RESUMO
Background: Extracellular heat-shock proteins (eHsp) are highly conserved molecules that play an important role in inflammatory diseases and have been quantified in plasma from patients with infectious diseases, including sepsis. There is a constant search for dependable biochemical markers that, in combination with conventional methods, could deliver a prompt and reliable diagnosis of early-onset neonatal sepsis. Objective: We sought to assess the level of eHsp-27, eHsp-60, eHsp-70, and tumor necrosis factor-alpha (TNFα) in plasma of healthy neonates at term and infants with early-onset neonatal sepsis. Methods: This study included 34 newborns that were classified as healthy neonates at term (blood samples from the umbilical cord, n = 23) or infants with early-onset neonatal sepsis (blood samples obtained from umbilical artery by standard sterile procedures before starting a systemic antibiotic intervention, n = 11). All blood samples were centrifuged, and the plasma recovered to determine eHsp-27, eHsp-60, eHsp-70, and TNFα levels by ELISA. Results: Our results indicate that the level of eHsp-27 in healthy neonates at term was 0.045 ± 0.024 pg/ml. This value decreased 2.5-fold in infants with early-onset neonate sepsis (0.019 ± 0.006 pg/ml, p = 0.004). In contrast, the levels of eHsp-60 and eHsp-70 in healthy neonates at term were 13.69 ± 5.3 and 4.03 ± 2.6 pg/ml, respectively. These protein levels increased significantly 1.8- and 1.9-fold in the plasma of infants with early-onset neonatal sepsis (p ≤ 0.001). The level of TNFα in healthy neonates at term was 2.94 ± 0.46 pg/ml, with a 3.0-fold increase in infants with early-onset neonatal sepsis (8.96 ± 0.72 pm/ml, p ≤ 0.001). The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of eHsp compared with that of C-reactive protein were 73.3, 60.0, 47.8, and 33.3%, respectively. Conclusion: This study demonstrated a consistent increase of eHsp-60 and eHsp-70 in the plasma of infants diagnosed with early-onset neonatal sepsis. These proteins showed higher sensitivity and specificity than C-reactive protein and blood culture test.
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It is estimated that 15% of all newborns admitted to the neonatal intensive care unit (NICU) for suspected sepsis receive multiple broad-spectrum antibiotics without pathogen identification. The gold standard for bacterial sepsis detection is blood culture, but the sensitivity of this method is very low. Recently, amplification and analysis of the 16S ribosomal DNA (rDNA) bacterial gene in combination with denaturing gradient gel electrophoresis (DGGE) has proven to be a useful approach for identifying bacteria that are difficult to isolate by standard culture methods. The main goal of this study was to compare two methods used to identify bacteria associated with neonatal sepsis: blood culture and broad range 16S rDNA-DGGE. Twenty-two blood samples were obtained from newborns with (n = 15) or without (n = 7) signs and symptoms of sepsis. Blood samples were screened to identify pathogenic bacteria with two different methods: (1) bacteriological culture and (2) amplification of the variable V3 region of 16S rDNA-DGGE. Blood culture analysis was positive in 40%, whereas 16S rDNA-DGGE was positive in 87% of neonatal sepsis cases. All 16S rDNA-DGGE positive samples were associated with some other signs of neonatal sepsis. CONCLUSION: Our study shows that the molecular approach with 16S rDNA-DGGE identifies twofold more pathogenic bacteria than bacteriological culture, including complex bacterial communities associated with the development of bacterial sepsis in neonates. What is Known: ⢠Neonatal sepsis affects 2.3% of birth in the NICU with a high mortality risk. ⢠Evidence supports the use of molecular methods as an alternative to blood culture for identification of bacterial associated neonatal sepsis. What is New: ⢠The DGGE gel is a good methodological approach for the identification of bacterial in neonatal blood samples. ⢠This study describes the pattern of electrophoretic mobility obtained by DGGE gels and allows to determine the type of bacteria associated in the development of neonatal sepsis.
