos T/imunologia , Animais , Anticorpos Antivirais/sangue , Antígenos CD/genética , Antígenos de Diferenciação de Linfócitos T/genética , Antígenos de Superfície/imunologia , Linfócitos B/imunologia , Antígeno B7-1 , Antígenos CD28 , Concanavalina A/farmacologia , Imunoglobulinas/sangue , Interleucina-2/biossíntese , Interleucina-2/farmacologia , Coriomeningite Linfocítica/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Mutantes , Mutação , Receptores de Interleucina-2/metabolismo , Linfócitos T Citotóxicos/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Vírus da Estomatite Vesicular Indiana/imunologia , Viroses/imunologiaRESUMO
OBJECTIVES: To analyse the injury-related content of children's television programmes preferred by boys and by girls, and to determine whether there are more televised models of unsafe behaviour in programmes preferred by boys. METHODS: Parents of 4-11-year-old children identified their children's favourite television programmes. Content analysis of 120 episodes of children's favourite programmes was used to quantify safe and risky behaviours, actual injuries and potential injuries. The gender of the characters portraying the behaviours was also analysed. RESULTS: More risky behaviour was portrayed in the boys' favourite programmes (mean per episode = 6.40) than in the girls' favourite programmes (mean = 2.57). There were almost twice as many potential injuries (n = 310) as actual injuries (n = 157). Potential injuries were portrayed more often by male characters (mean = 1.92) than female characters (mean = 0.98), mostly in the boys' favourite programmes. Actual injuries occurred more often to male characters (mean = 1.04) than to female characters (mean = 0.27) overall. CONCLUSIONS: Television programmes preferred by this sample of boys portrayed male role models engaging in risky behaviours and injuries more often than the programmes preferred by the sample of girls.
Assuntos
Assunção de Riscos , Televisão/estatística & dados numéricos , Ferimentos e Lesões/psicologia , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Segurança , Fatores Sexuais , Ferimentos e Lesões/etiologiaRESUMO
The mechanism of cargo coupling to kinesin motor proteins is a fundamental issue in organelle transport along microtubules. Kinectin has been postulated to function as a membrane anchor protein that attaches various organelles to the prototype motor protein kinesin. To verify the biological relevance of kinectin in vivo, the murine kinectin gene was disrupted by homologous recombination. Unexpectedly, kinectin-deficient mice were viable and fertile, and no gross abnormalities were observed up to 1 year of age. The assembly of the endoplasmic reticulum was essentially unaffected in kinectin-deficient cells. Mitochondria appeared to be correctly distributed throughout the cytoplasm along the microtubules. Furthermore, the stationary distribution and the bidirectional movement of lysosomes did not depend on kinectin. Kinectin-deficient phagocytes internalized and cleared bacteria, indicating that phagosome trafficking and maturation are functional without kinectin. Thus, these data unequivocally indicate that kinectin is not essential for trafficking of lysosomes, phagosomes, and mitochondria in vivo.
Assuntos
Proteínas Sanguíneas/fisiologia , Lisossomos/metabolismo , Proteínas de Membrana , Fagossomos/fisiologia , Alelos , Animais , Transporte Biológico , Proteínas Sanguíneas/genética , Linhagem Celular , Humanos , Membranas Intracelulares/metabolismo , Camundongos , Camundongos Knockout , Mutagênese , Organelas/metabolismo , Fagocitose , Fagossomos/metabolismo , Frações SubcelularesRESUMO
The major histocompatibility complex (MHC) glycoproteins, MHC1 and MHC2, play a key role in the presentation of antigen and the development of the immune response. In the current study we examined the regulation of the MHC2 in the mouse brain after facial axotomy. The normal facial motor nucleus showed very few slender and elongated MHC2+ cells. Transection of the facial nerve led to a gradual but strong upregulation in the number of MHC2+ cells, beginning at day 2 and reaching a maximum 14 days after axotomy, correlated with the induction of mRNA for tumor necrosis factor (TNF) alpha, interleukin (IL) 1beta and interferon-gamma (IFNgamma) and a peak in neuronal cell death. In almost all cases, MHC2 immunoreactivity was restricted to perivascular macrophages that colocalized with vascular basement membrane laminin and macrophage IBA1-immunoreactivity, with no immunoreactivity on phagocytic microglia, astrocytes or invading T-cells. Heterologous transplantation and systemic injection of endotoxin or IFNgamma did not affect this perivascular MHC2 immunoreactivity, and transgenic deletion of the IL1 receptor type I, or TNF receptor type 1, also had no effect. However, the deletion of IFNgamma receptor subunit 1 caused a significant increase, and that of TNF receptor type 2 a strong reduction in the number of MHC2+ macrophages, pointing to a counter-regulatory role of IFNgamma and TNFalpha in the immune surveillance of the injured nervous system.
