RESUMO
Systems biology has to increasingly cope with large- and multi-scale biological systems. Many successful in silico representations and simulations of various cellular modules proved mathematical modelling to be an important tool in gaining a solid understanding of biological phenomena. However, models spanning different functional layers (e.g. metabolism, signalling and gene regulation) are still scarce. Consequently, model integration methods capable of fusing different types of biological networks and various model formalisms become a key methodology to increase the scope of cellular processes covered by mathematical models. Here we propose a new integration approach to couple logical models of signalling or/and gene-regulatory networks with kinetic models of metabolic processes. The procedure ends up with an integrated dynamic model of both layers relying on differential equations. The feasibility of the approach is shown in an illustrative case study integrating a kinetic model of central metabolic pathways in hepatocytes with a Boolean logical network depicting the hormonally induced signal transduction and gene regulation events involved. In silico simulations demonstrate the integrated model to qualitatively describe the physiological switch-like behaviour of hepatocytes in response to nutritionally regulated changes in extracellular glucagon and insulin levels. A simulated failure mode scenario addressing insulin resistance furthermore illustrates the pharmacological potential of a model covering interactions between signalling, gene regulation and metabolism.
Assuntos
Redes Reguladoras de Genes , Modelos Biológicos , Transdução de Sinais , Calibragem , CinéticaRESUMO
Gene flow is particularly frequent in the genus Quercus (oaks), especially between closely related species. We focus here on Quercus ilex and the cork-producing Quercus suber, which occasionally hybridize although they are phylogenetically markedly separated. Morphological observations were combined with both allozymic and chloroplastic diagnostic markers to characterize hybridization and introgression and to infer their dynamics in two French regions (French Catalonia and Provence), which are separated by several hundred kilometres. Some hybrids were found in both regions, indicating recent hybridization events. As expected from previous studies, most hybrids resulted from female symbol Q. ilex x male symbol Q. suber crosses, but our data showed that the reciprocal cross is also possible. Partial independence between nuclear and chloroplastic introgression was observed in the two species. Nuclear introgression was limited in both species and both regions, with no preferred direction. In Provence, chloroplastic introgression was very rare in both species. Conversely, all Q. suber individuals from French Catalonia were introgressed by Q. ilex chlorotypes. This might be explained by introgression in the Iberian Peninsula antedating the first occurrence of the two species in French Catalonia. We also observed a new chlorotype that was created locally, and was exchanged between the two species. However, the two species still remain genetically differentiated. The dynamics and complexity of exchanges and the factors determining them (including human management of Q. suber) are discussed.
Assuntos
Cruzamentos Genéticos , Fluxo Gênico , Variação Genética , Hibridização Genética , Quercus/genética , DNA de Cloroplastos , França , Frequência do Gene , Genoma , Genótipo , GeografiaRESUMO
Recently, the amplified fragment length polymorphism (AFLP) technique has gained a lot of popularity, and is now frequently applied to a wide variety of organisms. Technical specificities of the AFLP procedure have been well documented over the years, but there is on the contrary little or scattered information about the statistical analysis of AFLPs. In this review, we describe the various methods available to handle AFLP data, focusing on four research topics at the population or individual level of analysis: (i) assessment of genetic diversity; (ii) identification of population structure; (iii) identification of hybrid individuals; and (iv) detection of markers associated with phenotypes. Two kinds of analysis methods can be distinguished, depending on whether they are based on the direct study of band presences or absences in AFLP profiles ('band-based' methods), or on allelic frequencies estimated at each locus from these profiles ('allele frequency-based' methods). We investigate the characteristics and limitations of these statistical tools; finally, we appeal for a wider adoption of methodologies borrowed from other research fields, like for example those especially designed to deal with binary data.
