RESUMO
Excessive neutrophil influx, their released neutrophil extracellular traps (NETs), and extracellular histones are associated with disease severity in influenza-infected patients. Neutrophil chemokine receptor CXC chemokine receptor 2 (CXCR2) is a critical target for suppressing neutrophilic inflammation. Herein, temporal dynamics of neutrophil activity and NETosis were investigated to determine the optimal timing of treatment with the CXCR2 antagonist, SCH527123 (2-hydroxy-N,N-dimethyl-3-[2-([(R)-1-(5-methyl-furan-2-yl)-propyl]amino)-3,4-dioxo-cyclobut-1-enylamino]-benzamide), and its efficacy together with antiviral agent, oseltamivir, was tested in murine and piglet influenza-pneumonia models. SCH527123 plus oseltamivir markedly improved survival of mice infected with lethal influenza, and diminished lung pathology in swine-influenza-infected piglets. Mechanistically, addition of SCH527123 in the combination treatment attenuated neutrophil influx, NETosis, in both mice and piglets. Furthermore, neutrophils isolated from influenza-infected mice showed greater susceptibility to NETotic death when stimulated with a CXCR2 ligand, IL-8. In addition, CXCR2 stimulation induced nuclear translocation of neutrophil elastase, and enhanced citrullination of histones that triggers chromatin decondensation during NET formation. Studies on temporal dynamics of neutrophils and NETs during influenza thus provide important insights into the optimal timing of CXCR2 antagonist treatment for attenuating neutrophil-mediated lung pathology. These findings reveal that pharmacologic treatment with CXCR2 antagonist together with an antiviral agent could significantly ameliorate morbidity and mortality in virulent and sublethal influenza infections.
Assuntos
Benzamidas/farmacologia , Ciclobutanos/farmacologia , Influenza Humana/mortalidade , Infecções por Orthomyxoviridae/patologia , Oseltamivir/farmacologia , Receptores de Interleucina-8B/efeitos dos fármacos , Animais , Armadilhas Extracelulares/microbiologia , Humanos , Influenza Humana/patologia , Elastase de Leucócito/efeitos dos fármacos , Pulmão/patologia , Camundongos , Ativação de Neutrófilo/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Neutrófilos/patologia , Infecções por Orthomyxoviridae/mortalidade , SuínosRESUMO
Excessive neutrophils recruited during influenza pneumonia contribute to severe lung pathology through induction of neutrophil extracellular traps (NETs) and release of extracellular histones. We have recently shown that activation of platelets during influenza enhances pulmonary microvascular thrombosis, leading to vascular injury and hemorrhage. Emerging evidence indicates that activated platelets also interact with neutrophils, forming neutrophil-platelet aggregates (NPAs) that contribute to tissue injury. Here, we examined neutrophil-platelet interactions and evaluated the formation of NPAs during influenza pneumonia. We also evaluated the efficacy of clopidogrel (CLP), an antagonist of the ADP-P2Y12 platelet receptor, alone or in combination with an antiviral agent (oseltamivir) against influenza infection in mice. Our studies demonstrated increased platelet activation and induction of NPAs in influenza-infected lungs, and that these NPAs led to NET release both in vitro and in vivo. Furthermore, neutrophil integrin Mac-1 (macrophage-1 antigen)-mediated platelet binding was critical for NPA formation and NET release. Administration of CLP reduced platelet activation and NPA formation but did not protect the mice against lethal influenza challenge. However, administration of CLP together with oseltamivir improved survival rates in mice compared with oseltamivir alone. The combination treatment reduced lung pathology, neutrophil influx, NPAs, NET release, and inflammatory cytokine release in infected lungs. Taken together, these results provide the first evidence that NPAs formed during influenza contribute to acute lung injury. Targeting both platelet activation and virus replication could represent an effective therapeutic option for severe influenza pneumonia.