Assuntos
Traumatismos do Nervo Facial/metabolismo , Genes MHC da Classe II/fisiologia , Macrófagos/metabolismo , Receptores de Interferon/fisiologia , Receptores do Fator de Necrose Tumoral/fisiologia , Animais , Axotomia/métodos , Nervo Facial/metabolismo , Traumatismos do Nervo Facial/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Interferon/deficiência , Receptores de Interferon/genética , Receptores do Fator de Necrose Tumoral/deficiência , Receptores do Fator de Necrose Tumoral/genética , Fator 1 Associado a Receptor de TNF/deficiência , Fator 1 Associado a Receptor de TNF/genética , Fator 1 Associado a Receptor de TNF/fisiologia , Fator 2 Associado a Receptor de TNF/deficiência , Fator 2 Associado a Receptor de TNF/genética , Fator 2 Associado a Receptor de TNF/fisiologia , Receptor de Interferon gamaRESUMO
Two protocols were examined for the ability to transfer a human T cell system into SCID mice. Upon intraperitoneal injection (i.p.) of human peripheral blood lymphocytes (PBL) into SCID mice the injected cells could be recovered over weeks from the peritoneal cavity, yet human T cells did not seed into secondary lymphoid organs such as the spleen, lymph nodes or bone marrow. In contrast, SCID mice grafted with human embryonal thymus tissue contained high numbers of CD4+CD8- and CD8+CD4- human T cells in their lymph nodes and spleen when they had been injected i.p. with human PBL.
Assuntos
Síndromes de Imunodeficiência/imunologia , Linfócitos T/transplante , Animais , Antígenos de Diferenciação de Linfócitos T/análise , Citotoxicidade Imunológica , Humanos , Imunidade Celular , Camundongos , Camundongos Mutantes/imunologia , Linfócitos T/imunologia , Timo/transplanteRESUMO
Freshly isolated human T lymphocytes were tested for their response to mycobacteria, mycobacterial lysates, 2 dimensional (2D) PAGE separated mycobacterial lysates, leishmania and defined leishmanial antigen preparations. While gamma delta T cells proliferated vigorously in the presence of mycobacteria and mycobacteria derived lysates, a significant stimulation from 2 D gel separated lysates was not detected. In addition gamma delta T cells failed to respond towards leishmania or leishmanial components. In the alpha beta T cell compartment some donors, presumably according to their state of immunity against mycobacteria, responded to mycobacteria, mycobacterial lysates and 2 D gel separated mycobacterial lysates. Neither freshly isolated gamma delta T cells nor alpha beta T cells from naive donors did mount a significant immune response against leishmania.