Assuntos
Técnicas Genéticas , Polimorfismo Genético , Evolução Biológica , Biologia Computacional/métodos , Simulação por Computador , Ecologia/métodos , Frequência do Gene , Marcadores Genéticos , Hibridização Genética , Fenótipo , SoftwareRESUMO
The detection of adaptive loci in the genome is essential as it gives the possibility of understanding what proportion of a genome or which genes are being shaped by natural selection. Several statistical methods have been developed which make use of molecular data to reveal genomic regions under selection. In this paper, we propose an approach to address this issue from the environmental angle, in order to complement results obtained by population genetics. We introduce a new method to detect signatures of natural selection based on the application of spatial analysis, with the contribution of geographical information systems (GIS), environmental variables and molecular data. Multiple univariate logistic regressions were carried out to test for association between allelic frequencies at marker loci and environmental variables. This spatial analysis method (SAM) is similar to current population genomics approaches since it is designed to scan hundreds of markers to assess a putative association with hundreds of environmental variables. Here, by application to studies of pine weevils and breeds of sheep we demonstrate a strong correspondence between SAM results and those obtained using population genetics approaches. Statistical signals were found that associate loci with environmental parameters, and these loci behave atypically in comparison with the theoretical distribution for neutral loci. The contribution of this new tool is not only to permit the identification of loci under selection but also to establish hypotheses about ecological factors that could exert the selection pressure responsible. In the future, such an approach may accelerate the process of hunting for functional genes at the population level.
Assuntos
Adaptação Biológica , Genômica/métodos , Ovinos/genética , Gorgulhos/genética , Animais , Frequência do Gene , Marcadores Genéticos , Sistemas de Informação Geográfica , Repetições de Microssatélites , Polimorfismo Genético , Análise de Regressão , Seleção Genética , Análise de Sequência de DNARESUMO
Genotyping errors occur when the genotype determined after molecular analysis does not correspond to the real genotype of the individual under consideration. Virtually every genetic data set includes some erroneous genotypes, but genotyping errors remain a taboo subject in population genetics, even though they might greatly bias the final conclusions, especially for studies based on individual identification. Here, we consider four case studies representing a large variety of population genetics investigations differing in their sampling strategies (noninvasive or traditional), in the type of organism studied (plant or animal) and the molecular markers used [microsatellites or amplified fragment length polymorphisms (AFLPs)]. In these data sets, the estimated genotyping error rate ranges from 0.8% for microsatellite loci from bear tissues to 2.6% for AFLP loci from dwarf birch leaves. Main sources of errors were allelic dropouts for microsatellites and differences in peak intensities for AFLPs, but in both cases human factors were non-negligible error generators. Therefore, tracking genotyping errors and identifying their causes are necessary to clean up the data sets and validate the final results according to the precision required. In addition, we propose the outline of a protocol designed to limit and quantify genotyping errors at each step of the genotyping process. In particular, we recommend (i) several efficient precautions to prevent contaminations and technical artefacts; (ii) systematic use of blind samples and automation; (iii) experience and rigor for laboratory work and scoring; and (iv) systematic reporting of the error rate in population genetics studies.
Assuntos
Genética Populacional , Genótipo , Projetos de Pesquisa , Viés de Seleção , Animais , Betula/genética , Humanos , Repetições de Microssatélites , Polimorfismo Genético , Rana temporaria/genética , Estatística como Assunto , Ursidae/genéticaRESUMO
CD83 is a marker molecule for mature dendritic cells (DCs) but is also substantially expressed on activated T cells in humans and mice. Its function is unknown, but CD83 knockout mice show an impaired thymic maturation of CD4-positive cells and soluble CD83 inhibits partially antigen-specific responses in vitro pointing to a role of CD83 in the immune system. Here we show that CD83-positive T cells produce strongly increased amounts of interferon-gamma and interleukin-2. In contrast, constitutive expression of CD83 on DCs alters neither the activation of DCs following addition of lipopolysaccharide nor the ability to present antigenic peptides. Thus, the expression of CD83 on T cells has direct functional consequences for tuning the activation threshold.