Assuntos
Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Infecções por Orthomyxoviridae/tratamento farmacológico , Oseltamivir/uso terapêutico , Ativação Plaquetária/efeitos dos fármacos , Pneumonia Viral/tratamento farmacológico , Replicação Viral/efeitos dos fármacos , Animais , Clopidogrel/uso terapêutico , Modelos Animais de Doenças , Sinergismo Farmacológico , Quimioterapia Combinada , Armadilhas Extracelulares , Feminino , Histonas/metabolismo , Vírus da Influenza A Subtipo H1N1/fisiologia , Pulmão/metabolismo , Pulmão/patologia , Pulmão/virologia , Camundongos , Neutrófilos/metabolismo , Neutrófilos/patologia , Infecções por Orthomyxoviridae/sangue , Infecções por Orthomyxoviridae/complicações , Oseltamivir/administração & dosagem , Oseltamivir/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/administração & dosagem , Inibidores da Agregação Plaquetária/farmacologia , Inibidores da Agregação Plaquetária/uso terapêutico , Pneumonia Viral/sangue , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Trombofilia/etiologiaRESUMO
Although exaggerated host immune responses have been implicated in influenza-induced lung pathogenesis, the etiologic factors that contribute to these events are not completely understood. We previously demonstrated that neutrophil extracellular traps exacerbate pulmonary injury during influenza pneumonia. Histones are the major protein components of neutrophil extracellular traps and are known to have cytotoxic effects. Here, we examined the role of extracellular histones in lung pathogenesis during influenza. Mice infected with influenza virus displayed high accumulation of extracellular histones, with widespread pulmonary microvascular thrombosis. Occluded pulmonary blood vessels with vascular thrombi often exhibited endothelial necrosis surrounded by hemorrhagic effusions and pulmonary edema. Histones released during influenza induced cytotoxicity and showed strong binding to platelets within thrombi in infected mouse lungs. Nasal wash samples from influenza-infected patients also showed increased accumulation of extracellular histones, suggesting a possible clinical relevance of elevated histones in pulmonary injury. Although histones inhibited influenza growth in vitro, in vivo treatment with histones did not yield antiviral effects and instead exacerbated lung pathology. Blocking with antihistone antibodies caused a marked decrease in lung pathology in lethal influenza-challenged mice and improved protection when administered in combination with the antiviral agent oseltamivir. These findings support the pathogenic effects of extracellular histones in that pulmonary injury during influenza was exacerbated. Targeting histones provides a novel therapeutic approach to influenza pneumonia.
Assuntos
Histonas/metabolismo , Pulmão/metabolismo , Infecções por Orthomyxoviridae/metabolismo , Pneumonia/metabolismo , Animais , Modelos Animais de Doenças , Humanos , Pulmão/patologia , Camundongos , Infecções por Orthomyxoviridae/patologia , Pneumonia/patologia , Trombose/metabolismo , Trombose/patologiaRESUMO
Francisella tularensis(Ft) is a highly virulent, intracellular Gram-negative bacterial pathogen. Acute Ft infection by aerosol route causes pneumonic tularemia, characterized by nodular hemorrhagic lesions, neutrophil-predominant influx, necrotic debris, fibrin deposition, and severe alveolitis. Ft suppresses activity of neutrophils by impairing their respiratory burst and phagocytic activity. However, the fate of the massive numbers of neutrophils recruited to the infection site is unclear. Here, we show that Ft infection resulted in prominent induction of neutrophil extracellular traps (NETs) within damaged lungs of mice infected with the live attenuated vaccine strain of Ft(Ft-LVS), as well as in the lungs of domestic cats and rabbits naturally infected with Ft. Further, Ft-LVS infection increased lung myeloperoxidase (MPO) activity, which mediates histone protein degradation during NETosis and anchors chromatin scaffolds in NETs. In addition, Ft infection also induced expression of peptidylarginine deiminase 4, an enzyme that causes citrullination of histones during formation of NETs. The released NETs were found largely attached to the alveolar epithelium, and disrupted the thin alveolar epithelial barrier. Furthermore, Ft infection induced a concentration-dependent release of NETs from neutrophils in vitro. Pharmacological blocking of MPO reduced Ft-induced NETs release, whereas addition of H2O2 (a substrate of MPO) significantly augmented NETs release, thus indicating a critical role of MPO in Ft-induced NETs. Although immunofluorescence and electron microscopy revealed that NETs could efficiently trap Ft bacteria, NETs failed to exert bactericidal effects. Taken together, these findings suggest that NETs exacerbate tissue damage in pulmonary Ft infection, and that targeting NETosis may offer novel therapeutic interventions in alleviating Ft-induced tissue damage.
