Pseudomonas aeruginosa induces spatio-temporal secretion of IL-1ß, TNFα, proMMP-9, and reduction of epithelial E-cadherin in human alveolar epithelial type II (A549) cells.

Pseudomonas aeruginosa, is an opportunistic bacterium with a high prevalence in diverse pulmonary infections. Although several genes are involved in the system of resistance and evasion of the immunological response of the host, little is known about the inflammatory, degradative, and cell-binding response induced by P. aeruginosa in human lung alveolar epithelial cells. The purpose of this study was to determine the cytokine expression (IL-1ß and TNFα), pro matrix metalloproteinases activation (proMMP-2 and proMMP-9), and the effects on the cell-binding adhesion protein (E-cadherin) in an in vitro model of human lung alveolar epithelial cells. A549 cells were stimulated with a different number of colony-forming units of P. aeruginosa for 3, 6, and 24 hours. Subsequently, the culture medium was collected, IL-1ß and TNFα levels were evaluated by ELISA; proMMP-2 and -9 levels were determined by substrate gel zymography, and the MMP-9 and E-cadherin assessed by immunostaining of A549 cells. Our results demonstrated that P. aeruginosa induces mainly the secretion of TNFα, increases actMMP-9 level, and significantly reduces the level of E-cadherin in the A549 cells. In summary, the inflammatory/degradative process induced by P. aeruginosa modulates the expression of the E-cadherin protein. The probable clinical implications of this study suggest the use of inhibitors that reduce the degradative activity of proMMP-9 which will be further explored in the next phase of this study.

Evaluation of the Light-Cycler® SeptiFast Test in Newborns With Suspicion of Nosocomial Sepsis.

BACKGROUND: Nosocomial sepsis (NS) in newborns (NBs) is associated with high mortality rates and low microbial recovery rates. To overcome the latter problem, new techniques in molecular biology are being used. OBJECTIVES: To evaluate the diagnostic efficacy of SeptiFast test for the diagnosis of nosocomial sepsis in the newborn. MATERIALS AND METHODS: 86 blood specimens of NBs with suspected NS (NOSEP-1 Test > 8 points) were analyzed using Light Cycler SeptiFast (LC-SF) a real-time multiplex PCR instrument. The results were analyzed with the Roche SeptiFast Identification Software. Another blood sample was collected to carry out a blood culture (BC). RESULTS: Sensitivity (Sn) and specificity (Sp) of 0.69 and 0.65 respectively, compared with blood culture (BC) were obtained for LC-SF. Kappa index concordance between LC-SF and BC was 0.21. Thirteen (15.11%) samples were BC positive and 34 (31.39%) were positive with LC-SF tests. CONCLUSIONS: Compared with BC, LC-SF allows the detection of a greater number of pathogenic species in a small blood sample (1 mL) with a shorter response time.

Progesterone elicits an inhibitory effect upon LPS-induced innate immune response in pre-labor human amniotic epithelium.

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.

Tissue-specific human beta-defensins (HBD)-1, HBD-2 and HBD-3 secretion profile from human amniochorionic membranes stimulated with Candida albicans in a two-compartment tissue culture system.

BACKGROUND: During intrauterine infection, amniochorionic membranes represent a mechanical and immunological barrier against dissemination of infection. Human beta defensins (HBD)-1, HBD-2, and HBD-3 are key elements of innate immunity that represent the first line of defense against different pathogen microorganisms associated with preterm labor. The aim of this work was to characterize the individual contribution of the amnion (AMN) and choriodecidua (CHD) regions to the secretion of HBD-1, HBD-2 and HBD-3, after stimulation with Candida albicans. METHODS: Full-thickness human amniochorionic membranes were obtained after delivery by elective cesarean section from women at 37-40 wk of gestation with no evidence of active labor. The membranes were cultured in a two-compartment experimental model in which the upper compartment is delimited by the amnion and the lower chamber by the choriodecidual membrane. One million of Candida albicans were added to either the AMN or the CHD face or to both and compartmentalized secretion profiles of HBD-1, HBD-2, and HBD-3 were quantified by ELISA. Tissue immunolocalization was performed to detect the presence of HBD-1, -2, -3 in tissue sections stimulated with Candida albicans. RESULTS: HBD-1 secretion level by the CHD compartment increased 2.6 times (27.30 [20.9-38.25] pg/micrograms protein) when the stimulus with Candida albicans was applied only on this side of the membrane and 2.4 times (26.55 [19.4-42.5] pg/micrograms protein) when applied to both compartments simultaneously. HBD-1 in the amniotic compartment remained without significant changes. HBD-2 secretion level increased significantly in the CHD when the stimulus was applied only to this region (2.49 [1.49-2.95] pg/micrograms protein) and simultaneously to both compartments (2.14 [1.67- 2.91] pg/micrograms protein). When the stimulus was done in the amniotic compartment HBD-2 remained without significant changes in both compartments. HBD-3 remained without significant changes in both compartments regardless of the stimulation modality. Localization of immune-reactive forms of HBD-1, HBD-2, and HBD-3 was carried out by immunohistochemistry confirming the cellular origin of these peptides. CONCLUSION: Selective stimulation of amniochorionic membranes with Candida albicans resulted in tissue-specific secretion of HBD-1 and HBD-2, mainly in the CHD, which is the first region to become infected during an ascending infection.

Preserved ex vivo inflammatory status in decidual cells from women with preterm labor and subclinical intrauterine infection.

OBJECTIVE: To compare the inflammatory response preserved ex vivo by decidual cells isolated from women who experienced preterm labor with and without subclinical intrauterine infection. METHODS: Fetal membranes were obtained after cesarean section from 35 women who delivered before 37 weeks of gestation following spontaneous preterm labor, with no clinical evidence of intrauterine infection. Decidua was microbiologically tested and cultured. Concentrations of anti-inflammatory cytokines (IL-2, IL-4, IL-10), pro-inflammatory cytokines (IL-6, IL-8, IL-1ß and TNF-α), and matrix metalloproteinases (MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9) were measured in the supernatants using Bio-Plex, and prostaglandin E(2) (PGE(2)) was measured by enzyme immunoassay. RESULTS: Subclinical infection was confirmed in 10 women (28.5%). Microorganisms isolated were Ureaplasma urealyticum (4), group B streptococci (3), Gardnerella vaginalis (1), and Escherichia coli (2). We found a significant increase of pro-inflammatory cytokines and a significant decrease of anti-inflammatory cytokines in supernatants from decidual cells obtained from women with preterm labor and subclinical intrauterine infection compared to women without infection. Secretion of MMP-1, MMP-8, MMP-9 and PGE(2) was significantly higher in infected women. Secretion of IL-8 by decidual cells from infected women persisted upon repeated in vitro culture passages. CONCLUSIONS: Almost 30% of idiopathic preterm labor cases were associated with subclinical intrauterine infection, and decidual cells isolated from these cases preserved an ex vivo inflammatory status after in vivo bacterial exposure.