Assuntos
Hemocultura , DNA Bacteriano/análise , Eletroforese em Gel de Gradiente Desnaturante , Sepse Neonatal/diagnóstico , RNA Ribossômico 16S/genética , Estudos de Casos e Controles , Feminino , Humanos , Recém-Nascido , Masculino , Sepse Neonatal/sangue , Sepse Neonatal/microbiologiaRESUMO
PROBLEM: Infection of human fetal membranes elicits secretion of pro-inflammatory modulators through its innate immune capacities. We investigated the effect of lipopolysacharide (LPS) and progesterone (P4) upon expression of TLR-4/MyD88, TNFα, IL-6, IL-8, IL-10, and HBD2 on the human amniotic epithelium. METHOD OF STUDY: Explants of the human amniotic epithelium were pre-treated with 0.01, 0.1, and 1.0 µM of P4; then cotreated with 1000 ng/mL LPS. TLR-4 was immuno-detected, and concentrations of MyD88, TNFα, IL-6, IL-8, IL-10, and HBD2 were quantified by ELISA. RESULTS: P4 significantly reduced the expression of LPS-induced TLR-4/MyD88. LPS increased the concentrations of TNFα, IL-6, IL-8, IL-10, and HBD2 by factors of 30-, eight, three, three, and fivefold, respectively. P4 at 1.0 µM was the most effective dose to blunt the secretion of TNFα, IL-6, and HBD-2. RU-486 blocks the effect of P4. CONCLUSION: P4 inhibited LPS-induced TLR-4/MyD88 and pro-inflammatory factors in the human amniotic epithelium. These results could explain partially how P4 can protect the amniotic region of fetal membranes and generate a compensatory mechanism that limits the secretion of pro-inflammatory modulators, which could jeopardize the immune privilege during pregnancy.
Assuntos
Âmnio/citologia , Epitélio/imunologia , Progesterona/imunologia , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Feminino , Regulação da Expressão Gênica , Antagonistas de Hormônios/farmacologia , Humanos , Tolerância Imunológica , Imunidade Inata , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos/imunologia , Mifepristona/farmacologia , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Gravidez , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , beta-Defensinas/genética , beta-Defensinas/metabolismoRESUMO
Rupture of the fetal membranes during human labor is associated with an inflammatory process localized to the maternal-fetal interface. There is evidence that specific leukocytes subsets are attracted to the choriodecidua, and that after homing they condition a local inflammatory microenvironment, possibly being directly involved in rupture of the membranes. In this study our aim was to compare the phenotypes and function of leukocytes located in the placental intervillous blood with peripheral leukocytes obtained before or after labor, including expression of modulators of inflammation in these cells. Flow cytometry revealed that the proportion of CD14(+) cells is increased in intervillous blood, suggesting the participation of monocytes/macrophages during labor. Real time qRT-PCR showed that at term gestation and particularly during labor, placental blood leukocytes adopt a different expression pattern of pro-inflammatory cytokines than leukocytes in peripheral blood, including IL-1beta and IL-1RA. During labor, both placental and peripheral leukocytes increase their secretion of matrix metalloproteinase-9. Moreover, we showed that placental leukocytes respond differently than peripheral leukocytes to bacterial lipopolysaccharide, secreting differential amounts of TNF-alpha, IL-1beta and IL-6. Finally, a preliminary proteomic characterization of placental leukocytes revealed a significantly higher number of individual proteins than in peripheral leukocytes. Our results support the existence of selective subsets of leukocytes recruited to the maternal-fetal interface that may participate in the triggering of parturition.