Assuntos
Células Epiteliais Alveolares/patologia , Armadilhas Extracelulares/metabolismo , Francisella tularensis/imunologia , Pulmão/patologia , Neutrófilos/imunologia , Tularemia/imunologia , Animais , Gatos , Células Cultivadas , Peróxido de Hidrogênio/metabolismo , Camundongos , Peroxidase/metabolismo , CoelhosRESUMO
Exaggerated host innate immune responses have been implicated in severe influenza pneumonia. We have previously demonstrated that excessive neutrophils recruited during influenza infection drive pulmonary pathology through induction of neutrophil extracellular traps (NETs) and release of extracellular histones. Chemokine receptors (CRs) are essential in the recruitment and activation of leukocytes. Although neutrophils have been implicated in influenza pathogenesis, little is known about their phenotypic changes, including expression of CRs occurring in the infected -lung microenvironment. Here, we examined CC and CXC CRs detection in circulating as well as lung-recruited neutrophils during influenza infection in mice using flow cytometry analyses. Our studies revealed that lung-recruited neutrophils displayed induction of CRs, including CCR1, CCR2, CCR3, CCR5, CXCR1, CXCR3, and CXCR4, all of which were marginally induced in circulating neutrophils. CXCR2 was the most predominant CR observed in both circulating and lung-infiltrated neutrophils after infection. The stimulation of these induced CRs modulated neutrophil phagocytic activity, ligand-specific neutrophil migration, bacterial killing, and NETs induction ex vivo. These findings indicate that neutrophils induce a novel CR repertoire in the infectious lung microenvironment, which alters their functionality during influenza pneumonia.
Assuntos
Neutrófilos/imunologia , Infecções por Orthomyxoviridae/imunologia , Orthomyxoviridae/imunologia , Pneumonia Viral/imunologia , Receptores de Quimiocinas/biossíntese , Animais , Modelos Animais de Doenças , Citometria de Fluxo , Camundongos , Neutrófilos/químicaRESUMO
OBJECTIVE: To determine effects of exercise performed while breathing cold air on expression of cytokines and influx of neutrophils in airways of horses. ANIMALS: 9 adult horses. PROCEDURES: In a crossover study, bronchoalveolar lavage fluid (BALF) was obtained 24 and 48 hours after each of 2 submaximal exercise sessions performed by horses while breathing warm (25 degrees C) or cold (-5 degrees C) air. Total and differential nucleated cell counts were determined for each BALF sample. Relative mRNA expression of cytokines in BALF cells was quantified by use of a reverse transcription-PCR assay. RESULTS: Horses had a modest but significant influx of neutrophils into the airways 24 hours after a single exercise session while breathing cold air. No other cell types were increased at 24 or 48 hours after exercising while breathing cold air. Continued increases in expression of cytokines interleukin (IL)-5 and-10 as well as proinflammatory cytokines IL-1, -6, and -8 were detected 24 hours after exercising while breathing cold air. Forty-eight hours after exercising while breathing cold air, expression of IL-10 was still higher than that for IL-10 after horses exercised while breathing warm air. Expression of tumor necrosis factor-alpha was significantly increased at 48 hours after exercising while breathing cold air. CONCLUSIONS AND CLINICAL RELEVANCE: Exposure of intrapulmonary airways to cold air alters immunologic responses of horses for at least 48 hours. The increased expression of cytokines that suppress cell-mediated immunity may predispose athletes to viral infections of the respiratory tract following exercise in cold weather.