Assuntos
Subpopulações de Linfócitos T/imunologia , Animais , Proteínas de Choque Térmico/imunologia , Humanos , Técnicas In Vitro , Leishmania/imunologia , Ativação Linfocitária , Mycobacterium/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta , Receptores de Antígenos de Linfócitos T gama-deltaRESUMO
Intestinal intraepithelial lymphocytes (IEL) appear to represent a peculiar set of immune cells compartmentalized at the interface between the organism and the external environment. In previous studies we observed that within human IEL TCR-tau/delta T cells represent a major fraction that predominantly express the CD8 molecule and preferentially uses the V-delta-1 gene segment. Thus these data suggested a preferential accumulation/homing of CD8+ V-delta-1+ IEL within the human intestinal epithelium. However, to date the functional role of these cells with regard to immune regulation at this most critical immunological site is poorly understood. In this study, the cytotoxic potential and proliferative capacity of human IEL in response to mitogenic stimuli has been characterized with respect to IEL T cell receptor type and TCR-tau/delta variable gene segment usage as determined by flowmetry. The frequency of TCR-1+ IEL expressing both CD56 and CD16 which are considered to be NK-cell markers was found to be much higher (38.9 +/- 12.4%) than within intestinal lamina propria lymphocytes (LPL) (9.1 +/- 4.8%) or peripheral blood lymphocytes (PBL) (6.4 +/- 3.3%). In contrast, the fractions of CD16-CD56+ cells within IEL, LPL and PBL were comparable. Surprisingly, IEL mediated NK-cell activity (K562 lysis) was virtually absent whereas within PBL it was within the normal range. Furthermore, in cytotoxicity assays employing 51Cr-labeled OKT3 hybridoma cells and P815 cells as targets, the cytotoxic potential of IEL was much lower than that of PBL.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Intestinos/imunologia , Subpopulações de Linfócitos/imunologia , Receptores de Antígenos de Linfócitos T gama-delta , Adulto , Antígenos CD , Colo/citologia , Colo/imunologia , Células Epiteliais , Epitélio/imunologia , Humanos , Intestinos/citologia , FenótipoRESUMO
Presentation of antigen is key to the development of the immune response, mediated by association of antigen with major histocompatibility complex glycoproteins abbreviated as MHC1 and MHC2. In the current study, we examined the regulation of MHC1 in the brain after facial axotomy. The normal facial motor nucleus showed no immunoreactivity for MHC1 (MHC1-IR). Transection of the facial nerve led to a strong and selective up-regulation of MHC1-IR on the microglia in the affected nucleus, beginning at day 2 and reaching a maximum 14 days after axotomy, coinciding with a peak influx of the T lymphocytes that express CD8, the lymphocyte coreceptor for MHC1. Specificity of the MHC1 staining was confirmed in beta2-microglobulin-deficient mice, which lack normal cell surface MHC1-IR. MHC1-IR was particularly strong on phagocytic microglia, induced by delayed neuronal cell death, and correlated with the induction of mRNA for tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and interferon-gamma and the influx of T lymphocytes. Mice with severe combined immunodeficiency (scid), lacking T and B cells, showed an increase in the number of MHC1-positive nodules but no significant effect on overall MHC1-IR. Transgenic deletion of the IL1 receptor type I, or the interferon-gamma receptor type 1 subunit, did not affect the microglial MHC1-IR. However, a combined deletion of TNF receptors 1 and 2 (TNFR1&2-KO) led to a decrease in microglial MHC1-IR and to a striking absence of the phagocytic microglial nodules. Deletion of TNFR2 (p75) did not have an effect; deletion of TNFR1 (p55) reduced the diffuse microglial staining for MHC1-IR but did not abolish the MHC1(+) microglial nodules. In summary, neural injury leads to the induction of MHC1-IR on the activated, phagocytic microglia. This induction of MHC1 precedes the interaction with the immune system, at least in the facial motor nucleus model. Finally, the impaired induction of these molecules, up to now, only in the TNFR-deficient mice underscores the central role of TNF in the immune activation of the injured nervous system.