Assuntos
Trabalho de Parto/imunologia , Leucócitos/imunologia , Placenta/imunologia , Membranas Extraembrionárias/imunologia , Feminino , Humanos , Inflamação/imunologia , Proteína Antagonista do Receptor de Interleucina 1/imunologia , Interleucina-1beta/imunologia , Interleucina-6/imunologia , Receptores de Lipopolissacarídeos/imunologia , Macrófagos/imunologia , Metaloproteinase 9 da Matriz/imunologia , Monócitos/imunologia , Polissacarídeos Bacterianos/imunologia , Gravidez , Fator de Necrose Tumoral alfa/imunologiaRESUMO
BACKGROUND: The human labor is an inflammatory process invading leukocytes modulated by gestational tissues. The local increase of cell adhesion molecules (CAMs) promotes the permanence of these leukocytes in the coriodecidua. Gestational tissues express ICAM-1, while circulating leukocytes expressing its ligand Mac-1. OBJECTIVE: To analyze, first, if the expression of CAMs in the fetal membranes is associated with progress of gestational age, and second, the expression of CAMs on circulating leukocytes in the uterus (placenta). MATERIAL AND METHOD: original and closed study conducted at the Instituto Nacional de Perinatologia Isidro Espinosa de los Reyes (Mexico City). We included samples from healthy women between 15 and 44 years of age with term pregnancies (> or =37 weeks gestation). RESULTS: Real time PCR analysis showed that the expression of CAMs in the fetal membranes remained constant before labor. ICAM3 and ICAM1 tended to increase during labor, while ICAM2, VCAM1, SELE and SELP decrease with advancing gestational age. Placental leukocytes showed a clear increase in the expression of ITGAM (Mac-1) during labor. CONCLUSIONS: These results show that the maternal-fetal interface expresses a specific combination of CAMs during labor, including ICAM1, ICAM-3 and Mac-1. The expression of these molecules could promote the retention of leukocytes in the local tissues to modulate the local inflammatory microenvironment during human labor.
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Moléculas de Adesão Celular/metabolismo , Membranas Extraembrionárias/metabolismo , Trabalho de Parto/metabolismo , Leucócitos/metabolismo , Terceiro Trimestre da Gravidez/metabolismo , Adolescente , Adulto , Distinções e Prêmios , Células Sanguíneas/metabolismo , Quimiotaxia de Leucócito , Sistemas Computacionais , Feminino , Idade Gestacional , Ginecologia , Humanos , Inflamação , México , Obstetrícia , Placenta/citologia , Placenta/metabolismo , Reação em Cadeia da Polimerase , Gravidez , Adulto JovemRESUMO
Premature rupture of chorioamniotic membranes complicated with intrauterine infection has been associated to degradation of extracellular matrix (ECM), which could explain local morphological changes. We used a culture system in which the chorioamniotic membranes form two independent chambers, allowing for the selective stimulation of either the amnion (AMN) and/or the choriodecidua (CHD) regions. Lipopolysaccharide (500 ng/ml) was added to the AMN and/or the CHD; secretions and gelatinolytic activity of matrix metalloproteinase (MMP)-2 and MMP-9 were measured in both compartments by enzyme-linked immunosorbent assay (ELISA) and zymography. Secretions of TIMP-1, TIMP-2 and TIMP-4 were measured by ELISA. Both metalloproteinases were immunolocalized in tissue sections. All stimulation modalities induced a similar proMMP-2 and proMMP-9 secretion pattern in the CHD with concentrations of 2.49 ng/ml and 90.91 pg/ml, respectively; the AMN showed no significant changes. The active forms of both enzymes did not change with any stimulation modality. TIMP-1, TIMP-2 and TIMP-4 secretions remained without significant changes (P = 0.41). ECM degradation and structural disarrangement were evident after stimulation. Secretion of proMMP-2 and proMMP-9 mainly in the CHD, presence of active forms associated to the tissue and minor changes in TIMPs secretion could favor ECM degradation and explain the weakening and thinning associated with the pathological rupture of chorioamniotic membranes.