Assuntos
Ar , Temperatura Baixa , Citocinas/metabolismo , Pulmão/metabolismo , Neutrófilos/metabolismo , Condicionamento Físico Animal/fisiologia , Animais , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Feminino , Masculino , RNA Mensageiro/metabolismoRESUMO
Ovarian follicular growth and dominance are controlled by a series of hormonal and intraovarian events including a decrease in intrafollicular IGF-binding proteins -2, -4 and -5 levels. Proteolytic enzymes such as pregnancy-associated plasma protein-A (PAPP-A) degrade IGFBPs and increase bioavailability of IGF-I and -II during follicular development. The objective of this study was to determine the effect of IGF-I, IGF-II, insulin (INS), LH, FSH, estradiol (E2), leptin or cortisol on ovarian PAPP-A mRNA levels. Granulosa (GC) from small (SM) (1-5 mm) and large (LG) (8-22 mm) follicles as well as theca cells (TC) from LG follicles were collected from bovine ovaries and cultured for 48 h in medium containing 10% FCS and then treated with various hormones in serum-free medium for an additional 24 h. Cells were treated with various concentrations (3-500 ng/ml) and combinations of IGF-I, IGF-II, FSH, LH, E2, INS, leptin and (or) cortisol for 24 h (Experiments 1-10). PAPP-A mRNA levels were measured using quantitative real-time RT-PCR. In SM-GC and LG-GC, none of the treatments significantly affected (P>0.10) PAPP-A mRNA abundance. In LG-TC, IGF-I, LH or cortisol did not affect (P>0.10) PAPP-A mRNA levels, whereas INS with or without LH decreased (P<0.05) PAPP-A mRNA. E2 alone decreased PAPP-A mRNA levels in LG-TC, and E2 amplified the insulin-induced inhibition of PAPP-A mRNA abundance in LG-TC. We conclude that control of PAPP-A mRNA abundance in granulosa and theca cells differs, and that E2 may be part of an intraovarian negative feedback system which may reduce the bioavailable IGFs in the theca layer during growth and selection of follicles.
Assuntos
Bovinos/metabolismo , Células da Granulosa/metabolismo , Proteína Plasmática A Associada à Gravidez/genética , RNA Mensageiro/genética , Células Tecais/metabolismo , Animais , Estradiol/farmacologia , Feminino , Hormônios/farmacologia , Hidrocortisona/farmacologia , Gravidez , Proteína Plasmática A Associada à Gravidez/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Somatomedinas/farmacologiaRESUMO
Athletes who perform repeated exercise while breathing cold air have a high prevalence of asthmalike chronic airway disease, but the mechanism linking such activity to airway inflammation is unknown. We used a novel animal model (exercising horses) to test the hypothesis that exercise-induced chronic airway disease is caused by exposure of intrapulmonary airways to unconditioned air, resulting in the upregulation of cytokine expression. Bronchoalveolar lavage fluid (BALF) was obtained from eight horses 5 h after submaximal exercise while they breathed room temperature or subfreezing air in a random crossover design. BALF total and differential nucleated cell counts were determined, and relative cytokine mRNA expression in BALF nucleated cells was quantified by real-time RT-PCR using primer and probe sequences specific for equine targets. There were no significant changes in total or differential cell concentrations between BALF recovered after warm and cold air exercise, although there was a strong trend toward increased concentrations of airway epithelial cells after cold air exercise (P = 0.0625). T(H)2 cytokines IL-4, IL-5, and IL-10 were preferentially upregulated after cold air exercise 12-, 9-, and 10-fold, respectively, compared with warm air exercise. Other cytokines (IL-2 and IL-6) were upregulated to a lesser extent (6- and 3-fold, respectively) or not at all (IL-1, IL-8, IFN-gamma, and TNF-alpha). These results suggest that cold weather exercise can lead to asthmalike airway disease through the local induction of cytokines typical of the T(H)2 phenotype.