Assuntos
Imunoglobulinas/imunologia , Ativação Linfocitária/imunologia , Glicoproteínas de Membrana/imunologia , Linfócitos T/imunologia , Animais , Antígenos CD , Diferenciação Celular/imunologia , Células Dendríticas/imunologia , Proteínas do Ovo/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Imunoglobulinas/biossíntese , Interferon gama/biossíntese , Interferon gama/imunologia , Interleucina-2/biossíntese , Interleucina-2/imunologia , Masculino , Glicoproteínas de Membrana/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ovalbumina/imunologia , Fragmentos de Peptídeos , Linfócitos T/metabolismo , Antígeno CD83RESUMO
Lipopolysaccharide (LPS) can activate human and murine T cells in vivo and in vitro. Here we analysed the effects of LPS on T cells with defined specificities in T-cell receptor (TCR)-transgenic systems. LPS rapidly induced high amounts of interferon (IFN)-gamma in a subpopulation of purified T cells from DO11.10 (OVA323-339/H2-Ad) and OT-1 (OVA257-264/H2-Kb) mice when coincubated with antigen-pulsed peritoneal exudate cells (PECs). LPS induced IFN-gamma in T cell cultures even when the number of antigenic major histocompatibility complex (MHC) class-I complexes was too small to stimulate the T cells. LPS, thus, overruled the unresponsiveness of the otherwise 'antigen-ignorant' T cells. The release of IFN-gamma strictly correlates with the PECs' ability to produce interleukin (IL)-12. In contrast to the induction of IFN-gamma, antigen-specific IL-2 secretion and proliferation of T cells were rather decreased in the presence of LPS. Only very few IFN-gamma-secreting natural killer (NK) cells and natural killer T (NKT) cells in the given experimental system could be detected using intracellular fluorescence-activated cell sorter (FACS) staining. Taken together, our results indicate that LPS has the potential to activate quiescent T cells and to specifically induce IFN-gamma in CD4 and CD8 T cells. This may have direct consequences for the activation of autoreactive T cells following bacterial infections.
Assuntos
Antígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Interferon gama/biossíntese , Lipopolissacarídeos/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Animais , Interleucina-12/fisiologia , Receptores de Lipopolissacarídeos/fisiologia , Camundongos , Receptores de Antígenos de Linfócitos T/fisiologiaRESUMO
Heat shock proteins (HSP) like Hsp60, Hsp70 and gp96 act directly on antigen-presenting cells (APC), e.g. by inducing the secretion of cytokines. Here we analyzed the impact of Hsp60 on the antigen-specific activation of CD8(+) T cells in a TCR transgenic system. Hsp60 induced low amounts of IFN-gamma in the absence of antigenic peptide; however, the release of IFN-gamma is increased by a factor of 3-10 following the addition of Hsp60 to purified populations of OT-1 [ovalbumin (OVA)257-264/H2-K(b)-restricted] T cells and antigen-pulsed peritoneal exudate cells (PEC) as APC. This effect is strictly correlated with the PEC ability to produce IL-12. In contrast, antigen-specific IL-2 secretion and T cell proliferation was not changed in the presence of Hsp60. Hsp60-containing OT-1 T cell cultures produced IFN-gamma even when the number of antigenic MHC class I complexes was too low to be stimulatory and could not be detected with specific mAb. Hsp60, thus, acts as a catalyzing molecule to initiate both innate and adaptive immune responses, and its presence (e.g. during an infection with cellular destruction) has direct consequences for the activation of otherwise 'ignorant' antigen-specific T cells.