Assuntos
Antígenos CD/fisiologia , Nervo Facial/fisiologia , Glicoproteínas/biossíntese , Antígenos de Histocompatibilidade Classe I/biossíntese , Microglia/fisiologia , Receptores do Fator de Necrose Tumoral/fisiologia , Animais , Antígenos CD/genética , Axotomia , Nervo Facial/química , Glicoproteínas/antagonistas & inibidores , Antígenos de Histocompatibilidade Classe I/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos SCID , Microglia/química , Receptores do Fator de Necrose Tumoral/deficiência , Receptores do Fator de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral , Receptores Tipo II do Fator de Necrose TumoralRESUMO
Analysis of the V beta-repertoire of antigen-reactive T cell populations can be approached using either flow-cytometry or PCR-based techniques. While the former method requires a complete set of V beta-specific monoclonal antibodies (mAbs) and large cell numbers for analysis, the latter is both time-consuming and labour-intensive. To circumvent the drawbacks of both these methods we have employed the recently developed technique of TaqManR PCR to analyse the V beta-usage of human T cell populations. TaqManR PCR is based on the 5'-->3' nuclease activity of Taq polymerase. During PCR amplification an internal oligonucleotide probe, that is labelled with a fluorescent reporter and a quencher dye, is cleaved by Taq polymerase. After cleavage, quenching of the reporter dye is lost and reporter fluorescence can be detected with a fluorescence plate reader. Using one C beta-specific fluorogenic probe and a panel of V beta-specific primers, we show that fluorescence-detected amplification of TCR beta cDNA is V beta-specific and linear within a 2-3-log range of template concentration. The sensitivity of TaqManR PCR is comparable to conventional detection of PCR-products by agarose gel staining, while processing time is reduced. Furthermore, superantigen-induced skewing of the V beta-repertoire of human T cells is readily detected with this method. Thus TaqManR PCR is a reliable and fast method for semiquantitative analysis of the V beta-repertoire of human T cell populations.
Assuntos
Família Multigênica/imunologia , Reação em Cadeia da Polimerase/métodos , Receptores de Antígenos de Linfócitos T alfa-beta/análise , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Subpopulações de Linfócitos T/metabolismo , Células Cultivadas , DNA Polimerase Dirigida por DNA , Humanos , Magnésio/fisiologia , Sondas de Oligonucleotídeos/normas , Reação em Cadeia da Polimerase/normas , Valores de Referência , Sensibilidade e Especificidade , Subpopulações de Linfócitos T/química , Taq PolimeraseRESUMO
Co-stimulatory factors are involved in different forms of brain pathology and play an important role in the activation of T-cells. In the current study, we explored the regulation of B7.2, a prominent member of the B7 family of costimulatory factors, in the facial motor nucleus (FMN) following facial axotomy and systemic application of lipopolysaccharide (LPS, endotoxin) using light and electron immunohistochemistry and cytokine-receptor-deficient mice. Facial axotomy led to a gradual increase of B7.2 immunoreactivity (IR) on microglial cell surface; similar effects were also observed following application of LPS, but both effects were not additive, suggesting overlapping or saturated signaling pathways. Some B7.2-IR was already present on activated microglia surrounding injured neurons at days 1-4 after injury, but became particularly intense during neuronal cell death, peaking at day 14. Previous studies revealed that these late microglial changes are accompanied by a strong increase in the expression of proinflammatory cytokines such as interleukin-1 beta (IL1beta) tumor necrosis factor-alpha (TNFalpha) and interferon gamma (IFNgamma) [J. Neurosci. 18 (1998a) 5804]. Here, deletion of the receptors for these cytokines-IL1R1, TNFR1 or TNFR2, but not IFNgammaR1-caused a strong and significant reduction in B7.2-IR in reactive microglial cells, compared with their wild type (WT) controls on the same genetic strain background, with a 31% decrease in IL1R1-/- , 39% in TNFR1-/- and 49% in TNFR2-/- mice. These data underscore the significance of IL1beta, TNFalpha and LPS, and their receptors, as potent inflammatory signals that regulate the cellular response in the injured brain as well as the interaction with the rapidly recruited immune system. The broad susceptibility of B7.2 regulation to a wide range of different inflammatory signals also points to its role as a sensor of molecular pathology, and a factor that plays an important accessory role in allowing and shaping the microglia/T-cell interaction in the injured central nervous system.