Assuntos
Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/imunologia , Temperatura Baixa , Citocinas/imunologia , Regulação da Expressão Gênica/imunologia , Condicionamento Físico Animal/métodos , Animais , Estudos Cross-Over , Eritroblastos/citologia , Eritroblastos/imunologia , Cavalos , Tempo (Meteorologia)RESUMO
To determine if (1) levels of pregnancy-associated plasma protein-A (PAPP-A) mRNA and insulin-like growth factor binding protein (IGFBP) (-2, -3, -4 and -5) mRNAs differ between the dominant and subordinate follicles during the follicular phase of an estrous cycle, and (2) these differences are associated with differences in follicular fluid (FFL) concentrations of steroids (estradiol, androstenedione, and progesterone), total and free IGF-I, or IGFBPs, estrous cycles of non-lactating Holstein dairy cows (n = 16) were synchronized with two injections of prostaglandin (PGF2 alpha) 11 days apart. Granulosa cells and FFL were collected either 24 h or 48 h after the second injection of PGF2 alpha. FFL from dominant follicles had lower concentrations of progesterone (P < 0.08) and higher concentrations of estradiol (P < 0.05), androstenedione (P < 0.0001), estradiol:progesterone ratio (P < 0.0001), free IGF-I (P < 0.0001), and calculated percentage free IGF-I (P < 0.01) than large subordinate follicles. Levels of IGFBP-2, -4, and -5 in FFL were 3.0- (P < 0.05), 2.4- (P < 0.06), and 3.4-fold (P < 0.05) greater, respectively, in subordinate than in dominant follicles. IGFBP-3, IGFBP-4 and PAPP-A mRNA expression and IGF-II concentration did not differ (P > 0.10) between dominant or subordinate follicles. Levels of IGFBP-2 and -5 mRNA were severalfold greater (P < 0.05) in subordinate than dominant follicles. IGFBP-5 mRNA in granulosa cells decreased (P < 0.05) 62% to 92%, between 24h and 48 h post-PGF2 alpha. We conclude that decreased levels of IGFBP-2 and -5 mRNA in granulosa cells may contribute to the decrease in FFL IGFBP-2 and -5 protein levels of preovulatory dominant follicles, and that changes in granulosa cell IGFBP-3 and -4 mRNA and PAPP-A mRNA levels do not occur during final preovulatory follicular development in cattle.
Assuntos
Bovinos , Células da Granulosa/química , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/genética , Folículo Ovariano/anatomia & histologia , Proteína Plasmática A Associada à Gravidez/genética , RNA Mensageiro/análise , Androstenodiona/análise , Animais , Estradiol/análise , Feminino , Líquido Folicular/química , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Proteína 4 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Proteína 5 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Fator de Crescimento Insulin-Like I/análise , Fator de Crescimento Insulin-Like II/análise , Folículo Ovariano/citologia , Folículo Ovariano/fisiologia , Progesterona/análiseRESUMO
This study was undertaken to test for differential gene expression in intramuscular adipocytes during fat deposition of feedlot steers. Angus x Hereford steers (n = 50) were fed a high-energy concentrate ration ad libitum for 20 (n = 5), 86 (n = 15), 121 (n = 15), and 146 days (n = 15) to obtain various degrees of intramuscular adipocyte development. Carcass traits were significantly different (P < 0.05) between the groups. Intramuscular adipose tissue was excised from the longissimus dorsi and snap frozen in liquid nitrogen. Pooled samples of total RNA representing each group were analyzed by differential-display polymerase chain reaction using 200 primer combinations comprising 20 arbitrary (5') and 10 anchor (3') oligonucleotides. Bands (n = 70) representing putative differences among treatment groups were excised, sequenced, and subjected to BLAST homology search. From these, 40 contained significant homology to known genes. One was of particular interest, the translational repressor NAT1 (novel APOBEC-1 target-1). NAT1 mRNA was quantified in individual animals to confirm differential expression among treatment groups. Results indicate that NAT1 message is more abundant (P < 0.05) in intramuscular adipocytes of younger/leaner animals.