Assuntos
Chaperonina 60/imunologia , Interferon gama/metabolismo , Linfócitos T Citotóxicos/imunologia , Animais , Apresentação de Antígeno , Chaperonina 60/genética , Células Eucarióticas , Humanos , Interleucina-12/imunologia , Células Matadoras Naturais/imunologia , Ativação Linfocitária , Camundongos , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T/genéticaRESUMO
The heat shock proteins (HSP) gp96, Hsp70 and Hsp60 activate professional antigen-presenting cells (APC) to secrete proinflammatory cytokines and to express costimulatory molecules. Here, we analyze the impact of Hsp60 as a hypothetical danger signal on the antigen-specific activation of T cells derived from DO11.10 TCR-transgenic mice. The release of IFN-gamma, induced by the antigenic OVA(323-339)-peptide, is increased and accelerated dramatically by the addition of Hsp60 to ex vivo purified populations of T cells and peritoneal macrophages (PEC), while the antigen-specific IL-2 production or proliferation of the T cells remain unchanged. In contrast, "effector" T cells, undergoing secondary stimulation, displayed almost unchanged activation kinetics in the presence of Hsp60. The presence of Hsp60 induces IFN-gamma and up-regulation of CD69 in T cell/PEC cocultures even in the absence of antigenic peptide and this induction of IFN-gamma is strictly dependent on the ability of the macrophages to produce IL-12. Taken together, our data strongly suggest that the presence of eukaryotic mitochondrial Hsp60 allows antigen-specific IFN-gamma secretion under conditions when an antigenic stimulus alone is not sufficient to activate T cells.
Assuntos
Chaperonina 60/farmacologia , Interferon gama/biossíntese , Linfócitos T/imunologia , Animais , Antígenos/imunologia , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Linhagem Celular , Células Cultivadas , Células Eucarióticas/metabolismo , Interleucina-2/biossíntese , Cinética , Lectinas Tipo C , Macrófagos Peritoneais/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Ovalbumina/imunologia , Fragmentos de Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T/genética , Regulação para CimaRESUMO
N-acyloxy-N-alkoxybenzamides are mutagenic in TA100 without the need for metabolic activation with S9. Electronic effects of substituents on both the benzamide ring in N-acetoxy-N-butoxybenzamides or the benzyloxy ring in N-acetoxy-N-benzyloxybenzamides do not influence mutagenicity levels. For N-benzoyloxy-N-benzyloxybenzamides, mutagenicity levels are inversely related to the electron-withdrawing effect of substituents on the benzoyloxy leaving group. Since reactivities increase with increasing electron-withdrawing effects, mutagenicity correlates with stability rather than reactivity of these mutagens. Hydrophobicity is the dominant factor controlling mutagenicity levels and data for all mutagens correlate with computed logP values with a lower dependence (h=0.22) than that recorded for indirect mutagens (h=1.0), except where a sterically demanding p-tert-butyl substituent or a naphthyl group is present. N-acetoxy-N-butoxynaphthamide exhibits a much higher level of mutagenicity than predicted by its logP value and activity may be ascribed to an intercalative binding process with DNA rather than straightforward hydrophobic binding in the major or minor groove. Since these are direct-acting mutagens, structural factors influence binding and reactivity towards DNA.
Assuntos
Benzamidas/toxicidade , Mutagênicos/toxicidade , Acetatos/toxicidade , Butiratos/toxicidade , Testes de Mutagenicidade , Relação Estrutura-AtividadeAssuntos
Blastocisto , Transferência Embrionária/métodos , Microinjeções/métodos , Animais , CamundongosRESUMO
Human CD83 is a cell surface protein expressed predominantly by dendritic cells (DC) and lymphoid cells. So far, there exists no information on the function and distribution of mCD83. Here we demonstrate that mCD83 is moderately expressed on resting T cells and DC, but strongly increases in its expression on T cells following activation with antigenic peptides or T cell receptor-specific mAb. When returning to the resting state, T cells down-regulate CD83 again. Ig fusion proteins which express the extracellular part of the mCD83 molecule (mCD83-Ig) specifically inhibit antigen-specific T cell proliferation and IL-2 secretion in spleen cell cultures from DO11.10 T cell receptor transgenic mice. Staining of spleen cells from BALB/c, XID and mu MT (B cell) knockout mice with mCD83-Ig proteins reveals the presence of a CD83 ligand predominantly expressed most likely by B220(+) cells since spleen cells from mu MT knockout mice do not bind mCD83-Ig. CD83, besides its established expression on human dendritic cells, thus, also represents a new marker molecule on activated T cells which with its specific ligand is involved in the regulation of T cell responses.