Assuntos
Membranas Extraembrionárias/enzimologia , Lipopolissacarídeos/farmacologia , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Âmnio/efeitos dos fármacos , Âmnio/enzimologia , Âmnio/ultraestrutura , Decídua/efeitos dos fármacos , Decídua/enzimologia , Decídua/ultraestrutura , Ensaio de Imunoadsorção Enzimática , Matriz Extracelular/metabolismo , Membranas Extraembrionárias/efeitos dos fármacos , Membranas Extraembrionárias/ultraestrutura , Feminino , Humanos , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Inibidores Teciduais de Metaloproteinases/metabolismo , Inibidor Tecidual 4 de MetaloproteinaseRESUMO
OBJECTIVE: This study evaluated the secretions of zymogen and active forms of matrix metalloproteinase (MMP)-9 and MMP-2 and their specific inhibitors, TIMP-1 and TIMP-2 by fetal membranes stimulated with group B Streptoccocci (GBS). METHODS: We used an in vitro experimental model that allowed us to estimate the individual contribution of the amnion (AM) and the choriodecidua (CHD) to the microbial insult. Membranes were obtained after delivery by elective cesarean delivery from women at 37 to 40 weeks of gestation without evidence of either active labor or intrauterine infection. Membranes were mounted in Transwell devices (Costar, New York, NY), physically separating the upper and lower chambers; 1 x 10(6) CFU of GBS was added to either AM or CHD and the secretions and gelatinolytic activity of MMP-2 and MMP-9 were measured in both compartments by enzyme-linked immunosorbent assay (ELISA) and zymography. TIMPs secretion was measured by ELISA. Both MMPs were immunolocalized in tissue sections. RESULTS: The simultaneous stimulation at both sides was followed by increases of proMMP-9 (85.0 +/- 18.63 pg/mL) and proMMP-2 (4.10 +/- 1.90 ng/mL) in the CHD (P <.05). When only one side of the membrane was stimulated, the secretion level of proMMP-2 increased 2.3-fold and that of proMMP-9 2.5-fold in the CHD. The active forms of both enzymes did not change with any modality of stimulation. The secretion level of both TIMPs remained without significant changes. CHD and AM were positive for immunoreactive MMP-2 and MMP-9. CONCLUSION: We propose that infection of fetal membranes with GBS is followed by active secretion of MMP and the CHD is the principal source of these mediators of extracellular matrix degradation.
Assuntos
Membranas Extraembrionárias/metabolismo , Membranas Extraembrionárias/microbiologia , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Complicações Infecciosas na Gravidez/fisiopatologia , Infecções Estreptocócicas/fisiopatologia , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Matriz Extracelular/metabolismo , Feminino , Ruptura Prematura de Membranas Fetais/microbiologia , Ruptura Prematura de Membranas Fetais/fisiopatologia , Humanos , Imuno-Histoquímica , Gravidez , Terceiro Trimestre da Gravidez , Streptococcus agalactiae , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Inibidor Tecidual de Metaloproteinase-2/metabolismoRESUMO
Ascendant colonization of pathogenic microorganisms from the vagina to the uterus is strongly associated to preterm labour and premature rupture of membranes. This study evaluated the secretion of interleukin (IL)-1beta, tumour necrosis factor (TNF)alpha, IL-6, prostaglandin E(2) (PGE(2)), and metalloproteinases 9 and 2 by the human chorioamnion stimulated with Candida albicans. Chorioamniotic membranes were obtained after delivery by elective Cesarean section from women at 37-40 weeks of gestation without evidence of active labour. The membranes were mounted in Transwell devices that form two independent compartments, which allow testing the individual responses and contributions of the amnion and choriodecidua. One million CFU ml(-1) of C. albicans was added to either the amniotic or choriodecidual surface and secretions of the markers were measured in both compartments using specific enzyme-linked immunosorbent assay and zymography. Fetal membranes followed different secretion patterns of proinflammatory cytokines depending on the side to which the stimulus was applied. IL-1beta was produced in higher amounts in the presence of C. albicans when applied to the choriodecidual side; TNFalpha and IL-6 secretion did not change in either the amnion or choriodecidual region. PGE(2) synthesis depicted a different pattern, the amniotic tissue was more responsive than the choriodecidual tissue, and this response tended to be higher even when only the amniotic side was stimulated. Matrix metalloproteinases (MMP)-9 increased after stimulation, being the choriodecidua its main source. Selective stimulation with C. albicans induced a differential secretion of IL-1beta, PGE(2), and MMP-9, resulting from a cooperative and bidirectional communication between the amnion and the choriodecidua.