Assuntos
Adipócitos/citologia , Ração Animal , Bovinos/genética , Fator de Iniciação Eucariótico 4G , Músculo Esquelético/crescimento & desenvolvimento , Fatores de Iniciação de Peptídeos/genética , Proteínas Repressoras/genética , Tecido Adiposo/citologia , Tecido Adiposo/crescimento & desenvolvimento , Animais , Bovinos/crescimento & desenvolvimento , Mapeamento Cromossômico , DNA , Grão Comestível , Perfilação da Expressão Gênica , Humanos , Masculino , Carne , Músculo Esquelético/citologia , Fatores de Iniciação de Peptídeos/biossíntese , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Proteínas Repressoras/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência do Ácido NucleicoRESUMO
During late peri-implantation development, porcine conceptuses undergo a rapid (2-3 hrs) morphological transformation from a 10 mm sphere to a thin filamentous form greater than 150 mm in length. Elongation of the conceptus is important for establishing adequate placental surface area needed for embryo and fetal survival throughout gestation. Genes involved with triggering this unique transition in conceptus development are not well defined. Objective of the present study was to utilize suppression subtractive hybridization (SSH) to characterize the change in gene expression during conceptus transformation from spherical (8-9 mm) to tubular (15-40 mm) to early filamentous (>150 mm) morphology. Spherical, tubular, and filamentous conceptuses were collected from pregnant gilts and subjected to SSH. Forward and reverse subtractions were performed to identify candidate genes differentially expressed during spherical to tubular and tubular to filamentous transition. A total of 384 transcripts were differentially screened to ensure unique expression. Of the transcripts screened, sequences were obtained for 142 that were confirmed to be differentially expressed among the various morphologies. Gene expression profiles during rapid trophoblastic elongation were generated for selected mRNAs using quantitative real-time PCR. During the transition from tubular to early filamentous conceptuses, s-adenosylhomocysteine hydrolase and heat shock cognate 70 kDa expression were significantly enhanced. A novel unknown gene was isolated and shown to be significantly up-regulated at the onset of rapid trophoblastic elongation and further enhanced in filamentous conceptuses.
Assuntos
Proteínas Fetais/biossíntese , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento , Hibridização de Ácido Nucleico/métodos , Suínos/genética , Trofoblastos/metabolismo , Adenosil-Homocisteinase/biossíntese , Adenosil-Homocisteinase/genética , Animais , DNA Complementar/genética , Feminino , Proteínas Fetais/genética , Proteínas de Choque Térmico HSP70/biossíntese , Proteínas de Choque Térmico HSP70/genética , Morfogênese/genética , Gravidez , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Técnica de Subtração , Suínos/metabolismo , Trofoblastos/ultraestruturaRESUMO
A molecular beacon (MB) array was designed based on unique regions of the 16S rRNA of the bacterium Francisella tularensis. Nucleic acid molecular beacons undergo a spontaneous fluorogenic conformational change when they hybridize to specific complementary targets. The array was printed on aldehyde glass or hydrogel slides and evaluated for functioning in presence of complementary oligonucleotide sequences, single-nucleotide mismatch sequences and multiple nucleotide mismatch sequences. Discriminating true target from mismatched targets was found to be dependent on type, number, and location of mismatches within the beacon (i.e. located in the stem or loop regions). Optimal conditions for molecular beacon deposition, and target hybridization were determined for oligonucleotide target mismatch discrimination. The beacon array was stable upon recharging by exposure to an alkaline solution, and repeatedly used. In addition, performance of the beacon array biosensor was compared with molecular beacons in homogeneous solution.