Assuntos
Linfócitos B/imunologia , Imunoglobulinas/análise , Glicoproteínas de Membrana/análise , Linfócitos T/imunologia , Animais , Antígenos CD , Células COS , Células Cultivadas , Primers do DNA , Eletroporação , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Imunoglobulinas/biossíntese , Imunoglobulinas/genética , Interleucina-2/análise , Células Jurkat , Ligantes , Ativação Linfocitária , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos Transgênicos , Ovalbumina/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Proteínas Recombinantes de Fusão/biossíntese , Baço/citologia , Transformação Genética , Antígeno CD83RESUMO
The permeabilities and genotoxicities of the Cr(III) complexes [Cr(en)(3)](3+), mer-[Cr(glygly)(2)](-), cis-[Cr(phen)(2)(OH(2))(2)](3+), and trans-[Cr(salen)(OH(2))(2)](+) and the Cr(V) analogues of cis-[Cr(phen)(2)(OH(2))(2)](3+) and trans-[Cr(salen)(OH(2))(2)](+) [en being 1,2-ethanediamine, glygly being glycylglycine, phen being 1,10-phenanthroline, and salen being N,N'-ethylenebis(salicylideneiminato)] have been studied in V79 Chinese hamster lung cells. Following exposure of approximately 10(6) cells to 0.4 mM Cr(III) for 4 h, the Cr uptake by single cells was less than 10(-)(14) g/cell (as determined by GFAAS analysis and as confirmed by PIXE analysis where the Cr concentration was below the limit of detection). Importantly, the Cr(V) analogue of cis-[Cr(phen)(2)(OH(2))(2)] was significantly more permeable than the Cr(III) complex. The cytotoxicity of the Cr(III) complexes increased in the following order: mer-[Cr(glygly)(2)](-) < [Cr(en)(3)](3+) approximately cis-[Cr(phen)(2)(OH(2))(2)](3+) < trans-[Cr(salen)(OH(2))(2)](+). No genotoxic effects were observed following exposure to mer-[Cr(glygly)(2)](-) or [Cr(en)(3)](3+) at concentrations up to 6 mM. The Cr(III) imine complexes trans-[Cr(salen)(OH(2))(2)](+) and cis-[Cr(phen)(2)(OH(2))(2)](3+), which could be oxidized to Cr(V) complexes, induced MN in vitro at rates of 13.6 and 3.3 MN/1000 BN cells/micromol of Cr, respectively. The comparative permeabilities and genotoxicities of trans-[Cr(salen)(OH(2))(2)](+) and [CrO(salen)](+) were similar due to the instability of the Cr(V) complex at physiological pH values (7.4). There was a substantial increase in the permeability of [Cr(O)(2)(phen)(2)](+), compared to that of the Cr(III) analogue, which was accompanied by a highly genotoxic response. Consequently, any Cr(III) complex that is absorbed by cells and can be oxidized to Cr(V) must be considered as a potential carcinogen. This has potential implications for the increased use of Cr(III) complexes as dietary supplements and highlights the need to consider the genotoxicities of a variety of Cr(III) complexes when determining the carcinogenic potential of Cr(III) particularly when "high" deliberately administered doses are concerned.
Assuntos
Compostos de Cromo/toxicidade , Pulmão/efeitos dos fármacos , Mutagênicos/toxicidade , Animais , Contagem de Células/efeitos dos fármacos , Linhagem Celular , Permeabilidade da Membrana Celular , Sobrevivência Celular/efeitos dos fármacos , Compostos de Cromo/farmacocinética , Cricetinae , Cricetulus , Relação Dose-Resposta a Droga , Microanálise por Sonda Eletrônica , Pulmão/citologia , Pulmão/metabolismo , Testes para Micronúcleos , Mutagênicos/farmacocinéticaRESUMO
CD26 or dipeptidylpeptidase IV (DPP IV) is a cell surface protease involved in T cell activation. It is a type II transmembrane glycoprotein consisting of a large extracellular part, a single transmembrane region and a short cytoplasmic tail without any common signalling motifs. To eluciate the mechanisms involved in CD26-mediated signalling we have constructed C-terminal deletion mutants of the human CD26 molecule and transfected them into murine T cell hybridomas. Stimulation experiments show that most of the extracellular part of CD26 can be deleted without affecting its costimulatory activity. The membrane proximal glycosylation rich region of CD26 is sufficient to transduce costimulatory signals. Activation of T cells via CD26, however, is not mediated by the important T cell receptor associated adaptor proteins LAT and TRIM as shown in colocalization assays.