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Âmnio/imunologia , Candida albicans/imunologia , Córion/imunologia , Dinoprostona/biossíntese , Interleucina-1/biossíntese , Metaloproteinase 9 da Matriz/biossíntese , Humanos , Interleucina-6/biossíntese , Técnicas de Cultura de ÓrgãosRESUMO
Extracellular matrix degradation in fetal membranes leading to its rupture is coupled to myometrial activity and cervical ripening during human normal labor. Mechanisms which modulate collagen degradation in amniochorion during labor have not been elucidated. Initial characterization of the effect of different blood compartments on connective tissue degradation in amniochorion during human labor was explored. Amniochorion explants were stimulated with plasma of maternal venous blood, umbilical cord blood or placental blood, obtained from women with pregnancies at term, with or without labor. MMP-2 and MMP-9 activities were quantified in conditioned media by gelatin-zymography as an index of connective tissue degradation. Collagen content was measured in tissue explants and collagen fibrils distribution was examined by electron microscopy. Placental plasma from term pregnancies, with or without labor, is enriched with soluble signals that enhance the in vitro MMP-9 production by amniochorion. Accompanying ultrastructural distortion of collagen fibers and demonstration of collagen degradation fragments confirmed induction of extracellular matrix degradation. Control experiments in which MMP-9 activity was blocked with TIMP-1 resulted in inhibition of all the above mentioned changes. These results suggest that placental intervillous space is a functional compartment in which mediators capable to induce collagen degradation in amniochorion are selectively expressed during human labor.
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Âmnio/metabolismo , Córion/metabolismo , Tecido Conjuntivo/metabolismo , Trabalho de Parto/metabolismo , Âmnio/citologia , Âmnio/enzimologia , Córion/citologia , Córion/enzimologia , Tecido Conjuntivo/enzimologia , Feminino , Humanos , Contagem de Leucócitos , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Gravidez , Técnicas de Cultura de TecidosRESUMO
OBJECTIVE: To identify whether soluble products from choriodecidual blood cells stimulated with group B streptococci (GBS) induce connective tissue degradation in human amniochorion. MATERIAL AND METHODS: Blood samples from choriodecidual compartment were collected by direct aspiration from placental cotyledons draining blood and represent local circulating cells. Samples were divided into two aliquots: one was stimulated with GBS (1 X 10(6) CFU/mL) and the other was kept free of bacteria as negative control. After overnight incubation, plasmas were separated. Chorioamnion explants were stimulated with 10% plasma for 12h at 37 degrees C in 5% CO2. MMP-9 proteolytic activity was measured in the supernatants by gelatin-zymography and IL-1beta and TNF-alpha were quantified by ELISA. Distribution of the collagenous fibrils in explants was examined by electron microscopy. STATISTICAL ANALYSIS: three independent experiments on duplicate were carried out and the statistical significance of experimental differences between groups was assessed with ANOVA test. RESULTS: MMP-9, IL-1beta and TNF-alpha production was significantly higher in supernatants from explants co-cultured with choriodecidual plasma from blood previously infected with GBS, compared with control plasma. Accompanying extensive changes of connective tissue arrangement confirm induction of extracellular matrix degradation. CONCLUSIONS: Choriodecidual plasma from blood stimulated with GBS is enriched with biochemical signals that enhance the MMP-9, IL-1alpha and TNF-beta production by amniochorion. These findings suggest that local circulating cells are capable to act in response to GBS choriodecidual infection through extracellular matrix degradation and the consequent rupture of membranes.