Assuntos
Técnicas Biossensoriais/instrumentação , Francisella tularensis/isolamento & purificação , Hibridização in Situ Fluorescente/instrumentação , RNA Ribossômico 16S/análise , Adsorção , Técnicas Biossensoriais/métodos , Desenho de Equipamento , Análise de Falha de Equipamento , Francisella tularensis/genética , Marcadores Genéticos/genética , Hibridização in Situ Fluorescente/métodos , Sondas RNA/análise , Sondas RNA/genética , RNA Ribossômico 16S/genética , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Conceptus-uterine communication is established during trophoblastic elongation when the conceptus synthesizes and releases estrogen, the maternal recognition signal in the pig. Interleukin-1beta (IL-1beta) is a differentially expressed gene during rapid trophoblastic elongation in the pig. The current investigation determined conceptus and endometrial changes in gene expression for IL-1beta, IL-1 receptor antagonist (IL-1Rant), IL-1 receptor type 1 (IL-1RT1), and IL-1 receptor accessory protein (IL-1RAP) in developing peri- and postimplantation conceptuses as well as uterine endometrium collected from cyclic and pregnant gilts. Conceptus IL-1beta gene expression was enhanced during the period of rapid trophoblastic elongation compared with earlier spherical conceptuses, followed by a dramatic decrease in elongated Day 15 conceptuses. IL-1RT1 and IL-1RAP gene expression was greater in Day 12 and 15 filamentous conceptuses compared with earlier morphologies while IL-1Rant gene expression was unchanged by conceptus development. The uterine lumenal content of IL-1beta increased during the process of trophoblastic elongation on Day 12. Uterine IL-1beta content declined on Day 15, reaching a nadir by Day 18 of pregnancy. IL-1beta gene expression in porcine conceptuses was temporally associated with an increase in endometrial IL-1RT1 and IL-1RAP gene expression in pregnant gilts. Endometrial IL-1beta and IL-1Rant gene expression were lowest during Days 10-15 of the estrous cycle and pregnancy. The temporal expression of IL-1beta during conceptus development and the initiation of conceptus-uterine communication suggests conceptus IL-1beta synthesis plays an important role in porcine conceptus elongation and the establishment of pregnancy in the pig.
Assuntos
Interleucina-1/fisiologia , Placenta/fisiologia , Prenhez , Trofoblastos/fisiologia , Animais , Desenvolvimento Embrionário , Feminino , Fertilização/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Proteína Antagonista do Receptor de Interleucina 1 , Placenta/citologia , Gravidez , Proteínas/genética , Receptores de Interleucina-1/genética , Receptores Tipo I de Interleucina-1 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sialoglicoproteínas/genética , Suínos , Trofoblastos/citologia , Útero/fisiologiaRESUMO
Molecular beacons (MBs) are oligonucleotide probes having a compact hairpin structure, with a fluorophore attached to one end and a quencher molecule attached to the other end. In its native state, the fluorophore is quenched by virtue of its proximity to the quencher molecule. Upon hybridization with its complementary oligonucleotide target, fluorescence is elicited due to a conformational change that results in separation of the fluorophore and quencher molecule. The present study describes the hybridization interaction of an MB to various complementary target sequences. The effects of temperature and length of complementary target sequences on hybridization were investigated using capillary electrophoresis and solution-based fluorescence techniques. Hybridization efficiency was dependent on the ability of the target sequences to destabilize the stem region by binding directly to the stem region. Optimal hybridization occurred between 40 and 50 degrees C for all targets tested, with the true target forming a more stable hybrid complex.
Assuntos
Eletroforese Capilar/métodos , Sondas de Oligonucleotídeos/química , Sequência de Bases , Corantes Fluorescentes , Francisella/genética , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico/métodos , Sondas de Oligonucleotídeos/genética , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Espectrometria de Fluorescência/métodos , TemperaturaRESUMO
During the period of attachment of the trophectoderm to the uterine lumenal surface in the pig, there is an increase in uterine blood flow and a localized hyperemic response induced by the developing conceptuses. The presence of tissue kallikrein in the porcine uterine lumen suggests that the kallikrein-kinin system may be functional during pregnancy in the pig. The objective of the present study was to determine the concentration of bradykinin within the uterine lumen during the estrous cycle and early pregnancy as well as endometrial gene expression and cellular localization of the bradykinin beta(2) receptor. Concentration of bradykinin in uterine flushings was greatest during estrus (Day 0) and Days 12-18 of the estrous cycle. However, there was a 5- to 10-fold increase in bradykinin content in pregnant uterine flushings on Days 12-18 of pregnancy compared with the estrous cycle. Endometrial bradykinin beta(2) receptor gene expression was greatest on Days 0, 12, 15, and 18 of the estrous cycle and pregnancy as gene expression decreased almost 6-fold on Days 5 and 10. Bradykinin beta(2) receptors were detected in the endometrial surface and glandular epithelium with greatest intensity of staining observed on Days 0, 12, 15, and 18 of the estrous cycle and pregnancy. Results from the present study suggest that the kallikrein-kinin system plays a role in the establishment of pregnancy in the pig.