Assuntos
Dipeptidil Peptidase 4/fisiologia , Transdução de Sinais/imunologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Animais , Antígenos CD/biossíntese , Antígenos de Diferenciação de Linfócitos T/biossíntese , Biomarcadores/análise , Dipeptidil Peptidase 4/imunologia , Dipeptidil Peptidase 4/metabolismo , Glicosilação , Humanos , Lectinas Tipo C , Ativação Linfocitária , Proteínas de Membrana/biossíntese , Camundongos , Camundongos Endogâmicos AKR , Mutagênese Sítio-Dirigida , Receptores de Antígenos de Linfócitos T/biossíntese , Receptores de Antígenos de Linfócitos T gama-delta/biossíntese , Células Tumorais Cultivadas , Regulação para Cima/imunologia , Proteínas ras/fisiologiaRESUMO
We elucidated the role of docosahexaenoic acid (DHA) on the increases in free intracellular calcium concentrations, [Ca(2+)]i, in human (Jurkat) T-cell lines. DHA evoked an increase in [Ca(2+)]i in a dose-dependent manner in these cells. Anti-CD3 antibody, known to stimulate increases in Ca(2+) from endoplasmic reticulum (ER) via the production of inositol trisphosphate, also evoked increases in [Ca(2+)]i in Jurkat T-cells. We also used thapsigargin which inhibits Ca(2+)-ATPase of the ER and, therefore, increases Ca(2+) in the cytosol. Interestingly, addition of DHA during the thapsigargin-induced peak response exerted an additive effect on the increases in [Ca(2+)]i in human T-cells, indicating that the mechanisms of action of these two agents are different. However, the DHA-induced calcium response was not observed when this agent was added during the anti-CD3-induced calcium peak, though its addition resulted in a prolonged and sustained calcium response as a function of time, suggesting that DHA recruits calcium, in part, from the ER pool and the prolonged response may be due to Ca(2+) influx. In the medium containing 0% Ca(2+), the DHA-evoked response on the increases in [Ca(2+)]i was significantly curtailed as compared to that in 100% Ca(2+) medium, supporting the notion that the response of the DHA is also due, in part, to the opening of calcium channels. Furthermore, preincubation of cells with tyrphostin A9, an inhibitor of Ca(2+) release-activated Ca(2+) (CRAC) channels also significantly curtailed the DHA-induced sustained response on the increases in [Ca(2+)]i in these cells. These results suggest that DHA induces an increase in [Ca(2+)]i via the ER pool and the opening of CRAC channels in human T-cells.
Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Ácidos Docosa-Hexaenoicos/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Anticorpos/farmacologia , Complexo CD3/metabolismo , Cálcio/metabolismo , ATPases Transportadoras de Cálcio/antagonistas & inibidores , Divisão Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Estrenos/farmacologia , Humanos , Inositol 1,4,5-Trifosfato/biossíntese , Líquido Intracelular/metabolismo , Células Jurkat , Potenciais da Membrana/efeitos dos fármacos , Pirrolidinonas/farmacologia , Linfócitos T/citologia , Tapsigargina/farmacologiaRESUMO
We have previously published results showing time-course data (Leigh et at., 1998) and a dose-response relationship between macrophage micronucleus formation and crystalline silica dose in intratracheally instilled rats at 2.5, 7.5, and 22.5 mg dosage, without an inert dust control (Wang et al., 1997). We here extend this study to low dose (0.025, 0.25, and 2.5 mg crystalline silica) with 2.5 mg TiO2 control. Specific-pathogen-free male Wistar rats were intratracheally instilled with 0.5 ml saline, and 0.025 mg, 0.25 mg, or 2.5 mg crystalline silica (Min-U-Sil 5) and 2.5 mg TiO2 suspended in 0.5 ml saline (5 rats in each group). Five days after instillation, rats were sacrificed and 10 ml of bronchial alveolar lavage fluid was obtained. A 100-µ1 volume was placed on slides by Cytospin centrifugation, stained with Diff-Quik, and 1000 macrophages were scored for micronuclei (defined by diameter < half main nucleus; same staining; round shape and complete separation). Micronucleus incidence was significantly elevated (p < .01) at the lowest crystalline silica dose compared with saline control. There was a dose-response relationship with crystalline silica exposure. Numbers (mean ± SEM) of micronucleated macrophages per 1000 macrophages scored were 1.5 ± 0.5 (saline), 3.3 ± 0.3 (0.025 mg crystalline silica), 7.1 ± 0.4 (0.25 mg crystalline silica), 10.1 ± 0.3 (2.5 mg crystalline silica), and 0.9 ± 0.3 (2.5 mg TiO2). We conclude that intratracheal instillation of low doses of crystalline silica can induce micronucleus formation in alveolar macrophages in a dose-related manner. We further believe that this is not a nonspecific effect, consistent with crystalline silica being a genotoxic carcinogen.
RESUMO
Heat shock proteins (HSP) like Hsp70 and gp96 are potent molecules to induce MHC class I-restricted cytotoxic T cells against antigens present in the cells from which the HSP were isolated. Fusion proteins consisting of mycobacterial Hsp70 covalently linked to antigenic peptide sequences are also capable of generating CTL specific for the peptide-encoded antigens. For effective CTL induction direct binding of the peptide or covalent association of the peptide in the case of antigenic fusion proteins is required. Since mycobacterial Hsp70 and eukaryotic Hsp70 differ significantly in their primary structure, and since gp96 compared to Hsp70 is more efficient in priming antigen specific CTL in our hands, we created fusion proteins consisting of His-tagged eukaryotic gp96 fused C-terminally to various peptide antigens. Here, we used antigenic sequences derived from the established ovalbumin (OVA) and beta-galactosidase (beta-GAL) model systems. We show that in vitro established OVA and beta-GAL specific CTL clones release TNF-alpha and IFN-gamma when incubated with recombinant gp96 irrespective of the antigenic peptide sequences hooked to the C-terminus of gp96. In contrast to gp96 preparations purified from beta-GAL expressing cell lines, recombinant gp96/beta-GAL fusion proteins were not able to generate beta-GAL-specific T cells in vivo. Possible explanations for the lack of antigen-specific immunogenicity of gp96 fusion proteins in vivo are discussed.
Assuntos
Antígenos de Neoplasias/farmacologia , Antígenos/imunologia , Ativação Linfocitária/efeitos dos fármacos , Fragmentos de Peptídeos/imunologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Animais , Antígenos/genética , Antígenos de Neoplasias/imunologia , Interferon gama/biossíntese , Fígado/química , Macrófagos Peritoneais/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteínas de Neoplasias/imunologia , Ovalbumina/imunologia , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Células Tumorais Cultivadas , beta-Galactosidase/imunologiaRESUMO
OBJECTIVE: The aim of this study was to explore the association between glycosylated hemoglobin concentrations during pregnancy and macrosomia. STUDY DESIGN: One hundred thirty-six pregnancies in 120 women with type 1 or type 2 diabetes were studied longitudinally between January 1, 1991, and December 31, 1996. Glycosylated hemoglobin concentration and several maternal variables of mothers of neonates who were large for gestational age were compared with those of neonates who were appropriate for gestational age. Receiver-operator characteristic curves and regression analyses were used to determine a threshold related to macrosomia and to assess its predictive value. RESULTS: Glycosylated hemoglobin concentrations throughout pregnancy were higher in mothers of neonates who were large for gestational age (n = 65) than in mothers of neonates who were appropriate for gestational age (n = 71, P <. 001). A first-trimester glycosylated hemoglobin concentration of >/=5.5% (3 SD above the normal mean) was established by receiver-operator characteristic curves as the strongest predictor of macrosomia and yielded an odds ratio of 24 in multiple logistic regression analysis. CONCLUSION: Macrosomia is determined mainly by first-trimester diabetes control.