Assuntos
Antígenos CD/fisiologia , Endotoxinas/farmacologia , Traumatismos do Nervo Facial/imunologia , Traumatismos do Nervo Facial/metabolismo , Glicoproteínas de Membrana/fisiologia , Microglia/fisiologia , Receptores de Interleucina-1/fisiologia , Receptores Tipo II do Fator de Necrose Tumoral/fisiologia , Receptores Tipo I de Fatores de Necrose Tumoral/fisiologia , Animais , Axotomia , Antígeno B7-2 , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/efeitos dos fármacos , Microglia/metabolismo , Fagócitos/efeitos dos fármacos , Fagócitos/metabolismo , Fagócitos/fisiologia , Receptores Tipo I de Interleucina-1RESUMO
OBJECTIVE: Infants with bronchopulmonary dysplasia (BPD) have been previously reported to have a decrease in growth velocity after stopping supplemental oxygen (SO). SO was stopped after a short-term recording (20-30 minutes) of pulse oxygen saturation (Sao2) of 92% or greater in room air. Other studies have documented that Sao2 decreases further during feedings and sleep in infants with BPD. Two questions were asked: (1) whether short-term, awake Sao2 studies would reliably predict prolonged sleep Sao2; and (2) how Sao2 sustained at 88% to 91% vs 92% or greater in room air would impact growth velocity in infants with BPD. METHODOLOGY: Short-term Sao2 studies were prospectively compared with prolonged sleep Sao2 (n = 63) and the growth velocity of infants who had SO discontinued after a prolonged sleep Sao2 recording of 88% to 91% (group 1; n = 14) versus 92% or greater (group 2; n = 34) in room air. RESULTS: Failure to maintain Sao2 at predetermined levels occurred in 18 (29%) of 63 infants during their first prolonged sleep study. There was no correlation between short-term awake Sao2 and prolonged sleep Sao2 recordings (r = .02). Body weight, height, weight for height, and rate of weight gain were similar for all study infants before SO was stopped and remained constant for group 2 infants after SO was stopped. However, group 1 infants had a significant decrease in the rate of weight gain (17.3 +/- 13.1 vs 3.7 +/- 6.1 g/kg per day), and the mean z scores for weight gain and weight for height also decreased significantly for group 1 infants. Energy intake, incidence of acute infection, hematocrit values, and medication use did not differ before or after stopping SO in either group. CONCLUSIONS: This study indicated that short-term, awake Sao2 measurements do not predict prolonged sleep Sao2, and overall, infants with BPD continued a positive growth trend when Sao2, remained greater than 92% during prolonged sleep.
Assuntos
Displasia Broncopulmonar/terapia , Crescimento/fisiologia , Hipóxia/terapia , Sono/fisiologia , Análise de Variância , Displasia Broncopulmonar/sangue , Displasia Broncopulmonar/fisiopatologia , Humanos , Hipóxia/sangue , Hipóxia/fisiopatologia , Lactente , Recém-Nascido , Oxigênio/sangue , Oxigenoterapia/estatística & dados numéricos , Estudos Prospectivos , Fatores de Tempo , Aumento de Peso/fisiologiaRESUMO
Acute lung injury represents a wide spectrum of pathologic processes, the most severe end of the spectrum being the acute respiratory distress syndrome. Reactive oxygen intermediates have been implicated as important in the pathobiochemistry of acute lung injury. The endogenous sources that contribute to the generation of reactive oxygen intermediates in acute lung injury are poorly defined but probably include the molybdenum hydroxylases, NAD(P)H oxidoreductases, the mitochondrial electron transport chain, and arachidonic acid-metabolizing enzymes. Our laboratory has focused, in particular, on the regulation of two of these enzyme systems, xanthine oxidoreductase (XDH/XO) and NAD(P)H oxidase. We observe that gene expression of XDH/XO is regulatory in a cell-specific manner and is markedly affected by inflammatory cytokines, steroids, and physiologic events such as hypoxia. Posttranslational processing is also important in regulating XDH/XO activity. More recently, the laboratory has characterized an NAD(P)H oxidase in vascular cells. The cytochrome components of the oxidase, gp91 and p22, appear similar to the components present in phagocytic cells that contribute to their respiratory burst. In human vascular endothelial and smooth muscle cells, oncostatin M potently induces gp91 expression. We believe that regulation of gp91 is a central controlling factor in expression of the vascular NAD(P)H oxidase. In summary, the studies support the concept that the oxidoreductases of vascular cells are expressed in a highly regulated and self-specific fashion.