Assuntos
Diabetes Mellitus Tipo 1/prevenção & controle , Diabetes Mellitus Tipo 2/prevenção & controle , Macrossomia Fetal/prevenção & controle , Hemoglobinas Glicadas/metabolismo , Adulto , Feminino , Humanos , Recém-Nascido , Estudos Longitudinais , Gravidez , Primeiro Trimestre da Gravidez , Gravidez em Diabéticas , Estudos Prospectivos , Análise de Regressão , Sensibilidade e EspecificidadeRESUMO
Heat shock proteins (HSP) Hsp70 and gp96 prime class I-restricted cytotoxic T cells against Ags present in the cells from which they were isolated. The immunization capacity of HSPs is believed to rely on their ability to bind antigenic peptides. In this study, we employed the well-established OVA and beta-galactosidase (beta-gal) antigenic model systems. We show that in vitro long-term established OVA and beta-gal-specific CTL clones release TNF-alpha and IFN-gamma when incubated with Ag-negative Hsp70 and gp96. In the absence of antigenic peptides, HSP-mediated secretion of TNF-alpha and IFN-gamma requires cell contact of the APC with the T cell but is not MHC-I restricted. Moreover, Hsp70 molecules purified from Ag-negative tissue, e.g., negative for antigenic peptide, are able to activate T cells in vivo, leading to significant higher frequencies in OVA-specific CD8+ T cells. In unprimed animals, these T cells lyse OVA-transfected cell lines and produce TNF-alpha and IFN-gamma after Ag stimulus. Taken together our data show that, besides the well-established HSP/peptide-specific CTL induction and activation, a second mechanism exists by which Hsp70 and gp96 molecules activate T cells in vivo and in vitro.
Assuntos
Antígenos de Neoplasias/administração & dosagem , Antígenos de Neoplasias/farmacologia , Proteínas de Choque Térmico HSP70/administração & dosagem , Proteínas de Choque Térmico HSP70/farmacologia , Ativação Linfocitária/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Antígenos de Neoplasias/imunologia , Linhagem Celular , Células Clonais , Epitopos de Linfócito T/imunologia , Proteínas de Choque Térmico HSP70/imunologia , Haplótipos , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Injeções Intraperitoneais , Interferon gama/biossíntese , Lipopolissacarídeos/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Ovalbumina/imunologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/metabolismo , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/biossínteseRESUMO
CD26 or dipeptidyl peptidase IV (DPP-IV) is a cell surface protease involved in T cell activation. Monoclonal antibodies (mAbs) directed against the CD26 molecule are able to stimulate CD26-expressing T cells. Although many different CD26-specific mAbs exist which are able to provide a triggering signal in T cells, little is known about their specific epitopes on the CD26 molecule. Whereas some mAbs were shown to compete with each other and to inhibit the association of adenosine deaminase (ADA) and human immunodeficiency virus 1 (HIV-1)-derived Tat protein with CD26, other CD26-specific mAbs obviously bind to distinct regions on DPP-IV. In the present study we have generated truncated versions of the human CD26 molecule and expressed them in COS-1 cells to study the binding pattern of a panel of 14 CD26-specific mAbs in confocal microscopy and, thus, correlated the CD26-specific mAbs epitopes with the binding region of ADA. We show that the majority of anti-CD26 mAbs is directed against the glycosylation-rich region of the molecule whereas the ADA-binding site could be located in the cysteine-rich region of DPP-IV. In contrast to binding experiments with purified ADA, which revealed a specific association with CD26 on CD26-positive Jurkat cells, HIV-derived Tat protein did not interact specifically with CD26 on transfected Jurkat cells, nor could Tat binding be competed by anti-CD26-specific mAbs.